In addition, the 244Pu/239Pu atomic ratio for the global falloutperiod between 1958 and 1965 is suggested to be (7.94 ± 0.31)·10–5. In the bottom layer of the sediment, dated 1953–1957,we detected a higher average 244Pu/239Pu ratioof (1.51 ± 0.11)·10–4, indicating thepossible impact of the first US thermonuclear tests (1952–1958).Plutonium distributionwas studied in an undisturbedsediment coresampled from the Tvären bay in the vicinity of the Studsviknuclear facility in Sweden. The complete analysis, including minorisotopes, of the Pu isotope composition  by analyzing a sedimentcore. The  Swedenwas no exception, and the Swedish nuclear program was initiated in1945. In the beginning, Sweden was working with the idea of usingheavy water reactors to produce nuclear power and Pu material forweapons. The nuclear program was first controlled by one state-ownedinstitution but later divided into two major organizations, the SwedishNational Defense Research Institute (FOA) and AB Atomenergi (AE).In 1959, AE started a large-scale nuclear research institute about100 km South of Stockholm, later in this paper referred to as Studsvik.Different research programs were conducted within this facility, includingwork on Pu separation and irradiated fuel. The Swedish nuclear weaponprogram, undertaken mainly by FOA, was decommissioned in 1972 whenSweden joined the Non-Proliferation Treaty (NPT). Equipment from theFOA facility was later transported to Studsvik for decontaminationand prepared for final storage.3 TheSwedish Radiation Safety Authority (SSM) regulates permissible levelsof these aquatic releases. Liquid discharges have been documentedsince the beginning of the operations in 1959, although the compositionof the releases had not been well described before the 1970s. Somegamma emitters and total alpha activities have been reported sincethe operation’s beginning, and since 2002, the reported radionuclidesare extended to include, for example, 238Pu and 239+240Pu. However, the release data do not include the minor Pu isotopeslike 241,242,244Pu or separate information on 239Pu and 240Pu. Nowadays, there are two operational dischargepoints with regular higher radioactive releases in the southern partof the Tvären bay and lower radioactive releases close to theStudsvik facility .Atmospheric nuclear weapon tests were conducted between 1945 and 1980.Radionuclide fallout from these tests constitutes a global input thathas covered the Earth’s surface, also called global fallout(GF). The GF peaked around in 1963 with maximum deposition in theNorthern Hemisphere (30–70° N) and is considered the mainPu input to the general environment.5 Inaddition, there are other sources with a local/regional impact, forexample, control releases from nuclear reprocessing facilities  and fallout coming from the Chernobyl accident in1986.7 The Pu isotopic composition varies accordingto the production mode, providing a unique “fingerprint”of different sources and becoming a key tool for many environmentalapplications (238Pu (t1/2 = 87.7 y), 239Pu (t1/2 =2.411 × 104 y), and 240Pu (t1/2 = 6.561 × 103 y), where 239Pu and 240Pu often are given as combined alpha activities,that is, 239+240Pu. However, more scarce data are availablefor the presence of minor Pu isotopes in the environment, that is, 241Pu (t1/2 = 14.2y), 242Pu (t1/2 = 3.75 ×105y), and 244Pu (t1/2 = 8.13 × 107 y). Thus, open questionsabout the origin of Pu could be solved by studying the complete Puisotopic composition.Tvären, as a part of the Baltic Sea, hasreceived Pu fromother sources than Studsvik. Naturally occurring Pu can be considerednegligible in the general environment.ications 1. The mo244Pu. Although this radionuclide has beenprimarily known for its applications in astrophysics,14 anthropogenic 244Pu could be an innovative tool due tothe recently developed techniques to assess this radionuclide at environmentallevels. 