extfieldsofstudy: Medicine Biology

text:
Antibacterial and Antifungal Activity of Essential Oils against Pathogens Responsible for Otitis Externa in Dogs and Cats

Background: Essential oils (EOs) are recommended by some veterinarians to treat otitis externa in pets, but data about their efficacy in scientific literature are very scant. Methods: Nine commercial EOs, from roman chamomile (Anthemis nobilis L.), star anise (Illicium verum), lavender (Lavandula hybrida), litsea (Litsea cubeba (Lour.) Pers.), basil (Ocimum basilicum L.), oregano (Origanum vulgare L. subsp. hirticum), rosemary (Rosmarinus officinalis L.), clary sage (Salvia sclarea L.), and thyme (Thymus vulgaris L.) were tested against bacterial and fungal pathogens previously isolated from dogs and cats with otitis externa. In particular, the analyses were carried out against Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus pseudointermedius, Aspergillus niger, Aspergillus fumigatus, Aspergillus terreus, Candida albicans, Candida tropicalis, Trichosporon sp., and Rhodotorula sp. Results: O. vulgare and S. sclarea showed superior antibacterial activity, even if not against all the strains. Trichosporon sp., C. albicans, and A. terreus were insensitive to most Eos, while other yeasts and molds showed different degrees of sensitivity. In particular, most fungi were inhibited by O. vulgare and R. officinalis. Conclusions: The obtained results suggest that some EOs could be included in treatment as an alternative therapeutic option in bacterial otitis complicated by fungi, in association with conventional drugs.

Introduction
Otitis externa is an inflammation of the external ear canal. Several predisposing factors in dogs and cats would be anatomic breed conformation, immunologic and endocrinopathic conditions, foreign material, trauma, and treatments. Various microorganisms can colonize the external ear canal, and they can proliferate with damage or inflammation due to primary factors [1]. Bacteria, yeasts, and parasites are common causes of otitis externa.
Among bacteria, staphylococci-mainly Staphylococcus pseudointermedius (formerly Staphylococcus intermedius [2]) and Staphylococcus aureusx-and Pseudomonas spp. are often isolated from ears of dogs and cats with acute or chronic otitis externa.
Antibiotic and/or antifungal treatments do not always resolve otitis externa in pets; conversely, they can worsen the situation, causing secondary and recurring infections.
The use of some essential oils (EOs) against otitis externa in pets is currently recommended by some veterinarians that-based on their experience-obtained good results in the treatment of these infections. However, data about the efficacy of EOs against bacteria and yeasts related to canine and/or feline otitis are not available in the scientific literature, with the exception of reports on M. pachydermatis otitis externa [10]; furthermore, information about human cases are very scant [11][12][13].
The aim of this study was to evaluate the in vitro antibacterial and antifungal activities of EOs against the most frequent pathogens isolated from the ears of dogs and cats with otitis externa. EOs have been selected for their effectiveness as reported in literature and for their availability on the market.
All EOs were acquired from the producer (FLORA ® , Pisa, Italy). They were maintained at 4 • C in dark glass vials and were microbiologically analyzed for quality control before use.

Gas Chromatography-Mass Spectrometry Analysis
The GC analysis was performed as previously described [14].

Bacterial Strains
Each EO was tested against three bacterial strains: Pseudomonas aeruginosa, S. aureus, and S. pseudointermedius. All the isolates were previously obtained from cases of otitis externa of dogs and/or cats, typed, and stored at −80 • C in glycerol broth.

Agar Disc Diffusion Method
Antibacterial activity of each EO was tested by Kirby-Bauer agar disc diffusion method following the previously described procedures [15,16].

Minimum Inhibitory Concentration
Each bacterial strain resulting sensitive to the EOs in the Kirby-Bauer test was submitted to the determination of the minimum inhibitory concentration (MIC).

Fungal Strains
Clinical isolates of A. niger, A. fumigatus, A. terreus, C. albicans, C. tropicalis, Trichosporon sp., and Rhodotorula sp. obtained from dogs and cats suffering from otitis externa were employed for testing. Molds were stored at −20 • C on potato dextrose agar, and yeasts were maintained in distilled water at room temperature.

Microdilution Test
MICs were determined by a microdilution test carried out as reported elsewhere [15], starting from a dilution of 5% (v/v), following the methods described by Clinical and Laboratory Standards Institute M38A 2 [18] for molds and Clinical and Laboratory Standards Institute M27A 3 [19] for yeasts.

Gas Chromatography-Mass Spectrometry Analysis
The composition of the assayed EOs is presented in Table 1. All these oils were rich in monoterpenes, except for A. nobilis, where non-terpenic esters such as isobutyl angelate (34.5%) and isoamyl angelate (18.7%) were the most represented constituents. The main terpenes identified in the EO of O. vulgare and T. vulgaris were carvacrol (65.9%) and thymol (52.6%), respectively, followed by p-cymene (15.3%) only in T. vulgaris. Linalyl acetate was evidenced in S. sclarea as the main constituent (54.7%). Linalool was the most important compound in the EO of O. basilicum and L. hybrida (46.0% and 31.5%, respectively). It is also important to highlight the presence of eugenol in O. basilicum with a significant percentage (11.5%) and linalyl acetate in L. hybrida (26.8%). The EO of I. verum was characterized by the presence of a good amount of anethol (89.8%). More represented constituents of L. cubeba were neral and geranial (32.5% and 36.4%, respectively), while R. officinalis was characterized by 37.9% of α-pinene, followed by 1,8-cineole (22.0%).

Antibacterial Activity
The results obtained in this investigation showed that the assayed EOs had different degrees of growth inhibition against the three tested bacterial isolates (Table 2). Table 2. The inhibition zones obtained testing the selected bacterial strains against the assayed EOs.  A. nobilis, I. verum, L. hybrida, and L. cubeba had no activity against the three tested bacteria. S. sclarea showed good activity against S. pseudointermedius, with an MIC value of 2.23 µg/µL and scarce activity against P. aeruginosa (MIC of 17.86 µg/µL). O. vulgare inhibited the growth of S. aureus and S. pseudointermedius, with MIC values of 2.36 µg/µL and 1.18 µg/µL, respectively; on the other hand, no activity was observed against P. aeruginosa. Very low antimicrobial activity was observed for the remaining EOs. DMSO, tested as negative control, did not cause growth inhibition.

Essential Oils
P. aeruginosa was resistant to eleven antibiotics and susceptible only to amikacin; S. aureus were susceptible to tetracycline, amoxicillin-clavulanic acid, erythromycin, and sulphametoxazoletrimethoprim; S. pseudointermedius was susceptible only to amoxicillin-clavulanic acid (Table 3). Table 3. The inhibition zones (expressed in mm) obtained testing the selected bacterial strains against different antibiotics (standard deviation was equal to 0).

Antimycotic Activity
Tested fungal isolates showed different sensitivity patterns to assayed EOs. The overall lowest MIC values were reported for O. vulgare and R. officinalis. S. sclarea, R. officinalis, and T. vulgaris had MIC values <0.3 µg/µL against C. tropicalis, while O. vulgare showed a good antifungal activity against aspergilli. Trichosporon sp. appeared to be sensitive to R. officinalis only, while Rhodotorula sp. was inhibited from R. officinalis and O. vulgare. This latter EO was the sole active when tested against C. albicans. Detailed results are reported in Table 4.

