Presence Of Nucleic Acids Research Articles

Artificial Insemination as a Possible Convenient Tool to Acquire Genome-Edited Mice via In Vivo Fertilization with Engineered Sperm

Advances in genome editing technology have made it possible to create genome-edited (GE) animals, which are useful for identifying isolated genes and producing models of human diseases within a short period of time. The production of GE animals mainly relies on the gene manipulation of pre-implantation embryos, such as fertilized eggs and two-cell embryos, which can usually be achieved by the microinjection of nucleic acids, electroporation in the presence of nucleic acids, or infection with viral vectors, such as adeno-associated viruses. In contrast, GE animals can theoretically be generated by fertilizing ovulated oocytes with GE sperm. However, there are only a few reports showing the successful production of GE animals using GE sperm. Artificial insemination (AI) is an assisted reproduction technology based on the introduction of isolated sperm into the female reproductive tract, such as the uterine horn or oviductal lumen, for the in vivo fertilization of ovulated oocytes. This approach is simpler than the in vitro fertilization-based production of offspring, as the latter always requires an egg transfer to recipient females, which is labor-intensive and time-consuming. In this review, we summarize the various methods for AI reported so far, the history of sperm-mediated gene transfer, a technology to produce genetically engineered animals through in vivo fertilization with sperm carrying exogenous DNA, and finally describe the possibility of AI-mediated creation of GE animals using GE sperm.

Structural Changes Likely Cause Chemical Differences between Empty and Full AAV Capsids.

Due to the success of adeno associated viruses (AAVs) in treating single-gene diseases, improved manufacturing technology is now needed to meet their demand. The largest challenge is creating a process to separate empty and full capsids. Patients received larger capsid doses than necessary due to the presence of empty capsids. By enabling the better separation of empty and full capsids, patients would receive the greatest therapeutic benefit with the least amount of virus capsids, thus limiting potential side effects from empty capsids. The two most common empty/full separation methods used in downstream processing are ultracentrifugation and anion exchange chromatography. Both processes have limitations, leading to a need for the identification of other structural differences that can be exploited to separate empty and full capsids. Here, we describe four possible theories of the structural changes that occur when AAV capsids envelop a genome. These theories include conformational changes occurring due to either the expansion or contraction of the capsid in the presence of nucleic acids, the constraining of the N-terminus into the five-fold pore when the genome is present, and the increased number of VP3 proteins in full capsids. These theories may reveal structural differences that can be exploited to separate full and empty capsids during manufacturing.

Rapid discrimination of African swine fever virus nucleic acid and virions using BenzoNuclease

African swine fever (ASF) is an acute and severe infectious disease caused by the African Swine Fever Virus (ASFV). ASFV exhibits significant resistance and stability in the environment, which, coupled with its double-stranded DNA and large genome, predisposes it to contaminate laboratory samples. This characteristic can lead to false-positive results in swine farm settings even days after disinfection, as detectable through polymerase chain reaction (PCR) or real-time fluorescent quantitative PCR (qPCR) assays. To meet the demand for efficient clinical methods capable of discriminating between ASFV nucleic acid and ASFV virions, this study aims to ascertain the efficacy of the nuclease “BenzoNuclease” in distinguishing ASFV nucleic acid (ASFV-DNA) from ASFV virions. BenzoNuclease is a versatile nucleic acid enzyme with the capacity to degrade nearly all forms of DNA and RNA. Initially, this research established a highly sensitive general PCR detection method for ASFV. Subsequently, a positive control was constructed using the M13 bacteriophage to substitute for active ASFV, facilitating the development of an improved qPCR method. It is important to note that common disinfectants have the potential to deactivate BenzoNuclease. However, in an environment simulating actual production applications, residual disinfectants do not interfere with the enzymatic efficacy of BenzoNuclease, thus not affecting the detection capabilities of this method. Positive clinical samples from pig farms, upon testing with the improved method, revealed that three samples were positive, indicating the presence of viral particles, while the remaining samples were negative, indicating the presence of nucleic acids. This provides an additional new option for sample testing in pig farms.

