DOK3 maintains intestinal homeostasis by suppressing JAK2/STAT3 signaling and S100a8/9 production in neutrophils.

How pathogenesis of inflammatory bowel disease (IBD) depends on the complex interplay of host genetics, microbiome and the immune system is not fully understood. Here, we showed that Downstream of Kinase 3 (DOK3), an adapter protein involved in immune signaling, confers protection of mice from dextran sodium sulfate (DSS)-induced colitis. DOK3-deficiency promotes gut microbial dysbiosis and enhanced colitis susceptibility, which can be reversed by the transfer of normal microbiota from wild-type mice. Mechanistically, DOK3 exerts its protective effect by suppressing JAK2/STAT3 signaling in colonic neutrophils to limit their S100a8/9 production, thereby maintaining gut microbial ecology and colon homeostasis. Hence, our findings reveal that the immune system and microbiome function in a feed-forward manner, whereby DOK3 maintains colonic neutrophils in a quiescent state to establish a gut microbiome essential for intestinal homeostasis and protection from IBD.

Multi-omics profiling of a CHO cell culture system unravels the effect of culture pH on cell growth, antibody titer, and product quality.

A robust monoclonal antibody (mAb) bioprocess requires physiological parameters such as temperature, pH, or dissolved oxygen to be well-controlled as even small variations in them could potentially impact the final product quality. For instance, pH substantially affects N-glycosylation, protein aggregation, and charge variant profiles, as well as mAb productivity. However, relatively less is known about how pH jointly influences product quality and titer. In this study, we investigated the effect of pH on culture performance, product titer, and quality profiles by applying longitudinal multi-omics profiling, including transcriptomics, proteomics, metabolomics, and glycomics, at three different culture pH set points. The subsequent systematic analysis of multi-omics data showed that pH set points differentially regulated various intracellular pathways including intracellular vesicular trafficking, cell cycle, and apoptosis, thereby resulting in differences in specific productivity, product titer, and quality profiles. In addition, a time-dependent variation in mAb N-glycosylation profiles, independent of pH, was identified to be mainly due to the accumulation of mAb proteins in the endoplasmic reticulum disrupting cellular homeostasis over culture time. Overall, this multi-omics-based study provides an in-depth understanding of the intracellular processes in mAb-producing CHO cell line under varied pH conditions, and could serve as a baseline for enabling the quality optimization and control of mAb production.

Multiomics analyses of cytokines, genes, miRNA, and regulatory networks in human mesenchymal stem cells expanded in stirred microcarrier-spinner cultures.

Mesenchymal stem cells (MSCs) are of great clinical interest as a form of allogenic therapy due to their excellent regenerative and immunomodulatory effects for various therapeutic indications. Stirred suspension bioreactors using microcarriers (MC) have been used for large-scale production of MSCs compared to planar cultivation systems. Previously, we have demonstrated that expansion of MSCs in MC-spinner cultures improved chondrogenic, osteogenic, and cell migration potentials as compared to monolayer-static cultures. In this study, we sought to address this by analyzing global gene expression patterns, miRNA profiles and secretome under both monolayer-static and MC-spinner cultures in serum-free medium at different growth phases. The datasets revealed differential expression patterns that correlated with potentially improved MSC properties in cells from MC-spinner cultures compared to those of monolayer-static cultures. Transcriptome analysis identified a unique expression signature for cells from MC-spinner cultures, which correlated well with miRNA expression, and cytokine secretion involved in key MSC functions. Importantly, MC-spinner cultures and conditioned medium showed increased expression of factors that possibly enhance pathways of extracellular matrix dynamics, cellular metabolism, differentiation potential, immunoregulatory function, and wound healing. This systematic analysis provides insights for the efficient optimization of stem cell bioprocessing and infers that MC-based bioprocess manufacturing could improve post-expansion cellular properties for stem cell therapies.

A Scalable Suspension Platform for Generating High-Density Cultures of Universal Red Blood Cells from Human Induced Pluripotent Stem Cells.

Universal red blood cells (RBCs) differentiated from O-negative human induced pluripotent stem cells (hiPSCs) could find applications in transfusion medicine. Given that each transfusion unit of blood requires 2 trillion RBCs, efficient bioprocesses need to be developed for large-scale in vitro generation of RBCs. We have developed a scalable suspension agitation culture platform for differentiating hiPSC-microcarrier aggregates into functional RBCs and have demonstrated scalability of the process starting with 6 well plates and finally demonstrating in 500 mL spinner flasks. Differentiation of the best-performing hiPSCs generated 0.85 billion erythroblasts in 50 mL cultures with cell densities approaching 1.7 × 107 cells/mL. Functional (oxygen binding, hemoglobin characterization, membrane integrity, and fluctuations) and transcriptomics evaluations showed minimal differences between hiPSC-derived and adult-derived RBCs. The scalable agitation suspension culture differentiation process we describe here could find applications in future large-scale production of RBCs in controlled bioreactors.

