The Preclinical Validation of 405 nm Light Parasiticidal Efficacy on Leishmania donovani in Ex Vivo Platelets in a Rag2-/- Mouse Model.

Violet-blue light of 405 nm in the visible spectrum at a dose of 270 J/cm2 alone has been shown to be an effective microbicidal tool for inactivating several bacteria, HIV-1, and Trypanosoma cruzi in ex vivo plasma and platelets. Unlike chemical- and ultraviolet (UV)-based pathogen inactivation methods for plasma and platelet safety, 405 nm light is shown to be less toxic to host cells at light doses that are microbicidal. In this report, we evaluated the parasiticidal activity of a 405 nm light treatment on platelets spiked with the Leishmania donovani parasite. Following the light treatment, parasite viability was observed to be near zero in both low- and high-titer-spiked platelets relative to controls. Furthermore, to test the residual infectivity after inactivation in vivo, the light-treated low-titer L. donovani-spiked platelets were evaluated in an immunodeficient Rag2-/- mouse model and monitored for 9 weeks. The parasiticidal efficacy of 405 nm light was evident from the lack of a presence of parasites in the mice spleens. Parasiticidal activity was confirmed to be mediated through 405 nm light-induced reactive oxygen species (ROS), as quantitatively measured by a 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA)-based assay. Overall, these results confirm the complete inactivation of L. donovani spiked in ex vivo platelets by 405 nm light treatment and exemplify the utility of the Rag2-/- mouse infection model for the preclinical validation of the parasiticidal efficacy of 405 nm light and this light-based technology as a potential PRT for ex vivo platelets.

Metabolomics evaluation of the photochemical impact of violet-blue light (405 nm) on ex vivo platelet concentrates.

INTRODUCTION: Microbicidal violet-blue light in the visible spectrum (405 nm) has been under evaluation for pathogen inactivation in ex vivo human plasma and platelets (PLTs) stored in plasma. Results to date have demonstrated that several blood-borne infectious disease-causing pathogens can be successfully reduced to significantly low levels in the light-treated plasma and PLTs. METHOD: In order to evaluate whether the microbicidal 405 nm light is safe for the treatment of PLT concentrates for pathogen inactivation, LC/MS-based metabolomics analyses were performed to evaluate the overall impact of 405 nm violet-blue light treatment on ex vivo PLT concentrates suspended in plasma and on plasma itself, and to identify metabolome changes in intra-platelet and extra-cellular medium (i.e., plasma). RESULTS: The metabolomics data identified that platelet activating factors (PAFs), agonists and prostaglandins, which can influence PLT basic functions such as integrity, activation, and aggregation potential were unaltered, suggesting that 405 nm light illumination is safe regarding PLT basic functions. Distinct increases in hydroxyl fatty acids and aldehydes, as well as decreases in antioxidant metabolites indicated that reactive oxygen species (ROS) were generated at high levels after only one hour of exposure to 405 nm light. Distinctly changed endogenous photosensitizer metabolites after 1 h of light exposure provided good evidence that 405 nm light was an effective microbicide acting through ROS mechanism and no external additive photosensitizers were required.

Antimicrobial 405 nm violet-blue light treatment of ex vivo human platelets leads to mitochondrial metabolic reprogramming and potential alteration of Phospho-proteome.

Continued efforts to reduce the risk of transfusion-transmitted infections (TTIs) through blood and blood components led to the development of ultraviolet (UV) light irradiation technologies known as pathogen reduction technologies (PRT) to enhance blood safety. While these PRTs demonstrate germicidal efficiency, it is generally accepted that these photoinactivation techniques have limitations as they employ treatment conditions shown to compromise the quality of the blood components. During ex vivo storage, platelets having mitochondria for energy production suffer most from the consequences of UV irradiation. Recently, application of visible violet-blue light in the 400-470 nm wavelength range has been identified as a relatively more compatible alternative to UV light. Hence, in this report, we evaluated 405 nm light-treated platelets to assess alterations in energy utilization by measuring different mitochondrial bioenergetic parameters, glycolytic flux, and reactive oxygen species (ROS). Furthermore, we employed untargeted data-independent acquisition mass spectrometry to characterize platelet proteomic differences in protein regulation after the light treatment. Overall, our analyses demonstrate that ex vivo treatment of human platelets with antimicrobial 405 nm violet-blue light leads to mitochondrial metabolic reprogramming to survive the treatment, and alters a fraction of platelet proteome.

