Assessing the effects of common topical exposures on skin bacteria associated with atopic dermatitis.

BACKGROUND: While patients and families struggling with atopic dermatitis (AD) have documented concerns for a contributory role of skin care products in AD pathology, nearly all the skin microbiome studies to date have asked participants to avoid topical products (such as soaps or select medications) for the preceding days to weeks prior to sample collection. Thus, given the established role of the microbiome in AD, the interactions between topical exposures, dysbiosis and AD remains underrepresented in the academic literature. OBJECTIVES: To address this knowledge gap, we expanded our previous evaluations to test the toxicological effects of a broader range of common chemicals, AD treatment lotions, creams and ointments using both health- and AD-associated strains of Roseomonas mucosa and Staphylococcus spp. METHODS: Use of in vitro culture techniques and mouse models were deployed to identify chemicals with dysbiotic or pre-biotic potential. A proof-of-concept study was subsequently performed in healthy volunteers to assess global microbiome shifts after exposure to select chemicals using dermatologic patch testing. RESULTS: Numerous chemicals possessed antibiotic properties, including many not marketed as anti-microbials. Through targeted combination of potentially beneficial chemicals, we identified combinations which promoted the growth of health-associated isolates over disease-associated strains in bacterial culture and enhanced microbe-specific outcomes in an established mouse model of AD; the most promising of which was the combination of citral and colophonium (often sold as lemon myrtle oil and pine tar). Additional studies would likely further optimize the combination of ingredients use. Similar results were seen in the proof-of-concept human studies. CONCLUSIONS: Our results could offer a systematic, multiplex approach to identify which products carry dysbiotic potential and thus may guide formulation of new topicals to benefit patients with AD.

K45A mutation of RIPK1 results in poor necroptosis and cytokine signaling in macrophages, which impacts inflammatory responses in vivo.

Receptor interacting protein kinase 1 (RIPK1) participates in several cell signaling complexes that promote cell activation and cell death. Stimulation of RIPK1 in the absence of caspase signaling induces regulated necrosis (necroptosis), which promotes an inflammatory response. Understanding of the mechanisms through which RIPK1 promotes inflammation has been unclear. Herein we have evaluated the impact of a K45A mutation of RIPK1 on necroptosis of macrophages and the activation of inflammatory response. We show that K45A mutation of RIPK1 results in attenuated necroptosis of macrophages in response to stimulation with LPS, TNFα and IFNß in the absence of caspase signaling. Impairment in necroptosis correlated with poor phosphorylation of RIPK1, RIPK3 and reduced trimerization of MLKL. Furthermore, K45A mutation of RIPK1 resulted in poor STAT1 phosphorylation (at S727) and expression of RANTES and MIP-1α following TNF-R engagement in the absence of caspase activation. Our results further indicate that in the absence of stimulation by pathogen-associated molecular patterns (PAMPs), cellular inhibitors of apoptotic proteins (cIAPs) prevent the K45-dependent auto-phosphorylation of RIPK1, leading to resistance against necroptosis. Finally, RIPK1(K45A) mice displayed attenuated inflammatory response in vivo as they were significantly resistant against endotoxin shock, but highly susceptible against a challenge with Salmonella typhimurium. This correlated with reduced expression of IL-1ß and ROS, and poor processing of caspase 8 by RIPK1(K45A) macrophages. Overall, these results indicate that K45 mediated kinase activity of RIPK1 is not only important for necroptosis but it also has a key role in promoting cytokine signaling and host response to inflammatory stimuli.

Intracellular nicotinamide adenine dinucleotide promotes TNF-induced necroptosis in a sirtuin-dependent manner.

Cellular necrosis has long been regarded as an incidental and uncontrolled form of cell death. However, a regulated form of cell death termed necroptosis has been identified recently. Necroptosis can be induced by extracellular cytokines, pathogens and several pharmacological compounds, which share the property of triggering the formation of a RIPK3-containing molecular complex supporting cell death. Of interest, most ligands known to induce necroptosis (including notably TNF and FASL) can also promote apoptosis, and the mechanisms regulating the decision of cells to commit to one form of cell death or the other are still poorly defined. We demonstrate herein that intracellular nicotinamide adenine dinucleotide (NAD(+)) has an important role in supporting cell progression to necroptosis. Using a panel of pharmacological and genetic approaches, we show that intracellular NAD(+) promotes necroptosis of the L929 cell line in response to TNF. Use of a pan-sirtuin inhibitor and shRNA-mediated protein knockdown led us to uncover a role for the NAD(+)-dependent family of sirtuins, and in particular for SIRT2 and SIRT5, in the regulation of the necroptotic cell death program. Thus, and in contrast to a generally held view, intracellular NAD(+) does not represent a universal pro-survival factor, but rather acts as a key metabolite regulating the choice of cell demise in response to both intrinsic and extrinsic factors.

