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During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determine the binding mode of HURP to microtubules using cryo-EM. The structure helps rationalize why HURP functions as a microtubule stabilizer. Additionally, HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observe that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in microtubule length control.

Innate immune responses to microbial pathogens are regulated by intracellular receptors known as nucleotide-binding leucine-rich repeat receptors (NLRs) in both the plant and animal kingdoms. Across plant innate immune systems, "helper" NLRs (hNLRs) work in coordination with "sensor" NLRs (sNLRs) to modulate disease resistance signaling pathways. Activation mechanisms of hNLRs based on structures are unknown. Our research reveals that the hNLR, known as NLR required for cell death 4 (NRC4), assembles into a hexameric resistosome upon activation by the sNLR Bs2 and the pathogenic effector AvrBs2. This conformational change triggers immune responses by facilitating the influx of calcium ions (Ca2+) into the cytosol. The activation mimic alleles of NRC2, NRC3, or NRC4 alone did not induce Ca2+ influx and cell death in animal cells, suggesting that unknown plant-specific factors regulate NRCs' activation in plants. These findings significantly advance our understanding of the regulatory mechanisms governing plant immune responses.

Cyanobacteria use large antenna complexes called phycobilisomes (PBSs) for light harvesting. However, intense light triggers non-photochemical quenching, where the orange carotenoid protein (OCP) binds to PBS, dissipating excess energy as heat. The mechanism of efficiently transferring energy from phycocyanobilins in PBS to canthaxanthin in OCP remains insufficiently understood. Using cryo-electron microscopy, we unveiled the OCP-PBS complex structure at 1.6- to 2.1-angstrom resolution, showcasing its inherent flexibility. Using multiscale quantum chemistry, we disclosed the quenching mechanism. Identifying key protein residues, we clarified how canthaxanthin's transition dipole moment in its lowest-energy dark state becomes large enough for efficient energy transfer from phycocyanobilins. Our energy transfer model offers a detailed understanding of the atomic determinants of light harvesting regulation and antenna architecture in cyanobacteria.

Most life scientists would agree that understanding how cellular processes work requires structural knowledge about the macromolecules involved. For example, deciphering the double-helical nature of DNA revealed essential aspects of how genetic information is stored, copied and repaired. Yet, being reductionist in nature, structural biology requires the purification of large amounts of macromolecules, often trimmed off larger functional units. The advent of cryogenic electron microscopy (cryo-EM) greatly facilitated the study of large, functional complexes and generally of samples that are hard to express, purify and/or crystallize. Nevertheless, cryo-EM still requires purification and thus visualization outside of the natural context in which macromolecules operate and coexist. Conversely, cell biologists have been imaging cells using a number of fast-evolving techniques that keep expanding their spatial and temporal reach, but always far from the resolution at which chemistry can be understood. Thus, structural and cell biology provide complementary, yet unconnected visions of the inner workings of cells. Here we discuss how the interplay between cryo-EM and cryo-electron tomography, as a connecting bridge to visualize macromolecules in situ, holds great promise to create comprehensive structural depictions of macromolecules as they interact in complex mixtures or, ultimately, inside the cell itself.

Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other hosts. We have previously demonstrated that IDV binds both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac). Bovines produce both Neu5,9Ac2 and Neu5Gc9Ac, while humans are genetically unable to synthesize Neu5Gc9Ac. 9-O-Acetylation of sialic acids is catalyzed by CASD1 via a covalent acetyl-enzyme intermediate. To characterize the role of Neu5,9Ac2 and Neu5Gc9Ac in IDV infection and determine which form of 9-O-acetylated sialic acids drives IDV entry, we took advantage of a CASD1 knockout (KO) MDCK cell line and carried out feeding experiments using synthetic 9-O-acetyl sialic acids in combination with the single-round and multi-round IDV infection assays. The data from our studies show that (i) CASD1 KO cells are resistant to IDV infection and lack of IDV binding to the cell surface is responsible for the failure of IDV replication; (ii) feeding CASD1 KO cells with Neu5,9Ac2 or Neu5Gc9Ac resulted in a dose-dependent rescue of IDV infectivity; and (iii) diverse IDVs replicated robustly in CASD1 KO cells fed with either Neu5,9Ac2 or Neu5Gc9Ac at a level similar to that in wild-type cells with a functional CASD1. These data demonstrate that IDV can utilize Neu5,9Ac2- or non-human Neu5Gc9Ac-containing glycan receptor for infection. Our findings provide evidence that IDV has acquired the ability to infect and transmit among agricultural animals that are enriched in Neu5Gc9Ac, in addition to posing a zoonotic risk to humans expressing only Neu5,9Ac2.IMPORTANCEInfluenza D virus (IDV) has emerged as a multiple-species-infecting pathogen with bovines as a primary reservoir. Little is known about the functional receptor that drives IDV entry and promotes its cross-species spillover potential among different hosts. Here, we demonstrated that IDV binds exclusively to 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and non-human 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac) and utilizes both for entry and infection. This ability in effective engagement of both 9-O-acetylated sialic acids as functional receptors for infection provides an evolutionary advantage to IDV for expanding its host range. This finding also indicates that IDV has the potential to emerge in humans because Neu5,9Ac2 is ubiquitously expressed in human tissues, including lung. Thus, results of our study highlight a need for continued surveillance of IDV in humans, as well as for further investigation of its biology and cross-species transmission mechanism.

