Avoidance response to CO2 in the lateral horn.

In flies, the olfactory information is carried from the first relay in the brain, the antennal lobe, to the mushroom body (MB) and the lateral horn (LH). Olfactory associations are formed in the MB. The LH was ascribed a role in innate responses based on the stereotyped connectivity with the antennal lobe, stereotyped physiological responses to odors, and MB silencing experiments. Direct evidence for the functional role of the LH is still missing. Here, we investigate the behavioral role of the LH neurons (LHNs) directly, using the CO2 response as a paradigm. Our results show the involvement of the LH in innate responses. Specifically, we demonstrate that activity in two sets of neurons is required for the full behavioral response to CO2. Tests of the behavioral response to other odors indicate the neurons are selective to CO2 response. Using calcium imaging, we observe that the two sets of neurons respond to CO2 in a different manner. Using independent manipulation and recording of the two sets of neurons, we find that the one that projects to the superior intermediate protocerebrum (SIP) also outputs to the local neurons within the LH. The design of simultaneous output at the LH and the SIP, an output of the MB, allows for coordination between innate and learned responses.

Generation of Fcabs targeting human and murine LAG-3 as building blocks for novel bispecific antibody therapeutics.

The immunoglobulin superfamily protein lymphocyte-activation gene 3 (LAG-3) participates in immune suppression and has been identified as a suitable target for cancer therapies. In order to generate bispecific antibodies targeting LAG-3, Fcabs (Fc-region with antigen binding) targeting human and murine LAG-3 were generated from phage libraries. These Fcabs bind to LAG-3, inhibiting its interaction with MHC class II, and induce IL-2 production in a T cell assay. Bispecific antibodies, known as mAb2, were produced by replacing the Fc region of a monoclonal antibody with Fcab sequences in the CH3 domain. mAb2 containing anti-LAG-3 Fcabs have mAb-like biophysical characteristics and retain LAG-3 binding and functional activity. mAb2 can thus be generated using multiple Fabs to investigate bispecific parings and develop novel therapeutics.

A HER2-specific Modified Fc Fragment (Fcab) Induces Antitumor Effects Through Degradation of HER2 and Apoptosis.

FS102 is a HER2-specific Fcab (Fc fragment with antigen binding), which binds HER2 with high affinity and recognizes an epitope that does not overlap with those of trastuzumab or pertuzumab. In tumor cells that express high levels of HER2, FS102 caused profound HER2 internalization and degradation leading to tumor cell apoptosis. The antitumor effect of FS102 in patient-derived xenografts (PDXs) correlated strongly with the HER2 amplification status of the tumors. Superior activity of FS102 over trastuzumab or the combination of trastuzumab and pertuzumab was observed in vitro and in vivo when the gene copy number of HER2 was equal to or exceeded 10 per cell based on quantitative polymerase chain reaction (qPCR). Thus, FS102 induced complete and sustained tumor regression in a significant proportion of HER2-high PDX tumor models. We hypothesize that the unique structure and/or epitope of FS102 enables the Fcab to internalize and degrade cell surface HER2 more efficiently than standard of care antibodies. In turn, increased depletion of HER2 commits the cells to apoptosis as a result of oncogene shock. FS102 has the potential of a biomarker-driven therapeutic that derives superior antitumor effects from a unique mechanism-of-action in tumor cells which are oncogenically addicted to the HER2 pathway due to overexpression.

A fuzzy description logic based IoT framework: Formal verification and end user programming.

The Internet of Things (IoT) has become one of the most popular technologies in recent years. Advances in computing capabilities, hardware accessibility, and wireless connectivity make possible communication between people, processes, and devices for all kinds of applications and industries. However, the deployment of this technology is confined almost entirely to tech companies, leaving end users with only access to specific functionalities. This paper presents a framework that allows users with no technical knowledge to build their own IoT applications according to their needs. To this end, a framework consisting of two building blocks is presented. A friendly interface block lets users tell the system what to do using simple operating rules such as "if the temperature is cold, turn on the heater." On the other hand, a fuzzy logic reasoner block built by experts translates the ambiguity of human language to specific actions to the actuators, such as "call the police." The proposed system can also detect and inform the user if the inserted rules have inconsistencies in real time. Moreover, a formal model is introduced, based on fuzzy description logic, for the consistency of IoT systems. Finally, this paper presents various experiments using a fuzzy logic reasoner to show the viability of the proposed framework using a smart-home IoT security system as an example.

TEMPO-conjugated tobacco mosaic virus as a magnetic resonance imaging contrast agent for detection of superoxide production in the inflamed liver.

