Neural network based integration of assays to assess pathogenic potential.

Limited data significantly hinders our capability of biothreat assessment of novel bacterial strains. Integration of data from additional sources that can provide context about the strain can address this challenge. Datasets from different sources, however, are generated with a specific objective and which makes integration challenging. Here, we developed a deep learning-based approach called the neural network embedding model (NNEM) that integrates data from conventional assays designed to classify species with new assays that interrogate hallmarks of pathogenicity for biothreat assessment. We used a dataset of metabolic characteristics from a de-identified set of known bacterial strains that the Special Bacteriology Reference Laboratory (SBRL) of the Centers for Disease Control and Prevention (CDC) has curated for use in species identification. The NNEM transformed results from SBRL assays into vectors to supplement unrelated pathogenicity assays from de-identified microbes. The enrichment resulted in a significant improvement in accuracy of 9% for biothreat. Importantly, the dataset used in our analysis is large, but noisy. Therefore, the performance of our system is expected to improve as additional types of pathogenicity assays are developed and deployed. The proposed NNEM strategy thus provides a generalizable framework for enrichment of datasets with previously collected assays indicative of species.

Antibody therapy reverses biological signatures of COVID-19 progression.

Understanding who is at risk of progression to severe coronavirus disease 2019 (COVID-19) is key to clinical decision making and effective treatment. We study correlates of disease severity in the COMET-ICE clinical trial that randomized 1:1 to placebo or to sotrovimab, a monoclonal antibody for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (ClinicalTrials.gov04545060). Laboratory parameters identify study participants at greater risk of severe disease, including a high neutrophil-to-lymphocyte ratio (NLR), a negative SARS-CoV-2 serologic test, and whole-blood transcriptome profiles. Sotrovimab treatment is associated with normalization of NLR and the transcriptomic profile and with a decrease of viral RNA in nasopharyngeal samples. Transcriptomics provides the most sensitive detection of participants who would go on to be hospitalized or die. To facilitate timely measurement, we identify a 10-gene signature with similar predictive accuracy. We identify markers of risk for disease progression and demonstrate that normalization of these parameters occurs with antibody treatment of established infection.

A toolkit for enhanced reproducibility of RNASeq analysis for synthetic biologists.

Sequencing technologies, in particular RNASeq, have become critical tools in the design, build, test and learn cycle of synthetic biology. They provide a better understanding of synthetic designs, and they help identify ways to improve and select designs. While these data are beneficial to design, their collection and analysis is a complex, multistep process that has implications on both discovery and reproducibility of experiments. Additionally, tool parameters, experimental metadata, normalization of data and standardization of file formats present challenges that are computationally intensive. This calls for high-throughput pipelines expressly designed to handle the combinatorial and longitudinal nature of synthetic biology. In this paper, we present a pipeline to maximize the analytical reproducibility of RNASeq for synthetic biologists. We also explore the impact of reproducibility on the validation of machine learning models. We present the design of a pipeline that combines traditional RNASeq data processing tools with structured metadata tracking to allow for the exploration of the combinatorial design in a high-throughput and reproducible manner. We then demonstrate utility via two different experiments: a control comparison experiment and a machine learning model experiment. The first experiment compares datasets collected from identical biological controls across multiple days for two different organisms. It shows that a reproducible experimental protocol for one organism does not guarantee reproducibility in another. The second experiment quantifies the differences in experimental runs from multiple perspectives. It shows that the lack of reproducibility from these different perspectives can place an upper bound on the validation of machine learning models trained on RNASeq data. Graphical Abstract.

Phenotype-Based Threat Assessment.

