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Highlights • Pseudopotentials of amino acids in a protein predict its photon wavelength emissions. • Spectral power density profiles for each protein in the JAK–STAT pathway converge. • Optimal distance for spectral predictions is between 0.70 and 0.80 nm. • Molecular pathways show functions of particles and waves., Cosic discovered that spectral analyses of a protein sequence after each constituent amino acid had been transformed into an appropriate pseudopotential predicted a resonant energy between interacting molecules. Several experimental studies have verified the predicted peak wavelength of photons within the visible or near-visible light band for specific molecules. Here, this concept has been applied to a classic signaling pathway, JAK–STAT, traditionally composed of nine sequential protein interactions. The weighted linear average of the spectral power density (SPD) profiles of each of the eight “precursor” proteins displayed remarkable congruence with the SPD profile of the terminal molecule (CASP-9) in the pathway. These results suggest that classic and complex signaling pathways in cells can also be expressed as combinations of resonance energies.

Highlights • Ubiquitin shows favorable in vivo properties after parenteral application. • Ubiquitin can bind unspecifically to CXCR4-expressing cell lines. • Ubiquitin does not elicit signaling via CXCR4-mediated pathways. • Ubiquitin is not a ligand of CXCR4. • Ubiquitin is suitable as scaffold for therapeutic applications., In the search for effective therapeutic strategies, protein-based biologicals are under intense development. While monoclonal antibodies represent the majority of these drugs, other innovative approaches are exploring the use of scaffold proteins for the creation of binding molecules with tailor-made properties. Ubiquitin is especially suited for this strategy due to several key characteristics. Ubiquitin is a natural serum protein, 100% conserved across the mammalian class and possesses high thermal, structural and proteolytic stability. Because of its small size and lack of posttranslational modifications, it can be easily produced in Escherichia coli. In this work we provide evidence that ubiquitin is safe as tested experimentally in vivo. In contrast to previously published results, we show that, in our hands, ubiquitin does not act as a functional ligand of the chemokine receptor CXCR4. Cellular assays based on different signaling pathways of the receptor were conducted with the natural agonist SDF-1 as a benchmark. In none of the assays could a response to ubiquitin treatment be elicited. Furthermore, intravenous application to mice at high concentrations did not induce any detectable effect on cytokine levels or hematological parameters.

Objective The aim of this study was to verify the hypothesis originated from bioinformatics and literature reviews that hnNRP A1 may be a new immune target of Behçet's disease (BD). Methods First, bioinformatics was used to show the correlation between hnRNP A1 and A2/B1 in amino acid sequences and three dimensional structures. Second, hnRNP A1 was expressed, purified, and immunologically confirmed by systematic immunology methods including: Western blotting, immunoprecipitation and Dot-ELISA. Then, ELISA was used to screen the anti-hnRNP A1 autoantibodies in newly confirmed clinical samples and the clinical significance was compared between anti-hnRNP A1 antibody positive and negative groups. Finally, the endothelial cells antigen profile of one anti-hnRNP A1 antibody positive BD patient was detected using immunoprecipitation with liquid chromatography tandem mass spectrometry (LC–TMS). Results In total 720 subjects enrolled and tested in this study. Our results demonstrated hnRNP A1 as a new immune target of BD. The reactivity of BD serum IgG antibodies against hnRNP A1 was significantly higher than healthy controls (P, Highlights • Bioinformatics was used to predict that hnRNP A1 may play a role in BD. • HnRNP A1 was immunologically confirmed as an autoantigen of BD. • Deep vein thrombosis has a close relationship with anti-hnRNP A1 antibody in patients' blood circulation. Behçet's disease (BD) is a chronic systemic autoimmune disease. The pathogenesis of BD is still not clear, and the diagnosis is based on typical clinical syndromes. Autoantigen identification was considered a key to solve this problem. This study was to verify the hypothesis suggested by bioinformatics that hnRNP A1 may be a new autoantigen of BD. Among the 720 subjects enrolled and systemic tested, our results demonstrated hnRNP A1 as a new autoantigen of BD, and associated with deep vein thrombosis.

