In in vivo studies, the mean plasma concentration of RPV in rats (431 ng/ml at the Day 7 time point) was approximately ten-fold greater than the trough concentration observed after a single-dose in previous clinical studies. These results are the first to indicate, by the determination of relative exposures between IM and MAP administration, that larger multi-array dissolving MAPs could potentially be used to effectively deliver human doses of RPV LA. Importantly, RPV was also detected in the lymph nodes, indicating the potential to deliver this ARV agent into one of the primary sites of HIV replication over extended durations. These MAPs could potentially improve patient acceptability and adherence to HIV prevention and treatment regimens and combat instances of needle-stick injury and the transmission of blood-borne diseases, which would have far-reaching benefits, particularly to those in the developing world.One means of combating the spread of human immunodeficiency virus (HIV) is through the delivery of long-acting, antiretroviral (ARV) drugs for prevention and treatment. The development of a discreet, self-administered and self-disabling delivery vehicle to deliver such ARV drugs could obviate compliance issues with daily oral regimens. Alternatives in development, such as long-acting intramuscular (IM) injections, require regular access to health care facilities and disposal facilities for sharps. Consequently, this proof of concept study was developed to evaluate the use of dissolving microarray patches (MAPs) containing a long-acting (LA) nanosuspension of the candidate ARV drug, rilpivirine (RPV). MAPs were mechanically strong and penetrated skin   In highvia the IM route, however, requires administration by a trained health care worker, which may limit access. Pain at the injection site has been reported in clinical studies, which could impact acceptability of a future product intended for use at either four or eight week intervals [Rilpivirine (RPV) is a next-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) that exhibits potent activity against wild-type HIV and some HIV mutants resistant to first-generation NNRTIs and has been approved for treatment since 2011 . The stantervals . Misuse ntervals , 83.8% intervals  and 86.1ntervals . Not allntervals .Fig. 1Thstratum corneum, to facilitate intradermal delivery of drugs and vaccines [The work presented here provides proof of principle for the use of an alternative, needle-free delivery system for LA nanosuspensions in the treatment of HIV, specifically focusing on determining the potential to use novel dissolving microarray patch (MAP) technologies  to facilvaccines ,17. MAPsvaccines  and is rvia MAP, could be deposited as a depot into the skin and as the nanoparticles dissolve in interstitial fluid, the drug would slowly be released and absorbed into the systemic circulation. If sufficient quantities of nanoparticles and accordingly, RPV, can be deposited into the skin, then therapeutic plasma levels of the drug could theoretically be maintained for prolonged periods [Infrequent application of MAPs could potentially obviate compliance issues with conventional delivery strategies, such as daily oral dosing regimens or IM injections, which require delivery by health care providers. This approach indicates that RPV, delivered  periods . Further periods . Sustain periods . With al22.1Poly(vinylpyrrolidone) (PVP) of molecular weight 360,000 Da; poly (PVA) of molecular weight 9000–10,000 Da; poly(ethylene glycol) (PEG) of varying molecular weights (200–600 Da) and glycerol were all purchased from Sigma-Aldrich, Gillingham, Dorset, UK. Gantrez™ S-97, copolymer of methyl vinyl ether and maleic acid (PMVE/MA), with a molecular weight of 1500,000 Da was a gift from Ashland, Kidderminster, UK. RPV base and long-acting RPV nanosuspension  were supplied by Janssen Pharmaceutical, Beerse, Belgium. Biopsy punches were purchased from Stiefel, Middlesex, UK and Tissue-Tek® optimal cutting temperature medium was obtained from Sakura Thatcham, UK.2.2w/w) RPV LA and 10% (w/w) PVA, as listed in Low molecular PVA  was used to formulate the microneedles of the arrays and due to the low water solubility of RPV, a long-acting RPV nanosuspension (RPV LA), developed and supplied by Janssen Pharmaceutical, was the drug source used throughout this work. MAPs were prepared using aqueous formulations containing varying amounts of PVA and RPV LA. The formulations were named according to their composition. For example, the 80R10P formulation was cast with 80% (w/w) glycerol.The baseplate used in all of the documented studies was formulated using aqueous solutions of 20% (w/w) PVP of molecular weight 360,000 Da and 1.5% (2.3w/w) PVP and 1.5% (w/w) glycerol, was then placed behind the microneedles. The moulds were placed inside a positive pressure compression chamber and a pressure of 3 bar was applied for 15 min. Finally, the MAPs were dried at room temperature for 24 h and were then removed from the moulds and used in mechanical tests. The RPV LA containing formulation was distributed between the needle tips and a thin baseplate behind the needles.Dissolving RPV LA MAPs with bioadhesive baseplates were prepared in a two-step process of microneedle casting, followed by baseplate adhesion behind the microneedles. The process is illustrated in w/w) RPV LA, 15% (w/w) PVA and 15% (w/w) water (see results section). These MAPs were produced containing a total of 30 mg of RPV LA. The RPV LA loading in the microneedles was calculated using Eq. Different formulations were prepared but it was established that the optimum formulation was that containing 70% , employed at a temperature of 25 °C, was used in these experiments. Two samples, namely, RPV LA diluted in water and RPV LA MAPs that were prepared, dried and then dissolved in water were analysed.2.6RPV quantification from excised neonatal porcine skin samples was performed using a Phenomenex™ Luna 5 μm C18  100 Å co2.7The dissolution rate of the RPV LA MAPs was investigated in excised neonatal porcine skin. Skin samples were carefully shaved using a disposable razor and then equilibrated in PBS (pH 7.4) before being mounted onto a styrofoam block, coated with PBS-soaked absorbent paper. MAPs were inserted into the skin with a pressure of 10 N/MAP over 30 s using a TA.XTPlus Texture Analyser  and the skin was then stored at 37 °C for the duration of the experiment. MAPS were removed at designated time points and digitally imaged. Optical coherence tomography (OCT)  was used to assess the successful insertion of the MAPs into the excised skin, as reported previously ,23.2.8in vivo conditions. Subsequently, the RPV LA MAPs were removed and a tissue sample, taken from that portion of the skin where the RPV LA MAP had been inserted, was obtained using a biopsy punch (6 mm diameter) . A diagrammatic representation of this procedure is presented in g, 10 min. Supernatants were analysed using the validated HPLC methodology.Full-thickness skin, collected from stillborn piglets, was shaved using a razor and then mounted onto a styrofoam block, coated with PBS-soaked absorbent paper. RPV LA MAPs were inserted into the skin using manual force, roughly equivalent to 20 N, over 30 s ,24. The 2.9v/v) trifluoroacetic acid (≥ 99% pure TFA), with a flow rate of 1 ml/min, and a run time of 10 min. The injection volume was 20 μl. The chromatograms obtained were analysed using Agilent ChemStation® Software B.02.01. Least squares linear regression analysis and correlation analysis were performed on the calibration curves produced, enabling determination of the equation of the line, its coefficient of determination and the residual sum of squares (RSS). The limits of detection (LOD) and quantification (LOQ) for this method were estimated according to the signal-to-noise ratio guidelines outlined in the European Pharmacopoeia, version 8.0 [i.e. 25 ng/ml).RPV quantification from rat plasma was performed using a Phenomenex SphereClone 5 μm (150 × 4.60 mm) column  and a RP-HPLC  with UV detection at 282 nm. The mobile phase was composed of 65:35; acetonitrile (ACN):water containing 0.1%  and all researchers carrying out the work held Personal Licences from the UK Home Office. Two cohorts were employed in in vivo studies: the intradermal (MAP) cohort and the control IM (IM) cohort. Experiments were conducted using female Sprague Dawley rats aged between 9 and 13 weeks upon commencement of the study and aged between 14 and 17 weeks upon completion of the study. Prior to the commencement of experiments, rats were acclimatised to laboratory conditions for a 7-day period. MAP applications were carried out as described previously and MAPs were removed after 24 h [Ethical permission for all ter 24 h . An imagThose rats in the IM cohort received 60 μl of 30 mg/ml RPV LA (1.8 mg dose) into the right hind thigh muscle.2.11.2via the lateral tail vein. Plasma was separated from the whole blood and prepared as described, prior to HPLC analysis.Blood samples were collected from the treated rats, at pre-determined time points , into heparinised tubes g for 10 min at 4 °C to remove any debris. A 35 μl volume of sample was then mixed with 65 μl acetonitrile (ACN), and a sample of this was analysed by HPLC. For data analysis purposes, arithmetic means were determined and plasma samples with RPV concentrations below the LOQ of the system were included in data tables as half the LOQ, namely 25 ng/ml.Vaginal washes were collected by washing the vaginal cavity with 200 μl sterile water for injection  at pre-determined time points . The samples were centrifuged at 14,000× 2.11.32 overdose and vaginal tissue, axillary, iliac and external lumbar lymph nodes were excised. The lymph nodes were weighed and then homogenised at 50 Hz for 10 min in a Qiagen TissueLyser LT . Samples were centrifuged at 14,000× g for 10 min at 4 °C and supernatants were collected. The nodes were then subjected to a second extraction procedure. Supernatants were dried under a stream of nitrogen at 37 °C for 50 min, samples were reconstituted in mobile phase and RPV content was quantified using the validated HPLC methodology. Vaginal tissue was treated similarly; however, the tissue was disrupted mechanically prior to homogenisation to increase the surface area in contact with the ACN, maximizing drug recovery. The amount of RPV in the organs was expressed as ng of drug per g of tissue (ng/g), in order to account for differences in tissue masses between rats. For data analysis purposes, arithmetic means were determined and plasma samples with RPV concentrations below the LOQ of the system were included in data tables as half the LOQ, namely 25 ng/ml.Animals were culled at the pre-determined experimental endpoints by CO2.11.4Estimation of human dose for RPV LA MAP were conducted to provide the same trough concentration as that observed after single-dose RPV LA IM, 600 mg, after 28 days (44 ng/ml), as this is the dosing regimen and monthly therapeutic RPV LA IM regimen in clinical development . Given t2.12p value <0.05 was denoted as a significant difference.Data was analysed using the One-Way ANOVA parametric test, followed by Tukey's multiple comparison post-hoc test. A 33.1MAPs were manufactured containing two layers. RPV LA was incorporated into the needle tips and into a thin film behind the needles. A rigid polymeric baseplate was added to facilitate insertion. Multiple published studies have previously used multi-layered MAPs . The novw/w) and baseplates cast from aqueous blends of 20% (w/w) poly(vinylpyrrolidone) (PVP), 1.5% (w/w) glycerol. The Parafilm® skin simulant model was employed, as previously described, as a means of determining the insertion ability of the RPV