Bacterial pathogen identification, which is critical for human health, has historically relied on culturing organisms from clinical specimens. More recently, the application of machine learning (ML) to whole-genome sequences (WGSs) has facilitated pathogen identification. However, relying solely on genetic information to identify emerging or new pathogens is fundamentally constrained, especially if novel virulence factors exist. In addition, even WGSs with ML pipelines are unable to discern phenotypes associated with cryptic genetic loci linked to virulence. Here, we set out to determine if ML using phenotypic hallmarks of pathogenesis could assess potential pathogenic threat without using any sequence-based analysis. This approach successfully classified potential pathogenetic threat associated with previously machine-observed and unobserved bacteria with 99% and 85% accuracy, respectively. This work establishes a phenotype-based pipeline for potential pathogenic threat assessment, which we term PathEngine, and offers strategies for the identification of bacterial pathogens.

MoS2 quantum dots based MoS2/HKUST-1 composites for the highly efficient catalytic oxidation of elementary mercury.

Due to the ever-tightening regulation on mercury emission in recent decades, there is an urgent need to develop novel materials for the removal of elemental mercury at coal-fired power plants. In this study, a series of MoS2 quantum dots (QDs)-based MoS2/HKUST-1 composite materials were prepared. It is found that MoS2 QDs were encapsulated by HKUST-1 and enhanced the crystallinity and specific surface area of HKUST-1. The MoS2/HKUST-1 showed excellent performance in catalytic oxidation of Hg0 as compared with pristine HKUST-1. It is found that surface layer of lattice oxygens is active and participates in Hg0 oxidation, while the consumption of surface oxygens then leads to the formation of oxygen vacancies on the surface. These vacancies are effective in the adsorption and dissociation of O2, which subsequently participates in the oxidation of Hg0. Moreover, the study on the influence of commonly seen gas components, such as SO2, NO, NH3 and H2O, etc., on Hg0 oxidation demonstrated that synergistic effects exist among these gas species. It is found that the presence of NO promotes the oxidation of Hg0 using oxygen as the oxidant.

Establishing Butyribacterium methylotrophicum as a Platform Organism for the Production of Biocommodities from Liquid C1 Metabolites.

Using the Wood-Ljungdahl pathway, acetogens can nonphotosynthetically fix gaseous C1 molecules, preventing them from entering the atmosphere. Many acetogens can also grow on liquid C1 compounds such as formate and methanol, which avoid the storage and mass transfer issues associated with gaseous C1 compounds. Substrate redox state also plays an important role in acetogen metabolism and can modulate products formed by these organisms. Butyribacterium methylotrophicum is an acetogen known for its ability to synthesize longer-chained molecules such as butyrate and butanol, which have significantly higher values than acetate or ethanol, from one-carbon (C1) compounds. We explored B. methylotrophicum's C1 metabolism by varying substrates, substrate concentrations, and substrate feeding strategies to improve four-carbon product titers. Our results showed that formate utilization by B. methylotrophicum favored acetate production and methanol utilization favored butyrate production. Cofeeding of both substrates produced a high butyrate titer of 4 g/liter when methanol was supplied in excess to formate. Testing of formate feeding strategies, in the presence of methanol, led to further increases in the butyrate to acetate ratio. Mixotrophic growth of liquid and gaseous C1 substrates expanded the B. methylotrophicum product profile, as ethanol, butanol, and lactate were produced under these conditions. We also showed that B. methylotrophicum is capable of producing caproate, a six-carbon product, presumably through chain elongation cycles of the reverse ß-oxidation pathway. Furthermore, we demonstrated butanol production via heterologous gene expression. Our results indicate that both selection of appropriate substrates and genetic engineering play important roles in determining titers of desired products. IMPORTANCE Acetogenic bacteria can fix single-carbon (C1) molecules. However, improvements are needed to overcome poor product titers. Butyribacterium methylotrophicum can naturally ferment C1 compounds into longer-chained molecules such as butyrate alongside traditional acetate. Here, we show that B. methylotrophicum can effectively grow on formate and methanol to produce high titers of butyrate. We improved ratios of butyrate to acetate through adjusted formate feeding strategies and produced higher-value six-carbon molecules. We also expanded the B. methylotrophicum product profile with the addition of C1 gases, as the organism produced ethanol, butanol, and lactate. Furthermore, we developed a transformation protocol for B. methylotrophicum to facilitate genetic engineering of this organism for the circular bioeconomy.

