Revealing the mechanism and function underlying pairwise temporal coupling in collective motion.

Coordinated motion in animal groups has predominantly been studied with a focus on spatial interactions, such as how individuals position and orient themselves relative to one another. Temporal aspects have, by contrast, received much less attention. Here, by studying pairwise interactions in juvenile zebrafish (Danio rerio)-including using immersive volumetric virtual reality (VR) with which we can directly test models of social interactions in situ-we reveal that there exists a rhythmic out-of-phase (i.e., an alternating) temporal coordination dynamic. We find that reciprocal (bi-directional) feedback is both necessary and sufficient to explain this emergent coupling. Beyond a mechanistic understanding, we find, both from VR experiments and analysis of freely swimming pairs, that temporal coordination considerably improves spatial responsiveness, such as to changes in the direction of motion of a partner. Our findings highlight the synergistic role of spatial and temporal coupling in facilitating effective communication between individuals on the move.

NKG2D receptor signaling shapes T cell thymic education.

The role of natural killer group 2D (NKG2D) in peripheral T cells as a costimulatory receptor is well established. However, its contribution to T cell thymic education and functional imprint is unknown. Here, we report significant changes in development, receptor signaling, transcriptional program, and function in T cells from mice lacking NKG2D signaling. In C57BL/6 (B6) and OT-I mice, we found that NKG2D deficiency results in Vß chain usage changes and stagnation of the double-positive stage in thymic T cell development. We found that the expression of CD5 and CD45 in thymocytes from NKG2D deficient mice were reduced, indicating a direct influence of NKG2D on the strength of T cell receptor (TCR) signaling during the developmental stage of T cells. Depicting the functional consequences of NKG2D, peripheral OT-I NKG2D-deficient cells were unresponsive to ovalbumin peptide stimulation. Paradoxically, while αCD3/CD28 agonist antibodies led to phenotypic T cell activation, their ability to produce cytokines remained severely compromised. We found that OT-I NKG2D-deficient cells activate STAT5 in response to interleukin-15 but were unable to phosphorylate ERK or S6 upon TCR engagement, underpinning a defect in TCR signaling. Finally, we showed that NKG2D is expressed in mouse and human thymic T cells at the double-negative stage, suggesting an evolutionarily conserved function during T cell development. The data presented in this study indicate that NKG2D impacts thymic T cell development at a fundamental level by reducing the TCR threshold and affecting the functional imprint of the thymic progeny. In summary, understanding the impact of NKG2D on thymic T cell development and TCR signaling contributes to our knowledge of immune system regulation, immune dysregulation, and the design of immunotherapies.

Targeting intracellular oncoproteins with dimeric IgA promotes expulsion from the cytoplasm and immune-mediated control of epithelial cancers.

Dimeric IgA (dIgA) can move through cells via the IgA/IgM polymeric immunoglobulin receptor (PIGR), which is expressed mainly on mucosal epithelia. Here, we studied the ability of dIgA to target commonly mutated cytoplasmic oncodrivers. Mutation-specific dIgA, but not IgG, neutralized KRASG12D within ovarian carcinoma cells and expelled this oncodriver from tumor cells. dIgA binding changed endosomal trafficking of KRASG12D from accumulation in recycling endosomes to aggregation in the early/late endosomes through which dIgA transcytoses. dIgA targeting of KRASG12D abrogated tumor cell proliferation in cell culture assays. In vivo, KRASG12D-specific dIgA1 limited the growth of KRASG12D-mutated ovarian and lung carcinomas in a manner dependent on CD8+ T cells. dIgA specific for IDH1R132H reduced colon cancer growth, demonstrating effective targeting of a cytoplasmic oncodriver not associated with surface receptors. dIgA targeting of KRASG12D restricted tumor growth more effectively than small-molecule KRASG12D inhibitors, supporting the potential of this approach for the treatment of human cancers.

Fine-scale collective movements reveal present, past and future dynamics of a multilevel society in Przewalski's horses.