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease1. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs2. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.

The implementation of educational programs that tackle specific contents and promote English skills is convenient, especially in areas where English skills are not properly developed at universities, such as Spain. Indeed, some subjects are currently presented in English for this purpose. CLIL (Content and Language Integrated Learning), where students learn English as a part of specific subject goals of their degree, could be a suitable way to improve English skills while acquiring the corresponding competences. Moreover, online lessons are becoming important, especially during the COVID-19 outbreak, when it was an alternative for face-to-face teaching. The aim of this work was the implementation of an online CLIL course about biorefineries for chemistry, chemical engineering, and mechanical industrial engineering students. The course was a first approach presented as a specialization course that could be merged with related subjects in the future. The main objective of this session was the gradual introduction of English grammar and vocabulary (specific terms) and the promotion of students' participation on oral discussions. The results were successful, showing that the students got motivated and obtained good grades. As possible improvements, the introduction of terms should be less continuous, otherwise the students could lose track of the lesson.

Marta L DeDiego,1,* Yadileiny Portilla,2,* Neus Daviu,2 Darío López-García,1 Laura Villamayor,1 Paula Vázquez-Utrilla,1 Vladimir Mulens-Arias,2 Sonia Pérez-Yagüe,2 Aitor Nogales,3 Jesús G Ovejero,4 Alvaro Gallo-Cordova,4 Luis Enjuanes,1 Sabino Veintemillas-Verdaguer,4 M Puerto Morales,4 Domingo F Barber2 1Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain; 2Department of Immunology, Oncology and Nanobiomedicine Initiative, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain; 3Center for Animal Health Research, CISA-INIA-CSIC, Madrid, Spain; 4Department of Nanoscience and Nanotechnology, Instituto de Ciencia de Materiales de Madrid (ICMM-CSIC), Madrid, Spain*These authors contributed equally to this workCorrespondence: Domingo F Barber; Marta L DeDiego, Email dfbarber@cnb.csic.es; marta.lopez@cnb.csic.esBackground: Severe Acute Respiratory syndrome coronavirus 2 (SARS-CoV-2) and Influenza A viruses (IAVs) are among the most important causes of viral respiratory tract infections, causing similar symptoms. IAV and SARS-CoV-2 infections can provoke mild symptoms like fever, cough, sore throat, loss of taste or smell, or they may cause more severe consequences leading to pneumonia, acute respiratory distress syndrome or even death. While treatments for IAV and SARS-CoV-2 infection are available, IAV antivirals often target viral proteins facilitating the emergence of drug-resistant viral variants. Hence, universal treatments against coronaviruses and IAVs are hard to obtain due to genus differences (in the case of coronavirus) or subtypes (in the case of IAV), highlighting the need for novel antiviral therapies. Interestingly, iron oxide nanoparticles (IONPs) with a 10 nm core size and coated with the biocompatible dimercaptosuccinic acid (DMSA: DMSA-IONP-10) display antiviral activity against SARS-CoV-2 in vitro.Methods: We analyzed the antiviral activity of DMSA-IONP-10 against SARS-CoV-2 infection in vivo, and against IAV infection in vitro and in vivo.Results: DMSA-IONP-10 treatment of mice after SARS-CoV-2 infection impaired virus replication in the lungs and led to a mildly reduced pro-inflammatory cytokine induction after infection, indicating that these IONPs can serve as COVID-19 therapeutic agents. These IONPs also had a prophylactic and therapeutic effect against IAV in tissue cultured cells at non-cytotoxic doses, and a therapeutic effect in IAV-infected-mice, inhibiting viral replication and slightly dampening the inflammatory response after viral infection. As an exacerbated inflammatory response to IAVs and SARS-CoV-2 is detrimental to the host, weakening this response in mice through IONP treatment may reduce disease severity. Interestingly, our data suggest that IONP treatment affects oxidative stress and iron metabolism in cells, which may influence IAV production.Conclusion: This study highlights the antiviral activity of DMSA-IONP-10 against important human respiratory viruses. Keywords: iron oxide nanoparticles, SARS-CoV-2, IAV, viral infection, oxidative stress, iron metabolism