RNA Solutions: Synthesizing Information to Support Transcriptomics (RNASSIST).

MOTIVATION: Transcriptomics is a common approach to identify changes in gene expression induced by a disease state. Standard transcriptomic analyses consider differentially expressed genes (DEGs) as indicative of disease states so only a few genes would be treated as signals when the effect size is small, such as in brain tissue. For tissue with small effect sizes, if the DEGs do not belong to a pathway known to be involved in the disease, there would be little left in the transcriptome for researchers to follow up with. RESULTS: We developed RNA Solutions: Synthesizing Information to Support Transcriptomics (RNASSIST), a new approach to identify hidden signals in transcriptomic data by linking differential expression and co-expression networks using machine learning. We applied our approach to RNA-seq data of post-mortem brains that compared the Alcohol Use Disorder (AUD) group with the control group. Many of the candidate genes are not differentially expressed so would likely be ignored by standard transcriptomic analysis pipelines. Through multiple validation strategies, we concluded that these RNASSIST-identified genes likely play a significant role in AUD. AVAILABILITY AND IMPLEMENTATION: The RNASSIST algorithm is available at https://github.com/netrias/rnassist and both the software and the data used in RNASSIST are available at https://figshare.com/articles/software/RNAssist_Software_and_Data/16617250. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genome-Wide Transcription Factor DNA Binding Sites and Gene Regulatory Networks in Clostridium thermocellum.

Clostridium thermocellum is a thermophilic bacterium recognized for its natural ability to effectively deconstruct cellulosic biomass. While there is a large body of studies on the genetic engineering of this bacterium and its physiology to-date, there is limited knowledge in the transcriptional regulation in this organism and thermophilic bacteria in general. The study herein is the first report of a large-scale application of DNA-affinity purification sequencing (DAP-seq) to transcription factors (TFs) from a bacterium. We applied DAP-seq to > 90 TFs in C. thermocellum and detected genome-wide binding sites for 11 of them. We then compiled and aligned DNA binding sequences from these TFs to deduce the primary DNA-binding sequence motifs for each TF. These binding motifs are further validated with electrophoretic mobility shift assay (EMSA) and are used to identify individual TFs' regulatory targets in C. thermocellum. Our results led to the discovery of novel, uncharacterized TFs as well as homologues of previously studied TFs including RexA-, LexA-, and LacI-type TFs. We then used these data to reconstruct gene regulatory networks for the 11 TFs individually, which resulted in a global network encompassing the TFs with some interconnections. As gene regulation governs and constrains how bacteria behave, our findings shed light on the roles of TFs delineated by their regulons, and potentially provides a means to enable rational, advanced genetic engineering of C. thermocellum and other organisms alike toward a desired phenotype.

Simultaneously Detecting Monoamine Oxidase A and B in Disease Cell/Tissue Samples Using Paper-Based Devices.

As enzymes in the outer membrane of the mitochondrion, monoamine oxidases (MAOs) can catalyze the oxidative deamination of monoamines in the human body. According to different substrates, MAOs can be divided into MAO-A and MAO-B. The imbalance of the MAO-A is associated with neurological degeneration, while excess MAO-B activity is closely connected with Parkinson's disease (PD) and Alzheimer's disease (AD); therefore, detection of MAOs is of great significance for the diagnosis and treatment of these diseases. This work reports the multiplexed detection of MAO-A and MAO-B using paper-based devices based on chemiluminescence (CL). The detection limits were 5.01 pg/mL for MAO-A and 8.50 pg/mL for MAO-B in human serum. In addition, we used paper-based devices to detect MAOs in human cells and tissue samples and found that the results of paper-based detection and Western blotting (WB) showed the same trend. While only one antibody can be incubated on the same membrane by WB, multiple antibodies incubated on the same paper enabled simultaneous detection of MAO-A and MAO-B by paper-based devices. The paper-based assay could be used for preliminary early screening of clinical samples for MAOs and can be extended as an alternative to WB for multiplexed detection of various proteins in disease cell or tissue samples.

