However, very little is known about the metabolic mechanisms and redox-sensitive proteins that influence cell cycle progression. We have identified cysteine residues on known cell cycle regulators in Arabidopsis that are potentially accessible, and could play a role in redox regulation, based on secondary structure and solvent accessibility likelihoods for each protein. We propose that redox regulation may function alongside other known posttranslational modifications to control the functions of core cell cycle regulators such as the retinoblastoma protein. Since our current understanding of how redox regulation is involved in cell cycle control is hindered by a lack of knowledge regarding both which residues are important and how modification of those residues alters protein function, we discuss how critical redox modifications can be mapped at the molecular level.Plant stem cells are the foundation of plant growth and development. The balance of quiescence and division is highly regulated, while ensuring that proliferating cells are protected from the adverse effects of environment fluctuations that may damage the genome. Redox regulation is important in both the activation of proliferation and arrest of the cell cycle upon perception of environmental stress. Within this context, reactive oxygen species serve as ‘ Core cell cycle genes and complexes, showing potentially accessible or exposed cysteines. Coloured boxes show the number of potentially exposed cysteines: Orange, 0 exposed; Green, 1–3 exposed; Blue, 4–12 exposed. •pro-life’ signals with positive roles in the regulation of the cell cycle.Reactive oxygen species serve as ‘•Increased oxidation of the cytosol and the nucleus occurs during the early stages of the cell cycle.•Post translational modification of cysteine residues may play a key role in cell cycle regulation.•We have identified a set of core of cell cycle proteins that have either accessible or potentially exposed cysteines.•New sensitive methods to characterise the redox proteome will identify redox-regulated cell cycle proteins. ROS are well-known regulators of plant growth and cell fate, as well as stress signalling molecules in plants and animals The plant redox signalling network constantly adjusts plant growth and development, as well as metabolism, to prevailing environmental conditions. Within this context reactive oxygen species (ROS) production controls numerous growth and developmental processes by modifying enzyme activity and protein-protein interactions. The accumulation of ROS, either through increased production or regulated decreases in antioxidant capacity, shifts the redox regulatory network to a more oxidising state, in which thiols are oxidised to protein disulfides, sulfenic or sulfinic acid derivatives, as well as glutathionylated and Stem cells within these zones are in a process of continuous renewal. They also generate the precursor cells that subsequently divide and differentiate to form all the specialized cells within the plant. A centrally located group of organising stem cells (four in Arabidopsis) within the RAM form the quiescent centre (QC). As in animal stem cell niches, the multipotent stem cells of the plant root QC maintain the surrounding cells in an undifferentiated state Upon leaving the meristematic stem cell niche, daughter cells of the multipotent initials undergo subsequent division, elongation, and differentiation to form the differentiated cells of the root, including the vasculature, epidermis and ground tissues The mitochondria in the SAM cells are organized in a perinuclear network suggesting a requirement for mitochondrial functions to drive the cell cycle In addition to ROS signalling and regulation of cellular redox state, stem cell identity, cell proliferation and fate are regulated in mammalian cells by a tight control of oxygen availability. The stem cell niche in mammals is maintained at low oxygen partial pressures, physiological hypoxia being central to the integrity of the stem cell niche In addition to control of oxygen gradients, oxidants such as hydrogen peroxide prime stem cell differentiation in animals and in plants , as well as inactivate several enzymes that are important in energy production and amino acid metabolism Superoxide is a one-electron reduction product of molecular oxygen with no reactivity to most biological molecules. It can, however, interact with nitric oxide (NO) and oxidise ascorbic acidIn plants, extracellular ROS interact with receptor-like kinases (RLKs) in the communication of information perceived in the cellular environment to the interior of the cell. The concurrent initiation of ROS-dependent and ROS-independent signalling linked to RLKs might also be critical in establishing overall cell fate and responses, the RLK-dependent modulation of apoplastic and intracellular conditions being important in regulating ROS perception and signalling pro-life’ signals serving as critical mediators of cytokine signalling with positive roles in survival is supported by a large body of evidence Nrf2 re-establishes a reduced intracellular redox state by inducing the expression of cytoprotective genes including those involved in the glutathione and thioredoxin systems. The ROS burst at G1 also triggers the expression of FOXO3, a transcription factor that plays essential roles in cell survival signalling with targets in the cell cycle such as the cyclin-dependent kinase inhibitor (CKI) p27 that is involved in cell cycle withdrawal, as well as defence against oxidative stress c-fos and c-jun as well as regulating the activities of protein kinases and phosphatases pro-life’ and survival signalling activities of ROS while also facilitating genetically programmed cell suicide pathways remain to be elucidated in plants and animals.ROS have multi-faceted effects on cell fate. In animals, the concept that ROS are ‘21/S transition. The binding of growth factors, such as epidermal growth factor (EGF) to their receptors (such as EGFR) is promoted by oxidation resulting from ROS accumulation Cells use proliferative signalling pathways and stress surveillance systems to regulate entry and progress through the cell cycle The A-type CDKs (CDKA) in plants regulate the G1/S and G2/M phase transitions and also function during the S phase, while the B7 1 type CDKs (CDKB) act on the G2/M transition and during the M phase in vivo probe provided the first evidence of a transient oxidation at G1 in the cytosol and nuclei of proliferating cells in the Arabidopsis embryonic root that is perturbed in mutants with low cellular antioxidant levels A recent study using a redox-sensitive Many cellular functions are controlled by redox processes. Local changes in the redox environment mediate the spatio-temporal regulation of protein functions and enzyme activities in a compartment-specific manner. At the molecular level, this is thought to be effected primarily via post translational modification (PTM) of cysteine residues (as discussed below). Redox regulation serves as a crucial PTM and modulator of protein function that is as yet unexplored in relation to the plant cell cycle.Cell cycle progression is regulated by the activity of cyclin dependent protein kinases (CDKs) and their regulatory partners, which are called cyclins (CYCs) Mitogenic signals promote RBR phosphorylation in plants through the action of CDKs in association with D-type cyclins, particularly CYCLIN D3:1 (CYCD3:1). RBR1 is a signal-dependent scaffold protein and a conserved regulator of cell proliferation, differentiation, and stem cell niche maintenance in Arabidopsis Upon phosphorylation, the E2FB transcription factors are released from RBR, enhancing cell cycle gene expression and stimulating cell proliferation, while E2FA remains associated with RBR and maintains meristems through repression of differentiation. The initial phosphorylations are thought to prime further CDK sites for phosphorylation 3http://www.geneontology.org/page/download-annotations) and then extracted the FASTA protein sequence for the representative gene model for each loci identified as cell-cycle related from Ensembl Plants (release-38). We subsequently used solvent accessibility prediction  including ROS/RNS/RSS and, whilst widespread redox modification of the proteome, high RES and formation of irreversible oxidations are considered hallmarks of damaging oxidative stress, low levels of RES are crucial for normal cellular function. There is growing appreciation that redox PTMs are site-specific, governed by the microenvironment of cysteine residues in vivo, and thus undergoes a very wide variety of redox-related PTMs S-sulfhydrate ; reaction with ROS to generate S-sulfenic acid (-SOH), and reaction with RNS in S-nitrosylation (-SNO) (S-sulfinic acid (-SO2H) and S-sulfonic acid (-SO3H). Historically this complex array of modifications was difficult to address in a global proteome context and with sufficient sensitivity to detect endogenous oxidative signalling PTMs. However, recent advances in redox proteomic technologies are delivering significant new insights into the scope and biology of redox regulation.Cysteine (-SH) is unique amongst amino acid residues in its ability to adopt oxidation states from -2 to + 6 n (-SNO) . Sulfeni5Mass spectrometry (MS)-based proteomics is unrivalled in its ability to identify and characterise large portions of the proteome at the level of individual residues and PTMs. Shotgun proteomics requires sample processing steps including protein denaturation and digest to release peptides for separation and MS detection. Direct MS detection of cysteine redox modifications is therefore not usually possible due to their low abundance and frequent instability. Most redox proteomics strategies therefore exploit the intrinsic chemical reactivity of thiols or cysteine PTMs to introduce labels enabling enrichment (via biotinylation or thiol-reactive resin). Quantification, including of PTM site occupancy, is becoming increasingly important and most redox proteomic approaches lend themselves well to isotope-based comparative quantification, for example exploiting labelling steps to introduce isotopic moieties. Methods can be broadly divided into two categories: those that exploit the intrinsic chemistry of redox PTMs to introduce a label directly at the modification site, and indirect methods that label free thiols then apply selective reduction to release specific cysteine PTM sites for differential labelling A&B.Fig. Here we review the most common and promising methods for profiling different redox PTMs and their applications in plant biology   and maleimide  E. MMTS ide, NEM) D. Providin vivo detection of cysteine residues.A limitation with biotinylation is that bulky reagents may not react efficiently with all thiols and site quantification is not always straightforward. Iodoacetamide-alkyne IAA; E, develo7Labelling thiols and detecting a reduction in labelling due to oxidation is rather indirect. Differential alkylation, or tag-switch, approaches have been widely applied to label sites of reversible cysteine oxidation . In the tag-switch approach, alkylation (or blocking) of free thiols is followed by reduction to release reversibly oxidised thiols and then a second labelling step is performed to introduce a means of detecting or enriching modified sites. In the biotin-switch method (BST) Although biotin is the most common affinity tag due to its extremely high binding affinity with streptavidin, enrichment introduces at least one additional step, resulting in quite lengthy protocols that reduce proteome coverage, and non-specific binding to the resin complicates analysis. To address these challenges, resin-based methods to capture thiols