Studying animal societies needs detailed observation of many individuals, but technological advances offer new opportunities in this field. Here, we present a state-of-the-art drone observation of a multilevel herd of Przewalski's horses, consisting of harems (one-male, multifemale groups). We track, in high spatio-temporal resolution, the movements of 238 individually identified horses on drone videos, and combine movement analyses with demographic data from two decades of population monitoring. Analysis of collective movements reveals how the structure of the herd's social network is related to kinship and familiarity of individuals. The network centrality of harems is related to their age and how long the harem stallions have kept harems previously. Harems of genetically related stallions are closer to each other in the network, and female exchange is more frequent between closer harems. High movement similarity of females from different harems predicts becoming harem mates in the future. Our results show that only a few minutes of fine-scale movement tracking combined with high throughput data driven analysis can reveal the structure of a society, reconstruct past group dynamics and predict future ones.

SMART-BARN: Scalable multimodal arena for real-time tracking behavior of animals in large numbers.

The SMART-BARN (scalable multimodal arena for real-time tracking behavior of animals in large numbers) achieves fast, robust acquisition of movement, behavior, communication, and interactions of animals in groups, within a large (14.7 meters by 6.6 meters by 3.8 meters), three-dimensional environment using multiple information channels. Behavior is measured from a wide range of taxa (insects, birds, mammals, etc.) and body size (from moths to humans) simultaneously. This system integrates multiple, concurrent measurement techniques including submillimeter precision and high-speed (300 hertz) motion capture, acoustic recording and localization, automated behavioral recognition (computer vision), and remote computer-controlled interactive units (e.g., automated feeders and animal-borne devices). The data streams are available in real time allowing highly controlled and behavior-dependent closed-loop experiments, while producing comprehensive datasets for offline analysis. The diverse capabilities of SMART-BARN are demonstrated through three challenging avian case studies, while highlighting its broad applicability to the fine-scale analysis of collective animal behavior across species.

Sézary syndrome originates from heavily mutated hematopoietic progenitors.

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains unclear. Using single-cell RNA or T-cell receptor (TCR) sequencing of 32 619 CD3+CD4+ and CD26+/CD7+ and 29 932 CD3+CD4+ and CD26-/CD7- lymphocytes from the peripheral blood of 7 patients with CTCL, coupled to single-cell ATAC-sequencing of 26,411 CD3+CD4+ and CD26+/CD7+ and 33 841 CD3+CD4+ and CD26-/CD7- lymphocytes, we show that tumor cells in Sézary syndrome and mycosis fungoides (MF) exhibit different phenotypes and trajectories of differentiation. When compared to MF, Sézary cells exhibit narrower repertoires of TCRs and exhibit clonal enrichment. Surprisingly, we identified ≥200 mutations in hematopoietic stem cells from multiple patients with Sézary syndrome. Mutations in key oncogenes were also present in peripheral Sézary cells, which also showed the hallmarks of recent thymic egression. Together our data suggest that CTCL arises from mutated lymphocyte progenitors that acquire TCRs in the thymus, which complete their malignant transformation in the periphery.

CCF-GNN: A Unified Model Aggregating Appearance, Microenvironment, and Topology for Pathology Image Classification.

Pathology images contain rich information of cell appearance, microenvironment, and topology features for cancer analysis and diagnosis. Among such features, topology becomes increasingly important in analysis for cancer immunotherapy. By analyzing geometric and hierarchically structured cell distribution topology, oncologists can identify densely-packed and cancer-relevant cell communities (CCs) for making decisions. Compared to commonly-used pixel-level Convolution Neural Network (CNN) features and cell-instance-level Graph Neural Network (GNN) features, CC topology features are at a higher level of granularity and geometry. However, topological features have not been well exploited by recent deep learning (DL) methods for pathology image classification due to lack of effective topological descriptors for cell distribution and gathering patterns. In this paper, inspired by clinical practice, we analyze and classify pathology images by comprehensively learning cell appearance, microenvironment, and topology in a fine-to-coarse manner. To describe and exploit topology, we design Cell Community Forest (CCF), a novel graph that represents the hierarchical formulation process of big-sparse CCs from small-dense CCs. Using CCF as a new geometric topological descriptor of tumor cells in pathology images, we propose CCF-GNN, a GNN model that successively aggregates heterogeneous features (e.g., appearance, microenvironment) from cell-instance-level, cell-community-level, into image-level for pathology image classification. Extensive cross-validation experiments show that our method significantly outperforms alternative methods on H&E-stained and immunofluorescence images for disease grading tasks with multiple cancer types. Our proposed CCF-GNN establishes a new topological data analysis (TDA) based method, which facilitates integrating multi-level heterogeneous features of point clouds (e.g., for cells) into a unified DL framework.