K+–Cl− co-transporter (KCC2) and Na+–K+–2Cl− co-transporter (NKCC1) are the main regulators of neuronal intracellular chloride concentration; altered expression patterns of KCC2 and NKCC1 have been reported in several neurodegenerative diseases. In this paper, we show the effect of repeated stress on KCC2, NKCC1, and serine 940 phosphorylated KCC2 (pKCC2ser940) immunoreactivity.The data were obtained from the hippocampus of female mice using single-plane confocal microscopy images. The mean fluorescence intensity of the perisomatic area of neurons, defined as raw fluorescence intensity (RFI) was calculated. Repeated stress (RS) resulted in a decrease in perisomatic area of immunoreactive (IR)-KCC2 and an increase of the IR-NKCC1. In addition, RS decreased perisomatic IR-pKCC2ser940, corresponding to that of KCC2. The data in this article support the results of a previous study [1] and provide the details of immunohistological methods. Interpretation of the data in this article can be found in “Repeated stress-induced expression pattern alterations of the hippocampal chloride transporters KCC2 and NKCC1 associated with behavioral abnormalities in female mice” by Tsukahara et al. [1]. Keywords: KCC2, NKCC1, repeated stress, IHC

Introduction Bone metastasis is the most common complication of advanced breast cancer. The associated cancer-induced bone disease is treated with bone-sparing agents like zoledronic acid. Clinical trials have shown that zoledronic acid also reduces breast cancer recurrence in bone; potentially by modifying the bone microenvironment surrounding disseminated tumour cells. We have characterised the early effects of zoledronic acid on key cell types of the metastatic niche in vivo, and investigated how these modify the location of breast tumour cells homing to bone. Methods Female mice were treated with a single, clinically achievable dose of zoledronic acid (100 μg/kg) or PBS. Bone integrity, osteoclast and osteoblast activity and number/mm trabecular bone on 1, 3, 5 and 10 days after treatment were assessed using μCT, ELISA (TRAP, PINP) and bone histomorphometry, respectively. The effect of zoledronic acid on osteoblasts was validated in genetically engineered mice with GFP-positive osteoblastic cells. The effects on growth plate cartilage were visualised by toluidine blue staining. For tumour studies, mice were injected i.c. with DID-labelled MDA-MB-231-NW1-luc2 breast cancer cells 5 days after zoledronic acid treatment, followed by assessment of tumour cell homing to bone and soft tissues by multiphoton microscopy, flow cytometry and ex vivo cultures. Results As early as 3 days after treatment, animals receiving zoledronic acid had significantly increased trabecular bone volume vs. control. This rapid bone effect was reflected in a significant reduction in osteoclast and osteoblast number/mm trabecular bone and reduced bone marker serum levels (day 3–5). These results were confirmed in mice expressing GFP in osteoblastic linage cells. Pre-treatment with zoledronic acid caused accumulation of an extra-cellular matrix in the growth plate associated with a trend towards preferential [1] homing of tumour cells to osteoblast-rich areas of bone, but without affecting the total number of tumour cells. The number of circulating tumour cells was reduced in ZOL treated animals. Conclusion A single dose of zoledronic acid caused significant changes in the bone area suggested to contain the metastatic niche. Tumour cells arriving in this modified bone microenvironment appeared to preferentially locate to osteoblast-rich areas, supporting that osteoblasts may be key components of the bone metastasis niche and therefore a potential therapeutic target in breast cancer., Highlights • A single, clinically relevant, dose of zoledronic acid rapidly reduces activity and number of both osteoblasts and osteoclasts in vivo. • This is accompanied by significant increased extra-cellular matrix content in areas of the metaphysis comprising the bone metastatic niche. • Breast cancer cells appear to preferentially home to osteoblast- and extra-cellular matrix rich regions of the metaphysis. • Osteoblasts are suggested to be a key component of the bone metastatic niche.

The cornea is the main refracting lens in the eye. As part of the outer tunic it has to be resilient, a property conferred by the organisation of the constituent collagen. It also has to be sufficiently elastic to regain its exact shape when deformed, in order not to distort the retinal image. The basis of this elasticity is not fully understood. The purpose of this study was to characterise in three dimensions the arrangement and distribution of elastic fibers in the human corneal stroma, using serial block face scanning electron microscopy. We have demonstrated that there exists a complex network of elastic fibers that appear to originate in the sclera or limbus. These appear as elastic sheets in the limbus and peripheral cornea immediately above the trabecular meshwork which itself appears to extend above Descemet's membrane in the peripheral stroma. From these sheets, elastic fibers extend into the cornea; moving centrally they bifurcate and trifurcate into narrower fibers and are concentrated in the posterior stroma immediately above Descemet's membrane. We contend that elastic sheets will play an important role in the biomechanical deformation and recovery of the peripheral cornea. The network may also have practical implications for understanding the structural basis behind a number of corneal surgeries., Graphical abstract, Highlights • We have characterised a complex system of elastic fibers in the posterior cornea. • These exist as sheets in the limbus that fenestrate and become fibers in the cornea. • We believe these fibers provide the elastic restoring force in the peripheral tissue. • This system should help explain some surgical properties of the cornea.