have been reported. Commercially available resin, thiopropyl Sepharose 6B, has been applied to directly capture free thiols via disulfide exchange, followed by on-resin protein digestion and multiplexed isobaric labelling 2O2-responsive residues in diatoms A popular quantitative method using a tag-switch strategy is OxiCAT, which can be applied to determine PTM site occupancy in a single sample Arabidopsis suspension cells treated with various oxidants or other exogenous agents There are several other reported isotopic/isobaric labelling methods for introducing quantification into tag-switch workflows. Like OxiCAT, some introduce isotopic labels at the modification site: these include cysTMT S-nitrosylation: free thiols are blocked via alkylation, then -SNO modifications reduced to the free thiol using ascorbate, sometimes in conjunction with copper(I) salts; the newly released thiols are labelled via biotinylation for enrichment and detection S-sulfenic acid S-sulfenylation (see below). This highlights the main limitation of tag-switch methods: their reliance on the selectivity of alkylating and reducing reagents, which is not complete. In a further example, a recent study investigating cross-reactivity between nitrosothiols and sulfinic acids found that MMTS may release methyl sulfinic acid that could react with S-nitrosylation sites and result in slower reduction to the free thiol, such that subsequent detection of -SNO is diminished S-acylation will also be reduced , which can complicate interpretation of results. Nevertheless, these strategies have revealed thousands of potentially redox-sensitive cysteines across diverse proteomes.Tag-switch methods can be adapted to address specific modifications by using a modification-selective reducing agent C. An exa8S-Sulfenylation (-SOH) is a transient PTM with signalling roles, for example in the regulation of protein tyrosine kinases and phosphatases S-sulfenylation profiling in plants S-sulfenylation of 226 proteins in Arabidopsis cell suspensions treated with H2O2, over half of which had not been previously reported to be modified in plants S-sulfenome.S-sulfenylome, down to specific proteins or protein families, might be targetable with different chemical probes A potential limitation of dimedone-based capture is the slow speed of the reaction in comparison to other reactions of sulfenic acids, such as disulfide formation or further oxidation S-sulfenylation detection Bicyclo[6.1.0]nonyne has been reported as alternative chemical probe to trap sulfenic acids, with reaction rates around 100-fold higher than dimedone S-sulfenic acid detection uses a cell-based genetic sensor for this PTM, based on the cysteine-rich domain of the yeast transcription factor YAP1, which forms disulfides with S-sulfenic acid modifications on its cognate signalling protein. Fusion of the cysteine-rich domain of YAP1 with an affinity tag creates a tool to capture and enrich S-sulfenylated proteins in vivo  at the site of modification, which is stable under further sample processing and MS analysis conditions SNOTRAP  for mutual detection of these two PTMs 2H was used to label -SNO, generating a thiosulfonate  and S-sulfonylation (-SO3H) have historically been considered mere markers of oxidative damage, rather than specific controlled signalling PTMs. However, increasing evidence suggests that this may be an over-simplification; for example, transient sulfinylation of peroxiredoxins was shown to be a conserved marker for circadian rhythms across all domains of life S-sulfonylation (-SO3H) is thus far limited to direct detection via MS S-sulfinic acid approaches have been reported recently.The more highly oxidised and  irreversible redox PTMs of S-sulfinic acids in lysates, following blocking of free thiols (necessary to avoid cross-reactivity)  S-sulfinated cysteines to form a sulfone adduct that is stable under acidic conditions Carroll et al. developed novel aryl-nitroso probe NO-Bio to directly capture ctivity) B [116]. Combined with quantitative MS, these recently developed direct labelling approaches should promote investigation of these understudied PTMs in different biological contexts.11S-sulfhydration, Zhang et al. developed chemistry to selectively displace the disulfide that is formed upon MSBT reaction with a CN-biotin reagent, enabling specific enrichment of these sites.  is challenging due to the similar chemical properties and reactivity of this PTM compared to free thiols. Modified tag-switch approaches have had some success, but site identification is difficult and these methods lack specificity e sites. A [119]. 12in situ a GSH analogue incorporating an azide bio-orthogonal tag, which is then metabolically incorporated into cellular proteins (Protein intra- or intermolecular disulfides are often detected using biotin-switch approaches (see above). The specific modification of cysteine with the low molecular weight thiol glutathione (GSH) and its homodimer disulfide (GSSG), however, can be monitored directly. The first of these direct approaches use biotin-functionalised GSH or GSSG analogues to identify sites susceptible to glutathionylation proteins C [123]. The final class of methods that have been developed to address disulfide-type redox PTMs exploit the activity of specific enzyme to classes, including thioredoxin (Trx), which reduces protein disulfides, and glutaredoxin (Grx), which reduces -SSG modifications. Qian, Thrall et al. used an enzymatic version of the tag-switch approach to detect glutathionylation: free thiols were first blocked and then a cocktail containing active Grx1 applied to selectively reduce glutathionylated proteins for subsequent capture on resin and identification D [77], . 13quantify endogenous redox PTMs and changes at the residue level are absolutely crucial for analysing subtle changes such as those occurring at different cell cycle stages. Thus the best methods incorporate both quantification and a means of identifying the precise sites of PTM into their workflow. Integrating chemical and enzymatic tools with state-of-the-art mass spectrometry-based instrumentation and optimised workflows also has a large impact. The potential increase in coverage is well-illustrated by the example of OxiCAT: the group who pioneered the method in vitro methods, although few reported studies have directly compared multiple techniques.Recent years have seen rapid evolution of methods to characterise the redox proteome. The key approaches are summarised, along with their advantages and limitations, in S-sulfenic acid modifications in live cells, and approaches to tackle PTMs that are difficult to distinguish from other cysteine redox states (e.g. S-sulfhydration). We anticipate that method development will also continue to underpin advances in redox biology.In addition to technical developments that advance our ability to interrogate the proteome with existing tools, exciting approaches are emerging to address previously understudied redox PTMs. Examples discussed above include the dimedone-based and more recently developed nucleophilic probes that are able to profile transient 1/S transition during the cell cycle. The RB family proteins regulate the G1/S transition in animals. Inactivation of RB cause oxidative stress by increasing ROS production in mitochondria, where RB is localized. The RB–E2F complex directly suppresses the expression of oxidative metabolism-related enzymes and mitochondrial protein translation genes. It will be intriguing to see if similar mechanisms of redox regulation of cell proliferation are present in plant cells, as well as other redox networks that link cell cycle regulation to metabolism and environmentally sensing. A better understanding of these processes will not only identify the similarities in the redox footprints between plants and animals but also clarify the mechanisms whereby plant stem cells respond to the cellular redox environment. Understanding the functional regulation of redox reactive cysteine residues in cell cycle proteins, and their interactions with mitochondrial and plastid signalling networks, will lead to a step change in our appreciation of how cell metabolism uses the redox reactions that harness energy for life to control cell proliferation and survival in plants and animals.In this review, we have highlighted the central roles of oxygen and redox signalling in regulating cell proliferation, tentatively identified a number of core cell cycle regulators that have the potential to be regulated in this way, and discussed the methods that can be used to analyse and quantify posttranslational redox modifications. Recent progress in increasing the limit and fidelity of detection make studies on the redox regulation of cell cycle control a realistic possibility, at least at the outset in plant cell cultures, where the cell cycle can be synchronised and cell cycle progression can easily be determined, with appropriate amounts of materials harvested for analysis. It will be interesting to determine whether redox processes influence the properties and functions of key cell cycle regulators such as RB and E2Fs, which control DNA replication and mitosis as well as the G


Mycobacterium tuberculosis. We identified mixed M. tuberculosis lineage infection in 2 captive elephants in Nepal by using spoligotyping and large sequence polymorphism. One elephant was infected with Indo-Oceanic and East African–Indian (CAS-Delhi) lineages; the other was infected with Indo-Oceanic and East Asian (Beijing) lineages.Tuberculosis in elephants is primarily caused by  Mycobacterium tuberculosis is a primary cause of tuberculosis (TB) in elephants  in Nepal.Elephant A was a female elephant ≈65–70 years old. She had been in retirement for 3 years before she died in February 2013. We observed TB-like lesions in the lungs postmortem  Figure 1http://chembio.com), a serologic test, on the postmortem lung fluid (an off-label use) of elephant A and the serum of elephant B; results were reactive in both cases, indicating the presence of antibodies to TB. We processed the suspected lung lesions according to standard guidelines  as described previously   and LSP detection PCR results (2 bands were observed). The spoligotyping results showed that the elephant A isolate had a new spoligotype that was not found in the international spoligotyping database. The elephant B isolate belonged to the Indo-Oceanic lineage  . The preS-Delhi)  Figure 2S-Delhi)  Figure 3M. tuberculosis lineages, whereas the second elephant was infected with the Indo-Oceanic and East Asian (Beijing) lineages. We previously identified the Indo-Oceanic lineage in 3 elephants from Nepal (Our study shows that the first elephant was infected with the Indo-Oceanic and East African–Indian (CAS-Delhi) We diagnosed the mixed lineage infection postmortem in both elephants. However, a successful antemortem diagnosis of mixed infection in a single elephant would enable a precise TB diagnosis and selection of an appropriate anti-TB treatment, which could eventually lead to the control of this disease at the herd level.The source of these mixed infections is unknown and could be from humans or elephants infected with these lineages. Infected elephant handlers who have daily close contact would be a likely human source. Genotyping of additional isolates from elephants and their handlers will help to determine the source of infection. We recommend regular TB screening of elephant handlers to safeguard human health and help prevent transmission of TB from humans to elephants.Mycobacterium tuberculosis lineage infection in 2 elephants, Nepal.Additional information regarding mixed