Discussion
The bacterial strains analyzed in this study have previously been isolated from pets with chronic otitis externa. These strains resulted multiresistant to several antibiotics, and for this reason they caused problems in therapy.
S. aureus has been isolated from several body sites of dogs and cats-in particular from skin, anterior nares, and anal region. Staphylococci seem to be transferred from the nose and mouth of the dog to its coat and ears by grooming and pruritic behaviors [20].
S. pseudointermedius is part of the microflora of the upper respiratory tract, mucosal, and skin surfaces of dogs and cats; moreover, it may be an invasive pathogen determining some pathologies, including otitis. Resistance of S. aureus and S. pseudintermedius to classical antibiotics used in clinical veterinary therapy is an increasing threat [21].
P. aeruginosa is an opportunistic, ubiquitous bacterial pathogen that can be isolated from the tissue of healthy animals, and has been determined to be the distinct cause of several infections, including chronic otitis externa. An increasing number of P. aeruginosa strains resistant to several antibiotics has been observed in recent years [22].
The results of this study show that not all EOs have activity against the tested bacterial strains. EO of O. vulgare gave good results only against staphylococci, in accordance with other studies that consider this EO as an effective antistaphylococcal natural product.
The major compounds of oregano EO-carvacrol, thymol, and terpenoids-seem to determine the antimicrobial activity [23], and it is speculated that they disrupt the membrane of bacteria; in particular, they sensitize the phospholipid bilayer of the cell membrane, increasing the permeability and leakage of vital intracellular constituents [24].
Previous studies found that S. sclarea is active against staphylococci, including S. aureus [25], whereas we found that the tested S. aureus strain was resistant. However, data about the antimicrobial activity of S. sclarea are mainly related to food pathogens, whereas no information is available about its clinical use.
Trichosporon sp., C. albicans, and A. terreus were insensitive to most EOs evaluated in the present study, while other fungi showed different degrees of sensitivity. C. tropicalis was reported as sensitive when tested against S. sclarea, R. officinalis, and T. vulgaris, confirming previously reported data concerning this latter EO [15,26].
O. vulgare has a known antimicrobial effect [26,27] due to the high amount of carvacrol, confirming the results of our study.
R. officinalis yielded interesting results showing its efficacy, which appear to be in agreement with Fu et al. [28] for A. niger, but not for C. albicans. Our results confirm the effectiveness of O. vulgare against A. niger and Rhodotorula sp., and the ineffectiveness of I. verum against A. niger, reported by Elgayyar et al. [27], but would not corroborate data referred to both O. basilicum and R. officinalis, as well as the efficacy of I. verum against Rhodotorula sp.
Molds and yeasts tested in the present report were cultured from affected animals' ears. Even if antifungal drug resistance is not a common feature involving these agents, such alternative treatment based on natural products could achieve the owners' approval [10], but the choice of an EOs-based treatment should take into account the identification of fungal organisms responsible for the disease in order to select the most suitable product for treatment. In fact, the results obtained in the present study would suggest that the analyzed EOs are not always able to replace antibiotics or antimycotic drugs to treat such infections, being that their effectiveness is strictly related to the pathogen involved. For example, in the case of P. aeruginosa isolate, the treatment with amikacin resulted better than the use of EOs that, although active, had high MIC values (from 17.86 µg/µL to 18.34 µg/µL).
In the light of the above considerations, the first choice option would take into account the causative agents of otitis with isolation and typing of the etiologic agent; successively, it would be important to carry out an antimicrobial susceptibility test to select an appropriate therapy. EOs can represent a good therapeutic tool, but considered that their efficacy is strongly related to the bacterial strain, it could be useful to evaluate the in vitro activity of different EOs in order to select a natural product effective against the pathogen and that could improve the efficacy treatment.
In conclusion, EOs could be scheduled as an alternative therapeutic option in bacterial otitis complicated by fungi in association with conventional drugs.


extfieldsofstudy: Biology Medicine

text:
Improvement of a rapid diagnostic application of monoclonal antibodies against avian influenza H7 subtype virus using Europium nanoparticles

The development of a sensitive and rapid diagnostic test is needed for early detection of avian influenza (AI) H7 subtype. In this study, novel monoclonal antibodies (mAbs) against influenza A H7N9 recombinant hemagglutinin (rHA)1 were developed and applied to a Europium nanoparticle–based rapid fluorescent immunochromatographic strip test (FICT) to improve the sensitivity of the rapid diagnostic system. Two antibodies (2F4 and 6D7) exhibited H7 subtype specificity in a dot-FICT assay by optimization of the conjugate and the pH of the lysis buffer. The subtype specificity was confirmed by an immunofluorescence assay and Western blot analysis. The limit of detection of the FICT employing novel mAbs 31 ng/mL for H7N9 rHA1 and 40 hemagglutination units/mL for H7 subtype virus. Sensitivity was improved 25-fold using Europium as confirmed by comparison of colloidal gold-based rapid diagnostic kit using the 2F4 and 6D7 mAbs.

difficulty in identifying an antibody that is sufficiently stable and maintains its antigen recognition functionality in non-optimal environments 18 .
In this study, two novel H7 subtype-specific monoclonal antibodies (mAbs) were developed and combined with a fluorescence molecule. The mAbs were adapted to POCT to develop a rapid fluorescent immunochromatographic strip test (FICT) assay. The assay was capable of producing results in 15 minutes.
The FICT assay is attractive as a rapid diagnostic test because of its greatly improved sensitivity for influenza RDT [19][20][21][22] . Europium is a widely-used and efficient fluorescence material, which has been incorporated in rapid diagnostic tests 23,24 . These tests require delicate biosensor equipment, such as microchip and microplate reader. Until now, no study has sought to apply the Europium to H7 subtype-specific rapid diagnostic test.
The present study introduces novel H7 subtype (H7N1 and H7N7)-specific mAbs and demonstrates their success in improving the performance of a FICT assay that utilizes Europium nanoparticles.

Results
Characterization of the mAbs. After immunization of mice with H7N9 rHA1 antigen, 10 hybridoma cell lines producing mAb were established. The antibody amounts ranged from 0.5 to 2 OD according to an ELISA for H7N9 rHA1 antigen (Fig. 1A). Indirect ELISA with all 10 mAbs was conducted for four influenza A subtype virus (H1N1, H5N3, H7N1, and H7N7) at 1,000 HAU/mL. Reactivity of four clones (2H1, 2C3, 6B2, and 6B9) to virus was relatively lower than other clones, although they showed positive signals in recombinant antigen-mediated indirect ELISA (Fig. 1B).
Six mAbs (6B7, 2F4, 6A9, 6F3, 6G5, and 6D7) reacted with the H7N1 and H7N7 subtypes and with H1N1 virus. One mAb (6G5) reacted with all subtypes. As positive control, anti-influenza A nucleoprotein (NP) (3G6) was used for confirmation of the equal amount of each subtype virus; an OD of about 2.5 was produced in the presence of 1,000 HA units (HAU)/mL.
A dot-FICT assay was used to select the pair of H7 subtype-specific mAbs, mimicking the strip test ( Fig. 2A). The broad detection (6G5) to 1,000 HAU/mL of each virus compelled its choice as the detection element. Five other mAbs (6A9, 6D7, 6F3, 6B7, and 2F4) with strong reaction to 1,000 HAU/mL of virus were dotted on the strip. The 6D7 mAb demonstrated the strongest reactivity with H7 subtypes with 6G5. The 6F3 and 2F4 mAbs, which had strong reactivity to virus, hardly reacted with 6G5, implying a shared epitope. As the strongest reactivity was observed by 6D7 for H7 subtypes, reactivity with other mAbs was examined to ascertain specificity (Fig. 2B). Further testing showed that three mAbs (6G5, 6F3, and 6B7) cross-reacted with H1N1 in addition to H7 subtypes. However, 2F4 showed the potential H7 subtype specificity with 6D7, showing strong reactivity to H7N1 and H7N7 virus. The 6A8 mAb failed to recognize H7N1 virus. The 2F4 and 6D7 mAbs were selected for subsequent experiments.
To further confirm the specificity, the viral reactivity of the mAbs was analyzed by Western blot assay and immunofluorescence assay (Fig. 3). Hybridoma cells were expanded in ascites and the purified antibody was confirmed by SDS-PAGE. The gels displayed two dominant bands at 50 kDa (heavy chain) and 25 kDa (light chain) (Fig. 3A). To assess the ability of these mAbs to recognize the linear epitope, 1,000 HAU/lane of virus was used for Western blot analysis. The 2F4 mAb reacted to both H7N1 and H7N7 subtypes (Fig. 3B). The 6D7 mAb did not react with linearized antigen, implying that this antibody would contribute to the specific reactivity through conformational structure of target antigen. In the immunofluorescence assay, the 2F4 and 6D7 mAbs both were reactive against the H7N1 and H7N7 virus subtypes and not against the other subtype tested (Fig. 3C). The positive signal of anti-influenza NP (3G6) showed the presence of virus in the two assays. Figure 1. Development of H7 subtype-specific antibodies. The fusions were performed using mouse spleen cells inoculated with H7N9 virus rHA1. Ten hybridomas were produced. The secreted antibodies from each hybridoma were tested for recombinant antigen (H7N9 HA1 (A) and different influenza subtype virus (B) by indirect ELISA. Pre-immune, serum from a healthy mouse; P.C., positive control antibody (anti-influenza A nucleoprotein antibody).
The test strip for FICT has a conjugate pad for conjugate and a sample pad for application of sample on a nitrocellulose membrane. Anti-influenza H7 subtype-specific antibody (6D7) and anti-mouse IgG Abs were coated on the test line (TL) and the control line (CL) of the nitrocellulose membrane, respectively. To perform the diagnostic assay, the conjugates were loaded on the conjugation pad in advance. Virus in lysis buffer was applied to the sample pad. In the absence of virus, the conjugates failed to react with mAb in the TL. However, in the presence of virus, conjugate was on TL line. At the CL, anti-mouse IgG recognized the antibody on conjugate. After the application of sample to the strip, the fluorescence intensity was digitalized by the light-emitting diode-based portable strip reader in 15 min.
The optimal performance of the FICT assay was obtained using 1,000 HAU/mL of H1N virus and H7N7 virus. The dot-FICT did not showed cross-reactivity with the Abs when H1N1 was present at 1,000 HAU/mL (Fig. 4B).
Nonspecific reaction of bioconjugate was explored by using different amounts of bovine serum albumin (BSA, 0.3125-5%) in conjugate storage buffer. BSA at 5% was able to decrease the cross-reaction of H1N. This concentration was chosen for the bioconjugate, even though H1N1 cross-reactivity persisted.
Instead of the increasing BSA further, we tested different pHs (8)(9)(10)(11)(12) of lysis buffer. The cross-reaction of H1N1 was decreased with reactivity of H7N7 virus (Fig. 4C). The remaining cross-reaction of H1N1 was not significantly different at pH 12 but the reactivity of H7N1 was decreased at that pH. Therefore, we chose the lysis buffer of pH 11. The results indicate that lysis buffer plays a key role in the antigen-Ab environment in addition to helping the migration of samples on a nitrocellulose membrane without non-specific reaction.
FICT assay performance. Serial dilutions of H7N9 HA1 and H5N1 HA1 (15-500 ng/mL were used to determine the limit of detection (LOD) of the FICT assay, based on the limit of blank (LOB), as described previously 25 . The FICT displayed H7N9 HA1 reactivity with good correlation (r 2 = 0.9826), but no reactivity with H5N1 rHA1 (Fig. 5). LOD for H7N9 rHA1 was 31 ng/mL. The proposed diagnostic system targeted the HA1 H7 subtype.
To confirm the virus specificity, serial dilutions (20-640 HAU/mL) of the H1N1, H5N3, H7N1, and H7N7 subtypes were tested in FICT using a 2F4 (conjugate)/6D7 (strip) Ab pair. The proposed assay showed good linear regression for H7N1 virus (r 2 = 0.9743) and H7N7 virus (r 2 = 0.9897). However, it could not recognize the other two subtypes tested (Fig. 6A). LOD was 40 HAU/mL of two H7 subtypes. Therefore, the assay had H7 subtype specificity. The RNA copy number of the LOD (40 HAU/mL) of FICT was determined (Fig. 6B). After preparing two-fold dilutions from of 10 HAU/mL to 80 plaque forming units (PFU)/mL of two H7 subtype viruses, 75 μL of sample was used for RNA extraction. Eluted RNA was used for rRT-PCR with the standard using plasmid dilution. To generate a calibration curve, serially-diluted RNA standard was used. A standard curve was drawn to show the starting copy number of the standard RNA vs the cycle threshold (Ct). RNA copy number/μL was determined by plasmid dilution as described previously 26 . The plot of a standard curve of Ct values against the logarithmic dilutions produced a r 2 value > 0.997 and the slope corresponded to efficiency in the range 87%, which was close to the optimized protocol. The 40 HAU/mL of H7N1 and H7N7 virus showed a Ct value of 30 and 31, respectively. The FICT RNA copy number LOD was 2.03 × 10 3 of RNA for H7N1 and 1.59 × 10 3 for H7N7.
The improved performance of the FICT assay was highlighted by comparison of RDT using colloidal gold nanoparticles. The 2F4/6DF Ab RDT showed a positive signal at 1000 HAU/mL of H7 subtype virus, but not with the other subtypes tested (Fig. 7A). This indicated that the fluorescent dye improved the performance of RDT by 25-fold. The conventional influenza A NP RDT displayed a LOD of 320, 160, 80, and 80 HAU/mL for H1N1, H5N3, H7N1, and H7N7 virus, respectively (Fig. 7B). The H7 subtype-specific FICT assay incorporating Europium nanoparticles markedly improved the performance of the RDT assay, surpassing the performance of the RDT detection of nucleoprotein, a highly expressed viral antigen, compared to HA.