Absence of RIPK3 predicts necroptosis resistance in malignant melanoma.

Acquired or intrinsic resistance to apoptotic and necroptotic stimuli is considered a major hindrance of therapeutic success in malignant melanoma. Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptotic and necroptotic cell death mediated by numerous cell death signalling platforms. In this report we investigated the impact of IAPs for cell death regulation in malignant melanoma. Suppression of IAPs strongly sensitized a panel of melanoma cells to death ligand-induced cell death, which, surprisingly, was largely mediated by apoptosis, as it was completely rescued by addition of caspase inhibitors. Interestingly, the absence of necroptosis signalling correlated with a lack of receptor-interacting protein kinase-3 (RIPK3) mRNA and protein expression in all cell lines, whereas primary melanocytes and cultured nevus cells strongly expressed RIPK3. Reconstitution of RIPK3, but not a RIPK3-kinase dead mutant in a set of melanoma cell lines overcame CD95L/IAP antagonist-induced necroptosis resistance independent of autocrine tumour necrosis factor secretion. Using specific inhibitors, functional studies revealed that RIPK3-mediated mixed-lineage kinase domain-like protein (MLKL) phosphorylation and necroptosis induction critically required receptor-interacting protein kinase-1 signalling. Furthermore, the inhibitor of mutant BRAF Dabrafenib, but not Vemurafenib, inhibited necroptosis in melanoma cells whenever RIPK3 is present. Our data suggest that loss of RIPK3 in melanoma and selective inhibition of the RIPK3/MLKL axis by BRAF inhibitor Dabrafenib, but not Vemurafenib, is critical to protect from necroptosis. Strategies that allow RIPK3 expression may allow unmasking the necroptotic signalling machinery in melanoma and points to reactivation of this pathway as a treatment option for metastatic melanoma.

Differential roles of RIPK1 and RIPK3 in TNF-induced necroptosis and chemotherapeutic agent-induced cell death.

Apoptosis is a key mechanism for metazoans to eliminate unwanted cells. Resistance to apoptosis is a hallmark of many cancer cells and a major roadblock to traditional chemotherapy. Recent evidence indicates that inhibition of caspase-dependent apoptosis sensitizes many cancer cells to a form of non-apoptotic cell death termed necroptosis. This has led to widespread interest in exploring necroptosis as an alternative strategy for anti-cancer therapy. Here we show that in human colon cancer tissues, the expression of the essential necroptosis adaptors receptor interacting protein kinase (RIPK)1 and RIPK3 is significantly decreased compared with adjacent normal colon tissues. The expression of RIPK1 and RIPK3 was suppressed by hypoxia, but not by epigenetic DNA modification. To explore the role of necroptosis in chemotherapy-induced cell death, we used inhibitors of RIPK1 or RIPK3 kinase activity, and modulated their expression in colon cancer cell lines using short hairpin RNAs. We found that RIPK1 and RIPK3 were largely dispensable for classical chemotherapy-induced cell death. Caspase inhibitor and/or second mitochondria-derived activator of caspase mimetic, which sensitize cells to RIPK1- and RIPK3-dependent necroptosis downstream of tumor necrosis factor receptor-like death receptors, also did not alter the response of cancer cells to chemotherapeutic agents. In contrast to the RIPKs, we found that cathepsins are partially responsible for doxorubicin or etoposide-induced cell death. Taken together, these results indicate that traditional chemotherapeutic agents are not efficient inducers of necroptosis and that more potent pathway-specific drugs are required to fully harness the power of necroptosis in anti-cancer therapy.

Characterization of GSK'963: a structurally distinct, potent and selective inhibitor of RIP1 kinase.