Global Analysis of Gene Expression Profiles Provides Novel Insights into the Development and Evolution of the Large Crustacean Eriocheir sinensis.

Chinese mitten crab (Eriocheir sinensis) is an important aquaculture species in Crustacea. Functional analysis, although essential, has been hindered due to the lack of sufficient genomic or transcriptomic resources. In this study, transcriptome sequencing was conducted on 59 samples representing diverse developmental stages (fertilized eggs, zoea, megalopa, three sub-stages of larvae, juvenile crabs, and adult crabs) and different tissues (eyestalk, hepatopancreas, and muscle from juvenile crabs, and eyestalk, hepatopancreas, muscle, heart, stomach, gill, thoracic ganglia, intestine, ovary, and testis from adult crabs) of E. sinensis. A comprehensive reference transcriptome was assembled, including 19,023 protein-coding genes. Hierarchical clustering based on 128 differentially expressed cuticle-related genes revealed two distinct expression patterns during the early larval developmental stages, demonstrating the distinct roles of these genes in "crab-like" cuticle formation during metamorphosis and cuticle calcification after molting. Phylogenetic analysis of 1406 one-to-one orthologous gene families identified from seven arthropod species and Caenorhabditis elegans strongly supported the hypothesis that Malacostraca and Branchiopoda do not form a monophyletic group. Furthermore, Branchiopoda is more phylogenetically closely related to Hexapoda, and the clade of Hexapoda and Branchiopoda and the clade of Malacostraca belong to the Pancrustacea. This study offers a high-quality transcriptome resource for E. sinensis and demonstrates the evolutionary relationships of major arthropod groups. The differentially expressed genes identified in this study facilitate further investigation of the cuticle-related gene expression networks which are likely associated with "crab-like" cuticle formation during metamorphosis and cuticle calcification after molting.

Production of H2-Rich Syngas From Lignocellulosic Biomass Using Microwave-Assisted Pyrolysis Coupled With Activated Carbon Enabled Reforming.

This study focuses on the use of a microwave reactor that combines biomass pyrolysis, at mild temperature, with catalytic reforming of the pyrolytic gas, using activated carbon, for generating hydrogen-rich synthesis gas. The traditional pyrolysis of biomass coupled with the reforming of its pyrolytic yields were also conducted using an electrically heated reactor. The bio-oil attained from conventional pyrolysis was higher in comparison to the yield from microwave pyrolysis. The reforming of the pyrolytic gas fraction led to reductions in bio-oil yield to <3.0 wt%, with a simultaneous increase in gaseous yields. An increase in the syngas and H2 selectivity was discovered with the reforming process such that the use of microwave pyrolysis with activated carbon reforming produced 85 vol% synthesis gas fraction containing 55 vol% H2 in comparison to the 74 vol% syngas fraction with 30 vol% H2 obtained without the reforming. Cracking reactions were improved with microwave heating, while deoxidation and dehydrogenation reactions were enhanced by activated carbon, which creates a reduction environment. Consequently, these reactions generated H2-rich syngas formation. The approach implemented in this study revealed higher H2, syngas yield and that the overall LHV of products has huge potential in the transformation of biomass into high-value synthesis gas.

InfoSAGE: Usage Pattern of a Family-Centric Care Coordination Online Platform.

Globally, there is an expanding elderly population, and families are finding it increasingly challenging to coordinate care for their older family members. This paper reports on the usage patterns of InfoSAGE, an online private social network that has tools for communication and care coordination for elders and their families. This descriptive analysis describes the types of family networks using the platform and types of functionality most used by elders and their family members.

Sustained Release Strategy Designed for Lixisenatide Delivery to Synchronously Treat Diabetes and Associated Complications.

