Emergent coexistence in multispecies microbial communities.

Understanding the mechanisms that maintain microbial biodiversity is a critical aspiration in ecology. Past work on microbial coexistence has largely focused on species pairs, but it is unclear whether pairwise coexistence in isolation is required for coexistence in a multispecies community. To address this question, we conducted hundreds of pairwise competition experiments among the stably coexisting members of 12 different enrichment communities in vitro. To determine the outcomes of these experiments, we developed an automated image analysis pipeline to quantify species abundances. We found that competitive exclusion was the most common outcome, and it was strongly hierarchical and transitive. Because many species that coexist within a stable multispecies community fail to coexist in pairwise co-culture under identical conditions, we concluded that multispecies coexistence is an emergent phenomenon. This work highlights the importance of community context for understanding the origins of coexistence in complex ecosystems.

Top-down and bottom-up cohesiveness in microbial community coalescence.

Microbial communities frequently invade one another as a whole, a phenomenon known as community coalescence. Despite its potential importance for the assembly, dynamics, and stability of microbial consortia, as well as its prospective utility for microbiome engineering, our understanding of the processes that govern it is still very limited. Theory has suggested that microbial communities may exhibit cohesiveness in the face of invasions emerging from collective metabolic interactions across microbes and their environment. This cohesiveness may lead to correlated invasional outcomes, where the fate of a given taxon is determined by that of other members of its community-a hypothesis known as ecological coselection. Here, we have performed over 100 invasion and coalescence experiments with microbial communities of various origins assembled in two different synthetic environments. We show that the dominant members of the primary communities can recruit their rarer partners during coalescence (top-down coselection) and also be recruited by them (bottom-up coselection). With the aid of a consumer-resource model, we found that the emergence of top-down or bottom-up cohesiveness is modulated by the structure of the underlying cross-feeding networks that sustain the coalesced communities. The model also predicts that these two forms of ecological coselection cannot co-occur under our conditions, and we have experimentally confirmed that one can be strong only when the other is weak. Our results provide direct evidence that collective invasions can be expected to produce ecological coselection as a result of cross-feeding interactions at the community level.

Functional attractors in microbial community assembly.

For microbiome biology to become a more predictive science, we must identify which descriptive features of microbial communities are reproducible and predictable, which are not, and why. We address this question by experimentally studying parallelism and convergence in microbial community assembly in replicate glucose-limited habitats. Here, we show that the previously observed family-level convergence in these habitats reflects a reproducible metabolic organization, where the ratio of the dominant metabolic groups can be explained from a simple resource-partitioning model. In turn, taxonomic divergence among replicate communities arises from multistability in population dynamics. Multistability can also lead to alternative functional states in closed ecosystems but not in metacommunities. Our findings empirically illustrate how the evolutionary conservation of quantitative metabolic traits, multistability, and the inherent stochasticity of population dynamics, may all conspire to generate the patterns of reproducibility and variability at different levels of organization that are commonplace in microbial community assembly.

Engineering complex communities by directed evolution.

Directed evolution has been used for decades to engineer biological systems at or below the organismal level. Above the organismal level, a small number of studies have attempted to artificially select microbial ecosystems, with uneven and generally modest success. Our theoretical understanding of artificial ecosystem selection is limited, particularly for large assemblages of asexual organisms, and we know little about designing efficient methods to direct their evolution. Here, we have developed a flexible modelling framework that allows us to systematically probe any arbitrary selection strategy on any arbitrary set of communities and selected functions. By artificially selecting hundreds of in silico microbial metacommunities under identical conditions, we first show that the main breeding methods used to date, which do not necessarily let communities reach their ecological equilibrium, are outperformed by a simple screen of sufficiently mature communities. We then identify a range of alternative directed evolution strategies that, particularly when applied in combination, are well suited for the top-down engineering of large, diverse and stable microbial consortia. Our results emphasize that directed evolution allows an ecological structure-function landscape to be navigated in search of dynamically stable and ecologically resilient communities with desired quantitative attributes.

