Application of Tendon Stem/Progenitor Cells and Platelet-Rich Plasma to Treat Tendon Injuries.

Tendon injuries like tendinopathy are a serious healthcare problem in the United States. However, current treatments for tendon injuries are largely palliative. Biologics treatments, including tendon stem/progenitor cells (TSCs) and platelet rich plasma (PRP) hold great potential to effectively treat tendon injuries. TSCs are tendon specific stem cells and have the ability to differentiate into tenocytes, the resident tendon cells responsible for tendon homeostasis and tendon repair in case of an injury. TSCs can also self-renew and thus can replenish the tendon with tendon cells (TSCs and tenocytes) to maintain a healthy tendon. The action of PRP can be complementary; PRP can augment and accelerate tendon healing by supplying abundant growth factors contained in platelets, and fibrin matrix, which functions as a natural conducive scaffold to facilitate tissue healing. This article provides a summary of the findings in recent basic and clinical studies on the applications of TSCs and PRP to the treatment of tendon injuries. It also outlines the challenges facing their applications in clinical settings. In particular, the controversy surrounding the efficacy of PRP treatment for tendon injuries are analyzed and solutions are suggested.

Pro-apoptotic gene regulation and its activation by gamma-irradiation in the Caribbean fruit fly, Anastrepha suspensa.

Transcriptional activation of pro-apoptotic genes in response to cytotoxic stimuli is a conserved feature of the cell death pathway in metazoans. However, understanding the extent of this conservation in insects has been limited by the lack of known pro-apoptotic genes in non-drosophilids. Recently, we described the pro-apoptotic genes, Asrpr and Ashid, from the tephritid, Anastrepha suspensa, that now allow us to explore the conservation of pro-apoptotic gene regulation between a tephritid and drosophilids. In this study, we determined the developmental profiles of Asrpr and Ashid transcripts during embryogenesis and in embryos exposed to γ-irradiation. Transcript levels of both genes determined by qRT-PCR were low throughout embryogenesis, with strong Ashid expression occurring during early to mid-embryogenesis and Asrpr expression peaking in late embryogenesis. This correlated to acridine orange stained apoptotic cells first appearing at 17 h and increasing over time. However, when irradiated at 16 h post-oviposition embryos exhibited significant levels of apoptosis consistent with strong induction of Asrpr and Ashid transcript levels by γ-irradiation in young embryos <24 h post-oviposition. Furthermore, embryos irradiated <24 h post-oviposition failed to hatch, those irradiated between 24 and 32 h had increased hatching rates, but between 48 and 72 h irradiation had no effect on egg hatching. This indicates a transition of embryos from an irradiation-sensitive to irradiation-resistance stage between 24 and 48 h. Throughout post-embryonic development, the two pro-apoptotic genes share similar patterns of up-regulated gene expression, which correlate to ecdysone-induced developmental events, especially during metamorphosis. Together these results provide the first direct evidence for a conserved molecular mechanism of the programmed cell death pathway in insects.

An EST database of the Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae).

Invasive tephritid fruit flies are a great threat to agriculture worldwide and warrant serious pest control measures. Molecular strategies that promote embryonic lethality in these agricultural pests are limited by the small amount of nucleotide sequence data available for tephritids. To increase the dataset for sequence mining, we generated an EST database by 454 sequencing of the caribfly, Anastrepha suspensa, a model tephritid pest. This database yielded 95,803 assembled sequences with 24% identified as independent transcripts. The percentage of caribfly sequences with hits to the closely related tephritid, Rhagoletis pomonella, transcriptome was higher (28%) than to Drosophila proteins/genes (18%) in NCBI. The database contained genes specifically expressed in embryos, genes involved in the cell death, sex-determination, and RNAi pathways, and transposable elements and microsatellites. This study significantly expands the nucleotide data available for caribflies and will be a valuable resource for gene isolation and genomic studies in tephritid insects.

Pro-apoptotic cell death genes, hid and reaper, from the tephritid pest species, Anastrepha suspensa.