Visible 405 nm Violet-Blue Light Successfully Inactivates HIV-1 in Human Plasma.

Despite significant advances in ensuring the safety of the blood supply, there is continued risk of transfusion transmitted infections (TTIs) from newly emerging or re-emerging infections. Globally, several pathogen reduction technologies (PRTs) for blood safety have been in development as an alternative to traditional treatment methods. Despite broad spectrum antimicrobial efficacy, some of the approved ultraviolet (UV) light-based PRTs, understandably due to UV light-associated toxicities, fall short in preserving the full functional spectrum of the treated blood components. As a safer alternative to the UV-based microbicidal technologies, investigations into the use of violet-blue light in the region of 405 nm have been on the rise as these wavelengths do not impair the treated product at doses that demonstrate microbicidal activity. Recently, we have demonstrated that a 405 nm violet-blue light dose of 270 J/cm2 was sufficient for reducing bacteria and the parasite in plasma and platelets suspended in plasma while preserving the quality of the treated blood product stored for transfusion. Drawn from the previous experience, here we evaluated the virucidal potential of 405 nm violet-blue light dose of 270 J/cm2 on an important blood-borne enveloped virus, the human immunodeficiency virus 1 (HIV-1), in human plasma. Both test plasma (HIV-1 spiked and treated with various doses of 405 nm light) and control plasma (HIV-1 spiked, but not treated with the light) samples were cultured with HIV-1 permissive H9 cell line for up to 21 days to estimate the viral titers. Quantitative HIV-1 p24 antigen (HIV-1 p24) levels reflective of HIV-1 titers were measured for each light dose to assess virus infectivity. Our results demonstrate that a 405 nm light dose of 270 J/cm2 is also capable of 4-5 log HIV-1 reduction in plasma under the conditions tested. Overall, this study provides the first proof-of-concept that 405 nm violet-blue light successfully inactivates HIV-1 present in human plasma, thereby demonstrating its potential towards being an effective PRT for this blood component safety.

MiRNA-103b Downregulates ITGB3 and Mediates Apoptosis in Ex Vivo Stored Human Platelets.

BACKGROUND: Blood bank-stored human platelets are one of the life-saving transfusion products to prevent bleeding in multiple clinical settings. In ex vivo storage, platelets undergo apoptosis and it is highly desirable to prevent this process to preserve platelet quality. However, underlying mechanisms of apoptosis are not well understood in stored platelets. Integrin beta 3 (ITGB3) glycoprotein plays multiple roles in platelet physiological processes, and it was reported in other cell types that downregulation of ITGB3 induces apoptosis. Small noncoding regulatory RNAs known as microRNAs (miRNAs), some of which are abundant in platelets such as miR-103b that belong to miR-103 family of miRNAs, known to play key roles in platelet functions both in vivo and during storage; Cellular miR-103 downregulates certain genes in other cell types and promotes apoptosis. However, whether miR-103b can target and downregulate ITGB3 in stored platelets and such miRNA regulation promotes apoptosis is not known. Here, we tested this working hypothesis. OBJECTIVE: Our objective of this study is to validate the abundance of miR-103b in stored platelets and identify whether ITGB3 is a target of miR-103b for the downregulation and this interaction promotes apoptosis. METHODS: RT-qPCR validation of miR-103b was performed in 11 donor samples at 3 different storage time points. In-silico analysis was performed to identify predicted targets of the miR-103b. The miRNA and messenger RNA interactions were confirmed using different biochemical approaches such as qRT-PCR, western blotting and, suppression of luciferase reporter gene expression by ectopic expression of miR-103b in HeLa cells. Final validation of the functional role of miR-103b in ITGB3 downregulation and resulting induction of apoptosis was assessed in stored platelets by FACS analysis following ectopic expression of miR-103b. RESULTS: Using the Target Scan Vert algorithm, we identified several integrin subunit-encoding mRNAs as potential targets of miR-103b. While ITGB3 and ITGB6 were found to have two targeting sites for miR-103b, since ITGB3 is known to play a role in apoptosis, we chose this for further validation in this study. Ectopic expression of miR-103b decreased the luciferase reporter activity in HeLa cells and decreased ITGB3 mRNA and protein levels in platelets, concomitant with an increase in apoptosis. CONCLUSION: The results demonstrate that in stored platelets, miR-103b is highly expressed and can interact with and downregulate ITGB3 and promote apoptosis in stored platelets.

