Stress‐induced increase of monoclonal antibody production in CHO cells

Monoclonal antibodies (mAbs) are of great interest to the biopharmaceutical industry due to their widely used application as human therapeutic and diagnostic agents. As such, mAb require to exhibit human‐like glycolization patterns. Therefore, recombinant Chinese hamster ovary (CHO) cells are the favored production organisms; many relevant biopharmaceuticals are already produced by this cell type. To optimize the mAb yield in CHO DG44 cells a corelation between stress‐induced cell size expansion and increased specific productivity was investigated. CO₂ and macronutrient supply of the cells during a 12‐day fed‐batch cultivation process were tested as stress factors. Shake flasks (500 mL) and a small‐scale bioreactor system (15 mL) were used for the cultivation experiments and compared in terms of their effect on cell diameter, integral viable cell concentration (IVCC), and cell‐specific productivity. The achieved stress‐induced increase in cell‐specific productivity of up to 94.94.9%–134.4% correlates to a cell diameter shift of up to 7.34 μm. The highest final product titer of 4 g/L was reached by glucose oversupply during the batch phase of the process.     

Identifying “vital attributes” for assessing disturbance–recovery potential of seafloor communities

Despite a long history of disturbance–recovery research, we still lack a generalizable understanding of the attributes that drive community recovery potential in seafloor ecosystems. Marine soft‐sediment ecosystems encompass a range of heterogeneity from simple low‐diversity habitats with limited biogenic structure, to species‐rich systems with complex biogenic habitat structure. These differences in biological heterogeneity are a product of natural conditions and disturbance regimes. To search for unifying attributes, we explore whether a set of simple traits can characterize community disturbance–recovery potential using seafloor patch‐disturbance experiments conducted in two different soft‐sediment landscapes. The two landscapes represent two ends of a spectrum of landscape biotic heterogeneity in order to consider multi‐scale disturbance–recovery processes. We consider traits at different levels of biological organization, from the biological traits of individual species, to the traits of species at the landscape scale associated with their occurrence across the landscape and their ability to be dominant. We show that in a biotically heterogeneous landscape (Kawau Bay, New Zealand), seafloor community recovery is stochastic, there is high species turnover, and the landscape‐scale traits are good predictors of recovery. In contrast, in a biotically homogeneous landscape (Baltic Sea), the options for recovery are constrained, the recovery pathway is thus more deterministic and the scale of recovery traits important for determining recovery switches to the individual species biological traits within the disturbed patch. Our results imply that these simple, yet sophisticated, traits can be effectively used to characterize community recovery potential and highlight the role of landscapes in providing resilience to patch‐scale disturbances.     

siRNA‐induced CD44 knockdown suppresses the proliferation and invasion of colorectal cancer stem cells through inhibiting epithelial–mesenchymal transition

CD44 has shown prognostic values and promising therapeutic potential in multiple human cancers; however, the effects of CD44 silencing on biological behaviors of cancer stem cells (CSCs) have not been fully understood in colorectal cancer. To examine the contribution of siRNA‐induced knockdown of CD44 to the biological features of colorectal CSCs, colorectal CSCs HCT116‐CSCs were generated, and CD44 was knocked down in HCT116‐CSCs using siRNA. The proliferation, migration and invasion of HCT116‐CSCs were measured, and apoptosis and cell‐cycle analyses were performed. The sensitivity of HCT116‐CSCs to oxaliplatin was tested, and xenograft tumor growth assay was performed to examine the role of CD44 in HCT116‐CSCs tumorigenesis in vivo. In addition, the expression of epithelial–mesenchymal transition (EMT) markers E‐cadherin, N‐cadherin and vimentin was quantified. siRNA‐induced knockdown of CD44 was found to inhibit the proliferation, migration and invasion, induce apoptosis, promote cell‐cycle arrest at the G1/G0 phase and increase the sensitivity of HCT116‐CSCs to oxaliplatin in HCT116‐CSCs, and knockdown of CD44 suppressed in vivo tumorigenesis and intrapulmonary metastasis of HCT116‐CSCs. Moreover, silencing CD44 resulted in EMT inhibition. Our findings demonstrate that siRNA‐induced CD44 knockdown suppresses the proliferation, invasion and in vivo tumorigenesis and metastasis of colorectal CSCs by inhibiting EMT.     