The maintenance and transition of cellular states are controlled by biological processes. Here we present a gene set-based transformation of single cell RNA-Seq data into biological process activities that provides a robust description of cellular states. Moreover, as these activities represent species-independent descriptors, they facilitate the alignment of single cell states across different organisms. Single cell RNA-Seq data can report on cellular types and states, but low signal-to noise and sparse data can make interpretation of cellular state difficult. Here the authors propose a transformation strategy to map RNA-Seq data to biological process activities that are species-agnostic and allow for comparison across species. However, the signal-to-noise ratio of scRNA-Seq data is usually poor, confounding cellular state interpretation. Considering that cellular states are controlled by genetic regulatory mechanisms2, we propose using biological process activities (BPAs) in place of the expression of individual genes in the scRNA-Seq data, which leverages an ensemble of dozens of related genes. In this way, discrepancies in individual genes can be averaged out, yielding reproducible measurements unaffected by common technical noises such as batch effects3 and drop-out events4  technologies has greatly advanced our understanding of cellular statests4 Fig. .Fig. 1Ov5.Gene sets have been used extensively over the past few years to infer the activity of biological processes in many applications. The various catalogs of gene sets, e.g., Molecular Signature Database (MSigDB), group genes into categories of related function. Such gene sets allow particular pathways to be associated with the results of high-throughput assays. Gene set enrichment analysis (GSEA) is a particularly successful approach that summarizes the putative importance of a biological process using an ensemble of expression levels for a set of genes documented to play a role in a specific process7 that extends GSEA to interpret scRNA-Seq data as inferred process activities. Compared to previous studies9, which used selected gene sets describing specific biological processes, e.g., cell cycle or TP53 pathway, for this study we use a comprehensive collection of gene sets, e.g., Gene Ontology (GO) Biological Process (BP), and the immunologic portion of the MSigDB collection called C710, contributing ~ 650 and ~ 1800 gene sets after filtering, respectively (see Methods). BPA’s only option is to decide what gene sets to use for the transformation . For an individual cell and a specific pathway, the BPA transform runs an aREA-based single-cell pathway enrichment analysis to create an activity score from the expression levels of the gene set members of the pathway  and esophagus (E) collection and labeled as original (Ori), whereas their counterparts containing simulated drop-out events were labeled drop-out (Dro)  scRNA-Seq data sets when gene expression profiles are used directly Fig. , but are16. However, as orthologs are usually determined by computational analysis of protein sequence alone17, the expression pattern of orthologous genes may not be the same across species18. The BPA transform, on the other hand, provides a common set of terms from which detailed gene sets can be retrieved in a species-specific manner10. Each species can be analyzed separately using their species-specific gene sets and then merged across species at the BPA level assuming the pathway categories are equivalent, giving an overarching perspective of cellular states and transitions across various organisms.We next performed cross-species single-cell state alignment using BPA profiles in place of gene expression profiles. A previous effort used the expression pattern of one-to-one orthologous genes to align single cells across species2. By analyzing human and mouse single cells separately, we find that the three steps were recapitulated. In human, the first step spans the oocyte to four-cell stage; the second step includes the eight-cell stage; and the third step includes only the morula stage. In mouse, the first step of the developmental timeline is relatively short compared with human, including the oocyte and pronuclear stages; the second step includes the two-cell and four-cell stages; and the last step includes the eight-cell and morula stages . Within each individual data set, cell types  and the variance within species (Vs) and found that the ratio (Vt/Vs) was indeed significantly higher than unity . In contrast, mapping genes to orthologs and using orthologous gene expression to combine the data sets resulted in a significantly lower level of species mixing within cell types (F test P = 0.0035). Visual inspection of the hierarchical clustering dendrogram of the BPA result 22, and mouse (GSE65565)23 embryo-related single cells. Single cells from human preimplantation embryos, especially in the early stages (E3 and E4), overlapped with mouse embryonic stem cells sh GSE666822, and 7 and SCENIC27, which infer the activity of transcription factors from their targets, might also be used. Other transforms such as deep autoencoders28 or network diffusion approaches29 are also possible. Methods that preserve the explicit association with genes  can be run prior to BPA transformation and thus offer the potential for exploring a combination of approaches. Finally, in addition to single-cell data sets, BPA is applicable to bulk expression analysis  data set5. In this study, the biological process subset of MSigDB’s GO gene sets (C5) and the MSigDB immunologic gene sets (C7) for Homo sapiens, Mus musculus, and Danio rerio were used in the presented analyses. C7 contains paired upregulated and downregulated gene sets. As single-cell RNA-sequencing profiles have high drop-out rates, the accuracy for quantifying underexpressed genes is low. To avoid using such genes, as well as redundancy caused by paired gene sets, we retrained only the up-regulated gene sets in C7 for analyses in the study. The gene sets were filtered by the number of genes in the gene set as aREA-based BPA tends to assign higher activities to larger gene sets. On the other hand, smaller gene sets yield less robust activities as indicated by an increase in the coefficient of variance as gene set sizes decrease  was used to quantify activity profiles from the original gene expression7. In this study, we used the log-transformed original single-cell expression as the signatures for inference instead of using a differential measure that would strongly depend on the composition of the data set and possibly impact cross-data set integration  across all cells may reflect that a gene set encodes cell type-related information. As shown in Supplementary Fig. Vs) and within cell types (Vt) and calculated their ratio Vt/Vs. Values higher than unity indicate cells of the same cell types are closer together (lower variance) compared with a cell of a different cell type from the same species (higher variance). The ratio follows an F-distribution with (N–Mt) and (N–Ms) degrees of freedom where N is the number of cells, Mt is the number of cell types (Mt = 3 for the PBMC data set) and Ms is the number of data sets . To combine the original gene expression vectors across species without transforming with BPA, we mapped all genes from human and mouse to their orthologous counterparts using the human–mouse mapping table available in the R Bioconductor biomaRt package.We used a statistical test to assess the degree to which data sets of different species are concordant either in the original gene expression space or upon transformation using BPA. Intuitively, cells of the same cell type but from different species should overlap thus maintaining the separation of distinct cell types yet mixing the species. To quantify this notion, we used an analysis of variance in which we measured the variance within species  dimensionality reduction using the cmdscale function in the R stats package. To detect cell types, DBSCANFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Following the 2018 European Cell Death Organization (ECDO) meeting in Saint Petersburg, high expectations preceeded the 2019 conference in Dresden, Germany. From the 25th to the 27th of September, more than 200 cell-death researchers from globally distributed institutions gathered in Dresden. An interesting mixture of basic scientists, clinician scientists, clinicians, postdocs, and students set the basis for vivid discussion and a lively atmosphere.In slight contrast to previously held training courses, the conference in Dresden was opened by a session of leading young scientists in our field termed “The future of cell death science—the young investigator presentations”, chaired by Markus Rehm. Mathieu Bertrand opened the session with a presentation on the molecular mechanisms of receptor-interacting protein kinase 1 (RIPK1) regulation that defines live and death decisions upon activation of the TNF-receptor 1 (TNFR1). In particular, the presenter focused on the role of the ubiquitin-editing enzyme A20 and the different zinc finger domains therein. These domains appear to define the type of ubiquitin chains that modify RIPK1 function. Mads Daugaard introduced current knowledge about prostate cancer progression with a focus on the role of glycosaminoglycan (GSG) enzymes, in particular CHST11. Mice deficient in this enzyme were not viable and human cancer cells die by anoikis. Christina Munoz-Pinedo presented mechanisms of starvation responses and demonstrated how glucose-deprived cancer cells release IL-8. Alexei Degterev showed details of the RIPK1 kinase, pocked and pointed out the differences between mice and humans within this structure. He also introduced CLSP37, a novel RIPK2 inhibitor. Bart Tummers demonstrated detailed studies about the origin of the autoimmune lymphoproliferative syndrome (ALPS), the pathophysiology of which has been an obstacle for decades in the cell-death research field. The integrated control of apoptosis, necroptosis, and pyroptosis appears to be dysregulated in this syondrome. Stephanie Kreis presented novel insights into drug combination therapies best to be used for melanoma treatments by combining miRNome profiling followed by qCLASH analysis , an innovative method to determine real miRNA–mRNA interactions, and explained a novel approach to overcome drug resistance in these tumors. Jose Pedro Friedmann-Angeli described a novel key inhibitor of ferroptotic cell death that appears to function alongside of the master regulator selenoprotein glutathione peroxidase 4 (GPX4).The second session on “Cell Death and Immunity/Inflammation” was chaired by Ivano Amelio and kicked off with the keynote lecture by Scott Lowe. He presented a fascinating overview on our understanding of the tumor suppressor p53 functions in and beyond cell death in his first part of the lecture. He subsequently pointed out novel insights about the involvement of p53 in the senescent-associated secretory phenotype (SASP), which he demonstrated to be controlled by nuclear factor kappa-B (NF-κB) and bromodomain-containing protein 4 (Brd4). Peter Vandenabeele compared the immunogenicity of apoptosis, necroptosis, and ferroptosis, challenging our current concept of immunogenic cell death and/or necroinflammation. Peter Krammer presented work on the “annexin check point system”, and Leonie Hartmann elaborated on the role of interferon-induced mixed lineage kinase domain like pseudokinase (MLKL) activation in gastrointestinal infection models. Tania Watts shed light on TNF-receptor signaling in the context of adaptive antiviral immune responses, and discussed a role for that system in cancer. Mads Gyrd-Hanssen presented on the mechanisms limiting ubiquitin-mediated post-translational modification by