Complete Inactivation of Blood Borne Pathogen Trypanosoma cruzi in Stored Human Platelet Concentrates and Plasma Treated With 405 nm Violet-Blue Light.

The introduction of pathogen reduction technologies (PRTs) to inactivate bacteria, viruses and parasites in donated blood components stored for transfusion adds to the existing arsenal toward reducing the risk of transfusion-transmitted infectious diseases (TTIDs). We have previously demonstrated that 405 nm violet-blue light effectively reduces blood-borne bacteria in stored human plasma and platelet concentrates. In this report, we investigated the microbicidal effect of 405 nm light on one important bloodborne parasite Trypanosoma cruzi that causes Chagas disease in humans. Our results demonstrated that a light irradiance at 15 mWcm-2 for 5 h, equivalent to 270 Jcm-2, effectively inactivated T. cruzi by over 9.0 Log10, in plasma and platelets that were evaluated by a MK2 cell infectivity assay. Giemsa stained T. cruzi infected MK2 cells showed that the light-treated parasites in plasma and platelets were deficient in infecting MK2 cells and did not differentiate further into intracellular amastigotes unlike the untreated parasites. The light-treated and untreated parasite samples were then evaluated for any residual infectivity by injecting the treated parasites into Swiss Webster mice, which did not develop infection even after the animals were immunosuppressed, further demonstrating that the light treatment was completely effective for inactivation of the parasite; the light-treated platelets had similar in vitro metabolic and biochemical indices to that of untreated platelets. Overall, these results provide a proof of concept toward developing 405 nm light treatment as a pathogen reduction technology (PRT) to enhance the safety of stored human plasma and platelet concentrates from bloodborne T. cruzi, which causes Chagas disease.

Amyloid precursor-like protein 2 interacts with claudin-7 and affects ovarian cancer cell survival.

AIM: In our previous report, we identified roles of CLDN7 in regulation of cell signaling. The goal of this study was to identify proteins interacting with CLDN7 in ovarian cancer. METHODS: The yeast two-hybrid system was used to identify proteins directly interacting with CLDN7 and cell survival was tested using colony formation assay. RESULTS: Amyloid precursor-like protein 2 (APLP2) was found directly associated with CLDN7 in ovarian cancer cell line OVCA420. In addition, APLP2 showed increased expression in ovarian cancer cell lines and tumor tissue samples compared with non-neoplastic ovarian tissues. Knockdown of CLDN7 led to increased expression of APLP2 at both the mRNA and protein levels. Knockdown of APLP2 was associated with decreased cell survival in ovarian cancer cells. CONCLUSION: We show a direct interaction of CLDN7 with APLP2. These findings suggest novel regulatory role for APLP2 in ovarian cancer, a role that appears to be mediated by CLDN7.

MiR-181a Reduces Platelet Activation via the Inhibition of Endogenous RAP1B.

