Background Sperm acrosomal SLLP1 binding (SAS1B) protein is found in oocytes, which is necessary for sperm-oocyte interaction, and also in uterine and pancreatic cancers. Anti-SAS1B antibody-drug conjugates (ADCs) arrested growth in these cancers. However, SAS1B expression in cancers and normal tissues has not been characterized. We hypothesized that SAS1B is expressed on the surface of other common solid cancer cells, but not on normal tissue cells, and might be selectively targeted therapeutically.Methods SAS1B expression in human normal and cancer tissues was determined by immunohistochemistry, and complementary DNA (cDNA) libraries were employed to PCR amplify human SAS1B and its transcripts. Monoclonal antibodies (mAbs) to human SAS1B were generated using mouse hybridomas. SAS1B deletion constructs were developed to map SAS1B’s epitope, enabling the creation of a blocking peptide. Indirect immunofluorescence (IIF) of human transfected normal and cancer cells was performed to assess SAS1B expression. SAS1B intracellular versus surface expression in normal and tumor tissues was evaluated by flow cytometry after staining with anti-SAS1B mAb, with specificity confirmed with the blocking peptide. Human cancer lines were treated with increasing mAb and ADC concentrations. ATP was quantitated as a measure of cell viability.Results SAS1B expression was identified in a subset of human cancers and the cytoplasm of pancreatic islet cells. Two new SAS1B splice variants were deduced. Monoclonal antibodies were generated to SAS1B splice variant A. The epitope for mAbs SB2 and SB5 is between SAS1B amino acids 32–39. IIF demonstrated intracellular SAS1B expression in transfected kidney cells and on the cell surface of squamous cell lung carcinoma. Flow cytometry demonstrated intracellular SAS1B expression in all tumors and some normal cells. However, surface expression of SAS1B was identified only on cancer cells. SB2 ADC mediated dose-dependent cytotoxic killing of multiple human cancer lines.Conclusion SAS1B is a novel cancer-oocyte antigen with cell surface expression restricted to cancer cells. In vitro, it is an effective target for antibody-mediated cancer cell lysis. These findings support further exploration of SAS1B as a potential therapeutic cancer target in multiple human cancers, either with ADC or as a chimeric antigen receptor-T (CAR-T) cell target.

The leaf-mining fly Hydrellia egeriae Rodrigues-Junior (Diptera: Ephydridae) was evaluated under quarantine conditions as a candidate biological control agent of the invasive aquatic weed Egeria densa Planchon (Hydrocharitaceae) in California, U.S.A. The objective of this study was to test the fly’s feeding preference for E. densa as compared to the North American native Elodea canadensis Michaux (Hydrocharitaceae), an important benchmark species. No choice testing revealed that H. egeriae can complete development on E. canadensis and choice testing indicated the fly presents little oviposition discrimination between E. densa and E. canadensis. It is concluded that the host range of H. egeriae is too broad for use as a biological control agent of E. densa in the U.S.A.

This report summarises efforts to establish Diorhabda carinulata (Desbrochers) and D. elongata (Brulle) in California for the control of invasive saltcedars (Tamarix spp.), which degrade riparian ecosystems in the western United States. Over 14,000 D. carinulata individuals were released in California among four locations between 1999 and 2002 but beetles only established at the Tinemaha Reservoir site, the most eastern release location. More than 236,000 D. elongata individuals were released between 13 sites from 2003-2009 and establishment was limited to two sites, along the Cache and Pope creeks in northwestern California. The D. carinulata population did not disperse beyond the release area despite the presence of nearby (ca. 20 km) patches of the host plant. In contrast, D. elongata spread along Cache Creek and branches of related tributaries within the same watershed at ca. 14 km per year. A survey of 122 Tamarix stands across 15 California counties revealed that neither introduced beetle colonised other host patches, including those in neighbouring watersheds. Despite exclusive use of T. parviflora for ca. 36 generations, field collected D. elongata adults demonstrated strong preferences for T. ramosissima over T. parviflora when selecting both resting and ovipositional sites in caged choice tests. The proportion of D. elongata ovipositing on T. parviflora varied over time but with no clear trend of shifting host preference despite strong selection pressure. Explanations for the limited establishment and spread of Diorhabda spp. as well as impact to the target weeds are discussed.

