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The peptide nucleic acid (PNA) is a chimeric molecule with the nucleobases connected by peptide bonds. This chimeric nature gives the PNA certain therapeutic advantages over natural antisense nucleic acid molecules. The PNA probes are known for its better and stronger complementation with target nucleic acids. However, cellular delivery of PNA is a major hurdle due to the charge-neutral nature of the PNA. For cellular delivery of PNA, peptide-PNA conjugates are used. This approach may face some practical limitation in terms of PNA antisense activity. In this study, we propose a novel RATH-2 peptide-based non-covalent PNA delivery mechanism. We observed RATH-2 shows a favorable molecular interaction with PNA at 16:1 (peptide:PNA) molar ratio resulting in co-centric nanoparticle formation. With this combination, we could achieve as high as 93% cellular delivery of the PNA. The proposed non-covalent RATH:PNA delivery model showed endocytic entrapment free delivery of PNA. The study further demonstrated the therapeutic application of PNA with in vitro antiviral intervention model. Using RATH-2 non-covalent PNA delivery system, we could inhibit 69.5% viral load. The present study demonstrates a cell-penetrating peptide:PNA interaction can lead to nanoparticle formations that facilitated cellular delivery of PNA.Key points• A novel cell-penetrating peptide (RATH-2) was identified for non-covalent delivery of PNA.• RATH-2 and PNA formed co-centric nanoparticles at appropriate molar combination.• PNA delivered through the RATH-2 inhibited the viral gene expression and reduced the viral load.

Background Fetuses affected by placental insufficiency do not receive adequate nutrients and oxygenation, become growth restricted and acidemic, and can demise. Preterm fetal growth restriction is a severe form of placental insufficiency with a high risk of stillbirth. We set out to identify maternal circulating mRNA transcripts that are differentially expressed in preterm pregnancies complicated by very severe placental insufficiency, in utero fetal acidemia, and are at very high risk of stillbirth. Methods We performed a cohort study across six hospitals in Australia and New Zealand, prospectively collecting blood from 128 pregnancies complicated by preterm fetal growth restriction (delivery Results In the Australia and New Zealand cohort, we identified five mRNAs that were highly differentially expressed among pregnancies with preterm fetal growth restriction: NR4A2, EMP1, PGM5, SKIL, and UGT2B1. Combining three yielded an area under the receiver operative curve (AUC) of 0.95. Circulating NR4A2 and RCBTB2 in the maternal blood were dysregulated in the presence of fetal acidemia in utero. We validated the association between preterm fetal growth restriction and circulating EMP1, NR4A2, and PGM5 mRNA in a cohort from Europe. Combining EMP1 and PGM5 identified fetal growth restriction with an AUC of 0.92. Several of these genes were differentially expressed in the presence of ultrasound parameters that reflect placental insufficiency. Circulating NR4A2, EMP1, and RCBTB2 mRNA were differentially regulated in another cohort destined for stillbirth, compared to ongoing pregnancies. EMP1 mRNA appeared to have the most consistent association with placental insufficiency in all cohorts. Conclusions Measuring circulating mRNA offers potential as a test to identify pregnancies with severe placental insufficiency and at very high risk of stillbirth. Circulating mRNA EMP1 may be promising as a biomarker of severe placental insufficiency.

Cellular gene delivery via polycations has wide implications for the potential of gene therapy, but it has remained a challenge due to the plethora of pre- and post-uptake barriers that must be overcome to reach desired efficiency. Herein we report poly(hexamethylene biguanide) (PHMB) as a nano-vector for intracellular delivery of plasmid DNA (pDNA) and oligodeoxynucleotides (ODNs). PHMB and pDNA or ODNs self-assembled into complex nanoparticles at different pH values (7.4 and 12). Their size, charge, cellular uptake, and gene-expression efficiency are assessed and compared to PEI analogues. The systematic results show that the nanoparticles are effective in delivering plasmid DNA and ODNs to model cell lines in culture (HepG2, HEK293T, HeLa), with measurable changes in gene expression levels, comparable to and, in some conditions, even higher than PEI. The well-accepted safety profile of PHMB makes it a valuable candidate for consideration as an effective intracellular DNA vector for further study and potential clinical translation.