244Pu can only be produced in thermonuclear explosions,where the fast neutron flux triggers its production after successiveneutron capture reactions on 239Pu.15 Such processes are very unlikely in civil nuclear reactors due tothe low neutron flux and the production of the short-lived 243Pu (t1/2 = 4.956 h) by neutron captureof 242Pu, which decays in the reactor before 244Pu is produced.16 This unique aspect of 244Pu makes it ideal to differentiate Pu originating from thermonuclearexplosions and other Pu sources, for example, fission weapon falloutand discharges from the nuclear fuel cycle. Several studies have beenreported17 establishing the first 244Pu/239Pu ratiosfor different sources .In this work, we used an undisturbedand facility 1.We use22.1210Pb (210Po). The model was fine-tuned by usinga clearly defined time marker in the sediment profile, that is, stablePb concentration that had a maximum in 1970, and the results havebeen validated by using additional time markers according to the Studsvikdischarge history records for 60Co and 152Eu. Thesedata will be published elsewhere.18A 46-cm-long sediment core,C3, was sampled in the deepest part (79 m) of the Tvären bay1 on the 2.219 Two groups of samples were processed: (i) the first setof samples (group 1) was intended for alpha spectrometry analysisand they were initially spiked with 242Pu to quantify thefinal Pu concentrations, and (ii) the second set of samples (group2) were processed to study the 240Pu/239Pu, 241Pu/239Pu, 242Pu/239Pu,and 244Pu/239Pu atom ratios by accelerator massspectrometry (AMS) and no 242Pu spike was added. Incineratedsediment samples were leached for 2 to 3 days using aqua regia, andactinides were coprecipitated with Fe(OH)3 from the dissolvedfraction. The Fe(OH)3 precipitate was dissolved in concentratedHCl and diluted with MQ H2O, and K2S2O5 was added to reduce Pu to Pu(III). Additionally, ascorbicacid was added to assure complete Fe reduction. Reduced forms of Puwere coprecipitated on Fe(OH)2, dried, and dissolved in8 M HNO3. The separation was performed on TEVA resin cartridgescoupled to a vacuum box. The cartridge was rinsed with 8 M HNO3 and 1 M HNO3 (Am and U elution) and 9 M HCl (Thelution). The Pu fraction was eluted using 2 M HCl/0.1 M NH2OH·HCl and, after decomposition, prepared for electrodepositionon stainless-steel discs from Na2SO4/H2SO4 solution adjusted to pH 2.1–2.4 with NH3·H2O.20 Finally,after alpha spectrometric measurements, samples were adapted for theiranalysis by AMS. Discs were leached in 3 M HNO3, and Puwas coprecipitated as Fe(OH)3 after adding 1 mg of Fe(III).Precipitates were dried and incinerated at 650 °C, mixed with3 mg of Nb powder, and pressed into aluminum cathodes.21 See Section S1 fromthe Supporting Information for more details.Beforeanalysis, all subsamples were again dried overnight at 80 °Cto ensure that possible adsorbed water during storage was removed.Sediment subsamples were calcined at a low temperature, 450 °C,to prevent the formation of refractory Pu species.2.3228Th in the 238Pu regionof interest using the 224Ra alpha peak for calculating 228Th. 238Pu and 239+240Pu measurementswere performed on a 1024-channel Alpha Analyst Canberra alpha spectrometer.The discs were measured between 15 and 33 days. MDA  values were 0.09 mBq·g–1 for 239+240Pu and 0.08 mBq·g–1 for 238Pu for 2 weeks measurements calculated using therevised Currie formula.22 The QA/QC ofthe method was controlled using the IAEA-135 reference material. See Section S1 from the Supporting Information formore details.The Pu discs were storedfor 2 weeks before the alpha spectrometric analysis to enable correctionsfor any remaining 240Pu/239Pu, 241Pu/239Pu, 242Pu/239Pu’, and 244Pu/239Pu isotopic ratio determinations were carriedout using the compact 1 MV AMS system at the CNA , following the technique for Pu measurementsdescribed in previous studies.23 For the control ofthe measurement stability and the necessary standard correction ofthe isotopic ratios, the ColPuS standard24 was also analyzed. Furthermore, instrumental  and procedural blanks were includedin the measurement sequence to control the background