BATF regulates progenitor to cytolytic effector CD8+ T cell transition during chronic viral infection.

During chronic viral infection, CD8+ T cells develop into three major phenotypically and functionally distinct subsets: Ly108+TCF-1+ progenitors, Ly108-CX3CR1- terminally exhausted cells and the recently identified CX3CR1+ cytotoxic effector cells. Nevertheless, how CX3CR1+ effector cell differentiation is transcriptionally and epigenetically regulated remains elusive. Here, we identify distinct gene regulatory networks and epigenetic landscapes underpinning the formation of these subsets. Notably, our data demonstrate that CX3CR1+ effector cells bear a striking similarity to short-lived effector cells during acute infection. Genetic deletion of Tbx21 significantly diminished formation of the CX3CR1+ subset. Importantly, we further identify a previously unappreciated role for the transcription factor BATF in maintaining a permissive chromatin structure that allows the transition from TCF-1+ progenitors to CX3CR1+ effector cells. BATF directly bound to regulatory regions near Tbx21 and Klf2, modulating their enhancer accessibility to facilitate the transition. These mechanistic insights can potentially be harnessed to overcome T cell exhaustion during chronic infection and cancer.

Tfh-cell-derived interleukin 21 sustains effector CD8+ T cell responses during chronic viral infection.

CD4+ T cell-derived interleukin 21 (IL-21) sustains CD8+ T cell responses during chronic viral infection, but the helper subset that confers this protection remains unclear. Here, we applied scRNA and ATAC-seq approaches to determine the heterogeneity of IL-21+CD4+ T cells during LCMV clone 13 infection. CD4+ T cells were comprised of three transcriptionally and epigenetically distinct populations: Cxcr6+ Th1 cells, Cxcr5+ Tfh cells, and a previously unrecognized Slamf6+ memory-like (Tml) subset. T cell differentiation was specifically redirected toward the Tml subset during chronic, but not acute, LCMV infection. Although this subset displayed an enhanced capacity to accumulate and some developmental plasticity, it remained largely quiescent, which may hinder its helper potential. Conversely, mixed bone marrow chimera experiments revealed that Tfh cell-derived IL-21 was critical to sustain CD8+ T cell responses and viral control. Thus, strategies that bolster IL-21+Tfh cell responses may prove effective in enhancing CD8+ T cell-mediated immunity.

The transcription factor IRF2 drives interferon-mediated CD8+ T cell exhaustion to restrict anti-tumor immunity.

Type I and II interferons (IFNs) stimulate pro-inflammatory programs that are critical for immune activation, but also induce immune-suppressive feedback circuits that impede control of cancer growth. Here, we sought to determine how these opposing programs are differentially induced. We demonstrated that the transcription factor interferon regulatory factor 2 (IRF2) was expressed by many immune cells in the tumor in response to sustained IFN signaling. CD8+ T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor and instead enabled sustained effector functions that promoted long-term tumor control and increased responsiveness to immune checkpoint and adoptive cell therapies. The long-term tumor control by IRF2-deficient CD8+ T cells required continuous integration of both IFN-I and IFN-II signals. Thus, IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a potential target to enhance cancer control.

Oxygen-evolving photosystem II structures during S1-S2-S3 transitions.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.

Architecture of the chloroplast PSI-NDH supercomplex in Hordeum vulgare.

The chloroplast NADH dehydrogenase-like (NDH) complex is composed of at least 29 subunits and has an important role in mediating photosystem I (PSI) cyclic electron transport (CET)1-3. The NDH complex associates with PSI to form the PSI-NDH supercomplex and fulfil its function. Here, we report cryo-electron microscopy structures of a PSI-NDH supercomplex from barley (Hordeum vulgare). The structures reveal that PSI-NDH is composed of two copies of the PSI-light-harvesting complex I (LHCI) subcomplex and one NDH complex. Two monomeric LHCI proteins, Lhca5 and Lhca6, mediate the binding of two PSI complexes to NDH. Ten plant chloroplast-specific NDH subunits are presented and their exact positions as well as their interactions with other subunits in NDH are elucidated. In all, this study provides a structural basis for further investigations on the functions and regulation of PSI-NDH-dependent CET.

The structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs.

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and the structure of the photosystem I-LHC (PSI-LHCI) supercomplex has been shown to be variable depending on the species of organisms. However, the structural and evolutionary correlations of red-lineage LHCs are unknown. Here, we determined a 1.92-Å resolution cryoelectron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137), which is an important taxon in the Cyanidiophyceae. We subsequently investigated the correlations of PSI-LHCIs from different organisms through structural comparisons and phylogenetic analysis. The PSI-LHCI structure obtained shows five LHCI subunits surrounding a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in the red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae, whereas no orthologous relationships were found between C. caldarium Lhcr1-3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

Structures and organizations of PSI-AcpPCI supercomplexes from red tidal and coral symbiotic photosynthetic dinoflagellates.

Marine photosynthetic dinoflagellates are a group of successful phytoplankton that can form red tides in the ocean and also symbiosis with corals. These features are closely related to the photosynthetic properties of dinoflagellates. We report here three structures of photosystem I (PSI)-chlorophylls (Chls) a/c-peridinin protein complex (PSI-AcpPCI) from two species of dinoflagellates by single-particle cryoelectron microscopy. The crucial PsaA/B subunits of a red tidal dinoflagellate Amphidinium carterae are remarkably smaller and hence losing over 20 pigment-binding sites, whereas its PsaD/F/I/J/L/M/R subunits are larger and coordinate some additional pigment sites compared to other eukaryotic photosynthetic organisms, which may compensate for the smaller PsaA/B subunits. Similar modifications are observed in a coral symbiotic dinoflagellate Symbiodinium species, where two additional core proteins and fewer AcpPCIs are identified in the PSI-AcpPCI supercomplex. The antenna proteins AcpPCIs in dinoflagellates developed some loops and pigment sites as a result to accommodate the changed PSI core, therefore the structures of PSI-AcpPCI supercomplex of dinoflagellates reveal an unusual protein assembly pattern. A huge pigment network comprising Chls a and c and various carotenoids is revealed from the structural analysis, which provides the basis for our deeper understanding of the energy transfer and dissipation within the PSI-AcpPCI supercomplex, as well as the evolution of photosynthetic organisms.

Engineered Nanoporous Frameworks for Adsorption Cooling Applications.

The energy demand for traditional vapor-compressed technology for space cooling continues to soar year after year due to global warming and the increasing human population's need to improve living and working conditions. Thus, there is a growing demand for eco-friendly technologies that use sustainable or waste energy resources. This review discusses the properties of various refrigerants used for adsorption cooling applications followed by a brief discussion on the thermodynamic cycle. Next, sorbents traditionally used for cooling are reviewed to emphasize the need for advanced capture materials with superior properties to improve refrigerant sorption. The remainder of the review focus on studies using engineered nanoporous frameworks (ENFs) with various refrigerants for adsorption cooling applications. The effects of the various factors that play a role in ENF-refrigerant pair selection, including pore structure/dimension/shape, morphology, open-metal sites, pore chemistry and possible presence of defects, are reviewed. Next, in-depth insights into the sorbent-refrigerant interaction, and pore filling mechanism gained through a combination of characterization techniques and computational modeling are discussed. Finally, we outline the challenges and opportunities related to using ENFs for adsorption cooling applications and provide our views on the future of this technology.

Bhlhe40 Promotes CD4+ T Helper 1 Cell and Suppresses T Follicular Helper Cell Differentiation during Viral Infection.