Necroptosis and signaling regulated by RIP1 kinase activity is emerging as a key driver of inflammation in a variety of disease settings. A significant amount has been learned about how RIP1 regulates necrotic cell death through the use of the RIP1 kinase inhibitor Necrostatin-1 (Nec-1). Nec-1 has been a transformational tool for exploring the function of RIP1 kinase activity; however, its utility is somewhat limited by moderate potency, off-target activity against indoleamine-2,3-dioxygenase (IDO), and poor pharmacokinetic properties. These limitations of Nec-1 have driven an effort to identify next-generation tools to study RIP1 function, and have led to the identification of 7-Cl-O-Nec-1 (Nec-1s), which has improved pharmacokinetic properties and lacks IDO inhibitory activity. Here we describe the characterization of GSK'963, a chiral small-molecule inhibitor of RIP1 kinase that is chemically distinct from both Nec-1 and Nec-1s. GSK'963 is significantly more potent than Nec-1 in both biochemical and cellular assays, inhibiting RIP1-dependent cell death with an IC50 of between 1 and 4 nM in human and murine cells. GSK'963 is >10 000-fold selective for RIP1 over 339 other kinases, lacks measurable activity against IDO and has an inactive enantiomer, GSK'962, which can be used to confirm on-target effects. The increased in vitro potency of GSK'963 also translates in vivo, where GSK'963 provides much greater protection from hypothermia at matched doses to Nec-1, in a model of TNF-induced sterile shock. Together, we believe GSK'963 represents a next-generation tool for examining the function of RIP1 in vitro and in vivo, and should help to clarify our current understanding of the role of RIP1 in contributing to disease pathogenesis.

Maladaptation and phenotypic mismatch in hatchery-reared Atlantic salmon Salmo salar released in the wild.

Changes in body shape, fluctuating asymmetry (FA) and crypsis were compared among Atlantic salmon Salmo salar fry kept as controls in captivity and those released and subsequently recaptured in the wild according to a before-after-control-impact (BACI) design. Hatchery fish that survived in the wild became more cryptic and displayed a much lower incidence of fin erosion and of asymmetric individuals than control fish kept in captivity. Significant differences in body shape were also apparent, and survivors had longer heads, thicker caudal peduncles and a more streamlined body shape than hatchery controls as early as 20 days following stocking, most likely as a result of phenotypic plasticity and non-random, selective mortality of maladapted phenotypes. Hatchery-reared fish typically perform poorly in the wild and the results of this study indicate that this may be due to phenotypic mismatch, i.e. because hatcheries generate fish that are phenotypically mismatched to the natural environment.

Silver spoons in the rough: can environmental enrichment improve survival of hatchery Atlantic salmon Salmo salar in the wild?

This study tested the 'silver spoon' hypothesis which posits that individuals that develop under favourable conditions should enjoy a fitness advantage later in life because they are more likely to recognize and settle in high-quality habitats. Atlantic salmon Salmo salar of two age classes (0+ and 1+ years) were reared in environmentally enriched or standard hatchery tanks for a short period (c. 10 weeks), were then released into a natural river and sampled on repeated occasions to test for silver-spoon effects. Compared with controls, enriched fish had a 6.4% higher recapture rate and settled in higher velocity habitats when they were stocked as 0+ year fry, but not when they were stocked as 1+ year parr. The opportunity for selection was generally higher for environmentally enriched fish than for controls, and also higher for 0+ than for 1+ year fish. Selection favoured individuals with high condition factor, extensive fat reserves and longer than average pectoral fins in both age classes but favoured a small body size in 1+ year and a large body size in 0+ year releases. Stomach analysis showed that enriched fish ate more, and adapted quicker to natural prey than controls. These results provide support for silver-spoon effects in fish and indicate that enrichment can improve post-release performance in conservation programmes, but seemingly only if fish are not kept in captivity for too long.

Patterns of metastatic spread in early breast cancer.

AIMS: The aim of this study was to prospectively investigate metastatic pathways of spread to lymph node versus bone marrow and identify biological characteristics that determine these patterns in early invasive breast cancer. PATIENTS AND METHODS: In all, 177 patients with early invasive breast cancer underwent surgical extirpation of the primary tumour with sentinel lymph node biopsy (SLNB). Bone marrow (BM) aspiration was performed to screen for cytokeratin-positive cells by immunocytochemistry. Lymphatic spread was assessed by histopathological examination of lymph nodes (LN). A representative subset of 87 tumours was analysed by tissue microarray (TMA) to evaluate expression of markers that potentially influence haematogenous vs. lymphatic spread. Patients were followed up for a median of 54.7 months. RESULTS: Of the 177 patients, 114 (64%) were BM-/LN-, 38 (22%) BM-/LN+, 19 (11%) BM+/LN- and 6 (3%) BM+/LN+. Multivariate analysis of histopathological characteristics revealed that increasing tumour size was significantly associated with both LN positivity (p = 0.003) and BM positivity (p = 0.01), the presence of lymphovascular invasion significantly correlated with LN+ (p = 0.01), whereas lower histological grade was significantly associated with BM+ (p = 0.03). LN+ and BM+ were non-significantly negatively related to each other. Univariate analysis of the TMA data showed differential expression patterns for several factors; significant differences between effects on the two metastatic pathways (lymphatic vs. haematogenous) were found for expression of CD54 (p = 0.03), osteopontin (p = 0.04), bone sialoprotein (p = 0.04) and CXCR4 (p = 0.009). High expression of CD54, osteopontin and bone sialoprotein (BSP) was positively associated with BM + but was either not associated, or negatively associated, with LN+. High CXCR4 expression was positively associated with LN+ and negatively with BM+. High VEGF-C expression was associated with both LN+ and BM+, although this did not attain statistical significance. Due to the small number of clinical events during clinical follow-up, no associations were identified between metastatic spread patterns, recurrence and/or death. CONCLUSION: These findings suggest that distinct lymphatic and haematogenous metastatic pathways exist in early breast cancer and that these pathways are governed by specific biological markers.