Myeloid cells make a pivotal contribution to tissue homeostasis during inflammation. Both tissue-specific resident populations and infiltrating myeloid cells can cause tissue injury through aberrant activation and/or dysregulated activity. Reliable identification and quantification of myeloid cells within diseased tissues is important to understand pathological inflammatory processes. Flow cytometry is a valuable technique for leukocyte analysis, but a standardized flow cytometric method for myeloid cell populations in the eye is lacking. Here, we validate a reproducible flow cytometry gating approach to characterize myeloid cells in several commonly used models of ocular inflammation. We profile and quantify myeloid subsets across these models, and highlight the value of this strategy in identifying phenotypic differences using Ccr2-deficient mice. This method will aid standardization in the field and facilitate future investigations into the roles of myeloid cells during ocular inflammation.

We report here the cleavage of the c-Jun N-terminal Kinase (JNK) pathway scaffold protein, JNK Interacting Protein-1 (JIP1), by caspases during both Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and staurosporine-induced apoptosis in HeLa cells. During the initiation of apoptosis, maximal JNK activation is observed when JIP1 is intact, whereas cleavage of JIP1 correlates with JNK inactivation and progression of apoptosis. JIP1 is cleaved by caspase-3 at two sites, leading to disassembly of the JIP1/JNK complex. Inhibition of JIP1 cleavage by the caspase-3 inhibitor DEVD.fmk inhibits this disassembly, and is accompanied by sustained JNK activation. These data suggest that TRAIL and staurosporine induce JNK activation in a caspase-3-independent manner and that caspase-3-mediated JIP1 cleavage plays a role in JNK inactivation via scaffold disassembly during the execution phase of apoptosis. Caspase-mediated cleavage of JIP scaffold proteins may therefore represent an important mechanism for modulation of JNK signalling during apoptotic cell death.

Benzene, a known occupational and environmental contaminant, is associated with increased risk of leukemia. The objectives of this study were to elucidate the regulatory mechanism of the hypomethylated STAT3 involved in benzene toxicity in vitro. As 1,4-benzoquinone (1,4-BQ) is one of benzene’s major toxic metabolites, AHH-1 cells were treated by 1,4-BQ for 24h with or without pretreatment of the antioxidant a-LA or the methyltransferase inhibitor, 5-aza-2′ deoxycytidine (5-aza). The cell viability was investigated using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ROS was determined via 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) flow cytometric assays. The level of oxidative stress marker 8-OHdG was measured by enzyme-linked immunosorbent assay. Methylation-specific PCR was used to detect the methylation status of STAT3. Results indicated the significantly increasing expression of ROS and 8-OHdG which accompanied with STAT3 hypomethylation in 1,4-BQ-treated AHH-1 cells. α-LA suppressed the expression of both ROS and 8-OHdG, simultaneously reversed 1,4-BQ-induced STAT3 hypomethylation. However, although the methylation inhibitor, 5-aza reduced the expression level of ROS and 8-OHdG, but had no obvious inhibiting effect on STAT3 methylation level. Taken together, oxidative stress are involved 1,4-BQ-induced STAT3 methylation expression.