Pro-apoptotic proteins from the reaper, hid, grim (RHG) family are primary regulators of programmed cell death in Drosophila due to their antagonistic effect on inhibitor of apoptosis (IAP) proteins, thereby releasing IAP-inhibition of caspases that effect apoptosis. Using a degenerate PCR approach to conserved domains from the 12 Drosophila species, we have identified the first reaper and hid orthologs from a tephritid, the Caribfly Anastrepha suspensa. As-hid is the first identified non-drosophilid homolog of hid, and As-rpr is the second non-drosophilid rpr homolog. Both genes share more than 50% amino acid sequence identity with their Drosophila homologs, suggesting that insect pro-apoptotic peptides may be more conserved than previously anticipated. Importantly, both genes encode the conserved IBM and GH3 motifs that are key for IAP-inhibition and mitochondrial localization. Functional verification of both genes as cell death effectors was demonstrated by cell death assays in A. suspensa embryonic cell culture, as well as in heterologous Drosophila melanogaster S2 cells. Notably, heterologous cell death activity was found to be higher for Anastrepha genes than their Drosophila counterparts. In common with the Drosophila cognates, As-hid and As-rpr negatively regulated the Drosophila inhibitor of apoptosis (DIAP1) gene to promote apoptosis, and both genes when used together effected increased cell death activity, indicating a co-operative function for As-hid and As-rpr. We show that these tephritid cell death genes are functional and potent as cell death effectors, and could be used to design improved transgenic lethality systems for insect population control.

Engineered resistance to Plasmodium falciparum development in transgenic Anopheles stephensi.

Transposon-mediated transformation was used to produce Anopheles stephensi that express single-chain antibodies (scFvs) designed to target the human malaria parasite, Plasmodium falciparum. The scFvs, m1C3, m4B7, and m2A10, are derived from mouse monoclonal antibodies that inhibit either ookinete invasion of the midgut or sporozoite invasion of salivary glands. The scFvs that target the parasite surface, m4B7 and m2A10, were fused to an Anopheles gambiae antimicrobial peptide, Cecropin A. Previously-characterized Anopheles cis-acting DNA regulatory elements were included in the transgenes to coordinate scFv production with parasite development. Gene amplification and immunoblot analyses showed promoter-specific increases in transgene expression in blood-fed females. Transgenic mosquito lines expressing each of the scFv genes had significantly lower infection levels than controls when challenged with P. falciparum.

Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens.

The Mexican fruit fly, Anastrepha ludens, is a highly significant agricultural pest species that has been genetically transformed with a piggyBac-based transposon vector system using independent vector and transposase helper plasmids. Minimum estimated germ-line transformation frequencies were approximately 13-21% per fertile G(0) individual, similar to previously reported frequencies using single vector-helper plasmids. Two vector constructs were tested with potential importance to transgenic strain development for mexfly biological control. The first allows post-integration stabilization of a transposon-vector by deletion of a terminal sequence necessary for mobilization. The complete pB[L1-EGFP-L2-DsRed-R1] vector was integrated into the Chiapas wild type strain with subsequent deletion of the L2-DsRed-R1 sub-vector carrying the piggyBac 3' terminal sequence. Quality control tests for three of the stabilization vector lines (previous to stabilization) assessed viability at all life stages, fertility, adult flight ability, and adult male sexual competitiveness. All three transgenic lines were less fit compared to the wild strain by approximately 5-10% in most tests, however, there was no significant difference in sexual competitiveness which is the major prerequisite for optimal strain release. The second vector, pB[XL-EGFP, Asß2-tub-DsRed.T3], has the DsRed.T3 fluorescent protein reporter gene regulated by the A. suspensa Asß2-tubulin promoter, that resulted in testis and sperm-specific DsRed fluorescence in transgenic male mexflies. Fluorescent sperm bundles were unambiguously observed in the spermathecae of non-transgenic females mated to transgenic males. One transgenic line apparently had a male-specific Y-chromosome insertion, having potential use for sexing by fluorescent-embryo sorting. All transgenic lines expressed easily detectable and stable fluorescence in adults allowing their identification after trapping in the field.

The beta2-tubulin gene from three tephritid fruit fly species and use of its promoter for sperm marking.

To isolate testis-specific regulatory DNA that could be used in genetically transformed insect pest species to improve their biological control, beta2-tubulin genes and their proximal genomic DNA were isolated from three economically important tephritid pest species, Anastrepha suspensa, Anastrepha ludens, and Bactrocera dorsalis. Gene isolation was first attempted by degenerate PCR on an A. suspensa adult male testes cDNA library, which fortuitously isolated the 2.85 kb beta1-tubulin gene that encodes a 447 amino acid polypeptide. Subsequent PCR using 5' and 3' RACE generated the 1.4 kb Asbeta2-tubulin gene that encodes a 446 amino acid polypeptide. Using primers to conserved sequences, the highly similar A. ludens and B. dorsalis beta2-tubulin genes, encoding identical amino acid sequences, were then isolated. To verify Asbeta2-tubulin gene identification based on gene expression, qRT-PCR showed that Asbeta2-tubulin transcript was most abundant in pupal and adult males, and specific to the testes. This was further tested in transformants having the DsRed.T3 reporter gene regulated by the Asbeta2-tubulin 1.3 kb promoter region. Fluorescent protein was specifically expressed in testes from third instar larvae to adults, and fluorescent sperm could be detected in the spermathecae of non-transgenic females mated to transgenic males.To confirm these matings, a PCR protocol was developed specific to the transgenic sperm DNA.