In response to acute infection, naive CD4+ T cells primarily differentiate into T helper 1 (Th1) or T follicular helper (Tfh) cells that play critical roles in orchestrating cellular or humoral arms of immunity, respectively. However, despite the well established role of T-bet and BCL-6 in driving Th1 and Tfh cell lineage commitment, respectively, whether additional transcriptional circuits also underlie the fate bifurcation of Th1 and Tfh cell subsets is not fully understood. In this article, we study how the transcriptional regulator Bhlhe40 dictates the Th1/Tfh differentiation axis in mice. CD4+ T cell-specific deletion of Bhlhe40 abrogates Th1 but augments Tfh differentiation. We also assessed an increase in germinal center B cells and Ab production, suggesting that deletion of Bhlhe40 in CD4+ T cells not only alters Tfh differentiation but also their capacity to provide help to B cells. To identify molecular mechanisms by which Bhlhe40 regulates Th1 versus Tfh lineage choice, we first performed epigenetic profiling in the virus specific Th1 and Tfh cells following LCMV infection, which revealed distinct promoter and enhancer activities between the two helper cell lineages. Furthermore, we identified that Bhlhe40 directly binds to cis-regulatory elements of Th1-related genes such as Tbx21 and Cxcr6 to activate their expression while simultaneously binding to regions of Tfh-related genes such as Bcl6 and Cxcr5 to repress their expression. Collectively, our data suggest that Bhlhe40 functions as a transcription activator to promote Th1 cell differentiation and a transcription repressor to suppress Tfh cell differentiation.

Quantitative Proteomics of Tissue-Infiltrating T Cells From CRC Patients Identified Lipocalin-2 Induces T-Cell Apoptosis and Promotes Tumor Cell Proliferation by Iron Efflux.

T cells play the most pivotal roles in antitumor immunity; the T-cell proteome and the differentially expressed proteins in the tumor immune microenvironment have rarely been identified directly from the clinical samples, especially for tumors that lack effective immunotherapy targets, such as colorectal cancer (CRC). In this study, we analyzed the protein expression pattern of the infiltrating T cells isolated from CRC patients using quantitative proteomics. CD4+ and CD8+ T cells were isolated from clinical samples and labeled by tandem mass tag reagents, and the differentially expressed proteins were quantified by mass spectrometry. The T-cell proteome profiling revealed dysfunctions in these tumor-infiltrating T cells. Specifically, antitumor immunity was suppressed because of differentially expressed metal ion transporters and immunity regulators. For the first time, lipocalin-2 (LCN2) was shown to be significantly upregulated in CD4+ T cells. Quantitative proteomic analysis of LCN2-overexpressed Jurkat cells showed that LCN2 damaged T cells by changes in iron transport. LCN2 induced T-cell apoptosis by reducing cellular iron concentration; moreover, the iron that was transported to the tumor microenvironment aided tumor cell proliferation, promoting tumor development. Meanwhile, LCN2 also influenced tumor progression through immune cytokines and cholesterol metabolism. Our results demonstrated that LCN2 has immunosuppressive functions that can promote tumor development; therefore, it is a potential immunotherapy target for CRC.

Quantitative Proteomic Analysis Reveals Functional Alterations of the Peripheral Immune System in Colorectal Cancer.

Colorectal cancer (CRC) is characterized by high morbidity, high mortality, and limited response to immunotherapies. The peripheral immune system is an important component of tumor immunity, and enhancements of peripheral immunity help to suppress tumor progression. However, the functional alterations of the peripheral immune system in CRC are unclear. Here, we used mass spectrometry-based quantitative proteomics to establish a protein expression atlas for the peripheral immune system in CRC, including plasma and five types of immune cells (CD4+ T cells, CD8+ T cells, monocytes, natural killer cells, and B cells). Synthesizing the results of the multidimensional analysis, we observed an enhanced inflammatory phenotype in CRC, including elevated expression of plasma inflammatory proteins, activation of the inflammatory pathway in monocytes, and increased inflammation-related ligand-receptor interactions. Notably, we observed tumor effects on peripheral T cells, including altered cell subpopulation ratios and suppression of cell function. Suppression of CD4+ T cell function is mainly mediated by high expression levels of protein tyrosine phosphatases. Among them, the expression of protein tyrosine phosphatase receptor type J (PTPRJ) gradually increased with CRC progression; knockdown of PTPRJ in vitro could promote T cell activation, thereby enhancing peripheral immunity. We also found that the combination of leucine-rich α-2 glycoprotein 1 (LRG1) and apolipoprotein A4 (APOA4) had the best predictive ability for colorectal cancer and has the potential to be a biomarker. Overall, this study provides a comprehensive understanding of the peripheral immune system in CRC. It also offers insights regarding the potential clinical utilities of these peripheral immune characteristics as diagnostic indicators and therapeutic targets.

