Development of an intravaginal ring for the topical delivery of Aurora kinase A inhibitor, MLN8237
Authors:
Yaman Tayyar aff001; Ryan Shiels aff001; Andrew C. Bulmer aff001; Alfred K. Lam aff002; Daniel Clarke aff001; Adi Idris aff001; Nigel A. McMillan aff001
Authors place of work:
School of Medical Science, Griffith University, Southport, Australia
aff001; Menzies Health Institute Queensland, Griffith University, Southport, Australia
aff002
Published in the journal:
PLoS ONE 14(11)
Category:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0225774
Summary
Human papilloma virus (HPV) is the main culprit in cervical cancers. Although the HPV vaccine is now available, the slow and gradual process for HPV cancers to form means little will change, even for vaccinated individuals. This warrants the development of new therapeutic strategies in both the newly diagnosed and recurrent patients. We have previously shown that Alisertib (MLN8237), an Aurora A kinase inhibitor, potently and selectively kills HPV-positive cervical cancer cells. However, Alisertib is known for its unfavorable side effects when administered systemically. A targeted delivery approach is therefore warranted. The topical delivery of drugs to the cervix for the treatment of cervical cancer is an underexplored area of research that has the potential to significantly improve therapeutic outcome. Here, we design a novel topical drug delivery system for localized delivery in the vaginal tract using intravaginal silicone rings loaded with Alisertib. We assessed the suitability of the drug for the application and delivery method and develop a high-performance liquid chromatography method, then show that the vaginal rings were effective at releasing Alisertib over an extended period of time. Furthermore, we showed that Alisertib-loaded vaginal rings did not induce overt inflammation in the mouse vaginal tract. Our work has major translational implications for the future development of vaginal ring devices for the topical treatment of cervical cancer.
Keywords:
inflammation – Cancer treatment – human papillomavirus – cervical cancer – Medical implants – High performance liquid chromatography – Drug delivery – Silicones
Introduction
Persistent infection with human papillomavirus (HPV) is the key risk factor for cervical cancer and is found in more than 99% of cervical cancer cases [1, 2]. Current treatments for cervical cancer involve a combination of surgery, radiotherapy, and/or chemotherapy. However, little improvement in patient outcomes has occurred with these treatments over the last 35 years [3]. We recently showed that Aurora A kinase inhibition using Alisertib (MLN8237) was selectively lethal for HPV-positive (+) cervical cancer cells, both in vitro and in vivo [4, 5]. Given that more than 75% of all cervical cancer cases are diagnosed within stages I–II [6, 7], where the cancer is still confined to epithelia of the cervix itself, it was rational to explore a potential localized delivery system for administering Alisertib in the cervical area. Intravaginal rings (IVRs) represent a topical delivery system for female genital disorders, that sit in the cervical area when administered [8]. IVRs are flexible, torus in shape and elastomeric drug carriers that maintain long-term delivery of a range of drugs currently in use, including microbicides, contraceptives and anti-HIV agents with demonstrated superior efficacy and convenient release profiles [9–16]. IVR polymers are commonly hydrophobic in nature and therefore provide a compatible carrier for hydrophobic molecules [17], such as Alisertib. Although local delivery of therapeutic drugs for cervical cancer may provide higher efficacy and better safety profile, little is known about the potential of IVRs in delivering Alisertib, let alone in the cervix. In this study, we explored the possibility of developing a topical delivery platform of Alisertib for the application in the vaginal tract. Matrix type silicone based IVRs were chosen as a starting point due to their compatibility with Alisertib, their safety profile, and expected favourable release profile. To our knowledge, this is the first demonstration of localizing the administration of Alisertib into the vaginal tract.
Materials and methods
Cells
The HPV+ cervical cancer cell line, CaSki, was obtained from the American Type Culture Collection (ATCC), and cultured as previously described [4]. CaSKi cells were grown in complete DMEM (Gibco-Invitrogen, Waltham, MA) supplemented with 10% heat inactivated foetal bovine serum (FBS) (Gibco- Invitrogen, Waltham, MA) and 1% of antibiotic/glutamine preparation (100 U/ml penicillin G, 100 U/ml streptomycin sulphate, and 2.9 mg/ ml of L-glutamine) (Gibco-Invitrogen, Waltham, MA). Immortalized normal human cervical keratinocytes, HCK1T, were kindly provided by Professor Tohru Kiyono (National Cancer Center Research Institute, Tokyo, Japan) and cultured as previously described [18]. HCK1T were grown in Keratinocyte serum-free media supplemented with 50μg/ml bovine pituitary extract and 5ng/ml human recombinant epidermal growth factor (Gibco-Invitrogen, Waltham, MA), 0.035mM CaCl2 and 1% of antibiotic preparation (100 U/ml penicillin G and 100 U/ml streptomycin sulphate) (Gibco-Invitrogen, Waltham, MA).
