Diverse and specialized metabolic capabilities of microbes in oligotrophic built environments.

BACKGROUND: Built environments (BEs) are typically considered to be oligotrophic and harsh environments for microbial communities under normal, non-damp conditions. However, the metabolic functions of microbial inhabitants in BEs remain poorly understood. This study aimed to shed light on the functional capabilities of microbes in BEs by analyzing 860 representative metagenome-assembled genomes (rMAGs) reconstructed from 738 samples collected from BEs across the city of Hong Kong and from the skin surfaces of human occupants. The study specifically focused on the metabolic functions of rMAGs that are either phylogenetically novel or prevalent in BEs. RESULTS: The diversity and composition of BE microbiomes were primarily shaped by the sample type, with Micrococcus luteus and Cutibacterium acnes being prevalent. The metabolic functions of rMAGs varied significantly based on taxonomy, even at the strain level. A novel strain affiliated with the Candidatus class Xenobia in the Candidatus phylum Eremiobacterota and two novel strains affiliated with the superphylum Patescibacteria exhibited unique functions compared with their close relatives, potentially aiding their survival in BEs and on human skins. The novel strains in the class Xenobia possessed genes for transporting nitrate and nitrite as nitrogen sources and nitrosative stress mitigation induced by nitric oxide during denitrification. The two novel Patescibacteria strains both possessed a broad array of genes for amino acid and trace element transport, while one of them carried genes for carotenoid and ubiquinone biosynthesis. The globally prevalent M. luteus in BEs displayed a large and open pangenome, with high infraspecific genomic diversity contributed by 11 conspecific strains recovered from BEs in a single geographic region. The versatile metabolic functions encoded in the large accessory genomes of M. luteus may contribute to its global ubiquity and specialization in BEs. CONCLUSIONS: This study illustrates that the microbial inhabitants of BEs possess metabolic potentials that enable them to tolerate and counter different biotic and abiotic conditions. Additionally, these microbes can efficiently utilize various limited residual resources from occupant activities, potentially enhancing their survival and persistence within BEs. A better understanding of the metabolic functions of BE microbes will ultimately facilitate the development of strategies to create a healthy indoor microbiome. Video Abstract.

Estrogen receptor-negative/progesterone receptor-positive breast cancer has distinct characteristics and pathologic complete response rate after neoadjuvant chemotherapy.

PURPOSE: The status of hormone receptors (HR) is an independent factor affecting survival and chemotherapy sensitivity in breast cancer (BC) patients, with estrogen receptor (ER) and progesterone receptor (PR) having the most significant effects. The ER-/PR + phenotype has been controversial in BC, and experts will face many challenges in determining treatment strategies. Herein, we systematically analyzed the clinicopathological characteristics of the ER-/PR + phenotype in BC patients and the response to chemotherapy. PATIENTS AND METHODS: We included two cohorts. The first cohort counted the relationship between clinicopathologic data and survival outcomes for 72,666 female patients in the Surveillance, Epidemiology, and End Results (SEER) database. The second cohort analyzed the relationship between clinicopathological data and pathologic complete response (pCR) rate in 879 patients at the Harbin Medical University Cancer Hospital. The classification data were compared by the chi-square test and Fister's exact test of the Logistic regression model, and predictor variables with P < 0.05 in the univariate analysis were included in the multivariate regression analysis. The Kaplan-Meier method evaluated breast cancer-specific survival (BCSS) and overall survival (OS) to investigate the relationship between different HR typing and survival and pCR. RESULTS: In the two cohorts, 704 (0.9%) and 11 (1.3%) patients had the ER-/PR + phenotype, respectively. The clinicopathologic features of patients with the ER-/PR + phenotype are more similar to those of the ER-/PR- phenotype. The ER-/PR + phenotype is more common in younger and premenopausal women, and most ER-/PR + phenotypes exhibit higher histological grades. Survival analysis showed that there were significant differences in OS and BCSS among patients with different HR states (P < 0.001). The survival results of patients with the ER + /PR + phenotype were the best. The prognosis of the ER-/PR + phenotype was similar to that of the ER-/PR- phenotype. On the other hand, we found that HR status was also an independent predictor of post-NAC pCR rate in BC patients. The ER + /PR- and ER-/PR- phenotypes were more sensitive to chemotherapy than the ER + /PR + phenotypes. CONCLUSION: HR status is the main factor affecting BC's survival outcome and pCR rate. Patients with the ER-/PR + phenotype possess more aggressive biological factors and can benefit significantly from chemotherapy. We need to pay more attention to this group and achieve individualized treatment, which will help us treat BC better and provide new targets and blueprints for our clinical treatment.