Gastric Cancer Immune Subtypes and Prognostic Modeling: Insights from Aging-Related Gene Analysis.

Gastric cancer (GC) is highly heterogeneous and influenced by aging-related factors. This study aimed to improve individualized prognostic assessment of GC by identifying aging-related genes and subtypes. Immune scores of GC samples from GEO and TCGA databases were calculated using ESTIMATE and scored as high immune (IS_high) and low immune (IS_low). ssGSEA was used to analyze immune cell infiltration. Univariate Cox regression was employed to identify prognosis-related genes. LASSO regression analysis was used to construct a prognostic model. GSVA enrichment analysis was applied to determine pathways. CCK-8, wound healing, and Transwell assays tested the proliferation, migration, and invasion of the GC cell line (AGS). Cell cycle and aging were examined using flow cytometry, ß-galactosidase staining, and Western blotting. Two aging-related GC subtypes were identified. Subtype 2 was characterized as lower survival probability and higher risk, along with a more immune-responsive tumor microenvironment. Three genes (IGFBP5, BCL11B, and AKR1B1) screened from aging-related genes were used to establish a prognosis model. The AUC values of the model were greater than 0.669, exhibiting strong prognostic value. In vitro, IGFBP5 overexpression in AGS cells was found to decrease viability, migration, and invasion, alter the cell cycle, and increase aging biomarkers (SA-ß-galactosidase, p53, and p21). This analysis uncovered the immune characteristics of two subtypes and aging-related prognosis genes in GC. The prognostic model established for three aging-related genes (IGFBP5, BCL11B, and AKR1B1) demonstrated good prognosis performance, providing a foundation for personalized treatment strategies aimed at GC.

Lymphocytic Choriomeningitis Virus Clone 13 Infection Results in CD8 T Cell-Mediated Host Mortality in Diacylglycerol Kinase α-Deficient Mice.

Diacylglycerol is a potent element of intracellular secondary signaling cascades whose production is enhanced by cell-surface receptor agonism and function is regulated by enzymatic degradation by diacylglycerol kinases (DGKs). In T cells, stringent regulation of the activity of this second messenger maintains an appropriate balance between effector function and anergy. In this article, we demonstrate that DGKα is an indispensable regulator of TCR-mediated activation of CD8 T cells in lymphocytic choriomeningitis virus Clone 13 viral infection. In the absence of DGKα, Clone 13 infection in a murine model results in a pathologic, proinflammatory state and a multicellular immunopathologic host death that is predominantly driven by CD8 effector T cells.

High-temperature superconductivity in monolayer Bi2Sr2CaCu2O8+δ.

Although copper oxide high-temperature superconductors constitute a complex and diverse material family, they all share a layered lattice structure. This curious fact prompts the question of whether high-temperature superconductivity can exist in an isolated monolayer of copper oxide, and if so, whether the two-dimensional superconductivity and various related phenomena differ from those of their three-dimensional counterparts. The answers may provide insights into the role of dimensionality in high-temperature superconductivity. Here we develop a fabrication process that obtains intrinsic monolayer crystals of the high-temperature superconductor Bi2Sr2CaCu2O8+δ (Bi-2212; here, a monolayer refers to a half unit cell that contains two CuO2 planes). The highest superconducting transition temperature of the monolayer is as high as that of optimally doped bulk. The lack of dimensionality effect on the transition temperature defies expectations from the Mermin-Wagner theorem, in contrast to the much-reduced transition temperature in conventional two-dimensional superconductors such as NbSe2. The properties of monolayer Bi-2212 become extremely tunable; our survey of superconductivity, the pseudogap, charge order and the Mott state at various doping concentrations reveals that the phases are indistinguishable from those in the bulk. Monolayer Bi-2212 therefore displays all the fundamental physics of high-temperature superconductivity. Our results establish monolayer copper oxides as a platform for studying high-temperature superconductivity and other strongly correlated phenomena in two dimensions.

