Kaposi’s sarcoma is a rare tumor caused by human herpesvirus 8 in the context of immunodeficiency, such as that induced by HIV infection or immunosuppressive therapy. In patients living with HIV (PLHIV), thanks to potent antiretroviral treatment that efficiently restores immunity and allows a better control of HIV infection, the occurrence of KS has decreased dramatically over the past 25 years. However, KS is still one the most frequent cancers in PLHIV, in particular in men who have sex with men and in sub-Saharan Africa, where it is still endemic. Even in the context of restored immunity, the risk of KS is still more than 30 times higher in PLHIV than in the general population.Kaposi’s sarcoma is an angioproliferative tumor caused by human herpesvirus 8 in the context of immunodeficiency, such as that induced by HIV infection or immunosuppressive therapy. Its incidence has dramatically fallen in patients living with HIV (PLHIV) since the introduction of potent antiretroviral combinations 25 years ago due to the restoration of immunity and better control of HIV replication. However, KS is still one of the most frequently occurring cancers in PLHIV, in particular in men who have sex with men and in sub-Saharan Africa, where it is still endemic. Even in the context of restored immunity, the risk of KS is still more than 30 times higher in PLHIV than in the general population. Recent evidence indicates that early initiation of antiretroviral treatment, which is recommended by current guidelines, may reduce the risk of KS but it needs to be accompanied by early access to care. This review mainly focuses on the recent epidemiological features of KS in the context of HIV infection. Kaposi’s sarcoma (KS) is an angioproliferative tumor that was first described in its “classic form” at the end of the XIX century by a Hungarian dermatologist, Moritz Kaposi, as a rare indolent cutaneous tumor affecting elderly individuals, mostly men from Eastern Europe and the area bordering the Mediterranean Sea. In the 1950s, a more aggressive and “endemic form” of KS was described in sub-Saharan Africa that affected young African adults and children . Then, iThe causative agent of KS, human herpesvirus 8 (HHV-8), also reported as KSHV (Kaposi’s sarcoma herpes virus), was identified in 1994 in KS lesions obtained from patients with acquired immunodeficiency syndrome (AIDS) . This brThe four forms of KS, the classic (Mediterranean), endemic (African), epidemic (HIV/AIDS-associated), and iatrogenic (transplant-related), share the same causative virus but have distinct epidemiological and clinical presentations. Some authors also hypothesize on the existence of a fifth form (see paragraph 5 below).Here, we will mostly focus on the epidemiology of KS in patients living with HIV (PLHIV) and on recent insights concerning its evolution. Certain insights on post-transplantation and other KS are also provided.HHV8 infection is not ubiquitous. Its prevalence is substantially higher in men who have sex with men (MSM) and in certain regions of the world, such as sub-Saharan Africa or the countries bordering the Mediterranean; whereas it has been estimated to affect < 5% of the general population of the USA and Europe . TransmiIn sub-Saharan Africa, the prevalence of HHV8 also varies depending on the region. It reaches 50% in Uganda ,12, wherA recent meta-analysis  estimateAnother meta-analysis  examinedAs a consequence of the heterogeneous distribution of HHV8, there are geographical disparities in the burden of KS. There were roughly 42,000 new KS cases and 20,000 deaths estimated in 2018 worldwide  and in tThe Globocan 2020 data , which cIn sub-Saharan Africa, KS is still one of the most common cancers ,26 and tOverall, these geographical differences are likely related to the combined effect of a higher prevalence of HHV8, a higher prevalence of HIV infection, and lower access to antiretroviral treatment (ART) in these regions. However, the incidence of KS has decreased in all regions of the world since the initiation of cART, as shown by a multiregional comparative study of PLHIV initiating combined ART (cART) . This suSince the very beginning of the HIV/AIDS epidemic and the first description of KS in a cluster of young MSM , KS has Prior to the introduction of potent antiretroviral treatment in 1996, which dramatically changed the course of HIV infection in PLHIV by increasing their life expectancy and reducing the incidence of opportunistic diseases, KS was among the most common AIDS-defining diseases in PLHIV. By 1989, 15% of the patients with AIDS in the US reported to the CDC had KS . In FranWith the advent of combined antiretroviral treatment (cART) in 1996, and the resulting virological and immunological control of HIV infection, the incidence rate of KS has dramatically declined over time ,33,34,35The most recent data from the USA indicate that the risk of KS is still decreasing ,38. In aKS is highly associated with immunodeficiency in PLHIV ,40,41,423), large cohorts have shown that the risk of KS is still high. Relative to the general population, the risk is 60-fold higher for patients with a recent CD4 level > 500/mm3 [3 for two years and an HIV viral load < 500 cp/mL [Although the risk of KS has declined over time in all transmission groups, the risk of KS is still 300–500-fold higher in PLHIV than in the general population ,33,37,43 500/mm3  and 35-f00 cp/mL . The ris00 cp/mL . Moreove00 cp/mL ,46,47.3. Indeed, the association was stronger in this population with restored immunity than in the whole population of virally suppressed PLHIV. The hazard ratio of KS ranged from 1.4 to 3.3 in PLHIV with restored immunity with a CD4/CD8 ratio between 0.8 and 0.3 relative to those with a CD4/CD8 ratio of 1, whereas the risk ranged from 1.2 to 2.6 in the whole population of virally suppressed PLHIV. This is an interesting finding, as the population of PLHIV with high CD4 levels is growing as a result of effective cART and early cART initiation.Recently, Caby et al.  showed lIn the context of IRIS, a paradoxical worsening or the first appearance of opportunistic infections and other conditions has been described . Several3 to immediate cART initiation or cART deferral until CD4 dropped < 350/mm3, immediate cART initiation reduced the risk of all cancer by 64%. The risk of KS was also significantly reduced by 91%  but these estimates were based on a very low number of events  and a relatively short follow-up of a median of 2.8 years (IQR 2.1–3.9). Recently, two large observational studies using causal inference methods have attempted to estimate the effect of early vs. deferred treatment on cancer risk on a larger population with a longer follow-up [Following the results of the INSIGHT START clinical trial , immediaollow-up ,55. In tollow-up  also estOverall, these results indicate that currently recommended earlier ART initiation may reduce the risk of developing KS. However, improvements in HIV diagnosis and in reducing the time to receive HIV care are needed to better measure the effect of earlier ART on KS risk.In PLHIV, KS risk is slightly associated with higher age see  in EuropIn resource-rich settings, KS in PLHIV is generally diagnosed at an age of approximately 40 years for PLHIV and 60 years for the general population. The fact that KS apparently occurs at lower age in PLHIV than in the general population is largely due to the differences in the age structure of the two populations and not the premature aging induced by prolonged inflammation/activation that puts PLHIV at higher risk of age-related comorbidities . Indeed,In PLHIV, the advent of combined ART has not only dramatically reduced the risk of KS but also changed the management of KS. Indeed, cART is essential in managing KS when immunosuppression is reversible and is the first KS treatment option in PLHIV ,59,60,61Immunosuppressed organ-transplanted recipients have an increased risk of developing cancer associated with viruses, such as Epstein–Barr virus, HHV8, hepatitis B and C, and human papillomavirus ,65,66,67Recently, a large American linkage study between a transplant registry and cancer registry  showed tPost transplantation KS is generally managed by reducing the immunosuppressive treatment to the lowest levels compatible with allograft function or by changing the immunosuppressive agent, such as changing from calcineurin inhibitors to mTOR inhibitors ,73.There are currently no specific recommendations for HHV8 screening (as there is no specific treatment for HHV8 infection) nor for cancer or a fortiori KS screening in transplanted patients. Furthermore, there is a consensus that neither donor nor recipient anti-HHV8 antibodies preclude transplantation.Apart from immunodeficiency induced by HIV/AIDS or by iatrogenic treatment, KS have drawn less attention in recent years. Classic KS occur mostly in elderly men living or emigrating from Eastern Europe and the Mediterranean Sea . Before the AIDS epidemic, classic KS incidence rate was estimated as 1.58/100,000 person-years in Sardinia  while itClassic KS are usually indolent and progress slowly while AIDS KS are more aggressive . In a reKS is a relatively rare tumor that is still endemic in southern and east Africa and has been associated with immunosuppression. KS is generally manageable by the introduction of antiretroviral treatment therapy for PLHIV or by reducing the immunosuppressive treatment for immunosuppressed organ-transplanted recipients. Current challenges in resource-poor settings are early HIV diagnosis, early treatment, and access to treatment to lower the KS burden in these contexts.