Successful therapeutic options remain elusive for pancreatic cancer. The exquisite sensitivity and specificity of humoral and cellular immunity may provide therapeutic approaches if antigens specific for pancreatic cancer cells can be identified. Here we characterize SAS1B (ovastacin, ASTL, astacin-like), a cancer-oocyte antigen, as an attractive immunotoxin target expressed at the surface of human pancreatic cancer cells, with limited expression among normal tissues. Immunohistochemistry shows that most pancreatic cancers are SAS1Bpos (68%), while normal pancreatic ductal epithelium is SAS1Bneg. Pancreatic cancer cell lines developed from patient-derived xenograft models display SAS1B cell surface localization, in addition to cytoplasmic expression, suggesting utility for SAS1B in multiple immunotherapeutic approaches. When pancreatic cancer cells were treated with an anti-SAS1B antibody-drug conjugate, significant cell death was observed at 0.01-0.1 μg/mL, while SAS1Bneg human keratinocytes were resistant. Cytotoxicity was correlated with SAS1B cell surface expression; substantial killing was observed for tumors with low steady state SAS1B expression, suggesting a substantial proportion of SAS1Bpos tumors can be targeted in this manner. These results demonstrate SAS1B is a surface target in pancreatic cancer cells capable of binding monoclonal antibodies, internalization, and delivering cytotoxic drug payloads, supporting further development of SAS1B as a novel target for pancreatic cancer.

Testis‐specific serine/threonine kinase 2 (TSSK2) is an important target for reversible male contraception. A high‐throughput screen of ≈17 000 compounds using a mobility shift assay identified two potent series of inhibitors having a pyrrolopyrimidine or pyrimidine core. The pyrrolopyrimidine 10 (IC50 22 nm; GSK2163632A) and the pyrimidine 17 (IC50 31 nm; ALK inhibitor 1) are the most potent TSSK2 inhibitors in these series, which contain the first sub‐100 nanomolar inhibitors of any TSSK isoform reported, except for the broad kinase inhibitor staurosporine. The novel, potent pyrimidine TSSK2 inhibitor compound 19 (IC50 66 nm; 2‐[[5‐chloro‐2‐[2‐methoxy‐4‐(1‐methylpiperidin‐4‐yl)anilino]pyrimidin‐4‐yl]amino]‐N‐methylbenzenesulfonamide) lacks the potential for metabolic activation. Compound 19 had a potency rank order of TSSK1>TSSK2>TSSK3>TSSK6, indicating that potent dual inhibitors of TSSK1/2 can be identified, which may be required for a complete contraceptive effect. The future availability of a TSSK2 crystal structure will facilitate structure‐based discovery of selective TSSK inhibitors from these pyrrolopyrimidine and pyrimidine scaffolds.

Microtubule-targeting drugs are widely used as cancer chemotherapeutic agents and they have been shown to inhibit mitotic progression and interphase signaling. Despite their use as a first line treatment for cancer, many patients develop resistance to microtubule-targeting drugs leading to early relapse and shorter survival. Moreover, the quality of life for patients who do survive is often substantially reduced due to the toxicity associated with these drugs. Therefore, identification of new factors that determine the effectiveness of microtubule-targeting agents will not only facilitate a better understanding of the mechanisms of acquired drug resistance but will also be amenable to therapeutic interventions. We discovered a novel microtubule associated protein “Matrin 3 (MATR3)” that is known to bind to RNA and play a critical role in RNA transport and RNA stabilization. Our results revealed that MATR3 acts as a potent tumor suppressor as it inhibits long-term growth, migration, invasion as well as tumor growth of triple negative breast cancer (TNBC) cells in vivo. We demonstrated that MATR3 overexpression induced cell cycle arrest and apoptosis in TNBC cells. Furthermore, analysis of breast cancer samples showed a significantly lower expression of MATR3 when compared to normal adjacent tissues. Importantly, our RNA immunoprecipitation (RIP)-seq analysis showed that MATR3 controls expression of several mitotic spindle organizing proteins by binding to their RNA. Interestingly, we found that these MATR3 regulated proteins were highly altered in breast cancer patients. In conclusion, we identified a novel RNA-binding protein that inhibits breast cancer growth and progression suppressor by regulating microtubule dynamics. Citation Format: Panneerdoss Subbarayalu, Subapriya Rajamanickam, Suryavathi Viswanadhapalli, Benjamin C. Onyeagucha, Vijay K. Eedunuri, Nicholas Dybdal-Hargreaves, Santosh Timilsina, Hima Bansal, Sanjay Bansal, Tabrez Mohammad, Yidong Chen, John C. Herr, Susan L. Mooberry, Manjeet K. Rao. A novel microtubule associated RNA binding protein matrin 3 act as a tumor suppressor by regulating mitotic spindle organizing proteins in triple negative breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3658.

