A Non-hydrolyzable ATP Derivative Generates a Stable Complex in a Light-inducible Two-component System

Isothermal calorimetry (ITC) measurements yielded the binding constants during complex formation of light-inducible histidine kinases (HK) and their cognate CheY-type response regulators (RR). HK-RR interactions represent the core function of the bacterial two-component system, which is also present in many bacterial phytochromes. Here, we have studied the recombinant forms of phytochromes CphA and CphB from the cyanobacterium Tolypothrix PCC7601 and their cognate RRs RcpA and RcpB. The interaction between the two reaction partners (HK and RR) was studied in the presence and absence of ATP. A complex formation was observable in the presence of ATP, but specific interactions were only found when a non-hydrolyzable ATP derivative was added to the mixture. Also, the incubation of the HK domain alone (expressed as a recombinant protein) with the RR did not yield specific interactions, indicating that the HK domain is only active as a component of the full-length phytochrome. Considering also previous studies on the same proteins (Hübschmann, T., Jorissen, H. J. M. M., Börner, T., Gärtner, W., and de Marsac, N. (2001) Eur. J. Biochem. 268, 3383–3389) we now conclude that the HK domains of these phytochromes are active only when the chromophore domain is in its Pr form. The formerly documented phosphate transfer between the HK domain and the RR takes place via a transiently formed protein-protein complex, which becomes detectable by ITC in the presence of a non-hydrolyzable ATP derivative. This finding is of interest also in relation to the function of some (blue light-sensitive) photoreceptors that carry the HK domain and the RR fused together in one single protein.     

Arachidonic Acid Stimulates Cell Adhesion through a Novel p38 MAPK-RhoA Signaling Pathway That Involves Heat Shock Protein 27

