Long-term efficacy and safety of porcine islet macrobeads in nonimmunosuppressed diabetic cynomolgus macaques.

Although human islet transplantation has proven to provide clinical benefits, especially the near complete amelioration of hypoglycemia, the supply of human islets is limited and insufficient to meet the needs of all people that could benefit from islet transplantation. Porcine islets, secreting insulin nearly identical to that of human insulin, have been proposed as a viable supply of unlimited islets. Further, encapsulation of the porcine islets has been shown to reduce or eliminate the use of immunosuppressive therapy that would be required to prevent rejection of the foreign islet tissue. The goal of the current study was to determine the long-term safety and efficacy of agarose encapsulated porcine islets (macrobeads) in diabetic cynomolgus macaques, in a study emulating a proposed IND trial in which daily exogenous insulin therapy would be reduced by 50% with no loss of glucose regulation. Four of six animals implanted with macrobeads demonstrated ≥ 30% reduction in insulin requirements in year 1 of follow-up. Animals were followed for 2, 3.5, and 7.4 years with no serious adverse events, mortality or evidence of pathogen transmission. This study supports the continued pursuit of encapsulated porcine islet therapy as a promising treatment option for diabetes mellitus.

Multiple clinically relevant immunotherapies prolong the function of microencapsulated porcine islet xenografts in diabetic NOD mice without the use of anti-CD154 mAb.

BACKGROUND: Our goal was to identify clinically relevant immunotherapies that synergize with microencapsulation to protect adult porcine islet (API) xenografts in diabetic NOD mice. We have shown previously that dual costimulatory blockade (CTLA4-Ig plus anti-CD154 mAb) combined with encapsulation protects APIs long-term in NOD mice. Since no anti-CD154 mAbs currently are approved for use in humans, we tested the efficacy of other targeted immunosuppression regimens that might be used for diabetic patients receiving encapsulated islets. METHODS: Microencapsulated APIs were transplanted i.p. in diabetic NOD mice given either no immunosuppression or combinations immunosuppressive reagents. Graft function was monitored by blood glucose levels, i.p. glucose tolerance tests, and histology. Mechanisms of rejection were investigated by phenotyping host peritoneal cells and measuring graft site cytokine and chemokine levels. RESULTS: New immunosuppressive therapies were compared to CTLA4-Ig plus anti-CD154 mAb, used here as a control. The most effective was triple treatment with CTLA4-Ig, anti-CD154 mAb, and intracapsular CXCL12, and the next most effective was a non-depleting anti-CD4 mAb (YTS177.9) plus intracapsular CXCL12. Three additional regimens (CTLA4-Ig plus YTS177.9, YTS177.9 alone, and anti-OX40-Ligand mAb alone) significantly prolonged encapsulated API function. Dual treatment with CTLA4-Ig plus anti-CD40 mAb was as effective as CTLA4-Ig plus anti-CD154 mAb. Five other monotherapies and three combination therapies did not augment encapsulated API survival. Most peritoneal cytokines and chemokines were either absent or minimal. At necropsy, the capsules were intact, not fibrosed, and clean when function was maintained, but were coated with host cells if rejection had occurred. CONCLUSIONS: Multiple different immunotherapies which specifically inhibit CD4+ T cells, modulate T-cell trafficking, or interfere with antigen presentation can substitute for anti-CD154 mAb to prolong encapsulated islet xenograft function in diabetic NOD mice.

Testing a Novel Attract-and-Kill Strategy for Drosophila suzukii (Diptera: Drosophilidae) Management.