Structural insights into the action mechanisms of artificial electron acceptors in photosystem II.

Photosystem II (PSII) utilizes light energy to split water, and the electrons extracted from water are transferred to QB, a plastoquinone molecule bound to the D1 subunit of PSII. Many artificial electron acceptors (AEAs) with molecular structures similar to that of plastoquinone can accept electrons from PSII. However, the molecular mechanism by which AEAs act on PSII is unclear. Here, we solved the crystal structure of PSII treated with three different AEAs, 2,5-dibromo-1,4-benzoquinone, 2,6-dichloro-1,4-benzoquinone, and 2-phenyl-1,4-benzoquinone, at 1.95 to 2.10 Å resolution. Our results show that all AEAs substitute for QB and are bound to the QB-binding site (QB site) to receive electrons, but their binding strengths are different, resulting in differences in their efficiencies to accept electrons. The acceptor 2-phenyl-1,4-benzoquinone binds most weakly to the QB site and showed the highest oxygen-evolving activity, implying a reverse relationship between the binding strength and oxygen-evolving activity. In addition, a novel quinone-binding site, designated the QD site, was discovered, which is located in the vicinity of QB site and close to QC site, a binding site reported previously. This QD site is expected to play a role as a channel or a storage site for quinones to be transported to the QB site. These results provide the structural basis for elucidating the actions of AEAs and exchange mechanism of QB in PSII and also provide information for the design of more efficient electron acceptors.

Electroacupuncture attenuates nociceptive behaviors in a mouse model of cancer pain.

Pain is a major symptom in cancer patients, and cancer-induced bone pain (CIBP) is the most common type of moderate and severe cancer-related pain. The current available analgesic treatments for CIBP have adverse effects as well as limited therapeutic effects. Acupuncture is proved effective in pain management as a safe alternative therapy. We evaluated the analgesic effect of acupuncture in treatment of cancer pain and try to explore the underlying analgesic mechanisms. Nude mice were inoculated with cancer cells into the left distal femur to establish cancer pain model. Electroacupuncture (EA) treatment was applied for the xenograft animals. Pain behaviors of mice were evaluated, followed by the detections of neuropeptide-related and inflammation-related indicators in peripheral and central levels. EA treatment alleviated cancer-induced pain behaviors covering mechanical allodynia, thermal hyperalgesia and spontaneous pain, and also down-regulated immunofluorescence expressions of neuropeptide CGRP and p75 in the skin of affected plantar area in xenograft mice, and inhibited expressions of overexpressed neuropeptide-related and inflammation-related protein in the lumbar spinal cord of xenograft mice. Overall, our findings suggest that EA treatment ameliorated cancer-induced pain behaviors in the mouse xenograft model of cancer pain, possibly through inhibiting the expressions of neuropeptide-related and inflammation-related protein in central level following tumor cell xenografts.

microRNA-486-5p Regulates DNA Damage Inhibition and Cisplatin Resistance in Lung Adenocarcinoma by Targeting AURKB.