Discussion
For a decade, many studies have sought to develop H7 subtype-specific rapid diagnostic immunoassays. Three ELISA-based H7 subtype-specific systems have been developed [27][28][29] . Recently, H7 subtype-specific Abs were reported to have vaccine efficacy 30,31 .
The LOD of the H7 rRT-PCR assay varies depending on primer, probe, and H7 subtype strain. Two reports achieved a LOD of 10 3 -10 4 copies of H7 RNA 32,33 . Using currently available probe and primers, Ct values of 35-37, corresponding to 20 and 200 copies per reaction, have been reported as the LOD of rRT-PCR of H7N1 virus 34 . Until now, only two studies have reported on a H7 subtype-specific diagnostic system using novel mAbs. In one study, one HA unit of H7 was detected using an ELISA system 35 . The other study developed a H7 subtype-specific RDT and reported the LOD H7-specificity varied widely from Cts of 26-31 by rRT-PCR, depending on the isolates and strain of H7N9 17 . In our study, the LOD of the FICT assay achieved the latter, with a Ct of 30 for H7N1 and 31 for H7N7 by rRT-PCR. Therefore, our proposed FICT assay that utilizes fluorescent Europium performs better in term of average of Cts values.
The outstanding improvement of this FICT assay was highlighted by comparison of RDT using the colloidal gold nanoparticle-conjugated mAbs (2F4/6D7). This RDT was able to detect only 1,000 HAU/mL of H7 subtype virus when we applied the pair of these Abs.
NP is a well-conserved antigen, which has a role in encapsidation of viral RNA and which is used to identify the presence of influenza A or B virus in RDT systems because of the presence of multiple copies 36 . The surface-oriented glycoprotein HA antigen is a component of a subtype-specific protein.
Present results from Western blot analysis and immunofluorescence assay indicated HA expression in AI virus was detected at low level compared to that of NP. Therefore, the RDT LOD of 1,000 HAU/mL achieved using 2F4/6D7 could be partially due to the relatively low expression of HA.
The finding is reasonable compared to NP because a commercial influenza A/B kit targeting NP has a LOD of 80-320 HAU/mL all four subtype virus (Fig. 7B).
Additionally, the improvement of performance of FICT employing Europium was confirmed by FICT targeting NP ( Figure S1). When we tested the Europium-conjugate with anti-influenza NP in FICT, LOD of RDT (LOD: 80 HAU/mL) was improved by 8-fold (LOD: 10 HAU/mL) in Europium-employing FICT assay. Therefore, our study supported that the low sensitivity of RDT could be improved by employing the fluorescence material. The main challenge in the present study was to find a method that would be suitably able to improve the ability of the rapid diagnostic assay. We achieved this goal using Europium containing nanoparticles.
Although coupling of fluorescent dye to antibody has a good record of improving RDT, this has not hitherto been explored using Europium, even though Europium is popular fluorescent material. This may have been because conjugating Europium nanoparticles to antibody can functionally inactivate the antibody 37 .
The currently optimized FICT can be easily exploited to apply Europium in RDTs for influenza virus and other diseases that require improved RDT sensitivity.
Virus infection could be diagnosed by immunofluorescence assay using fluorescent microscope. However, fluorescence microscopy requires the intensive high power light source including xenon, mercury or laser 38 . This light source limits the fluorescence microscopy to become a POCT.
Recently, modern smartphones are equipped with a lightweight, high-performance camera/imaging module, becoming the preferred tool for use in many POC applications 39,40 .
Therefore, the proposed fluorescent diagnostic system can contribute to the advance of the POCT diagnostic platform connected with modern smartphone-based diagnostic application.
BSA is a well-known blocker used to suppress non-specific reactions in an immunoassay 41 . pH can cause structural change of HA of influenza virus 42 . Therefore, we tested different pHs to find a more suitable condition of our FICT assay employing 6D7, which recognizes conformational structure of the HA1 epitope of H7 subtypes. H1N1 cross-reaction was suppressed by increasing pH, indicating that a non-physiologic pH could be efficient in discriminating HA subtypes. Antibody-antigen force is a weak interaction and the type of the weak interaction is composed of Van der Waals force, hydrogen bond, hydrophobic interaction, and ion-dipole 43 . Weak interaction is involved in the epitope-paratope binding and a few amino acids is comprised in the specific binding 44 . Many factors influence antigen-antibody reaction and one of the factors affecting antigen-antibody reaction is pH 43 . Therefore, we consider that modification of pH may be a good way to fine-tune the conformational antigen-antibody reaction.
The Europium-based assay includes strip (cost: <$2 USD/test strip) and LED device (Medisensor Inc., Daegu, South Korea, cost: ~$20,000). As test kit is generally sold at a price of 20 USD/test kit 45 , current Europium-based strip itself has a competition. Although the proposed assay requires an expensive reader, it still has advantage because device can be shared in one local area and reusable.
We tested the potential of performance in FICT with clinical samples by spiking virus and the proposed diagnostic method is considered to be useful as clinical application with human nasopharyngeal specimen ( Figure S2). Our study is limited by the lack of data of human H7N9-infected patients, although a recombinant antigen of H7N9 HA1 could allow detection of H7N9 virus. The detection sensitivity and specificity of this assay needs to be evaluated when H7N9 patient samples are available.
In conclusion, novel H7 subtype-specific mAbs were developed and applied to a rapid fluorescent diagnostic system that incorporated Europium nanoparticles. The system displayed H7 subtype-specificity and enhanced H7 subtype virus detection sensitivity.