The invasion of the spotted wing drosophila, Drosophila suzukii Matsumura, across the Americas and Europe has led to increased insecticide applications to protect fruit crops. This insecticide usage conflicts with integrated pest management programs, as well as harvest, export, and pollination services in the affected crops. A novel management tool was assessed against D. suzukii that may mitigate these conflicts. HOOK SWD, an attract-and-kill (A&K) formulation applied as a sprayable bait, was evaluated for three growing seasons in two berry crops in New Jersey and California. In blueberry crops treated with HOOK SWD, fruit infestations by D. suzukii were 2-8 times lower than in untreated crops. In trials in commercial raspberry fields, weekly or biweekly HOOK SWD applications combined with a single grower standard D. suzukii-targeted cover spray resulted in nearly 2-5 times fewer fruit infestations compared to the grower standard cover spray alone. Assays of the residual activity of HOOK SWD resulted in more than 78-93% adult D. suzukii mortality when exposed to raspberry leaves after the formulation had aged for 35 d in the field under plastic hoop houses. These results suggest that this A&K strategy can be integrated in D. suzukii management programs.

A model for determining an effective in vivo dose of transplanted islets based on in vitro insulin secretion.

BACKGROUND: Allogeneic islet transplantation for the treatment of type 1 diabetes often requires multiple implant procedures, from as many as several human pancreas donors, to achieve lasting clinical benefit. Given the limited availability of human pancreases for islet isolation, porcine islets have long been considered a potential option for clinical use. Agarose-encapsulated porcine islets (macrobeads) permit long-term culture and thus a thorough evaluation of microbiological safety and daily insulin secretory capacity, prior to implantation. The goal of this study was the development of a method for determining an effective dose of encapsulated islets based on their measured in vitro insulin secretion in a preclinical model of type 1 diabetes. METHODS: Spontaneously diabetic BioBreeding diabetes-prone rats were implanted with osmotic insulin pumps in combination with continuous glucose monitoring to establish the daily insulin dose required to achieve continuous euglycaemia in individual animals. Rats were then implanted with a 1×, 2× or 3× dose (defined as the ratio of macrobead in vitro insulin secretion per 24 hours to the recipient animal's total daily insulin requirement) of porcine islet macrobeads, in the absence of immunosuppression. In vivo macrobead function was assessed by recipient non-fasted morning blood glucose values, continuous glucose monitoring and the presence of peritoneal porcine C-peptide. At the end of the study, the implanted macrobeads were removed and returned to in vitro culture for the evaluation of insulin secretion. RESULTS: Diabetic rats receiving a 2× macrobead implant exhibited significantly improved blood glucose regulation compared to that of rats receiving a 1× dose during a 30-day pilot study. In a 3-month follow-up study, 2× and 3× macrobead doses initially controlled blood glucose levels equally well, although several animals receiving a 3× dose maintained euglycaemia throughout the study, compared to none of the 2× animals. The presence of porcine C-peptide in rat peritoneal fluid 3 months post-implant and the recurrence of hyperglycaemia following macrobead removal, along with the finding of persistent in vitro insulin secretion from retrieved macrobeads, confirmed long-term graft function. CONCLUSIONS: Increasing dosages of islet macrobeads transplanted into diabetic rats, based on multiples of in vitro insulin secretion matched to the recipient's exogenous insulin requirements, correlated with improved blood glucose regulation and increased duration of graft function. These results demonstrate the usefulness of a standardized model for the evaluation of the functional effectiveness of islets intended for transplantation, in this case using intraperitoneally implanted agarose macrobeads, in diabetic rats. The results suggest that some features of this islet-dosing methodology may be applicable, and indeed necessary, to clinical allogeneic and xenogeneic islet transplantation.

Microencapsulated adult porcine islets transplanted intraperitoneally in streptozotocin-diabetic non-human primates.