Sufficient sulfate supply has been linked to the development of sulfur induced resistance or sulfur enhanced defense (SIR/SED) in plants. In this study we investigated the effects of sulfate (S) supply on the response of genetically resistant tobacco (Nicotiana tabacum cv. Samsun NN) to Tobacco mosaic virus (TMV). Plants grown with sufficient sulfate (+S plants) developed significantly less necrotic lesions during a hypersensitive response (HR) when compared to plants grown without sulfate (−S plants). In +S plants reduced TMV accumulation was evident on the level of viral RNA. Enhanced virus resistance correlated with elevated levels of cysteine and glutathione and early induction of a Tau class glutathione S-transferase and a salicylic acid-binding catalase gene. These data indicate that the elevated antioxidant capacity of +S plants was able to reduce the effects of HR, leading to enhanced virus resistance. Expression of pathogenesis-related genes was also markedly up-regulated in +S plants after TMV-inoculation. On the subcellular level, comparison of TMV-inoculated +S and −S plants revealed that +S plants contained 55–132 % higher glutathione levels in mitochondria, chloroplasts, nuclei, peroxisomes and the cytosol than −S plants. Interestingly, mitochondria were the only organelles where TMV-inoculation resulted in a decrease of glutathione levels when compared to mock-inoculated plants. This was particularly obvious in −S plants, where the development of necrotic lesions was more pronounced. In summary, the overall higher antioxidative capacity and elevated activation of defense genes in +S plants indicate that sufficient sulfate supply enhances a preexisting plant defense reaction resulting in reduced symptom development and virus accumulation., Highlights ► Sulfate as possible enhancer of plant defense during virus infection. ► Sulfate fertilization reduces symptom severity and virus contents. ► Enhanced cysteine and glutathione metabolism up-regulates defense gene expression. ► Sulfate fertilization enhances plant defense during virus infection.

Leukocytes invade newly formed thrombi through interactions with platelets and fibrin and later contribute to the removal of fibrin deposits mainly through the action of neutrophil elastase. The present study attempts to express in quantitative terms the impact of neutrophils on the lytic processes in obliterative thrombi based on the local presence of elastase-specific fibrin degradation products (NE-FDP) in relation to the leukocyte, platelet and fibrin content of thrombi. Immunofluorescent detection of fibrin, NE-FDP and platelet antigens was performed in sections of thrombi from 28 patients subjected to thrombectomy in combination with DNA-staining for identification of nucleated cells. The digitalized fluorescent microscopic images were decomposed according to the color channel of each thrombus constituent. The integrated intensity values for all thrombus constituents were statistically evaluated with correlation, hierarchical agglomerative clustering , Hotelling'sT2 and F-statistics. Association between NE-FDP and leukocyte content of thrombi is evidenced by a significant Pearson correlation coefficient of 0.71 (p=0.00002). Cluster analysis reveals two classes of thrombi according to NE-FDP, leukocyte and platelet content and also two according to NE-FDP, leukocyte and fibrin content. When NE-FDP, fibrin and platelet content is normalized to the leukocyte count in the same thrombus, clusters with platelet-related thrombolytic resistance (inversely related NE-FDP and platelet content) and advanced cell-dependent thrombolysis (inversely related NE-FDP and fibrin content) are identified. These distinct patterns of thrombus constituents are snapshots of characteristic stages in the cell-dependent thrombolysis, which indicate a clot-stabilizing role for platelets in this process similar to their impact on the plasmin-dependent lysis.

Green algae have a great potential as biofactories for the production of proteins. Chlamydomonas reinhardtii, a representative of eukaryotic microalgae, has been extensively used as a model organism to study light-induced gene expression, chloroplast biogenesis, photosynthesis, light perception, cell–cell recognition, and cell cycle control. However, little is known about the glycosylation machinery and N-linked glycan structures of green algae. In this study, we performed mass spectrometry analysis of N-linked oligosaccharides released from total extracts of Chlamydomonas reinhardtii and demonstrated that C. reinhardtii algae possess glycoproteins with mammalian-like sialylated N-linked oligosaccharides. These findings suggest that C. reinhardtii may be an attractive system for expression of target proteins.

The human pentraxin proteins, serum amyloid P component (SAP) and C‐reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non‐specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL‐6 or IL‐8, nor does SAP cause release of IL‐1β or IL‐10. Furthermore neither of our preparations was pro‐inflammatory in mice in vivo.