the cylindromatosis lysine 63 deubiquitinase (CYLD). After the coffee break, Tom Vanden Berghe presented translational cell-death science by linking basic research to clinical trials to treat IL-1β and IL-18-driven sepsis. He additionally presented spectacular novel ferroptosis inhibitors that are on their way to clinical trials. Daniel Frank generated a K469R mutant of receptor-interacting protein kinase 3 (RIPK3), a polyubiquitin site close to the RHIM domain, and unexpectedly observed increased RIPK3 phosphorylation and necroptosis, downstream of RIPK1 kinase activity.A flash-talk session of eight-time 2-min presentations followed to especially announce some of the most interesting abstracts, before Carlo Croce set out for the 2019 CDD honorary lecture. In detail, he recapitulated the scientific endeavor to isolate BCL2. He explained the effort and roadblocks that needed to be overcome before the generation of venetoclax was actually possible. The first day ended with an open bar during the first poster session.Simone Fulda moderated the first session on the second day of the meeting: “Cell Death and Regeneration”. Kevin Ryan started a set of exciting presentations by demonstrating how cancers can be attacked when the glucose and mannose metabolism profiles are understood. Sudan He investigated the influence of necroptosis regulators on the tumor microenvironment. Elena Morgun focused her talk on the role of caspase-3 expression in a model of chronic wound healing. Jaewhan Song connected the protein Beclin-1, best known for its role in autophagy, to the necroptosis pathway and developed a model in which Beclin-1, independent of autophagy, interacts with MLKL. Edward Mocarski continued his work on herpesvirus proteins controlling necroptosis and thereby not only limiting the death of the host cell, but also the immune response against the virus. Mechanisms of mitochondrial permeability transition and connections between apoptotic and necrotic cell death controlled by Bax/Bak were discovered by Richard Kitsis, both in cell culture assays and mouse models of heart disease. Patricia Boya analyzed previously unrecognized links between autophagy and apoptosis, before Motti Gerlic shared his results on purified extracellular vesicles released from necroptotic cells. He used this material to perform a proteomic analysis of vesicles budding from dying cells. Florian Bock finished the morning session with the presentation of his results on paracrine resistance mechanisms upon cancer therapy. Participants thereafter enjoyed Poster Session 2, lunch, and vivid discussions.The afternoon session on “Cell Death and Cancer” was chaired by Klaus-Michael Debatin and started with a talk by Gerry Melino. He elaborated on the DNA damage response that his group discovered to be regulated by ZNF281, and defined this factor as a prognostic marker for neuroblastoma. Triona Ni Chonghaile works on HDAC6 inhibitors, which she found in a small-molecule screen in apoptosis-resistant cancer cells. Claudio Mauro introduced the metabolic control of immune-mediated inflammation and its affection upon cell death. Carol Prives presented insights on the regulation of ferroptosis by Mdm2/MdmX, a collaboration with the later keynote speaker Brent Stockwell. Andreas Villunger introduced the role of caspase-2 in cancer resistance, and Lisa Bouchier-Hayes, working on alternative functions of caspase-2, discovered this protease to function as an S-phase checkpoint regulator, potentially to allow DNA repair. Caitlin Brown showed the results on prominin 2 that regulates iron export and thereby sets the threshold for ferroptosis sensitivity. Finally, the second keynote lecture by Brent Stockwell on mechanisms and therapeutic applications of ferroptosis elegantly delineated the timeline of ferroptosis research, and introduced a novel antibody for the detection of ferroptosis. The day ended with the speakers dinner.The third day started when Chair Boris Zhivotovsky introduced Francis Chan as the first speaker in the session “Non-apoptotic Cell Death Mechanisms”. He introduced three ways of how the growing number of pathogens target RIPK3: “Tackle it! Chop it! Shred it!”. William Kaiser employed a virally inspired screen to discover the cytomegalous virus (CMV) entry receptor and a novel set of necroptosis regulators. Anne Hamacher-Brady established the role of endolysosomal interactions with Bax-mediated mitochondrial permeability transition. Benjamin Demarco clearly demonstrated a caspase-8-dependent cleavage of gasdermin D that appeared to be independent of the inflammasome. Again, coffee. Junying Yuan presented encouraging data on the benefit of necroptosis inhibition by RIPK1 inhibitors in a model of rodent stroke and neurodegeneration. Seamus Martin focused on the cell-death-independent functions of death molecules in regulating inflammation, and pointed out the importance to differentiate between cell death and cell stress functions. Feng Shao discovered a caspase cleavage motif in gasdermin molecules that is distinct from the commen tetrapeptide, and presented the structural basis for this novel mechanism. Douglas Green extended our knowledge on LC3-associated endocytosis (LANDO), a fundamentally important autophagy-independent pathway for microglial activation and neurodegeneration.The last session called “Structural and Systems Biology in Cell Death” was chaired by the president of the ECDO, Patrizia Agostinis. Peter Czabotar introduced his work on controlled conversion of Bax and Bak into membrane-permeabilizing executioners. Marion MacFarlane dissected the molecular architecture of FADD/caspase-8 signaling complexes in high-resolution imaging and structural modeling. Nadine Pollak, also working on extrinsic apoptosis, focused on the role of Mcl-1 in controlling caspase-8 activation during cell cycle progression. Jochen Prehn provided insights on how responses to chemotherapy can be predicted from systems analysis approaches in colorectal cancer and presented mechanistic insight into TRAIL-induced entosis as an escape mechanism of cancer cells. Last coffee of the meeting. Inna Lavrik used chemical probes designed to target the death receptor network to control extrinsic apoptosis. Judy Liberman demonstrated how microbes die by microptosis following cytotoxic lymphocyte stimulation.Finally, it was time for the 2019 ECDO honorary lecture, presented by Shigekazu Nagata. He took us on an exciting journey over several decades of cell-death research: from the discovery of phosphatidylserine as an eat-me signal to the molecular mechanisms of the phospholipid-scrambling machinery, explaining how billions of cells in each human body are dying each day. With the next few years of meeting announcement, the celebration of the poster prizes, and the conference dinner, the 27th ECDO meeting closed down. We are looking forward to the 28th ECDO conference in the United Kingdom.


To identify the vulnerable areas associated with systemic oxidative stress and further disruption of these vulnerable areas by measuring the associated morphology and functional network alterations in Parkinson’s disease (PD) patients with and without cognitive impairment.This prospective study was approved by the institutional review board of KCGMH, and written informed consent was obtained. Between December 2010 and May 2015, 41 PD patients with different levels of cognitive functions and 29 healthy volunteers underwent peripheral blood sampling to quantify systemic oxidative stress, as well as T1W volumetric and resting state functional MRI (rs-fMRI) scans. Rs-fMRI was used to derive the healthy intrinsic connectivity patterns seeded by the vulnerable areas associated with any of the significant oxidative stress markers. The two groups were compared in terms of the functional connectivity correlation coefficient (fc-CC) and gray matter volume (GMV) of the network seeded by the vulnerable areas.P < 0.005). We found that the MTL network of healthy subjects resembled the PD-associated atrophy pattern. Furthermore, reduced fc-CC and GMV were further associated with the aggravated cognitive impairment.The levels of oxidative stress markers, including leukocyte apoptosis and adhesion molecules, were significantly higher in the PD group. Using whole-brain VBM-based correlation analysis, the bilateral mesial temporal lobes (MTLs) were identified as the most vulnerable areas associated with lymphocyte apoptosis  contains supplementary material, which is available to authorized users. Parkinson’s disease (PD) is the second most common neurodegenerative movement disorder among the elderly and also presents as a spectrum of cognitive dysfunction, ranging from PD with normal cognition (PDN) to PD with mild cognitive impairment (PDMCI) to PD with dementia (PDD) . PDMCI iIn addition to structural changes, alterations of both dopaminergic and non-dopaminergic transmitter systems in the PD brain have also been found to modify several distinct functional networks underlying the different cognitive impairments . The funIn the large-scale study of human brain networks by Seeley et al., it was reported that spatial disease atrophy patterns reflect the healthy brain’s intrinsic functional network architecture. These findings support the network neurodegeneration hypothesis of Seeley et al.  and suggSystemic oxidative stress is an important etiology of the neuroinflammation seen in PD and is associated with further progression of the disease via various pathways, such as blood-brain barrier (BBB) dysfunction and the infiltration of peripheral immune cells and circulating cytokines , 13. TheThe main focus with respect to peripheral immune cells was on their role in dopamine neuron loss in the substantia nigra (SN) or striatum in previous studies. Although the peripheral immune cells are associated with the initial pathogenesis of PD , it is sIn the present study, we used voxel-based morphometry (VBM) and resting state functional MRI (rs-fMRI) to evaluate the effects of systemic oxidative stress on brain morphology and functional network alterations, respectively, in PD patients with different cognitive statuses. More specifically, the study’s aims were (1) to evaluate the differences in systemic oxidative stress and GM volume changes in PD patients and healthy controls, (2) to investigate the vulnerable areas and associated functional networks in the brain that are highly sensitive to systemic oxidative parameters, and (3) to evaluate the relationships between cognitive impairment and the structural and functional integrity of the networks of the vulnerable areas.This prospective study enrolled 41 patients who were diagnosed with idiopathic PD from December 2010 to May 2015 according to the United Kingdom Brain Bank criteria  by an exEach patient’s disease severity and cognitive functional status were evaluated using the Unified Parkinson’s Disease Rating Scale (UPDRS), the modified Hoehn and Yahr Staging Scale, the Schwab and England Activities of Daily Living Scale, and the MiniMental State Examination (MMSE). Neuropsychological evaluations of five cognitive domains  were conducted using subtests from the Cognitive Ability Screening Instrument  and the All the patients were classified as having PDN, PDMCI, or PDD. PDMCI was defined according to the Movement Disorder Society Task Force Guidelines . CognitiP < 0.05.Blood samples were drawn by venipuncture on the same day as the MRI study and neuropsychological testing were conducted. Systemic inflammation was evaluated in terms of the percentage of apoptotic peripheral leukocytes and the levels of cellular adhesion molecules. The level of leukocyte apoptosis was assessed using APO 2.7-phycoerythrin (PE). The quantities