Fine tuned modulation of the motor system by adjectives expressing positive and negative properties.

Using transcranial magnetic stimulation (TMS), motor evoked potentials (MEPs) were recorded from two antagonistic muscles, the first dorsal interosseus (FDI) of the hand and the extensor communis digitorum (EC) of the forearm. FDI is involved in grasping actions and EC in releasing. TMS pulses were delivered while participants were reading adjectives expressing either negative or positive pragmatic properties, at 150 ms after presentation of language material. Overall findings showed an interaction of adjective type (positive, negative) and muscle (FDI, EC), the effect being driven by a significant difference for negative adjectives. Further analysis aimed at investigating the effectiveness of positive adjectives showed a similar, but opposite, pattern of effects for the positive words in the initial two blocks. The present results indicate that, as for verbs and nouns, adjectives recruit the sensorimotor system, and their processing is best explained by an embodiment rather than an amodal approach to language.

Action observation versus motor imagery in learning a complex motor task: a short review of literature and a kinematics study.

Both motor imagery and action observation have been shown to play a role in learning or re-learning complex motor tasks. According to a well accepted view they share a common neurophysiological basis in the mirror neuron system. Neurons within this system discharge when individuals perform a specific action and when they look at another individual performing the same or a motorically related action. In the present paper, after a short review of literature on the role of action observation and motor imagery in motor learning, we report the results of a kinematics study where we directly compared motor imagery and action observation in learning a novel complex motor task. This involved movement of the right hand and foot in the same angular direction (in-phase movement), while at the same time moving the left hand and foot in an opposite angular direction (anti-phase movement), all at a frequency of 1Hz. Motor learning was assessed through kinematics recording of wrists and ankles. The results showed that action observation is better than motor imagery as a strategy for learning a novel complex motor task, at least in the fast early phase of motor learning. We forward that these results may have important implications in educational activities, sport training and neurorehabilitation.

Characterization of PEGylated biopharmaceutical products by LC/MS and LC/MS/MS.

PEGylation of peptide and proteins is an important method of improving their pharmacokinetic, pharmacodynamic, and immunological profiles, and thus enhancing their therapeutic effect. However, PEGylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG heterogeneity, site of PEG addition, and number of attached PEG moieties. Here, we present two methodologies for the structural characterization of PEGylated peptides and proteins. LC/MS methodology utilizing post-column addition of amines was developed to obtain accurate masses of PEGylated peptides and proteins, which can be used to assign the structures and number of attached PEGs. The PEGylated sites in PEGylated products could be elucidated with the tandem LC/MS methodology combining in-source fragmentation with CID-MS/MS. Both methodologies are applied to model PEGylated peptides to obtain the accurate masses and identify PEGylated sites.

Identification of racemization sites using deuterium labeling and tandem mass spectrometry.

Racemization of amino acids is a common chemical degradation pathway observed in biopharmaceuticals and is particularly prevalent in synthetic peptides. The identification of racemized amino acid residue(s) by mass spectrometry is particularly challenging due to isobaric mass between the isomeric forms. In this paper, we present a novel methodology combining stable deuterium labeling with collisionally induced dissociation-tandem mass spectrometry (CID-MS/MS) to elucidate racemized amino acid residues in immunoglobulin samples. Immunoglobulin G subclasses IgG1, IgG2, and IgG4 samples were first stressed in protonated or deuterated buffer (pH 8 or 9) at 40 or 50 degrees C storage for days or weeks. These forced degraded samples were reduced, S-carbamidomethylated, and digested with trypsin in protonated solution, and the tryptic digests were then analyzed via liquid chromatography/mass spectrometry (LC-MS) or sequenced via liquid chromatography/tandem mass spectrometry (LC-MS/MS) to detect racemized peptides and elucidate the location of racemized amino acid residues. The methodology successfully identified several racemized amino acid residues in the constant region of the heavy chains of the three IgG subclasses. Although the IgG subclasses have very similar primary protein sequences, our results interestingly indicated different racemization rates for specific amino acid residues.