Berberine (BBR) is a renowned natural compound that exhibits potent neuroprotective activities. However, the cellular and molecular mechanisms are still unclear. Hormesis is an adaptive mechanism generally activated by mild oxidative stress to protect the cells from further damage. Many phytochemicals have been shown to induce hormesis. This study aims to investigate whether the neuroprotective activity of BBR is mediated by hormesis and the related signaling pathways in 6-OHDA-induced PC12 cells and zebrafish neurotoxic models. Our results demonstrated that BBR induced a typical hormetic response in PC12 cells, i.e. low dose BBR significantly increased the cell viability, while high dose BBR inhibited the cell viability. Moreover, low dose BBR protected the PC12 cells from 6-OHDA-induced cytotoxicity and apoptosis, whereas relatively high dose BBR did not show neuroprotective activity. The hormetic and neuroprotective effects of BBR were confirmed to be mediated by up-regulated PI3K/AKT/Bcl-2 cell survival and Nrf2/HO-1 antioxidative signaling pathways. In addition, low dose BBR markedly mitigated the 6-OHDA-induced dopaminergic neuron loss and behavior movement deficiency in zebrafish, while high dose BBR only slightly exhibited neuroprotective activities. These results strongly suggested that the neuroprotection of BBR were attributable to the hormetic mechanisms via activating cell survival and antioxidative signaling pathways. Keywords: Berberine, Hormesis, Neuroprotection, PC12 cells, Zebrafish

The procyclic form of Trypanosoma brucei expresses procyclin surface glycoproteins with unusual glycosylphosphatidylinositol-anchor side chain structures that contain branched N-acetyllactosamine and lacto-N-biose units. The glycosyltransferase TbGT8 is involved in the synthesis of the branched side chain through its UDP-GlcNAc: βGal β1-3N-acetylglucosaminyltransferase activity. Here, we explored the role of TbGT8 in the mammalian bloodstream form of the parasite with a tetracycline-inducible conditional null T. brucei mutant for TbGT8. Under non-permissive conditions, the mutant showed significantly reduced binding to tomato lectin, which recognizes poly-N-acetyllactosamine-containing glycans. Lectin pull-down assays revealed differences between the wild type and TbGT8 null-mutant T. brucei, notably the absence of a broad protein band with an approximate molecular weight of 110 kDa in the mutant lysate. Proteomic analysis revealed that the band contained several glycoproteins, including the acidic ecto-protein phosphatase AcP115, a stage-specific glycoprotein in the bloodstream form of T. brucei. Western blotting with an anti-AcP115 antibody revealed that AcP115 was approximately 10 kDa smaller in the mutant. Enzymatic de-N-glycosylation demonstrated that the underlying protein cores were the same, suggesting that the 10-kDa difference was due to differences in N-linked glycans. Immunofluorescence microscopy revealed the colocalization of hemagglutinin epitope-tagged TbGT8 and the Golgi-associated protein GRASP. These data suggest that TbGT8 is involved in the construction of complex poly-N-acetyllactosamine-containing type N-linked and GPI-linked glycans in the Golgi of the bloodstream and procyclic parasite forms, respectively., Graphical abstract, Highlights • TbGT8 is involved in N-linked glycan synthesis in the bloodstream form. • AcP115 is a target glycoprotein of TbGT8-dependent glycan processing. • TbGT8 is localized in the Golgi and modified by N-linked glycan(s).

Highlights • α4β2∗ nAChRs stimulate IPSCs in FS interneurons, in layer V of the mouse PFC (Fr2). • In P16–P63 mice, nicotine increased both IPSC and mIPSC frequencies. • GABAergic terminals adjacent to PV+ cells expressed α4 nAChR. • The percentage of FS cells with somatic α4β2∗ currents decreased with age. • Hence, nAChRs may be able to induce local circuit disinhibition in Fr2 PFC., Nicotinic acetylcholine receptors (nAChRs) produce widespread and complex effects on neocortex excitability. We studied how heteromeric nAChRs regulate inhibitory post-synaptic currents (IPSCs), in fast-spiking (FS) layer V neurons of the mouse frontal area 2 (Fr2). In the presence of blockers of ionotropic glutamate receptors, tonic application of 10 μM nicotine augmented the spontaneous IPSC frequency, with minor alterations of amplitudes and kinetics. These effects were studied since the 3rd postnatal week, and persisted throughout the first two months of postnatal life. The action of nicotine was blocked by 1 μM dihydro-β-erythroidine (DHβE; specific for α4∗ nAChRs), but not 10 nM methyllycaconitine (MLA; specific for α7∗ nAChRs). It was mimicked by 10 nM 5-iodo-3-[2(S)-azetidinylmethoxy]pyridine (5-IA; which activates β2∗ nAChRs). Similar results were obtained on miniature IPSCs (mIPSCs). Moreover, during the first five postnatal weeks, approximately 50% of FS cells displayed DHβE-sensitive whole-cell nicotinic currents. This percentage decreased to ∼5% in mice older than P45. By confocal microscopy, the α4 nAChR subunit was immunocytochemically identified on interneurons expressing either parvalbumin (PV), which mainly labels FS cells, or somatostatin (SOM), which labels the other major interneuron population in layer V. GABAergic terminals expressing α4 were observed to be juxtaposed to PV-positive (PV+) cells. A fraction of these terminals displayed PV immunoreactivity. We conclude that α4β2∗ nAChRs can produce sustained regulation of FS cells in Fr2 layer V. The effect presents a presynaptic component, whereas the somatic regulation decreases with age. These mechanisms may contribute to the nAChR-dependent stimulation of excitability during cognitive tasks as well as to the hyperexcitability caused by hyperfunctional heteromeric nAChRs in sleep-related epilepsy.