Clinical characteristics and pathologic complete response (pCR) rate after neoadjuvant chemotherapy in postpartum women with breast cancer.

PURPOSE: Breast cancer (BC) is currently the leading cause of death in women worldwide. Studies have confirmed that pregnancy is an independent factor affecting the survival of BC patients. BC found during pregnancy, lactation, or shortly after delivery is what we used to think of as pregnancy-associated breast cancer (PABC). The current expert definition of this concept is not uniform; however, there is growing evidence that postpartum breast cancer (PPBC) differs from other types of BC in terms of both biological features and prognosis, with a slightly different focus on diagnosis and treatment. With the increase of female reproductive age population and changes in fertility policies in China, patients with PPBC are receiving increasing attention. Here, we systematically analyzed the clinicopathological characteristics and chemotherapeutic response of patients with PPBC. We retrospectively analyzed the clinicopathological data, molecular subtypes, chemotherapy regimens, and pathological complete remission (pCR) rates of 1343 patients with non-metastatic BC at Harbin Medical University Cancer Hospital from January 1, 2012 to May 31, 2023. The categorical data were compared by chi-square test and Fisher exact test using logistic regression model. Predictor variables with P < 0.05 in the univariate analysis were included in the multivariate regression analysis to investigate the relationship between different age groups and pCR. RESULTS: A total of 714 patients were eligible for analysis in this study, and 667 patients had a history of pregnancy, 40 (5.6%) of whom were PPBC patients. When diagnosed with BC, patients with PPBC were younger, more likely to undergo breast-conserving surgery (BCS), and more likely to achieve pCR (P < 0.05). In molecular typing, human epidermal growth factor receptor 2 (HER-2)-positive and triple-negative breast cancer (TNBC) were more frequent. In the entire cohort, HER-2 expression and delivery status were independent predictors of pCR rates in BC patients after neoadjuvant chemotherapy (NAC). CONCLUSION: Our findings suggest that postpartum status is an independent predictor of pCR attainment in BC patients. PPBC is more sensitive to chemotherapy than other patients.We need to pay more attention to this group and achieve individualized treatment, which will help us treat BC better and provide new targets and blueprints for our clinical therapy.

Autonomous self-burying seed carriers for aerial seeding.

Aerial seeding can quickly cover large and physically inaccessible areas1 to improve soil quality and scavenge residual nitrogen in agriculture2, and for postfire reforestation3-5 and wildland restoration6,7. However, it suffers from low germination rates, due to the direct exposure of unburied seeds to harsh sunlight, wind and granivorous birds, as well as undesirable air humidity and temperature1,8,9. Here, inspired by Erodium seeds10-14, we design and fabricate self-drilling seed carriers, turning wood veneer into highly stiff (about 4.9 GPa when dry, and about 1.3 GPa when wet) and hygromorphic bending or coiling actuators with an extremely large bending curvature (1,854 m-1), 45 times larger than the values in the literature15-18. Our three-tailed carrier has an 80% drilling success rate on flat land after two triggering cycles, due to the beneficial resting angle (25°-30°) of its tail anchoring, whereas the natural Erodium seed's success rate is 0%. Our carriers can carry payloads of various sizes and contents including biofertilizers and plant seeds as large as those of whitebark pine, which are about 11 mm in length and about 72 mg. We compare data from experiments and numerical simulation to elucidate the curvature transformation and actuation mechanisms to guide the design and optimization of the seed carriers. Our system will improve the effectiveness of aerial seeding to relieve agricultural and environmental stresses, and has potential applications in energy harvesting, soft robotics and sustainable buildings.