We found no statistical significant effect in the actigraphy of both groups  for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, wake after sleep onset, and total number of wake bouts. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. Dopamine has no effect on Per3 expression in healthy controls, but produces a significant difference in the ADHD group at ZT24 and ZT28. In the ADHD group, incubation with dopamine, either 1 µM or 10 µM, resulted in an adjustment of Per3 expression to control levels. A similar effect also was found in the expression of Per2. Statistical significant differences in the expression of Per2 (ZT4) in the control group compared to the ADHD group were found, following incubation with dopamine. The present study illustrates that dopamine impacts on circadian function. The results lead to the suggestion that dopamine may improve the sleep quality as well as ADHD symptoms by adjustment of the circadian gene expression, especially for Per2 and Per3.A link between dopamine levels, circadian gene expression, and attention deficit hyperactivity disorder (ADHD) has already been demonstrated. The aim of this study was to investigate the extent of these relationships by measuring circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after dopamine exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After  Per1b mutant zebrafish  by a rate-limiting enzyme, tyrosine hydroxylase (TH)  Roeder . A plasmDopamine is degraded by monoamine oxidase (MAO) in the cytosol and catechol-O methyl transferase (COMT) in the surrounding glial cells  and DBH confirm the heredity of ADHD syndromes  and Cryptochrome (Cry1/2) genes  for Parkinson's Disease, and dopamine antagonists like antipsychotic and anti-nausea agents.The psychostimulant methylphenidate and the selective norepinephrine transporter inhibitor atomoxetine, are two of the most frequently prescribed medications for ADHD. Methyphenidate blocks DAT, increasing levels of extracellular dopamine in the prefrontal cortex and striatum, as well as increasing concentrations of norepinephrine in the prefrontal cortex and hippocampus . Ide et al. observed that low-dose methylphenidate alters the reward system in wild-type mice via dopamine transporter inhibition. Dopamine transporter knockout mice do not exhibit such alterations. High-dose methylphenidate suppresses intracranial self-stimulation, suggesting the possibility that methylphenidate treatment does not increase the risk of drug dependence  is hypothesized. Therefore, in this study, the influence of dopamine on circadian rhythmicity are investigated in vitro.Based on the assumption of the effectiveness of dopamine for the treatment of ADHD, its influence on the expression of the core circadian genes  and written consent was obtained from each study participant. The study was conducted according to the ethical guidelines of the Declaration of Helsinki.ADHD patients and healthy controls participating in the study were recruited via the Department of Psychiatry and Psychotherapy, University Medical Centre Rostock. All ADHD patients were diagnosed by experienced psychiatrists in advance. The healthy control group was recruited of acquaintances of people involved in the study.Human dermal fibroblasts (HDF) were obtained from skin biopsies from dorsal forearm from ADHD patients and healthy control volunteers. Only adult individuals, able to give informed consent, were included. Healthy controls without a history of childhood and adult ADHD were matched for sex and age. Patients with more severe than ADHD symptoms, were excluded, as were shift workers. Screening for ADHD symptoms was done by using the WURS-k  as well as assessment of symptoms according to DSM-IV and ICD-10 criteria. Additional, the following psychometric tests were used to confirm ADHD diagnosis: SKIDI and II , DIVA 2.0 (Structured diagnostic interview), CAARS  and PSQI . The IQ of the healthy control group and volunteers with ADHD diagnosis were measured using MWT (Multiple-Choice Word Test). The chronotype of the participants were determined by the D-MEQ . No special cognitive testing was implemented in the study.Comorbidities were observed: 33.3% of participants with ADHD diagnosis has additionally adipositas, 8.3% has additionally addiction disorder, and 25.1% has additionally affective disorder. The remaining participants with ADHD diagnosis has no comorbidities.The four manuscripts of this special issue dealing with circadian rhythmicity describe unique research questions . Actigraphs were worn on the non-dominant wrist for a period of at least 7 consecutive days. The recording interval of the device was set at 60-s epochs. Data occurring before the first and after the final midnight of each record were excluded, ensuring at least 6 complete days for each participant, with a complete weekend included in each record.2) in Dulbecco's Modified Eagle Medium DMEM  /1 mg/ml Liberase TM  containing 100 units/ml penicillin, 100 µg/ml streptomycin  and 10% fetal bovine serum FBS .Human dermal fibroblasts (HDF) were isolated and cultured as described previously . Following 24 h cell viability was measured using the Trypan Blue Exclusion Test Strober .Clock, Bmal1, Per1, Per2, Per3 and Cry1 as well as housekeeping genes  was measured by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) with CFX Connect™ Real-Time PCR Detection System . The oligonucleotide sequences are presented in Table Upon confluency of the respective primary fibroblast cell culture from each participant, eight culture flask replicates were prepared and cells were incubated with either 1 µM or 10 µM dopamine . Cultures without dopamine were used as a negative control. After 24-h of incubation, the cells were synchronized with 100 nM dexamethasone  for 30 min. Samples were harvested every fourth hour after synchronization for a period of 28-h in solution D  and stored at -70 °C. Total RNA was isolated and purified with RNeasy Plus Mini Kit  as well as subjected to reverse transcription using the Superscript III First-Strand Synthesis System . Gene expression of www.euclock.org) to determine the best-fitting linear harmonic regression with an assumed period of 24-h and with α set at 0.05. The center-of-gravity of each best-fitting waveform in CircWave was used as the circadian acrophase, and the associated estimation error was used as the SD. Inferential statistics were carried out in SPSS (IBM Corporation).Circadian gene expression data were tested for significant circadian rhythmicity, using CircWave v. 1.4 software  with 3 measures . Although research in this field is generally scarce, we assumed that the influence of dopamine on the circadian gene expression will have an effect size d’ = 0.5, returning a required total sample size of 21. Taking into consideration an expected drop-out rate, n = 12 participants were allocated per each group. Data were analyzed via time series statistics adequately powered by 12 samples each, which in this statistical model is mathematically sufficient and thus representative , matched by healthy controls (HC) . All participants completed the Multiple-Choice Word Test , Morningness-Eveningness-Questionnaire, German Version  and Wender Utah Rating Scale, German Short Version . The demographic data are presented in Table Human dermal fibroblasts (HDF) were obtained via skin biopsy from volunteers with attention deficit hyperactivity disorder (ADHD) . No significant difference for mid-sleep on weekend days (p = 0.774), mid-sleep on weekdays (p = 0.169), social jetlag (p = 0.984), sleep efficiency (p = 0.833), WASO  and total number of wake bouts (p = 0.425) was shown  after dopamine incubation was compared with HDFs without dopamine. The viability of cells treated with Dopamine  was higher than compared to control cells without dopamine . After incubation with dopamine the cell numbers increased by 147% for 1 µM Dopamine and 154% for 10 µM Dopamine, compared to the cells without dopamine.The expression profiles of circadian genes after incubation with 1.0 µM and 10.0 µM dopamine concentrations were examined in primary fibroblasts cultured from skin biopsies collected from ADHD and healthy participants and synchronized with dexamethasone. Cultures without dopamine were used as a negative control.Bmal1, Cry1 and Per3 expression was strongly rhythmic in both groups . No rhythmicity was detected for Clock in both groups except for cultures incubated with 1 µM dopamine . In the ADHD group, 10 µM dopamine exposure dampened the rhythmicity of Clock . The same effect was observed for Per1/2 genes in cultures from healthy controls (HC) . Dopamine shifted the Clock circadian acrophase to 20.00 ± 3.37 h  compared to 5.68 ± 3.37 h  in cultures without dopamine. In the ADHD group exposure to dopamine shifted the Per1 circadian acrophase to 5.63 ± 2.22 h  compared to 7.07 ± 2.09 h  in cultures without dopamine.Per2 at ZT4  and Cry1 at ZT28 . A Bonferroni post hoc correction revealed a significant different expression of Per2  between the HC and ADHD cultures incubated with 10 µM dopamine. The expression and rhythm of Per2 gene in the ADHD group resulted in an adjustment to the HC group after 10 µM dopamine incubation  was different between HC and ADHD without dopamine incubation.Differences of clock gene expression levels among study groups were assessed using one-way ANOVA Fig. . When coCry1 at ZT28  and Per2 at ZT4 . A Bonferroni post hoc correction revealed a significant lower Cry1 expression in cultures incubated with 1 µM dopamine (p = 0.019) and 10 µM dopamine (p = 0.018) compared to HDF cultures without dopamine. 10 µM dopamine significant lowered the expression levels of Per2 at ZT4 (p = 0.040) compared to the cultures without dopamine. One-way ANOVA revealed in the ADHD group statistical significant Per2 expression at the same time-point, ZT4  between samples incubated with 1 µM and 10 µM dopamine. In the ADHD group, statistical significant differences were observed at ZT24 and ZT28 between cultures incubated with dopamine and negative controls, particularly, for period genes Per1  and Per3 . A Bonferroni post hoc correction revealed a significant higher expression of Per1 at ZT28 in cultures incubated with 1 µM dopamine (p = 0.049) and 10 µM dopamine (p = 0.016) compared to negative controls. The same effect was observed for Per3 gene expression for the cultures incubated with 1 µM dopamine . 1 µM (p = 0.003) and 10 µM (p = 0.010) dopamine increased the expression of Per3 at ZT24 compared to the negative controls.Gene expression in healthy participants revealed a statistical significant difference between cultures incubated with dopamine and negative controls (without dopamine incubation), as determined by one-way ANOVA for Clock gene. This effect was not observed in cultures incubated with 0 and 10 µM dopamine. Preliminary results from our work has shown that Clock gene rhythmicity is also linked with the neurotransmitter norepinephrine. Norepinephrine is a known synchronizer of the circadian rhythm . Additionally, dopamine significantly reduces the Per2 expression in cells of healthy controls. In the ADHD group, dopamine incubation results in an adjustment of Per2 expression to healthy controls without dopamine incubation. These results lead to the suggestion that dopamine may improve the sleep quality as well as ADHD symptoms by adjustment of the circadian gene expression, especially for Per2 and Per3.Dopamine showed no effect on It is to mention, that no special cognitive testing was implemented in this study. In addition, the participants of the ADHD group took no medication during the study. For further studies, a connection between circadian disturbances, cognitive deficits and the effect of medication would be suitable.


