Inhibiting Translation Elongation with SVC112 Suppresses Cancer Stem Cells and Inhibits Growth in Head and Neck Squamous Carcinoma.

Cancer stem cells (CSC) drive growth, therapy resistance, and recurrence in head and neck squamous cell carcinoma (HNSCC). Regulation of protein translation is crucial for normal stem cells and CSCs; its inhibition could disrupt stemness properties, but translation inhibitors are limited clinically due to toxicity. SVC112 is a synthetic derivative of bouvardin, a plant-derived translation elongation inhibitor. SVC112 had greater antiproliferative effects on HNSCC cells compared with the FDA-approved translation inhibitor omacetaxine mepesuccinate (HHT). SVC112 preferentially inhibited cancer cells compared with patient-matched cancer-associated fibroblasts, whereas HHT was equally toxic to both. SVC112 reduced sphere formation by cell lines and CSCs. SVC112 alone inhibited the growth of patient-derived xenografts (PDX), and SVC112 combined with radiation resulted in tumor regression in HPV-positive and HPV-negative HNSCC PDXs. Notably, CSC depletion after SVC112 correlated with tumor response. SVC112 preferentially impeded ribosomal processing of mRNAs critical for stress response and decreased CSC-related proteins including Myc and Sox2. SVC112 increased cell-cycle progression delay and slowed DNA repair following radiation, enhancing colony and sphere formation radiation effects. In summary, these data demonstrate that SVC112 suppresses CSC-related proteins, enhances the effects of radiation, and blocks growth of HNSCC PDXs by inhibiting CSCs. SIGNIFICANCE: Inhibiting protein elongation with SVC112 reduces tumor growth in head and neck squamous cell carcinoma and increases the effects of radiation by targeting the cancer stem cell pool.

Wnt signaling dynamics in head and neck squamous cell cancer tumor-stroma interactions.

Wnt pathway activation maintains the cancer stem cell (CSC) phenotype and promotes tumor progression, making it an attractive target for anti-cancer therapy. Wnt signaling at the tumor and tumor microenvironment (TME) front have not been investigated in depth in head and neck squamous cell carcinoma (HNSCC). In a cohort of 48 HNSCCs, increased Wnt signaling, including Wnt genes (AXIN2, LGR6, WISP1) and stem cell factors (RET, SOX5, KIT), were associated with a more advanced clinical stage. Key Wnt pathway proteins were most abundant at the cancer epithelial-stromal boundary. To investigate these observations, we generated three pairs of cancer-cancer associated fibroblast (CAF) cell lines derived from the same HNSCC patients. 3D co-culture of cancer spheres and CAFs mimicked these in vivo interactions, and using these we observed increased expression of Wnt genes (eg, WNT3A, WNT7A, WNT16) in both compartments. Of these Wnt ligands, we found Wnt3a, and less consistently Wnt16, activated Wnt signaling in both cancer cells and CAFs. Wnt activation increased CSC characteristics like sphere formation and invasiveness, which was further regulated by the presence of CAFs. Time lapse microscopy also revealed preferential Wnt activation of cancer cells. Wnt inhibitors, OMP-18R5 and OMP-54F28, significantly reduced growth of HNSCC patient-derived xenografts and suppressed Wnt activation at the tumor epithelial-stromal boundary. Taken together, our findings suggest that Wnt signaling is initiated in cancer cells which then activate CAFs, and in turn perpetuate a paracrine signaling loop. This suggests that targeting Wnt signaling in the TME is essential.

Salivary Gland Cancer Patient-Derived Xenografts Enable Characterization of Cancer Stem Cells and New Gene Events Associated with Tumor Progression.