BACKGROUND: Xenogeneic donors would provide an unlimited source of islets for the treatment of type 1 diabetes (T1D). The goal of this study was to assess the function of microencapsulated adult porcine islets (APIs) transplanted ip in streptozotocin (STZ)-diabetic non-human primates (NHPs) given targeted immunosuppression. METHODS: APIs were encapsulated in: (a) single barium-gelled alginate capsules or (b) double alginate capsules with an inner, islet-containing compartment and a durable, biocompatible outer alginate layer. Immunosuppressed, streptozotocin-diabetic NHPs were transplanted ip with encapsulated APIs, and graft function was monitored by measuring blood glucose, %HbA1c, and porcine C-peptide. At graft failure, explanted capsules were assessed for biocompatibility and durability plus islet viability and functionality. Host immune responses were evaluated by phenotyping peritoneal cell populations, quantitation of peritoneal cytokines and chemokines, and measurement of anti-porcine IgG and IgM plus anti-Gal IgG. RESULTS: NHP recipients had reduced hyperglycemia, decreased exogenous insulin requirements, and lower percent hemoglobin A1c (%HbA1c) levels. Porcine C-peptide was detected in plasma of all recipients, but these levels diminished with time. However, relatively high levels of porcine C-peptide were detected locally in the peritoneal graft site of some recipients at sacrifice. IV glucose tolerance tests demonstrated metabolic function, but the grafts eventually failed in all diabetic NHPs regardless of the type of encapsulation or the host immunosuppression regimen. Explanted microcapsules were intact, "clean," and free-floating without evidence of fibrosis at graft failure, and some reversed diabetes when re-implanted ip in diabetic immunoincompetent mice. Histology of explanted capsules showed scant evidence of a host cellular response, and viable islets could be found. Flow cytometric analyses of peritoneal cells and peripheral blood showed similarly minimal evidence of a host immune response. Preformed anti-porcine IgG and IgM antibodies were present in recipient plasma, but these levels did not rise post-transplant. Peritoneal graft site cytokine or chemokine levels were equivalent to normal controls, with the exception of minimal elevation observed for IL-6 or IL-1ß, GRO-α, I-309, IP-10, and MCP-1. However, we found central necrosis in many of the encapsulated islets after graft failure, and explanted islets expressed endogenous markers of hypoxia (HIF-1α, osteopontin, and GLUT-1), suggesting a role for non-immunologic factors, likely hypoxia, in graft failure. CONCLUSIONS: With donor xenoislet microencapsulation and host immunosuppression, APIs corrected hyperglycemia after ip transplantation in STZ-diabetic NHPs in the short term. The islet xenografts lost efficacy gradually, but at graft failure, some viable islets remained, substantial porcine C-peptide was detected in the peritoneal graft site, and there was very little evidence of a host immune response. We postulate that chronic effects of non-immunologic factors, such as in vivo hypoxic and hyperglycemic conditions, damaged the encapsulated islet xenografts. To achieve long-term function, new approaches must be developed to prevent this damage, for example, by increasing the oxygen supply to microencapsulated islets in the ip space.

Retention of gene expression in porcine islets after agarose encapsulation and long-term culture.

Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture.

A comprehensive microbiological safety approach for agarose encapsulated porcine islets intended for clinical trials.

BACKGROUND: The use of porcine islets to replace insulin-producing islet ß-cells, destroyed during the diabetogenic disease process, presents distinct challenges if this option is to become a therapeutic reality for the treatment of type 1 diabetes. These challenges include a thorough evaluation of the microbiological safety of the islets. In this study, we describe a robust porcine islet-screening program that provides a high level of confidence in the microbiological safety of porcine islets suitable for clinical trials. METHODS: A four-checkpoint program systematically screens the donor herd (Large White - Yorkshire × Landrace F1 hybrid animals), individual sentinel and pancreas donor animals and, critically, the islet macrobeads themselves. Molecular assays screen for more than 30 known viruses, while electron microscopy and in vitro studies are employed to screen for potential new or divergent (emergent) viruses. RESULTS: Of 1207 monthly samples taken from random animals over a 2-year period, only a single positive result for Transmissible gastroenteritis virus was observed, demonstrating the high level of biosecurity maintained in the source herd. Given the lack of clinical signs, positive antibody titers for Porcine reproductive and respiratory syndrome virus, Porcine parvovirus, and Influenza A confirm the efficacy of the herd vaccination program. Porcine respiratory coronavirus was found to be present in the herd, as expected for domestic swine. Tissue homogenate samples from six sentinel and 11 donor animals, over the same 2-year period, were negative for the presence of viruses when co-cultured with six different cell lines from four species. The absence of adventitious viruses in separate islet macrobead preparations produced from 12 individual pancreas donor animals was confirmed using validated molecular (n = 32 viruses), in vitro culture (cells from four species), and transmission electron microscopy assays (200 cell profiles per donor animal) over the same 2-year period. There has been no evidence of viral transmission following the implantation of these same encapsulated and functional porcine islets into non-immunosuppressed diabetic cynomolgus macaques for up to 4 years. Isolated peripheral blood mononuclear cells from all time points were negative for PCV (Type 2), PLHV, PRRSV, PCMV, and PERV-A, PERV-B, and PERV-C by PCR analysis in all six recipient animals. CONCLUSION: The four-checkpoint program is a robust and reliable method for characterization of the microbiological safety of encapsulated porcine islets intended for clinical trials.