Cell viability measurement
Cells were seeded overnight and treated the following day. At 72h, cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and half maximal inhibitory concentration (IC50) determined using GraphPad Prism v8.
Simulated vaginal fluid (SVF) preparation and Alisertib exposure
SVF was prepared as previously described [19]. For experiments involving SVF exposure to Alisertib (Jomar Life Research, Caribbean park, VIC, Australia), Alisertib/DMSO was incubated with SVF before diluting in cell media.
HPLC analysis of Alisertib
A stock solution of Alisertib (1 mg/mL) was prepared in 1% formic acid/acetonitrile for analysis via High Performance Liquid Chromatography (HPLC). The lambda max (λ max) of Alisertib was determined by scanning 200 μL of the stock solution from 200–650 nm on a MultiskanTM GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA) using 1% v/v formic acid in acetonitrile as a control. HPLC analysis was performed on a Shimadzu Prominence LC-20AT coupled to an SPD-M20A Diode Array Detector (DAD). Separation was achieved using a reverse phase C18 column (GraceSmart C18 150 × 4.6 mm, 3 μm, Grace Davidson, Australia; column oven set at 30°C) and a flow rate of 1.0 mL·min-1. The initial mobile phase consisted of 50% mobile phase B (0.1% v/v formic acid in HPLC grade acetonitrile (RCI Labscan, Thailand) and 50% mobile phase A (0.1% v/v formic acid in Milli-Q purified H2O). A linear gradient was applied: 0–0.25 minutes, 50% B; 0.25–4.75 minutes from 50% to 95% B; 4.75–6.75 minutes, 95% B. After 6.75 minutes, 50% B was run for 7 minutes to re-equilibrate the column in between analyses. A linear calibration curve was generated using 6 standard concentrations of Alisertib (3854.2 nM, 963.54 nM, 385.43 nM, 192.71 nM, 96.35 nM and 38.54 nM), and R2 was calculated. For experiments involving solubilizing Alisertib in isopropanol, 1 mg/ml (1% w/v) of Alisertib was prepared in 50% isopropanol for HPLC analysis.
Liquid chromatography/mass spectrometry (LCMS) analysis of Alisertib
HPLC coupled to Electrospray Ionisation Mass Spectrometry (HPLC-ESI/MS) was conducted using a Shimadzu Nexera X2 UHPLC system with SPD-M30A DAD coupled to a Shimadzu LCMS-2020 single quadrupole mass spectrometer in order to confirm the m/z of Alisterib. Separation was achieved using a reverse phase C18 column (Shimadzu Shim-pack GISS C18, 150 mm x 2.1 mm, 3 μm; column oven set at 30°C) and a flow rate of 0.375 mL·min-1. A linear gradient was applied: 0.0–2.5 minutes, from 50% to 95% B; 2.5–3.5 minutes, 95% B. After 3.5 minutes, 50% B was run for 3 minutes to re-equilibrate the column in between analyses. Alisterib was constituted as above, added to a HPLC vial, placed in the autosampler where 10 μL was injected for analysis with data acquired in positive ion mode. Qualitative ESI-MS operating conditions were: nebulizer and drying gas (N2 via Peak Scientific NM32LA nitrogen generator), desolvation line temperature 250 °C, drying gas flow 15 L·min−1, nebulizer gas flow 1.5 L·min−1, heat block temperature 200 °C, interface voltage 4.5 kV with the MS scan range set from 200–650 m/z with an event time of 0.07 sec. Single Ion Monitoring (SIM) was also used with 519.25 m/z with an event time of 0.05 sec.