Superoxide, an anionic dioxygen molecule, plays a crucial role in redox regulation within the body but is implicated in various pathological conditions when produced excessively. Efforts to develop superoxide detection strategies have led to the exploration of organic-based contrast agents for magnetic resonance imaging (MRI). This study compares the effectiveness of two such agents, nTMV-TEMPO and kTMV-TEMPO, for detecting superoxide in a mouse liver model with lipopolysaccharide (LPS)-induced inflammation. The study demonstrates that kTMV-TEMPO, with a strategically positioned lysine residue for TEMPO attachment, outperforms nTMV-TEMPO as an MRI contrast agent. The enhanced sensitivity of kTMV-TEMPO is attributed to its more exposed TEMPO attachment site, facilitating stronger interactions with water protons and superoxide radicals. EPR kinetics experiments confirm kTMV-TEMPO's faster oxidation and reduction rates, making it a promising sensor for superoxide in inflamed liver tissue. In vivo experiments using healthy and LPS-induced inflamed mice reveal that reduced kTMV-TEMPO remains MRI-inactive in healthy mice but becomes MRI-active in inflamed livers. The contrast enhancement in inflamed livers is substantial, validating the potential of kTMV-TEMPO for detecting superoxide in vivo. This research underscores the importance of optimizing contrast agents for in vivo imaging applications. The enhanced sensitivity and biocompatibility of kTMV-TEMPO make it a promising candidate for further studies in the realm of medical imaging, particularly in the context of monitoring oxidative stress-related diseases.

Murid herpesvirus-4 exploits dendritic cells to infect B cells.

Dendritic cells (DCs) play a central role in initiating immune responses. Some persistent viruses infect DCs and can disrupt their functions in vitro. However, these viruses remain strongly immunogenic in vivo. Thus what role DC infection plays in the pathogenesis of persistent infections is unclear. Here we show that a persistent, B cell-tropic gamma-herpesvirus, Murid Herpesvirus-4 (MuHV-4), infects DCs early after host entry, before it establishes a substantial infection of B cells. DC-specific virus marking by cre-lox recombination revealed that a significant fraction of the virus latent in B cells had passed through a DC, and a virus attenuated for replication in DCs was impaired in B cell colonization. In vitro MuHV-4 dramatically altered the DC cytoskeleton, suggesting that it manipulates DC migration and shape in order to spread. MuHV-4 therefore uses DCs to colonize B cells.

Human cytomegalovirus UL69 protein facilitates translation by associating with the mRNA cap-binding complex and excluding 4EBP1.

4EBP1 is phosphorylated by the mTORC1 kinase. When mTORC1 activity is inhibited, hypophosphorylated 4EBP1 binds and sequesters eIF4E, a component of the mRNA cap-binding complex, and blocks translation. As a consequence, mTORC1 activity is needed to maintain active translation. The human cytomegalovirus pUL38 protein preserves mTORC1 activity, keeping most of the E4BP1 in the infected cell in a hyperphosphorylated, inactive state. Here we report that a second viral protein, pUL69, also antagonizes the activity of 4EBP1, but by a separate mechanism. pUL69 interacts directly with eIF4A1, an element of the cap-binding complex, and the poly(A)-binding protein, which binds to the complex. When pUL69 accumulates during infection with wild-type virus, 4EBP1 is excluded from the complex. However, 4EBP1 is present in the cap-binding complex after infection with a pUL69-deficient virus, coincident with reduced accumulation of several late virus-coded proteins. We propose that pUL69 supports translation in human cytomegalovirus-infected cells by excluding hypophosphorylated 4EBP1 from the cap-binding complex.

Neuronal substrates of egg-laying behaviour at the abdominal ganglion of Drosophila melanogaster.

Egg-laying in Drosophila is the product of post-mating physiological and behavioural changes that culminate in a stereotyped sequence of actions. Egg-laying harbours a great potential as a paradigm to uncover how the appropriate motor circuits are organized and activated to generate behaviour. To study this programme, we first describe the different phases of the egg-laying programme and the specific actions associated with each phase. Using a combination of neuronal activation and silencing experiments, we identify neurons (OvAbg) in the abdominal ganglion as key players in egg-laying. To generate and functionally characterise subsets of OvAbg, we used an intersectional approach with neurotransmitter specific lines-VGlut, Cha and Gad1. We show that OvAbg/VGlut neurons promote initiation of egg deposition in a mating status dependent way. OvAbg/Cha neurons are required in exploration and egg deposition phases, though activation leads specifically to egg expulsion. Experiments with the OvAbg/Gad1 neurons show they participate in egg deposition. We further show a functional connection of OvAbg neurons with brain neurons. This study provides insight into the organization of neuronal circuits underlying complex motor behaviour.