Sucrose Improves Insecticide Activity Against Drosophila suzukii (Diptera: Drosophilidae).

The addition of sucrose to insecticides targeting spotted wing drosophila, Drosophila suzukii (Matsumura), enhanced lethality in laboratory, semifield, and field tests. In the laboratory, 0.1% sucrose added to a spray solution enhanced spotted wing drosophila feeding. Flies died 120 min earlier when exposed to spinosad residues at label rates enhanced with sucrose. Added sucrose reduced the LC50 for dried acetamiprid residues from 82 to 41 ppm in the spray solution. Laboratory bioassays of spotted wing drosophila mortality followed exposure to grape and blueberry foliage and/or fruit sprayed and aged in the field. On grape foliage, the addition of 2.4 g/liter of sugar with insecticide sprays resulted in an 11 and 6% increase of spotted wing drosophila mortality at 1 and 2 d exposures to residues, respectively, averaged over seven insecticides with three concentrations. In a separate experiment, spinetoram and cyantraniliprole reduced by 95-100% the larval infestation of blueberries, relative to the untreated control, 7 d after application at labeled rates when applied with 1.2 g/liter sucrose in a spray mixture, irrespective of rainfall; without sucrose infestation was reduced by 46-91%. Adding sugar to the organically acceptable spinosyn, Entrust, reduced larval infestation of strawberries by >50% relative to without sugar for five of the six sample dates during a season-long field trial. In a small-plot field test with blueberries, weekly applications in alternating sprays of sucrose plus reduced-risk insecticides, spinetoram or acetamiprid, reduced larval infestation relative to the untreated control by 76%; alternating bifenthrin and phosmet (without sucrose) reduced infestation by 65%.

Relative toxicity and residual activity of insecticides used in blueberry pest management: mortality of natural enemies.

A series of bioassays were conducted to determine the relative toxicities and residual activities of insecticides labeled for use in blueberry (Vaccinium corymbosum L.) on natural enemies, to identify products with low toxicity or short duration effects on biological control agents. In total, 14 insecticides were evaluated using treated petri dishes and four commercially available natural enemies (Aphidius colemani Viereck, Orius insidiosus [Say], Chrysoperla rufilabris [Burmeister], and Hippodamia convergens [Guérin-Menéville]). Dishes were aged under greenhouse conditions for 0, 3, 7, or 14 d before introducing insects to test residual activity. Acute effects (combined mortality and knockdown) varied by insecticide, residue age, and natural enemy species. Broad-spectrum insecticides caused high mortality to all biocontrol agents, whereas products approved for use in organic agriculture had little effect. The reduced-risk insecticide acetamiprid consistently caused significant acute effects, even after aging for 14 d. Methoxyfenozide, novaluron, and chlorantraniliprole, which also are classified as reduced-risk insecticides, had low toxicity, and along with the organic products could be compatible with biological control. This study provides information to guide blueberry growers in their selection of insecticides. Further research will be needed to determine whether adoption of a pest management program based on the use of more selective insecticides will result in higher levels of biological control in blueberry.