5‐Fluorouracil efficacy requires anti‐tumor immunity triggered by cancer‐cell‐intrinsic STING

5‐Fluorouracil (5‐FU) is a widely used chemotherapeutic drug, but the mechanisms underlying 5‐FU efficacy in immunocompetent hosts in vivo remain largely elusive. Through modeling 5‐FU response of murine colon and melanoma tumors, we report that effective reduction of tumor burden by 5‐FU is dependent on anti‐tumor immunity triggered by the activation of cancer‐cell‐intrinsic STING. While the loss of STING does not induce 5‐FU resistance in vitro, effective 5‐FU responsiveness in vivo requires cancer‐cell‐intrinsic cGAS, STING, and subsequent type I interferon (IFN) production, as well as IFN‐sensing by bone‐marrow‐derived cells. In the absence of cancer‐cell‐intrinsic STING, a much higher dose of 5‐FU is needed to reduce tumor burden. 5‐FU treatment leads to increased intratumoral T cells, and T‐cell depletion significantly reduces the efficacy of 5‐FU in vivo. In human colorectal specimens, higher STING expression is associated with better survival and responsiveness to chemotherapy. Our results support a model in which 5‐FU triggers cancer‐cell‐initiated anti‐tumor immunity to reduce tumor burden, and our findings could be harnessed to improve therapeutic effectiveness and toxicity for colon and other cancers.     

Collective colony growth is optimized by branching pattern formation in Pseudomonas aeruginosa

Branching pattern formation is common in many microbes. Extensive studies have focused on addressing how such patterns emerge from local cell–cell and cell–environment interactions. However, little is known about whether and to what extent these patterns play a physiological role. Here, we consider the colonization of bacteria as an optimization problem to find the colony patterns that maximize colony growth efficiency under different environmental conditions. We demonstrate that Pseudomonas aeruginosa colonies develop branching patterns with characteristics comparable to the prediction of modeling; for example, colonies form thin branches in a nutrient‐poor environment. Hence, the formation of branching patterns represents an optimal strategy for the growth of Pseudomonas aeruginosa colonies. The quantitative relationship between colony patterns and growth conditions enables us to develop a coarse‐grained model to predict diverse colony patterns under more complex conditions, which we validated experimentally. Our results offer new insights into branching pattern formation as a problem‐solving social behavior in microbes and enable fast and accurate predictions of complex spatial patterns in branching colonies.     

Broussonin A– and B–mediated inhibition of angiogenesis by blockade of VEGFR‐2 signalling pathways and integrin β1 expression

In the present study, we demonstrate the regulatory effects and mechanism of broussonin A and B, diphenylpropane derivatives isolated from Broussonetia kazinoki, on vascular endothelial growth factor‐A (VEGF‐A)–stimulated endothelial cell responses in vitro and microvessel sprouting ex vivo. Treatment with broussonin A or B suppressed VEGF‐A‐stimulated endothelial cell proliferation by regulating the expression of cell cycle–related proteins and the phosphorylation status of retinoblastoma protein. In addition, treatment with broussonin A or B abrogated VEGF‐A‐stimulated angiogenic responses including endothelial cell migration, invasion, tube formation and microvessel formation from rat aortic rings. These anti‐angiogenic activities of broussonin A and B were mediated through inactivation of VEGF‐A‐stimulated downstream signalling pathways, localization of vascular endothelial‐cadherin at cell‐cell contacts, and down‐regulation of integrin β1 and integrin‐liked kinase. Furthermore, treatment with broussonin A or B inhibited proliferation and invasion of non–small cell lung cancer and ovarian cancer cells. Taken together, our findings suggest the pharmacological potential of broussonin A and B in the regulation of angiogenesis, cancer cell growth and progression.     

A Silurian myodocope with preserved soft-parts: cautioning the interpretation of the shell-based ostracod record

Ostracod crustaceans are the most abundant fossil arthropods. The Silurian Pauline avibella gen. et sp. nov., from the Herefordshire Lagerstätte, UK, is an extremely rare Palaeozoic example with soft-part preservation. Based on its soft-part morphology, especially the exceptionally preserved limbs and presence of lateral eyes, it is assigned to the myodocopid myodocopes. The ostracod is very large, with an epipod on the fifth limb pair, as well as gills implying the presence of a heart and an integrated respiratory–circulatory system as in living cylindroleberidid myodocopids. Features of its shell morphology, however, recall halocyprid myodocopes and palaeocopes, encouraging caution in classifying ostracods based on the carapace alone and querying the interpretation of their shell-based fossil record, especially for the Palaeozoic, where some 500 genera are presently assigned to the Palaeocopida.     