Author Correction: Elephant shark genome provides unique insights into gnathostome evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TACI Constrains TH17 Pathogenicity and Protects against Gut Inflammation.

TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) plays critical roles in B cells by promoting immunoglobulin class switching and plasma cell survival. However, its expression and function in T cells remain controversial. We show here that TACI expression can be strongly induced in murine CD4+ T cells in vitro by cytokines responsible for TH17 but not TH1 or TH2 differentiation. Frequencies and numbers of TH17 cells were elevated in TACI-/ - compared with wild-type mice as well as among TACI-/ - versus wild-type CD4+ T cells in mixed bone marrow chimeras, arguing for a T cell-intrinsic effect in the contribution of TACI deficiency to TH17 cell accumulation. TACI-/ - mice were more susceptible to severe colitis induced by dextran sodium sulfate or adoptive T cell transfer, suggesting that TACI negatively regulates TH17 function and limits intestinal inflammation in a cell-autonomous manner. Finally, transcriptomic and biochemical analyses revealed that TACI-/ - CD4+ T cells exhibited enhanced activation of TH17-promoting transcription factors NFAT, IRF4, c-MAF, and JUNB. Taken together, these findings reveal an important role of TACI in constraining TH17 pathogenicity and protecting against gut disease.

Multi-omics profiling of CHO parental hosts reveals cell line-specific variations in bioprocessing traits.

Chinese hamster ovary (CHO) cells are the most prevalent mammalian cell factories for producing recombinant therapeutic proteins due to their ability to synthesize human-like post-translational modifications and ease of maintenance in suspension cultures. Currently, a wide variety of CHO host cell lines has been developed; substantial differences exist in their phenotypes even when transfected with the same target vector. However, relatively less is known about the influence of their inherited genetic heterogeneity on phenotypic traits and production potential from the bioprocessing point of view. Herein, we present a global transcriptome and proteome profiling of three commonly used parental cell lines (CHO-K1, CHO-DXB11, and CHO-DG44) in suspension cultures and further report their growth-related characteristics, and N- and O-glycosylation patterns of host cell proteins (HCPs). The comparative multi-omics and subsequent genome-scale metabolic network model-based enrichment analyses indicated that some physiological variations of CHO cells grown in the same media are possibly originated from the genetic deficits, particularly in the cell-cycle progression. Moreover, the dihydrofolate reductase deficient DG44 and DXB11 possess relatively less active metabolism when compared to K1 cells. The protein processing abilities and the N- and O-glycosylation profiles also differ significantly across the host cell lines, suggesting the need to select host cells in a rational manner for the cell line development on the basis of recombinant protein being produced.

Does Kidney Donor Risk Index implementation lead to the transplantation of more and higher-quality donor kidneys?

BACKGROUND: The Kidney Donor Risk Index (KDRI) is a quantitative evaluation of the quality of donor organs and is implemented in the US allocation system. This single-centre study investigates whether the implementation of the KDRI in our decision-making process to accept or decline an offered deceased donor kidney, increases our acceptance rate. METHODS: From April 2015 until December 2016, we prospectively calculated the KDRI for all deceased donor kidney offers allocated by Eurotransplant to our centre. The number of the transplanted versus declined kidney offers during the study period were compared to a historical set of donor kidney offers. RESULTS: After implementation of the KDRI, 26.1% (75/288) of all offered donor kidneys were transplanted, compared with 20.7% (136/657) in the previous period (P < 0.001). The median KDRI of all transplanted donor kidneys during the second period was 0.97 [Kidney Donor Profile Index (KDPI) 47%], a value significantly higher than the median KDRI of 0.85 (KDPI 34%) during the first period (P = 0.047). A total of 68% of patients for whom a first-offered donor kidney was declined during this period were transplanted after a median waiting time of 386 days, mostly with a lower KDRI donor kidney. CONCLUSIONS: Implementing the KDRI in our decision-making process increased the transplantation rate by 26%. The KDRI can be a supportive tool when considering whether to accept or decline a deceased donor kidney offer. More data are needed to validate this score in other European centres.

The seahorse genome and the evolution of its specialized morphology.

