CUR(E)ating a new approach to study fungal effectors and enhance undergraduate education through authentic research.

Course-based Undergraduate Research Experiences (CUREs) integrate active, discovery-based learning into undergraduate curricula, adding tremendous value to Biochemistry and Molecular Biology (BMB) education. There are multiple challenges in transforming a research project into a CURE, such as the readiness of students, the time commitment of the instructor, and the productivity of the research. In this article, we report a CURE course developed and offered in the University of Massachusetts Amherst BMB Department since 2018 that addresses these challenges. Our CURE focuses on fungal effectors which are proteins secreted by a destructive pathogenic fungus Fusarium oxysporum, one of the top five most devastating plant pathogens. By studying this group of proteins, students are connected to real-world problems and participate in the search for potential solutions. A 3-week "standard Boot Camp" is implemented to help students familiarize themselves with all basic techniques and boost their confidence. Next, molecular cloning, a versatile technique with modularity and repeatability, is used as the bedrock of the course. Our past 5 years of experience have confirmed that we have developed a novel and feasible CURE protocol. Measurable progress documented by students who took this course includes stimulated active learning and increased career trajectory to pursue hypothesis-based research to address societal needs. In addition, data generated through the course advance ongoing lab research. Collectively, we encourage the implementation of CURE among research-intensive faculty to provide a more inclusive research experience to undergraduate students, an important element in predicting career success.

Conservation and Expansion of Transcriptional Factor Repertoire in the Fusarium oxysporum Species Complex.

The Fusarium oxysporum species complex (FOSC) includes both plant and human pathogens that cause devastating plant vascular wilt diseases and threaten public health. Each F. oxysporum genome comprises core chromosomes (CCs) for housekeeping functions and accessory chromosomes (ACs) that contribute to host-specific adaptation. This study inspects global transcription factor profiles (TFomes) and their potential roles in coordinating CC and AC functions to accomplish host-specific interactions. Remarkably, we found a clear positive correlation between the sizes of TFomes and the proteomes of an organism. With the acquisition of ACs, the FOSC TFomes were larger than the other fungal genomes included in this study. Among a total of 48 classified TF families, 14 families involved in transcription/translation regulations and cell cycle controls were highly conserved. Among the 30 FOSC expanded families, Zn2-C6 and Znf_C2H2 were most significantly expanded to 671 and 167 genes per family including well-characterized homologs of Ftf1 (Zn2-C6) and PacC (Znf_C2H2) that are involved in host-specific interactions. Manual curation of characterized TFs increased the TFome repertoires by 3% including a disordered protein Ren1. RNA-Seq revealed a steady pattern of expression for conserved TF families and specific activation for AC TFs. Functional characterization of these TFs could enhance our understanding of transcriptional regulation involved in FOSC cross-kingdom interactions, disentangle species-specific adaptation, and identify targets to combat diverse diseases caused by this group of fungal pathogens.

CUR(E)ating a New Approach to Study Fungal Effectors and Enhance Undergraduate Education through Authentic Research.

Course-based Undergraduate Research Experiences (CUREs) integrate active, discovery-based learning into undergraduate curriculums, adding tremendous value to Biochemistry and Molecular Biology (BMB) education. There are multiple challenges in transforming a research project into a CURE, such as the readiness of students, the time commitment of the instructor, and the productivity of the research. In this article, we report a CURE course developed and offered in the University of Massachusetts Amherst BMB Department since 2018 that addresses these challenges. Our CURE focuses on fungal effectors which are proteins secreted by a destructive pathogenic fungus Fusarium oxysporum , one of the top five most devastating plant pathogens. By studying this group of proteins, students are connected to real-world problems and participate in the search for potential solutions. A three-week "standard Bootcamp" is implemented to help students familiarize themselves with all basic techniques and boost their confidence. Next, molecular cloning, a versatile technique with modularity and repeatability, is used as the bedrock of the course. Our past five years of experience have confirmed that we have developed a novel and feasible CURE protocol. Measurable progress documented by students who took this course includes stimulated active learning and increased career trajectory to pursue hypothesis-based research to address societal needs. In addition, data generated through the course advance ongoing lab research. Collectively, we encourage the implementation of CURE among research-intensive faculty to provide a more inclusive research experience to all students, an important element in predicting career success.

Conservation and Expansion of Transcriptional Factor Repertoire in the Fusarium oxysporum Species Complex.