Regulation of enzymatic reactions by chemical composition of peptide biomolecular condensates.

Biomolecular condensates are condensed intracellular phases that are formed by liquid-liquid phase separation (LLPS) of proteins, either in the absence or presence of nucleic acids. These condensed phases regulate various biochemical reactions by recruitment of enzymes and substrates. Developments in the field of LLPS facilitated new insights on the regulation of compartmentalized enzymatic reactions. Yet, the influence of condensate chemical composition on enzymatic reactions is still poorly understood. Here, by using peptides as minimalistic condensate building blocks and β-galactosidase as a simple enzymatic model we show that the reaction is restricted in homotypic peptide condensates, while product formation is enhanced in peptide-RNA condensates. Our findings also show that condensate composition affects the recruitment of substrate, the spatial distribution, and the kinetics of the reaction. Thus, these findings can be further employed for the development of microreactors for biotechnological applications.

Spontaneous Liquid Droplet-to-Gel Transition of Citrulline Polypeptide Complexed with Nucleic Acids.

Inside cells, proteins complex with nucleic acids to form liquid droplets resulting from liquid-liquid phase separation. The presence of mutated proteins can change the state of these liquid droplets to solids or gels, triggering neurodegenerative diseases. The mechanism of the liquid to solid or gel transition is still unclear. Solutions of poly(l-ornithine-co-l-citrulline) (PLOC) copolymers, which exhibit upper critical solution temperature-type behavior, change state upon cooling. In this study, we evaluated the effect of nucleic acids complexed with PLOC on phase changes. In the presence of nucleic acids, such as polyC and polyU, PLOC formed liquid droplets at low temperatures. The droplets dissolved at temperatures above the phase separation temperature. The phase separation temperature depended on the chemical structure of the nucleobase, implying that electrostatic and hydrogen bonding interactions between the nucleic acid and PLOC influenced phase separation. Furthermore, the liquid droplets spontaneously changed to gel-like precipitates due to spontaneous release of nucleic acids from the complex. The rate of the liquid droplet-to-gel transition depended on the magnitude of electrostatic and hydrogen bonding interactions between PLOC and nucleic acid. PLOC complexed with mRNA also underwent a liquid droplet-to-gel transition upon the release of mRNA. This work provides insights into the mechanism of pathogenic transitions of the cellular droplets.

Targeting nucleic acid phase transitions as a mechanism of action for antimicrobial peptides

Antimicrobial peptides (AMPs), which combat bacterial infections by disrupting the bacterial cell membrane or interacting with intracellular targets, are naturally produced by a number of different organisms, and are increasingly also explored as therapeutics. However, the mechanisms by which AMPs act on intracellular targets are not well understood. Using machine learning-based sequence analysis, we identified a significant number of AMPs that have a strong tendency to form liquid-like condensates in the presence of nucleic acids through phase separation. We demonstrate that this phase separation propensity is linked to the effectiveness of the AMPs in inhibiting transcription and translation in vitro, as well as their ability to compact nucleic acids and form clusters with bacterial nucleic acids in bacterial cells. These results suggest that the AMP-driven compaction of nucleic acids and modulation of their phase transitions constitute a previously unrecognised mechanism by which AMPs exert their antibacterial effects. The development of antimicrobials that target nucleic acid phase transitions may become an attractive route to finding effective and long-lasting antibiotics.

Discovery of microRNAs in Pyrus stigma exudates opens new research avenues in Horticulture.