Head and neck squamous cell carcinoma (HNSCC) is the most common cancer of the head and neck region with a low overall 5‐year survival rate of 50%, highlighting the need for more effective therapeutics. A novel cancer‐oocyte biomarker, SAS1B ( s perm a crosomal S LLP1 b inding protein), presents substantial opportunities for development of novel diagnostics and therapeutics for patients with HNSCC. SAS1B has been recently discovered as a membrane‐associated zinc metalloprotease expressed in growing oocytes housed in the secondary follicle stage through ovulation, and is virtually absent in other tissues. SAS1B has been identified in approximately 77% uterine tumors including 74% endometrioid and 87% malignant mixed Mullerian tumors (MMMT). Given the range of cancers expressing SAS1B, probing for SAS1B gene and protein may increase the number of possible cancer patients in which a therapeutic method targeting SAS1B would potentially be beneficial. Twenty‐four primary human tumor specimens and eight HNSCC human‐derived cell lines were analyzed for ASTL message and SAS1B protein. A PCR assay unique to ASTL/SAS1B, excluding all of the other 134 human metalloproteases, has been developed with validated primers to amplify various domains of ASTL/SAS1B transcripts. SAS1B was identified in all samples of cell lines and primary tumors [100% incidence] using primers spanning a part of the c‐terminus of SAS1B. In addition, 46kDa and 65kDa SAS1B isoforms have been identified by Western blot using anti‐SAS1B polyclonal antibodies. To test the possibility of SAS1B as an immunotherapeutic target for HNSCC, live cell staining of an HNSCC cell line, OSC19 was carried out using indirect immunofluorescence with anti‐SAS1B polyclonal antibodies revealing cell surface localization of SAS1B. Future experiments will focus on in‐vitro cell killing assays using a duocarmycin‐conjugated antibody to test internalization of the antibody drug conjugate and subsequent growth arrest capabilities. The restricted localization of SAS1B presents opportunities for development of novel diagnostics and highly selective treatment approaches that spare both normal tissues and the ovarian reserve, thus potentially preventing unwanted negative side effects common in currently available therapeutics. Support or Funding Information n/A

Spermatozoa must penetrate the outer investments of the oocyte, the cumulus oophorus and the zona pellucida (ZP), in order for fertilization to occur. This may require exposure of enzymes on the sperm's inner acrosomal membrane (IAM), one of which is matrix metalloproteinase (MMP) 2, to factors in oviductal fluid. Plasminogen is present in oviductal fluid and activates MMP2 in somatic tissues. The objectives of this study were: 1) to examine possible interactions between plasminogen and IAM-bound plasminogen activator receptor (SAMP14) and -MMP2, 2) to demonstrate plasminogen's presence in the extracellular environment at the site of fertilization, and 3) to provide evidence that plasminogen plays a role in fertilization. Zymographs of sonicated bull and rat sperm extracts incubated with plasmin and/or plasminogen (plasmin/ogen) showed acceleration of initiation of MMP2 activity in concentrations as low as 1 μg/ml. Immunohistochemical and immunofluorescence analysis of plasmin/ogen revealed its presence in the cytoplasm of mouse ovarian and oviductal oocytes, oviductal epithelium, around the ZP, and amongst the cumulus cells. We modified the standard in vitro fertilization (IVF) approach to more closely mimic natural fertilization by reducing sperm concentration during insemination by ∼100× and also comparing cumulus-intact and denuded oocytes. In mice, addition of plasminogen in IVF medium significantly improved fertilization, while MMP2 antibody significantly inhibited sperm penetration in these conditions. IVF improvement by plasminogen was blocked by SAMP14 antibody. Furthermore, MMP2 antibody inhibition was coincident with a failure by spermatozoa to disperse the cumulus oophorus. We provide evidence that plasminogen on its own and through an MMP2-related mechanism improves the ability of oocytes to be fertilized, and demonstrate its effect in sperm penetration of oocyte investments.

The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP K-m values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 mu M, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing as 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents. (C) 2016 Elsevier Inc. All rights reserved.