Purpose: Salivary gland cancers (SGC) frequently present with distant metastases many years after diagnosis, suggesting a cancer stem cell (CSC) subpopulation that initiates late recurrences; however, current models are limited both in their availability and suitability to characterize these rare cells.Experimental Design: Patient-derived xenografts (PDX) were generated by engrafting patient tissue onto nude mice from one acinic cell carcinoma (AciCC), four adenoid cystic carcinoma (ACC), and three mucoepidermoid carcinoma (MEC) cases, which were derived from successive relapses from the same MEC patient. Patient and PDX samples were analyzed by RNA-seq and Exome-seq. Sphere formation potential and in vivo tumorigenicity was assessed by sorting for Aldefluor (ALDH) activity and CD44-expressing subpopulations.Results: For successive MEC relapses we found a time-dependent increase in CSCs (ALDH+CD44high), increasing from 0.2% to 4.5% (P=0.033), but more importantly we observed an increase in individual CSC sphere formation and tumorigenic potential. A 50% increase in mutational burden was documented in subsequent MEC tumors, and this was associated with increased expression of tumor-promoting genes (MT1E, LGR5, and LEF1), decreased expression of tumor-suppressor genes (CDKN2B, SIK1, and TP53), and higher expression of CSC-related proteins such as SOX2, MYC, and ALDH1A1. Finally, genomic analyses identified a novel NFIB-MTFR2 fusion in an ACC tumor and confirmed previously reported fusions (NTRK3-ETV6 and MYB-NFIB)Conclusions: Sequential MEC PDX models preserved key patient features and enabled the identification of genetic events putatively contributing to increases in both CSC proportion and intrinsic tumorigenicity, which mirrored the patient's clinical course. Clin Cancer Res; 24(12); 2935-43. ©2018 AACR.

Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2.

Background: We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. Methods: We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. Results: CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation. Conclusions: The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.

Dead enzymes in the aldehyde dehydrogenase gene family: role in drug metabolism and toxicology.

INTRODUCTION: Dead enzymes are gene products (proteins) that lack key residues required for catalytic activity. In the pre-genome era, dead enzymes were thought to occur only rarely. However, they now have been shown to represent upwards of 10% of the total enzyme population in many families. The aldehyde dehydrogenase (ALDH) gene family encodes proteins that, depending on the isozyme, may be either catalytically-active or -inactive. Importantly, several ALDHs exhibit biological activities independent of their catalytic activity. For many of these, the physiological and pathophysiological functions remain to be established. AREAS COVERED: This article reviews the non-enzymatic functions of the ALDH superfamily. In addition, a search for additional non-catalytic ALDH records is undertaken. Our computational analyses reveal that there are currently 182 protein records (divided into 19 groups) that meet the criteria for dead enzymes. EXPERT OPINION: Dead enzymes have the potential to exert biological actions through protein-protein interaction and allosteric modulation of the activity of an active enzyme. In addition, a dead enzyme may also influence availability of substrate for other active enzymes by sequestering substrate, and/or anchoring the substrate to a particular subcellular space. A large number of putatively non-catalytic ALDH proteins exist that warrant further study.

Human ALDH1B1 polymorphisms may affect the metabolism of acetaldehyde and all-trans retinaldehyde--in vitro studies and computational modeling.