Extant diversity for phenotypic traits is an essential criterion to be considered when ordering priorities for conservation and improvement of animal genetic resources. Concretely, the characterisation of the distinctive body morphometry of a particular group of animals can aid in the design of selective breeding programs, given the strong correlation between body morphology and productive function. The present research aims to characterise an endangered autochthonous camel breed (Canarian camel), mainly relegated to leisure riding, for its body morphology, with a double objective: to explore the phenotypic diversity and structure of the breed for zoometric traits and assess the zoometric profile of this genetic resource that supports its differentiation from other camel breeds. Overall, the results highlight the existence of a high degree of diversity, which may be linked to genetic factors for zoometric traits in an endangered autochthonous breed with traditional in situ breeding schemes, which predicts the success of the implementation of genetic improvement schemes for such functional characteristics. This phenotypic diversity in body morphology could also be a tool for the evaluation of new functional niches within the efforts of functional valorisation of this camel breed for its sustainable conservation. Body morphology traits in the studied camel breed are significantly influenced by sex, physiological status and coat colour.

ABSTRACTWe introduce the innovative concept of Sustainable Laser Energy Consumption (SLEC, ε) that promotes sustainable metal additive manufacturing practices while maintaining productivity and tailored densification in the Selective Laser Melting (SLM) process. This study focuses on deriving analytical expressions through dimensional analysis and Buckingham’s π theorem to predict bulk density with high precision across studied materials: In718, W, AlSi10Mg, Ti6Al4V, and SS316L. This approach provides valuable insights and allows the setting of an appropriate combination of scanning speed and hatch distance for a range of laser power, enabling sustainable manufacturing of SLMed components with tailored density. Furthermore, the mathematical expression introduced as SLEC facilitates the tradeoff between energy consumption and productivity, promoting a greener and more sustainable future of metal additive manufacturing. Furthermore, the underlying fractal nature of SLM is explored through the investigation of the influence of fractal structures, present in the powder bed, on densification that provides information on how powder morphology and homogeneity (i.e. fractal dimension and lacunarity) of the powder bed are linked to a sustainable SLM process.

Abstract The redirection of T lymphocytes against tumor-associated or tumor-specific antigens, using bispecific antibodies or chimeric antigen receptors (CAR), has shown therapeutic success against certain hematological malignancies. However, this strategy has not been effective against solid tumors. Here, we describe the development of CAR T cells targeting p95HER2, a tumor-specific antigen found in HER2-amplified solid tumors. These CAR T cells display robust activity against p95HER2-expressing cell lines but demonstrate limited efficacy against patient-derived xenografts. As p95HER2 is invariably detectable on tumor cells that overexpress HER2, but not those that express HER2 at normal levels, we arm p95HER2-specific CAR T cells with affinity-tuned bispecific antibodies against HER2 and CD3 in order to redirect them only to HER2-amplified cells. The combination of p95HER2.CAR T cells and HER2 x CD3 bispecific antibodies lead to a complete regression in three HER2-positive, patient-derived mouse xenografts tumor models. This combination represents a promising strategy to redirect T cells against a subset of HER2-positive tumors.

Abstract During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determine the binding mode of HURP to microtubules using cryo-EM. The structure helps rationalize why HURP functions as a microtubule stabilizer. Additionally, HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observe that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in microtubule length control.

Structural studies aiming to visualize the interaction of Tau with microtubules (MTs) face several challenges, the main concerning the fact that Tau has multiple MT-interacting regions. In particular, the four (or three) pseudo-repeats of Tau bind to identical elements along the MT lattice but do it through non-identical residues. In addition, any given Tau molecule can use all its repeats or just one for its engagement with MTs. Finally, the binding of one Tau is not necessarily in register with respect to the next one. The mismatch in the MT and Tau repeats, therefore, challenges conventional modes of image analysis when visualizing these samples using cryo-electron microscopy. This commentary is dedicated to those challenges and ways to circumvent them while aiming for an atomic description of the Tau-tubulin interaction.

Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, including sample denaturation and preferential orientations that can occur due to the air-water interface. Streptavidin affinity grids, however, are currently utilized by few cryo-EM labs because they are not commercially available and require a careful fabrication process. Two-dimensional streptavidin crystals are grown onto a biotinylated lipid monolayer that is applied directly to standard holey-carbon cryo-EM grids. The high-affinity interaction between streptavidin and biotin allows for the subsequent binding of biotinylated samples that are protected from the air-water interface during cryo-EM sample preparation. Additionally, these grids provide a strategy for concentrating samples available in limited quantities and purifying protein complexes of interest directly on the grids. Here, a step-by-step, optimized protocol is provided for the robust fabrication of streptavidin affinity grids for use in cryo-EM and negative-stain experiments. Additionally, a trouble-shooting guide is included for commonly experienced challenges to make the use of streptavidin affinity grids more accessible to the larger cryo-EM community.

The enzyme rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the majority of biological carbon fixation on Earth. Although the vast majority of rubiscos across the tree of life assemble as homo-oligomers, the globally predominant form I enzyme-found in plants, algae, and cyanobacteria-forms a unique hetero-oligomeric complex. The recent discovery of a homo-oligomeric sister group to form I rubisco (named form I') has filled a key gap in our understanding of the enigmatic origins of the form I clade. However, to elucidate the series of molecular events leading to the evolution of form I rubisco, we must examine more distantly related sibling clades to contextualize the molecular features distinguishing form I and form I' rubiscos. Here, we present a comparative structural study retracing the evolutionary history of rubisco that reveals a complex structural trajectory leading to the ultimate hetero-oligomerization of the form I clade. We structurally characterize the oligomeric states of deep-branching form Iα and I'' rubiscos recently discovered from metagenomes, which represent key evolutionary intermediates preceding the form I clade. We further solve the structure of form I'' rubisco, revealing the molecular determinants that likely primed the enzyme core for the transition from a homo-oligomer to a hetero-oligomer. Our findings yield new insight into the evolutionary trajectory underpinning the adoption and entrenchment of the prevalent assembly of form I rubisco, providing additional context when viewing the enzyme family through the broader lens of protein evolution.

We provide an overview of the current state of knowledge of island frugivory and seed dispersal and identify knowledge gaps that are important for fundamental research on--and applied conservation of--island ecosystems. We conducted a systematic literature search of frugivory and seed dispersal on islands, omitting large, continental islands. This revealed a total of 448 studies, most (75%) published during the last two decades, especially after 2010. Nearly 65% of them were focused on eight archipelagos. There is a paucity of studies in Pacific archipelagos near Asia and Australia, and in the Indian Ocean. Data on island frugivory and seed dispersal are diverse but highly uneven in geographic and conceptual coverage. Despite their limited biodiversity, islands are essential reservoirs of endemic plants and animals and their interactions. Due to the simplicity of insular ecosystems, we can assess the importance of seed dispersal theory and mechanisms at species and community levels. These include the ecological and biogeographical meaning and prevalence of non-standard mechanisms of seed dispersal on islands; the seed dispersal effectiveness and the relative roles of different frugivore guilds (birds and reptiles being the most important); and patterns of community organization and their drivers as revealed by interaction networks. Island systems are characterized by the extinction of many natives and endemics, and high rates of species introductions. Therefore, understanding how these losses and additions alter seed dispersal processes has been a prevailing goal of island studies and an essential foundation for the effective restoration and conservation of islands. Keywords Conservation and restoration * Fleshy-fruited plants * Insular environments * Mutualistic interactions and ecological networks * Non-standard dispersal mechanisms * Seed dispersal effectiveness, Introduction By definition, islands are geographically isolated, which results in lower species richness of plants and animals, but greater levels of endemism and occurrence of relict species when compared to [...]

La minoría de edad de Enrique III de Castilla (1390-1393) se presenta como un escenario privilegiado para el estudio del conflicto y la negociación entre las distintas instancias de poder del reino, que permite un acercamiento a la cultura política de las elites y a los mecanismos de consenso puestos en marcha por estas. En este período, marcado por los debates sobre los modelos de regencia en torno al regimiento por vía de consejo o por tutores e regidores, las Cortes emergerán, en tanto que representación de la voluntad colectiva del reino, como principal instancia tutelar y arbitral.

