Pancreatic Cancer (PDAC): Introduction of Evidence-Based Complementary Measures into Integrative Clinical Management.

The most common form of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), which comprises some 85% of all cases. Currently, this is the fourth highest cause of cancer mortality worldwide and its incidence is rising steeply. Commonly applied clinical therapies offer limited chance of a lasting cure and the five-year survival rate is one of the lowest of the commonly occurring cancers. This review cultivates the hypothesis that the best management of PDAC would be possible by integrating 'western' clinical medicine with evidence-based complementary measures. Protecting the liver, where PDAC frequently first spreads, is also given some consideration. Overall, the complementary measures are divided into three groups: dietary factors, nutraceutical agents and lifestyle. In turn, dietary factors are considered as general conditioners, multi-factorial foodstuffs and specific compounds. The general conditioners are alkalinity, low-glycemic index and low-cholesterol. The multi-factorial foodstuffs comprise red meat, fish, fruit/vegetables, dairy, honey and coffee. The available evidence for the beneficial effects of the specific dietary and nutraceutical agents was considered at four levels (in order of prominence): clinical trials, meta-analyses, in vivo tests and in vitro studies. Thus, 9 specific agents were identified (6 dietary and 3 nutraceutical) as acceptable for integration with gemcitabine chemotherapy, the first-line treatment for pancreatic cancer. The specific dietary agents were the following: Vitamins A, C, D and E, genistein and curcumin. As nutraceutical compounds, propolis, triptolide and cannabidiol were accepted. The 9 complementary agents were sub-grouped into two with reference to the main 'hallmarks of cancer'. Lifestyle factors covered obesity, diabetes, smoking, alcohol and exercise. An integrative treatment regimen was devised for the management of PDAC patients. This involved combining first-line gemcitabine chemotherapy with the two sub-groups of complementary agents alternately in weekly cycles. The review concludes that integrated management currently offers the best patient outcome. Opportunities to be investigated in the future include emerging modalities, precision medicine, the nerve input to tumors and, importantly, clinical trials.

Targeting Tropomyosin Receptor Kinase in Cutaneous CYLD Defective Tumors With Pegcantratinib: The TRAC Randomized Clinical Trial.

Importance: There are no medical interventions for the orphan disease CYLD cutaneous syndrome (CCS). Transcriptomic profiling of CCS skin tumors previously highlighted tropomyosin receptor kinases (TRKs) as candidate therapeutic targets. Objective: To investigate if topical targeting of TRK with an existing topical TRK inhibitor, pegcantratinib, 0.5% (wt/wt), is safe and efficacious in CCS. Design, Setting, and Participants: A phase 1b open-label safety study, followed by a phase 2a within-patient randomized (by tumor), double-blind, placebo-controlled trial (the Tropomyosin Receptor Antagonism in Cylindromatosis [TRAC] trial). The setting was a single-center trial based at a tertiary dermatogenetics referral center for CCS (Royal Victoria Infirmary, Newcastle, United Kingdom). Patients who had germline mutations in CYLD or who satisfied clinical diagnostic criteria for CCS were recruited between March 1, 2015, and July 1, 2016. Interventions: In phase 1b, patients with CCS applied pegcantratinib for 4 weeks to a single skin tumor. In phase 2a, allocation of tumors was to either receive active treatment on the right side and placebo on the left side (arm A) or active treatment on the left side and placebo on the right side (arm B). Patients were eligible if they had 10 small skin tumors, with 5 matched lesions on each body side; patients were randomized to receive active treatment (pegcantratinib) to one body side and placebo to the other side once daily for 12 weeks. Main Outcomes and Measures: The primary outcome measure was the number of tumors meeting the criteria for response in a prespecified critical number of pegcantratinib-treated tumors. Secondary clinical outcome measures included an assessment for safety of application, pain in early tumors, and compliance with the trial protocol. Results: In phase 1b, 8 female patients with a median age of 60 years (age range, 41-80 years) were recruited and completed the study. None of the participants experienced any adverse treatment site reactions. Three patients reported reduced pain in treated tumors. In phase 2a (15 patients [13 female; median age, 51 years], with 150 tumors), 2 tumors treated with pegcantratinib achieved the primary outcome measure of response compared with 6 tumors treated with placebo. The primary prespecified number of responses was not met. The incidence of adverse events was low. Conclusions and Relevance: In this study, pegcantratinib, 0.5% (wt/wt), applied once daily appeared to be well tolerated and to penetrate the tumor tissue; however, the low tumor drug concentrations demonstrated are likely to account for the lack of response. Dose-escalation studies to assess the maximal tolerated dose may be beneficial in future studies of CCS. Trial Registration: isrctn.org Identifier: ISRCTN75715723.