Rho GTPases are critical components of cellular signal transduction pathways. Both hyperactivity and overexpression of these proteins have been observed in human cancers and have been implicated as important factors in metastasis. We previously showed that dietary n-6 fatty acids increase cancer cell adhesion to extracellular matrix proteins, such as type IV collagen. Here we report that in MDA-MB-435 human melanoma cells, arachidonic acid activates RhoA, and inhibition of RhoA signaling with either C3 exoenzyme or dominant negative Rho blocked arachidonic acid-induced cell adhesion. Inhibition of the Rho kinase (ROCK) with either small molecule inhibitors or ROCK II-specific small interfering RNA (siRNA) blocked the fatty acid-induced adhesion. However, unlike other systems, inhibition of ROCK did not block the activation of p38 mitogen-activated protein kinase (MAPK); instead, Rho activation depended on p38 MAPK activity and the presence of heat shock protein 27 (HSP27), which is phosphorylated downstream of p38 after arachidonic acid treatment. HSP27 associated with p115RhoGEF in fatty acid-treated cells, and this association was blocked when p38 was inhibited. Furthermore, siRNA knockdown of HSP27 blocked the fatty acid-stimulated Rho activity. Expression of dominant negative p115-RhoGEF or p115RhoGEF-specific siRNA inhibited both RhoA activation and adhesion on type IV collagen, whereas a constitutively active p115RhoGEF restored the arachidonic acid stimulation in cells in which the p38 MAPK had been inhibited. These data suggest that n-6 dietary fatty acids stimulate a set of interactions that regulates cell adhesion through RhoA and ROCK II via a p38 MAPK-dependent association of HSP27 and p115RhoGEF.The ability of tumor cells to metastasize to secondary sites is a hallmark of neoplastic disease. Unfortunately, this propensity to spread is the primary cause of morbidity and death in cancer patients (1). Metastasis is clearly a highly regulated, multistep process that occurs in a spatiotemporal manner (2–4). To escape the restrictive compartment boundaries characteristic of adult tissue, separate intravasation and extravasation steps requiring alterations in co-adhesion, adhesion, invasion, and migration must occur. Execution of these biological processes, involving multiple proteins and cellular organelles, require highly coordinated cell signaling mechanisms.The Rho family of small GTPases regulates many facets of cytoskeletal rearrangements that facilitate cell attachment and migration (5–7). Rho GTPases act as molecular switches by changing from an inactive GDP-bound conformation to an active GTP-bound conformation, thereby regulating a signaling pathway. These proteins are directly regulated by Rho guanine nucleotide exchange factors (GEFs),² Rho GTPase activating proteins, and Rho GDP-dissociation inhibitors (8–12). RhoGEFs bind to the GTPase to catalyze the dissociation of GDP, allowing the binding of GTP and thereby promoting Rho activation (8). The RGS (regulators of G protein signaling) domain-containing RhoGEFs are a recently described family of GEFs. Currently, there are three members of this family, PDZ-RhoGEF, LARG, and p115RhoGEF (13–15), in which the RGS domains function as a heterotrimeric GTPase-activating domain (13, 15, 16). The RGS family of RhoGEFs has been shown to regulate Rho during several processes including cytoskeletal rearrangements, cell adhesion, and cancer progression (17–21).There is significant interplay between the activity of small GTPases and signaling derived from fatty acid metabolism (22–28). Linoleic acid, which is metabolized to arachidonic acid, is an n-6 polyunsaturated fatty acid that is present at high levels in most western diets (29). In animal models, diets high in n-6 polyunsaturated fatty acids have been shown to enhance tumor progression and metastasis (30, 31). Additionally, arachidonic acid is stored in cell membranes and is made available by phospholipases under conditions of increased inflammatory response (32). Arachidonic acid is further metabolized by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 monooxygenases to yield bioactive products that have myriad effects on cells, and altered metabolism of arachidonic acid by COX, LOX, and P450 has been implicated in cancer progression (31, 33–36).We have studied mechanisms of cell adhesion using the MDA-MB-435 cells as a model of a highly metastatic human cancer cell line (37). These cells have been extensively studied for their ability to recapitulate the metastatic cascade in vivo and in vitro, although recent work indicates that the cells currently in use are most likely a human melanoma line (38). We initially observed that arachidonic acid (AA) enhanced adhesion of MDA-MB-435 cells to type IV collagen through specific integrin-mediated pathways (37). Exogenous AA led to the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase 2 and the phosphorylation of heat shock protein 27 (HSP27) via a p38 MAPK-dependent process (39). Inhibition of p38 MAPK activation blocked cell adhesion as did function-blocking antibodies specific for subunits of the collagen receptor (40). More recently, we identified the key metabolite of AA (15-(S)- hydroxyeicosatetraenoic acid) and the upstream kinases (TAK1 and MKK6) that are responsible for activation of p38 MAPK in this system (41).In this study we investigated the role of Rho activation in the MDA-MB-435 cells after exposure to arachidonic acid. Several aspects of the regulation of Rho signaling in these cells provide insights into the cross-talk between important signaling pathways.     

A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

BackgroundNatural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancerMethodsPatients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m² × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometryResultsTwenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30–65) years. Mean NK cell dose was 2.16 × 10⁷cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = < 0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansionConclusionsAdoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.     

Exploring movement patterns and changing distributions of baleen whales in the western North Atlantic using a decade of passive acoustic data

Six baleen whale species are found in the temperate western North Atlantic Ocean, with limited information existing on the distribution and movement patterns for most. There is mounting evidence of distributional shifts in many species, including marine mammals, likely because of climate‐driven changes in ocean temperature and circulation. Previous acoustic studies examined the occurrence of minke (Balaenoptera acutorostrata) and North Atlantic right whales (NARW; Eubalaena glacialis). This study assesses the acoustic presence of humpback (Megaptera novaeangliae), sei (B. borealis), fin (B. physalus), and blue whales (B. musculus) over a decade, based on daily detections of their vocalizations. Data collected from 2004 to 2014 on 281 bottom‐mounted recorders, totaling 35,033 days, were processed using automated detection software and screened for each species' presence. A published study on NARW acoustics revealed significant changes in occurrence patterns between the periods of 2004–2010 and 2011–2014; therefore, these same time periods were examined here. All four species were present from the Southeast United States to Greenland; humpback whales were also present in the Caribbean. All species occurred throughout all regions in the winter, suggesting that baleen whales are widely distributed during these months. Each of the species showed significant changes in acoustic occurrence after 2010. Similar to NARWs, sei whales had higher acoustic occurrence in mid‐Atlantic regions after 2010. Fin, blue, and sei whales were more frequently detected in the northern latitudes of the study area after 2010. Despite this general northward shift, all four species were detected less on the Scotian Shelf area after 2010, matching documented shifts in prey availability in this region. A decade of acoustic observations have shown important distributional changes over the range of baleen whales, mirroring known climatic shifts and identifying new habitats that will require further protection from anthropogenic threats like fixed fishing gear, shipping, and noise pollution.     

