Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques.

Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive 'fit-free' approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and longer spectral emission (580-625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550-585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells

Multiplexed immunofluorescence identifies high stromal CD68+PD‑L1+ macrophages as a predictor of improved survival in triple negative breast cancer

Triple negative breast cancer (TNBC) comprises 10–15% of all breast cancers and has a poor prognosis with a high risk of recurrence within 5 years. PD-L1 is an important biomarker for patient selection for immunotherapy but its cellular expression and co-localization within the tumour immune microenvironment and associated prognostic value is not well defined. We aimed to characterise the phenotypes of immune cells expressing PD-L1 and determine their association with overall survival (OS) and breast cancer-specific survival (BCSS). Using tissue microarrays from a retrospective cohort of TNBC patients from St George Hospital, Sydney (n = 244), multiplexed immunofluorescence (mIF) was used to assess staining for CD3, CD8, CD20, CD68, PD-1, PD-L1, FOXP3 and pan-cytokeratin on the Vectra Polaris™ platform and analysed using QuPath. Cox multivariate analyses showed high CD68+PD-L1+ stromal cell counts were associated with improved prognosis for OS (HR 0.56, 95% CI 0.33–0.95, p = 0.030) and BCSS (HR 0.47, 95% CI 0.25–0.88, p = 0.018) in the whole cohort and in patients receiving chemotherapy, improving incrementally upon the predictive value of PD-L1+ alone for BCSS. These data suggest that CD68+PD-L1+ status can provide clinically useful prognostic information to identify sub-groups of patients with good or poor prognosis and guide treatment decisions in TNBC

Phosphatidylserine Regulates Plasma Membrane Repair through Tetraspanin-Enriched Macrodomains

The integrity of the plasma membrane is critical to cell function and survival. Cells have developed multiple mechanisms to repair damaged plasma membranes. A key process during plasma membrane repair is to limit the size of the damage, which is facilitated by the presence of tetraspanin-enriched rings surrounding damage sites. Here, we identify phosphatidylserine-enriched rings surrounding damaged sites of the plasma membrane, resembling tetraspanin-enriched rings. Importantly, the formation of both the phosphatidylserine- and tetraspanin-enriched rings requires phosphatidylserine and its transfer proteins ORP5 and ORP9. Interestingly, ORP9, but not ORP5, is recruited to the damage sites, suggesting cells acquire phosphatidylserine from multiple sources upon plasma membrane damage. We further demonstrate that ORP9 contributes to efficient plasma membrane repair. Our results thus unveil a role for phosphatidylserine and its transfer proteins in facilitating the formation of tetraspanin-enriched macrodomains and plasma membrane repair

Distribution of raphespinal fibers in the mouse spinal cord

Background: Serotonergic raphespinal neurons and their fibers have been mapped in large mammals, but the non- serotonergic ones have not been studied, especially in the mouse. The present study aimed to investigate the termination pattern of fibers arising from the hindbrain raphe and reticular nuclei which also have serotonergic neurons by injecting the anterograde tracer BDA into them. Results: We found that raphespinal fibers terminate in both the dorsal and ventral horns in addition to lamina 10. There is a shift of the fibers in the ventral horn towards the dorsal and lateral part of the gray matter. Considerable variation in the termination pattern also exists between raphe nuclei with raphe magnus having more fibers terminating in the dorsal horn. Fibers from the adjacent gigantocellular reticular nucleus show similar termination pattern as those from the raphe nuclei with slight difference. Immunofluorescence staining showed that raphespinal fibers were heterogeneous and serotoninergic fibers were present in all laminae but mainly in laminae 1, 2, medial lamina 8, laminae 9 and 10. Surprisingly, immunofluorescence staining on clarified spinal cord tissue revealed that serotoninergic fibers formed bundles regularly in a short distance along the rostrocaudal axis in the medial part of the ventral horn and they extended towards the lateral motor neuron column area. Conclusion: Serotonergic and non-serotonergic fibers arising from the hindbrain raphe and reticular nuclei had similar termination pattern in the mouse spinal cord with subtle difference. The present study provides anatomical foundation for the multiple roles raphe and adjacent reticular nuclei play

TILs immunophenotype in breast cancer predicts local failure and overall survival : analysis in a large radiotherapy trial with long-term follow-up