AIM: Since RAP1B is critical for platelet functions, including hemostasis, this study was conducted to identify RAP1B regulating microRNAs (miRNAs) in ex vivo stored platelets. BACKGROUND: Previous studies with platelets identified factors affecting RAP1B activity but regulatory miRNAs that affect RAP1B protein expression have not been reported. OBJECTIVE: To understand the functional significance of miRNA mediated regulation of RAP1B in stored platelets, using microRNA, miR-181a as an example. METHODS: A Tagged RNA Affinity approach (MS2-TRAP) was employed to identify miRNAs that bound to the 3` untranslated region (3`UTR) of the RAP1B mRNA in HeLa cells as an assay system. And subsequently, the mRNA 3'UTR:miRNA interactions were verified in platelets through the ectopic expression of miR-181a mimic and appropriate controls. The interaction of such miRNAs with RAP1B mRNA was also validated by qRT-PCR and Western analysis. RESULTS: Sixty-two miRNAs from MS2 assay were then compared with already known 171 platelet abundant miRNAs to identify a common set of miRNAs. This analysis yielded six miRNAs (miR- 30e, miR-155, miR-181a, miR-206, miR-208a and miR-454), which are also predicted to target RAP1B mRNA. From this pool, miR-181a was selected for further study since RAP1B harbors two binding sites for miR-181a in its 3'UTR. Ectopic expression of miR-181a mimic in platelets resulted in lowering the endogenous RAP1B at both mRNA and protein levels. Further, miR-181a ectopic expression reduced the surface expression of the platelet activation marker, P-selectin. CONCLUSION: MicroRNA-181a can target RAP1B and this interaction has the potential to regulate platelet activation during storage.

RAP1 Downregulation by miR-320c Reduces Platelet Activation in Ex-vivo Storage.

BACKGROUND: A small GTPase Protein, the Ras-related Protein 1 (RAP1), abundant in platelets is known to be activated following agonist-induced platelet activation, suggesting that RAP1 downregulation could, in turn, reduce platelet activation in storage. Our objective of this study is to identify RAP1 regulating miRNAs and their role in platelet activation during storage. METHODS: We applied MS2-TRAP (tagged RNA affinity purification) methodology to enrich miRNAs that target the 3' untranslated region (3'UTR) of RAP1 mRNA in two mammalian cell lines followed by miRNA identification by microarray of total RNA samples enriched for miRNAs. Data analyses were done using different bioinformatics approaches. The direct miR:RAP1 3'UTR interaction was confirmed by using a dual luciferase reporter gene expression system in a mammalian cell line. Subsequently, platelets were transfected with one selected miR to evaluate RAP1 downregulation by this miRNA and its effect on platelet activation. RESULTS: Six miRNAs (miR-320c, miR-181a, miR-3621, miR-489, miR-4791 and miR-4744) were identified to be enriched in the two cell lines tested. We randomly selected miR-320c for further evaluation. The luciferase reporter assay system confirmed the direct interaction of miR-320c with RAP1 3'UTR. Further, in platelets treated with miR-320c, RAP1 protein expression was decreased and concomitantly, platelet activation was also decreased. CONCLUSION: Overall, the results demonstrate that miRNA-based RAP1 downregulation in ex vivo stored platelets reduces platelet activation.

MicroRNA-223 Regulates Septin-2 and Septin-6 in Stored Platelets.