In many plant species, flower stigma secretions are important in early stages of sexual reproduction. Previous chemical analysis and proteomic characterization of these exudates provided insights into their biological function. Nevertheless, the presence of nucleic acids in the stigma exudates has not been previously reported. Here, we studied the stigma exudates of Pyrus communis, Pyrus pyrifolia, and Pyrus syriaca and showed them to harbor extracellular RNAs of various sizes. RNA sequencing revealed, for the first time, the presence of known Rosaceae mature microRNAs (miRs), also abundant in the stigma source tissue. Predicted targets of the exudate miRs in the Arabidopsis thaliana genome include genes involved in various biological processes. Several of these genes are pollen transcribed, suggesting possible involvement of exudate miRs in transcriptional regulation of the pollen. Moreover, extracellular miRs can potentially act across kingdoms and target genes of stigma interacting organisms/microorganisms, thus opening novel applicative avenues in Horticulture.

Aptamer-controlled reversible colorimetric assay: High-activity bimetallic organic frameworks for the efficient sensing of marine biotoxins

Marine biotoxins poison seafood under conditions of eutrophication induced by harmful algal blooms, threatening life. It is challenging to construct efficient sensing technologies for trace marine biotoxins. Herein, we utilized Fe(III) and Zr(IV) to prepare bimetal-organic frameworks (ZrFe-MOF). The synergism between the two metals endowed ZrFe-MOF with high peroxidase-like activity, excellent stability, and good dispersity in aqua. Our results demonstrated that the inherent peroxidase-like activity of ZrFe-MOF was inhibited by the aptamers absorbed on this material surface. The effect mechanism was unraveled by clarifying the structural and electronic interactions. Meanwhile, the preferential binding between these specific aptamers and cognate targets triggered the release of rigid complexes from the surface of ZrFe-MOF, resulting in the reactivation of the peroxidase-like activity. Based on the reversible inhibition–activation nanozyme activity controlled by aptamers, a facile ZrFe–MOF-based colorimetric assay was proposed for the detection of marine biotoxins. This strategy was used to determine tetrodotoxin (TTX) as a proof of concept. The limit of detection (LOD) was as low as 0.07 ng/mL, and the detection range was 0.1–200 ng/mL. Furthermore, this assay could be used to successfully analyze natural TTX-contaminated puffer fish and clam samples. Efficient analytical performance was achieved. Notably, the discrimination of multiple marine biotoxins was successfully achieved in seawater based on the pattern recognition process. This study offers a paradigm for tuning MOF nanozyme activity in the presence of nucleic acids. The results bring new insights for developing multi-sensing platforms for various applications.

Osteoarthritis and the Microbiome: Knock, Knock, Anybody Home?