Aim: To determine the prognostic significance of the immunophenotype of tumour-infiltrating lymphocytes (TILs) within a cohort of breast cancer patients with long-term follow-up. Methods: Multiplexed immunofluorescence and automated image analysis were used to assess the expression of CD3, CD8, CD20, CD68, Fox P3, PD-1 and PD-L1 in a clinical trial of local excision and radiotherapy randomised to a cavity boost or not (n = 485, median follow-up 16 years). Kaplan–Meier and Cox multivariate analysis (MVA) methodology were used to ascertain relationships with local recurrence (LR), overall survival (OS) and disease-free survival (DFS). NanoString BC360 gene expression panel was applied to a subset of luminal patients to identify pathways associated with LR. Results: LR was predicted by low CD8 in MVA in the whole cohort (HR 2.34, CI 1.4–4.02, p = 0.002) and luminal tumours (HR 2.19, CI 1.23–3.92, p = 0.008) with associations with increased stromal components, decreased Tregs (FoxP3), inflammatory chemokines and SOX2. Poor OS was associated with low CD20 in the whole cohort (HR 1.73, CI 1.2–2.4, p = 0.002) and luminal tumours on MVA and low PD-L1 in triple-negative cancer (HR 3.44, CI 1.5–7, p = 0.003). Conclusions: Immunophenotype adds further prognostic data to help further stratify risk of LR and OS even in TILs low-luminal tumours

The parasitic lifestyle of an archaeal symbiont

DPANN archaea are a diverse group of microorganisms characterised by small cells and reduced genomes. To date, all cultivated DPANN archaea are ectosymbionts that require direct cell contact with an archaeal host species for growth and survival. However, these interactions and their impact on the host species are poorly understood. Here, we show that a DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. During these interactions, the nanohaloarchaeon appears to enter, or be engulfed by, the host cell. Our results provide experimental evidence for a predatory-like lifestyle of an archaeon, suggesting that at least some DPANN archaea may have roles in controlling host populations and their ecology

Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer

The interactions of platinum-anthraquinone complexes with cells and their intracellular components

In this study, we have explored how cisplatin, anthraquinones and platinum— anthraquinone complexes interact with cells and their intracellular components. Emphasis has been placed on the interactions the complexes have with DNA, the mechanisms by which they are trafficked into the cell and nucleus to access DNA, and how in turn this affects the cytotoxicity they induce in sensitive and resistant carcinoma cells, as well as healthy cells. The anthraquinone complexes that have been studied include; 103, 1,5C3, Pt(dien)103, Pt(dien)1,5C3 and Pt1C3 (Appendix 1). Examination of the affinity and specificity the complexes had for DNA, involved the use of UV-spectrophotometric and fluorometric titrations as well as two dimensional NMR. Titration of the complexes with DNA revealed hypochromic and bathochromic shifts in the absorption spectra, indicative of intercalation into DNA. The affinity for DNA was found to follow the order: 103 < 1,5C3 < Pt(dien)1C3 < Pt(dien)1,5C3, with Kapp values ranging from 1.4 x 104 to 5.5 x 105 M", indicating that attachment of the Pt(dien) moiety increased the affinity the anthraquinones have for DNA and that a threading intercalator has greater affinity for DNA than a classical intercalator. The DNA binding properties were also examined through monitoring the emission spectra of the anthraquinones. Enhancement of fluorescence intensity was observed for Pt(dien)103, 1,503 and Pt(dien)1,503 and all anthraquinones exhibited a hypsochromic shift in their emission maxima. The studies revealed that the anthraquinones are involved in more than one DNA binding mode over the DNA concentration range studied. Furthermore it was found that changes in emission profile of a chromophore may provide insight into sequence selectivity and/or whether a complex is intercalated or localised within the minor groove. The NMR experiments conducted on Pt(dien)1C3 and TGGCCA showed that the anthraquinone intercalated from the minor groove into the pyrimidine-purine sequences at each end of the hexamer. The intercalation of the complex resulted in the the hexamer retaining its 8- conformation and the Pt(dien) moiety residing in the minor groove in near proximity to G3. The cytotoxicity of the complexes was examined using the MTT assay in a number of cell lines, including; sensitive and resistant carcinoma cells and healthy cells. In A2780 ovarian carcinoma cells the |Cso values followed the trend cisplatin < 1,503 < 1C3 << Pt(dien)1,5C3 << Pt(dien)1C3 This trend was seen to differ between cell lines. The cell death pathway by which the complexes induced cytotoxicity in A2780 cells was examined through the use of Hoechst 33342, propidium iodide and confocal microscopy. Cisplatin and Pt1C3 were seen to trigger apoptosis, while 1,5-subsituted anthraquinones induce necrosis. The accumulation and intracellular distribution of the complexes was examined via two means; as a function of the intercalator, utilising the intrinsic fluorescence of the anthraquinone and using confocal microscopy, and as a function of the platinum attachment by quantifying cellular and nuclear platinum content though GF-AAS. In addition, the inhibitors antimycin, monensin, ouabain and wheat germ agglutinin were employed in both studies to reveal some of the mechanisms responsible for the intracellular distribution in sensitive and resistant carcinoma cells. Several results and conclusions came from these studies. Firstly, accumulation of the complexes is related to glycoproteins at the plasma membrane and/or intracellularly, but this interaction does not occur in cisplatin resistant A2780 cells. Secondly, organelles of the vacuolar system have roles in endocytosis, exocytosis and/or sequestration of the complexes and these pathways are altered in cisplatin resistant A2780 cells. Thirdly, the nuclear accumulation of cisplatin, Pt1C3, Pt(dien)1C3 and Pt(dien)1,5C3 does not occur via interaction with nuclear destined proteins and subsequent translocation through the nuclear pore complex, irrespective of whether the cell line is sensitive or resistant to cisplatin. Lastly, the cellular pathway of the fluorophore and of platinum is not always the same, indicating that cleavage of platinum groups occurs intracellularly