Actionable spontaneous antibody responses antagonize malignant progression in ovarian carcinoma.

OBJECTIVE: To demonstrate that shared antibody responses in endometriosis and endometriosis-associated ovarian cancer spontaneously antagonize malignant progression and can be leveraged to develop future immunotherapies. METHODS: B cells from cyopreserved clear cell ovarian carcinoma (CCC, n = 2), endometrioid ovarian carcinoma (EC, n = 2), and endometriomas (n = 2) were isolated, activated, and EBV-immortalized. Antibodies were purified from B cell supernatants and used for screening arrays containing most of the human proteome. Targets were prioritized based on accessibility (transmembrane or secreted proteins), expression in endometriosis and cancer, and concurrent IgA and IgG responses. We focused on antibodies targeting tumor-promoting syndecan binding protein (SDCBP) to demonstrate anti-tumor activity. Immunoblots and qPCR were performed to assess SDCBP expression in ovarian cancer and endometriosis cell lines and tumor samples. Recombinant IgG4 was generated using the variable heavy and light chains of dominant B cell receptors (BCRs) reacting against the extracellular domain of SDCBP, and used in in vivo studies in human CCC- and high-grade serous ovarian carcinoma (HGSOC)-bearing immunodeficient mice. RESULTS: Nine accessible proteins detected by both IgA and IgG were identified in all samples - including SDCBP, which is expressed in ovarian carcinomas of multiple histologies. Administration of α-SDCBP IgG4 in OVCAR3 (HGSOC), TOV21G and RMG-I (CCC) tumor-bearing mice significantly decreased tumor volume compared to control irrelevant IgG4. CONCLUSIONS: Spontaneous antibody responses exert suboptimal but measurable immune pressure against malignant progression in ovarian carcinomas. Using tumor-derived antibodies for developing novel immunotherapeutics warrants further investigation.

Inferring social influence in animal groups across multiple timescales.

Many animal behaviours exhibit complex temporal dynamics, suggesting there are multiple timescales at which they should be studied. However, researchers often focus on behaviours that occur over relatively restricted temporal scales, typically ones that are more accessible to human observation. The situation becomes even more complex when considering multiple animals interacting, where behavioural coupling can introduce new timescales of importance. Here, we present a technique to study the time-varying nature of social influence in mobile animal groups across multiple temporal scales. As case studies, we analyse golden shiner fish and homing pigeons, which move in different media. By analysing pairwise interactions among individuals, we show that predictive power of the factors affecting social influence depends on the timescale of analysis. Over short timescales the relative position of a neighbour best predicts its influence and the distribution of influence across group members is relatively linear, with a small slope. At longer timescales, however, both relative position and kinematics are found to predict influence, and nonlinearity in the influence distribution increases, with a small number of individuals being disproportionately influential. Our results demonstrate that different interpretations of social influence arise from analysing behaviour at different timescales, highlighting the importance of considering its multiscale nature. This article is part of a discussion meeting issue 'Collective behaviour through time'.

Anti-4-1BB immunotherapy enhances systemic immune effects of radiotherapy to induce B and T cell-dependent anti-tumor immune activation and improve tumor control at unirradiated sites.

Radiation therapy (RT) can prime and boost systemic anti-tumor effects via STING activation, resulting in enhanced tumor antigen presentation and antigen recognition by T cells. It is increasingly recognized that optimal anti-tumor immune responses benefit from coordinated cellular (T cell) and humoral (B cell) responses. However, the nature and functional relevance of the RT-induced immune response are controversial, beyond STING signaling, and agonistic interventions are lacking. Here, we show that B and CD4+ T cell accumulation at tumor beds in response to RT precedes the arrival of CD8+ T cells, and both cell types are absolutely required for abrogated tumor growth in non-irradiated tumors. Further, RT induces increased expression of 4-1BB (CD137) in both T and B cells; both in preclinical models and in a cohort of patients with small cell lung cancer treated with thoracic RT. Accordingly, the combination of RT and anti-41BB therapy leads to increased immune cell infiltration in the tumor microenvironment and significant abscopal effects. Thus, 4-1BB therapy enhances radiation-induced tumor-specific immune responses via coordinated B and T cell responses, thereby preventing malignant progression at unirradiated tumor sites. These findings provide a rationale for combining RT and 4-1bb therapy in future clinical trials.