Methods
Reagents. Europium nanoparticles were purchased from Bangs Laboratories Inc. (Fishers, IN, USA).   (H5N3, H7N1, and H7N7) were kindly provided from Professor Haan Woo Sung, Kangwon National University, Chuncheon, Republic of Korea. The egg-cultured virus stock was titrated by HA assay and plaque assays were performed as previously described 22, 46 . Production and characterization of mAbs. H7N9 (A/Anhui/1/2013) recombinant HA 1 (rHA1) (50 μg/100 μL) was mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich) and injected intraperitoneally into a 6-week-old female BALYSIS BUFFER/c mice (Daehan Bio-Link, Eumseong, Korea). Boosts consisted of the H7N9 rHA1 protein (25 μg/100 μL) mixed with an equal volume of Freund's incomplete adjuvant biweekly. After the third boost, adjuvant-free H7N9 rHA1 (5 μg/100 μL) was injected intravenously. The cell fusion technique was previously described 47 . In brief, after confirmation of the Ab in mouse serum, splenocytes were extracted from a selected immune mouse and fused with myeloma cell (F/0 cell line) at a ratio of 1:5 to 1:1 in 50% polyethylene glycol and then seeded in each well of a 96-well culture plate. Hybridoma cells were selected by subculture in HAT (hypoxanthine, aminopterin, and thymidine) and HT (hypoxanthine and thymidine) media in a 5% CO 2 incubator at 37 °C for 2 weeks. After colonies appeared in each well, Ab titers were determined by an enzyme-linked immunosorbent assay (ELISA). Hybridoma cells producing mAb were selected by ELISA and cultured with a complete DMEM in a 24-well culture plate. For scaled-up mAb Figure 7. Limit of detection of conventional RDT. The 2F4 mAb conjugated with colloidal gold nanoparticle (Au NP) was captured on the TL (6D7mAb) if there was virus during lateral flow reaction. After 20 min, the strip was read with the naked eye for a binary decision. Two non-H7 subtype viruses (H1N1 and H5N3) were applied in high titer (1,000 HAU/mL) and for two H7 subtype virus (H7N1 and H7N7), 500 and 1,000 HAU/mL of each virus were applied to RDT (A). Conventional influenza A RDT targeting influenza A nucleoprotein (NP) was tested with the two-fold serially diluted four subtype virus from 40 to 80 HAU/mL (B). The arrow indicates the LOD.
Scientific RepoRtS | 7: 7933 | DOI:10.1038/s41598-017-08328-9 production, mAb-producing cells were intraperitoneally injected into a 10-week-old female BALYSIS BUFFER/c mouse. Two weeks later, mouse ascites were collected and centrifuged at 5,000 × g for 15 min. The purified mAb from the ascites was obtained using a protein A agarose column (Amersham Biosciences, Uppsala, Sweden) and identified by Western blotting. For examination of the mAb isotypes, an Immunotype TM mouse monoclonal antibody isotyping kit (Sigma-Aldrich) was used.
Indirect ELISA assay. Indirect ELISA was performed as describe previously 46 . Briefly, the purified antigen or virus were diluted in 50 mM bicarbonate/carbonate coating buffer (pH 9.6) and 75 µL of sample were prepared and used to coat wells of a 96-well microtitre plate were prepared and used to coat wells of a 96-well microtitre plate (Greiner, Germany) 37 °C for 2 h. The plate was washed with 200 μL of PBS, 0.1% Tween 20 (PBS-T, pH 7.4) and then blocked with 5% non-fat dry milk at 37 °C for 2 h. To react the antibody, in case of recombinant antigen-mediated ELISA, pre-immune sera derived from healthy mice (0.5 μL of sera/100 μL/well) and mAb (1 μg/100 μL/well) were added to each well, respectively. In case of virus detection, mAbs (2 μg/100 μL/well) were added to each well and positive control (anti-influenza A NP, 2 μg/100 μL/well) to convince the each subtype virus per well were added to each well and incubated at 37 °C to detect antigens. After 1 h, secondary Ab in the form of horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Abcam, Cambridge, UK) was added to each well according to the manufacture's protocol. Stringent washing with PBS-T was performed five times to remove nonspecific biding and 100 μL of 3,3′,5,5′-tetra methyl benzidine (Sigma-Aldrich) substrate solution was added.
Western blot analysis. The four subtype virus (H5N, H1N1, H7N1, and H7N7) were subjected to 12% SDS-PAGE and a running gel at 100 V for 2 h. The gel was soaked in transfer buffer, and the resolved proteins were transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk for 2 h at 37 °C and each antibody diluted to a concentration of 10 μg/mL was added for 1 h. The HRP-conjugated antibody was used for detection according to the manufacturer's protocol. Anti-influenza NP (Clone 3G6) was used for loading control of each virus. Finally, Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) was used to visualize the band using the ChemiDoc MP System (Bio-Rad).
IFA. IFA was performed as described previously 48 . The first day, MDCK cells were coating on glass coverslips positioned in wells of a 6well plate and incubated at 37 °C overnight in an atmosphere of 5% CO 2 . The next day, cells were infected at a multiplicity of infection of 0.5 in DMEM containing 1% antibiotic, 0.3% BSA and 1 μg/mL trypsin from bovine pancreas for 12 h, 37 °C, and 5% CO 2 . Using fresh 4% paraformaldehyde in PBS (pH 6.9), cells were fixed for 15 min and after washing three times with 0.1% Tween 20 in PBS, a solution of 0.1% Triton X-100 was added to each well for permeabilization. Subsequently, cells were blocked with 5% BSA and 0.1% Tween 20 in PBS for 2 h at room temperature. To detect virus, mAbs (2F4 and 6D7) diluted 1:5000 in blocking buffer (0.5 μg/mL) were used. Subsequently, fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG secondary antibody was incubated for 1 h at room temperature. Finally, each coverslip was mounted on a slide including mounting medium and 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Images of fluorescent cells were acquired with a fluorescence microscope (Olympus, Tokyo, Japan) using a 400× objective. Anti-influenza NP (3G6) was used as positive control antibody.

Lateral flow test strips for fluorescent immunochromatographic test (FICT). The test strips
used consisted of four components: sample application pad, conjugate pad, nitrocellulose (NC) membrane and absorbent pad. The test line (TL) of the strip was prepared by dispensing a desired volume of 2.5 mg/mL mouse monoclonal (anti-influenza H7 subtype-specific mAb; 6D7) and 0.5 mg/ml polyclonal goat anti-mouse IgG on the control line (CL). The diagnostic strip was tested after drying the membrane at 30 °C for 2 days. To select the pair antibody, a dot-FICT assay was used. Briefly, Ab (0.1 µg/µL) was dotted on a NC membrane lacking a TL and dried at 30 °C for 2 days. To perform the FICT assay, 2 µL of Europium-conjugated 2F4 Ab from a stock solution was dropped onto conjugate pad and a mixture of 75 µL of samples with 75 µL of lysis buffer (100 mM Tris-HCl, pH 9.0, 0.025 M EDTA, 0.5% sodium deoxycholate, and 1% Triton X-100) were dropped onto sample pad for 15 minutes. The results of test strips were read with a portable fluorescent strip reader at excitation and emission wavelengths of at 355 nm and 612 nm, respectively (Medisensor, Daegu, South Korea) 49 . Both TL and CL signals were measured and the TL/CL ratio was calculated automatically.
Real-time RT-PCR (rRT-PCR). rRT-PCR was performed using a Quantitect Probe RT-PCR Kit (QIAGEN, Hilden, Germany) to determine the cycle threshold (Ct) values using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The H7 primers, probes, and RT-PCR condition were described previously 34 . For standard of RNA copy number, the template was generated in plasmid pGEM-T Easy (Promega, Madison, WI, USA) including a 132 base pair (bp) HA1 insert. In vitro transcription of HA1 RNA used a RiboMax (Promega) kit to determine the RNA copy number for the limit of detection of FICT. The standard curve was calculated automatically by plotting the Ct values against each standard of known RNA copy number and by extrapolating the linear regression line of this curve. The PCR products were analyzed in agarose gels (2%).
Scientific RepoRtS | 7: 7933 | DOI:10.1038/s41598-017-08328-9 Colloidal gold-based RDT assay. To evaluate the performance of FICT assay, colloidal gold nanoparticles conjugated to Ab was prepared as previously described 50 . After 0.02% chloroauric acid (HAuCl4·4H 2 O) (Sigma-Aldrich) was brought to a boil, 0.2% sodium citrate was added with constant stirring. After the mixture changed to a wine-reddish color, the solution was boiled for additional 5 min and then stirring for 10 min without boiling. Next, 1 mg of the mAb (2F4) was conjugated with the prepared colloidal gold particles (100 mL). The mAb-gold conjugate was precipitated by centrifugation (27,237 × g) and resuspended in PBS containing 0.1% of BSA to an optical density at 450 nm (OD 450 ) of 10. The TL of the strip was prepared by dispensing a desired volume of 2.5 mg/mL mouse mAb (6D7) and 0.5 mg/ml IgG mouse on the CL. Same lysis buffer was used for RDT. A commercial influenza virus A/B RDT (Standard Diagnostics, Yongin, South Korea) was used for comparison. Samples were applied following the manufacturer's instructions.
Ethical statement. The study was approved by the Institutional Review Board of the Wonkwang University Hospital (Approval No. WKUH201607-HRBR-078) and all methods were carried out in accordance with relevant guidelines and regulations.
Statistics. The mean ± standard deviation (SD) was calculated and all data were plotted using GraphPad Prism 5.0 (Graphpad, La Jolla, CA, USA).


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Fluorinated molecular beacons as functional DNA nanomolecules for cellular imaging† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc02819a Click here for additional data file.

Molecular beacons (MBs) are simple, but practical, fluorescent nanoprobes widely used to detect small molecules, nucleic acids and proteins.