Commentary The human microbiome—the ecosystem of bacteria living and interacting on (and in) human tissues—is enormously complex. Our understanding of its impact is evolving. Next-generation sequencing (NGS) techniques allow for the identification of microbial species and can be used to elaborately interrogate the composition of the bacterial community in specific anatomic sites. This has been extensively described in relation to the gut microbiome, the largest concentration of bacteria in the human body, where complex ecological interactions alter immunology1, pathogenesis2 and metabolic functions3. Far less is known about the microbiomes of anatomic sites previously considered sterile, such as the placenta, the central nervous system, and now the synovium. No one disputes that bacteria and bacterial DNA may transiently penetrate these protected spaces, but whether bacteria truly live there remains to be demonstrated. This preliminary study by Goswami et al. represents a relevant attempt to identify the microbiome of the human osteoarthritic hip and knee. The investigators obtained synovial fluid, tissue, and swabs from patients undergoing primary total hip or knee arthroplasty for osteoarthritis—patients with no overt signs of infection. Concordance in bacterial community composition among samples from the same patient was observed. Variations in diversity were seen depending on the hospital of enrollment and on whether patients had received a corticosteroid injection; these differences may be proxies for broader differences in patient-specific microbial composition. Surprisingly, comorbidities and demographic variables had no significant impact on the microbes found within a joint in this study. The detected microbes ranged from common to obscure—including some that are traditionally considered pathogenic and many others that are not. Some of the common sequences obtained included those of Staphylococcus, Streptococcus, and Pseudomonas. However, even though these are recognized orthopaedic pathogens, it would be surprising to confirm that they persist in clinically uninfected joints. Other bacteria found with frequency included some that are typically found in the environment but only occasionally cause opportunistic infections, such as Paenarthrobacter and Brevundimonas. The limitations of NGS techniques are important to consider. The presence of nucleic acids does not mean the presence of living microbes: remnants of dead bacteria, contamination introduced during sample collection and processing, and even miscategorized mammalian DNA in the vast NGS sequence libraries can all lead to positive findings even when no living bacteria are present. NGS findings are also difficult to confirm by alternate means (such as microscopy or culture), as the microbial burden is so low. A healthy degree of skepticism is warranted, given recent studies of low-biomass microbiomes in other tissues not known to harbor bacteria, such as the human fetus4,5. To their credit, the authors acknowledge these important limitations of their study. So, what can we learn from this study? First, the study serves as a reminder that the presence of microbial DNA in joints, in the absence of other findings of infection, is not diagnostic of infection. This is an important point particularly as it pertains to the molecular diagnosis of periprosthetic joint infection, which is gaining faster in popularity than in evidence. Optimizing molecular tests to diagnose infections is complicated by the implication that all joints may have subclinical quantities of bacterial DNA. Second, it is plausible that microbial nucleic acids are present in osteoarthritic joints. If the findings of this study are confirmed, further work should be done to establish whether these products are being produced intra-articularly by living bacteria, whether they are unique to diseased joints, and whether they represent a disease state that might benefit from treatment. The burden of proof should be high. Further confirmation of these findings with optimal controls, using alternate methodologies, and with replication across other populations (for instance, comparing osteoarthritic and healthy joints) will be needed to convince us that a stable microbiome exists within the joint capsule. Lastly, the techniques used in this study are less broadly available and much more complex than other existing diagnostic tools. The expertise to critically evaluate this type of methodology is beyond the scope of many clinicians. Further education and elaboration on clinically relevant findings are critical to realize the full clinical benefit of NGS in orthopaedics. In summary, this provocative paper offers evidence that microbial DNA is present in the osteoarthritic joint. The next steps that are needed will be to confirm these findings with appropriate controls at every juncture, and to evaluate whether these nucleic acids derive from living intra-articular bacterial communities or from transient circulation of dead microbes.

Characterization of benzopyrylium monomethine dyes as fluorescent probes for sensing and imaging of nucleic acids

In this study, a series of benzopyrylium monomethine dyes bearing a benzothiazole moiety and mainly differing in their substitution pattern were characterized regarding their ability for nucleic acid binding and their use as fluorescent probes for sensing and imaging of nucleic acids. Dyes were characterized with special focus on structure and substituent effects on dye binding towards nucleic acids. Absorption maxima of the free, unbound dyes ranged from 474 to 498 nm. Fluorescence intensities of the unbound dyes were extremely low with emission maxima in the range of 512 to 584 nm depending on the polarity of the used solvent. Quantum yields of the free dyes were below 1 %. Upon binding to nucleic acids, however, dyes became highly fluorescent. Dyes with sterically demanding 2-di- or -triamino substituents exhibited a significant enhancement of the fluorescence quantum yield in presence of nucleic acids. The formation of stable dye-nucleic acid complexes was confirmed by time- and spectrally-resolved fluorescence measurements using a streak camera-based setup. Dye complexation was associated with prolonged lifetimes. All dyes were cell permeable and able to stain nucleic acids present in nuclear structures in living as well as in fixed cells. Colocalization analysis with nuclear specific reporters revealed that all dyes bind selectively to nucleic acids, which confirms their applicability as highly specific fluorescent probes for sensing nucleic acids and cell nuclei imaging. In comparison to commercially available nucleic acid stains, the investigated dyes displayed superior staining properties with high cell viability and less cytoplasmic enrichment. In conclusion, the results obtained by different complementary methods demonstrate that studied dyes are useful fluorescent probes for sensing and imaging of nucleic acids in-vitro, in fixed as well as in living cells. Bulkier dye substituents like piperazinyl and the presence of quaternary ammonium salts in the 2-amino side chain enhance nucleic acid binding significantly, thereby making the parent dye structure attractive for sensing applications and ongoing studies of structure–function relationship of nucleic acid stains.