LA MAPs [w/w) PVA were brittle, however, and some microneedles broke off the arrays upon insertion, indicating a lack of mechanical strength.MAPs were prepared using RPV LA formulated from aqueous blends containing various PVA concentrations ranging from 0%–20%  PVP, 1.5% (w/w) glycerol in the baseplates. The MAPs were prepared as outlined schematically in The dissolving RPV LA MAPs were fabricated in two stages to avoid drug wastage . Followi3.3in vitro insertion depths obtained for the four formulations displayed similar trends and all were capable of piercing three layers of Parafilm®. The mean thickness of a Parafilm® layer is 126 ± 7 μm [w/w) PVA and results indicated that they could be inserted deeper into skin layers. Consequently, the 70R15P formulation was selected as the optimal formulation for MAP preparation.The percentage of holes created in Parafilm® layers for the different dissolving MAP formulations tested is shown in 6 ± 7 μm , thus su6 ± 7 μm ,25. MAPs3.4The properties of the nanosuspension should not be modified after being combined with PVA or dried to form the MAPs. Consequently, the particle size was evaluated before and after the formulation process. Even at the highest loading of PVA, there was no significant effect on RPV nanoparticle size or polydispersity C. It was3.5in situ in the skin. The pores created in the skin, 5 h post-MAP application are clearly defined in the OCT image presented in The dissolution of the needles of the RPV LA MAP in excised skin occurred slowly, likely due to the high content of hydrophobic RPV LA in the needles of the MAPs. The microscopic images presented in 3.63 of tissue, across three different application times, namely 30, 60 and 120 min  (four rats) or with simultaneous application of 4 × MAPs (of which 2 mg RPV LA/array was contained in the microneedles) (three rats). In the MAP cohort, MAPs were applied to the backs of the rats and removed after 24 h. It is evident from the exemplar digital image of a single MAP post-removal A that noPlasma samples were collected from the animals after MAP and IM administration and the mean concentrations of RPV in each cohort, at sampling time points of 1, 4, 7, 28 and 56 days, are presented in via MAP that had entered the systemic circulation. Four days post-MAP application, encapsulated RPV could have accounted for the majority of the drug in the animal and this would not have been quantifiable in the plasma, as the drug would not yet have reached the systemic circulation. Plasma concentrations of RPV subsequently decreased to 75.6 ± 71.5 ng/ml at 28 days and comparable RPV concentrations were maintained up to the experimental endpoint of 56 days (104.4 ± 51.9 ng/ml). Those rats treated with RPV LA via IM injection exhibited lower initial RPV plasma concentrations. Following IM injection, a peak concentration of 175.5 ± 197.6 ng/ml was detected 1 day post-injection and no samples had RPV concentrations above the LOQ of the HPLC system at 7 days (25 ± 7.2 ng/ml). Interestingly, the mean RPV plasma concentration at the experimental endpoint of 56 days (118.9 ± 54.2 ng/ml) was comparable to that achieved via RPV LA MAP at the same time point (104.4 ± 51.9 ng/ml). In terms of direct comparison of the results generated here with other experimental studies, this is challenging, as stated previously, due to the fact that not all of the drug encapsulated into the MAPs would effectively be delivered in vivo. Some preclinical work carried out in 2010 studied the pharmacokinetics, distribution and tolerability of RPV LA delivered via IM injection in rats and dogs [via IM or MAP exhibited comparable patterns of sustained drug in plasma over similar time frames. In the 2010 study, those rats given 5 mg/kg and 20 mg/kg RPV LA had Cmax of 71 ng/ml and 158 ng/ml, respectively at 7 h post-dose [via MAP, the plasma concentrations of drug post-MAP delivery of RPV LA must be considered in comparison to the IM control. In this regard, at 24 h, plasma levels of the drug in the MAP cohort (547.5 ± 298.1 ng/ml) exceeded those of the IM cohort (175.5 ± 197.6 ng/ml). When the differences in RPV LA dose administered are taken into consideration, this shows the potential intradermal delivery capabilities of this nanosuspension drug from MAPs. As stated earlier in the discussion, this assertion is further backed up by the fact that the two delivery routes resulted in equivalent mean plasma levels of the drug in rats at the experimental endpoint of 56 days.Specifically, the maximum mean plasma concentration of 547.5 ± 298.1 ng/ml was recorded just 1 day post-MAP application with 430.7 ± 608.1 ng/ml detected after 7 days. At the Day 4 time point, however, no samples within this cohort had RPV concentrations above the limit of quantification (LOQ) of the system. This was not unexpected, however, as the RPV detected 1 day post-MAP application would have consisted of rapidly dissolving RPV delivered intradermally and dogs , althougand dogs ), and coost-dose . In the in vitro viral replication (IC90) of 12.5 ng/ml has been suggested previously [via IM injection. Accordingly, convention dictates that an optimised MAP would be anticipated to also deliver RPV LA for 28-day exposure. RPV LA IM at a dosing regimen of 600 mg every 4 weeks is in Phase 3 clinical development for HIV prevention. The 2014 clinical study, carried out in HIV-negative volunteers, demonstrated that 600 mg IM dosing of RPV LA provided a mean plasma concentration of 44.2 ng/ml, 28-days after single-dose administration [2, is estimated to be needed to achieve similar duration of exposure after single-dose administration. A patch of this size would be unrealistic and inappropriate for patient usage however. Theoretically, an RPV LA MAP of approximately 28 cm2 could be cautiously predicted to maintain therapeutic concentrations of RPV in plasma over 7 days in humans, although further in vivo pharmacokinetic evaluations will now have to be carried out to bolster this proof of concept data. A patch of 28 cm2 would actually be smaller than commercially available transdermal patches including GlaxoSmithKline's (GSK) Nicotinell® nicotine patches of 30 cm2 [2 [2 MAP, following appropriate instruction, demonstrating clear feasibility of applying larger MAP patches [There is currently no consensus therapeutic plasma concentration for RPV but the documented drug plasma level above the protein-binding adjusted concentration required to inhibit 90% of eviously , we can f 30 cm2  and Jans0 cm2 [2 . In addi patches . ImportaWith reference to RPV in vaginal washes, no RPV was detected in the vaginal washes collected from rats 7, 28 or 56 days after administration of MAP or IM RPV LA. This is in contrast to clinical studies where RPV concentrations in vaginal fluid approximated those seen in plasma [3.8P = 0.0273).Vaginal tissue was extracted from all animals at the requisite experimental endpoints and RPV concentrations were expressed relative to the mass of the tissue removed from each animal (ng/g tissue). In animals treated with RPV LA MAP, RPV concentrations were highest in animals culled 7 days post-MAP application (1238 ± 347 ng/g tissue) C and decP = 0.0183). The concentrations in the 56-day IM cohort (197 ± 120 ng/g tissue) were similar to those determined at the same time point in the MAP study arm (169 ± 84 ng/g tissue). Once again, these results show that RPV LA can be successfully delivered intradermally via MAP. Interestingly, the localised tissue concentrations of RPV, when delivered from dissolving MAPs, were comparable to those reached via conventional IM delivery of the drug. The differences in RPV concentrations in vaginal tissue across the two treatment groups were also compared at each time point and there were no statistically significant differences between the treatment cohorts at common time points.In animals treated with RPV LA IM injection, the RPV concentrations in the vaginal tissue did not peak as high as those recorded in the MAP study arm B. RPV coSecondary lymph nodes are one of the principal sites of HIV replication and the restricted penetration of ARV drugs into these reservoir compartments may be one of the main mechanisms of viral persistence in individuals . Consequall of the animals, with concentrations of 1106 ± 278 ng/g tissue and 679 ± 382 ng/g tissue for the 28- and 56-day cohorts, respectively. RPV concentrations in the axillary nodes at 28 days and the external lumbar nodes at 56 days were all below the LOQ of the HPLC method. Irrespective of the cohort and as such, the length of time post-RPV LA MAP application, RPV was consistently found in the iliac nodes of all animals treated via these dissolving MAPs. The detection of RPV in lymph nodes over extended periods draws a parallel with a recent study using a long-acting ARV drug-combination which reported the same phenomenon [In the MAP arm of the study, two out of three rats in the 7-day cohort had quantifiable RPV in the iliac nodes, external lumbar nodes and axillary nodes. In the 28- and 56-day cohorts, however, only the iliac nodes had quantifiable RPV in enomenon . In the via IM, no drug was detectable in lymphoid tissues (thymus or spleen) of the rats 8 weeks post-dose [In contrast to the MAP study arm, after IM administration RPV was not quantified in the iliac nodes of all of the animals employed in the study. Rather, RPV concentrations were below the LOQ of the HPLC system in the iliac nodes of all of the animals in the 28-day cohort, with only one animal in the 56-day cohort having quantifiable RPV C. In conost-dose . No lympost-dose . The res2 MAPs) can be successfully self-administered by individuals, and to comparable depths as smaller patches with a single array, following appropriate instruction [In considering the possibility for self-application of MAPs, our research team and others have already published user feasibility studies, with results indicating that MAPs can be reproducibly applied to the skin without the need for complex applicators or specialist personnel . In additruction . The out4via MAP or IM led to comparable plasma, localised tissue and lymphoid tissue distribution results. Moreover, it has been cautiously estimated that an RPV LA MAP of approximately 28 cm2 could maintain therapeutic plasma levels of RPV over 7 days in humans. This work supports the potential future use of MAPs in the needle-free delivery of other long-acting ARV drugs, thus eradicating the necessity for daily oral medication regimens and eliminating the risks associated with needle-stick injuries. The outcomes of the current study have now been utilized to inform the target product profile for ARV MAPs. Formulation optimisation, stability evaluations, comprehensive preclinical and clinical pharmacokinetic studies, physiologically-based pharmacokinetic modelling and patient usability/acceptability studies are now necessary to fully realize the potential of these novel delivery platforms.This work documents a proof of principle study outlining the formulation of novel dissolving MAPs for intradermal delivery of RPV. This is the first time that an ARV nanosuspension has been incorporated into a MAP delivery format and the results illustrate the potential delivery capabilities of such a novel device. RPV LA administration