Introduction
Live cells are dened by an intricate network of interacting genes, mRNAs, proteins and small molecules. [1][2][3] To visualize and, hence, gain an understanding of intracellular interactions and processes, imaging techniques with high resolution and dynamic imaging capability are required. [4][5][6][7] However, the cell cytoplasm is an unfriendly environment for probes. For example, intracellular enzymes can digest the probe, resulting in false-positive signals. 8,9 Therefore, probes that are sensitive, but stable, are preferable for intracellular measurements.
DNA has been widely exploited as an intelligent and advanced material in construction of DNA-based nanodevices for biological applications. Molecular beacons (MBs) are duallabeled nanomolecules with a uorophore as a reporter at one end and a quencher at the other end. [10][11][12][13][14][15][16] In the absence of targets, MBs form a hairpin structure by intramolecular hydrogen bond interaction of Watson-Crick bases, and the uorescence is quenched by the close proximity of uorophore and quencher. Compared to other uorescent probes, MBs are highly sensitive for real-time monitoring based on their inherent uorescence signal transduction mechanism. Another advantage of these nanoprobes is specic target recognition based on their loop and stem structure. The simple but practical architecture of MBs has resulted in widespread applications that range from the detection of single-nucleotide polymorphisms (SNPs) to bacterial bioterrorism agents. [17][18][19][20][21][22] However, some challenges still remain when MBs are employed in complex biological environments, especially in living cells. The hairpin structure of an MB is oen disrupted by the interaction of stem sequences with nontarget small molecules, proteins or nucleic acids, thus giving false-positive signals. 23,24 The closed stem structure may also be opened in response to an acidic environment or temperature changes. 25 MBs composed of natural bases are not stable against nucleases and cell lysate, and such degradation in living cells also gives false-positive signals. 26 To address such challenges, researchers have developed advanced MBs by introducing articial nucleosides. For instance, to enhance thermostability of MBs, locked nucleic acid/base pairs have been modied in the stem region to allow a constant low uorescence background. To reduce falsepositive signals and improve specicity and stability of MBs, articial nucleoside pairs Z and P have been encoded into MBs to resist degradation by enzymes. [27][28][29][30][31][32] In a previous work, we incorporated 3,5-bis(triuoromethyl) benzene as a hydrophobic base into oligonucleotides and found that a stable planar duplex was formed merely through F-F base pairing. 33 Based on this observation, we hypothesized that an MB constructed through hydrophobic F-F base pairing would present the following advantages over conventional MBs: (1) enhanced stability against enzymatic degradation by the introduction of articial base F; (2) resistance to interference with other biomolecules by self-assembly of the stem through hydrophobic interactions instead of hydrogen bonding (Scheme 1). Articial DNAs may provide promising solutions for challenges strangling wide applications of DNA nanoprobes and nanodevices, and thus we illustrate here the construction of molecular beacons with hydrophobic bases for intracellular imaging.

Formation of beacon structure
To test our hypothesis, a series of triuoromethylated oligonucleotide molecular beacons (Table S1 †) were readily synthesized from our previously developed phosphoramidite F. Conventional MBs with six natural base pairs in the stem region (N6 MBs) and a series of oligonucleotides were also synthesized for the investigation. Both F4 MB (four F-F base pairs in the stem, see Table S1 † for detailed sequence) and F6 MB (six F-F base pairs in the stem, see Table S1 † for detailed sequence) form stable hairpin structure similar to conventional MBs. In comparison, F6 MB displays better signal-to-background ratio than F4 MB ( Fig. 1a and S1 †), and a somewhat better discriminatory power to a single-base mismatch of the target DNA sequence compared to N6 MB (Fig. S2 †).
Thermodynamics study. Conventional MBs maintain their closed hairpin conformation at room temperature. However, with increasing surrounding temperature, conventional MBs slowly change their conformation to the opened state, resulting in enhanced background uorescence intensity (Fig. 1b, black line). As shown in Fig. S4, † the S/B ratio of N6 MB was about 12.6-fold at 20 C, 11.3-fold at 30 C and only 8.4-fold at 37 C. Given preferable and reproducible detection under dynamic temperature change within the scope of room temperature, novel MB-based nanoprobes with better thermostability would provide more attractive performance in analytical detection. 28 Interestingly, unlike N6 MB, F6 MB maintained its conformation even at 90 C (Fig. 1b, red line) with constant S/B ratios of 10.6-fold at 20 C, 10.6-fold at 30 C and 10.0-fold at 37 C (Fig. S4 †). This phenomenon is explained by the following points. First, F-F base pairs display stronger binding free energies than natural base pairs, 33 providing tighter interaction in the stem region. Second, hydrophobic interactions between uorinated base pairs show stronger resistance against temperature change compared to hydrogen bonding between natural base pairs. 34, 35 We further veried that base pairing between F base pairs contributes to the super-thermostability of FMBs. To prove this, a molecular beacon combining six natural base pairs and six uorinated base pairs in the stem region was synthesized and termed F6N6 MB. F6N6 MB also shows thermostability in high surrounding temperatures (Fig. S6 †). Taken together, this evidence proved the super-thermostability of FMBs and, in particular, revealed that F6 MB could detect targets under relatively severe changes in ambient temperature.

Enhanced biostability
Intracellular biodegradation is a key factor affecting intracellular application of MB-based nanoprobes, as it can also result in false-positive signals. Therefore, we next investigated the resistance of F6 MB to digestion by endonuclease and cell lysate compared with N6 MB. As shown in Fig. 1c, a signicant uorescence increase of N6 MB at about 8.9-fold in 30 minutes was observed aer the addition of 0.25 U mL À1 DNase I, indicating that N6 MB suffered severely from enzymatic digestion. In contrast, F6 MB showed better resistance to enzymatic digestion of about 2.5-fold in 30 minutes. When stability against cell lysate was tested, results showed that F6 MB had better resistance to digestion compared to N6 MB (1.5-fold of F6 MB vs. 4.2-fold of N6 MB) (Fig. 1d). Compared with natural base pairs, uorinated base pairs cannot be recognized by endonuclease. Consequently, the replacement of natural base pairs by uorinated base pairs makes F6 MB more resistant to biodigestion  compared to N6 MB. Responses of both MBs to cDNA in cell lysate solution were investigated to determine their practical utility in complex in vitro systems. Because of severe digestion by cell lysate, N6 MB only had about 2.6-fold uorescence enhancement aer 30 minutes of incubation. For F6 MB, on the other hand, uorescence enhancement was about 5.8-fold in the same conditions (Fig. S7 †). This line of evidence demonstrates that F6 MB has better stability and, as a result, better uorescence recovery in complex intracellular biological environments.

Enhanced selectivity of FMBs
Next we compared the performance of F6 MB and N6 MB in the presence of nontarget nucleic acids. Fig. 2 shows signal enhancement aer hybridization of N6 MB and F6 MB with cDNA, Oligo 2, Oligo 3 and Oligo 4 in PBS buffer. The S/B ratios of N6 MB and F6 MB are both approximately 10-fold when they hybridize with cDNA. In the presence of nontarget nucleic acids containing sequences complementary to the stem sequence of N6 MB, such as Oligo 4, Oligo 3 and Oligo 2, the S/B ratios of N6 MB range from 8.2-fold to 9.6-fold, suggesting that false-positive signals could result from interference with nontarget sequences. Compared with N6 MB, only negligible signal enhancements were observed when F6 MB hybridized with Oligo 3 or Oligo 4. For example, the signal enhancement was under 3.6-fold for F6 MB hybridized with Oligo 2. These uorescence results verify that F6 MB is more resistant to interferences in complex biological environments than N6 MB.

Intracellular mRNA imaging
In addition to their degradation by nucleases, the cellular applications of MB-based nanoprobes, as oligonucleotides, are limited by their poor uptake by cells. For efficient delivery, MBs are typically injected into cells by microinjection technology or by complex delivery systems. In this paper, an extended internalized DNA strand AS1411 was linked at the terminus of F6 MB. AS1411, as a G-rich oligonucleotide, is capable of uptake into cytoplasm by the cell. The cellular internalization pathway was proved by a macropinocytosis process under the stimulation by a nucleolin-dependent mechanism. Because of its excellent target and internalization abilities to specic cancer cells, AS1411 and conjugated nanoparticles have been used widely in preclinical cancer diagnosis and therapy. Manganese Superoxide Dismutase (MnSOD) mRNA, which is overexpressed in MCF-7 breast cancer cells, [36][37][38] was used as a model target in our cellular imaging study. AS1411-linked F6 MnSOD MB effectively recognized the target DNA sequence, providing about 10-fold uorescence recovery in tube solution (Fig. 3b). AS1411-linked F6 MnSOD MBs with moderate concentration were then incubated with MCF-7 cells pretreated with lipopolysaccharide (LPS, 10 mg mL À1 ), followed by confocal uorescence microscopy imaging. As is shown in Fig. 3c and d, an obvious uorescence response (2.5-fold compared to control molecular beacon) was found aer one hour incubation. However, only negligible uorescence was observed for the same cells incubated with F6 control MB under the same conditions. These imaging results demonstrated that F6 MB is capable of imaging mRNA in the intracellular environment. To further assess the selectivity of AS1411-linked F6 MnSOD MBs to its targeted nucleic acids in cells, comparison of probes-incubated MCF-7 cells with and without mRNA overexpression was performed. As shown in Fig. S8, † an obvious uorescence shi was observed in the case of LPS-treated cells (about 1.7-fold in intensity) compared to the non-overexpressed cells. This result provided an additional proof of the specic recognition of F6 MB to its target in cells. Next, using different DNA concentrations, a cell cytotoxicity assay was performed to determine cell viability of MCF-7 cells aer binding with uorinated DNA hairpin. Results indicated that the F6 DNA hairpin had almost no effect on cell proliferation, thus demonstrating its biosafety (Fig. S9 †).