Peroxiredoxin 2 has immune regulatory functions, but its expression in human peripheral blood lymphocytes and levels in extracellular fluid in healthy subjects and rheumatoid arthritis patients are poorly described. In the present study, the median intracellular peroxiredoxin 2 protein content of lymphocytes from rheumatoid arthritis patients was more than two-fold higher compared with healthy subjects’ lymphocytes. Intracellular peroxiredoxin 3 levels were similar in healthy and rheumatoid arthritis lymphocytes. Flow cytometry detected peroxiredoxin 2 on the surface of ca. 8% of T cells and ca. 56% of B cells (median % values) of all subjects analyzed. Exofacial thioredoxin-1 was also observed. In the total lymphocyte population from rheumatoid arthritis patients, few cells (median, 6%) displayed surface peroxiredoxin 2. In contrast, a significantly increased proportion of interleukin-17+ve lymphocytes were exofacially peroxiredoxin 2+ve (median, 39%). Prdx2 was also detected in human extracellular fluids. We suggest that crucial inflammatory cell subsets, i.e. interleukin-17+ve T cells, exhibit increased exofacial redox-regulating enzymes and that peroxiredoxin 2 may be involved in the persistence of pro-inflammatory cells in chronic inflammation.

In a comparison of sialidase activities toward N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), we found that Salmonella typhimurium LT2 sialidase (STSA) hardly cleaved 4-methylumbelliferyl Neu5Gc (4MU-Neu5Gc). The kcat/Km value of STSA for 4MU-Neu5Gc was found to be 110 times lower than that for 4-methylumbelliferyl Neu5Ac (4MU-Neu5Ac). Additionally, STSA had remarkably weak ability to cleave α2-3-linked-Neu5Gc contained in gangliosides and equine erythrocytes. In silico analysis based on first-principle calculations with transition-state analogues suggested that the binding affinity of Neu5Gc2en is 14.3 kcal/mol more unstable than that of Neu5Ac2en. The results indicated that STSA preferentially cleaves Neu5Ac residues rather than Neu5Gc residues, which is important for anyone using this enzyme to cleave α2-3-linked sialic acids., Highlights • Salmonella typhimurium LT2 sialidase (STSA) hardly cleaved 4MU-Neu5Gc. • STSA hardly cleaved Neu5Gc residues in ganglioside and equine erythrocytes. • The kcat/Km value of STSA for 4MU-Neu5Gc was 110 times lower than that for 4MU-Neu5Ac. • Binding affinity of Neu5Gc2en is more unstable (by 14.3 kcal/mol) than that of Neu5Ac2en. • Amino acid residues that recognize sialyl α2-3 linkage also discriminate Neu5Gc and Neu5Ac.