Electric pulse exposure reduces AAV8 dosage required to transduce HepG2 cells.

We demonstrate that applying electric field pulses to hepatocytes, in vitro, in the presence of enhanced green fluorescent protein (EGFP)-expressing adeno-associated virus (AAV8) vectors reduces the viral dosage required for a given transduction level by more than 50-fold, compared to hepatocytes exposed to AAV8-EGFP vectors without electric field pulse exposure. We conducted 48 experimental observations across 8 exposure conditions in standard well plates. The electric pulse exposures involved single 80-ms pulses with 375 V/cm field intensity. Our study suggests that electric pulse exposure results in enhanced EGFP expression in cells, indicative of increased transduction efficiency. The enhanced transduction observed in our study, if translated successfully to an in vivo setting, would be a promising indication of potential reduction in the required dose of AAV vectors. Understanding the effects of electric field pulses on AAV transduction in vitro is an important preliminary step.

Monitoring of Spotted-Wing Drosophila (Diptera: Drosophilidae) Resistance Status Using a RAPID Method for Assessing Insecticide Sensitivity Across the United States.

Drosophila suzukii (Matsumura) has spread rapidly, challenging berry and cherry crop production due to its ability to lay eggs into ripening fruit. To prevent infestation by this pest, insecticides are applied during fruit ripening and harvest. We field-tested the Rapid Assessment Protocol for IDentification of resistance in D. suzukii (RAPID) on seventy-eight populations collected across eight U.S. states in 2017 and 2018. Exposure to LC50 rates of malathion, methomyl, spinetoram, spinosad, and zeta-cypermethrin led to average female fly mortality of 25.0% in 2017, and after adjusting concentrations the average was 39.9% in 2018. Using LC99 × 2 discriminating concentrations in 2017 and LC90 × 8 rates in 2018, average female mortalities were 93.3% and 98.5%, respectively, indicating high overall susceptibility. However, using these high concentrations we found 32.0% of assays with survival of some female flies in 2017 and 27.8% in 2018. The adjustment in discriminating dose from 2017 to 2018 also reduced the proportion of assays with <90% survival from 17.6 to 2.9%. Populations with low mortality when exposed to spinosad were identified using this assay, triggering more detailed follow-up bioassays that identified resistant populations collected in California coastal region berry crops. Widespread evaluations of this method and subsequent validation in California, Michigan, and Georgia in 2019-2021 show that it provides a quick and low-cost method to identify populations of D. suzukii that warrant more detailed testing. Our results also provide evidence that important insecticide classes remain effective in most U.S. regions of fruit production.

Entomopathogenic Nematodes for the Management of Plum Curculio in Highbush Blueberry.

Conotrachelus nenuphar Herbst (Coleoptera: Curculionidae) is a key pest of stone and pome fruits in the United States. Application of certain entomopathogenic nematode (EPN) species has shown efficacy in some crops when targeting the larval stage of C. nenuphar in soil. To date, however, no EPNs have been tested for the control of this pest in highbush blueberries. In 2020, laboratory and field studies were conducted to: (1) determine the persistence of Steinernema riobrave, S. carpocapsae, S. feltiae, and Heterorhabditis bacteriophora in acidic blueberry soil; (2) compare the virulence of these EPNs to C. nenuphar larvae and pupae; and (3) compare the efficacy of these EPN species to control this pest in blueberry fields. The greatest persistence in blueberry soil was exhibited by S. riobrave followed by S. carpocapsae. Superior virulence was observed in S. riobrave against C. nenuphar larvae and pupae. Promising levels of virulence were also observed in S. carpocapsae and S. feltiae against the larvae, but S. scarabaei had low virulence. In the field, S. riobrave provided significantly higher levels of C. nenuphar suppression (90%) than the other EPNs. The field efficacy of S. riobrave against C. nenuphar at low and high rates was confirmed in 2021. Steinernema riobrave has the potential to become an important component in the management of C. nenuphar in highbush blueberry.

