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Spatial analysis of microbiomes at single cell resolution with high multiplexity and accuracy has remained challenging. Here we present spatial profiling of a microbiome using sequential error-robust fluorescence in situ hybridization (SEER-FISH), a highly multiplexed and accurate imaging method that allows mapping of microbial communities at micron-scale. We show that multiplexity of RNA profiling in microbiomes can be increased significantly by sequential rounds of probe hybridization and dissociation. Combined with error-correction strategies, we demonstrate that SEER-FISH enables accurate taxonomic identification in complex microbial communities. Using microbial communities composed of diverse bacterial taxa isolated from plant rhizospheres, we apply SEER-FISH to quantify the abundance of each taxon and map microbial biogeography on roots. At micron-scale, we identify clustering of microbial cells from multiple species on the rhizoplane. Under treatment of plant metabolites, we find spatial re-organization of microbial colonization along the root and alterations in spatial association among microbial taxa. Taken together, SEER-FISH provides a useful method for profiling the spatial ecology of complex microbial communities in situ. Spatial analysis of microbiomes at single cell resolution is challenging. Here the authors report a highly multiplexed method for spatial profiling, sequential error-robust fluorescence in situ hybridisation (SEER-FISH), and show that this allows mapping of microbial communities at micron-scale.

Human gut Bacteroides and Parabacteroides species play crucial roles in human health and are known for their capacity to utilize diverse polysaccharides. Understanding how these bacteria utilize medicinal polysaccharides is foundational for developing polysaccharides-based prebiotics and drugs. Here, we systematically mapped the utilization profiles of 20 different medicinal polysaccharides by 28 human gut Bacteroides and Parabacteroides species. The growth profiles exhibited substantial variation across different bacterial species and medicinal polysaccharides. Ginseng polysaccharides promoted the growth of multiple Bacteroides and Parabacteroides species; in contrast, Dendrobium polysaccharides selectively promoted the growth of Bacteroides uniformis . This distinct utilization profile was associated with genomic variation in carbohydrate-active enzymes, rather than monosaccharides composition variation among medicinal polysaccharides. Through comparative transcriptomics and genetical manipulation, we validated that the polysaccharide utilization locus PUL34_ Bu enabled Bacteroides uniformis to utilize Dendrobium polysaccharides (i.e. glucomannan). In addition, we found that the GH26 enzyme in PUL34_ Bu allowed Bacteroides uniformis to utilize multiple plant-derived mannan. Overall, our results revealed the selective utilization of medicinal polysaccharide by Bacteroides and Parabacteroides species and provided insights into the use of polysaccharides in engineering the human gut microbiome. Here, the authors characterize the utilization of 20 medicinal polysaccharides by 28 human gut Bacteroides and Parabacteroides species, revealing substantial variability in bacterial growth responses, which they link to genomic differences in carbohydrate-active enzymes.

Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic to be isolated and crystallized, and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, some key biosynthetic mechanisms of such an old and important molecule have remained unclear. Here, we elucidate the MPA biosynthetic pathway that features both compartmentalized enzymatic steps and unique cooperation between biosynthetic and β-oxidation catabolism machineries based on targeted gene inactivation, feeding experiments in heterologous expression hosts, enzyme functional characterization and kinetic analysis, and microscopic observation of protein subcellular localization. Besides identification of the oxygenase MpaB′ as the long-sought key enzyme responsible for the oxidative cleavage of the farnesyl side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC′ and O-methyltransferase MpaG′, the Golgi apparatus-associated prenyltransferase MpaA′, the endoplasmic reticulum-bound oxygenase MpaB′ and P450-hydrolase fusion enzyme MpaDE′, and the peroxisomal acyl-coenzyme A (CoA) hydrolase MpaH′. The whole pathway is elegantly comediated by these compartmentalized enzymes, together with the peroxisomal β-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic steps, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.

Recent advancements in single‐cell genomic and transcriptomic sequencing, in situ sequencing, and molecular imaging‐based technologies have facilitated the examination of heterogeneity within microbial communities at the single‐cell level. These cutting‐edge methodologies permit the capture of phenotypic and genotypic heterogeneity, as well as the spatial organization within microbial communities. This enables in‐depth investigation into microbial dark matter, the evaluation of microbial responses to perturbations, and a comprehensive exploration of spatial functions involved in community assembly and social interactions within microbial communities. These interactions include inter‐microbial relationships, bacteria–phage interactions, and host–microbe interactions. Here, we highlight the key technological breakthroughs achieved, elucidating the perspectives from which these technologies enable us to interpret microbial heterogeneity at the single‐cell level. Additionally, we critically examine the limitations associated with these technologies. Furthermore, we explore how these methods could be combined and also their applications in future studies. The integration of these approaches holds great promise for increasing our understanding of the organization and function of microbes in complex ecosystems.

When hyperacute rejection is avoided by deletion of Gal expression in the pig, delayed xenograft rejection (DXR) becomes a major immunologic barrier to successful xenotransplantation. This study was to investigate the potential antigens involved in DXR. We isolated primary renal microvascular endothelial cells (RMEC) and aortic endothelial cells (AEC) from a GGTA1/CMAH double-knockout (DKO) pig (and a GGTA1-KO pig) and immunized cynomolgus monkeys with both of these cells. After sensitization, monkey serum antibody binding and cytotoxicity to RMEC was significantly higher than to AEC(p < 0.05), suggesting that RMEC are more immunogenic than AEC. Transcriptome sequencing of GGTA1/CMAH DKO pigs indicated that the expression of 1,500 genes was higher in RMEC than in AEC, while expression of 896 genes was lower. Next, we selected 101 candidate genes expressed only in pig RMEC, but not in pig AEC or in monkey or human RMEC. When these genes were knocked out individually in GGTA1/CMAH DKO RMEC, 32 genes were associated with reduced antibody binding, indicating that these genes might be primary immunologic targets involved in DXR. These genes may be important candidates for deletion in producing pigs against which there is a reduced primate immune response in pig kidney xenograft.