Expression, purification, and functional characterization of soluble recombinant full-length simian immunodeficiency virus (SIV) Pr55Gag

The simian immunodeficiency virus (SIV) precursor polypeptide Pr55Gag drives viral assembly and facilitates specific recognition and packaging of the SIV genomic RNA (gRNA) into viral particles. While several studies have tried to elucidate the role of SIV Pr55Gag by expressing its different components independently, studies using full-length SIV Pr55Gag have not been conducted, primarily due to the unavailability of purified and biologically active full-length SIV Pr55Gag. We successfully expressed soluble, full-length SIV Pr55Gag with His6-tag in bacteria and purified it using affinity and gel filtration chromatography. In the process, we identified within Gag, a second in-frame start codon downstream of a putative Shine-Dalgarno-like sequence resulting in an additional truncated form of Gag. Synonymously mutating this sequence allowed expression of full-length Gag in its native form. The purified Gag assembled into virus-like particles (VLPs) in vitro in the presence of nucleic acids, revealing its biological functionality. In vivo experiments also confirmed formation of functional VLPs, and quantitative reverse transcriptase PCR demonstrated efficient packaging of SIV gRNA by these VLPs. The methodology we employed ensured the availability of >95% pure, biologically active, full-length SIV Pr55Gag which should facilitate future studies to understand protein structure and RNA-protein interactions involved during SIV gRNA packaging.

Trimethine Cyanine Dyes as NA-Sensitive Probes for Visualization of Cell Compartments in Fluorescence Microscopy.

We propose symmetrical cationic trimethine cyanine dyes with β-substituents in the polymethine chain based on modified benzothiazole and benzoxazole heterocycles as probes for the detection and visualization of live and fixed cells by fluorescence microscopy. The spectral-luminescent properties of trimethine cyanines have been characterized for free dyes and in the presence of nucleic acids (NA) and globular proteins. The studied cyanines are low to moderate fluorescent when free, but in the presence of NA, they show an increase in emission intensity up to 111 times; the most pronounced emission increase was observed for the dyes T-2 in the presence of dsDNA and T-1 with RNA. Spectral methods showed the binding of all dyes to nucleic acids, and different interaction mechanisms have been proposed. The ability to visualize cell components of the studied dyes has been evaluated using different human cell lines (MCF-7, A2780, HeLa, and Hs27). We have shown that all dyes are cell-permeant staining nucleus components, probably RNA-rich nucleoli with background fluorescence in the cytoplasm, except for the dye T-5. The dye T-5 selectively stains some structures in the cytoplasm of MCF-7 and A2780 cells associated with mitochondria or lysosomes. This effect has also been confirmed for the normal type of cell line-human foreskin fibroblasts (Hs27). The costaining of dye T-5 with MitoTracker CMXRos Red demonstrates specificity to mitochondria at a concentration of 0.1 μM. Colocalization analysis has shown signals overlapping of dye T-5 and MitoTracker CMXRos Red (Pearson's Coefficient value = 0.92 ± 0.04). The photostability study shows benzoxazole dyes to be up to ∼7 times more photostable than benzothiazole ones. Moreover, studied benzoxazoles are less cytotoxic at working concentrations than benzothiazoles (67% of cell viability for T-4, T-5 compared to 12% for T-1, and ∼30% for T-2, T-3 after 24 h). Therefore, the benzoxazole T-4 dye is proposed for nucleic acid detection in vitro and intracellular fluorescence imaging of live and fixed cells. In contrast, the benzoxazole dye T-5 is proposed as a good alternative to commercial dyes for mitochondria staining in the green-yellow region of the spectrum.

Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A.

The maintenance of human mitochondrial DNA (mtDNA) is critical for proper cellular function as damage to mtDNA, if left unrepaired, can lead to a diverse array of pathologies. Of the pathways identified to participate in DNA repair within the mitochondria, base excision repair (BER) is the most extensively studied. Protein-protein interactions drive the step-by-step coordination required for the successful completion of this pathway and are important for crosstalk with other mitochondrial factors involved in genome maintenance. Human NEIL1 is one of seven DNA glycosylases that initiates BER in both the nuclear and mitochondrial compartments. In the current work, we scrutinized the interaction between NEIL1 and mitochondrial transcription factor A (TFAM), a protein that is essential for various aspects of mtDNA metabolism. We note, for the first time, that both the N- and C- terminal domains of NEIL1 interact with TFAM revealing a unique NEIL1 protein-binding interface. The interaction between the two proteins, as observed biochemically, appears to be transient and is most apparent at concentrations of low salt. The presence of DNA (or RNA) also positively influences the interaction between the two proteins, and molar mass estimates indicate that duplex DNA is required for complex formation at higher salt concentrations. Hydrogen deuterium exchange mass spectrometry data reveal that both proteins exchange less deuterium upon DNA binding, indicative of an interaction, and the addition of NEIL1 to the TFAM-DNA complex alters the interaction landscape. The transcriptional activity of TFAM appears to be independent of NEIL1 expression under normal cellular conditions, however, in the presence of DNA damage, we observe a significant reduction in the mRNA expression of TFAM-transcribed mitochondrial genes in the absence of NEIL1. Overall, our data indicate that the interaction between NEIL1 and TFAM can be modulated by local environment such as salt concentrations, protein availability, the presence of nucleic acids, as well as the presence of DNA damage.

Discrimination and surveillance of infectious severe acute respiratory syndrome Coronavirus 2 in wastewater using cell culture and RT-qPCR

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has been extensively detected in raw wastewater in studies exploring wastewater-based epidemiology (WBE) for early warning purposes. Nonetheless, only a few limited studies investigated the presence of SARS-CoV-2 in treated wastewaters to determine the potential health risks across the water cycle. The detection of SARS-CoV-2 has been done mostly by RT-qPCR and ddPCR, which only provides information on the presence of nucleic acids rather than information on potential infectivity. In this study, we set to develop and evaluate the use of viability RT-qPCR for the selective discrimination and surveillance of infectious SARS-CoV-2 in secondary-treated wastewater. Enzymatic (nuclease) and viability dye (Reagent D) pretreatments were applied to infer infectivity through RT-qPCR using porcine epidemic diarrhea virus (PEDV) as a CoV surrogate. Infectivity tests were first performed on PEDV purified RNA, then on infectious and heat-inactivated PEDV, and finally on heat inactivated PEDV spiked in concentrated secondary-treated wastewater. The two viability RT-qPCR methods were then applied to 27 secondary-treated wastewater samples positive for SARS-CoV-2 RNA at the outlet of five large urban wastewater treatment plants in Portugal. Reagent D pretreatment showed similar behavior to cell culture for heat-inactivated PEDV and both viability RT-qPCR methods performed comparably to VERO E6 cell culture for SARS-CoV-2 present in secondary-treated wastewater, eliminating completely the RT-qPCR signal. Our study demonstrated the lack of infectious SARS-CoV-2 viral particles on secondary-treated wastewater through the application of two pretreatment methods for the rapid inference of infectivity through RT-qPCR, showing their potential application in environmental screening. This study addressed a knowledge gap on the public health risks of SARS-CoV-2 across the water cycle.

Time-resolved infra-red studies of photo-excited porphyrins in the presence of nucleic acids and in HeLa tumour cells: insights into binding site and electron transfer dynamics.