Application of Plant Defense Elicitors Fails to Enhance Herbivore Resistance or Mitigate Phytoplasma Infection in Cranberries.

Synthetic elicitors of the salicylic acid (SA) and jasmonic acid (JA) plant defense pathways can be used to increase crop protection against herbivores and pathogens. In this study, we tested the hypothesis that elicitors of plant defenses interact with pathogen infection to influence crop resistance against vector and nonvector herbivores. To do so, we employed a trophic system comprising of cranberries (Vaccinium macrocarpon), the phytoplasma that causes false blossom disease, and two herbivores-the blunt-nosed leafhopper (Limotettix vaccinii), the vector of false blossom disease, and the nonvector gypsy moth (Lymantria dispar). We tested four commercial elicitors, including three that activate mainly SA-related plant defenses (Actigard, LifeGard, and Regalia) and one activator of JA-related defenses (Blush). A greenhouse experiment in which phytoplasma-infected and uninfected plants received repeated exposure to elicitors revealed that both phytoplasma infection and elicitor treatment individually improved L. vaccinii and L. dispar mass compared to uninfected, untreated controls; however, SA-based elicitor treatments reduced L. vaccinii mass on infected plants. Regalia also improved L. vaccinii survival. Phytoplasma infection reduced plant size and mass, increased levels of nitrogen (N) and SA, and lowered carbon/nitrogen (C/N) ratios compared to uninfected plants, irrespective of elicitor treatment. Although none of our elicitor treatments influenced transcript levels of a phytoplasma-specific marker gene, all of them increased N and reduced C/N levels; the three SA activators also reduced JA levels. Taken together, our findings reveal positive effects of both phytoplasma infection and elicitor treatment on the performance of L. vaccinii and L. dispar in cranberries, likely via enhancement of plant nutrition and changes in phytohormone profiles, specifically increases in SA levels and corresponding decreases in levels of JA. Thus, we found no evidence that the tested elicitors of plant defenses increase resistance to insect herbivores or reduce disease incidence in cranberries.

Interpreting Temporal and Spatial Variation in Spotted-Wing Drosophila (Diptera: Drosophilidae) Trap Captures in Highbush Blueberries.

Integrated pest management (IPM) programs for the spotted-wing drosophila Drosophila suzukii (Diptera: Drosophilidae) rely on insecticide applications to reduce adult populations and prevent fruit infestation. Although monitoring traps are used for early D. suzukii adult detection to time the start of insecticide applications, it remains unclear whether trap counts can be used to determine the efficacy of these programs and predict the risk of fruit infestation. To address this, a 2-yr study (2016-2017) was conducted in highbush blueberries in New Jersey (USA) to interpret D. suzukii trap count variation in relation to the frequency of insecticide applications and proximity to forest habitats. We also correlated trap counts with fruit infestation and used traps to determine the maximum dispersive distance traveled by D. suzukii adults within blueberry fields by using mark-release-capture studies. Using a trapping network across nine farms, we demonstrated that insecticide applications reduce D. suzukii trap counts, but this varied according to seasonality, and that traps placed closer to forest habitats within farms had higher fly counts than those placed in farm interiors. Moreover, blueberry fields that had zero fruit infestation also had predictably lower trap counts than fields with infested fruit, and the maximum dispersive distance for D. suzukii within blueberry fields was 90 m. In summary, while D. suzukii trap counts in blueberry farms could predict the frequency of insecticide applications and fruit infestation, the predictive power of our trap data was too variable across the blueberry harvest period to make it a reliable tool.

Sex pheromone of the scarab beetle Phyllophaga (Phytalus) georgiana (horn).

The sex pheromone of Phyllophaga (Phytalus) georgiana was characterized as valine methyl ester, tentatively the L-enantiomer. This is the first sex pheromone identified from the Phyllophaga subgenus Phytalus. The pheromone was extracted from female glands, the active component isolated by coupled gas chromatography-electroantennogram detection analysis, characterized by mass spectrometry, and shown to be active in field tests. The seasonal flight pattern was determined for P. georgiana as well as for three other species, P. anxia (both northern and southern genitalic forms), P. gracilis, and P. postrema. The latter three species were captured in traps baited with L-isoleucine methyl ester.