Additional file 1 Supplementary Table 1. Patient characteristics for the cases of Fetal Growth Restriction (FGR) and control cohorts as part of the EVVEREST study. Supplementary Table 2. Patient characteristics for the cohort of stillbirths and controls. Supplementary Table 3. Summary of the postulated biological functions of the proteins encoded by the five genes that were differentially regulated in the FOX cohort. Supplementary Fig. S1. Expression of circulating mRNAs among pregnancies with preterm fetal growth restriction in the FOX cohort. A-H shows qRT-PCR expression of eight genes identified by RNA-seq, and their respective receiver operating characteristic (AUC) curves. Controls were ongoing pregnancies unaffected by growth restriction where bloods were collected around the same gestational ages. **** p

A rapid label-free visual assay for the detection of viral RNA using peptide nucleic acid (PNA) probes and gold nanoparticles (AuNPs) is presented in this study. Diagnosis is a crucial step for the molecular surveillance of diseases, and a rapid visual test with high specificity could play a vital role in the management of viral diseases. In this assay, the specific agglomerative behavior of PNA with gold nanoparticles was manipulated by its complementation with viral RNA. The assay was able to detect 5-10 ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100 μl of test solution. The developed assay was more sensitive than a hemagglutination (HA) test, a routine platform test for the detection of Newcastle disease virus (NDV), and the developed assay was able to visually detect NDV with as little as 0.25 HA units of virus. In terms of the specificity, the test could discriminate single nucleotide differences in the target RNA and hence could provide visual viral genotyping/pathotyping. This observation was confirmed by pathotyping different known isolates of NDV. Further, the PNA-induced colorimetric changes in the presence of the target RNA at different RNA to PNA ratios yielded a standard curve with a linear coefficient of R(2)=0.990, which was comparable to the value of R(2)=0.995 from real-time PCR experiments with the same viral RNA. Therefore, the viral RNA in a given samples could be quantified using a simple visual spectrophotometer available in any clinical laboratory. This assay may find application in diagnostic assays for other RNA viruses, which are well known to undergo mutations, thus presenting challenges for their molecular surveillance, genotyping and quantification.

To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance.

Classical swine fever (CSF) is an economically important Office International des Epizooties (OIE) list A disease of swine characterized by high fever and multiple haemmorhages. The E2 glycoprotein of CSFV is immunogenic and induces neutralizing antibodies against CSFV. In the present study, complete coding region of the E2 gene from Indian virulent field isolate (Mathura) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subsequently cloned into a mammalian expression vector; pcDNA3.1(+) at BamHI and XbaI site. The recombinant plasmid; pcDNA.E2.CSFV. was confirmed by restriction enzyme digestion. The pcDNA.E2.CSFV. transfected Vero cell expressed E2 protein which was confirmed by western blotting, immunoperoxidase and indirect immunofluorescent tests. Additionally, flow cytometry analysis also confirmed that 15% of transfected Vero cells expressed the E2 glycoprotein compared to mock or vector alone transfected cells. Further study is under way to evaluate recombinant pcDNA.E2.CSFV. Mathura clone as DNA vaccine against CSFV.

In the present study, a novel cell penetrating peptide (CPP) named as Rath, has been identified from the avian infectious bursal disease virus. It has the potential to penetrate and translocate cargo molecules into cells independent of temperature. Additionally, it can deliver oligonucleotide in 30min and antibodies within an hour intracellular to chicken embryonic fibroblast primary cells. As an ideal delivery vehicle, it has the ability to protect the cargo molecules in the presence of serum, nucleases and has minimal or no cytotoxicity at even higher peptide concentrations studied. The biophysical characterizations showed that Rath has a dominant beta structure with a small alpha helix and has remarkable binding ability with protein and DNA. Thus, the characterization of unique Rath peptide to deliver protein or nucleic acid into the cells with non-covalent interaction could be used as an effective delivery method for various cell based assays.