Abstract Understanding how biotic interactions shape ecosystems and impact their functioning, resilience and biodiversity has been a sustained research priority in ecology. Yet, traditional assessments of ecological complexity typically focus on species-species interactions that mediate a particular function (e.g., pollination), overlooking both the synergistic effect that multiple functions might develop as well as the resulting species-function participation patterns that emerge in ecosystems that harbor multiple ecological functions. Here we propose a mathematical framework that integrates various types of biotic interactions observed between different species. Its application to recently collected data of an islet ecosystem—reporting 1537 interactions between 691 plants, animals and fungi across six different functions (pollination, herbivory, seed dispersal, decomposition, nutrient uptake, and fungal pathogenicity)—unveils a non-random, nested structure in the way plant species participate across different functions. The framework further allows us to identify a ranking of species and functions, where woody shrubs and fungal decomposition emerge as keystone actors whose removal have a larger-than-random effect on secondary extinctions. The dual insight—from species and functional perspectives—offered by the framework opens the door to a richer quantification of ecosystem complexity and to better calibrate the influence of multifunctionality on ecosystem functioning and biodiversity.

In this entry, the possibility of the implementation of a biorefinery based on multiple raw materials (from agricultural wastes, vegetable oils, etc.) is covered, pointing out the available technology to interconnect different processes so that the atom economy of the process is as high as possible, reducing the environmental impact and improving the efficiency of the energy or products obtained. For this purpose, this model is based on previous works published in the literature. The role of biorefineries is becoming more and more important in the current environmental scenario, as there is a global concern about different environmental issues such as climate change due to GHG emissions, among others. In this sense, a biorefinery presents several advantages such as the use of natural raw materials or wastes, with high atom economy values (that is, all the products are valorized and not released to the environment). As a consequence, the concept of a biorefinery perfectly fits with the Sustainable Development Goals, contributing to the sustainable growth of different regions or countries, regardless of their stage of development. The aim of this entry is the proposal of a biorefinery based on multiple raw materials, using different technologies such as transesterification to produce both biodiesel and biolubricants, steam reforming to produce hydrogen from glycerol or biogas, hydrothermal carbonization of sewage sludge to produce hydrochar, etc. As a result, these technologies have potential for the possible implementation of this biorefinery at the industrial scale, with high conversion and efficiency for most processes included in this biorefinery. However, there are some challenges like the requirement of the further technological development of certain processes. In conclusion, the proposed biorefinery offers a wide range of possibilities to enhance the production of energy and materials (hydrogen, biodiesel, biolubricants, different biofuels, hydrochar, etc.) through green technologies, being an alternative for petrol-based refineries.

Abstract A 3-D printing method to produce dental prostheses of complex shapes from a commercial, photocurable resin-ceramic slurry is developed and optimized. The microstructure, mechanical properties and wear behavior of the resulting material are evaluated and compared with a conventional/control sample and other ceramic-polymer dental composites. Commercial resin-ceramic dental slurries can be successfully extruded and appropriately photocured in a low cost 3-D printing system to produce cost-efficient complex dental parts that could be used in indirect restorations. The printing process does not appreciably introduce defects in the material and the 3-D printed composites exhibit mechanical properties (hardness, elastic modulus) and wear resistance comparable to the control material and analogous, conventional dental composites. The main wear mechanisms under sliding contact against a hard antagonist are plastic deformation at the asperity level and ceramic particle pull-out due to filler/matrix interfacial weakness. Graphical Abstract 3-D printing commercial resin-filler slurries creates cost-efficient tooth prostheses with properties akin to conventional dental composites

Abstract Background The selection of adequate indicators of tissue hypoxia for guiding the resuscitation process of septic patients is a highly relevant issue. Current guidelines advocate for the use of lactate as sole metabolic marker, which may be markedly limited, and the integration of different variables seems more adequate. In this study, we explored the metabolic profile and its implications in the response to the administration of a fluid challenge in early septic shock patients. Methods Observational study including septic shock patients within 24 h of ICU admission, monitored with a cardiac output estimation system, with ongoing resuscitation. Hemodynamic and metabolic variables were measured before and after a fluid challenge (FC). A two-step cluster analysis was used to define the baseline metabolic profile, including lactate, central venous oxygen saturation (ScvO2), central venous-to-arterial carbon dioxide difference (PcvaCO2), and PcvaCO2 corrected by the difference in arterial-to-venous oxygen content (PcvaCO2/CavO2). Results Seventy-seven fluid challenges were analyzed. Cluster analysis revealed two distinct metabolic profiles at baseline. Cluster A exhibited lower ScvO2, higher PcvaCO2, and lower PcvaCO2/CavO2. Increases in cardiac output (CO) were associated with increases in VO2 exclusively in cluster A. Baseline isolated metabolic variables did not correlate with VO2 response, and changes in ScvO2 and PcvaCO2 were associated to VO2 increase only in cluster A. Conclusions In a population of early septic shock patients, two distinct metabolic profiles were identified, suggesting tissue hypoxia or dysoxia. Integrating metabolic variables enhances the ability to detect those patients whose VO2 might increase as results of fluid administration.