Isolation of microsatellite markers and analysis of genetic diversity among east Atlantic populations of the sword razor shell Ensis siliqua: a tool for population management.

The sword razor shell Ensis siliqua (Linnaeus, 1758) is found mainly from Norway to the Atlantic coast of the Iberian Peninsula. It is intensively caught in Europe, being highly appreciated as seafood. To help in its conservation and management, five microsatellite markers were isolated and genetic variation was analyzed in samples from Ireland, Spain, and Portugal. The highly significant differentiation (θ = 0.287, P < 0.001) observed was mainly due to differences between samples from Irish and Iberian Peninsula localities, except Aveiro (its sample resembled the Irish samples, and it may be predominantly self-recruiting). These groups of samples showed significant differences in allelic richness that could be related to harvesting intensity. Moreover, microsatellites detected low but significant differentiation between Iberian localities (Celeiro and Olhão), and Aveiro differed significantly from Strangford Lough. Overall, results suggest that two independently evolving regions exist and that management strategies should be designed for each region.

Identification of Ensis siliqua samples and establishment of the catch area using a species-specific microsatellite marker.

European Council Regulation 104/2000 states that fishery products must be labeled to indicate commercial designation of species, the production method, and the catch area. Therefore, traceability of seafood implies knowledge of the species offered to retail and their origin. Ensis siliqua is a bivalve intensively fished in Europe and sold in fresh and canned forms. Although several published methods clearly differentiate Ensis genus species, none of those assess the origin of the commercial samples. In the present study, a microsatellite marker (Esi-UDC3055F) was developed to establish the catch area of E. siliqua samples. Amplification yielded a fragment of 275 or 302 base pairs, depending on whether they were Iberian or Irish populations. The usefulness of this method was also assessed in commercial samples. The results of this study provide a reliable methodology for the identification of catch area in European E. siliqua commercial samples. The coupling of this methodology with existing techniques for razor clam species identification provides a powerful tool for traceability and labeling enforcement.

Environmental quality assessment combining sediment metal levels, biomarkers and macrobenthic communities: application to the Óbidos coastal lagoon (Portugal).

Macroinvertebrate benthic communities are one of the key biological components considered for the assessment of benthic integrity in the context of the Water Framework Directive (WFD). However, under moderate contamination scenarios, the assessment of macrobenthic alterations at community level alone could be insufficient to discriminate the environmental quality of coastal and transitional waters. Keeping this in view, sediment quality of moderately contaminated sites in a coastal lagoon (Óbidos lagoon, Portugal) was assessed by the combination of sediment metal levels, Carcinus maenas biomarkers (accumulated metals and oxidative stress responses) and macrobenthic communities. Two sites were selected in confined inner branches (BS and BB) and a third one in the middle lagoon (ML). The site BB presented slightly higher levels of metals in sediment but biological variables calculated for macrobenthic data were not significantly different between sites. The biotic index M-AMBI that is being applied to assess environmental quality of transitional waters in the scope of the WFD pointed either to high (site ML) or good quality status (BS and BB) in the selected sites. However, crabs from BB site presented significantly higher levels of Ni in hepatopancreas than those from ML and macrobenthic community structure was significantly different between BB and ML. Additionally, spatial differences were obtained for oxidative stress parameters suggesting that BB site presented stressors for crabs (higher GST and lower GSH(t) at BB site). Factor analysis (PCA) integrating sediment contamination, biomarkers in crabs and macrobenthic data also distinguished BB site as the most environmentally disturbed. On the other hand, at ML site, some macrobenthic variables (equitability and polychaetes' diversity) were found to be enhanced by current environmental conditions, suggesting the existence of a better sediment quality. Current results pointed to the usefulness of integrating macrobenthic community alterations with responses at organism level (bioaccumulation and biochemical endpoints) in order to increase the accuracy of environmental quality assessment in lagoon systems. Moreover, the application of different statistical methods was also found to be recommendable.

Mating pair drives aggressive behavior in female Drosophila.