of cellular adhesion molecules were expressed as the mean fluorescence intensity of anti-LFA-1 or anti-macrophage-1 antigen (Mac-1) antibody-positive leukocytes. All the leukocytes and their subtypes were analyzed by flow cytometry. The statistical analyses were performed using the Statistical Package for Social Sciences . The group differences were compared by analysis of covariance (ANCOVA), with age and sex controlled for as potential confounding variables. Statistical significance was set at Bonferroni corrected For each subject, an MRI scan was performed using a 3.0 Tesla whole-body GE Signa MRI system equipped with an eight-channel head coil. A 3D high-resolution T1-weighted anatomical image was acquired using an inversion recovery fast spoiled gradient-recalled echo pulse sequence . A resting state functional image was also acquired using 300 contiguous echo planar imaging whole-brain functional scans . During the resting experiment, the scanner room was darkened and the participants were instructed to relax, with their eyes closed, without falling asleep. All patients were in the OFF-dopaminergic-medication state during the MRI scans.http://www.fil.ion.ucl.ac.uk/spm/) with default settings. All native space T1-weighted structural MRI scans were segmented into GM, white matter (WM), and cerebrospinal fluid (CSF) components. During normalization, the GM and WM images were affine-registered to the tissue probability maps in the Montreal Neurological Institute standard space. The resulting GM tissue segment images were transformed by the DARTEL registration procedure and were smoothed with a Gaussian kernel of full width at half maximum of 8  mm. The final GM images were analyzed within the framework of a full factorial design, whereas ANCOVA was performed with age, sex, and total intracranial volume (TIV) as covariates to investigate the gray matter volume (GMV) differences between the PD and control groups. The resulting statistical inferences were considered significant if the cluster-level family-wise error corrected P value was < 0.05. For the correlation analyses, whole-brain voxel-based multiple regression analysis with age and gender controlled for was used to identify the GM vulnerable areas associated with significant oxidative stress markers in PD.T1-weighted structural MRI data were analyzed using VBM and the Statistical Parametric Mapping software program  . For nett test embedded in SPM. Analyses of the correlations between the fc-CC and GMV of the network and the correlations among the fc-CC, disease severity, and cognitive impairment were also performed, with the former analyses controlling for TIV and the latter analyses controlling for age and sex. The cluster size was determined over 1000 Monte Carlo simulations using the AlphaSim program distributed with the REST software tool (http://resting-fmri.sourceforge.net/). A corrected significance level of P = 0.01 was obtained by a combined threshold of P = 0.01 for each voxel and an extent threshold of 43 voxels (cluster size > 1161 mm3). The threshold for statistical significance was set at P < 0.05.To test whether the changes in FC patterns were associated with the underlying structural atrophy, the GMV values of the four groups were calculated under the intrinsic connectivity networks of the healthy controls connecting to the vulnerable areas. ANCOVAs with age, sex, and TIV (only for GMV) as the covariates were then used to compare the groups in terms of the mean GMV and functional connectivity correlation coefficient (fc-CC) values of the network area. The intergroup differences in the seed-based functional connectivity to the vulnerable areas were tested using the voxel-wise two-sample P < 0.05)  were identified as the vulnerable areas associated with lymphocyte apoptosis    Fig. .Fig. 3Tht test conducted to compare all the PD patients and the NC group showed significant changes of the fc-CC values in the middle occipital lobe (MOL) and pons within the MTL functional network  values in the PD patients. Disruptions to the MTL network were seen in both its architecture and functional connectivity, and these architectural and functional disruptions were associated with each other.Within the MTL functional network, we found that the reduced GMV was associated with decreased fc-CC  and III  and total scores   and MMSE scores , as well as with greater cognitive impairment in the attention  and memory  domains  Fig. . In addiins Fig. .Fig. 4a r = − 0. 381, P = 0.024) and III  and total scores . The associations of the increased pons fc-CC values with greater disease severity as indicated by the UPDRS I  and MMSE scores , as well as with greater cognitive impairment in the attention  and memory  domains were emphasized. But the associations between the fc-CC values of the MOL and disease severity as indicated by the UPDRS I  and MMSE scores , as well as greater cognitive impairment in the attention  and memory  domains were weakened.In further analyses with education year, disease duration and medication as covariates, the reduced fc-CC values of the MTL functional network were less significantly associated with greater disease severity, including in terms of the UPDRS part I  (*Bonferroni corrected P < 0.05). (DOCX 376 kb)Additional file 2:Table S1. Oxidation parameters of the PD patients and normal controls after removal of outliers. (DOCX 18 kb)Additional file 3:Figure S2. The alternation of gray matter volume in PD. Gray matter atrophy patterns in the PD subgroups compared with the NC group and with each other. The PDD subgroup showed significantly diffuse GMV loss compared with the PDMCI and PDN subgroups, as well as the NC group. The PDN and PDMCI subgroups showed only small areas of GMV loss compared with the NC group, and there was no significant difference between them. . (DOCX 210 kb)Additional file 4:Figure S3. The alternation of functional connectivity within the MTL functional network in PD. The two-sample t test between all the PD patients and the NC group showed significant changes of the fc-CC values in the middle occipital lobe (MOL) and pons within the MTL functional network. . (DOCX 74 kb)


Several studies have been suggested that immunity plays a part in neurodevelopment and schizophrenia pathogenesis. Early age of onset in schizophrenia is associated with genetic factors which affect neurodevelopment. This study aims to identify immune abnormalities associated with neurodevelopmental impairments in early-onset schizophrenia (EOS) and adult-onset schizophrenia (AOS) patients. We determined the plasma levels of six cytokines  in schizophrenia patients and healthy controls. Measurements included neurological soft signs (NSS) to distinguish and subgroup those with neurodevelopmental impairments. The study included 210 schizophrenia patients, which were divided into 84 EOS and 126 AOS patients, as well as 122 healthy controls. We observed significant differences in levels of IL-4, IL-6 and IL-10 between EOS and AOS patients. The results demonstrated the area under ROC curve (AUC) of the IL-4 in EOS and healthy controls was 0.81. Moreover, these results indicated that AUC of the IL-4 and the combination of IL-4, IL-6 and IL-12 in EOS with NSS and healthy controls were 0.91 and 0.95. These cytokines are altered in EOS and schizophrenia patients with neurodevelopmental impairments and demonstrated good classification abilities. These findings manifested that both pro- and anti-inflammatory cytokines are contributed to the clinical and pathophysiological features of schizophrenia. Future works are expected to explore potential genetic effectors and predictors as well as therapeutic directions in personalized medicine for early-onset schizophrenia. Various studies indicated that psychosis is closely linked to immune responses The neurodevelopmental model of schizophrenia posits that environmental influences or genetic factors might initiate pathological processes before disease onset Despite these findings, it remains unclear whether specific immunological alterations in patients with neurodevelopmental disabilities are linked to a genetic vulnerability to EOS. Communications between the immune system and brain could contribute to the impaired neurodevelopment, and neurodegenerative symptoms observed in schizophrenia patients All schizophrenia patients were recruited between August 2011 and March 2016 from Chi Mei Medical Center, Jianan Mental Hospital, and Lok An Hospital in Taiwan. Patients were enrolled based on the criteria for schizophrenia in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The Chinese version of the Diagnostic Interview for genetic studies (DIGS-C), including positive and negative symptom scales, as well as neurodevelopmental evaluations were conducted with all participants. Positive symptom severity in this study was assessed using the scale for the assessment of positive symptoms (SAPS) and negative symptom severity was assessed using the scale for the assessment of negative symptoms (SANS). In this study, we recruited 210 chronic schizophrenia patients with different onset ages and 122 healthy individuals to serve as controls (HC) between 20 and 65 years old. The patients with chronic schizophrenia met the following criteria: (1) onset of psychotic symptoms more than 24 months earlier; and (2) currently receiving antipsychotic medication treatment. The healthy controls met the criteria: (1) absence of a current or past psychiatric disorder as confirmed by DIGS-C. There were 84 patients with early-onset schizophrenia , 126 patients with adult-onset schizophrenia  for the subsequent analyses. The age of schizophrenia onset was defined via consulting patients' family members and tracking the medical records. Patients with other major psychiatric disorders, severe neurological abnormalities, mental retardation, substance-related disorders, somatic disorders with neurological components, traumatic brain injuries, or prominent drug use problems were excluded from this study. The healthy controls with a personal or family history of psychiatric disorder were also excluded from the control group. This study's protocol, including its purpose, methods, and use of data collected, was approved by the institutional review board (IRB) of each participating hospital. All participants provided written informed consent to participate by signing the IRB-approved consent form.In order to determine the immune cytokines levels, we collected 10 ml of venous blood in VACUETTE® tubes containing EDTA from schizophrenia patients and healthy controls. Plasma samples were obtained by centrifugation at 3500 rpm (4℃) for 10 minutes and were stored at -80℃ until assay. Levels of six plasma cytokines  were measured using a sandwich enzyme-linked immunosorbent assay (ELISA)  according to the manufacturer's instructions. Samples from different groups were assayed on the same ELISA plate to minimize experimental variation.Neurological soft signs (NSS) are minor neurological abnormalities. The NSS scores were evaluated according to the NES Scale by Buchanan and Heinrichs (1989). A total of 26 items are assigned using four subscales according to dysfunction in sensory integration, motor coordination, motor sequencing, or other All analyses were conducted in SAS version 9.4 (SAS Institute Inc) and blinded to the study groups. The results were reported as the mean and standard deviation (SD). For categorical variables, comparisons between groups were performed using Pearson's chi-square test or Fisher exact test to determine the significant differences between expected and observed frequencies in one or more categories. In addition, Student's t-test was used for continuous variables. A p-value of less than 