Craig R. Brunetti

Myogenesis is a tightly regulated process essential for embryonic development, postnatal growth, and muscle regeneration. We recently identified that cytokinins (CTKs), a class of adenine-derived signaling molecules originally characterized in plants, are present in cultured skeletal muscle cells. The most abundant type of cytokinins detected within cultured muscle cells was isopentenyladenine (iP) in its nucleotide, riboside, and free base derivatives. The purpose of this study was to determine whether CTKs are also present in regenerating muscle tissue in vivo and to characterize the effects of iP and its riboside form, isopentenyladenosine (iPR), on muscle cell proliferation and differentiation. These effects were observed relative to adenine and adenosine, and to a second class of cytokinins with a large aromatic side chain, kinetin (the free base), and kinetin riboside. Cardiotoxin was used to induce muscle injury and repair processes in the gastrocnemius of 3- and 12-month-old mice. Samples were collected 3- and 7 days post-injury for ultra high-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-(ESI+)-HRMS/MS). Four CTKs (N⁶-benzyladenine (BA), dihydrozeatin-9-N-glucoside (DZ9G), isopentenyladenosine (iPR), and 2-methylthio-isopentenyladenosine (2-MeSiPR) were detected. 2-MeSiPR levels were significantly influenced by aging, as this CTK was increased in response to injury only in the younger mice. Treatment of C2C12 myoblasts with 10 µM of isopentenyladenosine (iPR) or kinetin riboside reduced cell proliferation, whereas iP (the free base) increased proliferation in a biphasic response. During differentiation, both iPR and kinetin riboside impaired myotube formation, while the free-base forms of iP and kinetin had no effect. Our data establishes that CTKs are present within muscle tissue and highly responsive to injury and aging. Furthermore, the biological activities of CTKs in muscle cells are influenced by structural modifications, including riboside conjugation and side chain composition. Understanding these differences provides insight into the distinct roles of CTKs in muscle cell metabolism and differentiation, offering potential implications for the use of exogenous CTKs in muscle biology and regenerative medicine.

Members of the Iridoviridae family, genus Ranavirus, represent a group of globally emerging pathogens of ecological and economic importance. In 2017, an amphibian die-off of wood frogs (Rana sylvatica) and boreal chorus frogs (Pseudacris maculata) was reported in Wood Buffalo National Park, Canada. Isolation and complete genomic sequencing of the tissues of a wood frog revealed the presence of a frog virus 3 (FV3)-like isolate, Rana sylvatica ranavirus (RSR), with a genome size of 105,895 base pairs, 97 predicted open reading frames (ORFs) bearing sequence similarity to FV3 (99.98%) and a FV3-like isolate from a spotted salamander in Maine (SSME; 99.64%). Despite high sequence similarity, RSR had a unique genomic composition containing ORFs specific to either FV3 or SSME. In addition, RSR had a unique 13 amino acid insertion in ORF 49/50L. No differences were found in the in vitro growth kinetics of FV3, SSME, and RSR; however, genomic differences between these isolates were in non-core genes, implicated in nucleic acid metabolism and immune evasion. This study highlights the importance of viral isolation and complete genomic analysis as these not only provide information on ranavirus spatial distribution but may elucidate genomic factors contributing to host tropism and pathogenicity.

Cytokinins (CKs) are a group of N⁶-substituted signaling molecules whose biosynthesis and metabolism have been documented in all kingdoms of life, including vertebrates. While their biological relevance in vertebrate systems continues to be elucidated, they have broadly been documented with therapeutic effects in exogenous applications. In this study, we evaluated the virostatic potential of four types of CKs including, N⁶-isopentenyladenine (iP), N⁶-isopentenyladenosine (iPR), N⁶-isopentenyladenosine-5′monophosphate (iPMP), and 2-methylthiol-N⁶-isopentenyladenosine (2MeSiPR) against the ranavirus type species, frog virus 3 (FV3). Following concurrent treatment and infection, iP and iPR reduced viral replication by 33.8% and 59.6%, respectively, in plaque formation assays. A decrease in viral replication was also observed when CK exposure was limited to 12 h prior to infection, where iP and iPR reduced viral replication by 31% and 23.75%, respectively. Treatment with iP and iPR was also marked by 48% and 60% decreases in viral load over 72 h, respectively, as measured in single step growth curves. Plaque morphology was altered in vitro, as iP and iPR treatment increased plaque area by 83% and 112% with lytic zone formation also becoming more prevalent in corresponding treatments. Treatment with iPMP and 2MeSiPR resulted in no effect on viral kinetics in vitro. The results of this study are the first to provide evidence of CK antiviral activity against a DNA virus and highlight the importance of their structure for therapeutic investigations.