Electrospinning is a novel and sophisticated technique for the production of nanofibers with high surface area, extreme porous structure, small pore size, and surface morphologies that make them suitable for biomedical and bioengineering applications, which can provide solutions to current drug delivery issues of poorly water-soluble drugs. Electrospun nanofibers can be obtained through different methods asides from the conventional one, such as coaxial, multi-jet, side by side, emulsion, and melt electrospinning. In general, the application of an electric potential to a polymer solution causes a charged liquid jet that moves downfield to an oppositely charged collector, where the nanofibers are deposited. Plenty of polymers that differ in their origin, degradation character and water affinity are used during the process. Physicochemical properties of the drug, polymer(s), and solvent systems need to be addressed to guarantee successful manufacturing. Therefore, this review summarizes the recent progress in electrospun nanofibers for their use as a nanotechnological tool for dissolution optimization and drug delivery systems for poorly water-soluble drugs. Nanofibers can be considered as nanomaterials based on their diameter, because they geometrically fall into the category of one-dimensional nanoscale elements, such as nanotubes and nanorods. However, their highly flexible nature makes them similar to two-dimensional elements and others like globular molecules as well. In addition to that, they can be viewed as nanostructured materials when filled with nanoparticles in order to form a composite. Therefore, nanofibers possess characteristics such as high surface area, extreme porous structure, small pore size and surface morphologies that make them suitable for drug development and biomedical applications, providing sophisticated and novel solutions to current drug delivery inconveniences -3.et al. based on their regenerative potential in spinal cord injuries [et al., their 3D architecture makes them very similar to the skin extracellular matrix structure, which allows their use as scaffolds for skin tissue engineering [et al. have worked with electrospun nanofibers loaded with bioactive molecules for improving wound healing [et al. for hydrophilic small molecule drugs [Electrospun nanofibers have been the target of different applications, due to their structure and physicochemical properties. These types of nanofibers have been studied by Schaub injuries , 5. Accoineering . In addi healing . Anotherle drugs . Recentlle drugs . Some otle drugs , 11.It is worth mentioning that a wide array of polymer types are being used in the creation of nanofibers, like synthetic, natural, biodegradable, non-degradable or a blend of them. Thanks to the nanotechnological boom, many techniques are available and employed for the manipulation of polymers and nanofibers’ preparation -14. MethHowever, among the mentioned applications of electrospun nanofibers, the most relevant is their capacity to act as drug delivery systems due to their high loading capacity, faster dissolution kinetics, APIs simultaneous delivery, and encapsulation. These outstanding properties make them attractive for the industry -30. Therth century, William Gilbert was the first in documenting research with high voltage direct current applied to a solution. He observed a cone shape when a drop of water was close to electrically charged amber [In the 17ed amber . By the ed amber , 33.Nevertheless, the first electrospinning patent ‘Process and apparatus for preparing artificial threads’ came in 1934 by Formhals, where he described an experimental setup characterized by a collecting device, drum wheel and a plate for the production of polymer filaments using electrostatic force, which later was going to be a novel technique in nanotechnology . NanotecAlthough electrospinning as a polymer-processing technology has been known for more than 80 years, it was not until 1994 when significant studies explored the production of nanofibers through this technique. By that year, the term ‘electrospinning’ was adopted among the scientific community thanks to the work of different researchers including Doshi, Reneker, and Chun, who demonstrated that many organic polymers can be electrospun into nanofibers , 39. SinIt is well known that electrospinning has its origin in ‘electrospraying’, wherein both cases the application of an electric potential to a polymer solution causes a charged liquid jet that moves downfield to an oppositely charged collector , 41. NevIn addition to that, nanofibers properties will depend on the type of polymer used and the method of manufacture, since there have been some modifications to the conventional electrospinning technique. The methods can be classified as needle or needleless electrospinning, where the latter is represented by roller and wire electrospinning, which offer some advantages over needle methods like the lack of clogging , 44. HowAlso known as co-electrospinning or blending electrospinning, it is conceived as a solid dispersion method that involves the dissolution of a drug in a polymer solution using a suitable solvent. It requires simpler equipment than other fiber manufacturing techniques, but it can create a great variety of products for different applications , 48. TheRegarding the process, the polymer solution is held by its surface tension at the end of the spinneret or capillary tip. Once the solution is subjected to high voltage by a direct current power supply, the droplet at the tip elongates to develop a conical form known as ‘Taylor Cone’ . When thAs it travels to the rotating or stationary collector two main events are worth highlighting. In the first place, the solvent evaporates but not completely, which represents a challenge for research groups due to solvent’s toxicity. On the other hand, the fine filament travels following a chaotic trajectory in a whip-like motion, known as bending instability. When it reaches the collector the continuous filament accumulates or deposits to form the nanofiber -60.et al. employed this technique to fabricate vascular grafts made of poly(ɛ-caprolactone) (PCL), gelatin, and poly (PVA). The obtained nanofibers rapidly degradated in vivo, also possessed appropriate properties for use in artery replacement, and offered scaffolds with high porosity enhancing cell proliferation and infiltration [et al. reported the creation of PCL /cellulose nanofibers for the use as biosensor strips [Tan ltration . In anotr strips .Coaxial electrospinning presents changes regarding the method described in solution electrospinning. In this multiple needle system, two polymer solutions or a polymer and a drug solution with different properties regarding solubility and hydrophilicity, are loaded into individual feeding units to simultaneously electrospin from coaxial spinnerets. Since the last ones share an axis, one solution is injected through the inner needle into the concentric outer one at the capillary tip, resulting in a continuous core-shell nanofiber. In addition to that, the principle can be applied to triaxial systems as well, where the electrospun nanofiber will contain a core, a middle layer, and sheath -65.The previously described process allows confining APIs inside the core and controlling drug delivery mainly through diffusion rather than desorption of the surface . Moreoveet al. provided an approach for the design of hypromellose (HPMC)-based hydrophilic composites fabricated through coaxial electrospinning. The core fluid of the electrospun nanofibers was prepared from an electrospinnable solution containing 13% (w/v) of HPMC, 2% (w/v) of ferulic acid and 3% (w/v) of poly(ethylene glycol) (PEG) in a 1:1 ethanol/dichloromethane (DCM) mixture, while ethanol was used as the sheat solvent for the process. The performance of the electrospun nanofibers in the dissolution test revealed to be 10 times faster than casting hydrophilic composites. Therefore, this approach can be useful as a means to obtain carriers for the delivery of poorly water-soluble drugs [et al. proposes an alternative approach. In this case, the sheath layer consisted of a solution of poly(vinylpyrrolidone) K60 (PVP) and citric acid, while the core was composed of a PEG 6000 and sodium hydroxide co-dissolving solution [Wang le drugs . Anothersolution .Like in solution electrospinning, the application of a high voltage results in the formation of a Taylor cone. However, in this case, it is compounded and formed by the core solution surrounded by the sheath polymer solution , 73. ThiA multiple spinnerets or nozzles set-up design is readily used for increasing the production rate of electrospun nanofibers due to its simplicity. This method can be classified into three categories: a) single spinneret with multiple jets, b) multiple spinnerets and each one with a single jet, and c) multiple spinnerets with multiple jets per each. Therefore, a mass production increase of nanofibers can be achieved by increasing not only the nozzles but also the tips or holes -77.Multi-jet electrospinning possesses a very similar process compared to the conventional method, where a continuously charged jet trajectory is defined by the electrical field. However, in this system, the jet direction towards the collector is