Diabetes and its complications have become a global challenge of public health. Herein, we aimed to develop a long-acting delivery system of lixisenatide (Lixi), a glucose-dependent antidiabetic peptide, based on an injectable hydrogel for the synchronous treatment of type 2 diabetes mellitus (T2DM) and associated complications. Two triblock copolymers, poly(ε-caprolactone-co-glycolic acid)-poly(ethylene glycol)-poly(ε-caprolactone-co-glycolic acid) and poly(d,l-lactic acid-co-glycolic acid)-poly(ethylene glycol)-poly(d,l-lactic acid-co-glycolic acid) possessing temperature-induced sol-gel transitions, were synthesized by us. Compared to the two single-component hydrogels, their 1/1 mixture hydrogel not only maintained the temperature-induced gelation but also exhibited a steadier degradation profile in vivo. Both in vitro and in vivo release studies demonstrated that the mixture hydrogel provided the sustained release of Lixi for up to 9 days, which was attributed to balanced electrostatic interactions between the positive charges in the peptide and the negative charges in the polymer carrier. The hypoglycemic efficacy of Lixi delivered from the mixture hydrogel after a single subcutaneous injection into diabetic db/db mice was comparable to that of twice-daily administrations of Lixi solution for up to 9 days. Furthermore, three successive administrations of the abovementioned gel system within a month significantly increased the plasma insulin level, lowered glycosylated hemoglobin, and improved the pancreatic function of the animals. These results were superior or equivalent to those of twice-daily injections of Lixi solution for 30 days, but the number of injections was markedly reduced from 60 to 3. Finally, an improvement in hyperlipidemia, augmentation of nerve fiber density, and enhancement of motor nerve conduction velocity in the gel formulation-treated db/db mice indicated that the sustained delivery of Lixi arrested and even ameliorated diabetic complications. These findings suggested that the Lixi-loaded mixture hydrogel has great potential for the treatment of T2DM with significant improvements in the health and quality of life of patients.

A Novel Cadherin-like Protein Mediates Adherence to and Killing of Host Cells by the Parasite Trichomonas vaginalis.

Trichomonas vaginalis, a prevalent sexually transmitted parasite, adheres to and induces cytolysis of human mucosal epithelial cells. We have characterized a hypothetical protein, TVAG_393390, with predicted tertiary structure similar to that of mammalian cadherin proteins involved in cell-cell adherence. TVAG_393390, renamed cadherin-like protein (CLP), contains a calcium-binding site at a position conserved in cadherins. CLP is surface localized, and its mRNA and protein levels are significantly upregulated upon parasite adherence to host cells. To test the roles of CLP and its calcium-binding dependency during host cell adherence, we first demonstrated that wild-type CLP (CLP) binds calcium with a high affinity, whereas the calcium-binding site mutant protein (CLP-mut) does not. CLP and CLP-mut constructs were then used to overexpress these proteins in T. vaginalis Parasites overexpressing CLP have ∼3.5-fold greater adherence to host cells than wild-type parasites, and this increased adherence is ablated by mutating the calcium-binding site. Additionally, competition with recombinant CLP decreased parasite binding to host cells. We also found that overexpression of CLP induced parasite aggregation which was further enhanced in the presence of calcium, whereas CLP-mut overexpression did not affect aggregation. Lastly, parasites overexpressing wild-type CLP induced killing of host cells ∼2.35-fold, whereas parasites overexpressing CLP-mut did not have this effect. These analyses describe the first parasitic CLP and demonstrate a role for this protein in mediating parasite-parasite and host-parasite interactions. T. vaginalis CLP may represent convergent evolution of a parasite protein that is functionally similar to the mammalian cell adhesion protein cadherin, which contributes to parasite pathogenesis.IMPORTANCE The adherence of pathogens to host cells is critical for colonization of the host and establishing infection. Here we identify a protein with no known function that is more abundant on the surface of parasites that are better at binding host cells. To interrogate a predicted function of this protein, we utilized bioinformatic protein prediction programs which allowed us to uncover the first cadherin-like protein (CLP) found in a parasite. Cadherin proteins are conserved metazoan proteins with central roles in cell-cell adhesion, development, and tissue structure maintenance. Functional characterization of this CLP from the unicellular parasite Trichomonas vaginalis demonstrated that the protein mediates both parasite-parasite and parasite-host adherence, which leads to an enhanced killing of host cells by T. vaginalis Our findings demonstrate the presence of CLPs in unicellular pathogens and identify a new host cell binding protein family in a human-infective parasite.