HIV‐1 infection of CD4 T cells impairs antigen‐specific B cell function

Failures to produce neutralizing antibodies upon HIV‐1 infection result in part from B‐cell dysfunction due to unspecific B‐cell activation. How HIV‐1 affects antigen‐specific B‐cell functions remains elusive. Using an adoptive transfer mouse model and ex vivo HIV infection of human tonsil tissue, we found that expression of the HIV‐1 pathogenesis factor NEF in CD4 T cells undermines their helper function and impairs cognate B‐cell functions including mounting of efficient specific IgG responses. NEF interfered with T cell help via a specific protein interaction motif that prevents polarized cytokine secretion at the T‐cell–B‐cell immune synapse. This interference reduced B‐cell activation and proliferation and thus disrupted germinal center formation and affinity maturation. These results identify NEF as a key component for HIV‐mediated dysfunction of antigen‐specific B cells. Therapeutic targeting of the identified molecular surface in NEF will facilitate host control of HIV infection.     

Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases

Mitogen‐activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less‐characterized disordered regions. We used a structurally consistent model on kinase‐docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under‐explored part of the human proteome and applied experimental tools specifically tailored to detect low‐affinity protein–protein interactions for their validation in vitro and in cell‐based assays. The combined computational and experimental approach enabled the identification of many novel MAPK‐docking motifs that were elusive for other large‐scale protein–protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase‐mediated partnerships evolved over time. The analysis suggests that most human MAPK‐binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK‐binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.     

Subcellular localization of Type VI secretion system assembly in response to cell–cell contact

Bacteria require a number of systems, including the type VI secretion system (T6SS), for interbacterial competition and pathogenesis. The T6SS is a large nanomachine that can deliver toxins directly across membranes of proximal target cells. Since major reassembly of T6SS is necessary after each secretion event, accurate timing and localization of T6SS assembly can lower the cost of protein translocation. Although critically important, mechanisms underlying spatiotemporal regulation of T6SS assembly remain poorly understood. Here, we used super‐resolution live‐cell imaging to show that while Acinetobacter and Burkholderia thailandensis can assemble T6SS at any site, a significant subset of T6SS assemblies localizes precisely to the site of contact between neighboring bacteria. We identified a class of diverse, previously uncharacterized, periplasmic proteins required for this dynamic localization of T6SS to cell–cell contact (TslA). This precise localization is also dependent on the outer membrane porin OmpA. Our analysis links transmembrane communication to accurate timing and localization of T6SS assembly as well as uncovers a pathway allowing bacterial cells to respond to cell–cell contact during interbacterial competition.     

Controlled X‐chromosome dynamics defines meiotic potential of female mouse in vitro germ cells

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X‐chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X‐inactivation and reactivation dynamics using a tailor‐made in vitro system of primordial germ cell‐like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X‐inactivation in PGCLCs in vitro and in germ cell‐competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X‐inactivation is followed by step‐wise X‐reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X‐inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine‐tuned X‐chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.     

Parasites rather than phoronts: Teratorhabditis synpapillata nematodes reduce lifespan of their Rhynchophorus ferrugineus host in a life stage‐dependent manner

Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) red palm weevils are often reported in association with different organisms including nematodes. The significance of this interaction and whether nematodes can influence their life‐history traits is unclear. We collected Rhynchophorus ferrugineus red palm weevils at different developmental stages and locations in Tunisia, observed and dissected them in search for nematodes and other interacting organisms, established laboratory colonies and identified the nematodes associated with them, and conducted nematode–insect interaction assays to determine the capacity of the nematodes to influence their life‐history traits. We observed Beauveria bassiana fungi in larvae, nymph, and adults; Centrouropoda and Uroobovella acari associated with the adults, and Teratorhabditis synpapillata nematodes associated with larvae and adults. Nematode–insect interaction bioassays revealed that T. synpapillata nematodes reduce the lifespan of the insect larvae in a population‐dependent manner, but do not influence the lifespan of adults. Our study uncovers an important factor that may determine population dynamics of this important palm pests and provides evidence to conclude that these organisms establish a parasitic relationship, rather than a phoretic relationship.     