Terahertz (THz) radiation, with frequencies spanning from 0.1 to 10 THz, has long been the most underdeveloped frequency band in electromagnetic waves, mainly due to the dearth of available high-power THz sources. Although the last decades have seen a surge of electronic and optical techniques for generating intense THz radiation, all THz sources reported until now have failed to produce above-millijoule (mJ) THz pulses. We present a THz source that enables a THz pulse energy up to tens of mJ, by using an intense laser pulse to irradiate a metal foil. Ultrahigh-power terahertz (THz) radiation sources are essential for many applications, for example, THz-wave-based compact accelerators and THz control over matter. However, to date none of the THz sources reported, whether based upon large-scale accelerators or high-power lasers, have produced THz pulses with energies above the millijoule (mJ) level. Here, we report a substantial increase in THz pulse energy, as high as tens of mJ, generated by a high-intensity, picosecond laser pulse irradiating a metal foil. A further up-scaling of THz energy by a factor of ∼4 is observed when introducing preplasmas at the target-rear side. Experimental measurements and theoretical models identify the dominant THz generation mechanism to be coherent transition radiation, induced by the laser-accelerated energetic electron bunch escaping the target. Observation of THz-field-induced carrier multiplication in high-resistivity silicon is presented as a proof-of-concept application demonstration. Such an extremely high THz energy not only triggers various nonlinear dynamics in matter, but also opens up the research era of relativistic THz optics. Terahertz (THz) radiation is usually utilized as a nonionizing probe in many science disciplines . With th2Currently, the most prominent approaches toward generating high-power THz pulses are based on conventional electron accelerators –13, and Recently, laser-produced plasmas have attracted considerable interest as a damage-free medium for achieving compact intense THz sources , 21. Las2227In this report, utilizing an ultraintense, picosecond (ps) laser pulse to irradiate a metal foil, we demonstrate the efficient generation of low-frequency (<3 THz) coherent THz radiation with pulse energies exceeding multimillijoules, surpassing other THz sources –32. The The experimental setup  at 75° was measured to be ∼2.3 mJ at frequencies below 20 THz. Spectral measurements, either with a set of low-pass filters or narrowband band-pass filters, showed that the THz radiation was low-frequency (<3 THz) dominated A. THz enTo identify the roles of sheath fields, ions, and electrons in the THz generation, we modified the sheath fields directly by controlling the preplasma at the target rear. An additional laser beam was introduced and focused to the target-rear surface, to generate an expanding preplasma ahead of the main pulse irradiating the front surface. The preplasma scale length was adjusted by varying the relative timing between the prepulse and main pulse. With increasing preplasma scale length, the maximum proton energy decreases significantly A, resultWCTR, generated by an electron bunch with a Boltzmann energy distribution, scales with the bunch charge Q and the electron temperature Te approximately as WCTR ∝Q2⋅Inset). With increasing laser energy, both Q and Te increase. According to the experimentally measured Te ∝Q1.4±0.6, it is anticipated that WCTR ∝Q2.3±0.1, which explains well the observed power-law index of 2.3 ± 0.7 (Te does not vary much because the target-front electron acceleration is not affected. Hydrodynamic simulations with the MULTI−fs code (Insets), one can calculate the radiation spectrum and angular distribution from the CTR theory (The coherent transition radiation (CTR) model , 37 can 2⋅Te0.22 C, Inset..3 ± 0.7 C. When i−fs code  show tha−fs code , and the−fs code B. In thi−fs code , and theR theory A and B.Ie(ω), emitted by a single electron in the sheath, depends mainly on the lifetime of electrons in the sheath, τs, which is determined by the electron kinetic energy and sheath-field strength. The coherent radiation intensity, Ib(ω), generated by an electron bunch, is approximately given as Ib(ω) ∝F2(ω)⋅Ie(ω), where F(ω) is the electron bunch form factor. For the picosecond electron bunch in our picosecond laser experiment, F(ω) gets rather weak at the frequency region over 1 THz, overlapping little with Ie(ω) especially at high sheath fields  wafers. If the THz field is adequately high, the interband luminescence will emerge  despite e-h) pairs. We attribute the generation of massive e-h carriers mainly to the process of impact ionization  and be available in university-scale laboratories. On the other hand, by comparing the broadband THz spectra (up to ∼30 THz) obtained previously in femtosecond laser-driven cases –32 with In conclusion, we have experimentally demonstrated the efficient generation of low-frequency THz pulses with an ultrahigh energy of ∼50 mJ, exceeding other state-of-the-art THz sources by nearly one order of magnitude. A further THz energy boost is obtained by introducing preplasmas to enhance the number of electrons escaping the sheath field generated at the target-rear surface. The application potential of the THz source is illustrated with a preliminary THz pump experiment, where a strong-field THz pulse induces the extraordinary multiplication of carriers in semiconductors. Multimillijoule THz sources reported here could enable the study of relativistic optics in the THz regime. Together with intrinsically synchronized energetic particles and photons generated concomitantly in laser–plasma interactions, more opportunities in the extreme THz science  will be 19 W/cm2 (a0 ∼ 6). The beam B8  as the prepulse, was focused by an f/15 OAP mirror onto the target-rear surface at an incidence angle of 75° and an enlarged focal spot size of ∼0.5 mm × 1.9 mm, corresponding to a laser intensity in the range of ∼(3−6) × 1013 W/cm2. The fairly large spot size of B8 produced a quasi–one-dimensional evolution of the preplasma, and ensured full overlap of the preplasmas with the B7-accelerated electron bunch. Neither THz radiation nor particle acceleration was detected when only B8 was applied on the target. Before the arrival of B7 at the target, the target-front surface was not perturbed by the target-rear prepulse. Evidences of this came from the fact that the target-front electron and optical diagnostic signals did not vary with the prepulse.The experiment was carried out at the Rutherford Appleton Laboratory using the Vulcan laser  operatinInset) with respect to the rear target normal was collected with similar lens configurations, and then relayed into THz energy and spectrum detection systems. To remove radiation at other wavelengths and avoid saturation of the THz detectors, THz filters and high-resistivity Si wafers were placed in the THz path. Owing to the high energies of the THz pulses produced in the experiment, a filter-based THz spectrometer was applied to measure the discretized THz spectrum in a single shot. In the THz spectrometer, the THz beam was splitted into eight beamlets. Low-pass or narrowband band-pass THz filters with varying cutoff or central frequencies were inserted in different beamlet paths, and the filtered THz radiation was measured by cross-calibrated pyroelectric detectors. The THz spectral intensity is retrieved as ITHz = Sdet/(Rdet⋅Tm⋅Δω), where Sdet is the detector signal, Rdet is the detector responsivity averaged over the transmission bandwidth, Δω, of band-pass filters, and Tm is the overall transmittance of THz components in the path, respectively. The spectrally integrated THz energy is obtained as WTHz = Sdet/(Rave⋅Tm), where Rave is the average responsivity within 10 THz. The inverse Fourier transform of the fitted THz spectrum (Inset) shows ∼83% of the total energy is distributed in a pulse duration of τTHz = 1.5 ps.The THz radiation at 75°, 45°, −20°, and −40° D, Inset spectrum A, Inset Inset), but it only highlighted the high-frequency components because of its decreasing sensitivity with decreasing radiation frequencies. Since the transverse formation length -based camera (DataRay Inc.) was used to image the THz source C, Inset,n length  of sub-TInset) at the pump laser energy of ∼60 J. In some shots a four-layered IP stack (Inset), showing a double-peak angular distribution with a divergence angle of ∼30°. A Cherenkov fiber array  detector, was used to detect the energy spectrum of electrons. The electron temperature was measured to be ∼2 MeV B, Inset IP stack , positioIP stack A, Inset,er array  was run Es, can be considered approximately to be uniform along the rear target normal for a specific electron. An electron leaves orthogonally the target surface with a momentum of p at t = 0, and returns back to the target at t = 2τs, where τs = p/eEs. By adopting a model of transient electric dipole consisting of the electron and its image charge, the resulting radiation spectral intensity is deduced asGiven the fact that both the lifetime and traveling distance of low-energy electrons in the sheath are quite small compared with those of sheath evolution, the sheath field, θ is the angle between the electron direction and the radiation wave vector.where WCTR, emitted per unit angular frequency dω and unit solid angle dΩ can be expressed asFor an electron bunch crossing an ideal conductor–vacuum interface, the CTR energy, Q is the electron bunch charge, g(p) is the momentum distribution function, which can be decomposed into two parts, the energy spectrum and the angular distribution. ξ is the normalized amplitude of radiation electric fields. D is the correction factor due to the finite target size. Expressions for ξ and D can be found in ref. F(ω) is the bunch form factor, defined as the Fourier transform of the normalized electron bunch temporal profile. For a collimated electron bunch with a Gaussian temporal distribution, F(ω) = exp. Spatially integral of the calculated angular distribution indicates that the total energy is ∼28 times the energy in 0.12 sr at 75°, which is measured to be 1.96 mJ (±20%) within 3 THz in the experiment. Hence the total THz energy emitted from the target-rear surface is evaluated to be ∼55 mJ.Typically the electron bunch duration is on the order of the laser pulse duration (FWHM∼1.5 ps in this experiment). By substituting the measured spatial and energy spectral distributions A and B ohttps://edata.stfc.ac.uk/handle/edata/747.Data associated with research published in this paper can be accessed at