The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation.Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus.A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes.Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement.A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death.The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathway, and tumor cell growth arrest induced by antibody-toxin conjugates suggest therapeutic approaches that would selectively target tumors while limiting adverse drug effects in healthy cells.The SAS1B metalloproteinase is proposed as a prototype cancer-oocyte tumor surface neoantigen for development of targeted immunotherapeutics with limited on-target/off tumor effects predicted to be restricted to the population of growing oocytes.

Abstract Microtubules are highly dynamic components of the cytoskeleton that play an important role in a wide range of cellular processes including cell division, cell motility and intracellular transport. Increasing evidence suggests that alterations in microtubule dynamics are critical for cancer growth and metastasis. Microtubule associated proteins (MAPs) regulate microtubule dynamics and consequently can affect sensitivity of cancer cells to microtubule targeting drugs. We discovered a novel microtubule associated protein "Matrin 3 (MATR3)" that is known to bind to RNA and play a critical role in RNA transport and RNA stabilization. Immunofluorescence analyses revealed that although MATR3 is predominantly a nuclear protein, it translocates to the cytoplasm and interacts with microtubules when breast cancer cells are treated with paclitaxel. We show that MATR3 associates with stabilized microtubules and it mediates microtubule polymerization in a taxol-dependent manner. Interestingly, our results reveal that MATR3 acts as a effective tumor suppressor as it inhibits breast cancer colony formation, migration and invasion of breast cancer cells in addition to suppressing breast tumor growth in vivo. Analysis of breast cancer samples showed a significantly lower expression of MATR3 when compared to normal adjacent tissues. Our results indicate the possibility that MATR3 mediates its tumor suppressor function by binding and regulating proteins that are known to affect microtubule dynamics. We believe that nuclear-cytoplasmic shuttling of MATR3 is critical for stability of key proteins that might regulate paclitaxel-dependent microtubule dynamics and subsequently cellular effects in cancer cells. Citation Format: Panneerdoss Subbarayalu, Subapriya Rajamanickam, Suryavathi Viswanadhapalli, Nicholas Dybdal-Hargreaves, Santosh Timilsina, Sanjay Bansal, Hima Bansal, Tabrez Mohammad, Yidong Chen, John C Herr, Susan L Mooberry, Manjeet K Rao. Matrin 3: A novel micro-tubule associated RNA binding protein that acts as a potent tumor suppressor [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-09.

SAS1B (or Ovastacin) is considered an ideal candidate for both female contraception and for cancer therapies. SAS1B first appears in oocytes of follicles at the primary‐secondary transition in pre‐pubertal rodent ovaries and beyond. Quiescent oocytes within primordial and primary follicles are SAS1B negative. In 7 eutherian adult ovaries, the precise temporal and spatial onset of SAS1B expression was conserved. Crystal studies are a vital tool for drug design and for better understanding the molecular structure of targetable proteins, such as SAS1B. Full length SAS1B and its mature form (lacking the N terminus) were PCR‐cloned using gene specific primers sequence containing a kozak sequence at the N‐terminus and six histidine tags at the C‐terminus, using the TOPO cloning strategy. The PCR products were introduced into a pcDNA 3.4 vector and transformed into chemically competent E. Coli cells. Cells were plated on ampicillin infused Luria agar plates and colonies were allowed to grow overnight. Colonies were inoculated in fresh ampicillin containing Luria broth and DNA was extracted using a miniprep kit. Eluted DNA was sent for DNA sequencing. One of the full length clones, clone #9, showed the correct insert for full length SAS1B while screening of E. Coli colonies for the correct sequence for matured SAS1B is ongoing. Subsequently, once the correct sequences have been confirmed through sequencing, transfection of the correct vector‐insert species will be conducted using Expi293 mammalian cells, followed by protein isolation, detection and purification for the purpose of obtaining soluble SAS1B to generate crystals.

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization.