greatly influenced by Coulomb forces caused by neighboring jets. Precisely, one of the major issues of this method is a jet deviation that is exerted by the interaction of electrostatic forces between them, which provokes defects in the nanofibers , 78, 79.et al. proposed placing an auxiliary grounded electrode to solve the problem, which improves the production rate as well [In order to reduce jet repulsion, many approaches have been performed such as increasing the applied voltage, different spinnerets arrangements, and adjusting the distance between them. Regarding that, Liu  as well . Later w as well .Sometimes it is necessary to combine blends of polymers using one solvent or different polymer solutions for nanofiber production through electrospinning. However, it is not always possible to manufacture nanofibers this way, since both polymers are required to be thermodynamically miscible when dissolving them in the same solvent. In addition, for polymer solution mixtures, polymer-polymer, polymer-solvent and solvent-solvent interactions must be analyzed , 83.Thus, side by side electrospinning introduces itself as a means for overcoming these difficulties. This method focuses on controlling viscosity and conductivity of two polymer solutions that do not get into physical contact until they reach the tip of the spinneret, where they are electrospun simultaneously. Therefore, the resulting nanofiber consists of a bicomponent system with good mechanical strength and great thermal stability. In addition to that, it has different properties on both sides, based on the corresponding component placed on each , 85.et al. have employed this technique for the fabrication of Janus fibers using a teflon-coated parallel spinneret, which allowed the formation of a Janus Taylor cone for obtaining high quality integrated Janus structures. The electrospun nanofibers had one side composed of PVP K60 and ketoprofen, while the other consisted of ethyl cellulose (EC) and the drug as well. The mentioned biphasic design provided a fast dissolution on PVP K60 side and a sustained release of the remaining drug on the EC side [Yu  EC side . Later,  EC side .et al., it has been developing recently as an alternative technique of electrospinning [Proposed by Xu spinning . This nospinning -91.For this purpose, either water-in-oil or oil-in-water emulsions can be electrospun . During et al. investigated the influence of Span 80, sodium dodecyl sulfate (SDS) and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer towards the morphological properties of an electrospun poly (ɛ-caprolactone) /bovine serum albumin nanofibers. It was found that a 0.4% (w/v) SDS emulsion produced the most uniform electrospun nanofibers through emulsion electrospinning [et al. employed the technique for the production of core-shell PVA/dextran sulfate nanofibers loaded with ciprofloxacin, which demonstrated to improve the sustained release of the drug compared to the nanofibers obtained by co-electrospinning [Hu spinning . In anotspinning .This technique is considered one of the most relevant variations of the conventional electrospinning method because instead of using a polymer solution it employs molten polymers for achieving nanofiber production , 97. AltMelt electrospinning stands out for being considered as a green technology in nanofibers manufacturing, which allows exploring new approaches regarding biomedical and bioengineering applications without the restrictions of using solvents, and related issues like incomplete elimination and their toxicity , 101. Inet al. proposed in 2014 a variant of this method called ‘Polymer Melt Differential Electrospinning’ (PMDES) [Larrondo and Manley were the pioneers of this method three decades ago when they tried to produce nanofibers from molten polypropylene (PP) but ended obtaining fibers with diameters greater than 50 μm due to high melt viscosity of the polymer -106. Des (PMDES) .In PMDES scale-up, it was found out that producing multiple jets from one umbrella-shaped spinneret resulted in the obtention of nanofibers with an average diameter of 300 nm, with an efficiency 500 – 1000 times higher than solution electrospinning, and about 80 times higher compared to conventional melt electrospinning . HoweverAmong the most frequently used materials in nanotechnology, polymers are actively employed in this field for the production of nanofibers by electrospinning. A wide variety of them, from natural and synthetic origins, have been electrospun under different required manufacturing conditions. One can also use polymer blends to obtain nanofibers with different structures such as core-shell, ribbon-like, porous, and aligned. Therefore, polymer properties and functionalities provide nanofibers with desirable characteristics and performance to fulfill the general demands for specialized applications , 110.Properties such as hydrophilic and hydrophobic nature of the raw material need to be addressed since they have a major effect on drug release from polymeric nanofiber matrix. Hydrophobic polymers provide limited diffusion of aqueous solutions into the matrix due to their restricted contact angle allowing for long-term release, while hydrophilic polymers cause the diffusion of the API to the medium by their swelling mechanism and their dissolution . Furtherco-glycolic acid) (PLGA), and poly(lactide-co-ε-caprolactone) (PLCL). Regarding the natural raw materials, the use of chitosan, gelatin, collagen, chitin, fibrinogen, and silk fibroin have been reported [The most popular synthetic polymers that have been electrospun are hydroxypropylmethyl cellulose (HPMC), PEO, PVP, sodium carboxymethyl cellulose (NaCMC), PVA, alginates, dextran, PCL, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly, polyethylene (PE), and poly(phenylene sulfide) (PPS) can’t be electrospun satisfactorily , 117.G) [Polymer solubility is complex due to its molecular weight, size difference compared to the solvent, viscosity of the system and changes in terms of structure. The dissolution process is considered to occur in two stages. First, solvent molecules diffuse slowly through the polymer to produce a swollen gel, where polymer-solvent interactions are expected to break intermolecular polymeric bonds. Then, when all polymer-polymer bonds are broken, a true solution is formed , 119. InG) :H is the enthalpy of the process, T stands for temperature and ΔS is the entropy. An adequate solvent is able to make polymer molecules to expand, while a poor one causes them to collapse [i.e. the lowest temperature at which a polymer of infinite molecular weight is completely soluble in a determined solvent) induce polymer’s molecules to expand, which reduces Gibbs free energy of the system until it reaches a negative value where the polymer is soluble [where Δcollapse . Also, t soluble -123.However, in order to evaluate a solvent for a determined drug-polymer system, researchers frequently employ the triangular diagram or solubility map as the main tool for the estimation, as shown in Since different solvents display different levels of electrospinnability, choosing one entails important features to be taken into consideration besides basic parameters like dielectric constant and relative permittivity. In the first place, there must be a balance in the evaporation rate, so that it is enough to allow the fiber to maintain its integrity when arriving at the collector, but not too fast for causing hardening before it reaches the nanometer range. Furthermore, solvent viscosity and surface tension must not prevent the jet formation or allow the polymer solution to drawn freely from the spinneret , 128.A polymer solution constituted by multiple solvents may present manufacturing problems, since solvents with different solubility properties can affect several factors from the polymer and the nanofibers. Polymer chain conformation, viscoelasticity, and critical minimum concentration can be influenced by the selection of the solvent . On the et al. worked with a biodegradable polymer blend composed of PLA and butylene adipate‐co‐terephthalate (PBAT) for the manufacturing of electrospun nanofibers. In this opportunity, they explored the effects of solution parameters such as types of binary solvents, solvent mixing ratio, and polymer blend concentration. They performed solubility tests of the PLA/PBAT blend for the selection of a suitable binary solvent system that guarantees electrospinnability, resulting in the use of a 3:2 dichloromethane (DCM)/ dimethylformamide (DMF) mixture [Kathsee  mixture . Althoug mixture . However mixture .Currently, most of the drug products available do not address the growing demand for personalized therapies. Many of them face several formulation challenges due to the physicochemical properties of the APIs, which may prevent the development of a suitable pharmaceutical form that exerts an adequate therapeutic effect -135. Howet al. state that poor water solubility is one of the major pharmaceutical challenges for drug development [et al. classified drug molecules as a function of their aqueous solubility and intestinal permeability in the Biopharmaceutics Classification System (BCS) [in vivo dissolution, and low bioavailability [It is well known that drug molecules have to be released from the matrix of the pharmaceutical