Rickettsiales are often found in association with ciliated protists. An interesting case is the bacterial endosymbiont “Candidatus Megaira”, which is phylogenetically closely related to the pathogen Rickettsia. “Candidatus Megaira” was first described as an intracellular bacterium in several ciliate species. Since then it has been found in association with diverse evolutionary distantly-related hosts, among them other unicellular eukaryotes, and also algae, and metazoa, such as cnidarians. We provide the characterization of several new strains of the type species “Candidatus Megaira polyxenophila”, and the multidisciplinary description of a novel species, “Candidatus Megaira venefica”, presenting peculiar features, which highlight the diversity and variability of these widespread bacterial endosymbionts. Screening of the 16S rRNA gene short amplicon database and phylogenetic analysis of 16S rRNA gene hypervariable regions revealed the presence of further hidden lineages, and provided hints on the possibility that these bacteria may be horizontally transmitted among aquatic protists and metazoa. The phylogenetic reconstruction supports the existence of at least five different separate species-level clades of “Candidatus Megaira”, and we designed a set of specific probes allowing easy recognition of the four major clades of the genus.Members of the order  Rickettsiaceae  comprises obligate intracellular bacteria hosted by eukaryotic organisms. Rickettsia and Orientia are the most studied genera and they are etiological agents of many human diseases, such as spotted fever or typhus1. Within the Rickettsiaceae family, several other bacteria are not of direct health concern and have been also referred to as “neglected Rickettsiaceae”2. These bacteria are important for understanding key evolutionary and ecological phenomena, such as host-symbiont interactions4, the emergence of pathogenicity6, and even the evolution of mitochondria, which are considered by several authors related to Rickettsiales7.The family Rickettsiaceae family were known to colonize only arthropods and vertebrates8, but with the discovery and characterization of new species and genera phylogenetically affiliated to this family, this conviction vanished. Indeed, recent studies14 unraveled the existence of many “neglected Rickettsiaceae”, often hosted by protists. Considering the interwoven phylogenetic relationships among bacteria harbored by diverse hosts15, these data provided indirect evidence that some Rickettsiaceae can be frequently transmitted among hosts from evolutionary far related lineages9.Traditionally, members of the Rickettsiaceae” is the endosymbiont “Candidatus Megaira”2. The first molecular report of this endosymbiont was in 2005 from the ciliate Diophrys oligothrix, where it was mentioned as a member of Rickettsiaceae16. Subsequently, this bacterium was found in many different hosts ranging from multicellular organisms, like cnidarians18 to green algae including chlorophytes and streptophytes21, and protists - amoebae11, and numerous ciliates22. In some cases, bacteria were associated to the gut content of animals, such as ascidians23, fish24, or worms25. As these studies dealt mainly with molecular approaches, the true nature of these associations was not clarified. Some full-length 16S rRNA gene sequences belonging to the genus “Ca. Megaira” have been retrieved from environmental samples, such as freshwater lakes26, aquaria27, wastewater treatment plants28, and soil29, but these studies do not allow conclusions regarding their association with possible hosts.An interesting bacterial genus within “neglected Ca. Megaira polyxenophila” was retrieved in diverse species of ciliates belonging to different classes2, including the fish parasite Ichthyophthirius multifiliis12, and in a wide range of additional, evolutionary distant, organisms21. In ciliates, this bacterial endosymbiont is able to colonize different host cell compartments (e.g. macronucleus or cytoplasm) according to the host species. Colonization of ciliate cells by bacteria can be tentatively explained accounting that these protists, as phagotrophic predators, can engulf bacteria, which may escape from digestion and colonize one of many cell compartments available31. Considering that ciliates and other protists are at the basis of many trophic chains32, it is tempting to hypothesize that they may transmit their symbionts to other hosts at higher trophic levels2.Notably, the type species “Ca. Megaira polyxenophila” and describe the novel species “Ca. Megaira venefica”. We also provide a critical revision of the genus “Ca. Megaira”, the already described species, and new clades it comprises. We designed and validated a new genus-specific probe for fluorescence in situ hybridization (FISH) experiments in order to allow an easy recognition of members of the genus. Moreover, we designed in silico four clade/species specific probes targeting the same hypervariable 16S rRNA region, and validated two of them for which strains of “Ca. Megaira polyxenophila” and “Ca. Megaira venefica” were available. Screening of 16S rRNA gene amplicons applying the Integrated Microbial Next Generation Sequencing (IMNGS)33 platform demonstrates the high diffusion of this bacterial genus in many ecosystems.Herein we report 14 new strains of “Paramecium hosts were infected and then molecularly characterized. A list of ciliate cultures harbouring bacterial endosymbionts affiliated to “Ca. Megaira” is provided .A preliminary screening for the presence of bacterial symbionts was performed by Differential Inference Contrast (DIC) microscopy on paramecia residing in the Core Facilities Centre “Culture Collections of Microorganisms”. When a bacterial symbiont was detected, usually all ed Table . As for 34. With this perspective, in this study, ciliates were identified up to morphospecies level, combining morphological investigation on features, such as the cell shape, size, and micronuclear appearance and number (data not shown) with molecular characterization through 18S rRNA gene sequencing  gene and internal transcribed spacer (ITS1-5.8S-ITS2) was also performed to identify the species .18S rRNA gene sequence of algal endosymbionts of Ca. Megaira polyxenophila” . This observation suggests that representatives of two “Ca. Megaira” clades were co-occurring in the same host/sample. Some of these chimeras have been included in previous phylogenies of the genus ; these sequences were excluded from the following analyses , and these values are below the threshold established (98.65-98.7%) to discriminate different bacterial species according to 16S rRNA gene sequence35. The situation of Clade B was more heterogeneous; indeed, all members shared a minimum identity of 96.5%, which may reveal further inner species level subdivision. On the contrary, Clade C and Clade E were more homogeneous and had identity range higher than 98.6% and 99.5%, respectively. Identities of members of Clade C with other “Ca. Megaira” clades did not exceed 98.3%. Therefore, the newly characterized symbionts together with previously available sequences allowed to define Clade C as a new species, which we named “Ca. Megaira venefica” , which is clearly not affiliated to any of the described clades, but instead forms a sister group of Clade E, thus suggesting the possible existence of an additional clade  and 1.00 of posterior probability), and includes all our characterized symbionts assigned to this species. Clade D, sister group of Clade A, is fully supported , and consists of two sequences isolated from aquatic plants and a freshwater lake, respectively. Clade B is well supported , and contains almost only bacteria associated to marine hosts, such as Bryopsis19, corals18, and a Haplosporidium species infecting a marine mollusc36. Clade C is moderately supported , and includes the new species “Ca. Megaira venefica”, represented by symbionts found in Paramecium (current study), bacteria associated to Hydra oligactis, I. multifiliis, and by uncultured environmental bacteria. Finally, Clade E comprises exclusively bacteria detected in the fish parasite I. multifiliis and is highly supported .The clade comprising the type species “in silico all available members of “Ca. Megaira” present in our phylogenetic reconstruction , or chimeric sequences  could be experimentally tested with FISH experiments using available strains  at different formamide concentrations . For the other untested probes, we assumed that they should work properly as they were designed on the same 16S rRNA gene region, which should be accessible as in the tested closely related “Ca. Megaira” species. Nevertheless, future controls are recommended once strains of these clades will be available. The signal for the two tested probes resulted visible at all formamide levels, and signal intensity was optimal at 0% formamide .“tum Fig. . Infectieus Fig. , while sasm Fig. , while cups Fig. . A furthCa. Megaira polyxenophila” strains studied presented the classical “Ca. Megaira polyxenophila” ultrastructure21, consisting of double membrane , homogeneous cytoplasm, a clear halo surrounding the cells, and size dimensions in the range (~1.5 µm × 0.6 µm). Cytoplasm of P. primaurelia strain ThK-1 and macronucleus of P. caudatum strain Sp 9-5 were densely packed with bacteria  originating from 4673 samples. After removal of short and low quality sequences, all remaining 11306 sequences were pooled and re-clustered in 194 OTUs using a 99% identity threshold. OTUs were divided in three different groups according to which hypervariable region of 16S rRNA gene they belonged to and phylogenetic trees were constructed for each hypervariable region. In all trees, the five major clades of “Ca. Megaira” are distinguishable and their relationships are in agreement with full-length 16S rRNA gene phylogeny: Clade A and D are sister groups, as well as Clade C and Clade E, whereas Clade B is always separate  and relative abundance  of the five “Ca. Megaira” clades were estimated for environments and host categories . In most cases, relative abundances were comprised between 0.01% and 0.06% including also environments where bacteria had a high frequency of occurrence  or in in which they had a minimal frequency of occurrence . Clade E showed very low relative abundances in all categories considered . In general, relative abundance, which is higher in aquatic animals, suggests that, in these samples, if present, “Ca. Megaira” is also relatively abundant. Intriguingly, this is true also for terrestrial animals in which “Ca. Megaira” is seldom found (frequency of occurrence <1%), but, when present, has relative abundance higher than in soil . The high relative abundance of clades A and E in seawater if compared to low frequency of occurrence could be explained as a specific abundance in unidentified microeukaryote present in those specific samples. Unfortunately, no reliable estimate is available on the potential presence of microeukaryotes existing in water samples used for these metabarcoding studies. In general, the comparison of data on frequency occurrence with those on relative abundance are coherent with the idea that “Ca. Megaira” is associated to eukaryotic hosts.We failed to assign directly each clade of the 16S rRNA gene hypervariable region phylogenies to a certain environment or host by checking OTUs ecological origins. A more detailed environmental investigation of a 16S rRNA gene short amplicon database was applied considering frequencies of occurrence and relative abundances for each “Ca. Megaira” presence dealt with aquatic environments, and these bacteria were reported as endosymbionts of diverse hosts, both unicellular, namely ciliates22 and amoebae11, and multicellular ones, such as cnidarians18, and green algae19. Protozoa and unicellular green algae are known to be frequent hosts of “Ca. Megaira” symbionts2. However, these hosts are so far underrepresented in 16S rRNA gene metabarcoding studies and, interestingly, our results revealed the absence of “Ca. Megaira” in the few samples associated to unicellular organisms. At the same time, we strongly suppose that many positive environmental hits from aquatic samples or gut samples of aquatic organisms might be explained by presence of unnoticed protists hosting “Ca. Megaira” endosymbionts, especially considering that “Ca. Megaira” – like all Rickettsiales - has never been shown to have a free-living stage. On the contrary, terrestrial organisms’ samples are very numerous, but “Ca. Megaira” sequences were extremely rare in these datasets. Relative abundance of “Ca. Megaira” sequence is generally low, as it is an endosymbiotic bacterium and sampling procedures were not aimed to isolate only host organisms. The relative abundance of endosymbionts in ciliates can reach high values only in studies specifically designed to analyse the microbiome of single cells49. Although aquatic ecosystems appear to be preferential environments for these bacteria , or their associated microbiomes, are other ecological niches where “Ca. Megaira” can occur . For P. bursaria illumination regime of 10 hours darkness and 14 hours light by 2000 lx lamps was used. Cultures are available and maintained at the RR CCM Core Facilities Centre “Culture Collections of Microorganisms” of