Label-free focusing of viral particles under a temperature gradient coupled with continuous swirling flow.

The advances of biomedicine and biotechnology demand new approaches to enrich biological nanoparticles, such as viruses, viral vectors and nanovesicles, in an easy-to-operate fashion. Conventional methods, such as ultracentrifugation and ultrafiltration, require bulky instruments and extensive manual operation. Inspired by recent research of thermophoresis of biomolecules and bio-nanoparticles in aqueous solutions, we present a microfluidic design that directly focuses nanoparticles in a label-free and flow-through process by coupling an engineered swirling flow and a moderate, one-dimensional temperature gradient. Enrichment of polystyrene particles, HIV and bacteriophage samples was quantitatively determined, indicating the compatibility of the microfluidic approach with synthetic and biological samples. The focusing results are well predicted using a numerical model. As thermophoresis is ubiquitous, the microfluidic approach can be applied broadly to bio-nanoparticle enrichment without the necessity of labeling, buffer exchange, or sheath fluids, permitting continuous retrieval of concentrated species in a simple, controlled flow with little infrastructure needs.

Morphing pasta and beyond.

Morphing structures are often engineered with stresses introduced into a flat sheet by leveraging structural anisotropy or compositional heterogeneity. Here, we identify a simple and universal diffusion-based mechanism to enable a transient morphing effect in structures with parametric surface grooves, which can be realized with a single material and fabricated using low-cost manufacturing methods (e.g., stamping, molding, and casting). We demonstrate from quantitative experiments and multiphysics simulations that parametric surface grooving can induce temporary asynchronous swelling or deswelling and can transform flat objects into designed, three-dimensional shapes. By tuning the grooving pattern, we can achieve both zero (e.g., helices) and nonzero (e.g., saddles) Gaussian curvature geometries. This mechanism allows us to demonstrate approaches that could improve the efficiency of certain food manufacturing processes and facilitate the sustainable packaging of food, for instance, by creating morphing pasta that can be flat-packed to reduce the air space in the packaging.

Population differentiation of Rhodobacteraceae along with coral compartments.

Coral mucus, tissue, and skeleton harbor compositionally different microbiota, but how these coral compartments shape the microbial evolution remains unexplored. Here, we sampled bacteria inhabiting a prevalent coral species Platygyra acuta and sequenced genomes of 234 isolates comprising two populations in Rhodobacteraceae, an alphaproteobacterial lineage representing a significant but variable proportion (5-50%) of the coral microbiota. The Ruegeria population (20 genomes) contains three clades represented by eight, six, and six isolates predominantly sampled from the skeleton (outgroup), mucus (clade-M), and skeleton (clade-S), respectively. The clade-M possesses functions involved in the utilization of coral osmolytes abundant in the mucus (e.g., methylamines, DMSP, taurine, and L-proline), whereas the clade-S uniquely harbors traits that may promote adaptation to the low-energy and diurnally anoxic skeleton (e.g., sulfur oxidation and swimming motility). These between-clade genetic differences were largely supported by physiological assays. Expanded analyses by including genomes of 24 related isolates (including seven new genomes) from other marine environments suggest that clade-M and clade-S may have diversified in non-coral habitats, but they also consolidated a key role of distinct coral compartments in diversifying many of the above-mentioned traits. The unassigned Rhodobacteraceae population (214 genomes) varies only at a few dozen nucleotide sites across the whole genomes, but the number of between-compartment migration events predicted by the Slatkin-Maddison test supported that dispersal limitation between coral compartments is another key mechanism diversifying microbial populations. Collectively, our results suggest that different coral compartments represent ecologically distinct and microgeographically separate habitats that drive the evolution of the coral microbiota.