HOTAIR DNAme score in ovarian cancer cell lines and MSCs depending on number of passages after stable HOTAIR transfection (ovarian cancer cells) or after starting in vitro culture (MSCs). The HOTAIR DNAme score is the Pearson correlation coefficient between the 67-CpG HOTAIR DNAme signature and the corresponding DNAme profile of each of the cell lines. Whereas neither HOTAIR expression nor passage number had an impact on the correlation coefficient in ovarian cancer cell lines, early passage (multipotent) MSCs (irrespective of whether they had been irradiated or not) showed a much stronger association with the 67-CpG HOTAIR signature than higher passage (senescent) MSCs (from the same individuals). (PDF 128 kb)

Ten-fold internal cross-validations to identify an optimal DNAme signature. Upper panel shows the total misclassification error (y-axis) as a function of the shrinkage threshold (x-axis) used. Lower panel shows the misclassification error for each phenotype (1 = low HOTAIR expression, 2 = high HOTAIR expression) as a function of the same shrinkage threshold. The optimal minimal classifier was found at a threshold of approximately 1.47, corresponding to a 67-CpG signature at an estimated false discovery rate (FDR) of approximately 0.17 (not shown). The FDR was estimated using a permutation scheme as implemented in the pamr R-package, and the relatively low FDR (only about 17 % of the 67 CpGs are expected to be false positives) demonstrates the presence of a genuine DNAme signal associated with HOTAIR expression. (PDF 13 kb)

Background Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed. Methodology/Principal Findings Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB’s DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB. Conclusions Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators., Author Summary Intracellular pathogens are difficult to kill because their localization inside host cells provides protection against immunity and chemotherapies. Thus, successful treatment of diseases caused by intracellular pathogens may need combination therapies and effective delivery systems. Cutaneous leishmaniasis (CL) is caused by intracellular protozoan pathogens of the genus Leishmania. CL is a long established global problem, yet there are no suitable vaccine or chemotherapy options. We found that the well-tolerated cationic polymer PHMB is able to directly kill Leishmania major (L. major) and to enhance host-directed killing by improving the delivery of immunomodulatory nucleic acids. The study exemplifies parallel host- and pathogen-directed killing of an intracellular pathogen in the presence of effective drug delivery platforms. This general strategy holds great promise for therapy of a range of diseases caused by intracellular pathogens.

Newcastle disease virus (NDV), an avian paramyxovirus, induces apoptosis in chicken embryo fibroblast (CEF) cells. In the present investigation, the ability of haemagglutinin-neuraminidase (HN) protein of NDV to cause apoptosis in CEF cells was examined. The results revealed that cells expressing the HN protein demonstrated decreased DNA content, phosphatidylserine exposure and increased cytoplasmic vacuolation. Up-regulation of caspase-1, -9, -8, -3, loss of mitochondrial transmembrane potential and an increase in oxidative stress were also observed in cells expressing the HN protein. Based on the above results it can be concluded that HN protein of NDV causes apoptosis in CEF cells.

Aim: This study was designed to develop peptide analogs of Infectious Bursal Disease (IBD) virus VP5 protein segment having cell penetrating ability to improve their interaction with cargo molecule (Nucleic acid) without affecting the backbone conformation. Materials and Methods: IBDV VP5 protein segment designated as RATH peptide were synthesized using solid phase peptide synthesis and their solution conformation was elucidated using CD spectroscopy in polar (water) and apolar (TFE) solvents. Cell penetrating ability of RATH-CONH2 was observed using FITC labeled peptide internalization in to HeLa cells under fluorescent microscopy. The efficacy of RATH analog interactions with nucleic acids was evaluated using FITC labeled oligonucleotides by fluorescence spectroscopy and plasmid constructs in gel retardation assay. Results: CD spectra of RATH analogs in water and apolar trifluroethanol (TFE) helped to compare their secondary structures which were almost similar with dominant beta conformations suggesting successful induction of positive charge in the analogs without affecting back bone conformation of CPP designed. Cell penetrating ability of RATH CONH2 in HeLa cell was more than 90%. The fluorescence spectroscopy and plasmid constructs in gel retardation assay demonstrated successful interaction of amide analogs with nucleic acid. Conclusion: Intentional changes made in IBDV derived peptide RATH COOH to RATH CONH2 did not showed major changes in backbone conformation and such modifications may help to improve the cationic charge in most CPPs to interact with nucleic acid. [Vet World 2013; 6(6.000): 307-312]