The Fusarium oxysporum species complex (FOSC) includes both plant and human pathogens that cause devastating plant vascular wilt diseases and threaten public health. Each F. oxysporum genome comprises core chromosomes (CCs) for housekeeping functions and accessory chromosomes (ACs) that contribute to host-specific adaptation. This study inspected global transcription factor profiles (TFomes) and their potential roles in coordinating CCs and ACs functions to accomplish host-specific pathogenicity. Remarkably, we found a clear positive correlation between the sizes of TFome and proteome of an organism, and FOSC TFomes are larger due to the acquisition of ACs. Among a total of 48 classified TF families, 14 families involved in transcription/translation regulations and cell cycle controls are highly conserved. Among 30 FOSC expanded families, Zn2-C6 and Znf_C2H2 are most significantly expanded to 671 and 167 genes per family, including well-characterized homologs of Ftf1 (Zn2-C6) and PacC (Znf_C2H2) involved in host-specific interactions. Manual curation of characterized TFs increased the TFome repertoires by 3%, including a disordered protein Ren1. Expression profiles revealed a steady expression of conserved TF families and specific activation of AC TFs. Functional characterization of these TFs could enhance our understanding of transcriptional regulation involved in FOSC cross-kingdom interactions, disentangle species-specific adaptation, and identify targets to combat diverse diseases caused by this group of fungal pathogens.

Degradable ZnS-Supported Bioorthogonal Nanozymes with Enhanced Catalytic Activity for Intracellular Activation of Therapeutics.

Bioorthogonal catalysis using transition-metal catalysts (TMCs) provides a toolkit for the in situ generation of imaging and therapeutic agents in biological environments. Integrating TMCs with nanomaterials mimics key properties of natural enzymes, providing bioorthogonal "nanozymes". ZnS nanoparticles provide a platform for bioorthogonal nanozymes using ruthenium catalysts embedded in self-assembled monolayers on the particle surface. These nanozymes uncage allylated profluorophores and prodrugs. The ZnS core combines the non-toxicity and degradability with the enhancement of Ru catalysis through the release of thiolate surface ligands that accelerate the rate-determining step in the Ru-mediated deallylation catalytic cycle. The maximum rate of reaction (Vmax) increases ∼2.5-fold as compared to the non-degradable gold nanoparticle analogue. The therapeutic potential of these bioorthogonal nanozymes is demonstrated by activating a chemotherapy drug from an inactive prodrug with efficient killing of cancer cells.

Intracellular Activation of Anticancer Therapeutics Using Polymeric Bioorthogonal Nanocatalysts.

Bioorthogonal catalysis provides a promising strategy for imaging and therapeutic applications, providing controlled in situ activation of pro-dyes and prodrugs. In this work, the use of a polymeric scaffold to encapsulate transition metal catalysts (TMCs), generating bioorthogonal "polyzymes," is presented. These polyzymes enhance the stability of TMCs, protecting the catalytic centers from deactivation in biological media. The therapeutic potential of these polyzymes is demonstrated by the transformation of a nontoxic prodrug to an anticancer drug (mitoxantrone), leading to the cancer cell death in vitro.

Heterogenous Internalization of Nanoparticles at Ultra-Trace Concentration in Environmental Individual Unicellular Organisms Unveiled by Single-Cell Mass Cytometry.

The application and consumption of nanoparticles (NPs) inevitably result in the contamination of environmental water. The internalized NPs in unicellular organisms could travel to human bodies along food chains and raise health concerns. Current research failed to determine the characteristics of cellular uptake of NPs by unicellular organisms at extremely low concentration in the real environment. We here developed a label-free high-throughput mass cytometry method to investigate gold NP (AuNP) uptake in a unicellular organism (Tetrahymena thermophila) at the single-cell level. The limit of detection for Au is as low as to 6.67 × 10-18 g/cell, which equals ∼5.3 5 nm AuNPs. We demonstrated that active engulfment pathways were responsible for the cellular accumulation of AuNPs and T. thermophila could also eliminate the cellular AuNPs rapidly. The interaction between AuNPs and T. thermophila is highly dependent on the sizes of nanoparticles; i.e., the population of T. thermophila containing AuNPs decreased with the increment of the diameters of AuNPs when exposed to the same mass concentration. For each type of AuNP, distinct heterogeneous cellular uptake of AuNPs by T. thermophila was observed. Intriguingly, for 5 nm AuNP, even at 0.001 ng/mL, some T. thermophila cells could concentrate AuNPs, indicating a real environmental concern even when water was contaminated by only trace level of NPs. This method represents a promising tool for simultaneous determination of physiological status of cells together with the intracellular level of heavy metal or metallic NPs in study of biological effects.

Intracellular Activation of Bioorthogonal Nanozymes through Endosomal Proteolysis of the Protein Corona.

Bioorthogonal activation of prodrugs provides a strategy for on-demand on-site production of therapeutics. Intracellular activation provides a strategy to localize therapeutics, potentially minimizing off-target effects. To this end, nanoparticles embedded with transition metal catalysts (nanozymes) were engineered to generate either "hard" irreversible or "soft" reversible coronas in serum. The hard corona induced nanozyme aggregation, effectively inhibiting nanozyme activity, whereas only modest loss of activity was observed with the nonaggregating soft corona nanozymes. In both cases complete activity was restored by treatment with proteases. Intracellular activity mirrored this reactivation: endogenous proteases in the endosome provided intracellular activation of both nanozymes. The role of intracellular proteases in nanozyme reactivation was verified through treatment of the cells with protease inhibitors, which prevented reactivation. This study demonstrates the use of intracellular proteolysis as a strategy for localization of therapeutic generation to within cells.