Seahorses have a specialized morphology that includes a toothless tubular mouth, a body covered with bony plates, a male brood pouch, and the absence of caudal and pelvic fins. Here we report the sequencing and de novo assembly of the genome of the tiger tail seahorse, Hippocampus comes. Comparative genomic analysis identifies higher protein and nucleotide evolutionary rates in H. comes compared with other teleost fish genomes. We identified an astacin metalloprotease gene family that has undergone expansion and is highly expressed in the male brood pouch. We also find that the H. comes genome lacks enamel matrix protein-coding proline/glutamine-rich secretory calcium-binding phosphoprotein genes, which might have led to the loss of mineralized teeth. tbx4, a regulator of hindlimb development, is also not found in H. comes genome. Knockout of tbx4 in zebrafish showed a 'pelvic fin-loss' phenotype similar to that of seahorses.

The genome of the largest bony fish, ocean sunfish (Mola mola), provides insights into its fast growth rate.

BACKGROUND: The ocean sunfish (Mola mola), which can grow up to a length of 2.7 m and weigh 2.3 tons, is the world's largest bony fish. It has an extremely fast growth rate and its endoskeleton is mainly composed of cartilage. Another unique feature of the sunfish is its lack of a caudal fin, which is replaced by a broad and stiff lobe that results in the characteristic truncated appearance of the fish. RESULTS: To gain insights into the genomic basis of these phenotypic traits, we sequenced the sunfish genome and performed a comparative analysis with other teleost genomes. Several sunfish genes involved in the growth hormone and insulin-like growth factor 1 (GH/IGF1) axis signalling pathway were found to be under positive selection or accelerated evolution, which might explain its fast growth rate and large body size. A number of genes associated with the extracellular matrix, some of which are involved in the regulation of bone and cartilage development, have also undergone positive selection or accelerated evolution. A comparison of the sunfish genome with that of the pufferfish (fugu), which has a caudal fin, revealed that the sunfish contains more homeobox (Hox) genes although both genomes contain seven Hox clusters. Thus, caudal fin loss in sunfish is not associated with the loss of a specific Hox gene. CONCLUSIONS: Our analyses provide insights into the molecular basis of the fast growth rate and large size of the ocean sunfish. The high-quality genome assembly generated in this study should facilitate further studies of this 'natural mutant'.

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons.

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

The p53-Mdm2 interaction and the E3 ligase activity of Mdm2/Mdm4 are conserved from lampreys to humans.

The extant jawless vertebrates, represented by lampreys and hagfish, are the oldest group of vertebrates and provide an interesting genomic evolutionary pivot point between invertebrates and jawed vertebrates. Through genome analysis of one of these jawless vertebrates, the Japanese lamprey (Lethenteron japonicum), we identified all three members of the important p53 transcription factor family--Tp53, Tp63, and Tp73--as well as the Mdm2 and Mdm4 genes. These genes and their products are significant cellular regulators in human cancer, and further examination of their roles in this most distant vertebrate relative sheds light on their origin and coevolution. Their important role in response to DNA damage has been highlighted by the discovery of multiple copies of the Tp53 gene in elephants. Expression of lamprey p53, Mdm2, and Mdm4 proteins in mammalian cells reveals that the p53-Mdm2 interaction and the Mdm2/Mdm4 E3 ligase activity existed in the common ancestor of vertebrates and have been conserved for >500 million years of vertebrate evolution. Lamprey Mdm2 degrades human p53 with great efficiency, but this interaction is not blocked by currently available small molecule inhibitors of the human HDM2 protein, suggesting utility of lamprey Mdm2 in the study of the human p53 signaling pathway.

Ancient Duplications and Expression Divergence in the Globin Gene Superfamily of Vertebrates: Insights from the Elephant Shark Genome and Transcriptome.

Comparative analyses of vertebrate genomes continue to uncover a surprising diversity of genes in the globin gene superfamily, some of which have very restricted phyletic distributions despite their antiquity. Genomic analysis of the globin gene repertoire of cartilaginous fish (Chondrichthyes) should be especially informative about the duplicative origins and ancestral functions of vertebrate globins, as divergence between Chondrichthyes and bony vertebrates represents the most basal split within the jawed vertebrates. Here, we report a comparative genomic analysis of the vertebrate globin gene family that includes the complete globin gene repertoire of the elephant shark (Callorhinchus milii). Using genomic sequence data from representatives of all major vertebrate classes, integrated analyses of conserved synteny and phylogenetic relationships revealed that the last common ancestor of vertebrates possessed a repertoire of at least seven globin genes: single copies of androglobin and neuroglobin, four paralogous copies of globin X, and the single-copy progenitor of the entire set of vertebrate-specific globins. Combined with expression data, the genomic inventory of elephant shark globins yielded four especially surprising findings: 1) there is no trace of the neuroglobin gene (a highly conserved gene that is present in all other jawed vertebrates that have been examined to date), 2) myoglobin is highly expressed in heart, but not in skeletal muscle (reflecting a possible ancestral condition in vertebrates with single-circuit circulatory systems), 3) elephant shark possesses two highly divergent globin X paralogs, one of which is preferentially expressed in gonads, and 4) elephant shark possesses two structurally distinct α-globin paralogs, one of which is preferentially expressed in the brain. Expression profiles of elephant shark globin genes reveal distinct specializations of function relative to orthologs in bony vertebrates and suggest hypotheses about ancestral functions of vertebrate globins.