St. Petersburg State University, Russia. Identification of ciliate species was performed morphologically54, and then confirmed by molecular analysis.Several strains of ciliates were used in this study Table . CiliateApproximately 100 starved ciliate cells were repeatedly washed in sterile distilled water to minimize bacterial contamination, and then fixed in 70% ethanol. Total genomic DNA was extracted employing the NucleoSpin™ Plant II kit  using the protocol for fungal DNA extraction.55. PCR products were estimated through electrophoresis on 1% agarose, purified with EuroGold Cycle Pure Kit , and then sequenced with appropriate internal primers at GATC Biotech  (for further details see Supplementary Text S2).Polymerase chain reactions (PCRs) were performed in a C1000™ Thermal Cycler  with the high-fidelity TaKaRa Ex Taq . All PCRs consisted of 35 cycles with a preliminary denaturation step at 94 °C for 3 min, then for each cycle denaturation at 94 °C for 30 seconds, annealing at 55 °C for 45 seconds and elongation at 72 °C for 90 seconds, and a final elongation step at 72 °C for 5 minutes56 using the SILVA database release 128, and then manually edited in order to optimize base-pairing in the predicted rRNA stem regions. A total number of 130 sequences were selected from the family Rickettsiaceae . Firstly, 119 sequences longer than 1397 bp were used to build the character matrix, then 11 short sequences were added, and phylogeny was inferred.The obtained 16S rRNA gene sequences were aligned automatically with the automatic aligner of the ARB software package57 according to the Akaike Information Criterion. Phylogenetic analyses were performed using both ML and BI methods. ML tree was inferred with 1000 bootstrap pseudoreplicates using PhyML software 3.058, while Bayesian Inference was carried out with MrBayes 3.259 employing three runs, with one cold and three heated Monte Carlo Markov chains each, for 1000000 generations with a burn-in of 25%. Runs were stopped after checking that the average standard deviation of split frequencies reached a value below 0.01.The optimal substitution model was determined with jModelTestCa. Megaira” genus, and for each of the most common clades A + D, B, C, and E, respectively (Table in silico both on RDP35 and on TestProbe tool 3.0 (SILVA rRNA database60) allowing 0 and 1 mismatches  experiments was the one described by Szokoli et al.61. Experiments always included negative controls, namely experiments without the use of any probe, and hybridizations were performed using several formamide concentrations in the hybridization buffer  to test the different stringency levels for the newly designed FISH probes. The almost universal bacterial probe EUB338 (5′-GCTGCCTCCCGTAGGAGT-3′62) was used to verify the presence of other intracellular bacteria in the host cells.The protocol used for all fluorescence P. primaurelia ThK-1, P. caudatum Sp 9-5, P. bursaria strains 1M-2(T), VL3-1, VL12-10, P. nephridiatum Sr 2-6, and P. putrinum ETu 7-4 were prepared for electron microscopy as described by Nitla et al.63. Briefly, cells were fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde in phosphate buffer , with a post-fixation in 1.5% OsO4. Afterwards cells were dehydrated at increasing percentages of ethanol solutions, and finally embedded in Epoxy embedding medium . Ultrathin sections were stained with uranyl acetate followed by lead citrate. Samples were observed using JEOL JEM-1400  electron microscope.P. bursaria VL12-10 cells were briefly washed in water, collected in a small drop, squashed on a cover slip and air-dried. The images were obtained with NTEGRA AURA microscope in a semi-contact mode.33, in order to use the results for investigating both diversity and environmental distribution of “Ca. Megaira” in the environments and in potential hosts. The almost complete 16S rRNA genes of Clade A - “Ca. Megaira polyxenophila” , Clade B , Clade C - “Ca. Megaira venefica” (1M-2(T), KT851791), Clade D , Clade E , and the stand-alone sequence from wastewater (CU466797) were selected as representatives of “Ca. Megaira” genus and used as queries.Raw sequencing 16S rRNA gene reads derived from more than 111000 microbiome samples were screened using the platform IMNGS64. Afterwards, OTUs were aligned with MUSCLE65, and phylogenetic analyses were performed using FastTree66. To attribute an environmental provenance to the OTUs, each sequence clustering in the same OTU was screened and assigned to its environment, according to the provenance of the original sample. Thus, a single environment was assigned to the respective OTU when more than 50% of sequences had the same origin, while two environments were considered when two ecosystems were equally dominant. The category “Unclassified” was used when more than two environments were dominant.Sequences longer than 300 bp, and having at least 95% of identity with the queries were selected for analysis. Sequences retrieved from all queries were pooled together, and were separated in three datasets according to hypervariable regions V1-2, V4-6, V7-8, and then clustered in OTUs with a threshold at 99% using UCLUSTCa. Megaira” clade , two diverse indices were calculated: frequency of occurrence and relative abundance. Frequency of occurrence was estimated as the number of microbiome samples positive for “Ca. Megaira” in each category divided the total number of samples of the category . Relative abundance of “Ca. Megaira” for each specific environment was calculated as the average of relative abundances in “Ca. Megaira” positive samples (i.e. the number of “Ca. Megaira” reads divided total number of reads for each sample; for further details see Supplementary Text In order to further investigate environmental distribution of each “Supplementary InformationSupplementary TablesEnvironmental distribution primary data


Ventilation rate and rating of perceived exertion (RPE) were not significantly different between cadence conditions with one-tailed paired t-test analysis . Respiratory exchange ratio and caloric unit cost were significantly greater with lower cadence condition . Mean power at self-selected cadence was 4.4 ± 0.5 W·kg−1, and at lowered cadence was 4.4 ± 0.5 W·kg−1. Mean form power at self-selected cadence was 1.1 ± 0.1 W·kg−1, and at lowered cadence was 1.1 ± 0.1 W·kg−1. There were positive, linear correlations between running economy and power ; running economy and form power . The findings suggest running economy is positively correlated with Stryd’s power and form power measures yet the footpod may not be sufficiently accurate to estimate differences in the running economy of competitive runners.A novel running wearable called the Stryd Summit footpod fastens to a runner’s shoe and estimates running power. The footpod separates power output into two components, Stryd power and form power. The purpose of this study was to measure the correlations between running economy and power and form power at lactate threshold pace. Seventeen well-trained distance runners, 9 male and 8 female, completed a running protocol. Participants ran two four-minute trials: one with a self-selected cadence, and one with a target cadence lowered by 10%. The mean running economy expressed in terms of oxygen cost at self-selected cadence was 201.6 ± 12.8 mL·kg Use of wearable technologies (wearables) like Global Positioning System (GPS) watches, fitness trackers, and pedometers has increased in recent years . Wearabl2) [2) is transmitted into useful mechanical power (cycling power) [Cycling power meters have been available for years. Cyclists use these in training and racing and the relationships between cycling power output and oxygen consumption (VO2) , cycling2) , and cyc2)  have beeg power) . Most ofg power) . A variety of methods for measuring or estimating running power have been explored. In these methods different assumptions were made when calculating power and the power calculations required ground reaction force data or kinematic data for input. Due to these differences in assumptions and input data, the values resulting from the power calculations varied widely ,10,11,122·kg−1·km−1) is calculated using measurements of steady-state oxygen consumption, participant body mass, and distance traveled [Running economy is defined as the energy demand required to sustain a submaximal velocity . Runningtraveled ,15. Runntraveled ,16. 2max among similarly capable distance runners [Running economy is considered an essential component of distance running performance and is modulated by both biomechanical and physiological factors ,18. It i runners . Since r runners , improvi runners ,19,20,21Running economy is related to biomechanical factors including vertical oscillation, ground contact time, stride length, and stride frequency ,23. It h2). The researchers found that power and VO2 had a weak positive relationship (r = 0.29). Since running economy is considered central to running performance [Measurement of running economy requires expensive equipment  that most runners do not have free access to. As wearable devices have become more sophisticated, athletes and coaches have had access to more data . Wearablformance , an alteIt is unknown if power as measured by the Footpod has a stronger relationship with running economy than power as measured by the Stryd Pioneer chest strap. Furthermore, it is unknown if changes in cadence will cause significant change in power or form power, as they can with running economy ,25,26. IA total of 17 participants were recruited and took part in the study . Participants were either current or alumni members of the university’s cross country or track and field team. The study was reviewed and approved by the university’s Institutional Review Board. Informed consent was obtained, then running history and physical activity readiness questionnaires were completed before the running trials. 2max values were estimated using recent (within 12 months) race performance times (distances from 800 m to marathon) [2max values were then utilized to prescribe a workload estimated to be at lactate threshold pace corresponding to 85–89% of velocity at VO2max (vVO2max), which was a training intensity all subjects were familiar with [2max has been established as a measure that more closely correlates with running performance than either running economy or VO2max individually [2max value that correlated with an estimated VO2max was not exceeded . Participant characteristics are reported in The focus of the investigation was on testing the efficacy of the Footpod power metrics as measures related to running economy. Participant VOarathon) ,29. Estiiar with ,29. vVO2vidually ,31. All 2 and CO2 sensors were calibrated based on known gas tank concentrations and room air measurements. Flowmeter calibrations were then completed using a 3.0 L syringe. Stryd metrics were recorded by a Stryd Summit footpod  paired to a Garmin Fenix 3 watch  with the Stryd Run Data Field. Testing was completed on a Trackmaster TMX425c treadmill .A Parvo Medics TrueOne 2400 metabolic analyzer  was used to record gas exchange measures. Gas exchange data were recorded on a Dell desktop computer. Prior to each testing session, the metabolic analyzer was powered on to warm up and then OPrior to testing, participants were given a brief overview of the protocol. Participants then put on the metabolic analyzer mouthpiece and nose clip. The Footpod was attached to the runner’s left shoelace equidistant from the participant’s malleolus and the toe of their shoe. Participant height and weight values were entered in the Stryd Android app and Garmin watch.Participants completed a two-stage discontinuous running protocol. Data collection began during a