Manufacturing intravaginal rings (IVRs) containing Alisertib
Mouse-sized molds were 3D printed on a Ultimaker 2+ printer (Cambridge, MA). The dimensions of the mold cavities were 4.2 mm diameter (including a 1.4 mm inner filled cylinder) x 1.4 mm depth. Biomedical grade SILASTIC MDX4-4210 silicone elastomer base (Dow Corning, Thailand) was used as a base material to manufacture the rings. Both the curing agent (1 part by weight) and the elastomer base (10 parts by weight) were heated to 80 °C to facilitate the curing process and to decrease silicone viscosity. Three sets of Alisertib rings were manufactured and tested; containing either 0.5%, 1% or 2% (w/w) Alisertib. The required amount of Alisertib powder was added to the silicone blend and mixed thoroughly using a stainless-steel spatula on a glass board for 10 minutes at room temperature. After mixing, the resulting blend was moved to a 1 ml syringe and injected through a 19-gauge medical needle with a removed bevel into the mold. The silicone was allowed to cure at room temperature overnight, then the mass of the rings recorded. A one-way ANOVA test was performed to identify any significant differences in weight between the three groups.
Alisertib release profile assessment
IVRs were then suspended in scintillation vials containing release medium (50% isopropanol/water) to maintain sink conditions (Alisertib < 10% saturation) and hung using thin glass tubes to avoid floating. The vials were incubated in an orbital shaker at 60 round per minute (RPM), at 37 °C to simulate vaginal tract conditions. The medium was sampled every 24h for 5 days, and then every 48h for the remainder of the experiment for HPLC analysis. The cumulative release was calculated by integrating the area under the curve in the release graph (LabSolutions Analysis Data System, Shimadzu, Kyoto, Japan).
Intravaginal ring implantation in mice
IVRs loaded with or without 1% ALISERTIB were pre-lubricated with K-Y Jelly (Johnson & Johnson, New Brunswick, NJ) before being implanted into the vaginal tract of 8 to 12-week- old FVB mice. On Day 7, mice were euthanized, and genital organs collected. Tissues were then embedded in paraffin and stained with haemotoxylin and eosin. Animal work was approved by the Griffith University Animal Ethics Committee (GU Ref No: MSC/06/16/AEC).
Statistical analysis
Statistical analyses were done on the IBM SPSS Statistics 22 software. One-way ANOVA tests were performed where relevant and a 95% confidence interval (p-value < 0.05) was applied to compare controls and treated samples.
Results
Alisertib can be detected by HPLC and LC-MS analysis
Lambda max of the Alisertib standard was identified at 314–316 nm and the ion in greatest abundance under the peak of interest was 519.25 m/z [M+H]+. Thus, 316nm was used to quantify Alisertib via HPLC (Fig 1a). The mean AUC was calculated from triplicate injections and a calibration curve was generated (Fig 1b). Linear regression analysis was used to generate a line of best fit, which had an R2 value of 0.9999. This standard curve was used in the subsequent experiments to quantify Alisertib present in samples. LCMS analysis confirmed the m/z of the prominent Alisertib ion as 519.25 [M+H]+ which was consistent when Alisertib was constituted in both acetonitrile and isopropanol (Fig 1c). An electron ion chromatogram (EIC) for the 519.25+ m/z [M+H]+ ion after the Alisertib was injected is shown (Fig 1e and 1f). The LCMS mode confirmed the reliability of the developed method in detecting Alisertib in isopropanol.
HPV-transformed cervical cancer cells were sensitive to Alisertib treatment and Alisertib bioactivity unchanged upon exposure to simulated vaginal fluid and high temperature conditions
We previously showed that the presence of HPV major oncogene, E7, sensitized cervical cancer cells to killing by Alisertib (4). To assess the optimal dose for Alisertib to be therapeutically effective, the IC50 of Alisertib of CaSKi cells (194.9 nM), an HPV+ cervical line, was determined. Compared to the IC50 of HCK1T cells (IC50 = 29.5 mM), HPV-negative (-) immortalized primary cervical keratinocytes, CaSki cells were roughly 100-fold more sensitive to Alisertib treatment (Fig 2a). Next, we assessed whether Alisertib would retain its biological activity in the often hydrophilic and low pH vaginal microenvironment. To simulate these conditions, we incubated Alisertib in SVF before treating CaSki cells. SVF is commonly used to test vaginal rings [20]. Cytotoxicity of SVF-exposed Alisertib was comparable to that of Alisertib itself or exposed to PBS, irrespective of incubation time, suggesting that Alisertib is able to maintain its biological activity in SVF (Fig 2b). As IVR manufacturing and Alisertib embedding process involved high temperature conditions, we confirmed that heating Alisertib to 80 °C for 30 min did not affect its biological activity (Fig 2c). Another important facet to consider is the stability of Alisertib during the downstream extraction process for quantifying the Alisertib in isopropanol. HPLC analysis revealed minimal degradation of Alisertib in these conditions, even after 48h (Fig 2d). Overall, we show that Alisertib bioactivity remains unchanged upon its exposure to SVF and high temperatures and its stability is maintained in isopropanol over time.