dosage form and then, dissolve in order to be absorbed and to cause the intended therapeutic effect. Nevertheless, both processes are greatly dependent on the physicochemical properties of the API, and the ones from the excipients as well . Consequelopment . Amidon em (BCS) . Classesem (BCS) -142. A glability , 143-145Therefore, due to the recently increasing number of poorly water-soluble drugs, many methods have been employed for improving dissolution . Electroet al., a major challenge for electrospinning is the scale-up process and this issue is currently being addressed by both academic and industrial researchers [Nevertheless, drug-polymer systems are not exempt from presenting incompatibilities, not only chemical but also physical, such as a low degree of miscibility that may lead to an unstable amorphous phase . On the earchers -153.Synthetic, natural and biologically active substances can be loaded into electrospun nanofibers and it has been widely reported by many research papers. Electrospinning provides nanofibers with a large surface area to volume ratio, which combined with the possibility of choosing an adequate solvent for solubilizing the drug of interest, makes it a technique with high loading capacity. In addition to that, the method is considered as an economic, suitable, stable and improved medium for drug loading that provides control over drug release kinetics -156.et al., coaxial electrospinning was employed to load flurbiprofen for obtaining electrospun PVP/PLGA core-shell nanofibers [Drug loading into electrospun nanofibers can be performed in different ways based on the techniques previously explained. In the first place, the drug can be dissolved directly with the polymer by the same solvent or it may require to be previously dissolved in a small amount of another one in order to be added to the polymer solution, wherein both cases the drug will end embedded in the fabricated nanofiber through co-electrospinning , 155. A nofibers . Also, fnofibers -161.Nanotechnology has made the use of nanoparticles and nanostructures a trend for increasing the solubility of APIs, which is known to be a limiting step for bioavailability. Because of that, the dissolution test has extended its application not only to solid pharmaceutical dosage forms but to novel drug delivery systems such as electrospun nanofibers. It is well known that solubility is greatly dominated by the nature of the substance functional groups and their interactions with those of the solvent . ElectroHowever, it is clearly stated in the literature that the enhanced solubility and dissolution of drugs exhibited by electrospun nanofibers is mainly due to the presence of the compound in the amorphous state. This can be explained by the fact that many APIs have higher kinetic energy in that arrangement than in the crystalline form . Aside fet al. worked with solution electrospinning to fabricate different indomethacin buccal films made of Ethocel, HPMC and Tween 80. According to the differential scanning calorimetry (DSC) and X-ray diffraction analysis (XRD), the drug was present in the amorphous state. Dissolution performance was evaluated in vitro in a medium of a buffer at pH 6.8, in which it was found that the presence of Tween 80 accompanied with 5% (w/w) of HPMC caused a fast release in the first five minutes, which increased the dissolution rate of the drug in about 62% compared to the nanofiber produced with the water-insoluble polymer Ethocel [et al. produced terbinafine hydrochloride buccal films using PVA and chitosan for the electrospinning process, where the nanofibers showed fast and complete dissolution of the drug [Nazari  Ethocel . Also, Sthe drug . In anotthe drug .Drug delivery systems are designed for improving significantly the therapeutic efficacy and safety of a drug through controlling the targeted-site of action and the release rate. The development of this type of system involves the understanding of drug release kinetics in order to select the most appropriate compounds for the intended purpose. Therefore, controlled release is useful for modifying delivery kinetics, reducing toxicity and side effects, as well as for adjusting therapies according to patients’ convenience -174.et al. developed a tetrahydro curcumin transdermal patch through electrospinning solution, where the poorly water-soluble active was loaded at a concentration of 8.7% (w/w of dry polymers) into a 2:1 mix of solutions of PCL 10% (w/v) and PEG 5% (w/v) in a chloroform and acetone solvent system, obtaining nanofibers with an average diameter of 400 ± 20 nm. The cumulative % release was 95.11% at 24 hours, exhibiting first-order kinetics that matches Higuchi’s diffusion model for which this development might be feasible for once in a day transdermal drug delivery [Electrospun nanofibers have been considered as suitable structures that can control spatially and temporally drug delivery of more than one drug. The first aspect can be achieved by placing the nanofiber at the targeted site by invasive or non-invasive methods to avoid systematic exposure -177. Ravdelivery .The obtained nanofibers offer different outstanding properties for hydrophobic active substances, which are explained by their nanostructured nature that improves not only physicochemical and pharmacokinetic properties but also provides protection from enzymatic or chemical degradation. This nanostructure overcomes high drug uptake limitations, exhibits efficient drug transport and has the capacity of encapsulating chemical or biological therapeutic agents during the manufacturing process , 180.et al. successfully created supersaturated electrospun nanofibers for controlled release of piroxicam made of HPMC and 1,1,1,3,3,3-hexa-fluoro-2-propanol (HFIP) as the solvent. The dry diameter of the nanofibers was in the range of 400-600 nm based on scanning electron microscopy studies (SEM). Solid-state studies of the nanofibers carried by DSC and XRD confirmed the presence of the drug in the amorphous state, but after three months they showed a slow tendency to recrystallize into form III. Physicochemical properties of the nanofibers were strongly dependent on the concentration of HPMC, exhibited short lag-time, no initial burst release and zero-order dissolution kinetics [Paaver kinetics .Electrospun nanofibers are able to achieve controlled release at the targeted site by making use of several polymers, which can be biodegradable or non-degradable. According to that, drug release from non-degradable polymers is performed by diffusion, while release from degradable polymers occurs mainly by matrix erosion. However, the selection of the polymeric matrix is conditioned by the requirements of the application , 183. WhDrug release from hydrophilic polymers is possible through surface desorption, diffusion and matrix erosion due to the degradation of the nanofiber caused by enzymes. When nanofibers are constituted by this type of polymers, direct contact with an aqueous solution alters its glassy state and forms a rubbery gel, causing swelling of the polymeric matrix, which transports a drug through its network until it reaches the medium , 186. Foet al. developed research for the delivery of celecoxib lipophilic nanoparticles, which were formed by loading the poorly water-soluble drug dissolved in a volatile oil-solvent system into the electrospinning aqueous polymer solution composed of a high molecular weight PVA. The obtained hydrophilic electrospun nanofibers enabled fast dissolution and release of celecoxib nanoparticles, since the drug was present in the amorphous state and the average size of the nanoparticles was in the range of 21-93 nm [Gordon 21-93 nm .et al. designed an implantable tissue-engineered scaffold through electrospinning made of a self-coated interfacial layer developed between an inorganic and an organic matrix for time-programmed multi-drug release. The scaffold’s design propitiated the absence of interactions; the inside-located drug was ibuprofen, which experienced short-term release for 30 days, while doxorubicin was loaded outside the interfacial layer and showed a sustained long-term release during 90 days [Polymer, solvent and drug compatibility need to be addressed for achieving reproducible drug release and thus, drug delivery. When the electrospun nanofiber system carries multiple drugs, their affinity to the polymer as well as the interaction between them have to be considered. If any physicochemical interaction is present it may lead to problems regarding the diffusion of the drug, which will result in different release profiles -192. Zha 90 days .et al., the chosen electrospinning method and the process itself allow controlling the release kinetics through some critical parameters such as matrix properties, nanofiber diameter, porosity and of course their morphology. However, if the different drugs are loaded by the same electrospinning method, the release will depend on each diffusion coefficient [Also, as stated by Haider fficient . Thus, dfficient .However, recent research is focusing on developing electrospun nanofibers that are responsive to certain stimuli or feedback factors that start the controlled release of the drug at the targeted site. These novel drug delivery systems are called smart electrospun nanofibers , 