five-minute warm-up at a self-selected “easy” pace. Participant self-selected cadence was then determined during a one-minute run at the participant’s assigned threshold pace immediately after the five-minute warm-up. Following the warm-up and self-selected cadence measurement, participants took a three-minute standing rest on the treadmill. During the rest, researchers calculated a cadence value 10% lower than the participants’ self-selected cadence for them to replicate during the experimental protocol. A coin was flipped to determine the order of testing protocol stages. For the self-selected cadence stage, participants ran four minutes at assigned threshold pace while attempting to match their cadence with a metronome chirping at their self-selected cadence from the warm-up period. For the lower cadence stage, participants ran four minutes at assigned threshold pace while attempting to match their cadence with the metronome set to a value 10% lower than their self-selected cadence .2 cost and caloric unit cost), caloric unit cost, VO2, respiratory exchange ratio, ventilation, average power, average form power, and average cadence were calculated from the last minute of each running stage.After the rest period, participants began the first stage of the experimental protocol (self-selected or lowered cadence). Upon completion of their first stage, participants rested for three minutes. After the first stage rest period, participants began their second stage (self-selected or lowered cadence). Upon completion of the second stage, participants rested for one minute and data collection ceased. Ratings of perceived exertion (RPE) were recorded at the completion of the self-selected and lowered cadence running stages. Running economy . For all statistical analyses, significance was set at ’s scale : <0.10, ormation .2, respiratory exchange ratio, RPE and ventilation separated by cadence condition are reported in t(15) = 6.573, p < 0.001). Physiological variables of VO2, t(16) = −1.856, p = 0.041, and respiratory exchange ratio, t(16) = −1.962, p = 0.034 were both significantly greater at the lowered cadence condition. There were no statistically significant differences between cadence conditions for the following variables: RPE, t(16) = −1.515, p = 0.075, and ventilation, t(16) = −1.495, p = 0.77.Mean data (± standard deviation (SD)) for cadence, VOt(16) = −2.017, p = 0.031), caloric unit cost (t(16) = −2.093, p = 0.027), power (t(16) = −6.349, p < 0.001), and form power (t(16) = −5.664, p < 0.001).Mean data (± SD) for running economy, caloric unit cost, power, and form power are reported in −1); however, the decrease in cadence was significantly less during the lowered cadence condition ; cadence and form power (r = −0.9 to −0.6). Values were statistically significant and positive for the following relationships: running economy and caloric unit cost (r = 0.9 to 1.0); running economy and power (r = 0.2 to 0.8); running economy and form power (r = 0.1 to 0.8); caloric unit cost and power (r = 0.2 to 0.8); caloric unit cost and form power (r = 0.1 to 0.8); power and form power (r = 0.5 to 0.9).r = 0.9 to 1.0); running economy and power (r = 0.2 to 0.8); running economy and form power (r = 0.2 to 0.8); caloric unit cost and power (r = 0.2 to 0.8); caloric unit cost and form power (r = 0.2 to 0.8); power and form power (r = 0.4 to 0.9). Correlation coefficient values (90% confidence limits) were statistically significant and negative for the following relationships: cadence and running economy (r = −0.8 to −0.1); cadence and caloric unit cost (r = −0.8 to −0.2); cadence and form power (r = −1.0 to −0.7). In general, power was positively related to running economy for both cadence conditions. Correlation scatterplots for running economy and power metrics are shown in r = 0.2 to 0.8; form power and running economy r = 0.1 to 0.8) and lowered cadence  conditions. Since the lower and upper confidence limits of the correlations were positive, an inference can be made that there is a clear, substantial relationship between the Stryd Summit’s power metrics and running economy [The Footpod represents a novel method of estimating running power. The purpose of this study was to determine the relationship between established measures of running economy  and Stryd’s proprietary power metrics . Positive correlations  were measured between running economy and Stryd’s power and form power for both self-selected cadence  pooling data from recreational and elite runners using the Stryd Pioneer chest-mounted running power meter. The authors measured a significant difference in metabolic cost while running on different surfaces, yet this did not elicit a difference in estimated power. The current study was able to measure a significant difference in metabolic cost (paired t-test statistic) by altering cadence and a concomitant significant increase in estimated power. Aubry, Power, and Burr  found a −1) was significantly lower (p < 0.001) than self-selected cadence (179.6 ± 8.4 strides·min−1). Since the relationship between cadence and running economy has been well established [−1·min−1), power , and form power . For the purposes of determining the efficacy of the Footpod, the ~4% decrease in cadence was enough to elicit a significant increase in running economy (~1%) and a significant increase (~5%) in form power. This study aimed to elicit a significant change in participant running economy and power by attempting to impose a 10% decrease in cadence. The actual decrease in cadence was only 3.9%, yet, one-way paired t-tests reported the lowered cadence  was smaller than ideal for correlation analysis. Although statistically significant correlations were determined for power and running economy, it is not possible to evaluate the strength of the relationship as 90% confidence intervals were large , these correlations should be viewed with caution as the corresponding rre large . Also, tp < 0.05 level, (p = 0.08), it is of interest that runners perceived the lowered cadence condition as a greater effort. The change in ventilation between cadence conditions was also nonsignificant (p = 0.77), but the mean difference of 2.2 L·min−1 greater ventilation with lower cadence suggest a higher physiological demand. Taking this all into account, lowering cadence from a self-selected value elicited poorer running economy, along with higher power and form power. While the change in RPE did not reach significance at a Cadence had negative correlations with form power and negative correlations with both measures of running economy. Previous studies have found a negative relationship between cadence and running economy ,24,26, aShould running power have a large positive correlation with running economy, it may be desirable for runners to monitor power values for improvement both acutely and longitudinally. Previous studies have used various strength-oriented interventions for improving running economy , and simThe present study suggests there is a positive relationship between power and form power, and running economy expressed in terms of oxygen cost and caloric unit cost. It may be that form power is more sensitive to changes in cadence and other biomechanical factors than running economy or caloric unit cost. This is logical since form power is based on mechanical measures from the inertial measurement unit of the Footpod. While target-lowered cadence values were not met, they were statistically less than self-selected cadence values. As expected, the lower cadence resulted in significantly poorer running economy and significantly greater power output at a given treadmill speed. The positive correlation between power and running economy suggests the Footpod may reflect changes in running economy from alterations in cadence yet is not clear if the Footpod is sufficiently accurate to account for differences in the running economy of competitive runners.


ObjectiveThis study aimed to assess the knowledge and attitudes of pregnant females in Al-Ahsa city, Kingdom of Saudi Arabia (KSA) toward hepatitis B virus infection.MethodsA cross-sectional study was done at the Maternity and Children’s Hospital, Al-Ahsa. A total of 422 of every third pregnant women were recruited from 6/12/2019 to 20/12/2019. Self-administered questionnaire was provided that contained three aspects: sociodemographic, perception and source of information about hepatitis B, and attitude toward hepatitis B infection. Analysis was performed using SPSS version 21 .ResultsA total of 422 pregnant women participated in this study with a response rate of 93.7%. Among them, 44.79% had a university degree or higher education level, about 82% had information about hepatitis B virus (HBV) during their pregnancy, 0.9% knew a person with HBV, 48.1% knew that hepatitis B is caused by virus, 72% knew that hepatitis B has vaccine, 41.9% knew that hepatitis B spreads via mother, 79.6% were willing to do hepatitis B test during pregnancy, 80.1% were willing to allow for kids’ vaccination against HBV, and 83.4% were willing to allow their kids for hepatitis B testing. There was a significant relationship between the level of education and the knowledge score. And there was a significant relationship between the level of education and attitudes score.ConclusionThere is insufficient knowledge among pregnant women regarding hepatitis B infection, while pregnant women showed remarkably positive attitudes regarding therapy and immunization. So, we highly recommend for awareness campaigns about viral hepatitis regarding means of transmission, and possible treatment options. Chronic hepatitis B virus (HBV) infections remain a major public health issue worldwide despite availability of effective vaccine and potent antiviral treatments . ChronicPerinatal transmission is one of the commonest modes of HBV transmission worldwide. This perinatal transmission of HBV leads to severe long-term sequelae . ChildreThe Global Advisory Group on the Expanded Program on Immunisation recommended that countries with a more than 2% prevalence of HBV carriers should add hepatitis B vaccine into their routine infant immunization schedules, a recommendation which was endorsed by the World Health Assembly . ConsequSeveral studies reported the prevalence of HBV in Saudi Arabia among the general public, school students, blood donors, health care workers, and pregnant women. It has shown that HBV infection in Saudi Arabia is acquired mainly through horizontal and vertical transmission similar to what is observed in other endemic countries. It has been reported that 5-10% of the population were infected with HBV -10. EpidPregnant women are vulnerable and if infected can transmit infection to infants, children, health workers during delivery as well as to sexual partners. Major risk factors identified in studies carried out among pregnant women and women of childbearing age include, level of education, history of blood transfusion, surgery, abortions, sexual transmitted infection, higher mean parity, early sexual debut, polygamy and higher numbers of sexual partners ,13.So, the level of awareness of the pregnant females about HBV infection plays an important role in HBV prevalence. We suspect that one of the explanations of this high HBV prevalence in eastern region of Saudi Arabia is deficient knowledge on infection with the HBV, especially regarding its prevention in the fertile female population. Hence, this study aimed to assess the knowledge and attitudes of pregnant females in Al-Ahsa city, Kingdom of Saudi Arabia (KSA) toward hepatitis B virus infection.A cross-sectional study was conducted in antenatal care clinics in Al-Ahsa region. The hospital that included was Maternity and Children’s Hospital