Directed Evolution of Microbial Communities.

Directed evolution is a form of artificial selection that has been used for decades to find biomolecules and organisms with new or enhanced functional traits. Directed evolution can be conceptualized as a guided exploration of the genotype-phenotype map, where genetic variants with desirable phenotypes are first selected and then mutagenized to search the genotype space for an even better mutant. In recent years, the idea of applying artificial selection to microbial communities has gained momentum. In this article, we review the main limitations of artificial selection when applied to large and diverse collectives of asexually dividing microbes and discuss how the tools of directed evolution may be deployed to engineer communities from the top down. We conceptualize directed evolution of microbial communities as a guided exploration of an ecological structure-function landscape and propose practical guidelines for navigating these ecological landscapes.

Artificially selecting bacterial communities using propagule strategies.

Artificial selection is a promising approach to manipulate microbial communities. Here, we report the outcome of two artificial selection experiments at the microbial community level. Both used "propagule" selection strategies, whereby the best-performing communities are used as the inocula to form a new generation of communities. Both experiments were contrasted to a random selection control. The first experiment used a defined set of strains as the starting inoculum, and the function under selection was the amylolytic activity of the consortia. The second experiment used multiple soil communities as the starting inocula, and the function under selection was the communities' cross-feeding potential. In both experiments, the selected communities reached a higher mean function than the control. In the first experiment, this was caused by a decline in function of the control, rather than an improvement of the selected line. In the second experiment, this response was fueled by the large initial variance in function across communities, and stopped when the top-performing community "fixed" in the metacommunity. Our results are in agreement with basic expectations from breeding theory, pointing to some of the limitations of community-level selection experiments that can inform the design of future studies.

Generation and characterization of avian-derived anti-human CD19 single chain fragment antibodies.

The human cluster of differentiation 19 (CD19) is highly expressed in most leukemia, rendering is a promising therapeutic target. In this study, we generated anti-CD19 single-chain variable fragments (scFv) from immunized chickens by phage display technology. After constructing a scFv antibody library with 2.5 × 108 compositional diversity for panning, one representative scFv clone S2 which can specifically recognize to the CD19 protein was isolated and characterized. The binding reactivity of the scFv S2 to the endogenous CD19 protein of the ARH-77 leukemia cancer cell was verified through flow cytometry and the binding affinity of scFv S2 is 6.9 × 10-8 M determined by the surface plasmon resonance system. Compared with the chicken germline, hyper mutation in the complementarity-determining regions (CDRs) suggested that scFv S2 could be generated through an antigen-driven humoral response. By molecular modeling, the possible CDR configurations of scFv S2 were constructed rationally. Furthermore, the characteristics of chicken antibodies of a protein database were investigated. The findings in this study contribute to antibody development and engineering because they reveal the geometric structures and properties of the CDRs in chicken antibodies.

Interaction of S17 Antibody with the Functional Binding Region of the Hepatitis B Virus Pre-S2 Epitope.

To understand the mechanism for inhibition of hepatitis B virus (HBV) infection is important. In this study, single-chain variable fragment (scFv) antibodies were generated and directed to the pre-S2 epitope of HBV surface antigen (HBsAg). These human scFvs were isolated from a person with history of HBV infection by phage display technology. An evaluation of panning efficiency revealed that the eluted phage titer was increased, indicating that specific clones were enriched after panning. Selected scFvs were characterized with the recombinant HBsAg through Western blotting and enzyme-linked immunosorbent assay to confirm the binding ability. Flow cytometry analysis and immunocytochemical staining revealed that one scFv, S17, could recognize endogenous HBsAg expressed on the HepG2215 cell membrane. Moreover, the binding affinity of scFv S17 to the pre-S2 epitope was determined to be 4.2 × 10-8 M. Two ion interactions were observed as the major driving forces for scFv S17 interacting with pre-S2 by performing a rational molecular docking analysis. This study provides insights into the structural basis to understand the interactions between an antibody and the pre-S2 epitope. The functional scFv format can potentially be used in future immunotherapeutic applications.