0.05 was considered statistically significant.The analysis of the receiver operating characteristic (ROC) curves was conducted using logistic regression to plot the accuracy, sensitivity and specificity with multivariate cytokine levels in schizophrenia patients and healthy controls We also applied a partial least squares-linear discriminant analysis (PLS-LDA) to perform multivariate analyses for group classification by using plasma cytokines as independent variables to further distinguish the study groups Patient demographic characteristics are summarized in Table We determined the plasma levels of cytokines in schizophrenia patients and healthy controls Table . In EOS We further evaluated whether the severity of schizophrenia in all patients as well as early-onset and adult-onset would affect the cytokines levels. We determined the plasma levels of cytokines in all schizophrenia patients and even divided them into subgroups based on the median of the summary SAPS and SANS score Table . The resLogistic regression analysis was used to evaluate the classification efficiency of differential diagnoses of schizophrenia patients and healthy controls. AUCs of ROC curves were calculated to evaluate the performance of the classifier. Table Table The Partial least squares-linear discriminant analysis was performed to identify differences between the groups, with the best class functions showing clear separations by partial-least-squares (PLS) factors Figure a. The paWe further conducted the discriminant function in schizophrenia, EOS and AOS patients with NSS versus healthy controls. The classification accuracy of combined cytokines, IL-4, IL-6 and IL-12, was 79% and cross-validation accuracy was 79% for schizophrenia patients with NSS compared with healthy controls Table . AdditioThis study highlights the association between immune cytokines and neurodevelopmental markers for schizophrenia patients. These cytokines involved in peripheral immune system-brain communication are thought to play roles in schizophrenia pathogenesis. Neurological soft signs have been consistently reported as endophenotypes in patients with schizophrenia and their relatives Levels of IL-1β, IL-4, IL-6, IL-10, IL-12 and TNF-α have previously been studied in a meta-analysis There are several limitations of our study. First, this study was cross-sectional, and we can't determine the course of immune markers from the time of psychosis onset to later phases of schizophrenia. Longitudinal studies are required to determine cytokines dysregulations in schizophrenia across the disease duration. In previous studies, IL-1β and IL-4 levels did not change during antipsychotic treatment In conclusion, our results provide evidence of lower plasma IL-4 levels in EOS patients compared with healthy controls. Plasma IL-4 levels were also significantly different between EOS patients with and those without neurodevelopmental impairments. In summary, the findings may support the use of cytokines as potential biomarkers to identify EOS or AOS in patients. Alteration in IL-4 levels in schizophrenia patients is reportedly linked to neurodevelopmental outcomes with immune dysregulation. This study demonstrated that the discriminatory abilities of cytokines are sufficient to identify EOS patients with neurodevelopmental impairments, which helps explain some of the clinical and pathophysiological features of schizophrenia. Future studies may seek to clarify potential biomarkers and therapeutic strategies for personalized medicine in EOS patients by incorporating information about molecular mechanisms and genetic determinants.Supplementary figures and tables.Click here for additional data file.


Our meta-analysis of the non-wood archaeobotanical data puts into question the concept of the wholesale introduction of a crop “package” by pioneer settler groups arriving from the East. It is proposed instead that selective cereal crop introduction formed part of a complex pattern of sociocultural interactions that brought together indigenous and immigrant groups into new communities.The multi-period  site of Franchthi Cave, located in the Argolid peninsula of southern mainland Greece, is unique in the Eastern Mediterranean for preserving a long archaeological sequence extending from the Upper Palaeolithic through to the end of the Neolithic period. In this paper, we present new anthracological (carbonized fuel wood waste) evidence from Franchthi Cave with which we reconstruct the changing ecology of woodland vegetation in its environs during the late Pleistocene and the early-mid Holocene. The integrated archaeobotanical record (charred wood and non-wood macro-remains) demonstrates that in the Lateglacial the now-submerged coastal shelf of the southern Argolid peninsula was covered by steppe grassland vegetation dominated by junipers, almonds, cereals and legumes. The rapid climatic amelioration that marked the start of the Holocene brought about the disappearance of juniper and the expansion of deciduous woodland, cereals and lentils. This woodland-grassland biome bears no analogues in the modern and historical vegetation ecology of the Aegean basin. Instead, it is directly comparable to the steppe woodland biomes exploited by late Pleistocene and early Holocene hunter-gatherers in Southwest Asia, and points to the convergent evolution of late Pleistocene and early Holocene plant exploitation strategies between the two regions. Continuous sea-level rise during the early Holocene led to the gradual extinction of this unique palaeohabitat, which acted as the catalyst for the selective introduction of domesticated cereal crops at Franchthi Cave in the early 9 During the Lateglacial and the early Holocene fuel wood and plant-food procurement targeted the open grassland-woodland biome that dominated the still exposed coastal shelf of the southern Argolid peninsula. Late Palaeolithic and Mesolithic plant exploitation focused on the intensive harvesting of almonds  and terebinth nuts supplemented by large-seeded annuals including oats, barley and lentils. This plant resource spectrum  overlaps significantly with those known from a majority of late Pleistocene and early Holocene habitation sites located in the semi-arid steppe woodlands of continental Southwest Asia –22, 29. th millennium cal BP by the increasing salinization of the Kiladha bay area due to saltwater upstream movement and intrusion of the aquifer via the coastal karst systems. Thus, it is likely that the introduction at ~8700 cal BP of domesticated emmer wheat and 2-row hulled barley could have been motivated, at least in part, by these irreversible shifts in local plant ecologies and palaeohabitats that brought about a significant reduction in the availability of hitherto exploited plant resources. The integrated wood and non-wood botanical datasets point to a complete shift in the ecology of fuel wood gathering and food production at the very beginning of the 8th millennium cal BP . As the FC coastline approached its modern configuration, fuel wood gathering targeted Mediterranean maquis woodlands that were also routinely cleared for the establishment of cereal crop fields. Cereal cultivation provided the bulk of plant-derived subsistence in the Neolithic period, with a concurrent significant reduction in the consumption of nuts and other fruit species gathered from the wild.The loss of a large portion of the coastal shelf to SLR between the LGM and the start of the Younger Dryas at ~12,900 cal BP was counterbalanced by the dramatic expansion of grassland vegetation caused by the abrupt increases in temperature and precipitation that marked the start of the Holocene at ~11,700 cal BP . This clMedicago seeds). This non-rectifiable recovery bias precludes a more precise evaluation of the floristic composition of the FC grassland vegetation and how it changed through time, including the ruderal floras associated with intensively gathered  crop progenitor species such as barley, oats and lentils. A new program of carbon and nitrogen stable isotope analyses of crop progenitor and other large legume seeds could also help elucidating plant growth conditions including identifying the impacts of a range of possible plant management regimes . The hypothesis of cultivation without domestication at FC  must therefore remain open to future empirical testing. However, it should also be noted here that the FC archaeobotanical record is compatible with the pattern of pre-agricultural plant exploitation known from early Holocene habitation sites in Southwest Asia in which the selective introduction of crop domesticates was preceded by long periods during which wild-type grasses and/or legumes were managed without evidence for local domestication events or even “predomestication” cultivation [Whether or not the prehistoric inhabitants of FC cultivated wild cereals and legumes during the late Palaeolithic and the Mesolithic  is a question that cannot be addressed at present without new excavation and primary archaeobotanical data collection. Despite the large scale and the intensity of sampling at FC in the 1970s, the large flotation mesh size used at the time has resulted, at best, in the sporadic recovery of certain elements of the small-seeded wild flora  which took place soon after 9000 cal BP and was followed in later Neolithic phases by the introduction of einkorn wheat . Unlike th millennium cal BP [th millennium cal BP took place against a background of SLR-induced cumulative habitat erosion that led to the decimation of the traditionally exploited plant and faunal resources. Domesticated cereals and caprines thus acted as catalysts for the reorientation of the local economies, in what was by then a low-yield and ecologically highly fragmented Mediterranean coastal landscape that could no longer sustain the exploitation of wild plants and game as staple foods by human foragers. The deep history of such processes of selective plant resource adoption and spread can be traced in the increasing circulation of population groups, resources and knowledge in the Eastern Mediterranean during the early Holocene. Such movements were already underway during the “long” 11th millennium cal BP  and peaked with the intensification of maritime interaction networks  in the Aegean basin from the 9th millennium cal BP [A broadly comparable pattern of selective crop adoption has been recently proposed for interpreting the introduction of domesticated crop species at the site of Boncuklu in the Konya plain of central Anatolia during the 11m cal BP . A key dm cal BP  also sugm cal BP . By contm cal BP   were exported in 1982 for analysis outside Greece with the permission of the Hellenic Ministry of Culture (permit no. 39573/468). The wood charcoal specimens reported in this paper  and FAS (excavation stopped at a depth of -11.2m due to reaching sea level) . Taxon diversity saturation curves were obtained for every wood charcoal fraction containing more than 100 fragments >4mm with counting stopped once curves had levelled off. An exception was made for the >4mm charcoal fractions of samples FAS:173–175. These were the only excavation units available for analysis from stratum W2 (Lower Mesolithic) that had been sampled with large-scale tank flotation. Hence, their charcoal counts were raised as much as feasible in order to enhance the representativeness of taxon diversity for this segment of the charcoal stratigraphy.Quercus pubescens-type [Amygdalus instead. This fact  positively disproves previously published claims [Depending on their size, wood charcoal specimens were either hand- or pressure-fractured with a carbon steel razor blade to produce fresh sections of all three anatomical planes . These were