DNA synthesis
All DNA strands used in this work were synthesized on the Polygen 12-column DNA/RNA solid synthesizer on a 1.0 micromolar scale, using the corresponding controlled pore glass (CPG). As-synthesized 3,5-bis(triuoromethyl)benzoyl phosphoramidite monomer was dissolved in anhydrous dichloromethane in 0.1 M concentration and coupled on the synthesizer with 600 s coupling time. Aer synthesis, the obtained oligonucleotides were cleaved and deprotected from the CPG, followed by precipitation in cold ethanol solution at À20 C overnight. Aer centrifugation to remove supernatant solution, the DNA products were dissolved with 0.1 M TEAA and puried by reversed phase HPLC using a BioBasic4 column. Finally, the 4,4 0 -dimethytribenzyl group was removed from DNA by adding 80% acetic acid aqueous solution, and the DNA was again precipitated in cold ethanol. Aer drying in vacuum and the desalting step, the synthesized oligonucleotides were quantied by measuring their absorbance at 260 nm.

Fluorescence measurements
A JY uorimeter was used for all uorescence measurements. For FAM group, the solution was excited at 488 nm and scanned from 500 to 700 nm with a bandwidth of 5 nm. For the kinetic mode of uorescence measurements, the excitation wavelength was 488 nm, and emission wavelength was 520 nm with a bandwidth of 5 nm.

Melting curve measurement
All measurements were performed in an Applied-Biosystems 7500 RT-PCR instrument with a sample concentration of 200 nM. The temperature was increased from 20 to 90 C in 1 degree increments per minute. The FAM uorescence signal was measured.
Cell culture MCF-7 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum and 0.5 mg mL À1 penicillinstreptomycin at 37 C in 5% CO 2 . CCRF-CEM (human leukemia) cells were maintained in 1640 medium, supplemented with 10% fetal bovine serum and 0.5 mg mL À1 penicillin-streptomycin at 37 C in 5% CO 2 .

Cell lysate preparation
About three million CCRF-CEM cells were washed twice with DPBS buffer and then dispersed in 1 mL DPBS buffer solution. The mixture was lysed with a sonicator for 2 minutes, and the resulting cell lysate solution was used immediately.

Cytotoxicity assay
The cytotoxicity of uorinated molecular beacons was evaluated using a 96-well proliferation assay at 37 C in 5% CO 2 atmosphere. A sample of 5000 MCF-7 cells in 100 mL fresh cell culture medium was seeded into each test well. Aer 24 h, cell culture medium was removed, and then another 100 mL of fresh culture medium without serum was added, followed by adding uorinated DNA molecular beacons at the desired concentration. Aer 6 h treatment, culture medium without serum was removed and 100 mL of fresh cell culture medium was added for an additional 48 h treatment. Finally, 10 mL of CCK-8 was added to the well, and the 96-well plate was subjected to absorption measurement at 450 nm using a microplate reader.

FACS analysis
MCF-7 cells were placed in a 35 mm cell culture dish and grown to around 80% conuence for 48 h before the experiment. Before incubation with molecular beacons, MCF-7 cells were pretreated with or without 10 mg mL À1 LPS (lipopolysaccharide) for 30 minutes. Then the solution was removed and cells were allowed to incubate with probes for one hour. Aer incubation, cells were washed twice with PBS buffer and then subjected to FACS assay.
Confocal uorescence microscopy imaging MCF-7 cells were placed in a 35 mm cell culture dish and grown to around 80% conuence for 48 h before the experiment. Cells were washed with 1 mL PBS buffer containing 5 mM MgCl 2 . Before incubation with molecular beacons, MCF-7 cells were pretreated with 10 mg mL À1 LPS for 30 minutes. Then LPS solution was removed by washing with PBS. Treated cells were incubated with molecular beacon probes at the desired concentration in PBS buffer containing 5 mM MgCl 2 and 4.5 mM glucose for one hour at 37 C in 5% CO 2 . Aer incubation, cells were washed twice with PBS buffer and then subjected to confocal microscopy imaging.

Conclusions
In summary, this study illustrates the synthesis and characterization of FMB-based nanoprobes. The introduction of the base surrogate F into MBs signicantly increases their thermostability and improves stability against cell lysate over N6 MB. By imaging mRNA in MCF-7 cells with F6 MnSOD MB, we demonstrated the potential of this nanoprobe for dynamic monitoring of cell-specic targets.

Conflicts of interest
The authors declare no competing nancial interests.


text:
From skeletal muscle to stem cells: an innovative and minimally-invasive process for multiple species

Bone marrow and adipose tissue represent the two most commonly exploited sources of adult mesenchymal stem cells for musculoskeletal applications. Unfortunately the sampling of bone marrow and fat tissue is invasive and does not always lead to a sufficient number of cells. The present study describes a novel sampling method based on microbiopsy of skeletal muscle in man, pigs, dogs and horses. The process includes explant of the sample, Percoll density gradient for isolation and subsequent culture of the cells. We further characterized the cells and identified their clonogenic and immunomodulatory capacities, their immune-phenotyping behavior and their capability to differentiate into chondroblasts, osteoblasts and adipocytes. In conclusion, this report describes a novel and easy-to-use technique of skeletal muscle-derived mesenchymal stem cell harvest, culture, characterization. This technique is transposable to a multitude of different animal species.

Results
As we were initially seeking an alternative method for collecting equine pluripotent stem cells, we basically developed our technique on equine muscles. Subsequently, we have evaluated this concept on dogs, pigs and humans.
The sampling method: muscular microbiopsy. To initiate the culture of pluripotent muscle-derived stem cells, we use muscular microbiopsies of approximately 15 to 20 mg of tissue. The sampling procedure is performed with a semi-automatic 14 gauge microbiopsy needle. The sampling site is shaved and aseptically prepared, a local anesthetic is injected subcutaneously and the microbiopsy is collected through a small skin puncture. Immediately after collection, each sample is placed in culture medium and maintained at 4 °C until use.
To date, we have sampled 45 horses, 3 pigs, 10 dogs and 2 humans with this method. All of these samples, except 3 of the dogs, were successfully cultured as described below. We neither observed any contamination of the sampling site, nor any adverse effect on muscle function except for humans who showed some painless muscle twitching for approximately 12 hours. With one microbiopsy of about 15 to 20 mg, we had sufficient tissue to easily initiate a culture. We showed also that microbiopsies can be done by veterinarians in clinical practice and do not require the hospitalisation of the animal. The absence of adverse effects and the facility of sampling enabled us to apply this technique in exercising high-performance horses. This aspect is also relevant as regards human patients.
Initiation of the cell culture. Culture preparation was performed using sterile equipment, in the controlled environment of a biosafety cabinet. Microbiopsy specimens were washed twice in phosphate buffer saline solution (PBS), carefully dissected and then cut into small pieces. Each piece was placed individually into the 16 central wells of 24-multi-well dish pre-filled with culture medium. The multi-well dish was incubated at 37 °C in a CO 2 incubator.
After three to four days in culture, the first cells started to appear around the muscle pieces. Depending on the species, about 10 to 18 days (13.2 days ± 2.63) after initiating the culture, a halo of cells was visible around the tissue and the number of cells was sufficient to allow for pluripotent stem cells isolation. Before the isolation step, we obtained a mean of 63000 ± 30675 cells from the 16 wells pooled together.
Pluripotent stem cell isolation: discontinuous Percoll density gradient centrifugation. The cells emerging from explants are detached using tryspin-EDTA, centrifuged (200 × g, 10 min, 37 °C) and the pellet was suspended in Hank's Balanced Salt Solution (HBSS). The cellular suspension was then placed on a 3 layers discontinuous Percoll density gradient (15%, 25% and 35%). The cell fractions with different densities appeared at the interfaces between each Percoll fraction after centrifugation at 1250 × g (25 °C, 20 min). To continue the culture, we chose to work with the 15-25% fraction. The fraction was washed once with HBSS and centrifuged at 200 × g, 10 min at 37 °C. The supernatant was discarded and the pellet was suspended in 1 ml of culture medium. The cells were then cultured in T-25 cm 2 Flask.
We observed that the cells reached 80% confluence in T-25 cm 2 Flask within 7 ± 1 days. The cells could then be frozen in liquid nitrogen or further passed for other experiments.
Validation. The clonogenic capacities of the cells were assessed for the cells coming from each Percoll fraction (<15%, 15-25%, 25-35% and >35%) in horses (n = 3) and humans (n = 2). Their abilities to differentiate into adipocytes, chondroblasts and osteoblasts were evaluated for the cells coming from each Percoll fraction in horses (n = 5), but only for the 15-25% fraction in humans (n = 2) and dogs (n = 3). Their immunomodulatory capacities in horses (n = 2) and humans (n = 2) were also assessed. The expressions of CD90, CD105, CD44, CD45 and MHCII were studied for the cells from each Percoll fraction in 2 horses and for the 15-25% fraction in humans (n = 2) and 6 more horses. In humans (n = 2), their expression of CD34, CD19 and CD73 was also shown. Some equine bone marrow mononuclear cells were used to validate the cross-reactivity of all the antibodies used with the equine epitopes.
Clonogenic capacities. The clonogenic capacities of the cells were evaluated with a "fibroblast-colony forming units" assay (CFU-F). The morphological aspect of the CFU's observed was not different from those habitually observed with pluripotent stem cells isolated from bone marrow or Wharton Jelly (Fig. 1). The best clonogenic capacities were observed for the 15-25% cells.
Trilineage differentiation. Adipogenic, osteogenic, and chondrogenic differentiations of equine, human and canine cells were performed in adapted specific media. The 15-25% cells obtained from each species were able to differentiate into adipocytes, chondroblasts and osteoblasts when cultured with the corresponding induction media (Fig. 2). For horses, the cells coming from each Percoll fraction were able to trilineage differentiation.
Immunophenotyping. First, we validated the cross reactivity of the antibodies by using equine bone marrow mononuclear cells. As shown in Fig. 3, the CD73, CD19 and CD34 did not recognize the equine epitopes. Therefore, the equine cells were only evaluated for their expression of CD44, CD45, CD90, CD105 and MHCII. This evaluation was performed for cells from each Percoll fraction. For human cells, only those from the 15-25% fraction were used but they were further evaluated for their expression of CD73, CD19 and CD34. For the 15-25% cells from horses and humans, over 60% of the cells expressed CD105, CD44 and CD90 whereas less than 2% expressed CD45 and MHCII. Furthermore, for humans, more than 80% of cells were positive for CD73 but less than 2% expressed CD34 and CD19 (Figs 3 and 4).
For 2 horses, we characterized the cells of the different Percoll fractions. We observed that less than 2% of the cells of all fractions expressed MHCII and CD45 and more than 60% of the cells of all fractions expressed CD105 Scientific RepoRts | 7: 696 | DOI:10.1038/s41598-017-00803-7 and CD44 whereas the expression of CD90 varied between the fractions. From the 2 horses tested, CD90 was expressed by 48% of the <15% cells, 73% of the 15-25% cells, 36% of the 25-35% cells and 10% of the >35% cells.
Immunomodulatory capacities. The immunomodulatory capacities of the cells were evaluated by their ability to inhibit the proliferation of purified T-lymphocytes (LT).
An inhibition of LT proliferation (%) was observed in the presence of stem cells from the 2 species. For horses, the greatest inhibition was observed with the ratio stem cells/LT of 1/8. For human, the optimal ratio of stem cells/ LT was 1/4. These patterns were similar to the ones observed with stem cells from bone marrow for the 2 species.