197.Although these systems have experienced considerable progress in the past years, all of them face the same challenges regarding the manufacturing process, quality control and cytotoxicity, which imply a great barrier to faster development . HoweverpH-responsive: Potential targeted sites such as tissues and organs differ in their physiological pH value, which implies a lot of interest for research in this stimulus for triggering and modulating drug release. Ideally, pH-responsive electrospun nanofibers should be designed and developed in a way that makes them able to release the drug at a certain pH close to that caused by a condition or a disease in the biological surroundings [oundings , 212.Nanofiber drug release is expected to be reduced or completely stopped whenever the condition is improved and the microenvironment pH shifts back to the physiological value. Most polymers used for this purpose are biocompatible and biodegradable containing carboxylic acids or amine groups, which undergo protonation and deprotonation as well as changes in size, shape, and hydrophobicity causing the drug release mostly through a swelling mechanism . AlthougThermo-responsive: These systems are composed of polymers that suffer solubility modifications based on the lower critical solution temperature (LCST)  that is usually close to normothermia (37 °C). Thus, below LCST the polymer is completely miscible, but above that parameter, it is only partially due to a reversible phase transition from a hydrophilic form to a dehydrated or hydrophobic state. The aforementioned transition is an entropy-driven process caused by the release of water molecules upon heating [e.g. a locally heated tumor) enhances the release because of nanofibers shrinking, caused by the polymer conversion into a hydrophobic form [ heating . The polbic form -218.Magnetic field-responsive: Magnetic field stimulus provides advantages compared to the other options since it is well known that it does not affect tissues and its penetration in the body is greater than heat or light, causing neither cell death nor DNA damage [3O4 (magnetite) or Fe2O3 (maghemite) into the polymers or polymer solution used during the electrospinning process [A damage , 220. ThA damage . For the process , 223. Ma process .in vivo administration, conferring all of the superparamagnetic related properties to the entire nanocomposite [A common developing strategy involves coating the surface of SPIONs with polymers or encapsulating them in a biodegradable matrix to form a nanocomposite known as magnetic responsive polymer composites (MRPCs) . The foromposite . Magnetiomposite , 226.Electric field-responsive: Electrospun nanofibers constituted by electric-responsive polymers experience changes in their volume and swelling mechanism when exposed to an electric field. Electrospinning of ion-doped conducting polymers and polymer composites or coatings has been applied for manufacturing nanofibers intended for electric field-responsive drug delivery system [y system -229.Multi stimuli-responsive: This kind of system represents a new opportunity for electrospun nanofibers with enhanced performance under the influence of two or more signals. Thus, in the case of multiple drug-loaded nanofibers, the release of each drug can respond to a different stimulus, according to the respective polymeric matrix and the manufacturing electrospinning technique. In addition to that, drug release can be triggered at the same time or sequentially, due to the influence of combining multiple stimuli [ stimuli , 231.in vitro dissolution performance compared to the conventional solid dispersion systems. Their most important property is related to their large surface area to volume ratio, which is responsible for the optimized performance in combination with particle size reduction and amorphization of the API. Therefore, electrospinning as a formulation strategy that makes use of electrostatic and mechanical forces to spin fibers, is being positioned as a novel and useful method for local and systemic drug delivery of poorly water-soluble drugs. Although the potential benefits from this technique are known, there is still work to do for overcoming safety and scale-up challenges.Nanotechnological development through electrospinning is giving the world the opportunity of overcoming the obstacles presented during the design, formulation, and manufacturing of pharmaceutical, biomedical and bioengineering applications. Currently, electrospun nanofibers are at the forefront of nanotechnology, providing the ability to control and manipulate the properties of the obtained nanostructures, which exhibit improved


The results of the ANOVA yielded R2 and adjusted R2 values of 0.96 and 0.93 respectively, this affirmed that the predictive model fitted well with the experimental data. Furthermore, the process parameters were optimized using the CCD method and a maximum desirability function of 226 nm was achieved for the diameter of the nanofibers. This is very close to the 233 nm diameter obtained from a confirmatory experiment using the optimum conditions. Therefore, the model is representative of the process, and it could be used for future studies for the reduction of the diameter of electrospun nanofibers.The present research aimed to investigate the effect of working parameters on the electrospinning of niobium–tungsten oxide nanofibers and optimize the process using central composite design (CCD) based on the response surface methodology (RSM). An experiment was designed to assess the effects of five variables including the applied voltage (V), spinning distance (D), polymer concentration (P), flow rate (F), and addition of NaCl (N) on the resulting diameter of the nanofibers. Meanwhile, a second-order prediction model of nanofibers diameter was fitted and verified using analysis of variance (ANOVA). The results show that the diameter of the nanofibers was significantly influenced by all the variables except the flow rate. Some second-order and cross factor interactions such as VD, DP, PF, PN, and P Over the last few years, researchers have been focusing on the fabrication of ceramic nanofibers with large surface-to-volume ratios, as these materials have potential applications where high porosity is desirable . In the Electrospinning has been widely used for fabricating nanofibers from most organic polymers because it is easier to prepare a polymer solution with the required rheological properties for electrospinning ,11. This−1 [2Ti12O25 (229 mAh g−1) [4Ti5O12 (175 mAh g−1) [2Ti3O7 (198 mAh g−1) [Most recently, ceramic nanowires are being explored as potential electrode materials for electrochemical energy storage devices. One of the ceramic nanowires currently being explored for this purpose is niobium–tungsten oxide nanowires . This ma−1 , this ismAh g−1) , Li4Ti5OmAh g−1) , and Li2mAh g−1) . Besides[4Ti5O12 5 mAh g−1[4Ti5O12 5 mAh g−1mAh g−1) ,19,20,21mAh g−1)  made the[4Ti5O12 5 mAh g−12 [Over the last few decades, various attempts had been made to understand the effects of working parameters such as applied voltage, solution composition, electric field strength, type of collector, and polymer solution flow rate on the diameter and morphology of electrospun ceramic nanofibers such as TiO2 . HoweverThe use of various statistical experimental design methods for studying the effects of variables on chemical processes had been reported by several authors ,24,25,26The process of fabricating niobium–tungsten oxide nanofibers and their potential as an anode material for LIBs had been reported ,17. To tThe experimental design and results analysis in this research were carried out using JMP Pro version 9.s.2 software . Potential parameters that can influence the electrospinning process are numerous and it is difficult to investigate all of them in one single research due to time and cost. In this regard, series of preliminary investigations and extensive literature review were carried out to select the most influential factors. Furthermore, the factors selected in this research are similar to those investigated by other authors ,27,33.Before optimization, experiments were conducted using a screening design that generated sixteen experimental runs. The screening experiment was much more useful because it provided the feasibility range for each factor to obtain uniform nanofibers. The results obtained from the screening experiments are summarized in The CCD generated thirty-six experimental runs including ten as the replication of the central points. All the experiments were executed in random order and the corresponding values of the diameter of the nanofibers were recorded as the response.