Alisertib is released from intravaginal rings in a controlled and sustained kinetic over 3 weeks in vitro
To investigate Alisertib release kinetics, intravaginal rings were suspended in a scintillation vial in an appropriate volume of release medium and in vitro release profile from the rings into 50% isopropanol/water was measured over 21 days. Alisertib-embedded rings (0.5%, 1% or 2% (w/w) Alisertib) were manufactured with no significant weight differences (ANOVA test, F = 1.03, p > 0.05). Release media was replaced, and Alisertib concertation measured every 24h with fresh media for the initial 5 days and then every 48h after 5 days. As expected, a high amount of Alisertib was released on Day 1 and that the cumulative Alisertib release profile was gradual over 21 days (Fig 3). Importantly, Alisertib release kinetics obeyed root time (t1/2) kinetics when plotted on a linear cumulative release versus square-root time profile (R2: 0.5% = 0.9963, 1% = 0.9883, 2% = 0.9972). Notably, Alisertib release profiles for both the 1% and 2% Alisertib rings were identical most likely due to over-saturation of Alisertib for concentrations above 1% (w/w).
Alisertib-loaded intravaginal rings did not cause any local inflammation in the vaginal tract
Finally, we wanted to address the general safety of administering this delivery device in the mouse vaginal tract. Previous studies that implanted vaginal rings in mice used sutures to secure the rings in place [21, 22]. No suturing was done in our experiments to minimize procedure-related disturbance to the vaginal tract. All rings were confirmed to be in the vaginal tracts on Day 3 or 4, but not after Day 7, most likely due to displacement of the rings out of the vaginal tract over time. Given that the highest amount of ALISERTIB is released within the first few days (Fig 3), we decided that it was important to assess the effect of this short-term high loading dose on the implanted genital region. We confirmed that there is no notable local inflammation associated with our implants, nor any effect on the weight of the mice (Fig 4).
Discussion
In this study, we developed a potential topical vaginal delivery platform of Alisertib for the potential treatment of cervical cancer. Although, topical delivery to the cervix is feasible, it was necessary to explore and optimize biological, chemical and technical aspects of the chosen delivery platform. Oral administration of Alisertib can have severe adverse clinical effects following phase I/II trials (See review by Tayyar et al., [23]). In this study, we have chosen silicone intra-vaginal rings for their ability to deliver hydrophobic drugs over an extended period of time based on the reliability achieved by those currently commercially available [8, 20]. As Alisertib is a highly hydrophobic compound (logP = 5.74, predicted using MarvinSketch Software, ChemAxon), it was logical to first begin with testing the ability to incorporate it in silicone, the historical polymer of choice.
We first developed a sensitive HPLC method for the detection and quantifying of Alisertib in its solutions and performed LCMS analysis to ensure the identity of the standard. We then ensured that Alisertib was able to retain activity in the vaginal tract by simulating the same conditions. Interestingly, the biological efficacy of Alisertib showed high tolerance to all conditions related to exposure to SVF (i.e. acidic conditions) and the process of embedding the drug into silicone rings (i.e. high temperature conditions) (Fig 2b and 2c). An important consideration when studying intra-vaginal rings is measuring and optimizing their drug release profiles. The medical grade silicone elastomer used in fabrication is limited to external use or short-term implant applications over 29 days, hence why Alisertib release profile was initially tested over 21 days (Fig 3). We were also interested in testing the safety of a high loading dose followed by a continuous release over 3 weeks as we believe this will lead to a more rapid initiation of the biological effects of Alisertib. Here we demonstrate the “burst effect” phenomenon with Alisertib (Fig 3), typically seen in matrix type vaginal rings [20]. This was expected and observed in all Alisertib ring concentrations, where a relatively high amount of drug was released over the first 2 days. The burst effect could easily be overcome by employing reservoir-type or shell-type rings. However, we anticipate beneficial anti-cancer effect due to the initial high loading dose. We also showed that Alisertib-loaded rings did not result in any overt inflammation or anatomical disturbances over the first week of application in vivo (Fig 4). These preliminary results encourage us to take this platform forward for future efficacy testing and longer treatment cycles in orthotopic patient-derived xenograft cervical cancer in vivo models.