Suppressor of hairy-wing, modifier of mdg4 and centrosomal protein of 190 gene orthologues of the gypsy insulator complex in the malaria mosquito, Anopheles stephensi.

DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy-like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric-a-brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood-fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes.

Mosquito salivary allergen Aed a 3: cloning, comprehensive molecular analysis, and clinical evaluation.

BACKGROUND: Allergic reactions to mosquito bites are an increasing clinical concern. Due to the lack of availability of mosquito salivary allergens, they are underdiagnosed. Here, we reported a newly cloned mosquito Aedes (Ae.) aegypti salivary allergen. METHODS: A cDNA encoding a 30-kDa Ae. aegypti salivary protein, designated Aed a 3, was isolated from an expression library. The full-length cDNA was cloned into a baculovirus expression vector, and recombinant Aed a 3 (rAed a 3) was expressed, purified, and characterized. Skin prick tests with purified rAed a 3 and Ae. aegypti bite tests were performed in 43 volunteers. Serum rAed a 3-specific IgE levels were measured in 28 volunteers. RESULTS: The primary nucleotide sequence, deduced amino acid sequence, and IgE-binding sites of Aed a 3 were identified. rAed a 3-selected antibodies recognized a 30-kDa Ae. aegypti saliva protein. rAed a 3 bound IgE in mosquito-allergic volunteers and the binding could be inhibited by the addition of natural mosquito extract dose dependently. Immediate skin test reactions to rAed a 3 correlated significantly with mosquito bite-induced reactions. Of the bite test-positive volunteers, 32% had a positive rAed a 3 skin test and 46% had specific IgE. No bite test-negative volunteers reacted to rAed a 3 in either the skin tests or the IgE assays, confirming the specificity of the assay. CONCLUSIONS: Aed a 3 that corresponds to the Aegyptin protein is a major mosquito salivary allergen. Its recombinant form has biological activity and is suitable for use in skin tests and specific IgE assays in mosquito-allergic individuals.

Transcription activator-like effector nuclease (TALEN)-mediated female-specific sterility in the silkworm, Bombyx mori.

Engineering sex-specific sterility is critical for developing transgene-based sterile insect technology. Targeted genome engineering achieved by customized zinc-finger nuclease, transcription activator-like effector nuclease (TALEN) or clustered, regularly interspaced, short palindromic repeats/Cas9 systems has been exploited extensively in a variety of model organisms; however, screening mutated individuals without a detectable phenotype is still challenging. In addition, genetically recessive mutations only detectable in homozygotes make the experiments time-consuming. In the present study, we model a novel genetic system in the silkworm, Bombyx mori, that results in female-specific sterility by combining transgenesis with TALEN technologies. This system induces sex-specific sterility at a high efficiency by targeting the female-specific exon of the B. mori doublesex (Bmdsx) gene, which has sex-specific splicing isoforms regulating somatic sexual development. Transgenic animals co-expressing TALEN left and right arms targeting the female-specific Bmdsx exon resulted in somatic mutations and female mutants lost fecundity because of lack of egg storage and abnormal external genitalia. The wild-type sexual dimorphism of abdominal segment was not evident in mutant females. In contrast, there were no deleterious effects in mutant male moths. The current somatic TALEN technologies provide a promising approach for future insect functional genetics, thus providing the basis for the development of attractive genetic alternatives for insect population management.

piRNA pathway gene expression in the malaria vector mosquito Anopheles stephensi.