Transcriptional profiling of androgen receptor (AR) mutants suggests instructive and permissive roles of AR signaling in germ cell development.

The androgen receptor (AR) is a transcription factor that plays a critical role in male sexual development, spermatogenesis, and maintenance of hormonal homeostasis. Despite the extensive knowledge of the phenotypic consequences of mutations in Ar, very little is known about the transcriptional targets of AR within the testis. To identify potential targets of androgen signaling in the testis, we have analyzed the transcriptional profile of adult testes from Ar hypomorphs alone or in combination with Sertoli cell-specific Ar ablation. Using Affymetrix MOE430A mouse genome arrays we interrogated more than 22,000 transcripts. We found the expression level of 62 transcripts in the Ar mutants differed by greater than 2-fold compared with wild type. We also found that more transcripts were up-regulated than down-regulated, highlighting AR's role as a transcriptional repressor in the testis. Twelve transcripts were uniquely affected, and 16 transcripts were more severely affected in Sertoli cell-specific Ar ablation compared with hypomorphic Ar mutants. Using a comparative genomic approach, we analyzed the 6 kb around the transcriptional start sites of affected transcripts for conserved AREs (androgen response elements). We identified at least one conserved ARE in 65% of the genes misregulated in our microarray analysis where clear mouse-human orthologs were available. We used a reporter assay in cell culture to functionally verify the AREs for the kallikrein 27 gene. This suggests that the majority of the misregulated transcripts have a high probability of being direct AR targets. The transcripts affected by these Ar mutations encode a diverse array of proteins whose molecular functions support the contention that AR supports spermatogenesis in both a permissive and instructive fashion.

Mechanical stimulation increases collagen type I and collagen type III gene expression of stem cell-collagen sponge constructs for patellar tendon repair.

Our group has shown that mechanical stimulation increases the stiffness of stem cell-collagen sponge constructs at 14 days in culture and subsequent rabbit patellar tendon repairs at 12 weeks postsurgery. What remains unclear is which genes might be responsible for this increase in stiffness. Therefore, the objective of this study was to determine how a tensile stimulus affects the gene expression of stem cell-collagen sponge constructs used to repair rabbit central patellar tendon defects. Tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 10 adult rabbits at 0.14 x 10(6) cells/construct in type I collagen sponges. Half of the constructs were mechanically stimulated once every 5 min for 8 h/d to a peak strain of 2.4% for 2 weeks. The other half remained in an incubator without mechanical stimulation for 2 weeks. After 14 days in culture, half of the stimulated and nonstimulated constructs were prepared to determine the expression of collagen type I, collagen type III, decorin, fibronectin, and glyceraldehyde-3-phosphate dehydrogenase genes using real-time quantitative reverse transcriptase polymerase chain reaction. The remaining constructs were mechanically tested to determine their mechanical properties. Two weeks of in vitro mechanical stimulation significantly increased collagen type I and collagen type III gene expression of the stem cell-collagen sponge constructs. Stimulated constructs showed 3 and 4 times greater collagen type I (p = 0.0001) and collagen type III gene expression (p = 0.001) than nonstimulated controls. Stimulated constructs also had 2.5 times the linear stiffness and 4 times the linear modulus of nonstimulated constructs. However, mechanical stimulation did not significantly increase decorin or fibronectin gene expression (p = 0.2) after 14 days in culture. This study shows that mechanical stimulation of cell-sponge constructs produces similar increases in the expression of 2 structural genes, as well as linear stiffness and linear modulus.

Transcriptional profiles of intestinal tumors in Apc(Min) mice are unique from those of embryonic intestine and identify novel gene targets dysregulated in human colorectal tumors.