A Usability Evaluation of the InfoSAGE App for Family-Based Medication Management.

The design of a mobile medication manager within a broader family and elder-centric collaboration platform faces challenges of usability and wide applicability. To inform the development and use cases of eldercare apps, we present the preliminary results of a usability study of an iOS and Android app intended for both family members and aging adults for the mobile management of medication lists. Seven participants were recorded during the performance of eight typical use-case scenarios of the medication portion of the InfoSAGE app. Audio and video recordings were analyzed for themes and events. The aim of this paper is to help inform future design choices for eldercare mobile apps.

Thermogel Loaded with Low-Dose Paclitaxel as a Facile Coating to Alleviate Periprosthetic Fibrous Capsule Formation.

Medical-grade silicones as implants have been utilized for decades. However, the postoperative complications, such as capsular formation and contracture, have not yet been fully controlled and resolved. The aim of the present study is to elucidate whether the capsular formation can be alleviated by local and sustained delivery of low-dose paclitaxel (PTX) during the critical phase after the insertion of silicone implants. A biocompatible and thermogelling poly(lactic acid- co-glycolic acid)- b-poly(ethylene glycol)- b-poly(lactic acid- co-glycolic acid) triblock copolymer was synthesized by us. The micelles formed by the amphiphilic polymers in water could act as a reservoir for the solubilization of PTX, a very hydrophobic drug. The concentrated polymer aqueous solution containing PTX exhibited a sol-gel transition upon heating and formed a thermogel depot at body temperature. In vitro release tests demonstrated that the entrapped microgram-level PTX displayed a sustained release manner up to 57 days without a significant initial burst effect. Customized silicone implants coated with the PTX-loaded thermogels at various drug concentrations were inserted into the pockets of the subpanniculus carnosus plane of rats. The histological observations performed 1 month postoperation showed that the sustained release of PTX with an appropriate dose significantly reduced the peri-implant capsule thickness, production and deposition of collagen, and expression of contracture-mediating factors compared with bare silicone implants. More importantly, such an optimum dose had an excellent repeatability for the suppression of the capsular formation. Therefore, this study provides a strategic foothold regarding the sustained release of low-dose PTX to alleviate fibrotic capsule formation after implantation, and the microgram-level PTX-loaded thermogel holds great potential as an "all-purpose antifibrosis coating" for veiling the surfaces of various implantable medical devices.

Trichomonas vaginalis Macrophage Migration Inhibitory Factor Mediates Parasite Survival during Nutrient Stress.

Trichomonas vaginalis is responsible for the most prevalent non-viral sexually transmitted disease worldwide, and yet the mechanisms used by this parasite to establish and maintain infection are poorly understood. We previously identified a T. vaginalis homologue (TvMIF) of a human cytokine, human macrophage migration inhibitory factor (huMIF). TvMIF mimics huMIF's role in increasing cell growth and inhibiting apoptosis in human host cells. To interrogate a role of TvMIF in parasite survival during infection, we asked whether overexpression of TvMIF (TvMIF-OE) confers an advantage to the parasite under nutrient stress conditions by comparing the survival of TvMIF-OE parasites to that of empty vector (EV) parasites. We found that under conditions of serum starvation, overexpression of TvMIF resulted in increased parasite survival. Serum-starved parasites secrete 2.5-fold more intrinsic TvMIF than unstarved parasites, stimulating autocrine and paracrine signaling. Similarly, we observed that addition of recombinant TvMIF increased the survival of the parasites in the absence of serum. Recombinant huMIF likewise increased the parasite survival in the absence of serum, indicating that the parasite may use this host survival factor to resist its own death. Moreover, TvMIF-OE parasites were found to undergo significantly less apoptosis and reactive oxygen species (ROS) generation under conditions of serum starvation, consistent with increased survival being the result of blocking ROS-induced apoptosis. These studies demonstrated that a parasitic MIF enhances survival under adverse conditions and defined TvMIF and huMIF as conserved survival factors that exhibit cross talk in host-pathogen interactions.IMPORTANCE Macrophage migration inhibitory factor (MIF) is a conserved protein found in most eukaryotes which has been well characterized in mammals but poorly studied in other eukaryotes. The limited analyses of MIF proteins found in unicellular eukaryotes have focused exclusively on the effect of parasitic MIF on the mammalian host. This was the first study to assess the function of a parasite MIF in parasite biology. We demonstrate that the Trichomonas vaginalis MIF functions to suppress cell death induced by apoptosis, thereby enhancing parasite survival under adverse conditions. Our research reveals a conserved survival mechanism, shared by a parasite and its host, and indicates a role for a conserved protein in mediating cross talk in host-pathogen interactions.

CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis.

The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T. vaginalis cell. We then created DNA constructs required to express several components essential to drive CRISPR/Cas9-mediated DNA modification: guide RNA (gRNA), the Cas9 endonuclease, short oligonucleotides and large, linearized DNA templates. Using these technical advances, we have established CRISPR/Cas9-mediated repair of mutations in genes contained on circular DNA plasmids harbored by the parasite. We also engineered CRISPR/Cas9 directed homologous recombination to delete (i.e. knock out) two non-essential genes within the T. vaginalis genome. This first report of the use of the CRISPR/Cas9 system in T. vaginalis greatly expands the ability to manipulate the genome of this pathogen and sets the stage for testing of the role of specific genes in many biological processes.

Structure and function of the alternatively spliced isoforms of the ecdysone receptor gene in the Chinese mitten crab, Eriocheir sinensis.

Alternative splicing is an essential molecular mechanism that increase the protein diversity of a species to regulate important biological processes. Ecdysone receptor (EcR), an essential nuclear receptor, is essential in the molting, growth, development, reproduction, and regeneration of crustaceans. In this study, the whole sequence of EcR gene from Eriocheir sinensis was obtained. The sequence was 45,481 bp in length with 9 exons. Moreover, four alternatively spliced EcR isoforms (Es-EcR-1, Es-EcR-2, Es-EcR-3 and Es-EcR-4) were identified. The four isoforms harbored a common A/B domain and a DNA-binding region but different D domains and ligand-binding regions. Three alternative splicing patterns (alternative 5' splice site, exon skipping, and intron retention) were identified in the four isoforms. Functional studies indicated that the four isoforms have specific functions. Es-EcR-3 may play essential roles in regulating periodic molting. Es-EcR-2 may participate in the regulation of ovarian development. Our results indicated that Es-EcR has broad regulatory functions in molting and development and established the molecular basis for the investigation of ecdysteroid signaling related pathways in E. sinensis.

Reductive defluorination of perfluorooctanoic acid by titanium(III) citrate with vitamin B12 and copper nanoparticles.

Perfluorooctanoic acid (PFOA) is widespread in the environment, which causes serious health and safety concerns. A mechanistic study on reductive defluorination of PFOA by titanium(III) citrate in the presence of catalysts was conducted. Vitamin B12 was used to catalyze reduction reactions by shuttling electrons from a reducing agent (electron donor) to PFOA to produce a Co-carbon bond intermediates. In the presence of copper nanoparticles, a precursor complex, B12-C7F14COOH, adsorbed on the metal surface, followed by a hydrogenolytic reaction to form less-fluorinated products. The synergistic effect between vitamin B12 and copper nanoparticles enhances the reductive activities by electron-transfer reactions and hydrogenolysis. The efficient reduction of PFOA to less-noxious compounds was demonstrated with a copper dose of 2gL-1, titanium(III) citrate (45mM), and vitamin B12 (0.2mM) with an initial pH of 9.0 and 70°C. In this anoxic aqueous solution, the biomimetic reductive system effectively removed 65% of PFOA. The mass balance on fluoride matched the observed degradation of PFOA, while no short-chain intermediates were detected.