PURPOSE: To elucidate additional substrate specificities of ALDH1B1 and determine the effect that human ALDH1B1 polymorphisms will have on substrate specificity. METHODS: Computational-based molecular modeling was used to predict the binding of the substrates propionaldehyde, 4-hydroxynonenal, nitroglycerin, and all-trans retinaldehyde to ALDH1B1. Based on positive in silico results, the capacity of purified human recombinant ALDH1B1 to metabolize nitroglycerin and all-trans retinaldehyde was explored. Additionally, metabolism of 4-HNE by ALDH1B1 was revisited. Databases queried to find human polymorphisms of ALDH1B1 identified three major variants: ALDH1B1*2 (A86V), ALDH1B1*3 (L107R), and ALDH1B1*5 (M253V). Computational modeling was used to predict the binding of substrates and of cofactor (NAD(+)) to the variants. These human polymorphisms were created and expressed in a bacterial system and specific activity was determined. RESULTS: ALDH1B1 metabolizes (and appears to be inhibited by) nitroglycerin and has favorable kinetics for the metabolism of all-trans retinaldehyde. ALDH1B1 metabolizes 4-HNE with higher apparent affinity than previously described, but with low throughput. Recombinant ALDH1B1*2 is catalytically inactive, whereas both ALDH1B1*3 and ALDH1B1*5 are catalytically active. Modeling indicated that the lack of activity in ALDH1B1*2 is likely due to poor NAD(+) binding. Modeling also suggests that ALDH1B1*3 may be less able to metabolize all-trans retinaldehyde and that ALDH1B1*5 may bind NAD(+) poorly. CONCLUSIONS: ALDH1B1 metabolizes nitroglycerin and all-trans-retinaldehyde. One of the three human polymorphisms, ALDH1B1*2, is catalytically inactive, likely due to poor NAD(+) binding. Expression of this variant may affect ALDH1B1-dependent metabolic functions in stem cells and ethanol metabolism.

Effects of Cutrine-Plus® algaecide and predators on wood frog (Lithobates sylvaticus) tadpole survival and growth.

Copper contamination is increasing in many aquatic ecosystems. One mode by which copper can be introduced into aquatic ecosystems is as an algaecide, such as Cutrine-Plus®. Using a mesocosm experiment, we examined the effects of Cutrine-Plus® on wood frog (Lithobates sylvaticus) tadpoles. In addition, we examined how the presence of a nonnative predator the Western mosquitofish (Gambusia affinis) may interact with exposure to Cutrine-Plus®. Exposure to our low and high Cutrine-Plus® treatments had a strong negative effect on the wood frog tadpoles, and survivorship was greatly decreased in the low treatment, and no tadpoles survived in the high treatment. Additionally, the tadpoles that survived the low treatment were significantly smaller than those in the control treatment. Mosquitofish had no effect on the survivorship or growth of wood frog tadpoles, and mosquitofish presence did not have a significant interaction with the Cutrine-Plus® treatments. Cutrine-Plus® clearly had a negative effect on wood frog tadpoles at the concentrations used in our experiment, which were at and below the label-recommended dosages, suggesting that the use of Cutrine-Plus® in natural ponds may have negative consequences for wood frog populations and possibly other amphibians.

Update of the human and mouse SERPIN gene superfamily.

The serpin family comprises a structurally similar, yet functionally diverse, set of proteins. Named originally for their function as serine proteinase inhibitors, many of its members are not inhibitors but rather chaperones, involved in storage, transport, and other roles. Serpins are found in genomes of all kingdoms, with 36 human protein-coding genes and five pseudogenes. The mouse has 60 Serpin functional genes, many of which are orthologous to human SERPIN genes and some of which have expanded into multiple paralogous genes. Serpins are found in tissues throughout the body; whereas most are extracellular, there is a class of intracellular serpins. Serpins appear to have roles in inflammation, immune function, tumorigenesis, blood clotting, dementia, and cancer metastasis. Further characterization of these proteins will likely reveal potential biomarkers and therapeutic targets for disease.

ALDH16A1 is a novel non-catalytic enzyme that may be involved in the etiology of gout via protein-protein interactions with HPRT1.