Gene loss through pseudogenization contributes to the ecological diversification of a generalist Roseobacter lineage.

Ecologically relevant genes generally show patchy distributions among related bacterial genomes. This is commonly attributed to lateral gene transfer, whereas the opposite mechanism-gene loss-has rarely been explored. Pseudogenization is a major mechanism underlying gene loss, and pseudogenes are best characterized by comparing closely related genomes because of their short life spans. To explore the role of pseudogenization in microbial ecological diversification, we apply rigorous methods to characterize pseudogenes in the 279 newly sequenced Ruegeria isolates of the globally abundant Roseobacter group collected from two typical coastal habitats in Hong Kong, the coral Platygyra acuta and the macroalga Sargassum hemiphyllum. Pseudogenes contribute to ~16% of the accessory genomes of these strains. Ancestral state reconstruction reveals that many pseudogenization events are correlated with ancestral niche shifts. Specifically, genes related to resource scavenging and energy acquisition were often pseudogenized when roseobacters inhabiting carbon-limited and energy-poor coral skeleton switched to other resource-richer niches. For roseobacters inhabiting the macroalgal niches, genes for nitrogen regulation and carbohydrate utilization were important but became dispensable upon shift to coral skeleton where nitrate is abundant but carbohydrates are less available. Whereas low-energy-demanding secondary transporters are more favorable in coral skeleton, ATP-driven primary transporters are preferentially kept in the energy-replete macroalgal niches. Moreover, a large proportion of these families mediate organismal interactions, suggesting their rapid losses by pseudogenization as a potential response to host and niche shift. These findings illustrate an important role of pseudogenization in shaping genome content and driving ecological diversification of marine roseobacters.

Ancestral niche separation and evolutionary rate differentiation between sister marine flavobacteria lineages.

Marine flavobacteria are specialists for polysaccharide degradation. They dominate in habitats enriched with polysaccharides, but are also prevalent in pelagic environments where polysaccharides are less available. These niches are likely occupied by distinct lineages, but evolutionary processes underlying their niche differentiation remain elusive. Here, genomic analyses and physiological assays indicate that the sister flavobacteria lineages Leeuwenhoekiella and Nonlabens likely explore polysaccharide-rich macroalgae and polysaccharide-poor pelagic niches respectively. Phylogenomic analyses inferred that the niche separation likely occurred anciently and coincided with increased sequence evolutionary rate in Nonlabens compared with Leeuwenhoekiella. Further analyses ruled out the known mechanisms likely driving evolutionary rate acceleration, including reduced selection efficiency, decreased generation time and increased mutation rate. In particular, the mutation rates were determined using an unbiased experimental method, which measures the present-day populations and may not reflect ancestral populations. These data collectively lead to a new hypothesis that an ancestral and transient mutation rate increase resulted in evolutionary rate increase in Nonlabens. This hypothesis was supported by inferring that gains and losses of genes involved in SOS response, a mechanism known to drive transiently increased mutation rate, coincided with evolutionary rate acceleration. Our analyses highlight the evolutionary mechanisms underlying niche differentiation of flavobacteria lineages.

Comparative transcriptome analysis explores maternal to zygotic transition during Eriocheir sinensis early embryogenesis.

The maternal genome directs almost all aspects of early animal development. As development proceeds, the elimination of maternal gene products and zygotic genome activation (ZGA) occur during the maternal to zygotic transition (MZT). To study the molecular mechanisms regulating this developmental event in Eriocheir sinensis, RNA-Seq technology was applied to generate comprehensive information on transcriptome dynamics during early embryonic stages. In total, 32,088 annotated unigenes were obtained from the transcriptomes of fertilized eggs and embryos at the cleavage (2-4 cell) and blastula stage. A total of 566 maternal genes and 1165 zygotic genes were isolated, among which 103 and 266 genes were predicted conserved maternal transcripts (COMATs) and conserved zygotic transcripts (COZYTs), respectively. The COMATs performed housekeeping gene functions and may be essential for initiating early embryogenesis of the Bilateria. Furthermore, 87, 76 and 117 differentially expressed genes associated with the MZT, morphogenesis and immunity were identified when compared the three transcriptomic datasets. We also unmask that the MZT takes place around the cleavage stage, when the genes involved in the clearance of maternal gene products and the ZGA were significantly up-regulated. Taken together, these datasets provide a valuable resource for understanding the mechanisms of early developmental events in E. sinensis, and facilitate further studies on molecular mechanisms of asynchronous development in crabs.