−1, 100% purity, Sigma Aldrich, Oakville, ON, Canada), ethanol  and nitric acid . All the materials used in this research were of analytical grade. Firstly, a certain amount of NaCl , ammonium metatungstate hydrate , polyvinylpyrrolidone  was applied to the droplet of the precursor solution at the tip of the needle, the application of high voltage results in the deformation of the droplet. The polymer jet is ejected into the electric field, and this causes the jet to undergo a bending movement under columbic repulsion and the polymer jet is stretched into nanofibers before being deposited on the collector. A schematic diagram of the electrospinning process is shown in The niobium–tungsten oxide nanofibers were fabricated based on the experimental design in Micrographs of the nanofibers fabricated under different experimental conditions were obtained using a Scanning Electron Microscope (SEM) . The average nanofiber diameter for each of the experiments was determined by measuring the diameter of 50 randomly selected nanofibers  using Imdf) as a function of the five factors  was determined in terms of the coded factors by performing multiple regression analysis on the experimental data.A model Equation (1) that describes the diameter of the niobium–tungsten oxide nanofibers  of the response was also conducted to evaluate the full second-order polynomial approximation of the response surface model. The significance of each coefficient of the model equation was determined using the corresponding df) and the factors in a coded unit:The result of the multiple regression analysis  yielded p-values) from Fisher’s (F) exact test. At 95% confidence level, model equation parameters with p-values less than 0.05 are significant while the model equation parameters with p-values greater than 0.05 are non-significant [V, D, P, N, VD, DP, PF, PN, and P2 were significant in the model. The f-value was also used to confirm the level of significance of the model terms. The level of significance was based on the magnitude of f-values, with a higher value representing a larger influence on the process being studied [f-values are in agreement with those of the p-values.The significance of each parameter in the model equation including quadratic, cross-factor interactions, and linear was evaluated to affirm the effect of each term in the model together with their interactions using ANOVA at 95% confidence level and probability values  and the adjusted R2 for the model by plotting the diameter of the experimental nanofibers against the model predicted nanofibers diameter. The value of the (R2 = 0.96) shows that only 4.0% of the total variations are not explained by the model. Additionally, the value obtained for the adjusted R2 (0.93) is high, R2 and adjusted R2 close to 1.0 indicate that there are minor discrepancies between the predicted and experimental nanofibers diameter [The efficiency of the developed model was verified by computing the linear correlation coefficient as shown in diameter ,24,28. Tp-value. The insignificant lack of fit also affirms that the predictive model fitted well with the observed data [The accuracy of the prediction model was also verified by ANOVA. The results presented in ved data ,27,28. AThe three-dimensional (3D) response surfaces and two-dimensional (2D) contour plots of the cross-factor interaction effects between the model parameters are presented in V), spinning distance (D), polymer concentration (P), flow rate (F), and NaCl concentration (N) have been determined to meet the previously set goal of minimizing the diameter of the nanofibers. The maximum desirability function was achieved with nanofiber diameter of 226 nm under optimum conditions of 24 kV applied voltage, 20 cm spinning distance, 8.5 wt % polymer concentration, 1.7 mL h−1, flow rate, and 0.8 wt % NaCl concentration. The model has been validated by conducting another experiment using the obtained optimum conditions. The experimental result obtained under the optimum conditions is 233 nm, which is very close to the 226 nm predicted value. This further confirms the reliability of the developed model. It also implies that this method can be successfully employed for fabricating niobium–tungsten oxide nanofibers with controlled morphology and diameter for various applications.The optimum experimental conditions of the five variables; the applied voltage (AMA) using Equation (2) [Xi is the experimental nanofibers diameter at the run (i), Yi is the predicted nanofibers diameter at the run (i), and n is the total number of experimental runs.Furthermore, the model was verified using the graphical representation of the experimental and predicted values of the diameter of the nanofibers shown in tion (2) ,44 whereThe result obtained from Equation (2) shows an average model accuracy of 98%, this is considered as an acceptable model ,38.R2) and the adjusted R2 of 0.96 and 0.93 were obtained, with an average model accuracy of 98%. Furthermore, the result of the ANOVA test performed on the model indicates that the applied voltage (V), spinning distance (D), polymer concentration (P), NaCl Concentration (N), and other cross factor interactions such as VD, DP, PF, PN, and P2 have a significant effect on the diameter of the nanofibers. The CCD method was utilized to optimize the process parameters. Under the optimum conditions of the applied voltage (24 kV), spinning distance (20 cm), polymer concentration (8.5 wt %), flow rate (1.7 mL h−1), and NaCl concentration (0.8 wt %), experimental nanofibers of 233 nm were obtained. This is very close to the 226 nm diameter predicted by the model. Therefore, the proposed model is representative of the process, and it could be employed as a base for future studies for the reduction of nanofiber diameter within the range of the factors used in the research. Furthermore, the approach presented in this study can be employed as a basis for fabricating uniform ceramic nanofibers for various applications. Nevertheless, it should be noted that the electrospinning of ceramic nanofibers is dependent on the choice of precursor salt and type of polymer. In terms of future work, it is suggested that the influence of other relevant working parameters on the electrospinning of niobium–tungsten oxide nanofibers are investigated using other approaches such as machine learning and artificial neural networks.The influence of process parameters on the electrospinning of niobium–tungsten oxide nanofibers was investigated and optimized using the response surface method. The predictive model developed using RSM together with CCD was found to be accurate and reliable for representing the diameter of the nanofibers. The reliability of the predictive model was assessed using ANOVA and linear correlation coefficient. The determination coefficient (


Compared with WT, deletion of sEH inhibited vascular calcification induced by AP. sEH deletion also abolished high phosphorus (Pi)-induced phenotypic transition of vascular smooth muscle cells (VSMCs) independent of its epoxyeicosatrienoic acids (EETs) hydrolysis. Further gene expression analysis identified the potential role of Sirtuin 3 (Sirt3) in the sEH-regulated VSMC calcification. Under high Pi treatment, sEH interacted with Sirt3, which might destabilize Sirt3 and accelerate the degradation of Sirt3. Deletion of sEH may preserve the expression of Sirt3, and thus maintain the mitochondrial adenosine triphosphate (ATP) synthesis and morphology, significantly suppressing VSMC calcification. Our data supported that sEH deletion inhibited vascular calcification and indicated a promising target of sEH inhibition in vascular calcification prevention.Vascular calcification is common in chronic kidney disease (CKD) and contributes to cardiovascular disease (CVD) without any effective therapies available up to date. The expression of soluble epoxide hydrolase (sEH) is different in patients with and without vascular calcification. The present study investigates the role of sEH as a potential mediator of vascular calcification in CKD. Both Ephx2 Vascular calcification is a highly regulated process sharing a similar mechanism with bone formation . It is cSoluble epoxide hydrolase (sEH), an enzyme with both epoxide hydrolase and phosphatase activities, plays an important role in regulating multiple physiological and pathological functions . One of Herein, we investigated the characteristics and potential mechanisms of sEH in vascular calcification through in vivo and in vitro experiments. We found that deletion of sEH may inhibit vascular calcification. Further gene expression analysis identified the possible role of Sirt3 in the sEH-regulated vascular calcification. Deletion of sEH may preserve the expression of Sirt3 independent of EETs hydrolysis, and thus maintain the mitochondrial adenosine triphosphate (ATP) production and morphology, significantly suppressing the vascular smooth muscle cell (VSMC) calcification. Therefore, sEH may represent a promising preventive target for CKD-related vascular calcification.