BACKGROUND: Septins have been identified to play important roles in platelets, but their regulation in platelets is unknown. Human platelet being an enucleated and terminally differentiated cell, mRNA downregulation by miRs is one of the posttranscriptional mechanisms operative in platelets. OBJECTIVE: Since platelets are known to have miR-223 in abundance, the objective of this study is to test whether a) platelet septins have miR-223 interacting target sites in their mRNA 3'UTRs, b) septin mRNAs and miR-223 form complexes with Argonaute 2 (AGO2) protein in platelets, which is the catalytic component of an RNA Induced Silencing Complex (RISC), c) a reporter gene with septin mRNA 3' untranslated region (UTR) is subjected to downregulation by miR-223 and d) anti-miR-223 can suppress miR-223 activity and enhance septin-2 expression in platelets. METHOD: Bioinformatics tools were used to screen mRNA 3'UTRs of septin-2 and septin-6 for miR- 223 target sites. Subsequently, platelet extracts were immunoprecipitated by AGO2 antibodies to identify that the two septin mRNAs and miR-223 were in complex with AGO2. A luciferase reporter chimeric- gene expression system was utilized to monitor miR-223 mediated downregulation luciferase gene containing the 3'UTR of either septin-2 or septin-6. Further, anti-miR-223 was utilized in platelets to directly demonstrate the role of miR-223 on the expression of septin-2. RESULTS: Our results demonstrate that in stored platelets a) septine-2 and septin-6 mRNAs have miR- 223 target sites, b) septin-2 and septin-6 are in complex with Ago-2, c) in luciferase reporter gene system, the interaction of miR-223 with 3' UTRs of septin-2 and septin-6 leads to downregulation of luciferase expression and d) anti-miR-223 downregulated miR-223 activity and thereby the expression of septin-2 is upregulated. CONCLUSION: The results demonstrate that like in nucleated cells, enucleated platelets also have miRbased mechanisms for the regulation of their septins.

miR-570 interacts with mitochondrial ATPase subunit g (ATP5L) encoding mRNA in stored platelets.

Loss of platelet quality during ex vivo storage is a major concern in the transfusion medicine field and it has been known that platelet mitochondrial dysfunction is associated with storage time. In the last decade, small noncoding RNAs also known as microRNAs (miRNAs) have been reported to regulate key cellular processes through their target sequence interactions with selected mRNAs. In this study, we focused on understanding the mechanisms of platelet mitochondrial dysfunction during storage through miRNA regulation of mRNAs. RNA was isolated from day 0, day 5, and day 9 of stored human leukocyte-depleted platelets and subjected to differential miRNA and mRNA profiling. The miRNA profiling identified several miRNAs at low levels including a set of 12 different miR-548 family members (miR-548a-3p, miR-548aa, miR-548x, miR-548ac, miR-548c-3p, miR-603, miR-548aj, miR-548ae, miR-548z, miR-548u, miR-548al, and miR-570-3p). The mRNA profiling identified, among many, the mitochondrial ATP synthase subunit g (ATP5L) mRNA at high levels during storage. Target Scan algorithm for potential targets of miR-570-3p also identified ATP5L as one of its targets. We further identified two target sites for miR-570-3p in the 3' untranslated region (3'UTR) of ATP5L mRNA. While ATP5L is a subunit of F0ATPase complex, its function is not established yet. Overexpression of miR-570-3p in platelets resulted in reduced levels of ATP5L mRNA and concomitant ATP loss. These experimental results provide first-time insights into the miRNA-mRNA interactions underlying mitochondrial dysfunction in ex vivo stored platelets and warrants further investigation.

Platelet MicroRNAs: An Overview.

MicroRNAs (miRNAs) are short ~22-nucleotide noncoding RNA that have been found to influence the expression of many genes and cellular processes by either repressing translation or degrading messenger RNA transcripts. Platelet miRNA expression has been shown to be perturbed during ex vivo storage of platelets and in platelet-associated disorders. Although bioinformatics-based miRNA target predictions have been established, direct experimental validation of the role of miRNAs in platelet biology has been rather slow. Target prediction studies are, nonetheless, valuable in directing the design of appropriate experiments to test specific miRNA:messenger RNA interactions relevant to the underlying mechanisms of platelet function in general and in disease as well as in ex vivo storage-associated "storage lesions," a collective term used to include physiologic, biochemical, and morphologic changes that occur in stored platelets. This brief review will focus on emerging human platelet miRNA studies to emphasize their potential role relevant to transfusion medicine field in terms of regulating platelet signaling pathways, markers of platelet associated disorders, and remote impactors of gene expression (intercellular biomodulators) as well as potential platelet quality markers of storage and pathogen reduction treatments.

Small ncRNA Expression-Profiling of Blood from Hemophilia A Patients Identifies miR-1246 as a Potential Regulator of Factor 8 Gene.