Elucidation of PEGylation site with a combined approach of in-source fragmentation and CID MS/MS.

Poly(ethylene glycol) (PEG)ylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG heterogeneity, site of addition and number of attached PEGylated moieties. Recently, we published a novel LC/MS methodology with a post-column addition of amines to obtain accurate masses of PEGylated peptides and proteins. The accurate masses can be used to assign the structures and number of attached PEGs [15], but the PEGylation site remains unclear in situations where multiple potential attachments are involved. Here, we present a methodology combining in-source fragmentation (ISF) with CID-MS/MS to elucidate the PEGylated sites in PEGylated products. All PEGylated samples, either prepared in acidic solution, or collected from a RP-HPLC stream, were first ionized via ISF to produce products containing small PEG fragment attachment, and then those fragment ions obtained were sequenced via CID MS/MS to deduce the PEGylation site. The methodology was successfully applied to PEGylated glucagon and IgG4 antibody light chain, which demonstrated that the small PEG fragments attached were stable during the CID activation.

Legumain and cathepsin-L expression in human unstable carotid plaque.

OBJECTIVE: The cysteine protease, legumain, is thought to have a role in the processing and activation of proteases such as cathepsin-L, which have been implicated in plaque rupture. This study aimed to determine: if legumain activity is up-regulated in unstable areas of plaque; the effect of legumain over-expression on the activity of cathepsin-L and the effect of mutation of the legumain RGD sequence on its cellular location. METHODS AND RESULTS: Legumain was measured in human carotid plaque extracts (n=17) using a novel ELISA and modified activity assay. Unstable regions of plaque contained more than twice the amount of legumain protein (P<0.001) and activity (P<0.03) compared with stable regions of the same plaque. Over-expression of legumain in THP-1 macrophages using an adenoviral construct resulted in the processing of cathepsin-L from its 30kDa to its 25kDa form compared with controls. CONCLUSION: Unstable regions of plaque contain increased levels of active legumain. Over-expression of legumain in macrophages alters intracellular processing of cathepsin-L to its mature 25kDa form. This may be a means by which legumain could contribute to plaque instability.

Characterization of poly(ethylene glycol) and PEGylated products by LC/MS with postcolumn addition of amines.

PEGylation of peptides and proteins presents significant challenges for structural characterization due to the heterogeneity of the poly(ethylene glycol) (PEG), the number of PEG moieties attached, and the site(s) of PEGylation. In this work, a novel and powerful methodology using a postcolumn addition combined with LC/MS was developed and applied to examine high molecular weight (>/=20 kDa) PEG as well as PEGylated peptide and protein products. The PEG and PEGylated compounds were eluted from RP-HPLC, and the HPLC stream was mixed with diethylmethylamine (DEMA) or triethylamine (TEA) through a T mixer coupled to a time-of-flight mass spectrometer. With these conditions, PEG is diethylmethyl- or triethyl-ammoniated instead of protonated while the protein or peptide remains protonated. The charges for PEG and the PEGylated compounds were greatly reduced, and there was no convolution among differently charged ions, even for 40 kDa PEG or tri-20 kDa PEGylated IgG4 heavy chain. Mass accuracies (<0.01%) obtained are similar to large molecular weight proteins. By selecting specific amines, such as DEMA, commercially available software was used to deconvolute the spectra composed of the diethylmethylammoniated PEG or the diethylmethylammoniated and protonated PEGylated peptide or protein to obtain accurate masses. The examples presented in this report demonstrate that the methodology can be used to elucidate different PEG structures and modifications, such as oxidation and maleimide ring opening of PEGylated peptides or proteins.

Gendered policies and practices that increase older men's risk of elder mistreatment.

We aim to detail some of the ways that social policy and gendered practices put older men at risk of elder mistreatment. Research on the abuse and neglect that older adults experience has often focused on the characteristics of the victims and the dynamics within families, emphasizing factors such as the likelihood of an intergenerational cycle of violence, substance abuse and dependency, and older men's financial status as key risks in elder abuse. The effect on men from this type of analysis is that elder mistreatment remains an individual or family problem rather than being viewed as a larger societal concern. This article challenges the individualistic focus by outlining the importance of societal forces affecting older men's risk of mistreatment.