Age-related Macular Degeneration (AMD) is a common, irreversible blinding condition that leads to the loss of central vision. AMD has a complex aetiology with both genetic as well as environmental risks factors, and share many similarities with Alzheimer's disease. Recent findings have contributed significantly to unravelling its genetic architecture that is yet to be matched by molecular insights. Studies are made more challenging by observations that aged and AMD retinas accumulate the highly pathogenic Alzheimer's-related Amyloid beta (Aβ) group of peptides, for which there appears to be no clear genetic basis. Analyses of human donor and animal eyes have identified retinal Aβ aggregates in retinal ganglion cells (RGC), the inner nuclear layer, photoreceptors as well as the retinal pigment epithelium. Aβ is also a major drusen constituent; found correlated with elevated drusen-load and age, with a propensity to aggregate in retinas of advanced AMD. Despite this evidence, how such a potent driver of neurodegeneration might impair the neuroretina remains incompletely understood, and studies into this important aspect of retinopathy remains limited. In order to address this we exploited R28 rat retinal cells which due to its heterogeneous nature, offers diverse neuroretinal cell-types in which to study the molecular pathology of Aβ. R28 cells are also unaffected by problems associated with the commonly used RGC-5 immortalised cell-line, thus providing a well-established model in which to study dynamic Aβ effects at single-cell resolution. Our findings show that R28 cells express key neuronal markers calbindin, protein kinase C and the microtubule associated protein-2 (MAP-2) by confocal immunofluorescence which has not been shown before, but also calretinin which has not been reported previously. For the first time, we reveal that retinal neurons rapidly internalised Aβ1-42, the most cytotoxic and aggregate-prone amongst the Aβ family. Furthermore, exposure to physiological amounts of Aβ1-42 for 24 h correlated with impairment to neuronal MAP-2, a cytoskeletal protein which regulates microtubule dynamics in axons and dendrites. Disruption to MAP-2 was transient, and had recovered by 48 h, although internalised Aβ persisted as discrete puncta for as long as 72 h. To assess whether Aβ could realistically localise to living retinas to mediate such effects, we subretinally injected nanomolar levels of oligomeric Aβ1-42 into wildtype mice. Confocal microscopy revealed the presence of focal Aβ deposits in RGC, the inner nuclear and the outer plexiform layers 8 days later, recapitulating naturally-occurring patterns of Aβ aggregation in aged retinas. Our novel findings describe how retinal neurons internalise Aβ to transiently impair MAP-2 in a hitherto unreported manner. MAP-2 dysfunction is reported in AMD retinas, and is thought to be involved in remodelling and plasticity of post-mitotic neurons. Our insights suggest a molecular pathway by which this could occur in the senescent eye leading to complex diseases such as AMD., Graphical abstract Image 1, Highlights • Molecular basis of complex retinopathies such as AMD is incompletely understood. • The Alzheimer's-related Aβ peptides are rapidly internalised by retinal neurons. • Internalised Aβ is retained within neurons and transiently impairs MAP-2. • Subretinally injected Aβ mimics its naturally-occurring distribution in aged retinas.

Highlights • Drug sensitivity testing identified novel drugs like clofarabine effective in treating gastric cancer. • mRNA sequencing can be used to identify agents with synergistic activity to a reference compound. • Pictilisib sensitizes gastric cancer to clofarabine treatment through AKT inhibition. • The combination of clofarabine and pictilisib inhibits tumor growth in cell lines and PDX models., Gastric cancer (GC) is frequently characterized by resistance to standard chemotherapeutic regimens and poor clinical outcomes. We aimed to identify a novel therapeutic approach using drug sensitivity testing (DST) and our computational SynerySeq pipeline. DST of GC cell lines was performed with a library of 215 Federal Drug Administration (FDA) approved compounds and identified clofarabine as a potential therapeutic agent. RNA-sequencing (RNAseq) of clofarabine treated GC cells was analyzed according to our SynergySeq pipeline and identified pictilisib as a potential synergistic agent. Clonogenic survival and Annexin V assays demonstrated increased cell death with clofarabine and pictilisib combination treatment (P

The membrane bound metalloprotease aminopeptidase N (APN, CD13, EC 3.4.11.2) is a well established marker of normal and malignant cells of the myelo-monocytic lineage. It is also expressed by leukaemic blasts of a small group of patients suffering from acute or chronic lymphoid leukaemia. Recently, the expression of the APN gene in T cell lines as well as the induction of APN gene and surface expression in human peripheral T cells by mitogenic activation have been demonstrated. Here, by means of cytofluorimetric analysis evidence is provided, that the induction of APN surface expression is partially resistent to the action of the inhibitors of protein biosynthesis, puromycin and cycloheximide, and is not prevented by tunicamycin, an inhibitor of glycosylation. These data suggest that the rapid mitogen-induced surface expression of APN, detectable 20 hours after stimulation is dominated by mechanisms not dependent on de novo protein biosynthesis or glycosylation. As shown by simultaneous analyses, the inhibitors used did also differently modify the induction of surface expression of other inducible glycosylated leukocyte surface antigens, namely CD25, CD69 and CD95.