Hemophilia A (HA) is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). Genetic mutations in the gene encoding FVIII (F8) have been extensively studied. Over a thousand different mutations have been reported in the F8 gene. These span a diverse range of mutation types, namely, missense, splice-site, deletions of single and multiple exons, inversions, etc. There is nonetheless evidence that other molecular mechanisms, in addition to mutations in the gene encoding the FVIII protein, may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors) and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. We identified several ncRNAs, and among them hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-à-vis the role of nc-RNAs in the regulation of F8 expression. These hypotheses have not been exhaustively tested in this study as they require carefully curated clinical samples.

MicroRNAs and Major Blood-borne Infectious Viral Diseases.

The presence of microRNAs (miRNAs) and their ease of detection in body fluids including serum and whole blood have opened new avenues for developing novel non-invasive methods for diagnostics and therapeutic applications for both infectious and noninfectious diseases. Blood-borne infectious viral diseases pose challenge to public health at large and, especially to health care workers, emergency responders and public safety personnel. Several studies have explored these newly identified miRNAs in blood borne infectious diseases for various purposes. This review highlights and focuses only on some of the available literature on the patient associated cellular miRNAs in blood-borne viral diseases and its occasional extrapolation to infected cell cultures as it relates to blood-borne hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV). Where appropriate, this review further points to the potential of miRNAs as non-invasive early disease detection biomarkers for these viral infections as well as possible prospects and challenges of miRNA-based therapies in treating these viral infections.

Molecular requirements for transformation of fallopian tube epithelial cells into serous carcinoma.

Although controversial, recent studies suggest that serous ovarian carcinomas may arise from fallopian tube fimbria rather than ovarian surface epithelium. We developed an in vitro model for serous carcinogenesis in which primary human fallopian tube epithelial cells (FTECs) were exposed to potentially oncogenic molecular alterations delivered by retroviral vectors. To more closely mirror in vivo conditions, transformation of FTECs was driven by the positive selection of growth-promoting alterations rather antibiotic selection. Injection of the transformed FTEC lines in SCID mice resulted in xenografts with histologic and immunohistochemical features indistinguishable from poorly differentiated serous carcinomas. Transcriptional profiling revealed high similarity among the transformed and control FTEC lines and patient-derived serous ovarian carcinoma cells and was used to define a malignancy-related transcriptional signature. Oncogene-treated FTEC lines were serially analyzed using quantitative reverse transcription-polymerase chain reaction and immunoblot analysis to identify oncogenes whose expression was subject to positive selection. The combination of p53 and Rb inactivation (mediated by SV40 T antigen), hTERT expression, and oncogenic C-MYC and HRAS accumulation showed positive selection during transformation. Knockdown of each of these selected components resulted in significant growth inhibition of the transformed cell lines that correlated with p27 accumulation. The combination of SV40 T antigen and hTERT expression resulted in immortalized cells that were nontumorigenic in mice, whereas forced expression of a dominant-negative p53 isoform (p53DD) and hTERT resulted in senescence. Thus, our investigation supports the tubal origin of serous carcinoma and provides a dynamic model for studying early molecular alterations in serous carcinogenesis.

Claudin-7 is frequently overexpressed in ovarian cancer and promotes invasion.

BACKGROUND: Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC. METHODOLOGY/PRINCIPAL FINDINGS: A total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression. In real-time RT-PCR analysis, the gene for claudin-7, CLDN7, was found to be upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Small interfering RNA-mediated knockdown of claudin-7 in ovarian cancer cells led to significant changes in gene expression as measured by microarrays and validated by RT-PCR and immunoblotting. Analyses of the genes differentially expressed revealed that the genes altered in response to claudin-7 knockdown were associated with pathways implicated in various molecular and cellular functions such as cell cycle, cellular growth and proliferation, cell death, development, and cell movement. Through functional experiments in vitro, we found that both migration and invasion were altered in cells where CLDN7 had been knocked down or overexpressed. Interestingly, claudin-7 expression was associated with a net increase in invasion, but also with a decrease in migration. CONCLUSION/SIGNIFICANCE: Our work shows that claudin-7 is significantly upregulated in EOC and that it may be functionally involved in ovarian carcinoma invasion. CLDN7 may therefore represent potential marker for ovarian cancer detection and a target for therapy.