In conclusion, we show that Alisertib is a suitable candidate for vaginal delivery using intra-vaginal rings, and the use of this molecule for topical treatment is justified. We developed a sensitive HPLC method for the quantification of Alisertib in its solutions then tested the release of the manufactured rings over 21 days. In vitro, the manufactured rings achieved relatively reasonable release kinetics over 21 days and exhibited the “burst effect” with root time kinetics over an extended period of time. Importantly, Alisertib-loaded rings did not cause any short-term local inflammation within the mouse vaginal tract.
Zdroje
1. Forman D, de Martel C, Lacey CJ, Soerjomataram I, Lortet-Tieulent J, Bruni L, et al. Global burden of human papillomavirus and related diseases. Vaccine. 2012;30 Suppl 5:F12–23.
2. zur Hausen H. Papillomavirus infections—a major cause of human cancers. Biochim Biophys Acta. 1996;1288(2):F55–78. doi: 10.1016/0304-419x(96)00020-0 8876633
3. Cancer of the Cervix Uteri (Invasive), 5-Year Relative and Period Survival by Race, Diagnosis Year, Age and Stage at Diagnosis [Internet]. The National Cancer Institute. 2016 [cited 24 October 2016]. http://seer.cancer.gov/csr/1975_2013/browse_csr.php?sectionSEL=5&pageSEL=sect_05_table.08.html#b.
4. Gabrielli B, Bokhari F, Ranall MV, Oo ZY, Stevenson AJ, Wang W, et al. Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer. Mol Cancer Ther. 2015;14(12):2753–61. doi: 10.1158/1535-7163.MCT-15-0506 26516156
5. Martin D, Fallaha S, Proctor M, Stevenson A, Perrin L, McMillan N, et al. Inhibition of Aurora A and Aurora B Is Required for the Sensitivity of HPV-Driven Cervical Cancers to Aurora Kinase Inhibitors. Mol Cancer Ther. 2017;16(9):1934–41. doi: 10.1158/1535-7163.MCT-17-0159 28522591
6. Pecorelli S, Odicino F. Cervical cancer staging. Cancer J. 2003;9(5):390–4. doi: 10.1097/00130404-200309000-00009 14690314
7. Seiden MV. Chapter 97. Gynecologic Malignancies. In: Longo DL, Fauci AS, Kasper DL, Hauser SL, Jameson JL, Loscalzo J, editors. Harrison's Principles of Internal Medicine, 18e. New York, NY: The McGraw-Hill Companies; 2012.
8. Malcolm RK, Edwards KL, Kiser P, Romano J, Smith TJ. Advances in microbicide vaginal rings. Antiviral Res. 2010;88 Suppl 1:S30–9.