Discussion
With this study, we propose for the first time an effective and patented (Mammalian muscle-derived stem cells, WO2015091210) method to obtain mesenchymal stem cells from muscles. Furthermore, this process is easily transposable to multiple species including humans.
First, the sampling method that we propose, using muscular microbiopsy is less invasive. In horses, harvesting muscle tissue is easier than bone marrow or periosteal tissue. Skeletal muscles represent about one third of body mass and the risk of inducing a lesion at the sampling site is low. However, the required quantities described by Radtke et al. 11 or even by Tamaki et al. 28 for their method could be prohibitive for competing horses and especially for dogs and humans. With our microbiopsy method, only 10 to 20 mg of tissue are required as against approximately 6 g according to Radtke 16 . A study performed by Votion et al. 30 demonstrated that the microbiopsy technique can easily be performed by veterinarians in the field. Because of the ease of sampling and the absence of adverse effects, we can consider this method safe for the use in man, performance horses and other domestic animals.
Numerous authors have reported using enzymatic digestion to isolate stem cells from muscular sample 11,13,15,28,31,32 . The quantity of tissue collected using our microbiopsy technique is insufficient for the purpose of enzymatic digestion 33 . The technique of enzymatic digestion not only requires larger quantities of tissue but also bears the risk of proteolytic contamination which can damage the cell surface ligands and receptors necessary for stem cell function after in vivo transplantation 28,34 . The mechanical steps related to the method of enzymatic digestion may also be damaging for sensitive cells such as stem cells. Therefore we developed a technique that simply uses muscular microbiopsies as explants and involves waiting until progenitor cells grow from them. In this way, we reduce the number of steps involving manipulation and the potential sources of contamination. Further, sampling and cutting the biopsies to form explants mimics muscle fiber trauma and represents a trigger for stem cell activation, migration and proliferation [35][36][37] . The three-dimensional structure of the parent tissue during the crucial early stages of cell outgrowth is maintained while providing the outgrowing cells with a rich nutritive media in which they proliferate. Skeletal muscle explants thus mimic the in vivo environment of the regenerating muscle and stimulate stem cell migration and division 35 .
Muscle-derived cells are a mixture of subpopulations from different lineages and different developmental stages 38,39 . Different techniques are used to isolate muscle-derived stem cells which can be marker profile-dependent or not. This last type of methods selects the cells on the basis of their adhesion characteristics (modified preplate technique 40 ) or their densities, which are reliable in their expression of specific molecular markers 32 . Ultimately, from the perspective of clinical applications, the isolation technique must allow easy and rapid acquisition of stem cells, while respecting financial concerns in veterinary medicine. In our study, we decided to adapt a discontinuous Percoll gradient method 32 on the cells obtained from the muscle explants. Essentially, two kinds of stem cells exist in muscle tissue: satellite cells and muscle-derived stem cells. Satellite cells, referred to by many as 'muscle stem cells' , are 'myogenic precursors' capable of regenerating muscle tissue and demonstrating self-renewal properties; however, they are considered to be committed to the myogenic lineage 41 . Muscle-derived stem cells, which may represent a predecessor of the satellite cell, are considered to exhibit a higher regeneration capacity and better cell survival and a broader range of multilineage capabilities 17 . In a previous study using muscular microbiopsies as explants, our group has obtained a primary equine myoblast culture 42   that the isolation step is essential to select stem cells among the muscular cell populations and suggest that applying only enzymatic digestion may be insufficient.
At first, we worked with equine samples and isolated 4 subpopulations of cells with the Percoll gradient (ie cells <15%, between 15 and 25%, between 25 and 35% and >35%). We characterized the 4 subpopulations of cells from 2 horses and observed that all of them were able to trilineage differentiation but the greatest expression of CD90 was found for the cells from the 15-25% fraction. Furthermore, the 15-25% cells showed the greatest proliferative and clonogenic capacities. Regarding these results and the data from the literature 32 and to simplify our process, we finally choose to keep only the cells from the 15-25% Percoll fraction. This was also applied when we worked with samples from other species.
As defined by the ISCT 29 , our method allows the isolation of pluripotent mesenchymal muscle-derived stem cells from horse, human, dog and pig microbiopsies. Unfortunately, we were not allowed to use the entire panel of antibodies described by the ISCT for equine cells because the cross reactivity study showed us that CD73, CD19 and CD34 did not recognize the equine epitope. For humans, the results obtained were in accordance with the recommendations while for equine cells, only positivity for CD90, CD105 and CD44 and negativity for CD45 and MHCII was demonstrated. Even while it is known for human cells 44 , this is the first time that the expression of CD105 is described for equine skeletal muscle-derived stem cells. CD105, which is a component of the TGF beta-1 complex, has different important biological functions such as angiogenesis 45 and growth induction at the joint/articular level. TGF beta-1 stimulates chondrocyte division as well as cartilage matrix synthesis but decreases the release of PGE2 by osteoarthritic synovial fibroblasts and hence decreases PGE2 stimulated matrix degradation in osteoarthritis 46 . CD105 expression thus appears to show excellent tissue regeneration characteristics to the obtained stem cells.
We also showed that our cells can form fibroblast-like colonies in culture, with a similar aspect to what is generally observed with other mesenchymal stem cell sources. The isolated cells can also differentiate into adipocytes, chondroblasts and osteoblasts and support a plurality of freeze-thaw cycles without losing their pluripotency. Another characteristic of the isolated cells, which is also interesting from a clinical point of view, is that they show immunomodulatory capacities towards T lymphocytes. If this is well known as regards human and equine stem cells 47-49 , it has never been shown specifically with equine skeletal muscle-derived stem cells. This anti-inflammatory property confers a real advantage in the clinical use of the cells.   Table 1. Open histograms show nonspecific isotype control staining and solid histograms show specific staining for the indicated marker. Cross-reactivity was not found for CD73, CD34 and CD19 markers.
Scientific RepoRts | 7: 696 | DOI:10.1038/s41598-017-00803-7 To our knowledge, this is the first time such a comprehensive characterization has been described for skeletal muscle-derived stem cells from numerous species. Even if further investigations are indicated, we are now able to propose a suitable process to quickly and easily obtain fully characterized pluripotent stem cells from minimally-invasive muscular microbiopsies. These cells have distinct properties that make them particularly attractive for clinical use.

Reagents
General. DMEM Antibodies. All information relating to the antibodies used can be found in Table 1.