Tumor formation constitutes a major obstacle to the clinical application of embryonic stem cell-derived (ESC-derived) cells. In an attempt to find major extracellular signaling and intrinsic factors controlling tumorigenicity and therapeutic functionality of transplanted ESC-derived retinal progenitor cells (ESC-RPCs), we evaluated multiple kinds of ESC-RPCs in a mouse retinal degeneration model and conducted genome-wide gene expression profiling. We identified canonical WNT signaling as a critical determinant for the tumorigenicity and therapeutic function of ESC-RPCs. The function of WNT signaling is primarily mediated by TCF7, which directly induces expression of Sox2 and Nestin. Inhibition of WNT signaling, overexpression of dominant-negative Tcf7, and silencing Tcf7, Sox2, or Nestin all resulted in drastically reduced tumor formation and substantially improved retinal integration and visual preservation in mice. These results demonstrate that the WNT signaling cascade plays a critical role in modulating the tumorigenicity and functionality of ESC-derived progenitors.

Introduction Degenerative retinal diseases like age-related macular degeneration (AMD) are the leading cause of blindness. Cell transplantation showed promising therapeutic effect for such diseases, and embryonic stem cell (ESC) is one of the sources of such donor cells. Here, we aimed to generate retinal progenitor cells (RPCs) from rat ESCs (rESCs) and to test their therapeutic effects in rat model. Methods The rESCs (DA8-16) were cultured in N2B27 medium with 2i, and differentiated to two types of RPCs following the SFEBq method with modifications. For rESC-RPC1, the cells were switched to adherent culture at D10, while for rESC-RPC2, the suspension culture was maintained to D14. Both RPCs were harvested at D16. Primary RPCs were obtained from P1 SD rats, and some of them were labeled with EGFP by infection with lentivirus. To generate Rax::EGFP knock-in rESC lines, TALENs were engineered to facilitate homologous recombination in rESCs, which were cotransfected with the targeting vector and TALEN vectors. The differentiated cells were analyzed with live image, immunofluorescence staining, flow cytometric analysis, gene expression microarray, etc. RCS rats were used to mimic the degeneration of retina and test the therapeutic effects of subretinally transplanted donor cells. The structure and function of retina were examined. Results We established two protocols through which two types of rESC-derived RPCs were obtained and both contained committed retina lineage cells and some neural progenitor cells (NPCs). These rESC-derived RPCs survived in the host retinas of RCS rats and protected the retinal structure and function in early stage following the transplantation. However, the glia enriched rESC-RPC1 obtained through early and longer adherent culture only increased the b-wave amplitude at 4 weeks, while the longer suspension culture gave rise to evidently neuronal differentiation in rESC-RPC2 which significantly improved the visual function of RCS rats. Conclusions We have successfully differentiated rESCs to glia enriched RPCs and retinal neuron enriched RPCs in vitro . The retinal neuron enriched rESC-RPC2 protected the structure and function of retina in rats with genetic retinal degeneration and could be a candidate cell source for treating some degenerative retinal diseases in human trials.

Spatial analysis of microbiomes at single cell resolution with high multiplexity and accuracy has remained challenging. Here we present spatial profiling of a microbiome using sequential error-robust fluorescence in situ hybridization (SEER-FISH), a highly multiplexed and accurate imaging method that allows mapping of microbial communities at micron-scale. We show that multiplexity of RNA profiling in microbiomes can be increased significantly by sequential rounds of probe hybridization and dissociation. Combined with error-correction strategies, we demonstrate that SEER-FISH enables accurate taxonomic identification in complex microbial communities. Using microbial communities composed of diverse bacterial taxa isolated from plant rhizospheres, we show that SEER-FISH can quantify the abundance of each taxon and map microbial biogeography on roots. SEER-FISH should enable accurate spatial profiling of the ecology and function of complex microbial communities. Competing Interest Statement The authors have declared no competing interest.

Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic in human history and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, its biosynthetic mechanisms have remained a long-standing mystery. Here, we elucidate the MPA biosynthetic pathway that features both compartmentalized enzymatic steps and unique cooperation between biosynthetic and β-oxidation catabolism machineries based on targeted gene inactivation, feeding experiments in heterologous expression hosts, enzyme functional characterization and kinetic analysis, and microscopic observation of protein subcellular localization. Besides identification of the oxygenase MpaB′ as the long-sought key enzyme responsible for the oxidative cleavage of sesquiterpene side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC' and O-methyltransferase MpaG', the Golgi apparatus-associated prenyltransferase MpaA', the endoplasmic reticulum-bound oxygenase MpaB' and P450-hydrolase fusion enzyme MpaDE', and the peroxisomal acyl-CoA hydrolase MpaH'. The whole pathway is elegantly co-mediated by these compartmentalized enzymes, together with the peroxisomal β-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic pathway, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.