Cationic porphyrins based on the 5,10,15,20-meso-(tetrakis-4-N-methylpyridyl) core (TMPyP4) have been studied extensively over many years due to their strong interactions with a variety of nucleic acid structures, and their potential use as photodynamic therapeutic agents and telomerase inhibitors. In this paper, the interactions of metal-free TMPyP4 and Pt(II)TMPyP4 with guanine-containing nucleic acids are studied for the first time using time-resolved infrared spectroscopy (TRIR). In D2O solution (where the metal-free form exists as D2TMPyP4) both compounds yielded similar TRIR spectra (between 1450-1750 cm-1) following pulsed laser excitation in their Soret B-absorption bands. Density functional theory calculations reveal that vibrations centred on the methylpyridinium groups are responsible for the dominant feature at ca. 1640 cm-1. TRIR spectra of D2TMPyP4 or PtTMPyP4 in the presence of guanosine 5'-monophosphate (GMP), double-stranded {d(GC)5}2 or {d(CGCAAATTTGCG)}2 contain negative-going signals, 'bleaches', indicative of binding close to guanine. TRIR signals for D2TMPyP4 or PtTMPyP bound to the quadruplex-forming cMYC sequence {d(TAGGGAGGG)}2T indicate that binding occurs on the stacked guanines. For D2TMPyP4 bound to guanine-containing systems, the TRIR signal at ca. 1640 cm-1 decays on the picosecond timescale, consistent with electron transfer from guanine to the singlet excited state of D2TMPyP4, although IR marker bands for the reduced porphyrin/oxidised guanine were not observed. When PtTMPyP is incorporated into HeLa tumour cells, TRIR studies show protein binding with time-dependent ps/ns changes in the amide absorptions demonstrating TRIR's potential for studying light-activated molecular processes not only with nucleic acids in solution but also in biological cells.

Accumulation of endogenous and exogenous nucleic acids in "Touch DNA" components on hands.

Successful forensic DNA profiling from handled items is increasingly routine in casework. This "touch DNA" is thought to contain both cellular and acellular nucleic acid sources. However, there is little clarity on the origins or characteristics of this material. The cellular component consists of anucleate, terminally differentiated corneocytes (assumed to lack DNA), and the occasional nucleated cell. The acellular DNA source is fragmentary, presumably cell breakdown products. This study examines the relative contributions each component makes to the hand-secretions (endogenous) and hand-accumulations (exogenous) by recovering rinses from the inside and outside of worn gloves. Additionally, cellular and acellular DNA was measured at timepoints up to 2 h after hand washing, both with and without interim contact. Microscopic examination confirmed cell morphology and presence of nucleic acids. Following the novel application of a hair keratinocyte lysis method and plasma-DNA fragment purification to hand rinse samples, DNA profiles were generated from both fractions. Exogenous cell-free DNA is shown to be a significant source of touch DNA, which reaccumulates quickly, although its amplifiable nuclear alleles are limited. Endogenous DNA is mostly cellular in origin and provides more allelic information consistently over time.

Peptide-Based Nanoparticles for Therapeutic Nucleic Acid Delivery.

Gene therapy offers the possibility to skip, repair, or silence faulty genes or to stimulate the immune system to fight against disease by delivering therapeutic nucleic acids (NAs) to a patient. Compared to other drugs or protein treatments, NA-based therapies have the advantage of being a more universal approach to designing therapies because of the versatility of NA design. NAs (siRNA, pDNA, or mRNA) have great potential for therapeutic applications for an immense number of indications. However, the delivery of these exogenous NAs is still challenging and requires a specific delivery system. In this context, beside other non-viral vectors, cell-penetrating peptides (CPPs) gain more and more interest as delivery systems by forming a variety of nanocomplexes depending on the formulation conditions and the properties of the used CPPs/NAs. In this review, we attempt to cover the most important biophysical and biological aspects of non-viral peptide-based nanoparticles (PBNs) for therapeutic nucleic acid formulations as a delivery system. The most relevant peptides or peptide families forming PBNs in the presence of NAs described since 2015 will be presented. All these PBNs able to deliver NAs in vitro and in vivo have common features, which are characterized by defined formulation conditions in order to obtain PBNs from 60 nm to 150 nm with a homogeneous dispersity (PdI lower than 0.3) and a positive charge between +10 mV and +40 mV.