Aggression is an adaptive set of behaviors that allows animals to compete against one another in an environment of limited resources. Typically, males fight for mates and food, whereas females fight for food and nest sites.1 Although the study of male aggression has been facilitated by the extravagant nature of the ritualized displays involved and the remarkable armaments sported by males of many species,2,3,4 the subtler and rarer instances of inter-female aggression have historically received much less attention. In Drosophila, females display high levels of complex and highly structured aggression on a food patch with conspecific females.5,6,7,8,9 Other contexts of female aggression have not been explored. Indeed, whether females compete for mating partners, as males do, has remained unknown so far. In the present work, we report that Drosophila melanogaster females reliably display aggression toward mating pairs. This aggressive behavior is regulated by mating status and perception of mating opportunities and relies heavily on olfaction. Furthermore, we found that food odor in combination with OR47b-dependent fly odor sensing is required for proper expression of aggressive behavior. Taken together, we describe a social context linked to reproduction in which Drosophila females aspiring to mate produce consistent and stereotyped displays of aggression. These findings open the door for further inquiries into the neural mechanisms that govern this behavior.

Epibiont assemblages on limpet shells: Biodiversity drivers in intertidal rocky shores.

Limpet shells can harbour a high diversity of species. Individuals of four limpet species (Patella depressa, Patella ulyssiponensis, Patella vulgata and Siphonaria pectinata) were collected monthly during one year in southern Portugal. Epibiont organisms were identified, counted and the percentage cover of facilitator taxa was also recorded. A total of 86 taxa were identified with abundance reaching 674 epibionts on a single basibiont shell. P. ulyssiponensis showed the highest epibiont species diversity and richness while P. depressa and P. vulgata showed similar diversity and richness. P. depressa had a more even epibiotic community mainly due to higher densities of Chthamalus sp. Overall, basibiont species was the key factor determining the epibiotic community, followed by month/season and erect algae. The presence of erect algae potentiated the epibionts diversity on limpet shells, whereas the occurrence of barnacles tended to decrease it and the presence of crustose algae had no significant effect on epibionts diversity. These findings shed further light on the biological and ecological complex relationships among keystone species inhabiting intertidal rocky shores.

Targeting DNA damage response components induces enhanced STING-dependent type-I IFN response in ATM deficient cancer cells and drives dendritic cell activation.

The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example, ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However, additional antitumor benefits from targeting the DDR pathways, which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal, inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here, we demonstrate that ATM -/- FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However, STING deficiency in tumor cells does not prevent DC activation, suggesting that transactivation of the STING pathway occurs within DCs. Furthermore, depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells, indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary, in this study, we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation, which is enhanced in ATM-deficient cells.

Combined use of Nassarius reticulatus imposex and statolith age determination for tracking temporal evolution of TBT pollution in the NW Portuguese continental shelf.

Spatial and temporal trends of tributyltin (TBT) pollution on the northern continental shelf of Portugal (5-34 m depth) were assessed using Nassarius reticulatus as a bioindicator. Imposex levels were determined in 2006 and 2010, and integrated with age readings from statoliths for a better chronological analysis of the data. Females affected with imposex were detected in 84% of the sites surveyed in 2006, with levels higher than the Ecological Quality Objective (EcoQO) set by the OSPAR Commission for N. reticulatus (VDSI < 0.3) in approximately half of those sites. In 2010, imposex was only detected in 20% of the surveyed sites and, when detected, the VDSI were lower than the EcoQO, except for one location. The comparison between the current results and the levels previously reported for 2004 and 2005 in the same offshore area evidences a clear decline of imposex in 2010 that confirms the efficacy of Regulation (EC) No. 782/2003 and the IMO AFS-Convention (entered into force in 2003 and 2008, respectively). Moreover, the age of the whelks used in the monitoring is estimated in 3-5 years, indicating that actual TBT water contamination in 2010 may in fact be lower than that extrapolated from the imposex levels. We conclude that the legislation was indeed very effective in reducing TBT pollution in the NW Portuguese continental shelf, not only in terms of the magnitude but also in terms of fast field repercussion.

Climate change vulnerability assessment of the main marine commercial fish and invertebrates of Portugal.

This is the first attempt to apply an expert-based ecological vulnerability assessment of the effects of climate change on the main marine resources of Portugal. The vulnerability, exposure, sensitivity, adaptive capacity, and expected directional effects of 74 species of fish and invertebrates of commercial interest is estimated based on criteria related to their life-history and level of conservation or exploitation. This analysis is performed separately for three regions of Portugal and two scenarios of climate change (RCP 4.5 and RCP 8.5). To do that, the fourth assessment report IPCC framework for vulnerability assessments was coupled to the outputs of a physical-biogeochemical model allowing to weight the exposure of the species by the expected variability of the environmental variables in the future. The highest vulnerabilities were found for some migratory and elasmobranch species, although overall vulnerability scores were low probably due to the high adaptive capacity of species from temperate ecosystems. Among regions, the highest average vulnerability was estimated for the species in the Central region while higher vulnerabilities were identified under climate change scenario RCP 8.5 in the three regions, due to higher expected climatic variability. This work establishes the basis for the assessment of the vulnerability of the human activities relying on marine resources in the context of climate change.