Discovery of Potent Cysteine-Containing Dipeptide Inhibitors against Tyrosinase: A Comprehensive Investigation of 20 × 20 Dipeptides in Inhibiting Dopachrome Formation.

Tyrosinase is an essential copper-containing enzyme required for melanin synthesis. The overproduction and abnormal accumulation of melanin cause hyperpigmentation and neurodegenerative diseases. Thus, tyrosinase is promising for use in medicine and cosmetics. Our previous study identified a natural product, A5, resembling the structure of the dipeptide WY and apparently inhibiting tyrosinase. Here, we comprehensively estimated the inhibitory capability of 20 × 20 dipeptides against mushroom tyrosinase. We found that cysteine-containing dipeptides, directly blocking the active site of tyrosinase, are highly potent in inhibition; in particular, N-terminal cysteine-containing dipeptides markedly outperform the C-terminal-containing ones. The cysteine-containing dipeptides, CE, CS, CY, and CW, show comparative bioactivities, and tyrosine-containing dipeptides are substrate-like inhibitors. The dipeptide PD attenuates 16.5% melanin content without any significant cytotoxicity. This study reveals the functional role of cysteine residue positional preference and the selectivity of specific amino acids in cysteine-containing dipeptides against tyrosinase, aiding in developing skin-whitening products.

Aptamer-conjugated polymeric nanoparticles for the detection of cancer cells through "turn-on" retro-self-quenched fluorescence.

We have developed a simple, sensitive, and rapid fluorescence assay for the detection of cancer cells, based on "turn-on" retro-self-quenched fluorescence inside the cells. 1,3-Phenylenediamine resin (DAR) nanoparticles (NPs) containing rhodamine 6G (R6G) are conjugated with aptamer (apt) sgc8c to prepare sgc8c-R6GDAR NPs, while that containing rhodamine 101 (R101) are conjugated with TD05 for the preparation of TD05-R101DAR NPs. The sgc8c-R6GDAR and TD05-R101DAR NPs separately recognize CCRF-CEM and Ramos cells. The fluorescence intensities of the two apt-DAR NPs are both weak due to self-quenching, but they increase inside the cells as a result of release of the fluorophores from the apt-DAR NPs. The apt-DAR NPs' structure becomes less compact at low pH value, leading to the release of the fluorophores. The sgc8c-R6GDAR and TD05-R101DAR NPs allow detection of as low as 44 CCRF-CEM cells and 79 Ramos cells mL(-1), respectively, using a commercial reader within 10 min. Practicality of the two probes have been validated by the quantitation and identification of CCRF-CEM and Ramos cells spiked in blood samples through conventional fluorescence and flow cytometry analysis, with advantages of sensitivity, selectivity, and rapidity.

Single chain antibody fragment with serine protease inhibitory property capable of neutralizing toxicity of Trimeresurus mucrosquamatus venom.

Trimeresurus mucrosquamatus (TM) is one of majorities of snake envenomation with necrotic and hemorrhagic toxin in Taiwan. In this study, chickens were used as an alternative animal model for immunization with TM venom. Using phage display technology to process four rounds of panning, selected single chain variable fragments (scFv) could specifically recognize TM venom proteins, which were later identified as a group of homogeneous venom serine protease. The specific scFv antibodies showed various inhibitory effects on sheep RBC lysis induced by TM venom using an indirect hemolytic assay in vitro. In addition, the survival times of mice were extended to certain degrees when treated with these scFv antibodies individually or in a combination. To elucidate the inhibitory mechanism, we used molecular modeling to build up the serine protease structure to simulate the possible interactions with scFv antibodies. The results suggested that the CDR-loop of the scFv antibodies (3S10 or 4S1) might bind at the 99-loop of venom serine protease so as to affect substrate access due to the partial collapse of the subsite S2 and the partial movement of the subsite S4. It is hoped these chicken-derived antibodies could be applied to develop diagnostic and therapeutic agents against snakebites.