9. Malcolm K, Woolfson D, Russell J, Andrews C. In vitro release of nonoxynol-9 from silicone matrix intravaginal rings. J Control Release. 2003;91(3):355–64. doi: 10.1016/s0168-3659(03)00260-8 12932713
10. Stafford MK, Ward H, Flanagan A, Rosenstein IJ, Taylor-Robinson D, Smith JR, et al. Safety study of nonoxynol-9 as a vaginal microbicide: Evidence of adverse effects. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology. 1998;17(4):327–31. doi: 10.1097/00042560-199804010-00006 9525433
11. Van Damme L, Ramjee G, Alary M, Vuylsteke B, Chandeying V, Rees H, et al. Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet. 2002;360(9338):971–7. doi: 10.1016/s0140-6736(02)11079-8 12383665
12. Malcolm RK, Woolfson AD, Toner CF, Morrow RJ, McCullagh SD. Long-term, controlled release of the HIV microbicide TMC120 from silicone elastomer vaginal rings. J Antimicrob Chemother. 2005;56(5):954–6. doi: 10.1093/jac/dki326 16155060
13. Woolfson AD, Malcolm RK, Morrow RJ, Toner CF, McCullagh SD. Intravaginal ring delivery of the reverse transcriptase inhibitor TMC 120 as an HIV microbicide. Int J Pharm. 2006;325(1–2):82–9. doi: 10.1016/j.ijpharm.2006.06.026 16884869
14. Bell SE, Dennis AC, Fido LA, Malcolm RK, Sirimuthu NM, Toner CF, et al. Characterization of silicone elastomer vaginal rings containing HIV microbicide TMC120 by Raman spectroscopy. J Pharm Pharmacol. 2007;59(2):203–7. doi: 10.1211/jpp.59.2.0007 17270074
15. Fetherston SM, Boyd P, McCoy CF, McBride MC, Edwards KL, Ampofo S, et al. A silicone elastomer vaginal ring for HIV prevention containing two microbicides with different mechanisms of action. Eur J Pharm Sci. 2013;48(3):406–15. doi: 10.1016/j.ejps.2012.12.002 23266465
16. Morrow RJ, Woolfson AD, Donnelly L, Curran R, Andrews G, Katinger D, et al. Sustained release of proteins from a modified vaginal ring device. Eur J Pharm Biopharm. 2011;77(1):3–10. doi: 10.1016/j.ejpb.2010.10.010 21055465
17. Dang TTN. Polymeric matrix devices for controlled delivery of agents to prevent bacterial and viral infection: The University of Queensland, School of Pharmacy; 2014.
18. Yugawa T, Nishino K, Ohno S, Nakahara T, Fujita M, Goshima N, et al. Noncanonical NOTCH signaling limits self-renewal of human epithelial and induced pluripotent stem cells through ROCK activation. Mol Cell Biol. 2013;33(22):4434–47. doi: 10.1128/MCB.00577-13 24019071
19. Owen DH, Katz DF. A vaginal fluid simulant. Contraception. 1999;59(2):91–5. doi: 10.1016/s0010-7824(99)00010-4 10361623
20. Malcolm RK, Boyd PJ, McCoy CF, Murphy DJ. Microbicide vaginal rings: Technological challenges and clinical development. Adv Drug Deliv Rev. 2016;103:33–56. doi: 10.1016/j.addr.2016.01.015 26829289
21. Wyatt TL, Whaley KJ, Cone RA, Saltzman WM. Antigen-releasing polymer rings and microspheres stimulate mucosal immunity in the vagina. J Control Release. 1998;50(1–3):93–102. doi: 10.1016/s0168-3659(97)00114-4 9685876
22. Radomsky ML, Whaley KJ, Cone RA, Saltzman WM. Controlled vaginal delivery of antibodies in the mouse. Biol Reprod. 1992;47(1):133–40. doi: 10.1095/biolreprod47.1.133 1637941
23. Tayyar Y, Jubair L, Fallaha S, McMillan NAJ. Critical risk-benefit assessment of the novel anti-cancer aurora a kinase inhibitor alisertib (MLN8237): A comprehensive review of the clinical data. Crit Rev Oncol Hematol. 2017;119:59–65. doi: 10.1016/j.critrevonc.2017.09.006 29065986
Článek vyšel v časopise
PLOS One
2019 Číslo 11
- Řekněte mi, co jíte, a já vám řeknu, jaká je úroveň vaší kognice
- Duševní onemocnění se u teenagerů přenáší v rámci školní třídy
- Od kostiček k lidské kůži. Jak si postavit 3D biotiskárnu?
- Pomohou nám s detekcí karcinomu plic i včely?
- Původní český test ALBA pro odhalení mírných kognitivních deficitů získal certifikaci MZ ČR
Nejčtenější v tomto čísle
- A daily diary study on maladaptive daydreaming, mind wandering, and sleep disturbances: Examining within-person and between-persons relations
- A 3’ UTR SNP rs885863, a cis-eQTL for the circadian gene VIPR2 and lincRNA 689, is associated with opioid addiction
- A substitution mutation in a conserved domain of mammalian acetate-dependent acetyl CoA synthetase 2 results in destabilized protein and impaired HIF-2 signaling
- Molecular validation of clinical Pantoea isolates identified by MALDI-TOF