Imposex in Bolinus brandaris from the Ria formosa lagoon (southern Portugal): usefulness of "single-site baselines" for environmental monitoring.

The present study aimed at creating a so called "single-site baseline" (SSB) of imposex in the purple dye murex (Bolinus brandaris) from the Ria Formosa lagoon (Algarve coast, southern Portugal), at the moment that the IMO Antifouling System Convention (banning globally the use of organotin compounds in antifouling paints on ships' hulls) entered into force (September 2008). In the context of monitoring long-term temporal trends of pollution levels, SSBs are proposed whenever it is difficult to obtain adequate numbers of bioindicator specimens in most of the study area or if significant short-term variability in imposex levels is likely to occur. In order to deal with these constraints, this study created a SSB by sampling on a monthly-basis (from October 2008 to September 2009) in a single site considered to be representative of the Ria Formosa as a whole (vicinities of the Culatra Island) and where B. brandaris is abundant all year round. The SSB provided a good characterisation of the monthly variation in the imposex indices, which ranged throughout the year between 72.7 < I% < 100; 0.08 < FPLI < 1.73; 1.20 < RPLI < 23.27; 1.29 < VDSI < 3.54. Only a SSB could have detected this large variability, which otherwise might have been unnoticed and would have produced biased temporal and spatial comparisons, leading to erroneous interpretations of environmental monitoring data. Therefore, this SSB of imposex in B. brandaris constitutes valuable reference data to assess the future trend of organotin pollution in the Ria Formosa, after the benchmark date of the complete eradication of organotin from antifouling agents was implemented worldwide.

Ovipositor Extrusion Promotes the Transition from Courtship to Copulation and Signals Female Acceptance in Drosophila melanogaster.

Communication between male and female fruit flies during courtship is essential for successful mating, but, as with many other species, it is the female who decides whether to mate. Here, we show a novel role for ovipositor extrusion in promoting male copulation attempts in virgin and mated females and signaling acceptance in virgins. We first show that ovipositor extrusion is only displayed by sexually mature females, exclusively during courtship and in response to the male song. We identified a pair of descending neurons that controls ovipositor extrusion in mated females. Genetic silencing of the descending neurons shows that ovipositor extrusion stimulates the male to attempt copulation. A detailed behavioral analysis revealed that during courtship, the male repeatedly licks the female genitalia, independently of ovipositor extrusion, and that licking an extruded ovipositor prompts a copulation attempt. However, if the ovipositor is not subsequently retracted, copulation is prevented, as it happens with mated females. In this study, we reveal a dual function of the ovipositor: while its extrusion is necessary for initiating copulation by the male, its retraction signals female acceptance. We thus uncover the significance of the communication between male and female that initiates the transition from courtship to copulation.

CD137/OX40 Bispecific Antibody Induces Potent Antitumor Activity that Is Dependent on Target Coengagement.

Following the success of immune checkpoint blockade therapy against cancer, agonistic antibodies targeting T-cell costimulatory pathways are in clinical trials. The TNF superfamily of receptors (TNFRSF) members CD137 and OX40 are costimulatory receptors that stimulate T-cell proliferation and activation upon interaction with their cognate ligands. Activating CD137 and OX40 with agonistic mAbs stimulates the immune system due to their broad expression on CD4+ and CD8+ T cells and natural killer cells and has antitumor effects in preclinical models. Most TNFRSF agonist antibodies require crosslinking via Fcγ receptors (FcγR), which can limit their clinical activity. FS120 mAb2, a dual agonist bispecific antibody targeting CD137 and OX40, activated both CD4+ and CD8+ T cells in an FcγR-independent mechanism, dependent on concurrent binding. A mouse surrogate version of the bispecific antibody displayed antitumor activity in syngeneic tumor models, independent of T regulatory cell depletion and of FcγR interaction, but associated with peripheral T-cell activation and proliferation. When compared with a crosslink-independent CD137 agonist mAb, the FS120 surrogate induced lower liver T-cell infiltration. These data support initiation of clinical development of FS120, a first-in-class dual agonist bispecific antibody for the treatment of human cancer.