Cytokinins (CKs) are small signaling molecules present in all kingdoms of life; however, their biological relevance outside of plants is poorly characterized. CK detection across kingdoms has been shown to be inconsistent and is often organism or cell line dependent. To ensure optimal detection in epithelioma papulosum cyprini (EPC), a cold-blooded vertebrate cell line, a range of biomasses were evaluated. CKs were extracted by solid-phase extraction and then detected using high-performance liquid chromatography and tandem mass spectrometry. The objectives were to establish endogenous CK profiles and characterize their biosynthesis in EPC cells. Among the 14 CKs that were scanned for, only 4 forms were detected overall within the samples analyzed, including, N6-(Δ2-isopentenyl) adenine-9-riboside (iPR), N6-(Δ2-isopentenyl) adenine-9-riboside-5′ (mono/di/tri)phosphate (iPNT), and cis-zeatin-9-riboside-5′ (mono/di/tri)phosphate ( cZNT) and 2‐methylthio‐zeatin (2MeSZ). The total CK concentrations ranged from 0.03 pmol/g to 681.025 pmol/gfwt. The results from this study provided evidence that EPC cells can produce and process CKs.

Cytokinins (CKs) are a class of growth‐promoting signaling molecules that affect multiple cellular and developmental processes. These phytohormones are well studied in plants, but their presence continues to be uncovered in organisms spanning all kingdoms, which poses new questions about their roles and functions outside of plant systems. Cytokinin production can be initiated by one of two different biosynthetic enzymes, adenylate isopentenyltransfases (IPTs) or tRNA isopentenyltransferases (tRNA‐IPTs). In this study, the social amoeba, Dictyostelium discoideum , was used to study the role of CKs by generating deletion and overexpression strains of its single adenylate‐IPT gene, iptA . The life cycle of D. discoideum is unique and possesses both single‐ and multicellular stages. Vegetative amoebae grow and divide while food resources are plentiful, and multicellular development is initiated upon starvation, which includes distinct life cycle stages. CKs are produced in D. discoideum throughout its life cycle and their functions have been well studied during the later stages of multicellular development of D. discoideum . To investigate potential expanded roles of CKs, this study focused on vegetative growth and early developmental stages. We found that iptA ‐deficiency results in cytokinesis defects, and both iptA ‐deficiency and overexpression results in dysregulated tricarboxylic acid (TCA) cycle and amino acid metabolism, as well as increased levels of adenosine monophosphate (AMP). Collectively, these findings extend our understanding of CK function in amoebae, indicating that iptA loss and overexpression alter biological processes during vegetative growth that are distinct from those reported during later development.

Viruses are obligate intracellular parasites that alter host metabolic machinery to obtain energy and macromolecules that are pivotal for replication. Ranavirus, including the type species of the genus frog virus 3 (FV3), represent an ecologically important group of viruses that infect fish, amphibians, and reptiles. It was established that fatty acid synthesis, glucose, and glutamine metabolism exert roles during iridovirus infections; however, no information exists regarding the role of purine metabolism. In this study, we assessed the impact of exogenously applied purines adenine, adenosine, adenosine 5′-monophosphate (AMP), inosine 5′-monophosphate (IMP), inosine, S-adenosyl-L-homocysteine (SAH), and S-adenosyl-L-methionine (SAM) on FV3 replication. We found that all compounds except for SAH increased FV3 replication in a dose-dependent manner. Of the purines investigated, adenine and adenosine produced the most robust response, increasing FV3 replication by 58% and 51%, respectively. While all compounds except SAH increased FV3 replication, only adenine increased plaque area. This suggests that the stimulatory effect of adenine on FV3 replication is mediated by a mechanism that is at least in part independent from the other compounds investigated. Our results are the first to report a response to exogenously applied purines and may provide insight into the importance of purine metabolism during iridoviral infection.