Is the Kidney Donor Risk Index a step forward in the assessment of deceased donor kidney quality?

The allocation of deceased donor kidneys has become more complex because of the increasing spectrum of donors and recipients age and comorbidities. Several scoring systems have been proposed to evaluate the donor quality of deceased donor kidneys, based on clinical, pathological or combined parameters to predict the risk of renal allograft failure. Nonetheless, besides the dichotomous extended criteria donor (ECD) score, none of the others have been used in clinical practice because of numerous reasons, ranging from lack of robust validation to the technical challenges associated with the evaluation of donor biopsies. Recently, the Kidney Donor Risk Index (KDRI) and Profile Index (KDPI) were introduced in the USA as a refined version of the ECD score. This scoring system is based on 10 donor factors, therefore providing a finely granulated evaluation of donor quality without the need of a kidney biopsy.Here, we review the advantages and drawbacks of the main scoring systems, and we describe the components of the KDRI and KDPI. It is an easily accessible online tool, based solely on donor factors readily available at the moment of the donor offer. Importantly, the KDPI has also been made part of the 'longevity matching' allocation in the USA, where the best kidneys are allocated to the recipients with the longest predicted post-transplant survival. The KDRI should provide us with a robust qualitative evaluation of deceased donor quality, and therefore will probably play a role in deceased donor kidney allocation policies across Europe in the near future. Hopefully, the KDRI and the KDPI should help transplant programmes to better allocate the scarce resource of deceased donor kidneys.

The genomic substrate for adaptive radiation in African cichlid fish.

Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.

Elephant shark genome provides unique insights into gnathostome evolution.

The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates was accompanied by major morphological and physiological innovations, such as hinged jaws, paired fins and immunoglobulin-based adaptive immunity. Gnathostomes subsequently diverged into two groups, the cartilaginous fishes and the bony vertebrates. Here we report the whole-genome analysis of a cartilaginous fish, the elephant shark (Callorhinchus milii). We find that the C. milii genome is the slowest evolving of all known vertebrates, including the 'living fossil' coelacanth, and features extensive synteny conservation with tetrapod genomes, making it a good model for comparative analyses of gnathostome genomes. Our functional studies suggest that the lack of genes encoding secreted calcium-binding phosphoproteins in cartilaginous fishes explains the absence of bone in their endoskeleton. Furthermore, the adaptive immune system of cartilaginous fishes is unusual: it lacks the canonical CD4 co-receptor and most transcription factors, cytokines and cytokine receptors related to the CD4 lineage, despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity.

Evidence for at least six Hox clusters in the Japanese lamprey (Lethenteron japonicum).

Cyclostomes, comprising jawless vertebrates such as lampreys and hagfishes, are the sister group of living jawed vertebrates (gnathostomes) and hence an important group for understanding the origin and diversity of vertebrates. In vertebrates and other metazoans, Hox genes determine cell fate along the anteroposterior axis of embryos and are implicated in driving morphological diversity. Invertebrates contain a single Hox cluster (either intact or fragmented), whereas elephant shark, coelacanth, and tetrapods contain four Hox clusters owing to two rounds of whole-genome duplication ("1R" and "2R") during early vertebrate evolution. By contrast, most teleost fishes contain up to eight Hox clusters because of an additional "teleost-specific" genome duplication event. By sequencing bacterial artificial chromosome (BAC) clones and the whole genome, here we provide evidence for at least six Hox clusters in the Japanese lamprey (Lethenteron japonicum). This suggests that the lamprey lineage has experienced an additional genome duplication after 1R and 2R. The relative age of lamprey and human paralogs supports this hypothesis. Compared with gnathostome Hox clusters, lamprey Hox clusters are unusually large. Several conserved noncoding elements (CNEs) were predicted in the Hox clusters of lamprey, elephant shark, and human. Transgenic zebrafish assay indicated the potential of CNEs to function as enhancers. Interestingly, CNEs in individual lamprey Hox clusters are frequently conserved in multiple Hox clusters in elephant shark and human, implying a many-to-many orthology relationship between lamprey and gnathostome Hox clusters. Such a relationship suggests that the first two rounds of genome duplication may have occurred independently in the lamprey and gnathostome lineages.