Aneuploid senescent cells activate NF‐κB to promote their immune clearance by NK cells

The immune system plays a major role in the protection against cancer. Identifying and characterizing the pathways mediating this immune surveillance are thus critical for understanding how cancer cells are recognized and eliminated. Aneuploidy is a hallmark of cancer, and we previously found that untransformed cells that had undergone senescence due to highly abnormal karyotypes are eliminated by natural killer (NK) cells in vitro. However, the mechanisms underlying this process remained elusive. Here, using an in vitro NK cell killing system, we show that non‐cell‐autonomous mechanisms in aneuploid cells predominantly mediate their clearance by NK cells. Our data indicate that in untransformed aneuploid cells, NF‐κB signaling upregulation is central to elicit this immune response. Inactivating NF‐κB abolishes NK cell‐mediated clearance of untransformed aneuploid cells. In cancer cell lines, NF‐κB upregulation also correlates with the degree of aneuploidy. However, such upregulation in cancer cells is not sufficient to trigger NK cell‐mediated clearance, suggesting that additional mechanisms might be at play during cancer evolution to counteract NF‐κB‐mediated immunogenicity.     

P21 activated kinase‐1 (PAK1) in macrophages is required for promotion of Th17 cell response during helminth infection

CD4⁺T cells differentiate into distinct functional effector and inhibitory subsets are facilitated by distinct cytokine cues present at the time of antigen recognition. Maintaining a balance between T helper 17 (Th17) and regulatory T (Treg) cells are critical for the control of the immunopathogenesis of liver diseases. Here, by using the mouse model of helminth Schistosoma japonicum (S japonicum) infection, we show that the hepatic mRNA levels of P21‐activated kinase 1 (PAK1), a key regulator of the actin cytoskeleton, adhesion and cell motility, are significantly increased and associated with the development of liver pathology during S japonicum infection. In addition, PAK1‐deficient mice are prone to suppression of Th17 cell responses but increased Treg cells. Furthermore, PAK1 enhances macrophage activation through promoting IRF1 nuclear translocation in an NF‐κB‐dependent pathway, resulting in promoting Th17 cell differentiation through inducing IL‐6 production. These findings highlight the importance of PAK1 in macrophages fate determination and suggest that PAK1/IRF1 axis‐dependent immunomodulation can ameliorate certain T cell–based immune pathologies.     

AMPK regulates cell shape of cardiomyocytes by modulating turnover of microtubules through CLIP‐170

AMP‐activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP‐170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell–cell junction present between cardiomyocytes. A contractile inhibitor, MYK‐461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK‐461, suggesting that the localization of AMPK is regulated by mechanical stress. Time‐lapse imaging analysis revealed that the inhibition of CLIP‐170 Ser‐311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK‐461 increased the individual cell area of cardiomyocytes in CLIP‐170 phosphorylation‐dependent manner. Moreover, heart‐specific CLIP‐170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP‐170 at the intercalated disks.     

Paleovirology of ‘syncytins’, retroviral env genes exapted for a role in placentation

The development of the emerging field of ‘paleovirology’ allows biologists to reconstruct the evolutionary history of fossil endogenous retroviral sequences integrated within the genome of living organisms and has led to the retrieval of conserved, ancient retroviral genes ‘exapted’ by ancestral hosts to fulfil essential physiological roles, syncytin genes being undoubtedly among the most remarkable examples of such a phenomenon. Indeed, syncytins are ‘new’ genes encoding proteins derived from the envelope protein of endogenous retroviral elements that have been captured and domesticated on multiple occasions and independently in diverse mammalian species, through a process of convergent evolution. Knockout of syncytin genes in mice provided evidence for their absolute requirement for placenta development and embryo survival, via formation by cell–cell fusion of syncytial cell layers at the fetal–maternal interface. These genes of exogenous origin, acquired ‘by chance’ and yet still ‘necessary’ to carry out a basic function in placental mammals, may have been pivotal in the emergence of mammalian ancestors with a placenta from egg-laying animals via the capture of a founding retroviral env gene, subsequently replaced in the diverse mammalian lineages by new env-derived syncytin genes, each providing its host with a positive selective advantage.