Functional characterization of the promoter of the vitellogenin gene, AsVg1, of the malaria vector, Anopheles stephensi.

Some genetic strategies for controlling transmission of mosquito-borne diseases call for the introgression of antipathogen effector genes into vector populations. Endogenous mosquito promoter and other cis-acting DNA sequences are needed to direct the expression of the effector molecules to maximize their efficacy. Vitellogenin (Vg)-encoding gene control sequences are candidates for driving tissue-, stage- and sex-specific expression of exogenous genes. One of the Anopheles stephensi Vg genes, AsVg1, was cloned and a full-length cDNA, as well as 850 base pairs adjacent to the 5'-end, were sequenced and characterized. Expression of AsVg1 is restricted to the fat body tissues of blood-fed females, and the amino acid sequence of the conceptual translation product is >85% identical to those of other anopheline Vgs. These characteristics support the conclusion that AsVg1 is a Vg-encoding gene. Functional analyses of the AsVg1 putative cis-regulatory sequences were performed using transgenic mosquitoes. The results showed that DNA fragments encompassing the 850 base pairs immediately adjacent to the 5'-end of the gene and the 3'-end untranslated region are sufficient to direct sex-, stage- and tissue-specific expression of a reporter gene. These data indicate that the AsVg1 promoter is a good candidate for controlling the expression of anti-pathogen effector molecules in this malaria vector mosquito.

Structure and expression of the lipophorin-encoding gene of the malaria vector, Anopheles gambiae.

Lipophorin is the major hemolymph protein responsible for lipid transport among tissues of insects. This protein may be a lipid source for the development and reproduction of human malaria parasites in mosquitoes, and therefore could be a target to disrupt malaria parasite development in the vector. The lipophorin of Anopheles gambiae was purified by KBr gradient ultracentrifugation and showed variation in density from 1.111 to 1.143 g/ml during development. The amount and density of lipophorin increase in blood-fed females, indicating an adaptation of vitellogenic mosquitoes to an elevated rate of lipid transport to the developing eggs. The A. gambiae lipophorin gene is composed of eight exons and transcribes an mRNA that is 10,516 nucleotides in length. The predicted initial translation product is a preproapoliphorin consisting of 3332 amino acids, which is processed by proteolysis to generate two mature apolipophorins: apolipophorin-I (Mr = 280,000) and apolipophorin-II (Mr = 81,000). The gene is expressed in the fat body tissues throughout development. An elevated transcriptional activity of the lipophorin gene during vitellogenesis is consistent with the presence of putative cis-regulatory elements (GATA and ecdysone responsive elements) in its 3'-end flanking DNA sequence.

Structurally unique recombinant Kazal-type proteinase inhibitor retains activity when terminally extended and glycosylated.

Recombinant derivatives of the Kazal-type serine proteinase inhibitor GmSPI2 (36 amino acid residues), which is a component of insect silk, were prepared in the expression vector Pichia pastoris. The rhSPI2 had a C-terminal hexahistidine tag attached to the GmSPI2 sequence, rtSPI2 was extended with GluAlaAla at the N-terminus, and rfSPI2 included this N-terminal extension and a C-terminal tail of 22 residues (myc epitope and hexahistidine). A portion of the secreted rfSI2 was O-glycosylated with a trimannosyl or hexamannosyl. The native inhibitor was active slightly on trypsin and highly on subtilisin and proteinase K. The extended C-terminus in rhSPI2 and rfSPI2 enhanced activity on the two latter enzymes and rendered rfSPI2 active on elastase and pronase, but abolished the inhibition of trypsin. The glycosylation of rfSPI2 reduced its inhibitory activity to a level comparable with the native inhibitor. The rtSPI2 with tripeptide extension at the N-terminus and no C-terminal modification was clearly less active than the native inhibitor. None of the tested compounds inhibited alpha-chymotrypsin and the non-serine proteinases.

Engineering Plasmodium-refractory phenotypes in mosquitoes.

A remarkable number of effector mechanisms have been developed for interfering with malaria parasite development in mosquitoes. These effector mechanisms affect different aspects of parasite biology and therefore could be targeted synergistically to reduce the probability of emergence of parasite resistance to any one mechanism. The use of these mechanisms will depend on how efficiently and safely they can be introduced into existing mosquito populations.