Lung adenocarcinoma (LUAD) severely affects human health, and cisplatin (DDP) resistance is the main obstacle in LUAD treatment, the mechanism of which is unknown. Bioinformatics methods were utilized to predict expression and related pathways of AURKB in LUAD tissues, as well as the upstream regulated microRNAs. qRT-PCR assayed expression of AURKB and microRNA-486-5p. RIP and dual-luciferase experiments verified the binding and interaction between the two genes. CCK-8 was used to detect cell proliferation ability and IC50 values. Flow cytometry was utilized to assess the cell cycle. Comet assay and western blot tested DNA damage and γ-H2AX protein expression, respectively. In LUAD, AURKB was upregulated, but microRNA-486-5p was downregulated. The targeted relationship between the two was confirmed by RIP and dual-luciferase experiments. Cell experiments showed that AURKB knock-down inhibited cell proliferation, reduced IC50 values, induced cell cycle arrest, and caused DNA damage. The rescue experiment presented that high expression of microRNA-486-5p could weaken the impact of AURKB overexpression on LUAD cell behavior and DDP resistance. microRNA-486-5p regulated DNA damage to inhibit DDP resistance in LUAD by targeting AURKB, implying that microRNA-486-5p/AURKB axis may be a possible therapeutic target for DDP resistance in LUAD patients.

Metabolic heterogeneity affects trastuzumab response and survival in HER2-positive advanced gastric cancer.

BACKGROUND: Trastuzumab is the only first-line treatment targeted against the human epidermal growth factor receptor 2 (HER2) approved for patients with HER2-positive advanced gastric cancer. The impact of metabolic heterogeneity on trastuzumab treatment efficacy remains unclear. METHODS: Spatial metabolomics via high mass resolution imaging mass spectrometry was performed in pretherapeutic biopsies of patients with HER2-positive advanced gastric cancer in a prospective multicentre observational study. The mass spectra, representing the metabolic heterogeneity within tumour areas, were grouped by K-means clustering algorithm. Simpson's diversity index was applied to compare the metabolic heterogeneity level of individual patients. RESULTS: Clustering analysis revealed metabolic heterogeneity in HER2-positive gastric cancer patients and uncovered nine tumour subpopulations. High metabolic heterogeneity was shown as a factor indicating sensitivity to trastuzumab (p = 0.008) and favourable prognosis at trend level. Two of the nine tumour subpopulations associated with favourable prognosis and trastuzumab sensitivity, and one subpopulation associated with poor prognosis and trastuzumab resistance. CONCLUSIONS: This work revealed that tumour metabolic heterogeneity associated with prognosis and trastuzumab response based on tissue metabolomics of HER2-positive gastric cancer. Tumour metabolic subpopulations may provide an association with trastuzumab therapy efficacy. CLINICAL TRIAL REGISTRATION: The patient cohort was conducted from a multicentre observational study (VARIANZ;NCT02305043).

Quantifying the Energy Spillover between Photosystems II and I in Cyanobacterial Thylakoid Membranes and Cells.

The spatial separation of photosystems I and II (PSI and PSII) is thought to be essential for efficient photosynthesis by maintaining a balanced flow of excitation energy between them. Unlike the thylakoid membranes of plant chloroplasts, cyanobacterial thylakoids do not form tightly appressed grana stacks that enforce strict lateral separation. The coexistence of the two photosystems provides a ground for spillover-excitation energy transfer from PSII to PSI. Spillover has been considered as a pathway of energy transfer from the phycobilisomes to PSI and may also play a role in state transitions as means to avoid overexcitation of PSII. Here, we demonstrate a significant degree of energy spillover from PSII to PSI in reconstituted membranes and isolated thylakoid membranes of Thermosynechococcus (Thermostichus) vulcanus and Synechocystis sp. PCC 6803 by steady-state and time-resolved fluorescence spectroscopy. The quantum yield of spillover in these systems was determined to be up to 40%. Spillover was also found in intact cells but to a considerably lower degree (20%) than in isolated thylakoid membranes. The findings support a model of coexistence of laterally separated microdomains of PSI and PSII in the cyanobacterial cells as well as domains where the two photosystems are energetically connected. The methodology presented here can be applied to probe spillover in other photosynthetic organisms.