Individual Nanoporous Carbon Spheres with High Nitrogen Content from Polyacrylonitrile Nanoparticles with Sacrificial Protective Layers.

Functional nanoporous carbon spheres (NPC-S) are important for applications ranging from adsorption, catalysis, separation to energy storage, and biomedicine. The development of effective NPC-S materials has been hindered by the fusion of particles during the pyrolytic process that results in agglomerated materials with reduced activity. Herein, we present a process that enables the scalable synthesis of dispersed NPC-S materials by coating sacrificial protective layers around polyacrylonitrile nanoparticles (PAN NPs) to prevent interparticle cross-linking during carbonization. In a first step, PAN NPs are synthesized using miniemulsion polymerization, followed by grafting of 3-(triethoxysilyl)propyl methacrylate (TESPMA) to form well-defined core-shell structured PAN@PTESPMA nanospheres. The cross-linked PTESPMA brush layer suppresses cross-linking reactions during carbonization. Uniform NPC-S exhibiting diameters of ∼100 nm, with relatively high accessible surface area (∼424 m2/g), and high nitrogen content (14.8 wt %) was obtained. When compared to a regular nanoporous carbon monolith (NPC-M), the nitrogen-doped NPC-S demonstrated better performance for CO2 capture with a higher CO2/N2 selectivity, an increased efficiency in catalytic oxygen reduction reactions, as well as improved electrochemical capacitive behavior. This miniemulsion polymerization-based strategy for the preparation of functional PAN NPs provides a new, facile approach to prepare high-performance porous carbon spheres for diverse applications.

Photocatalytic Active Mesoporous Carbon/ZnO Hybrid Materials from Block Copolymer Tethered ZnO Nanocrystals.

Severe water pollution issues present an important contemporary challenge that drives the development and advancement of efficient and environmentally benign photocatalysts that enable the degradation of pollutants upon visible light irradiation. One example is zinc oxide/carbon (ZnO/C) hybrid materials that have been shown to be effective photocatalysts. To maximize the effectiveness of ZnO/C hybrids, materials with high accessible surface area of ZnO are required. Here, a novel strategy is presented to enable the synthesis of fine dispersions of ZnO nanoparticles within a porous carbon matrix. The synthesis entails the grafting of ZnO nanparticles with polystyrene-b-poly(styrene-co-acrylonitrile) (PS-b-PSAN) block copolymer and subsequent pyrolysis of the material under inert gas (N2) atmosphere. During the pyrolysis process, the PS block effectively prevents agglomeration of ZnO particles, thus resulting in a fine dispersion of ZnO nanocrystals within a prorous C matrix. Materials are found to exhibit a dye adsorption capacity of 125 mg g-1 (from a methylene blue aqueous solution with a concentration of 305 mg L-1) and dye degradation rate constant of 0.021 min-1. The significant increase of effective surface area and degradation efficacy (as compared to ZnO/C synthesized by the pyrolysis of binary PSAN/ZnO blends) is rationalized as a consequence of the increased porosity that promotes dye adsorption and transport within the hybrid material.

Two spliced isoforms of the sex-determination gene fruitless in the Chinese mitten crab Eriocheir sinensis.