−/− and wild-type (WT) mice were fed with AP diet for overall 16 weeks  were significantly lower in Ephx2−/− than in WT mice while the serum calcium was unchanged  induced CKD. Ephx2eks Fig. . As showsed Fig. . The sersed Fig. . Howeverged Fig. . As knowged Fig. . sEH delged Fig. . These d−/− mice  to induce in vitro calcification, separately. High Pi significantly increased the calcium deposition of WT VSMCs estimated by Alizarin Red S staining and calcium content assay in a time-dependent manner, but this increase was abolished by sEH deletion , a contractile marker of VSMC, was markedly reduced in aortas of WT mice, whereas the level was nearly unchanged in Ephx2ice Fig. . It indiion Fig. . Consistion Fig. , whereasion Fig. . This phN‐[1‐(1‐oxopropyl)‐4‐piperidinyl]‐N′‐[4‐(trifluoromethoxy)phenyl]‐urea (TPPU), to inhibit the epoxide hydrolase activity of sEH, and to explore whether the VSMC calcification was affected. Our results showed that neither t-AUCB nor TPPU was able to decrease the calcium deposition of VSMCs estimated by Alizarin S Red staining , among which 405 were downregulated and 391 were upregulated in Ephx2−/− VSMCs with high Pi treatment  in VSMCs. Knockdown of Sirt3 abolished the inhibitory effect of sEH deletion on the calcium nodule formation and calcium deposition in VSMCs Fig. . Sirt3 s−/− cells, the degradation became much slower  was the most important downstream target of Sirt3. Sirt3 was able to deacetylate PGC-1α to enhanced mitochondrial biogenesis and energy generating . Herein,ent Fig. . Furtherion Fig. . It indills Fig. . Consistent Fig. . The knoent Fig. . These dThis study provided the first evidence that sEH deletion may attenuate CKD/high Pi-induced vascular calcium deposit and VSMC osteogenic transition. Furthermore, we identified Sirt3 as the possible regulator in the sEH-related vascular calcification. sEH deletion may set Sirt3 free from sEH–Sirt3 complex and stabilize Sirt3, which ameliorated mitochondrial abnormality under CKD/high Pi condition Fig. .Fig. 6Sc−/− mice were less vulnerable to CKD-induced vascular calcification than WT ones. The VSMC from Ephx2−/− mice also resisted to the high Pi-induced calcium deposit and phenotypic transition. All these data indicated the protective role of deletion of sEH in vascular calcification. Indeed, the protective effect of sEH inhibition on vascular remodeling has been confirmed. Previous studies demonstrated that sEH inhibition greatly reduced the increasement of cytokines, chemokines and adhesion molecules, impaired mitochondrial dysfunction, and thus suppressed the development of atherosclerosis and hypertension  was supplemented to 5 mL, followed by static culture for 5 days. All cells were cultured at 37 °C, 5% CO2 in a saturation humidified incubator. Then primary VSMCs growing out of the tissue chips were trypsinized and transferred into a new flask for further experiments. Before performing further experiments, VSMCs were tested by immunofluorescence for α-SMA for phenotypic confirmation. The cells up to passages 4–8 were used for experiments. Calcification of VSMC was induced by incubation in calcifying medium  for 3–7 days as previously reported [The primary mouse VSMC was isolated from thoracic aorta of Ephx2reported . Brieflyreported . The culFor knockdown of Sirt3 in VSMC, siRNA targeting Sirt3 was used, and a nontargeting scramble siRNA was used as a control. Two siRNA oligonucleotides (RIBOBIO) previously verified for the efficient knockdown of Sirt3 expression in VSMCs were used for final experiment  to rupture the membrane for 20 min followed by blocking with 5% bovine serum albumin (BSA). Sections were incubated with primary antibodies against BMP2 , SM22α , or rabbit IgG alone (negative control) overnight at 4 °C. After incubation with an HRP-labeled anti-rabbit IgG secondary antibody, sections were stained with a DAB Kit . Nuclei were counterstained with hematoxylin before sealing with neutral resin.Total protein samples were extracted from either tissues or whole-cell extracts with RIPA lysis buffer  adding a protease and phosphatase inhibitor cocktail . The concentrations of protein were determined by BCA Protein Assay kit . The protein was mixed with the 4× Laemmli Sample Buffer  containing β-mercaptoethanol  and boiled at 95 °C for 10 min. Total proteins were loaded in SDS polyacrylamide gels followed by electrophoresis and then blotted onto 0.2 μm PVDF  membranes. The membranes were then blocked by 5% BSA for 1 h at room temperature. The primary and secondary antibodies used are listed in Supplementary Table 2 plate were harvested and lysed with IP lysis containing a protease and phosphatase inhibitor cocktail . Then lysates were centrifuged, and the supernatants were collected for protein concentration determination and further Co-IP analysis. The protein samples were incubated with antibody-coupled resin overnight at 4 °C. After washing with wash buffer for three times, the IP products were eluted and added the 5× Lane Marker Sample Buffer containing 100 mM dl-dithiothreitol . The samples were heated at 95 °C for 5 min and cooled to room temperature before further applying to the gel. The antibodies for Co-IP are shown in Supplementary Table Co-IP analysis was performed using Co-Immunoprecipitation Kit Pierce, 26149) according to the manufacturer’s protocol. Briefly, after planned incubation, VSMCs grown in a 15 cm6149 acco−/− and WT VSMC after high Pi treatment by using TRIzol™ Reagent . About 1 μg of RNA was used to create the sequencing library by using the KAPA Stranded RNA-seq Library Prep Kit (Illumina). Then the quality of library was checked by an Aglient 2100 Bioanalyzer, and the final quantification of the library was performed by the qPCR method. The final sequencing library of different samples was denatured by 0.1 M NaOH to generate single-stranded DNA, and then amplified in situ on TruSeq SR Cluster Kit v3-cBot-HS . The ends of the generated fragments were sequenced for 150 cycles using an Illumina X-ten/NovaSeq sequencer.Total RNA was extracted from Ephx2P value <0.05. Next, GSEA were analyzed and visualized with GSEA software . Venn diagram was performed on the web‐tools Venn on Bioinformatics & Evolutionary Genomics site (http://bioinformatics.psb.ugent.be/webtools/Venn).For gene expression analysis, the quantity of mRNA expression of each gene was presented as FPKM (Fragments Per Kilobase of gene/transcript model per Million mapped fragments). The DEGs, analyzed by R software (version 3.5), were identified when the log2FoldChange ≥2 or ≤−2, and the corrected To evaluate vascular calcification, Von Kossa and Alizarin Red S staining were performed in mice artery tissues. For Von Kossa staining, Von Kossa staining kit  from Leagene was used. Paraffin-embedded cross-sections were prepared. Each sample was sliced at 3 μm thickness. After deparaffinizing, samples were incubated with 5% silver nitrate under ultraviolet light for 2 h. Then un-reacted sliver was removed by incubation in 5% sodium thiosulfate for 5 min. Nuclei were counterstained with hematoxylin. For Alizarin Red S staining, the whole aorta already fixed with 4% paraformaldehyde was incubated with Alizarin Red S solution  for 5 min. A reddish color was displayed if positively stained.To characterize and estimate the calcific nodules of treated VSMCs, Alizarin Red S staining was performed. After incubating with calcifying medium, the culture medium was discarded, and the treated VSMCs were washed with phosphate-buffered saline (PBS) for three times. Then 4% paraformaldehyde was used to fix the VSMCs for 30 min. After washing three times with PBS again, the fixed VSMCs were exposed to Alizarin Red S staining solution for about 20 min. At last, wash three times with PBS. Positively stained cells displayed a reddish color. To quantify Alizarin red S staining, 10% hexadecylpyridinium chloride monohydrate (CPC)  was used for decolorization. The plate was shaken at a speed of 60 r.m.p. for 60 min at room temperature and the decolorizing solution was collected for the detection of the spectrophotometry at a wavelength of 562 nm.After the incubation periods with calcifying culture medium, the treated VSMCs were incubated with 0.6 N hydrogen chloride at 4 °C. After 24-h incubation, the hydrogen chloride supernatant was collected to test the calcium content using the calcium deposition kit from Nanjing Jiancheng Bioengineering Institute (C004-2). The remaining cells were used to detect the protein concentration. The cellular calcium content standardized by protein concentration was calculated according to the formula on the instruction.ATP production was detected by the Enhanced ATP Assay Kit  according to the manufacturer’s instructions. The results were normalized to the total protein concentrations.4 in 0.1 M PBS (pH 7.4) for 2 h at room temperature. Then the samples were dehydrated with graded ethanol and embedded with resin. The embedding models with resin and samples were moved into an oven at 65 °C to polymerize for over 48 h. The resin blocks were cut to 60–80 nm thin on the ultramicrotome (Leica), and the tissues were fished out onto the 150 meshes cuprum grids with formvar film. Then the sections were stained with uranyl acetate in pure ethanol and leas citrate. After air-drying sections at room temperature overnight, the ultrastructure was observed under a TEM .After planned incubation, VSMCs were harvested and fixed by the TEM fixative  for 2–4 h at 4 °C. Transfer cells to centrifuge tube and spin to get cell pellet. After pre-embedded with 1% agarose solution, cell pellets were post-fixed with 1% OsOt-test was applied for comparisons between two groups. One-way ANOVA was used to analyze differences between multiple groups and post hoc analysis (Bonferroni method) was performed to analyze the difference between the groups. P < 0.05 were considered statistically significant . Statistical analysis was performed with SPSS version 25.0  or Graphpad Prism version 8.0.All experiments were independently repeated at least three times. All values in the text and figures were expressed as mean ± standard deviation (SD). Data were evaluated by analysis of variance. Student’s Supplementary Materials