examined using incident-light (darkfield/brightfield) materials optical microscopy at magnifications x50, xl00, x200, x400-x500. Botanical identifications (to genus or family level) were made using the wood anatomical identification criteria established for European and Eastern Mediterranean arboreal floras. Select charcoal fragments were also compared to specimens held in modern wood charcoal reference collections in Greece and at Liverpool. Part of microscopy analysis also involved the evaluation of some of the charcoal identifications previously published by Hansen. Wood charcoals previously reported as ens-type  were foud claims  for the Juniperus by the present study exhibited ray height (TLS) predominantly in the range of 1–4 cells. Based on the ubiquity of this anatomical feature we classified them as Juniperus sp. instead of Cupressus (cypress) which belongs to the same family (Cupressaceae) and in charred form exhibits almost identical wood anatomical characters to Juniperus. All Pistacia wood charcoals exhibited ray width (TLS) predominantly in the range of (2)3-4-5(6) cells. We found no specimens with predominantly narrow rays (1-2-3seriate) that would have indicated the presence of Pistacia lentiscus, the species originally proposed by Hansen based on her observations of the surface morphology of charred Pistacia nutlets [Pistacia nutlets is highly variable. Although in principle both P. terebinthus and P. lentiscus could be represented at FC, it is improbable that fuel wood was procured exclusively from P. terebinthus and nuts from P. lentiscus. For this reason, it is more likely that the species represented in the wood and non-wood charred plant assemblage is Pistacia terebinthus.All charcoal fragments identified as  nutlets . The morAmygdalus and Prunus has its basis on modern wood anatomical studies that separate between two broad taxonomic groups: (1) Amygdalus webbii, A. dulcis  [A. orientalis, A. korshinskyi, P. cerasifera and P. microcarpa, and has also been verified in other Eastern Mediterranean late Pleistocene and early Holocene charcoal assemblages in which RP only, or RP and DP morphotypes have been detected [Amygdalus from Prunus based on wood anatomy also agrees with previous and more recent botanical and genetic work in the Eastern Mediterranean and Southwest Asia that treats Amygdalus as a distinct genus from Prunus within the Rosaceae family [Amygdalus and diffuse-porous = DP, corresponding to Prunus). We thus concluded that both Amygdalus (RP) and Prunus (DP) were present in the charcoal samples. All intermediate (semi-ring porous) types that could not be characterized more precisely due to the small size of the analyzed specimens were classified as Prunoideae . Further separation between individual species of Amygdalus is not possible based on wood anatomy. Hansen’s analysis of Amygdalus charred nutshell remains suggested that Amygdalus webbii is more likely to be represented rather than A. communis [A. webbii (syn. Prunus webbii (Spach) Vierh.) is native to the southern Balkan peninsula, including mainland Greece, the eastern Aegean islands, and the island of Crete. It is thus likely (if unprovable) that A. webbii is present in the FC charred plant assemblage both as charred wood and nutshell fragments.The distinction drawn between –5 or 6–8seriate) seriate) . The samdetected , 41–42. e family . In the communis . Wild A.Amygdalus, to explore if the occurrence of specific ray width classes could be related to genetic differences or is associated instead with wood caliber. Vitrification was ubiquitous in Amygdalus charcoals hence, although this feature was recorded, it was not quantified. TS planes of select specimens from the dominant taxa  were photographed using a Leica S8APO stereozoom microscope fitted with a GXCAM HICHROME-LITE HDMI 5MP camera at the Liverpool Archaeobotany Laboratory to create a set of reference images of the eco-anatomical features observed on the TS planes of the major charcoal taxa present at FC  is currently underway and will be published elsewhere.Several wood anatomical characters and eco-anatomical features were recorded for each botanically identified fragment >4mm including curvature degree  and the presence/absence of pith and bark, resin/gum ducts, fungal hyphae, scar/callus tissue, and narrow/false rings. The aim was to explore woodland growth conditions  and potential preferences for the gathering of small-size and dead/decayed wood as fuel . Kabukcunt at FC . A systeThe primary (sample-by-sample) charcoal taxon counts are presented in spreadsheet form in To date, there has been no detailed published description of the archaeological features excavated at FC. For this reason, all sampled excavation units were considered, in principle, to represent generalized charcoal scatters found in variable concentrations across the sampled sequence, except for a few units that were unequivocally identified as hearths by the excavators . HoweverQuercus spp. which  were impossible to determine as either deciduous (D) or evergreen (E) oak. These were mostly present in Neolithic (strata Y2-3) samples, in which both evergreen and deciduous oak charcoals are well represented. Thus, including them in the anthracological diagram could not add something significant to palaeoenvironmental reconstruction. Another Quercus spp. fragment recovered from H1A:207 is almost certainly intrusive from stratigraphically later units hence it was also excluded. By contrast, other highly likely intrusive fragments (1 fragment of Arbutus in H1A:213 and 1 fragment of Quercus (E) in each of H1A:207 and H1A:160) were kept in since the same taxa also appear in Upper Mesolithic (evergreen oak) and Neolithic (both taxa) CSUs. Their status as intrusive is clearly marked in the data tables  with which to construct the FC anthracological diagram. Further data reduction was necessary involving the exclusion of indeterminate (Indet.) fragments from the per-CSU charcoal counts. In all cases, these were charcoal fragments that were unidentifiable due to their very small size and/or poor preservation. They could only be determined as Indet. conifers (Indet. C) or angiosperms (Indet. A). Including them in the quantified charcoal counts would have provided no useful information for palaeoenvironmental reconstruction  recorded for the entire anthracological sequence). The Indet. category does not include unidentifiable taxa . The occasional bark fragments were also excluded from the per-CSU charcoal counts, alongside fragments of a tables .rioja’, function ‘strat.plot’) and edited by Asouti (with the addition of labels and phasing boundaries) with Adobe Photoshop CC 2018. It represents the FC anthracological sequence from the earliest (H1A:219–212 –Stratum R) to the latest CSU (FAS N.V.2 –Stratum Y3) and covers a timespan of ~22,000 years including all FC strata, except for W3 and X1  and X2  from which no charcoal samples were available for analysis .Kabukcu designed and performed all multivariate statistical analyses. To gain a better understanding of vegetation change through time, it was necessary to integrate the results of the anthracological analysis with those of the analysis of non-wood botanical remains previously published by Hansen . To thisAmygdalus and Pistacia nutshell Hansen’s original MNI  converted counts were kept in, while any unconverted fragment counts were rejected. For the wood charcoal counts, only positively identified fragments (to genus or family level) were included . Data reduction at the sample level involved limiting analysis only to samples that contained >20 identified items  to reduce redundancy and noise in the resulting data matrix.To minimize the impact of taphonomic biases on the multivariate data matrix a series of data reduction actions were performed on the FC non-wood botanical dataset. All taxa that were demonstrably preferentially preserved  were excluded from the data matrix. Only whole item  counts were used. Counts of chaff, pod fragments, and unconverted seed or other plant part fragment counts were also excluded from the data matrix, in order to minimize the risk of taxon over-representation through these particular data categories. Likewise, for FactoMineR’ [The dendroanthracological dataset was analyzed with Multiple Correspondence Analysis (MCA) using R package ‘toMineR’ . MCA is vegan’, function ‘metaMDS’, a 3D plot of the individual samples and projected species values was produced [Given the comparatively low number of samples from which both wood and non-wood botanical data were available, and the inherent recovery biases affecting the greater part of the Lower and Upper Mesolithic botanical sequence from H1A and FAS, it was necessary to expand multivariate analyses to include archaeobotanical samples from which no charcoal samples were available for analysis, thus incorporating materials originating from trenches FAN and H1B. The aim was to enhance as much as possible our understanding of changes in non-arboreal flora and vegetation through time, and the types of plant palaeohabitats exploited by the FC inhabitants through its long occupation history. The same data reduction methodologies outlined above were repeated for this sample population. In the resulting data matrix, all taxa and samples counting <10 identified items were excluded, in order to reduce the degree of redundancy and prevent patterning forced by extremely rare taxa and/or low-density samples. A pervasive feature of the FC archaeobotanical assemblage is the enormous variation observed in botanical finds density  across the different occupation phases. This characterized especially the Lower Mesolithic samples (strata W1-2) that contained >28,000 identified items, contrasting with the considerably lower botanical densities retrieved from both later and earlier samples . For thiproduced . The resproduced  stress vS1 Data File(A) Absolute charcoal fragment counts for all excavation units sampled from FC Trenches H1A and FAS; (B-1) Botanical zones as defined by Hansen  and thei(XLSX)Click here for additional data file.S2 Data File(XLSX)Click here for additional data file.S3 Data File(XLSX)Click here for additional data file.S4 Data File(A) Dendroanthracological data for 1344 charcoal fragments from trenches H1A and FAS; (B-1) Results of Multiple Correspondence Analysis (MCA) run on dendroanthracological features on all fragments: eigenvalues, percentage of variance and cumulative percentage of variance for each dimension; (B-2) Results of Multiple Correspondence Analysis (MCA) run on dendroanthracological features: contribution of variables to dimensions, and cos2 values.(XLSX)Click here for additional data file.S5 Data FileAmygdalus charcoal fragments; (B-1) Results of Multiple Correspondence Analysis (MCA) run on Amygdalus dendroanthracological features: eigenvalues, percentage and cumulative percentage of variance for each dimension; (B-2) Results of MCA run on Amygdalus dendroanthracological features: contribution of variables to dimensions, and cos2 values.(A) Dendroanthracological data  for 502 (XLSX)Click here for additional data file.S6 Data File(A) Data matrix (taxon counts) for FC samples containing both wood charcoal and non-wood charred plant remains ; (B-1) Results of Correspondence Analysis (CA) run on the per-sample charcoal and non-wood botanical taxon counts: contribution to dimensions and cos2 values; (B-2) Results of CA run on the per-sample charcoal and non-wood botanical taxon counts: eigenvalues and percentage of variance for dimensions.(XLSX)Click here for additional data file.S7 Data FileData matrix (taxon counts) for all FC samples containing non-wood charred plant remains .(XLSX)Click here for additional data file.