Method
The sampling method: muscular microbiopsy. To initiate the culture of pluripotent muscular derived stem cells, we used muscular microbiopsies which represented each approximately 15 to 20 mg of tissue. The microbiopsy procedure on animals is approved by the Animal Ethical Commission of the University of Liège and is performed in accordance with the relevant guidelines. Human samples are derived from healthy volunteer donors and informed consent is obtained. All experiments are approved by the Regional Ethical Research Committee of the Bordet Institute and conducted according to the principles expressed in the Helsinki Declaration. In this study all horses and humans were sampled without sedation, dogs were sedated with dexmedetomidine 5-10 µg/kg intramuscularly and pigs have been sampled under general anesthesia. Samples were performed by means of a 14G-sized semiautomatic microbiopsy needle in all species except for dogs for which a 16G needle was used. For horses, samples were taken from the triceps brachii muscle at a point situated halfway on a line joining the point of the elbow and the point of the shoulder. For dogs, samples were taken from the long head of the triceps brachii muscle. In pigs the samples were taken from the semitendinosus muscle. For humans,   Table 1. Antibodies for analyzing the cellular proteins on equine and human cells.
we sampled once in the biceps brachii and once in the vastus lateralis of the quadriceps femoris. For dogs and horses, the sample site is shaved and surgically scrubbed for 5 minutes. Only the surgically scrub was performed for human and pigs. A local anesthesic (lidocaine 2%) was injected subcutaneously at the sample site. After having put on the sterile gloves, a full thickness skin puncture is performed using the introducing sheath with its mandarin in place. After arming the biopsy needle (2 notches), it is slid into the introducing sheath from which the mandarin was previously removed. The biopsy needle together with the introducing sheath is inserted through the skin puncture into the muscle and advanced for 2 to 3 cm. After releasing the spring loaded mechanism, the needle with its sheath is withdrawn from the muscle. The sample is removed from the biopsy needle using a sterile disposable injection needle. The sample is carefully placed in a tube which containing DF20 medium [DMEM/ Ham's F12 culture medium with Hepes and Glutamine, 20% of heat inactivated (FBS), 5 ml penicillin (1000 U/ ml)-streptomycin (10000 µg/ml) and 2.5 ml amphotericin B (250 µg/ml)]. Prior to delivery to the laboratory, the microbiopsies are kept in the DF20 at 4 °C.
Initiation of the cell culture. Culture preparation was performed using sterile equipment, in the controlled environment of a biosafety cabinet. Two half 10 cm Petri dishes were prepared with 5 ml of PBS to wash the microbiopsy sample. A drop of PBS was placed apart in one of the half dish. The 16 central wells of a 24-multiwell dish were filled with 150 µl of DF20 and the outer wells with PBS (1 ml/well) to prevent drying out of wells containing explants. The medium containing the microbiopsy sample was poured into an empty half 10 cm Petri dish.
With the help of forceps, the microbiopsy specimen was successively washed twice in the 2 different half 10 cm Petri dish containing 5 ml of PBS. Each microbiopsy specimen was carefully dissected using the sterile forceps and the scalpel blade, while attempting to retain as far as possible only muscular tissue (light pink tissue) in a half Petri dish containing 5 ml of PBS solution. The microbiopsy specimens were cut into small pieces (size of the tip of the scalpel blade) and placed apart in the drop of PBS. Each piece of muscle was individually placed into the 16 central wells of 24-mutliwell dish and incubated at 37 °C under controlled atmosphere (5% CO 2 and 21% O 2 ). The wells were monitored each day and when necessary, some DF20 was added to avoid the cells drying out (typically a single time between the initiation of culture and the isolation step).

Pluripotent stem cells isolation.
First, the discontinuous Percoll gradient was prepared. Each layer was prepared in a separate sterile 15 ml conical bottom centrifuge tube using a 9 g/l NaCl solution. For one multi-well dish containing explants, 2.5 ml of each density of Percoll (15%, 25% and 35%) were prepared by adding for 15% : 375 µl Percoll + 2.125 ml NaCl; for 25% : 625 µl Percoll + 1.875 ml NaCl and for 35% : 875 µl Percoll + 1.625 ml NaCl. Two ml of the 15% Percoll solution were placed in the bottom of a 15 ml conical bottom centrifuge tube. From below, 2 ml of the 25% Percoll solution and then 2 ml of the 35% Percoll solution were placed successively. Within the multi-well dish, the culture medium and the pieces of muscle present in each culture well were discarded before rinsing with 500 µl of PBS. The PBS was discarded and 150 µl of trypsin added to each cultured well. The multi-well plate was placed for 2-5 minutes in the 37 °C and 5% CO 2 incubator. When cells were detached, 300 µl of DF20 were added in each well to stop the action of trypsin. The cells were harvested from all the culture wells in a 15 ml conical bottom centrifuge tube and centrifuge for 10 min at 200 × g and 20 °C. The supernatant was removed and the cells were suspended in 1 ml of HBSS. The cellular suspension (cells in 1 ml of HBSS) was carefully placed on the Percoll gradient and centrifuge for 20 minutes at 1250 × g and 20 °C without brake. For 2 horses, each fraction (ie cells <15%, between 15 and 25%, between 25 and 35% and >35%) was harvest separately in order to characterize them for immunophenotype, clonogenic capacities and trilineage differentiation capabilities. Finally, it was chosen to work only with the cells from the 15-25% fraction so to harvest this fraction of interest, the first 2 ml were carefully removed, the following 2 ml were retained and the rest of the solution was discarded. The cellular suspension was diluted by adding 6 ml of HBSS and centrifuged for 10 min at 200 × g and 20 °C. The supernatant was discarded and the cells suspended in 1 ml of DF20. The cellular suspension was placed in a 25 cm 2 T-Flask, 5 ml of fresh DF20 were added and the T-Flask was incubated at 37 °C in the 5% CO 2 incubator. The medium was changed for new DF20 the day after the isolation step and again 3 days later. The isolated cells can then further be passed classically using trypsin and multiplied for up to 8 passages or cryopreserved in liquid nitrogen.
Validation. Clonogenic capacities. The clonogenic capacities of the human and equine cells were evaluated with a "fibroblast-CFU" assay. Cells in primo-culture or after one passage, were seeded at low density (5000 cells/ flask) and grown for 10 days. Fibroblastic colonies of more than 50 cells were scored using an inverted microscope after May Grünwald Giemsa staining.
Trilineage differentiation. These differentiations were carried on human, equine and canine cells.
-Adipogenic differentiation: For the adipogenic differentiation, 5000 cells/well were plated in a 24-well plate or 50000 cells/well in a 6-well plate in NH AdipoDiff Medium. After 21 days, cells were colored using Oil Red O. Briefly, cells were washed with PBS and fixed with 8% formaldehyde or 100% ethanol before staining with Oil Red O solution.
-Chondrogenic differentiation: To induce chondrogenesis, cells were transferred in the bottom of 15 ml conical tubes and differentiated into chondroblasts in pellet culture (250000 cells/pellet) in 1 ml specific chondrocyte induction medium. Tubes were incubated for 21 days at 37 °C in a 5% CO 2 incubator, and the medium was replaced every week. Briefly, after 21 days, the micro-masses were fixed with methanol and whole mount stained with Alcian Blue.
-Osteogenic differentiation: For the osteogenic differentiation, the cells were plated in DF20 in a 24-well plate at a density of 5000 cells/well or 50000 cells/well of a 6 well multi-well dish. After 24-48 h, the osteogenic medium was added to the adherent cells. Every week, cells were fed with complete replacement of the medium. At days 21, the calcium mineralization was assessed by coloration with Alizarin Red. Briefly, cells were washed in PBS and fixed in 70% ethanol. Cells were finally stained with Alizarin Red.
Immunophenotyping. Human and equine muscle derived stem cells and bone marrow mononuclear cells were analyzed by flow cytometry. Briefly, the cells (10 5 ) were washed with PBS and incubated with the following monoclonal antibodies (Table 1): CD105, CD44, MCH II, CD45, CD90 (equine and human cells), CD73, CD19 and CD34 (human cells only). After washing with MACSQuant Running Buffer, the cells were fixed with 4% formaldehyde solution. Data were acquired using MACSQuant Analyzer and evaluated using FCS Express 4 Flow Cytometry Software (De Novo Software, Los Angeles, CA, USA).
Immunomodulatory capacities. The cells obtained from human or equine muscles were plated at 4000 cells/ cm 2 in a flat-bottomed 24-well plate. After a short period of adherence, allogeneic LT purified from peripheral blood and stimulated by phyto-hemagglutinin (PHA/IL2) were incubated with the plated stem cells for 5 days of co-culture in RPMI-1640 medium supplemented with 10% FBS. We tested several stem cell:LT ratios (from 1:80 to 1:4) to investigate their importance in the stem cell mediated effects.
Lymphocyte proliferation was evaluated after carboxyfluorescein diacetate_succinimidyl ester (CFDA-SE) labeling. For CFDA-SE labeling (CellTraceTM CFSE cell proliferation kit), 10 mM CFDA-SE dye was used to stain 10 7 LT before co-incubation with stem cells. After 5 days of co-culture CFSE fluorescence was analyzed by flow cytometry. The CFSE profile of the labeled cells was composed of several distinctive peaks representing the number of cell divisions that the proliferated lymphocytes had undergone after activation. LT proliferation was expressed by the percentage of positive LT.