The ability of transposons to mobilize to new places in a genome enables them to introgress rapidly into populations. The piRNA pathway has been characterized recently in the germ line of the fruit fly, Drosophila melanogaster, and is responsible for downregulating transposon mobility. Transposons have been used as tools in mosquitoes to genetically transform a number of species including Anopheles stephensi, a vector of human malaria. These mobile genetic elements also have been proposed as tools to drive antipathogen effector genes into wild mosquito populations to replace pathogen-susceptible insects with those engineered genetically to be resistant to or unable to transmit a pathogen. The piRNA pathway may affect the performance of such proposed genetic engineering strategies. In the present study, we identify and describe the An. stephensi orthologues of the major genes in the piRNA pathway, Ago3, Aubergine (Aub) and Piwi. Consistent with a role in protection from transposon movement, these three genes are expressed constitutively in the germ-line cells of ovaries and induced further after a blood meal.

Comparison of transgene expression in Aedes aegypti generated by mariner Mos1 transposition and ΦC31 site-directed recombination.

Transgenic mosquitoes generated by transposable elements (TEs) often poorly express transgenes owing to position effects. To avoid these effects, the ΦC31 site-directed recombination system was used to insert transgenes into a locus favourable for gene expression in Aedes aegypti. We describe phenotypes of mariner Mos1 TE and ΦC31 transgenic mosquitoes expressing the enhanced green fluorescent protein (EGFP) reporter in midguts of blood-fed females. Mosquitoes of nine TE-generated lines [estimated transformation frequency (TF): 9.3%] clearly expressed the eye-specific selection marker but only 2/9 lines robustly expressed the EGFP reporter. The piggyBac TE-generated ΦC31 docking strain, attP26, supported recombination with attB site containing donors at an estimated TF of 1.7-4.9%. Using a codon-optimized ΦC31 integrase mutant instead of the 'wild-type' enzyme did not affect TF. Site-directed recombination of line attP26 with an attB-containing donor expressing EGFP from the Ae. aegypti carboxypeptidase promoter produced one transgenic line with blood-fed females expressing the reporter in midgut tissue. Docking strain attP26 also supported robust expression of Flock House virus B2 from the Ae. aegypti polyubiquitin promoter. Our data confirm that eye-specific selection marker expression alone is not a reliable indicator for robust gene-of-interest expression in Ae. aegypti and that the ΦC31 system can ensure predictable transgene expression in this mosquito species.

A role for endosomal proteins in alphavirus dissemination in mosquitoes.

Little is known about endosomal pathway proteins involved in arthropod-borne virus (arbovirus) assembly and cell-to-cell spread in vector mosquitoes. UNC93A and synaptic vesicle-2 (SV2) proteins are involved in intracellular transport in mammals. They show amino acid sequence conservation from mosquitoes to humans, and their transcripts are highly enriched in Aedes aegypti during arbovirus infection. Transient gene silencing of SV2 or UNC93A in mosquitoes infected with the recombinant alphavirus Sindbis MRE16-enhanced green fluorescent protein (SINV; family Togaviridae) resulted in the accumulation of viral positive- and negative-strand RNA, congregation of virus envelope antigen in intracellular networks, and reduced virus dissemination outside of the midgut. Further, UNC93A silencing, but not SV2 silencing, resulted in a 10-fold reduction in viral titres at 4 days post-infection. Together, these data support a role for UNC93A and SV2 in virus assembly or budding. Cis-regulatory elements (CREs) were identified at the 5'-ends of genes from the original data set in which SV2 and UNC93A were identified. Common CREs at the 5'-end genomic regions of a subset of enriched transcripts support the hypothesis that UNC93A transcription may be co-regulated with that of other ion transport and endosomal trafficking proteins.

Transgene-mediated suppression of dengue viruses in the salivary glands of the yellow fever mosquito, Aedes aegypti.

Controlled sex-, stage- and tissue-specific expression of antipathogen effector molecules is important for genetic engineering strategies to control mosquito-borne diseases. Adult female salivary glands are involved in pathogen transmission to human hosts and are target sites for expression of antipathogen effector molecules. The Aedes aegypti 30K a and 30K b genes are expressed exclusively in adult female salivary glands and are transcribed divergently from start sites separated by 263 nucleotides. The intergenic, 5'- and 3'-end untranslated regions of both genes are sufficient to express simultaneously two different transgene products in the distal-lateral lobes of the female salivary glands. An antidengue effector gene, membranes no protein (Mnp), driven by the 30K b promoter, expresses an inverted-repeat RNA with sequences derived from the premembrane protein-encoding region of the dengue virus serotype 2 genome and reduces significantly the prevalence and mean intensities of viral infection in mosquito salivary glands and saliva.