The fruitless (fru) gene plays an important role in the sex determination pathway and courtship behavior of Drosophila melanogaster. In the present study, two fru isoforms (Esfru1 and Esfru2) were identified from the Chinese mitten crab Eriocheir sinensis. Sequence analysis showed that Esfru1 and Esfru2 were encoded by the same genomic locus and generated by alternative splicing of pre-mRNA. Esfru1 had all introns completely spliced out, while Esfru2 had a longer exon1 with an additional 78bp sequence. They both contained a conserved BTB domain which was also found in D. melanogaster Fru isoforms. Analysis on temporal expression profiles of Esfru1-2 (a common region of Esfru1 and Esfru2) and Esfru2 (a specific region of Esfru2) showed that they expressed in similar patterns during embryonic development but in different patterns during larval development. The expression of Esfru1-2 decreased gradually from zoea III stage, however, Esfru2 increased from zoea IV stage and reached the peak at megalopa stage. Expression distribution in tissues and in situ hybridization analysis revealed that they showed sexually dimorphic expression in gonads, hepatopancreas and brains. Esfru1-2 showed significantly higher expression in female gonads and hepatopancreas than in males, but highly expressed in male brains than that in females. Interestingly, Esfru2 was displayed in a male-specific manner in each tissue, especially in gonads and hepatopancreas. Our results indicate that Esfru1 might be involved in both sexual brain neuronal structure development and related to female-specific character development. Esfru2 may participate in male-specific character development. This is the first report that characterizes two spliced variants of fru in crustaceans and provides basic information for further functional studies of the crab sex-determination mechanism.

PECTIN METHYLESTERASE34 Contributes to Heat Tolerance through Its Role in Promoting Stomatal Movement.

Pectin, a major component of the primary cell wall, is synthesized in the Golgi apparatus and exported to the cell wall in a highly methylesterified form, then is partially demethylesterified by pectin methylesterases (PMEs; EC 3.1.1.11). PME activity on the status of pectin methylesterification profoundly affects the properties of pectin and, thereby, is critical for plant development and the plant defense response, although the roles of PMEs under heat stress (HS) are poorly understood. Functional genome annotation predicts that at least 66 potential PME genes are contained in Arabidopsis (Arabidopsis thaliana). Thermotolerance assays of PME gene T-DNA insertion lines revealed two null mutant alleles of PME34 (At3g49220) that both consistently showed reduced thermotolerance. Nevertheless, their impairment was independently associated with the expression of HS-responsive genes. It was also observed that PME34 transcription was induced by abscisic acid and highly expressed in guard cells. We showed that the PME34 mutation has a defect in the control of stomatal movement and greatly altered PME and polygalacturonase (EC 3.2.1.15) activity, resulting in a heat-sensitive phenotype. PME34 has a role in the regulation of transpiration through the control of the stomatal aperture due to its cell wall-modifying enzyme activity during the HS response. Hence, PME34 is required for regulating guard cell wall flexibility to mediate the heat response in Arabidopsis.

Transparent and High Refractive Index Thermoplastic Polymer Glasses Using Evaporative Ligand Exchange of Hybrid Particle Fillers.

Development of high refractive index glasses on the basis of commodity polymer thermoplastics presents an important requisite to further advancement of technologies ranging from energy efficient lighting to cost efficient photonics. This contribution presents a novel particle dispersion strategy that enables uniform dispersion of zinc oxide (ZnO) particles in a poly(methyl methacrylate) (PMMA) matrix to facilitate hybrid glasses with inorganic content exceeding 25% by weight, optical transparency in excess of 0.8/mm, and a refractive index greater than 1.64 in the visible wavelength range. The method is based on the application of evaporative ligand exchange to synthesize poly(styrene-r-acrylonitrile) (PSAN)-tethered zinc oxide (ZnO) particle fillers. Favorable filler-matrix interactions are shown to enable the synthesis of isomorphous blends with high molecular PMMA that exhibit improved thermomechanical stability compared to that of the pristine PMMA matrix. The concurrent realization of high refractive index and optical transparency in polymer glasses by modification of a thermoplastic commodity polymer could present a viable alternative to expensive specialty polymers in applications where high costs or demands for thermomechanical stability and/or UV resistance prohibit the application of specialty polymer solutions.