Riparian zones are reputed to be effective at preventing export of agricultural groundwater nitrogen (N) from local ecosystems. This is one impetus behind riparian zone regulations and initiatives. However, riparian zone function can vary under different conditions, with varying impacts on the regional  environment. Rates of groundwater delivery to the surface appear to have significant effects on the N-removing capabilities of a riparian zone. Research conducted at a first-order agricultural watershed with a well-defined riparian zone in the Maryland coastal plain indicates that more than 2.5 kg/day of nitrate-N can be exported under moderate-to-high stream baseflow conditions. The total nitrate-N load that exits the system increases with increasing flow not simply because of the greater volume of water export. Stream water nitrate-N concentrations also increase by more than an order of magnitude as flow increases, at least during baseflow. This appears to be largely the result of changes in dominant groundwater delivery mechanisms. Higher rates of groundwater exfiltration lessen the contact time between nitrate-carrying groundwater and potentially reducing riparian soils. Subsurface preferential flow paths, in the wetland and adjacent field, also strongly influence N removal. Simple assumptions regarding riparian zone function may be inadequate because of complexities observed in response to changing hydrologic conditions.


Antibody-drug conjugates (ADCs) combine the tumor selectivity of antibodies with the potency of cytotoxic small molecules thereby constituting antibody-mediated chemotherapy. As this inherently limits the adverse effects of the chemotherapeutic, such approaches are heavily pursued by pharma and biotech companies and have resulted in four FDA (Food and Drug Administration)-approved ADCs. However, as with other cancer therapies, durable responses are limited by the fact that under cell stress exerted by these drugs, tumors can acquire mechanisms of escape. Resistance can develop against the antibody component of ADCs by down-regulation/mutation of the targeted cell surface antigen or against payload toxicity by up-regulation of drug efflux transporters. Unique resistance mechanisms specific for the mode of action of ADCs have also emerged, like altered internalization or cell surface recycling of the targeted tumor antigen, changes in the intracellular routing or processing of ADCs, and impaired release of the toxic payload into the cytosol. These evasive changes are tailored to the specific nature and interplay of the three ADC constituents: the antibody, the linker, and the payload. Hence, they do not necessarily endow broad resistance to ADC therapy. This review summarizes preclinical and clinical findings that shed light on the mechanisms of acquired resistance to ADC therapies. The basic principle of antibody-drug conjugates (ADCs) is to enhance the tumor selectivity of cancer treatment with highly cytotoxic small molecules by covalently linking them to antibody molecules directed against tumor-specific cell surface antigens. Although it sounds simple, this underlying principle has proven very difficult to implement in practice, as illustrated by the fact that two decades of ADC development efforts by biotech and pharma companies have so far only yielded four FDA (Food and Drug Administration)-approved, commercially available products. Currently, more than 150 ADC programs are being actively pursued in different stages of preclinical and clinical development  ~2 µM) than in the extracellular space . The net result is early dissociation of the ADC from its target, which can then be recycled to the cell surface and capture and internalize more ADC molecules. This could be a smart way to curb resistance mechanisms that diminish cytosolic escape of the payload since it only enhances target-mediated but not target-independent payload uptake and hence should also increase the therapeutic window.A conceptually intriguing approach to increase the efficiency of payload delivery to the cytosol, and hence the cytotoxic potency of ADCs, has recently been patented by Ward and coworkers from Texas University . They enPurposely selected combination therapies seem to be a more promising strategy to overcome or avoid resistance mechanisms. Simultaneous or frequently alternating administration of ADCs with other cytotoxic agents with a different mode of action reduces the likelihood that individual tumor clones become simultaneously resistant to both agents, while in combination therapy with immune-checkpoint inhibitors, the ADC component is intended to enhance anti-tumor immunity by immunogenic cell death and enhanced tumor antigen release.For combining an ADC with another cytotoxic agent, the two drugs should not have overlapping adverse effect profiles in order to avoid additive toxicities. This is illustrated by a phase 1b/2a metastatic breast cancer trial that tested combining T-DM1 with docetaxel (with and without pertuzumab). While docetaxel plus T-DM1 appeared efficacious, nearly half of the patients experienced severe adverse effects that required dose reductions . EquallyObviously, the less two agents have in common, the less likely cross-resistance will occur. A recombinant immunotoxin consisting of the anti-HER2 diabody C6.5 fused to a de-immunized form of the plant toxin bouganin has been shown to overcome cross-resistance to both T-DM1 and a trastuzumab-MMAE conjugate due to its completely different mode of action . AlthougTAF15, a gene encoding an RNA-binding factor that regulates transcription and RNA processing. Once ATACs have proven their clinical utility, their ability to broadly inhibit resistance against other drugs could make them promising combination agents for other ADCs. However, the first ATAC from Heidelberg Pharma is expected to enter the clinic only later this year.Another drug class with a unique mode of action are antibody targeted amanitin conjugates (ATACs), ADCs with α-amanitin as payload. By studying drug-tolerant cancer cell colonies that survive in the presence of different cytotoxic drugs, Kume et al. found that the RNA-polymerase II inhibitor, α-amanitin, consistently and potently inhibited the drug-tolerant phenotype . MechaniClinical evaluation of ADC combinations is ongoing for a multitude of targeted agents and chemotherapeutics that are either already approved or in late-stage clinical development. For example, there is great interest in extending the clinical success of CDK4/6 inhibitors in hormone receptor-positive luminal breast cancer to metastatic HER2+ breast cancer by combination therapy with T-DM1. Immune checkpoint inhibiting (ICI) antibodies have revolutionized cancer therapy by achieving long-lasting responses and even cures in subsets of patients. They do so by enhancing anti-tumor immune responses of effector T-cells and stimulating anti-tumor immunological memory in patients. ICI antibody therapy works well for “inflamed” (hot) tumors that have already an ongoing anti-tumor T-cell response, but not for “immune desert” or “immune excluded” (cold) tumors, where there is no anti-tumor T-cell response or T-cells are excluded from the tumor, respectively . ChemothCombining ADCs with ICI antibody therapy could increase the percentage of patients with complete and durable responses by turning immunologically cold tumors into hot ones using the three mechanisms outlined above. Looking at it the other way round, the key attribute that makes ICI antibodies attractive as combination agents for enhancing ADC therapy is that a strong anti-tumor immune response leads to antigen-cross-presentation, which then allows the immune system to destroy tumor cells irrespective of whether they have down-regulated or completely lost the surface antigen against which the ADC is directed. In a HER2-positive orthotopic tumor model that was inherently resistant to treatment with a combination of ICI antibodies, co-treatment with T-DM1 led to tumor rejections accompanied by massive infiltration of T-cells into tumors . The samAlthough few, the currently FDA-approved ADCs provide valuable treatment options for difficult to treat patient populations. The recent establishment of site-specific conjugation methods promises to increase the rather poor success rate for drug development of ADCs to date. Primary and acquired resistance are frequently encountered limitations for this very potent class of agents. Their complex mode of action requires a multi-step cascade of events to occur efficiently for a sufficient amount of toxic payload to be delivered inside the targeted cancer cells. This provides tumor cells with many opportunities to thwart an ADC attack. The most promising avenues for achieving more long-term therapeutic benefits for a larger percentage of patients are deliberately chosen combination therapies.


Objectives: Studies have been increased on daily lives of older adults; however, little is known about daily interactions between parents and children, especially both are in their later lives. Utilizing parent-child dyadic data, this study aims to investigate the joint daily interactions of the very old parents and their old children with focus on the relationship type and differences in parent’s and child’s report. Methods: A sample of 105 parents (aged ≥ 80) and 105 children (aged ≥ 65) were interviewed to assess frequency on daily interactions . Structural Equation Modeling was used to examine differences in amounts of daily interactions by relationship type . Results: Both parents and children reported high level of daily interactions; however, frequency of watching TV and going out together were relatively lower, compared to other forms of daily interactions. Parents and children showed high concordance in reports; 84% of dyads had the same reports in interactions (absolute mean discrepancy score < 0.1). Mother-daughter relationship reported significantly higher frequency on daily interactions, comparted to mother-son relationship. No significant differences were found between the mother-daughter dyads and the mother-in-law and daughter-in-law dyads. Discussion: Findings suggest that very old parents and old children tend to have high levels of daily interactions, with high concordance in reports. It also highlights its importance of considering relationship type in the dynamic of daily lives and experiences between very old parents and old children.