Catalysis and specificity of the polycondensation of aminopropyltrimethoxysilane on nucleic acids

The polycondensation of a silane derivative such as aminopropyltrimethoxysilane (ATMS) in the presence of nucleic acids has never been investigated. Our group has previously demonstrated that in chloroform ATMS hydrolysis and polycondensation were faster when the reaction were carried out in the presence of double stranded DNA (146 bp). The results showed that the kinetics of ATMS hydrolysis was affected by the base type used, a fast hydrolysis reaction rate being observed with nucleotide molecules containing adenosine group, and that in the absence of water the amino group of deoxyadenosine units, and not the hydroxylic group of the sucrose residue, can react with ATMS methoxy groups. The present work was initiated aiming at providing a better understanding of this effect. It was observed that the polymerization degree of oligodeoxyadenylate has a clear impact on the kinetic of reaction this effect being as much important as the polymerization degree of the oligodeoxyadenylate was high. Structural investigation by molecular modeling showed that this enhanced reactivity can be explained by conformational effects. Altogether, these results are accounted for assuming that DNA can act as a specific template for ATMS polycondensation, in organic medium such as chloroform, opening the way to possible DNA encapsulation, and a new way for DNA chemical modification in organic solvent.

Serosurvey of West Nile virus (WNV) in free-ranging raptors from Brazil.

West Nile virus (WNV) is a mosquito-borne Flavivirus that can affect birds, horses, and humans, and is the only zoonotic Flavivirus that has been identified in six continents. In Brazil, until 2010, there was no evidence of WNV circulation. Recently, the virus was isolated from a horse with encephalitis, and the first human cases were registered in Brazil. Despite that, there is still no information on the enzootic cycle of this virus in birds or wildlife. This study aimed to investigate whether there is evidence of WNV circulation among wild birds from Southern Brazil. For this, we used free-living wild raptors (live-trapped or rescued) as potential sentinels to investigate the presence of WNV antibodies using ELISA and plaque reduction neutralization test (PRNT) assay. In addition, the presence of nucleic acids from Flavivirus family members was investigated. None of the birds sampled presented clinical findings compatible with WNV. Of the 200 serum samples from birds of prey belonging to 21 species, ten (5%) were positive for the presence of WNV antibodies on ELISA testing. The PRNT test did not confirm the ELISA results, but indicated that three birds had possibly been exposed to Saint Louis encephalitis virus (SLEV). All samples were negative for Flavivirus RNA. The results presented here evince the need for permanent surveillance for emerging flaviviruses in Brazil, as well as for a contingency policy in the case of human/animal outbreaks, particularly in high-risk areas.

Spectroscopic and molecular docking studies of the interactions of monomeric unsymmetrical polycationic fluorochromes with DNA and RNA

The photophysical properties of a series of unsymmetrical di- and tricationic monomethine cyanine dyes were studied in the presence of nucleic acids using combined spectroscopic techniques and molecular docking. The analysis of the UV–visible absorption and the fluorescence spectra revealed a strong association of the title cyanines with nucleic acids, while the interaction magnitude is notably higher for the double-stranded DNA, compared to that of the single-stranded RNA. The binding parameters of the cyanine dyes were determined in terms of the McGhee & von Hippel neighboring site-exclusion model. Cumulatively, the results from the optical spectroscopy measurements along with those from the molecular docking analysis were found to be consistent, presuming minor groove binding motif as predominant between the examined cyanines and deoxyribonucleic acid, whereas external binding upon biocomplexation with ribonucleic acid.