Head-tracking of freely-behaving pigeons in a motion-capture system reveals the selective use of visual field regions.

Using a motion-capture system and custom head-calibration methods, we reconstructed the head-centric view of freely behaving pigeons and examined how they orient their head when presented with various types of attention-getting objects at various relative locations. Pigeons predominantly employed their retinal specializations to view a visual target, namely their foveas projecting laterally (at an azimuth of ± 75°) into the horizon, and their visually-sensitive "red areas" projecting broadly into the lower-frontal visual field. Pigeons used their foveas to view any distant object while they used their red areas to view a nearby object on the ground (< 50 cm). Pigeons "fixated" a visual target with their foveas; the intervals between head-saccades were longer when the visual target was viewed by birds' foveas compared to when it was viewed by any other region. Furthermore, pigeons showed a weak preference to use their right eye to examine small objects distinctive in detailed features and their left eye to view threat-related or social stimuli. Despite the known difficulty in identifying where a bird is attending, we show that it is possible to estimate the visual attention of freely-behaving birds by tracking the projections of their retinal specializations in their visual field with cutting-edge methods.

Reactions of 1-C-acceptor-substituted glycals with nucleophiles under acid promoted (Ferrier-rearrangement) conditions.

The reactivity of O-peracetylated and O-perbenzoylated 1-COOMe, 1-CONH2 and 1-CN-substituted glycals was studied against O-, S-, N- and C-nucleophiles in the presence of Lewis acids. Allylic substituted products with exclusive axial stereoselectivity were formed with simple alcohols, N3-, and Cl- ions, but with benzyl thiol the Ferrier rearrangement took place and thioglycosides were obtained. The use of a sugar derived thiol resulted in the formation of both the allylic substituted and the rearranged products.

[Minimally invasive and O-arm assisted en bloc spinal tumor resections]. / Minimálisan invazív és O-arm asszisztált en bloc gerincdaganat-reszekciók.

BACKGROUND AND PURPOSE: The en bloc resection of spinal tumors is required in primary spine tumors and in selected cases of secondary spine tumors, where the primary disease is under control and long survival time is expected. Three cases are presented, applying O-arm assisted navigation or minimally invasive anterior approaches for en bloc tumor removal. METHODS: O-arm navigation assisted osteotomies were carried out to remove a Th.V. breast tumor metastasis en bloc, intact bony part of the Th.V. vertebra was spared. Vertebral corpectomies of a patient with L.IV. chordoma and of a patient with L.V. carcinoid were also performed using minimally invasive, microscope assisted, anterior approaches to the lumbar spine. RESULTS: No morbidity or local recurrence were detected in the patient with breast cancer 1 year after the operation. Nevertheless, new spinal metastasis were revealed 1 year after surgery despite the appropriate oncological treatment. The patient with L.IV. chordoma is still tumor free (last follow-up: 18 month after surgery), but post operatively detected lower limb paresis and gait disturbances are persisted. The posterior healthy bony parts of the spinal column remained intact, since only anterior approaches were used for en bloc L.IV. corpectomy. No morbidity or recurrence was detected in patient with L.V. carcinoid tumor on 1 year follow-up. CONCLUSION: Both the O-arm navigation assisted surgery and the minimally invasive anterior approaches to the spine can help to reduce surgical morbidity and to spare healthy bony structures of the spine. The later could play important role to provide long term spine stability. The presented new surgical technologies can be accepted only, if they produce at least the same oncological results on longer follow-ups as conventional surgical approaches.

Genetic Subtyping and Phenotypic Characterization of the Immune Microenvironment and MYC/BCL2 Double Expression Reveal Heterogeneity in Diffuse Large B-cell Lymphoma.