Background: The diversity of butterfly color patterns can be attributed to a relatively small number of pattern elements that are homologous across Lepidoptera. Although genes involved in patterning some of these elements have been identified, the development of several major elements remains poorly understood. To identify genes underlying wing pupal cuticle markings and wing margin color patterns, we examined expression of the candidate transcription factors Engrailed/Invected (En/Inv), Distal-less (Dll), Cubitus interruptus (Ci), and Spalt in two nymphalids:Junonia coeniaandBicyclus anynana.Results: We found that En/Inv, Dll, and Ci mark domains on theJ. coenialast-instar forewing disc that closely correspond to the position and shape of pupal cuticle markings. We also found that Spalt demarcates wing margin color patterns in bothJ. coeniaandB. anynana, and that CRISPR/Cas9 deletions in thespaltgene result in reduction and loss of wing margin color patterns inJ. coenia. These data demonstrate a role forspaltin promoting wing margin color patterning, in addition to its previously described role in eyespot patterning.Conclusion: Our observations support the model that a core set of regulatory genes are redeployed multiple times, and in multiple roles, during butterfly wing pattern development. Of these genes,spaltis of special interest as it plays a dual role in both eyespot and margin color pattern development.

Cytokinins (CKs) are a family of evolutionarily conserved growth regulating hormones. While CKs are well-characterized in plant systems, these N⁶-substituted adenine derivatives are found in a variety of organisms beyond plants, including bacteria, fungi, mammals, and the social amoeba, Dictyostelium discoideum. Within Dictyostelium, CKs have only been studied in the late developmental stages of the life cycle, where they promote spore encapsulation and dormancy. In this study, we used ultra high-performance liquid chromatography-positive electrospray ionization-high resolution tandem mass spectrometry (UHPLC-(ESI+)-HRMS/MS) to profile CKs during the Dictyostelium life cycle: growth, aggregation, mound, slug, fruiting body, and germination. Comprehensive profiling revealed that Dictyostelium produces 6 CK forms (cis-Zeatin (cZ), discadenine (DA), N⁶-isopentenyladenine (iP), N⁶-isopentenyladenine-9-riboside (iPR), N⁶-isopentenyladenine-9-riboside-5′ phosphate (iPRP), and 2-methylthio-N⁶-isopentenyladenine (2MeSiP)) in varying abundance across the sampled life cycle stages, thus laying the foundation for the CK biosynthesis pathway to be defined in this organism. Interestingly, iP-type CKs were the most dominant CK analytes detected during growth and aggregation. Exogenous treatment of AX3 cells with various CK types revealed that iP was the only CK to promote the proliferation of cells in culture. In support of previous studies, metabolomics data revealed that DA is one of the most significantly upregulated small molecules during Dictyostelium development, and our data indicates that total CK levels are highest during germination. While much remains to be explored in Dictyostelium, this research offers new insight into the nature of CK biosynthesis, secretion, and function during Dictyostelium growth, development, and spore germination.

The characterization of the function of conserved viral genes is central to developing a greater understanding of important aspects of viral replication or pathogenesis. A comparative genomic analysis of the iridoviral genomes identified 26 core genes conserved across the family Iridoviridae. Three of those conserved genes have no defined function; these include the homologs of frog virus 3 (FV3) open reading frames (ORFs) 88R, 91R, and 94L. Conserved viral genes that have been previously identified are known to participate in a number of viral activities including: transcriptional regulation, DNA replication/repair/modification/processing, protein modification, and viral structural proteins. To begin to characterize the conserved FV3 ORFs 88R, 91R, and 94L, we cloned the genes and determined their intracellular localization. We demonstrated that 88R localizes to the cytoplasm of the cell while 91R localizes to the nucleus and 94L localizes to the endoplasmic reticulum (ER).