Gout, a common form of inflammatory arthritis, is strongly associated with elevated uric acid concentrations in the blood (hyperuricemia). A recent study in Icelanders identified a rare missense single nucleotide polymorphism (SNP) in the ALDH16A1 gene, ALDH16A1*2, to be associated with gout and serum uric acid levels. ALDH16A1 is a novel and rather unique member of the ALDH superfamily in relation to its gene and protein structures. ALDH16 genes are present in fish, amphibians, protista, bacteria but absent from archaea, fungi and plants. In most mammalian species, two ALDH16A1 spliced variants (ALDH16A1, long form and ALDH16A1_v2, short form) have been identified and both are expressed in HepG-2, HK-2 and HK-293 human cell lines. The ALDH16 proteins contain two ALDH domains (as opposed to one in the other members of the superfamily), four transmembrane and one coiled-coil domains. The active site of ALDH16 proteins from bacterial, frog and lower animals contain the catalytically important cysteine residue (Cys-302); this residue is absent from the mammalian and fish orthologs. Molecular modeling predicts that both the short and long forms of human ALDH16A1 protein would lack catalytic activity but may interact with the hypoxanthine-guanine phosphoribosyltransferase (HPRT1) protein, a key enzyme involved in uric acid metabolism and gout. Interestingly, such protein-protein interactions with HPRT1 are predicted to be impaired for the long or short forms of ALDH16A1*2. These results lead to the intriguing possibility that association between ALDH16A1 and HPRT1 may be required for optimal HPRT activity with disruption of this interaction possibly contributing to the hyperuricemia seen in ALDH16A1*2 carriers.

Comparative genomics, molecular evolution and computational modeling of ALDH1B1 and ALDH2.

Vertebrate ALDH2 genes encode mitochondrial enzymes capable of metabolizing acetaldehyde and other biological aldehydes in the body. Mammalian ALDH1B1, another mitochondrial enzyme sharing 72% identity with ALDH2, is also capable of metabolizing acetaldehyde but has a tissue distribution and pattern of activity distinct from that of ALDH2. Bioinformatic analyses of several vertebrate genomes were undertaken using known ALDH2 and ALDH1B1 amino acid sequences. Phylogenetic analysis of many representative vertebrate species (including fish, amphibians, birds and mammals) indicated the presence of ALDH1B1 in many mammalian species and in frogs (Xenopus tropicalis); no evidence was found for ALDH1B1 in the genomes of birds, reptiles or fish. Predicted vertebrate ALDH2 and ALDH1B1 subunit sequences and structures were highly conserved, including residues previously shown to be involved in catalysis and coenzyme binding for human ALDH2. Studies of ALDH1B1 sequences supported the hypothesis that the ALDH1B1 gene originated in early vertebrates from a retrotransposition of the vertebrate ALDH2 gene. Given the high degree of similarity between ALDH2 and ALDH1B1, it is surprising that individuals with an inactivating mutation in ALDH2 (ALDH2*2) do not exhibit a compensatory increase in ALDH1B1 activity. We hypothesized that the similarity between the two ALDHs would allow for dominant negative heterotetramerization between the inactive ALDH2 mutants and ALDH1B1. Computational-based molecular modeling studies examining predicted protein-protein interactions indicated that heterotetramerization between ALDH2 and ALDH1B1 subunits was highly probable and may partially explain a lack of compensation by ALDH1B1 in ALDH2(∗)2 individuals.

Aldehyde dehydrogenases in cellular responses to oxidative/electrophilic stress.

Reactive oxygen species (ROS) are continuously generated within living systems and the inability to manage ROS load leads to elevated oxidative stress and cell damage. Oxidative stress is coupled to the oxidative degradation of lipid membranes, also known as lipid peroxidation. This process generates over 200 types of aldehydes, many of which are highly reactive and toxic. Aldehyde dehydrogenases (ALDHs) metabolize endogenous and exogenous aldehydes and thereby mitigate oxidative/electrophilic stress in prokaryotic and eukaryotic organisms. ALDHs are found throughout the evolutionary gamut, from single-celled organisms to complex multicellular species. Not surprisingly, many ALDHs in evolutionarily distant, and seemingly unrelated, species perform similar functions, including protection against a variety of environmental stressors such as dehydration and ultraviolet radiation. The ability to act as an "aldehyde scavenger" during lipid peroxidation is another ostensibly universal ALDH function found across species. Upregulation of ALDHs is a stress response in bacteria (environmental and chemical stress), plants (dehydration, salinity, and oxidative stress), yeast (ethanol exposure and oxidative stress), Caenorhabditis elegans (lipid peroxidation), and mammals (oxidative stress and lipid peroxidation). Recent studies have also identified ALDH activity as an important feature of cancer stem cells. In these cells, ALDH expression helps abrogate oxidative stress and imparts resistance against chemotherapeutic agents such as oxazaphosphorine, taxane, and platinum drugs. The ALDH superfamily represents a fundamentally important class of enzymes that contributes significantly to the management of electrophilic/oxidative stress within living systems. Mutations in various ALDHs are associated with a variety of pathological conditions in humans, highlighting the fundamental importance of these enzymes in physiological and pathological processes.