Al-Ahsa. The study was carried out between 6/12/2019 and 20/12/2019. In this study, 422 pregnant women were included. The sampling technique of this study was systematic sampling of every third pregnant female visitor to the antenatal care clinic of the hospital who was included in the study based upon the two weeks visits to the clinics. Participants were included from four antenatal clinics of Maternity and Children’s Hospital Al-Ahsa based upon the number of data collection. The data were collected in this study by self-administered questionnaire which was adopted from a relevant literature to answer the study objective .The questionnaire was divided into three main parts: the first part was regarding sociodemographic data including age, occupation, education, and marital status, etc., while the second part was about perception and source of information about hepatitis B, and the third part was about attitude toward hepatitis B infection.Data were stored as database with no access except for those who were authorized. Then the data were coded, entered into Microsoft Excel sheet, and analysis was performed using Statistical Package for Social Sciences (SPSS) version 21 . Descriptive analysis was used for participants’ characteristics, Chi-square was used to compare participants’ attitude of HBV and the independent variables to determine the association, logistic regression was used to estimate the adjusted odd ratio (OR), and inferential statistics were represented by P value (<0.05) and confidence interval (Null hypothesis = 1 for OR).Of the 450 pregnant women who were approached, 422 agreed to participate with a response rate of 93.7%. The mean age of the participants was 30.89, while the majority of participants’ education levels were high school and university as shown in Figure Regarding the awareness data, about 82% of participants reported that they received information about HBV during their pregnancy, around 15.9% reported that they did not receive any information about it. Also, 18.5% said they know a person with HBV, while 76.5% said they do not know a person affected with HBV. Moreover, 0.9% reported to having HBV while 95.3% denied having HBV as shown in Table Educational level and awarenessFor Question 1 (Heard about hepatitis B?), one illiterate participant (100%) said ‘no’, eight elementary level participants (50%) said ‘no’ and eight (50%) said ‘yes’ from a total of 16 (100%) participants, 12 intermediate level participants (35.2%) said ‘no’ and 22 (64.8%) said ‘yes’, 144 high school level participants (82.7%) said ‘yes’ from a total of 174 (100%) participants, 166 university level participants (88.2%) said ‘yes’ out of 180 (100%) participants as shown in Figure Regarding the knowledge dataFor Question 1 (Hepatitis B caused by virus?), 203 (48.1%) of the participants said ‘yes’, 27 (6.4%) said ‘no’ and 190 (45%) said ‘I don’t know’ with a total of 420 participants and a missing of two participants. For Question 5 (Hepatitis B has vaccine?), 304 (72%) said ‘yes’, 13 (3.1%) said ‘no’ and 104 (24.6) said ‘I don’t know’ with a total of 421 participants and a missing of one participant. For Question 6 (Hepatitis B spreads via blood?), 200 (47.4%) said ‘yes’, 43 (10.2%) said ‘no’ and 172 (40.8%) said ‘I don’t know’ with a total of 415 participants and a missing of seven participants. For Question 7 (Hepatitis B spreads via sex?), 119 (28.2%) said ‘yes’, 102 (24.2%) said ‘no’ and 198 (46.9%) said ‘I don’t know’ with a total of 419 participants and missing of three participants. For Question 8 (Hepatitis B spreads via mother?), 177 (41.9%) said ‘yes’, 38 (9.0%) said ‘no’ and 206 (48.8%) said ‘I don’t know’ with a total of 421 participants and a missing of one participant. For Question 9 (Hepatitis B spreads via needle?), 187 (44.3%) said ‘yes’, 47 (11.1%) said ‘no’ and 185 (43.8%) said ‘I don’t know’ with a total of 419 (99.3%) participants and a missing of three (0.7) participants as shown in Table About the attitude dataFor Question 1 (Hepatitis B testing during pregnancy), 336 (79.6%) said ‘yes’, 48 (11.4%) said ‘no’ and 34 (8.1%) said ‘I don’t know’ with a total of 418 (99.1%) and a missing of four (0.9%) participants. For Question 2 (Allow kids for vaccine for HBV), 338 (80.1%) said ‘yes’, 40 (9.5%) said ‘no’ and 40 (9.5) said ‘I don’t know’ with a total of 418 (99.1%) and a missing of four (0.9%) participants. For Question 3 (Allow kids for antibodies for hepatitis B), 227 (53.8) said ‘yes’, 108 (25.6%) said ‘no’ and 78 (18.5%) said ‘I don’t know’, with a total of 413 (97.9%) and a missing of nine (2.1%) participants. For Question 4 (Having drugs to prevent HBV), 359 (85.1%) said ‘yes’, 22 (5.2%) said ‘no’ and 35 (8.3%) said ‘I don’t know’ with a total of 416 (98.6%) and a missing of six (1.4%) participants. For Question 5 (Allow kids for hepatitis B testing), 352 (83.4%) said ‘yes’, 28 (6.6%) said ‘no’ and 40 (9.5%) said ‘I don’t know’, with a total of 420 (99.5%) and a missing of two (0.5%) participants as shown in Table 2  = 29.619, p = .001. Also, a statistically significant relationship was found between education and some of HBV transmission modes like blood, and needles. Moreover, there was a statistically significant relationship between education and the willing of participants to let their newborn to receive an HBV vaccine of, X2  = 18.538, p = .047.A chi-square test of independence was performed to examine the relation between education level and knowledge about HBV vaccine. There was a statistically significant relationship between these variables, XThis study aimed to measure the knowledge and attitudes of pregnant females in Al-Ahsa city, KSA toward hepatitis B virus infection. As a result, we collected the responses of 422 participants, and recorded and compared demographical, awareness, and health attitudes-related data.This study revealed that there is a significant relationship between the level of education and the knowledge score, that is to say, the number of questions answered correctly. This correlation was noted when comparing the level of education and attitudes score, i.e., positive attitudes toward vaccination and treatment, where those with higher education level were more likely to answer correctly. This is consistent with the current literature as multiple studies revealed that low health literacy was observed among those with high school or lower levels of education, and among those, the rate of disease and poor health was higher ,16. On tOverall, we observed a lack of knowledge regarding viral transmission. For instance, only 47% of participants recognized that HBV is transmitted by blood compared to 85.8% in Vietnam , 46% to Up to 40% of the cases of HBV are transmitted perinatally, and this particular type of acquisition of the disease is associated with significantly higher infection-related morbidity and mortality ,24. FurtA 2012 study revealed that treatment with Telbivudine during pregnancy significantly improved disease outcomes regarding fetal transmission, viral load, and liver enzymes normalization . FurtherWe found a high rate of positive attitudes regarding vaccination. That is to say, 80.1% of the participants were likely to allow their children’s vaccination. This attitude is similar to that of Vietnam and China, 86% and 89%, respectively ,28. ThisAs speculated, the present study revealed that the degree of knowledge regarding hepatitis B infection is poor among the subjects involved. On the other hand, the participants showed remarkably positive attitudes regarding therapy and immunization. Thus, we highly recommend awareness campaigns on viral hepatitis regarding means of transmission, and possible treatment options, and also emphasizing the importance of vaccination to newborns as well as to nonimmune mothers postpartum.


The ability of crops to overcome Al toxicity varies among crop species and cultivars. Here, we report for a first time the simultaneous responses of photosystem II (PSII) and photosystem I (PSI) to Al3+ phytotoxicity. The responses of PSII and PSI in the durum wheat (Triticum turgidum L. cv. ‘Appulo E’) and the triticale  were evaluated by chlorophyll fluorescence quenching analysis and reflection spectroscopy respectively, under control  and 148 μM Al  conditions. During control growth conditions the high activity of PSII in ‘Appulo E’ led to a rather higher electron flow to PSI, which induced a higher PSI excitation pressure in ‘Appulo E’ than in ‘Dada’ that presented a lower PSII activity. However, under 148 μM Al the triticale ‘Dada’ presented a lower PSII and PSI excitation pressure than ‘Appulo E’. In conclusion, both photosystems of ‘Dada’ displayed a superior performance than ‘Appulo E’ under Al exposure, while in both cultivars PSII was more affected than PSI from Al3+ phytotoxicity.Aluminium (Al) the most abundant metal in the earth’s crust is toxic in acid soils (pH < 5.5) mainly in the ionic form of Al Altho at pH < .5 or as  at pH > .5 [3+, dynthesis ,18,19,20ynthesis ,22,23, rynthesis ,18,24,253+, that is toxic to most plants at relatively low concentrations, it is the main limiting factor in the world’s arable non-irrigated crop production to over 40% [3+ toxicity effects on photosynthesis has the potential to increase cereal yields by understanding the factors that influence negatively the molecular mechanism of absorbed light energy utilization.In the form of the trivalent cation Alover 40% ,27. Withover 40% . Consequover 40% . Since p2O to NADP+ through the formation of strong reductants, and of a proton gradient that is used to drive ATP synthesis [Photosynthesis is the process by which organisms convert the absorbed solar energy into chemical energy via photosystem II (PSII) and photosystem I (PSI). Light reactions of photosynthesis are driven by the cooperation of PSII and PSI that work coordinately to transfer photosynthetic electrons efficiently and are located in the photosynthetic membranes of chloroplasts, the thylakoids ,30. Chloynthesis ,34.A) and the quinone B (QB) [3+ concentrations resulted in a reduction of the energy transfer from light harvesting complex (LHCI) to RCs of PSI, followed by an impairment of PSI RCs and electron transfer of PSI [Aluminium toxicity has been shown to reduce the photochemical efficiency of PSII in several plant species ,38,39,40e B (QB) , and cloe B (QB) ,38,41,42e B (QB) ,42,43). r of PSI .Absorption of more light than what can be used to drive photosynthesis, causes photodamage to the photosynthetic apparatus and the light-processing structures, primarily PSII, resulting in a decrease in the photosynthetic activity causing reduced plant growth and productivity ,46. AmonChlorophyll fluorescence measurements and in particular measurements of PSII excitation pressure, that is the redox state of the plastoquinone (PQ) pool, have been proposed as a sensitive bio-indicator to measure Al effects on plants . Chlorop3+ toxicity, in the durum wheat (Triticum turgidum L. cv. ‘Appulo E’) and the triticale . Triticale is considered as more Al-tolerant species than durum wheat (Triticum turgidum); this difference is largely attributed to its superior ability to grow better under acidic conditions [A number of studies as already mentioned have examined the functioning of PSII under Al toxicity ,41,42,43nditions .Triticum turgidum L. cv. ‘Appulo E’) and the triticale  were used to compare the tolerance of the two photosystems to Al toxicity. Seeds obtained from the Institute of Plant Breeding and Genetic Resources, Thermi, Greece, germinated at 22 ± 1 °C for 2 d in the dark. The germinated seeds were transferred in a growth chamber and mounted on nylon-mesh floats on plastic vessels filled with nutrient solution at pH 6.5 [Durum wheat (t pH 6.5 . The