Comparative fitness assessment of Anopheles stephensi transgenic lines receptive to site-specific integration.

Genetically modified mosquitoes that are unable to transmit pathogens offer opportunities for controlling vector-borne diseases such as malaria and dengue. Site-specific gene recombination technologies are advantageous in the development of these insects because antipathogen effector genes can be inserted at integration sites in the genome that cause the least alteration in mosquito fitness. Here we describe Anopheles stephensi transgenic lines containing phi C31 attP'docking' sites linked to a fluorescent marker gene. Chromosomal insertion sites were determined and life-table parameters were assessed for transgenic mosquitoes of each line. No significant differences in fitness between the transgenic and nontransgenic mosquitoes were detected in this study. These transgenic lines are suitable for future site-specific integrations of antiparasite transgenes into the attP sites.

Tumor mapping in 2 large multigenerational families with CYLD mutations: implications for disease management and tumor induction.

OBJECTIVES: To comprehensively ascertain the extent and severity of clinical features in affected individuals from 2 large families with proven heterozygous mutations in the CYLD locus and to correlate these findings with the 3 appendageal tumor predisposition syndromes (familial cylindromatosis, Brooke-Spiegler syndrome, and multiple familial trichoepitheliomas) known to be associated with such germline mutations. DESIGN: Interfamilial and intrafamilial observational study. SETTING: Tertiary genetic and dermatology referral center. PARTICIPANTS: Thirty-four individuals recruited from 2 large multigenerational families with CYLD mutations. Clinical details, history, and tumor maps were obtained from all participants; in 18, the information was corroborated by detailed clinical examination. MAIN OUTCOME MEASURES: Tumor density, distribution and histologic findings, associated medical conditions, patient symptoms, and impact of disease on quality of life. RESULTS: The severity of penetrance and phenotype varied within families. Although an approximately equal female to male predisposition was noted, 5 women and 1 man (of 26 patients surveyed [23%]) had undergone total scalp removal. The average age at onset was 16 years (range, 8-30 years). Symptoms reported by affected patients included painful tumors (in 12 of 23 patients [52%] who answered the question), conductive deafness, and sexual dysfunction. Of the 26 surveyed patients, tumors were noted on the scalp in 21 (81%), on the trunk in 18 (69%), and in the pubic area in 11 (42%). Tumor mapping provided clinical evidence that correlated with hormonally stimulated hair follicles being particularly vulnerable to loss of heterozygosity and tumor induction. CONCLUSIONS: The burden of disease at sites other than the head and neck appears to be underreported in the literature and greatly affects quality of life. Differentiation between the clinical diagnoses has little prognostic or clinical utility in genetic counseling, even within individuals from the same family. Thus, we suggest an encompassing diagnosis of "CYLD cutaneous syndrome." Finally, the clinical distribution of tumors suggests that hormonal factors may play an important role in tumor induction in these patients.

Stability and loss of a virus resistance phenotype over time in transgenic mosquitoes harbouring an antiviral effector gene.

Transgenic Aedes aegypti were engineered to express a virus-derived, inverted repeat (IR) RNA in the mosquito midgut to trigger RNA interference (RNAi) and generate resistance to dengue virus type 2 (DENV2) in the vector. Here we characterize genotypic and phenotypic stabilities of one line, Carb77, between generations G(9) and G(17). The anti-DENV2 transgene was integrated at a single site within a noncoding region of the mosquito genome. The virus resistance phenotype was strong until G(13) and suppressed replication of different DENV2 genotypes. From G(14)-G(17) the resistance phenotype to DENV2 became weaker and eventually was lost. Although the sequence of the transgene was not mutated, expression of the IR effector RNA was not detected and the Carb77 G(17) mosquitoes lost their ability to silence the DENV2 genome.