Update of the human secretoglobin (SCGB) gene superfamily and an example of 'evolutionary bloom' of androgen-binding protein genes within the mouse Scgb gene superfamily.

The secretoglobins (SCGBs) comprise a family of small, secreted proteins found in animals exclusively of mammalian lineage. There are 11 human SCGB genes and five pseudogenes. Interestingly, mice have 68 Scgb genes, four of which are highly orthologous to human SCGB genes; the remainder represent an 'evolutionary bloom' and make up a large gene family represented by only six counterparts in humans. SCGBs are found in high concentrations in many mammalian secretions, including fluids of the lung, lacrimal gland, salivary gland, prostate and uterus. Whereas the biological activities of most individual SCGBs have not been fully characterised, what already has been discovered suggests that this family has an important role in the modulation of inflammation, tissue repair and tumorigenesis. In mice, the large Scgb1b and Scgb2b gene families encode the androgen-binding proteins, which have been shown to play a role in mate selection. Although much has been learned about SCGBs in recent years, clearly more research remains to be done to allow a better understanding of the roles of these proteins in human health and disease. Such information is predicted to reveal valuable novel drug targets for the treatment of inflammation, as well as designing biomarkers that might identify tissue damage or cancer.

Aldehyde dehydrogenase 1B1: molecular cloning and characterization of a novel mitochondrial acetaldehyde-metabolizing enzyme.

Ethanol-induced damage is largely attributed to its toxic metabolite, acetaldehyde. Clearance of acetaldehyde is achieved by its oxidation, primarily catalyzed by the mitochondrial class II aldehyde dehydrogenase (ALDH2). ALDH1B1 is another mitochondrial aldehyde dehydrogenase (ALDH) that shares 75% peptide sequence homology with ALDH2. Recent population studies in whites suggest a role for ALDH1B1 in ethanol metabolism. However, to date, no formal documentation of the biochemical properties of ALDH1B1 has been forthcoming. In this current study, we cloned and expressed human recombinant ALDH1B1 in Sf9 insect cells. The resultant enzyme was purified by affinity chromatography to homogeneity. The kinetic properties of purified human ALDH1B1 were assessed using a wide range of aldehyde substrates. Human ALDH1B1 had an exclusive preference for NAD(+) as the cofactor and was catalytically active toward short- and medium-chain aliphatic aldehydes, aromatic aldehydes, and the products of lipid peroxidation, 4-hydroxynonenal and malondialdehyde. Most importantly, human ALDH1B1 exhibited an apparent K(m) of 55 µM for acetaldehyde, making it the second low K(m) ALDH for metabolism of this substrate. The dehydrogenase activity of ALDH1B1 was sensitive to disulfiram inhibition, a feature also shared with ALDH2. The tissue distribution of ALDH1B1 in C57BL/6J mice and humans was examined by quantitative polymerase chain reaction, Western blotting, and immunohistochemical analysis. The highest expression occurred in the liver, followed by the intestinal tract, implying a potential physiological role for ALDH1B1 in these tissues. The current study is the first report on the expression, purification, and biochemical characterization of human ALDH1B1 protein.

Update of human and mouse forkhead box (FOX) gene families.