PURPOSE: Diffuse large B-cell lymphoma (DLBCL) is molecularly and clinically heterogeneous, and can be subtyped according to genetic alterations, cell-of-origin, or microenvironmental signatures using high-throughput genomic data at the DNA or RNA level. Although high-throughput proteomic profiling has not been available for DLBCL subtyping, MYC/BCL2 protein double expression (DE) is an established prognostic biomarker in DLBCL. The purpose of this study is to reveal the relative prognostic roles of DLBCL genetic, phenotypic, and microenvironmental biomarkers. EXPERIMENTAL DESIGN: We performed targeted next-generation sequencing; IHC for MYC, BCL2, and FN1; and fluorescent multiplex IHC for microenvironmental markers in a large cohort of DLBCL. We performed correlative and prognostic analyses within and across DLBCL genetic subtypes and MYC/BCL2 double expressors. RESULTS: We found that MYC/BCL2 double-high-expression (DhE) had significant adverse prognostic impact within the EZB genetic subtype and LymphGen-unclassified DLBCL cases but not within MCD and ST2 genetic subtypes. Conversely, KMT2D mutations significantly stratified DhE but not non-DhE DLBCL. T-cell infiltration showed favorable prognostic effects within BN2, MCD, and DhE but unfavorable effects within ST2 and LymphGen-unclassified cases. FN1 and PD-1-high expression had significant adverse prognostic effects within multiple DLBCL genetic/phenotypic subgroups. The prognostic effects of DhE and immune biomarkers within DLBCL genetic subtypes were independent although DhE and high Ki-67 were significantly associated with lower T-cell infiltration in LymphGen-unclassified cases. CONCLUSIONS: Together, these results demonstrated independent and additive prognostic effects of phenotypic MYC/BCL2 and microenvironment biomarkers and genetic subtyping in DLBCL prognostication, important for improving DLBCL classification and identifying prognostic determinants and therapeutic targets.

Percutaneous Spine Fusion Combined with Whole-Body Traction in the Acute Surgical Treatment of AO A- and C-Type Fractures: A Technical Note.

BACKGROUND: A diminished level of pain following the operation and shortened hospitalization are the distinct advantages of minimally invasive spine surgery (MISS). However, manipulating the spine with additional MISS tools (e.g., distraction and compression devices) is often cumbersome. Our paper draws attention to a cost-free, fast, indirect decompression method that can be used in the acute treatment of thoracolumbar spine fractures. The presented method involves ligamentotaxis by whole-body traction in the operating room combined with percutaneous spine fixation. METHODS: Fifteen patients with thoracolumbar injuries A type and C type (without distraction) by AO classification were operated sequentially with the combination of whole-body traction and percutaneous minimally invasive spine fixation. Data were analyzed retrospectively. RESULTS: A total of 139 screws were implanted into 70 segments in 6 female and 9 male patients. The average clinical follow-up was 16 months. Average preoperative traumatic kyphosis was 17 degrees, and an average postoperative kyphosis was 1.8 degrees. The fractured vertebrae's height gain was an average of 11.0 mm (range 3.9-21.9 mm) ventrally and an average of 5.4 mm (range 1-11.2 mm) dorsally after the surgeries. The spinal canal space narrowing showed an average 6.5 mm improvement postoperatively. Operative time averaged 2 hours and 34 minutes, and blood loss averaged 250 mL (range 150-400 mL). No neurologic complications and wound healing problems were observed. CONCLUSIONS: The combination of MISS and whole-body traction provided successful anatomical correction in thirteen of the fifteen cases of compression type thoracolumbar fractures without extensive surgical exploration.

N-glycosylation structure - function characterization of omalizumab, an anti-asthma biotherapeutic product.