Preparation of Well-Defined Poly(styrene-co-acrylonitrile)/ZnO Hybrid Nanoparticles by an Efficient Ligand Exchange Strategy.

Poly(styrene-co-acrylonitrile) (PSAN)-capped ZnO nanoparticles (NPs) were synthesized by a "ligand exchange" method. First, octylamine (OA)-capped ZnO NPs were prepared by reaction of OA and zinc 2-ethylhexanoate (Zn(EH)2). Then PSAN polymer ligands were synthesized by activators regenerated by electron transfer (ARGET) atom transfer radical polymerization (ATRP) and were efficiently exchanged with OA ligands on the ZnO particle surface benefiting from the relatively low boiling point of OA (175 °C). The morphology, content of ZnO, and grafting density of the nanocomposite were well controlled by altering the ratio between OA and polymer ligands as well as the molecular weight of PSAN-NH2 used in the exchange reaction. The resulting ZnO/polymer nanocomposites were stable in THF with narrow size distributions and varying grafting densities from 0.9 to 2.5 nm-2. With excess amount of polymer ligands, individual dispersed ZnO NPs were observed. However, with a limited amount of ligands, NPs clusters were formed, as confirmed by TEM and DLS.

Comparative Transcriptome Analysis Reveals Sex-Biased Gene Expression in Juvenile Chinese Mitten Crab Eriocheir sinensis.

Sex-biased genes are considered to account for most of phenotypic differences between males and females. In order to explore the sex-biased gene expression in crab, we performed the whole-body transcriptome analysis in male and female juveniles of the Chinese mitten crab Eriocheir sinensis using next-generation sequencing technology. Of the 23,349 annotated unigenes, 148 were identified as sex-related genes. A total of 29 candidate genes involved in primary sex determination pathways were detected, indicating the sex determination cascade of the mitten crab might be more complex than previously supposed. Differential expression analysis showed 448 differentially expressed genes (DEGs) between the two transcriptomes. Most of DEGs were involved in processes such as metabolism and immunity, and not associated with obvious sexual function. The pathway predominantly enriched for DEGs were related to lysosome, which might reflect the differences in metabolism between males and females. Of the immune DGEs, 18 up-regulated genes in females were humoral immune factors, and eight up-regulated genes in males were pattern recognition receptors, suggesting sex differences of immune defense might exist in the mitten crab. In addition, two reproduction-related genes, vitellogenin and insulin-like androgenic gland factor, were identified to express in both sexes but with significantly higher level in males. Our research provides the first whole-body RNA sequencing of sex-specific transcriptomes for juvenile E. sinensis and will facilitate further studies on molecular mechanisms of crab sexual dimorphism.

PtPLC, a pacifastin-related inhibitor involved in antibacterial defense and prophenoloxidase cascade of the swimming crab Portunus trituberculatus.

Pacifastin-related inhibitor is a new family of serine protease inhibitors that regulate the proteolytic cascade in multiple biological processes. Contrary to the knowledge on the structure and inhibitory mechanism of pacifastin-like members in locust, very little is known about their functions. Here, we report the inhibitory activities in relation to the structural characteristics of pacifastin light chain (PtPLC) gene identified from the swimming crab Portunus trituberculatus. The mature PtPLC and five PLD-related domains with critical residues were expressed in Escherichia coli, and assayed for their activities. The recombinant PtPLC (rPtPLC) displayed inhibitory activities against trypsin and chymotrypsin in a dose dependent manner, with a preference for trypsin. Except for rPtPLC-D4, the other four rPtPLC-related domains could inhibit at least one of serine proteases. The enzyme specificity of PtPLC domains generally corresponded to the nature of the P1 residue at the reactive site. rPtPLC was able to inhibit the growth of Gram-negative bacteria Vibrio alginolyticus and Pseudomonas aeruginosa, but not the Gram-positive bacterium and fungus tested. Further phenoloxidase (PO) assay showed the rPtPLC could depress the crab proPO system activation in vitro, and lead to 72.8% inhibition of PO activity at the concentration of 9.11 µM. It also suppressed proPO activation induced by rPtcSP and rPtSPH1. As the first functional study of the recombinant PLC protein in crustaceans, the present results together indicate that PtPLC functions in the crab immune response possibly via inhibiting bacterial growth and regulating the proPO system.