The forkhead box (FOX) proteins are transcription factors that play complex and important roles in processes from development and organogenesis to regulation of metabolism and the immune system. There are 50 FOX genes in the human genome and 44 in the mouse, divided into 19 subfamilies. All human FOX genes have close mouse orthologues, with one exception: the mouse has a single Foxd4 , whereas the human gene has undergone a recent duplication to a total of seven ( FOXD4 and FOXD4L1 --> FOXD4L6 ). Evolutionarily ancient family members can be found as far back as the fungi and metazoans. The DNA-binding domain, the forkhead domain, is an example of the winged-helix domain, and is very well conserved across the FOX family and across species, with a few notable exceptions in which divergence has created new functionality. Mutations in FOX genes have been implicated in at least four familial human diseases, and differential expression may play a role in a number of other pathologies - ranging from metabolic disorders to autoimmunity. Furthermore, FOX genes are differentially expressed in a large number of cancers; their role can be either as an oncogene or tumour suppressor, depending on the family member and cell type. Although some drugs that target FOX gene expression or activity, notably proteasome inhibitors, appear to work well, much more basic research is needed to unlock the complex interplay of upstream and downstream interactions with FOX family transcription factors.

Update of human and mouse matrix metalloproteinase families.

Matrix metalloproteinases (MMPs) are a family of zinc proteases that degrade most of the components of the extracellular matrix (ECM). MMPs also have a number of non-traditional roles in processing factors related to cell growth/proliferation, inflammation and more. There are 23 human MMPs and 23 mouse MMPs, most of which share orthology among most vertebrates; other examples have been found in invertebrates and plants. MMPs are named in order of discovery, but also have been grouped by domain structure or by phylogenetic analysis. MMPs are multi-domain proteins which generally contain a signal sequence; propeptide (which keeps the protein inactive until cleaved); catalytic domain; and a hemopexin-like domain (which provides substrate specificity). MMPs are thought to play a role in many disease states, including arthritis, vascular disease, lung injury, wound repair, cancer and various neurodegenerative disorders. Although there has been much clinical interest in MMP inhibitors (MMPIs), few trials have been successful - often due to the broad nature of inhibition and the complex role of different MMPs in a given disease state.

Impact of duration versus frequency of probing by Homalodisca vitripennis (Hemiptera: Cicadellidae) on inoculation of Xylella fastidiosa.

Successful infection of the plant pathogenic bacterium Xylella fastidiosa (Wells) from an infected plant to a new host involves three main steps: 1) acquisition of the bacterium by a vector; 2) inoculation of a noninfected host plant by the vector; and 3) establishment of sufficient titers of X. fastidiosa in the host plant to sustain a chronic infection. Understanding the basic biology of the transmission process is a key to limiting the spread of plant diseases induced by X. fasdidiosa and reducing agricultural losses, especially those experienced in California since the introduction of a new vector, Homalodisca vitripennis (Germar) (Hemiptera, Cicadellidae) (formerly H. coagulata Say), the glassy-winged sharpshooter. In this study, H. vitripennis adults that acquired X. fastidiosa were allowed access to chrysanthemum plant cuttings for 30, 60, 90, or 120 min. The numbers of X. fastidiosa acquired (i.e., cells present in the insect foregut) and the number inoculated to the plant cuttings were separately determined using quantitative real-time polymerase chain reaction (PCR). In addition, the number of times glassy-winged sharpshooter stylets probed plant cuttings and the amount of time glassy-winged sharpshooter spent actively ingesting were monitored using video surveillance. Linear regression did not indicate a relationship between the number of X. fastidiosa cells inoculated into the plant cutting and either the titer of pathogen present in the insect or amount of time spent ingesting per probe. However, the number of probes significantly influenced the number of X. fastidiosa cells inoculated. Due to the highly variable nature of transmission, our model could not account for all observed variation as indicated by low R2 values. However, our results suggest that the mechanism of transmission is dependent on probing behaviors more than ingestion duration.