Omalizumab, a glycoprotein based biotherapeutics, is one of the most frequently used targeted antibody biopharmaceutical to reduce asthma exacerbations, improve lung function and reduce oral corticosteroid use. The effector function and clearance time of such glycoprotein drugs is affected by their N-glycosylation, that defines the required administration frequency to improve the quality of life in appropriately selected patients. Therefore, the glycosylation of biologics is an important critical quality attribute (CQA). The profile of asparagine linked carbohydrates is greatly dependent on the manufacturing process. Even a small deviation may have a major effect on the structure and therefore the function of the biotherapeutic product. For this reason, comprehensive N-glycosylation analysis is of high importance during production and release. Capillary electrophoresis (CE) is one of the frequently used tools to characterize protein therapeutics and utilized by the biopharmaceutical industry for protein and glycan level analysis, which are key parts both for drug development and quality control. To reveal important structure - function relationships, characterization of omalizumab is presented using capillary SDS gel electrophoresis with UV detection at the protein level and capillary gel electrophoresis with laser induced fluorescent detection at the N-linked carbohydrate level. This latter technique was also used for oligosaccharide sequencing for glycan structure validation. The results suggested no ADCC function - structure relationship due to the mostly core fucosylated biantennary glycans found. However, the presence of the high mannose structures probably affects the clearance rate of the drug.

The geometry of decision-making in individuals and collectives.

Choosing among spatially distributed options is a central challenge for animals, from deciding among alternative potential food sources or refuges to choosing with whom to associate. Using an integrated theoretical and experimental approach (employing immersive virtual reality), we consider the interplay between movement and vectorial integration during decision-making regarding two, or more, options in space. In computational models of this process, we reveal the occurrence of spontaneous and abrupt "critical" transitions (associated with specific geometrical relationships) whereby organisms spontaneously switch from averaging vectorial information among, to suddenly excluding one among, the remaining options. This bifurcation process repeats until only one option-the one ultimately selected-remains. Thus, we predict that the brain repeatedly breaks multichoice decisions into a series of binary decisions in space-time. Experiments with fruit flies, desert locusts, and larval zebrafish reveal that they exhibit these same bifurcations, demonstrating that across taxa and ecological contexts, there exist fundamental geometric principles that are essential to explain how, and why, animals move the way they do.

Genomic complexity is associated with epigenetic regulator mutations and poor prognosis in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma with high mutation burdens but a low response rate to immune checkpoint inhibitors. In this study, we performed targeted next-generation sequencing and fluorescent multiplex immunohistochemistry, and investigated the clinical significance and immunological effect of mutation numbers in 424 DLBCL patients treated with standard immunochemotherapy. We found that KMT2D and TP53 nonsynonymous mutations (MUT) were significantly associated with increased nonsynonymous mutation numbers, and that high mutation numbers (MUThigh) were associated with significantly poorer clinical outcome in germinal center B-cell-like DLBCL with wild-type TP53. To understand the underlying mechanisms, we identified a gene-expression profiling signature and the association of MUThigh with decreased T cells in DLBCL patients with wild-type TP53. On the other hand, in overall cohort, MUThigh was associated with lower PD-1 expression in T cells and PD-L1 expression in macrophages, suggesting a positive role of MUThigh in immune responses. Analysis in a whole-exome sequencing dataset of 304 patients deposited by Chapuy et al. validated the correlation of MUT-KMT2D with genomic complexity and the significantly poorer survival associated with higher numbers of genomic single nucleotide variants in activated B-cell-like DLBCL with wild-type TP53. Together, these results suggest that KMT2D inactivation or epigenetic dysregulation has a role in driving DLBCL genomic instability, and that genomic complexity has adverse impact on clinical outcome in DLBCL patients with wild-type TP53 treated with standard immunochemotherapy. The oncoimmune data in this study have important implications for biomarker and therapeutic studies in DLBCL.

Vortex phase matching as a strategy for schooling in robots and in fish.

It has long been proposed that flying and swimming animals could exploit neighbour-induced flows. Despite this it is still not clear whether, and if so how, schooling fish coordinate their movement to benefit from the vortices shed by others. To address this we developed bio-mimetic fish-like robots which allow us to measure directly the energy consumption associated with swimming together in pairs (the most common natural configuration in schooling fish). We find that followers, in any relative position to a near-neighbour, could obtain hydrodynamic benefits if they exhibit a tailbeat phase difference that varies linearly with front-back distance, a strategy we term 'vortex phase matching'. Experiments with pairs of freely-swimming fish reveal that followers exhibit this strategy, and that doing so requires neither a functioning visual nor lateral line system. Our results are consistent with the hypothesis that fish typically, but not exclusively, use vortex phase matching to save energy.