Hazard ratios (HR) for death of patients who received carboplatin-based treatment in the GRONINGEN set and had RNA available to analyze HOTAIR. (PDF 138 kb)

Clinicopathological characteristics of ovarian cancer patients from TCGA set. (PDF 178 kb)

Clinicopathological characteristics of ovarian cancer patients (INNSBRUCK set) for which HOTAIR expression and DNAme have been done. (PDF 189 kb)

Hazard ratios for relapse and death for patients who received carboplatin treatment (a) and patients who did not receive carboplatin treatment (b) in the original INNSBRUCK set. (PDF 183 kb)

Kaplan-Meier survival estimates in patients in TCGA set whose tumours had a high (a) and a low (b) HOTAIR DNA methylation score. Survival analysis was performed according to the type of chemotherapy patients received: carboplatin based (Carboplatin), carboplatin-cisplatin (Carbo-Cisp) or cisplatin chemotherapy (Cisplatin). Hazard ratios (HR) and P values were calculated comparing cisplatin- with carboplatin-based (non-cisplatin-containing) chemotherapy regimens. (PDF 8 kb)

Boxplots of beta methylation values of the 67 CpGs (INNBSRUCK set) which demonstrate the largest difference between HOTAIR -negative (indicated as â 1 â and green boxes ) and HOTAIR -positive (indicated as â 2 â and red boxes ) ovarian cancer samples. (PDF 144 kb)

Clinicopathological characteristics of ovarian cancer patients from the EUROPE set. (PDF 177 kb)

Additional description of methods and sample sets. (PDF 221 kb)

Clinicopathological characteristics of ovarian cancer patients (INNSBRUCK set) for which HOTAIR expression and DNAme have been done. (PDF 189 kb)

Hazard ratios (HR) for death of patients who received carboplatin-based treatment in the GRONINGEN set and had RNA available to analyze HOTAIR. (PDF 138 kb)

Chemosensitivity of A2780 and OVCAR8 ovarian cancer cells which are stably transfected with LacZ (control) or HOTAIR. Cells were treated with cisplatin (0.5–25 μM) or carboplatin (10–160 μM) for 3 days and analysed by the cell survival MTT assay (Sigma). (PDF 397 kb)

Performance of the DNAme signature in the primary INNSBRUCK carboplatin-treated ovarian cancer set and TCGA set. (PDF 215 kb)

Kaplan-Meier survival estimates in patients from the BERGEN set who received no chemotherapy (untreated group, n = 9) (a) or carboplatin-based chemotherapy (n = 40) (b) and stratified according to HOTAIR expression. Patients (n = 49) treated in Bergen (Norway) and whose cancers had >25 % stroma component were analyzed; 8, 2, 34 and 5 had stage 1, 2, 3 and 4 disease, respectively; 32, 6, 9 and 2 had a serous, mucinous, endometrioid and clear cell cancer, respectively. In the untreated group, significantly more patients had stage 1 disease (44 % versus 10 % in the carboplatin group) and no residual disease after primary surgery (75 % versus 38 % in the carboplatin group). The top tertile expressing samples were deemed as high (positive) HOTAIR expressors and compared with low/absent (negative) HOTAIR expressors. There is no significant difference between high and low HOTAIR expressors with regards to grade, stage or residual disease. Comparing HOTAIR-positive with HOTAIR-negative patients, the hazard ratio is 2.55 (95 % confidence interval 1.14–5.68), P value 0.018. (PDF 244 kb)

Kaplan-Meier survival estimates in patients from the BERGEN set who received no chemotherapy (untreated group, n = 9) (a) or carboplatin-based chemotherapy (n = 40) (b) and stratified according to HOTAIR expression. Patients (n = 49) treated in Bergen (Norway) and whose cancers had >25 % stroma component were analyzed; 8, 2, 34 and 5 had stage 1, 2, 3 and 4 disease, respectively; 32, 6, 9 and 2 had a serous, mucinous, endometrioid and clear cell cancer, respectively. In the untreated group, significantly more patients had stage 1 disease (44 % versus 10 % in the carboplatin group) and no residual disease after primary surgery (75 % versus 38 % in the carboplatin group). The top tertile expressing samples were deemed as high (positive) HOTAIR expressors and compared with low/absent (negative) HOTAIR expressors. There is no significant difference between high and low HOTAIR expressors with regards to grade, stage or residual disease. Comparing HOTAIR-positive with HOTAIR-negative patients, the hazard ratio is 2.55 (95 % confidence interval 1.14–5.68), P value 0.018. (PDF 244 kb)