MicroRNAs in ovarian carcinomas.

The molecular mechanisms involved in epithelial ovarian cancer initiation and progression are just beginning to be elucidated. In particular, it has become evident that microRNAs (miRNAs or miRs), a class of molecules that post-transcriptionally regulate gene expression, play a major role in ovarian tumorigenesis. Several microRNA profiling studies have identified changes in microRNA patterns that take place during ovarian cancer development. While most deregulated microRNAs are down-regulated in cancer, and may therefore act as tumor suppressors, others are elevated and may represent novel oncogenes in this disease. A number of microRNAs identified as aberrantly expressed in ovarian carcinoma have been shown to have important functional roles in cancer development and may therefore represent targets for therapy. In addition, some of the microRNA patterns may have prognostic significance. The identification of functional targets represents a major hurdle in our understanding of microRNA function in ovarian carcinoma, but significant progress is being made. It is hoped that a better understanding of the microRNA expression and roles in ovarian cancer may provide new avenues for the detection, diagnosis, and therapy of this deadly disease.

MicroRNA expression and identification of putative miRNA targets in ovarian cancer.

BACKGROUND: MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Emerging evidence suggests the potential involvement of altered regulation of miRNA in the pathogenesis of cancers, and these genes are thought to function as both tumor suppressors and oncogenes. METHODOLOGY/PRINCIPAL FINDINGS: Using microRNA microarrays, we identify several miRNAs aberrantly expressed in human ovarian cancer tissues and cell lines. miR-221 stands out as a highly elevated miRNA in ovarian cancer, while miR-21 and several members of the let-7 family are found downregulated. Public databases were used to reveal potential targets for the highly differentially expressed miRNAs. In order to experimentally identify transcripts whose stability may be affected by the differentially expressed miRNAs, we transfected precursor miRNAs into human cancer cell lines and used oligonucleotide microarrays to examine changes in the mRNA levels. Interestingly, there was little overlap between the predicted and the experimental targets or pathways, or between experimental targets/pathways obtained using different cell lines, highlighting the complexity of miRNA target selection. CONCLUSION/SIGNIFICANCE: Our results identify several differentially expressed miRNAs in ovarian cancer and identify potential target transcripts that may be regulated by these miRNAs. These miRNAs and their targets may have important roles in the initiation and development of ovarian cancer.

Immunogenicity study of recombinant human sperm-associated antigen 9 in bonnet macaque (Macaca radiata).

BACKGROUND: The human sperm-associated antigen 9 (hSPAG9) is of special interest attributing to the findings indicating that SPAG9 is an acrosomal molecule. SPAG9 is not only restricted to acrosomal compartment but also persists in equatorial segment post-acrosome reaction, which is a key location in sperm-egg interaction. METHODS AND RESULTS: Immunogenicity studies in macaques were carried out with recombinant hSPAG9 (rhSPAG9) adsorbed on alum, which resulted in high titres of anti-rhSPAG9 antibodies as determined by enzyme-linked immunosorbent assay (ELISA). Immunoblotting analysis employing anti-rhSPAG9 antibodies generated in monkeys indicated that antibodies specifically reacted with native SPAG9 from macaque and human sperm and rhSPAG9 protein. Furthermore, indirect immunofluorescence experiments demonstrated SPAG9 localization in the acrosomal compartment of macaque and human sperm. In addition, monkey antibodies against rhSPAG9 significantly inhibited the human spermatozoa adherence or penetration in zona-free hamster oocytes. CONCLUSION: These results demonstrate that rhSPAG9 adsorbed on alum is highly immunogenic in subhuman primate model and therefore represents a suitable sperm-based vaccine immunogen for fertility trials in macaque.

Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein.

Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.