Binding of [Pt(1C3)(dien)]²⁺ to the duplex DNA oligonucleotide 5´-d(TGGCCA)-3´ : the effect of an appended positive charge on the orientation and location of anthraquinone intercalation

The binding of a platinum intercalator complex [Pt(1C3)(dien)]²⁺ (1C3 = 1-[(3-aminopropyl)amino]-anthracene-9,10-dione, dien = 3-azapentane-1,5-diamine) to DNA and to the self-complementary oligonucleotide 5´-d(TGGCCA)-3´ has been investigated by UV-visible spectrophotometry and 2D NMR spectroscopy, respectively. The uncomplexed anthraquinone, 1C3, has an apparent DNA binding constant of 1.4 X 10⁴, similar to that of ethidium bromide. Addition of the coordinatively saturated {Pt(dien)} moiety increases the binding constant to 3.7 X 10⁵ M⁻¹, showing the effect of the increased positive charge introduced by this moiety. Multiple binding modes are evident from the lack of isosbestic points in the titration spectra and the non-linear nature of the half-reciprocal plot used to calculate the binding constant. [Pt(1C3)(dien)]²⁺ forms a 2:1 adduct with 5´-d(TGGCCA)-3´ and is shown by 2D NMR to intercalate primarily in the TG:CA base pairs at the ends of the oligonucleotide with the side chain and {Pt(dien)} situated in the minor groove.8 page(s

Application of Rapid Fluorescence Lifetime Imaging Microscopy (RapidFLIM) to Examine Dynamics of Nanoparticle Uptake in Live Cells

A key challenge in nanomedicine stems from the continued need for a systematic understanding of the delivery of nanoparticles in live cells. Complexities in delivery are often influenced by the biophysical characteristics of nanoparticles, where even subtle changes to nanoparticle designs can alter cellular uptake, transport and activity. Close examination of these processes, especially with imaging, offers important insights that can aid in future nanoparticle design or translation. Rapid fluorescence lifetime imaging microscopy (RapidFLIM) is a potentially valuable technology for examining intracellular mechanisms of nanoparticle delivery by directly correlating visual data with changes in the biological environment. To date, applications for this technology in nanoparticle research have not been explored. A PicoQuant RapidFLIM system was used together with commercial silica nanoparticles to follow particle uptake in glioblastoma cells. Importantly, RapidFLIM imaging showed significantly improved image acquisition speeds over traditional FLIM, which enabled the tracking of nanoparticle uptake into subcellular compartments. We determined mean lifetime changes and used this to delineate significant changes in nanoparticle lifetimes (&gt;0.39 ns), which showed clustering of these tracks proximal to both extracellular and nuclear membrane boundaries. These findings demonstrate the ability of RapidFLIM to track, localize and quantify changes in single nanoparticle fluorescence lifetimes and highlight RapidFLIM as a valuable tool for multiparameter visualization and analysis of nanoparticle molecular dynamics in live cells