Parallels,differences and lessons: a comparison of the management of foot-and-mouth disease and COVID-19 using UK 2001/2020 as points of reference

Foot-and-mouth disease (FMD) is an extremely infectious viral infection of cloven-hoofed animals which is highly challenging to control and can give rise to national animal health crises, especially if there is a lack of pre-existing immunity due to the emergence of new strains or following incursions into disease-free regions. The 2001 FMD epidemic in the UK was on a scale that initially overwhelmed the national veterinary services and was eventually controlled by livestock lockdown and slaughter on an unprecedented scale. In 2020, the rapid emergence of COVID-19 has led to a human pandemic unparalleled in living memory. The enormous logistics of multi-agency control efforts for COVID-19 are reminiscent of the 2001 FMD epidemic in the UK, as are the use of movement restrictions, not normally a feature of human disease control. The UK experience is internationally relevant as few countries have experienced national epidemic crises for both diseases. In this review, we reflect on the experiences and lessons learnt from UK and international responses to FMD and COVID-19 with respect to their management, including the challenge of preclinical viral transmission, threat awareness, early detection, different interpretations of scientific information, lockdown, biosecurity behaviour change, shortage of testing capacity and the choices for eradication versus living with infection. A major lesson is that the similarity of issues and critical resources needed to manage large-scale outbreaks demonstrates that there is benefit to a ‘One Health’ approach to preparedness, with potential for greater cooperation in planning and the consideration of shared critical resources.     

A novel allele of the Arabidopsis thaliana MACPF protein CAD1 results in deregulated immune signaling

Immune recognition in plants is governed by two major classes of receptors: pattern recognition receptors (PRRs) and nucleotide-binding leucine-rich repeat receptors (NLRs). Located at the cell surface, PRRs bind extracellular ligands originating from microbes (indicative of “non-self”) or damaged plant cells (indicative of “infected-self”), and trigger signaling cascades to protect against infection. Located intracellularly, NLRs sense pathogen-induced physiological changes and trigger localized cell death and systemic resistance. Immune responses are under tight regulation in order to maintain homeostasis and promote plant health. In a forward-genetic screen to identify regulators of PRR-mediated immune signaling, we identified a novel allele of the membrane-attack complex and perforin (MACPF)-motif containing protein CONSTITUTIVE ACTIVE DEFENSE 1 (CAD1) resulting from a missense mutation in a conserved N-terminal cysteine. We show that cad1-5 mutants display deregulated immune signaling and symptoms of autoimmunity dependent on the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), suggesting that CAD1 integrity is monitored by the plant immune system. We further demonstrate that CAD1 localizes to both the cytosol and plasma membrane using confocal microscopy and subcellular fractionation. Our results offer new insights into immune homeostasis and provide tools to further decipher the intriguing role of MACPF proteins in plants.     

Common Factors in Biofeedback Administered by Psychotherapists

Common factors are nonspecific therapeutic elements common across different varieties of psychotherapy. In a recent study, 68 expert psychotherapy researchers with a variety of allegiances collectively rated biofeedback as being negatively associated with many common factors (Tschacher et al. in Clin Psychol Psychother 21(1):82–96, 2014), including the therapeutic alliance. However, it seems implausible that biofeedback could benefit so many people while being incompatible with the therapeutic alliance and other common factors. The present study investigated the experiences of biofeedback clients who participated in a brief heart rate variability biofeedback protocol in order to explore the potential roles of common factors in biofeedback. The results of this study offer preliminary evidence that many common factors—including therapeutic alliance, self-efficacy expectation, mastery experiences, provision of explanatory scheme, mindfulness, and even cognitive restructuring—may play a role in biofeedback outcomes. Future research on this topic should include mediation and moderation models investigating the role of specific common factors on outcome and process studies to help determine what clinician behaviors are most helpful. Deeper investigation of common factors in biofeedback may benefit future biofeedback research and practice and address the concerns of colleagues outside of the biofeedback community who believe that biofeedback is at odds with common factors.