ESP1/SPESP1 is a testis-specific, postmeiotic gene expressed in round spermatids that encodes equatorial segment protein 1, an intra-acrosomal protein found in the acrosomal matrix and on the luminal surface of the inner and outer acrosomal membranes within the equatorial segment domain of mature spermatozoa. A comparison of testicular protein extracts with caput, corpus, and caudal epididymal sperm proteins revealed striking differences in the apparent masses of SPESP1 isoforms. The predominant isoforms of SPESP1 in the testis were 77 and 67 kDa, with 47-kDa forms present to a minor degree. In contrast, SPESP1 isoforms of 47 and 43 kDa were found in caput, corpus, and caudal sperm, indicating that SPESP1 undergoes noticeable mass changes during spermiogenesis and/or subsequent transport to the epididymis. On two-dimensional (2D) SDS-PAGE, testicular SPESP1 isoforms resolved as a train of pI values from 4.9 to 5.2. Immunoprecipitated 77-kDa SPESP1 from testis reacted with the glycoprofile stain after one-dimensional and 2D gel electrophoresis, indicating that the 77-kDa testicular isoform was highly glycosylated. One charge variant of the 67-kDa isoform was also glycoprofile positive after 2D gel resolution. The 47- and 43-kDa isoforms of SPESP1 from epididymal sperm did not stain with glycoprofile, suggesting an absence of, or few, glycoprofile-sensitive glycoconjugates in epididymal SPESP1. Treatment of testicular extracts with a variety of glycosidases resulted in mass shifts in immunoreactive SPESP1, indicating that testicular SPESP1 was glycosylated and that terminal sialic acid, N- and O-glycans were present. A mixture of deglycosidase enzymes (including PNGase-F, neuraminidase, beta1-4 galactosidase, endo-alpha-N-acetylgalactosaminidase, and beta N-acetyl-glucosaminidase) completely eliminated the 77- and 67-kDa SPESP1 bands and resulted in the appearance of 75-, 60-, 55-, 50-, 47-, and 43-kDa forms, confirming that both the 77- and 67-kDa testicular forms of SPESP1 contain complex carbohydrate residues. Treatment of caudal epididymal sperm with PNGase-F enzymes showed a faint deglycosylated band at 30 kDa, but neuraminidase did not result in any molecular shift, indicating that epididymal sperm SPESP1 did not contain sialic acid/N-acetylglucosamine residues. These findings are consistent with the hypothesis that SPSPESP1 undergoes significant glycosylation in the testis and that the majority of these glycoconjugates are removed by the time sperm reach the caput epididymis. Studies of the fate of SPESP1 after the acrosome reaction localized SPESP1 to the equatorial segment region in both noncapacitated and capacitated, acrosome-reacted sperm. During capacitation, SPESP1 underwent proteolysis, resulting in a 27-kDa fragment. Zona-free oocytes incubated with recSPESP1 protein showed complementary binding sites on the microvillar oolemmal domain. Both recSPESP1 and anti-recSPESP1 antibody inhibited in vitro fertilization.

When laboratory host specificity tests on weed biological control agents produce ambiguous results or are suspected of producing false-positive findings, field cage or open field tests can be used to help determine the true ecological host range of the agent. The leaf beetle Diorhabda elongata (Brullé) from Crete, imported to the United States for the control of saltcedar (Tamarix spp., Tamaricaceae), showed a low but variable ovipositional response to nontarget Frankenia spp. (Frankeniaceae) in previous laboratory tests conducted in small cages, where up to 11.4% of eggs were laid on these native plants. Results from field tests presented in this article show that no eggs were laid on Frankenia palmeri S. Watson and significantly more eggs were always laid on Tamarix ramosissima Ledebour than Frankenia salina (Molina) I. M. Johnston. Furthermore, the ovipositional response to F. salina was substantially lower than that recorded in laboratory tests. The percent of eggs laid on F. salina in field tests was 3.7 in a paired choice cage test, 4.3 in a multiple choice cage test, and 2.5 in a multiple choice open field test, suggesting that the true acceptance rate of the nontarget by D. elongata in the field will be lower than laboratory tests predicted. However, some damage was caused to F. salina by adult and larval feeding in the field, although this occurred only at the very end of the open field test, when D. elongata densities were extremely high, and all of the surrounding saltcedar had been totally defoliated. Scientific representatives from various stakeholder organizations (state, county, university, and environmental groups) viewed the open field test when in progress and reviewed the final results before advising State regulatory agencies on beetle redistribution. These test results, and the open review process, led regulators to conclude that redistribution of D. elongata in California was warranted owing to its significant ability to defoliate saltcedar, and its low rate of feeding on nontarget Frankenia spp. The introduction of D. elongata provides an interesting case study for risk assessment of a potentially efficacious weed biocontrol agent that may also be capable of using nontarget native plants.