Clinicopathological characteristics of ovarian cancer patients from the EUROPE set. (PDF 177 kb)

Clinicopathological features of patients (GRONINGEN set) stratified according to HOTAIR expression. (PDF 161 kb)

Performance of the DNAme signature in the primary INNSBRUCK carboplatin-treated ovarian cancer set and TCGA set. (PDF 215 kb)

Performance of the DNAme signature in the carboplatin (a) and cisplatin (b) treated EUROPE set. (PDF 212 kb)

Kaplan-Meier survival estimates in patients in TCGA set whose tumours had a high (a) and a low (b) HOTAIR DNA methylation score. Survival analysis was performed according to the type of chemotherapy patients received: carboplatin based (Carboplatin), carboplatin-cisplatin (Carbo-Cisp) or cisplatin chemotherapy (Cisplatin). Hazard ratios (HR) and P values were calculated comparing cisplatin- with carboplatin-based (non-cisplatin-containing) chemotherapy regimens. (PDF 8 kb)

Additional description of methods and sample sets. (PDF 221 kb)

Performance of the DNAme signature in the carboplatin-treated set from the ROCHESTER-MAYO set. (PDF 201 kb)

Boxplots of beta methylation values of the 67 CpGs (INNBSRUCK set) which demonstrate the largest difference between HOTAIR -negative (indicated as â 1 â and green boxes ) and HOTAIR -positive (indicated as â 2 â and red boxes ) ovarian cancer samples. (PDF 144 kb)

Clinicopathological features of patients (GRONINGEN set) stratified according to HOTAIR expression. (PDF 161 kb)

Performance of the DNAme signature in the carboplatin-treated set from the ROCHESTER-MAYO set. (PDF 201 kb)

Kaplan-Meier survival estimates for patients whose tumours expressed HOTAIR (a, c) and whose tumours did not express HOTAIR (b, d) in the INNSBRUCK (a, b) and GRONINGEN sets (c, d). Survival analysis was performed according to the type of chemotherapy patients received: carboplatin monotherapy (Carbo-Mono), carboplatin-paclitaxel (Carbo-Pacli), carboplatin-cyclophosphamide (Carbo-Cyclo) or cisplatin-based chemotherapy (Cisplat). Hazard ratios (HR) and P values were calculated comparing cisplatin- with carboplatin-based chemotherapy regimens. (PDF 281 kb)

Kaplan-Meier survival estimates for patients whose tumours expressed HOTAIR (a, c) and whose tumours did not express HOTAIR (b, d) in the INNSBRUCK (a, b) and GRONINGEN sets (c, d). Survival analysis was performed according to the type of chemotherapy patients received: carboplatin monotherapy (Carbo-Mono), carboplatin-paclitaxel (Carbo-Pacli), carboplatin-cyclophosphamide (Carbo-Cyclo) or cisplatin-based chemotherapy (Cisplat). Hazard ratios (HR) and P values were calculated comparing cisplatin- with carboplatin-based chemotherapy regimens. (PDF 281 kb)

Performance of the DNAme signature in the carboplatin (a) and cisplatin (b) treated EUROPE set. (PDF 212 kb)

Hazard ratios for relapse and death for patients who received carboplatin treatment (a) and patients who did not receive carboplatin treatment (b) in the original INNSBRUCK set. (PDF 183 kb)

Chemosensitivity of A2780 and OVCAR8 ovarian cancer cells which are stably transfected with LacZ (control) or HOTAIR. Cells were treated with cisplatin (0.5–25 μM) or carboplatin (10–160 μM) for 3 days and analysed by the cell survival MTT assay (Sigma). (PDF 397 kb)

Clinicopathological characteristics of ovarian cancer patients from TCGA set. (PDF 178 kb)