PtSerpin from the swimming crab Portunus trituberculatus, a putative regulator of prophenoloxidase activation with antibacterial activity.

Serpin or serine protease inhibitor is the largest family of protease inhibitors involved in many innate immune pathways, particularly the prophenoloxidase (proPO) activating system in arthropod. Here, we report the molecular and functional characterization of PtSerpin identified from the swimming crab Portunus trituberculatus. The genomic sequence encoding mature peptide of PtSerpin gene contained two exons of 84 and 1098 bp separated by one intron of 111 bp. The recombinant PtSerpin (rPtSerpin) with a predicted size of 44 kDa was expressed in Escherichia coli system, purified and assayed for its activities. The rPtSerpin exhibited inhibitory activity against trypsin in a dose-dependent manner, but did not affect chymotrypsin, which could define a role for PtSerpin as a trypsin inhibitor. The rPtSerpin could inhibit the growth of Gram-negative bacterium Vibrio alginolyticus, but not the tested Gram-positive bacterium and fungus. Further phenoloxidase (PO) assay showed PO activity was dramatically increased in hemocyte lysate supernatant of P. trituberculatus upon bacterial challenge. The rPtSerpin could depress the crab proPO system activation in vitro, and it could lead to 100% inhibition of PO activity under the concentration of 8.62 µM. Moreover, the rPtSerpin was able to inhibit the PO activity induced by rPtcSP and rPtSPH1. These results together indicate that PtSerpin is a potential trypsin inhibitor and may participate in crab innate immunity by the inhibition of bacterial growth and the regulation of proPO system.

Transcriptome profiling analysis on whole bodies of microbial challenged Eriocheir sinensis larvae for immune gene identification and SNP development.

To study crab immunogenetics of individuals, newly hatched Eriocheir sinensis larvae were stimulated with a mixture of three pathogen strains (Gram-positive bacteria Micrococcus luteus, Gram-negative bacteria Vibrio alginolyticus and fungi Pichia pastoris; 10(8) cfu·mL(-1)). A total of 44,767,566 Illumina clean reads corresponding to 4.52 Gb nucleotides were generated and assembled into 100,252 unigenes (average length: 1,042 bp; range: 201-19,357 bp). 17,097 (26.09%) of 65,535 non-redundant unigenes were annotated in NCBI non-redundant protein (Nr) database. Moreover, 23,188 (35.38%) unigenes were assigned to three Gene Ontology (GO) categories, 15,071 (23.00%) to twenty-six Clusters of orthologous Groups (COG) and 8,574 (13.08%) to six Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. Numerous genes were further identified to be associated with multiple immune pathways, including Toll, immune deficiency (IMD), janus kinase (JAK)-signal transducers and activators of transcription (STAT) and mitogen-activated protein kinase (MAPK) pathways. Some of them, such as tumor necrosis factor receptor associated factor 6 (TRAF6), fibroblast growth factor (FGF), protein-tyrosine phosphatase (PTP), JNK-interacting protein 1 (JIP1), were first identified in E. sinensis. TRAF6 was even first discovered in crabs. Additionally, 49,555 single nucleotide polymorphisms (SNPs) were developed from over 13,309 unigenes. This is the first transcriptome report of whole bodies of E. sinensis larvae after immune challenge. Data generated here not only provide detail information to identify novel genes in genome reference-free E. sinensis, but also facilitate our understanding on host immunity and defense mechanism of the crab at whole transcriptome level.