seet pH 6.5 .4)212H2O for 14 days. Al-containing pots (nutrient solution plus 148 μM Al) were acidified initially to pH 4.5 with 1N HCl [3+ activities were calculated to be 16.8 μM [Al was supplied at 148 μM as KAl content, as described previously [−1 fresh weight.The level of lipid peroxidation of controls and 14-days Aleviously , accordieviously . The cona fluorescence (Fo) was measured by application of a weak modulated light beam (L1) followed by a saturating light pulse (L2) to measure maximal chlorophyll a fluorescence (Fm) in the dark adapted (20 min) samples. Then, by application of the actinic light (LA) and saturating light pulses, maximum chlorophyll a fluorescence in the light (Fm′) was measured, while to assess steady-state photosynthesis (Fs) values, the actinic light (LA) alone was applied. Minimum chlorophyll a fluorescence in the light (Fo′) was measured immediately after turning off the actinic light (LA) (Chlorophyll a fluorescence was measured in dark-adapted (20 min) leaf samples, using a pulse amplitude modulation fluorometer , as described before ,43. Firsght (LA) . The cal700+ measuring system was employed to monitor light-induced changes in leaf absorbance at around 820 nm according to Havaux et al. [1) was calculated as: B1 = ΔS/(ΔS)max = (Rfr − R’)/(Rfr − R).A Hansatech Px et al. , as descx et al. . The frat-test. Differences were considered statistically significant at p < 0.05.Data are presented as the mean ± SD. Statistical analysis was performed using the Student’s PSΙΙ) (NPQ) (NO) (PSΙΙ and lower ΦNPQ (NO (that did not differ from control conditions)  a and lowΙ) (NPQ) b, with nPQ) (NO) a, comparwer ΦNPQ , but higditions) a, than tnditions a.The triticale ‘Dada’ had higher non-photochemical fluorescence quenching (NPQ) under control growth conditions (pH 6.5) than the durum wheat ‘Appulo E’ but under 148 μM Al at pH 4.5 it was the reverse b.qp) (qp) (Under control growth conditions the durum wheat ‘Appulo E’ presented higher electron transport rate (ETR) a and a mqp) b, than tqp) a, but thqp) (qp) b with duFv/Fm) under normal growth conditions was higher in the durum wheat ‘Appulo E’, but under 148 μM Al it was higher in the triticale ‘Dada’ (Fv’/Fm’) was similar under control growth conditions  content and expressed as nmol (MDA) gn ‘Dada’ .1) or PSI excitation pressure under both control growth conditions and Al exposure was higher in ‘Appulo E’  precipitated with other elements and essentially non-toxic to plant growth; (c) different hydroxyl Al monomers also non-toxic to roots [3+ in solution [3+ activities in the nutrient solutions were calculated to be 16.8 μM.In a hydroponic solution as summarized by Famoso et al. , Al may to roots ; or (d) solution . In our solution , the frePSII) under control growth conditions (NPQ) (NO) in both ‘Dada’ and ‘Appulo E’ (NPQ) in ‘Appulo E’ (PSII) (NO) compared to ‘Dada’ (The significant lower quantum efficiency of PSII photochemistry in ‘Dada’ (Φnditions a was comns (NPQ) b, that wppulo E’ a. Under ppulo E’ b not onl’ (PSII) a, but evo ‘Dada’ a.Fv/Fm value  decreased significantly (1O2) production. The quantum yield of non-regulated non-photochemical energy loss (ΦNO) consists of chlorophyll fluorescence internal conversions and intersystem crossing, which leads to the formation of 1O2 via the triplet state of chlorophyll (3chl*) [The high excitation pressure in PSII (1 − qp) under Al exposure, observed in ‘Appulo E’ , indicatficantly a, thus e (3chl*) ,76,77. T (3chl*) ,79,80,813+ phytotoxicity in both cultivars.In conclusion, we confirmed that the triticale cv. ‘Dada’ was more tolerant to Al phytotoxicity than durum wheat ‘Appulo E’, as reflected by the better PSII functionality under Al acidic conditions. However, under normal growth conditions , durum wheat ‘Appulo E’ displayed a better PSII functionality. Yet, under Al exposure, PSII was more affected than PSI from Al


The stability for a class of generalized Minty variational-hemivariational inequalities has been considered in reflexive Banach spaces. We demonstrate the equivalent characterizations of the generalized Minty variational-hemivariational inequality. A stability result is presented for the generalized Minty variational-hemivariational inequality with  X be a real Banach space with its dual J. Recall that the variational-hemivariational inequality [Let mentclass2pt{minimUnder some suitable conditions,  is equivLet It is well known that the variational inequality theory has wide applications in finance, economics, transportation, optimization, operations research, and engineering sciences, see –25. In 2Inspired and motivated by the above work of Zhong and Huang , we invend Huang  from theX be a real reflexive Banach space. Let X. Clarke’s generalized directional derivative of J at x in the direction y, denoted by f and Clarke’s generalized gradient of J (see [Let f J (see ), respec(0), see .LetXbe a Banach space andJbe a locally Lipschitz functional on X. Then we have: (i)The functionis finite, convex, positively homogeneous, and subadditive;(ii)is upper semicontinuous and is Lipschitz continuous on the second variable;(iii)(iv)is a nonempty, convex, bounded, and weak∗-compact subset of(v)For every(vi)The graph ofis closed intopology, wheredenotes the spaceequipped with weak∗topology, i.e., ifandare sequences such thatinXandweakly∗inthenK be a nonempty, closed, and convex subset of X. Let Y be a topological space. We use K which is defined by K, denoted by K is defined by K is defined as Let f is defined byLet X converging weakly to x and It is known thatn 2.5 in , we dedu(i)upper semicontinuous at (ii)lower semicontinuous at A set-valued mapping F is said to be continuous at F is continuous on K iff it is both upper and lower semicontinuous at every point of K.F is said to be (i)K iff, for all monotone on (ii)K iff, for all pseudomonotone on (iii)K with respect to a set F and K for every stably pseudomonotone on (iv)f-pseudomonotone on K iff, for all (v)K iff, for all The mapping Let ψ is said to be bi-sequentially weakly lower semicontinuous iff, for any sequences Let Letbe a nonempty, closed, and convex set withThen there exists no sequencesatisfyingandIfKis a cone, then there exists no sequencewithsatisfyingLetbe a nonempty, closed, and convex set withThen there exists no sequencewithsatisfyingLetbe a metric space andbe a given point. Letbe a set-valued mapping with nonempty values, and letLbe upper semicontinuous atThen there exists a neighborhoodUofsuch thatfor allLetEbe a Hausdorff topological vector space andbe a nonempty and convex set. Letbe a set-valued mapping satisfying the following conditions: (i)Gis a KKM mapping, i.e., for every finite subsetAofK, (ii)is closed inEfor every(iii)is compact inEfor someThenD of In this section, we introduce several characterizations for the solution set Let SupposeThenΦ is a proper, convex, and lower semicontinuous function and so X.Define a function D is nonempty. According to formula (2.29) in [Let 2.29) in , \documeConversely, if SupposeThenIf It is known that if m 3.1 in  is a genm 3.1 in .Suppose the following statements hold: (i)Dis nonempty and bounded;(ii)(iii)There exists a bounded setsuch that, for everythere exists somesatisfyingThen (i)⇒(ii). (ii)⇒(iii) ifhas nonempty interior. (iii)⇒(i) ifFispseudomonotone onK.The relationship (i)⇒(ii) can be deduced from Theorem d. Then Next, we first prove that (ii)⇒(iii). If (iii) does not hold, then there exists a sequence her with  implies F is K. Indeed, let K. In fact, for any f and the Lipschitz continuity of It remains to prove that (iii) implies (i) under the assumption that F, we getG is a KKM mapping.Next we prove that C is a bounded, closed, and convex . Let K, and let M is weakly compact and convex. Let M and Assume that Let Suppose the following statements hold: (i)Dis nonempty and bounded;(ii)(iii)There exists a bounded setsuch that, for everythere exists somesatisfyingThen (i)⇒(ii). (ii)⇒(iii) ifhas nonempty interior. (iii)⇒(i) ifFispseudomonotone onK.It is known that if (iii) in  is a gen(iii) in .In this section, we will establish the stability of solution sets for the generalized Minty variational-hemivariational inequality Let Ifthen there exists a neighborhoodofwithsuch thatfor allL and Lemma n and so n. Since L, there exists F, there exists Assume that the conclusion does not hold. Then there exists a sequence Ifthen there exists a neighborhoodofwithsuch thatfor allWhenever is case,  and for eachthe mappingispseudomonotone onM;(ii)the solution set ofis nonempty and bounded.Then there exists a neighborhoodofwithsuch that, for everythe solution set ofis nonempty and bounded. Moreover, iffis continuous onandis continuous, thenω-whereandare the solution sets ofandrespectively.F is By Theorem ω-ω-n. By the definition of n. From the above discussion, we need only to prove that ω-x. We claim that there exists X and hence weakly closed. Next we prove that L is lower semicontinuous and F is lower semicontinuous, it follows that there exists a sequence of elements f is continuous on Next, we prove that Assume that all the conditions of Corollary are satisfied. Suppose that(i)for eachthe mappingisf-pseudomonotone onM;(ii)the solution set ofis nonempty and bounded.Then there exists a neighborhoodofwithsuch that, for everythe solution set ofis nonempty and bounded. Moreover, iffis continuous onthenω-whereandare the solution sets ofandrespectively.F in the first variable reduces to the f-pseudomonotonicity of F in the first variable. Utilizing Theorem Whenever It is known that if orem 4.2  generali=f=0, He  obtained=f=0, He  when eit=f=0, He ). Theref=f=0, He  is a gen=f=0, He .In the following, as an application of Theorem If F is single-valued, then  x is a solution of ∗ open neighborhood of F is upper hemicontinuous, setting f, we havex is a solution of (i) The conclusion is obvious. Now we prove (ii). Suppose that IfFisf-pseudomonotone onK, then every solution ofsolvesIfFis upper hemicontinuous onKwith nonempty values, then every solution ofsolves(i) F reduces to the f-pseudomonotonicity of F. Utilizing Lemma Whenever LetKbe a nonempty, closed, and convex subset in a reflexive Banach spaceX, be a convex and lower semicontinuous function, andbe a locally Lipschitz functional. Suppose thatFis upper hemicontinuous andpseudomonotone onKwith nonempty values. Consider the following statements: (i)the solution set ofis nonempty and bounded;(ii)the solution set ofis nonempty and bounded;(iii)Then (i)⇔(ii) and (ii)⇒(iii); moreover, ifthen (iii)⇒(ii) and hence they all are equivalent.F, the equivalence of (i) and (ii) is stated in Lemma Under the assumptions of LetKbe a nonempty, closed, and convex subset in a reflexive Banach spaceXandbe a convex and lower semicontinuous function. Suppose thatFis upper hemicontinuous andf-pseudomonotone onKwith nonempty values. Consider the following statements: (i)the solution set ofis nonempty and bounded;(ii)the solution set ofis nonempty and bounded;(iii)Then (i)⇔(ii) and (ii)⇒(iii); moreover, ifthen (iii)⇒(ii) and hence they all are equivalent.F reduces to the f-pseudomonotonicity of F, and statements (i), (ii), and (iii) in Lemma Whenever It is known that if a 4.2 in  reduces a 4.2 in . Therefoa 4.2 in  generalia 4.2 in  from theFrom Theorem Assume that all the conditions of Theorem 4.1 are satisfied. Suppose that(i)for eachthe mappingis upper hemicontinuous andpseudomonotone onM;(ii)the solution set ofis nonempty and bounded.Then there exists a neighborhoodofwithsuch that, for everythe solution set ofis nonempty and bounded. Moreover, iffis continuous onandis continuous, thenω-whereandare the solution sets ofandrespectively.F is upper hemicontinuous with nonempty values and M, it follows from Lemma Since