The adenomatous polyposis coli (APC) tumor suppressor is a major regulator of the Wnt signaling pathway in normal intestinal epithelium. APC, in conjunction with AXIN and GSK-3beta, forms a complex necessary for the degradation of beta-catenin, thereby preventing beta-catenin/T-cell factor interaction and alteration of growth-controlling genes such as c-MYC and cyclin D1. Inappropriate activation of the Wnt pathway, via Apc/APC mutation, leads to gastrointestinal tumor formation in both the mouse and human. In order to discover novel genes that may contribute to tumor progression in the gastrointestinal tract, we used cDNA microarrays to identify 114 genes with altered levels of expression in Apc(Min) mouse adenomas from the duodenum, jejunum, and colon. Changes in the expression of 24 of these 114 genes were not observed during mouse development at embryonic day 16.5, postnatal day 1, or postnatal day 14 (relative to normal adult intestine). These 24 genes are not previously known Wnt targets. Seven genes were validated by real-time reverse transcription-PCR analysis, whereas four genes were validated by in situ hybridization to mouse adenomas. Real-time reverse transcription-PCR analysis of human colorectal cancer cell lines and adenocarcinomas revealed that altered expression levels were also observed for six of the genes Igfbp5, Lcn2, Ly6d, N4wbp4 (PMEPA1), S100c, and Sox4.

Tempo-Spatial Dynamics of Adult Plum Curculio (Coleoptera: Curculionidae) Based on Semiochemical-Baited Trap Captures in Blueberries.

Plum curculio, Conotrachelus nenuphar (Herbst), has become an important pest of highbush blueberries in the northeastern United States. Here, we conducted experiments in 2010-2013 to compare the efficacy of semiochemical-baited traps for C. nenuphar versus conventional (beating cloth) sampling methods in blueberries, and to understand the seasonal abundance and distribution of C. nenuphar adults within and among blueberry fields using these traps. Black pyramid traps baited with the C. nenuphar aggregation pheromone grandisoic acid and the fruit volatile benzaldehyde caught three to four times more adults than unbaited traps without causing an increase in injury to berries in neighboring bushes. Numbers of adult weevils caught in traps correlated with those on bushes (beating cloth samples), indicating that trap counts can predict C. nenuphar abundance in the field. Early in the season, traps placed 20 m from field edges near a forest caught higher C. nenuphar numbers than traps placed at farther distances, suggesting movement of overwintered weevils from outside fields. Using a trapping network across multiple fields in an organic farm, we found evidence of C. nenuphar aggregation in "hotspots"; early in the season, C. nenuphar numbers in traps were higher in the middle of fields, and there was a correlation between these numbers and distance from the forest in 2013 but not in 2012. These results show that semiochemical-baited traps are effective in capturing C. nenuphar adults in blueberries, and that these traps should be placed in the interior of fields preferably, but not exclusively, near wooded habitats to maximize their efficacy.

Pheromone Autodetection: Evidence and Implications.

Olfactory communication research with insects utilizing sex pheromones has focused on the effects of pheromones on signal receivers. Early pheromone detection studies using the silkworm moth, Bombyx mori L., and Saturniids led to the assumption that emitters, especially females, are unable to detect their own pheromone. Pheromone anosmia, i.e., the inability of females to detect their conspecific sex pheromone, was often assumed, and initially little attention was paid to female behaviors that may result from autodetection, i.e., the ability of females to detect their sex pheromone. Detection of conspecific pheromone plumes from nearby females may provide information to improve chances of mating success and progeny survival. Since the first documented example in 1972, numerous occurrences of autodetection have been observed and verified in field and laboratory studies. We summarize here a significant portion of research relating to autodetection. Electrophysiological and behavioral investigations, as well as expression patterns of proteins involved in pheromone autodetection are included. We discuss problems inherent in defining a boundary between sex and aggregation pheromones considering the occurrence of autodetection, and summarize hypothesized selection pressures favoring autodetection. Importance of including autodetection studies in future work is emphasized by complications arising from a lack of knowledge combined with expanding the use of pheromones in agriculture.