from the AMSmeasurement and from the laboratory . An additional correction was included in the case of 241Pu to subtract the in-grown 241Am on the overalldetected 241 mass counts. For this, the elapsed time between the samplepreparation and the AMS analysis was taken into account and a 10%higher production of AmO– ions was considered inthe Cs sputter ion source compared to that of PuO–.25 Thus, between 0.5 and 16% of the overalldetected 241 mass counts were subtracted following this proxy .33.1238Pu (α-spectrometry)and 239Pu, 240Pu, 241Pu, 242Pu, and 244Pu (AMS) for the studied sediment core aregiven in 238Pu and 241Pu have partially decayedsince their initial deposition, two depth profiles are shown in thosecases: one decay corrected to the sampling date (2016-10-13) and onedecay corrected to the date of the layer according to the 210Pb dating method. Additionally, the overall detonated yield duringthe atmospheric testing period (1945–1980) is shown to comparethe plutonium isotope concentrations with the intensity of the historicalnuclear tests. The 238Pu/239+240Pu activityratios and 240Pu/239Pu, 241Pu/239Pu, 242Pu/239Pu, and 244Pu/239Pu atomic ratios are shown in Section S1 and Figure S1).Concentrations depth profilesof 2 and (802.5 ± 8.1) Bq/m2 for 238Pu and 239+240Pu, respectively.These values are significantly higher than the previously reportedinventories from different stations along the Baltic Sea, rangingfrom (63 ± 1) to (159 ± 3) Bq/m2 for 239+240Pu and from (2.8 ± 0.1) to (7.8 ± 0.3) Bq/m2 for 238Pu.26 For the remainingPu isotopes measured by AMS, atomic inventories of (579.7 ± 6.4)·1012 atoms/m2 for 239Pu, (83.18 ±0.91)·1012 atoms/m2 for 240Pu,(1.685 ± 0.026)·1012 atoms/m2 for 241Pu, and (1.793 ± 0.051)·1010 atoms/m2 for 244Pu have been obtained.The Pu activity inventory at thesampling locationhas been estimatedto be (383.5 ± 4.3) Bq/m44.1244Pu is produced only in the thermonuclearexplosions, no 244Pu is expected to be present in the Studsvikdischarges or in any other sources in the vicinity of the samplingsite, 244Pu results are suitable to calculate the GF Pufraction.Pu discharges from the Studsvik nuclear researchfacility started in 1959, which coincides with the most intensivenuclear test period 2g. Due t27The atmospheric nuclear testing can be divided intotwo periods according to the fission yield and the number of explosionsfor the most powerful tests (>4 Mt). Thus, the earlier thermonuclearweapons testing phase (1952–1958) was dominated by the U.S.program (from now on the first GF period), whereas the later yearsup the Treaty Banning Nuclear Weapon Tests in the Atmosphere in 1963 were mostly dominated bythe USSR tests, with the global deposition peaking in 1963.244Pu concentrations in thesediment core agree withthis pattern  is observed . Knowing the Pu isotopic ratios, itis possible to identify the sediment layers that are only affectedby the GF. Obvious deviations from the expected Pu characteristicvalues from the GF ·10–4, and (3.70 ± 0.13)·10–3 for 240Pu/239Pu, 241Pu/239Pu, and 242Pu/239Pu, respectively. These ratios are in goodagreement with the previously reported characteristic integrated GFratios ·10–5 derived from these sediment layerscan be considered representative of the GF 244Pu/239Pu ratio in the second GF period. In a previous work,9244Pu/239Pu ratios were reportedfrom two soil samples collected at Salzburg (Austria), only affectedby GF. The reported values of (6.35 ± 0.11)·10–5 and (5.09 ± 0.16)·10–5 are in reasonableagreement with our GF value considering the scarcity of the resultsand being integrated values for soil samples from a different latitude.To the best of our knowledge, these are the only reported values regardingthe 244Pu/239Pu signal from GF.9 For further discussion, our derived 244Pu/239Pu characteristic atom ratio, that is, (7.94 ± 0.31)·10–5, is considered the most representative for the secondGF period and from our sediment core since the layer dated 1959 tothe top layer. pattern 2f. A 244m the GF 1 are obsobserved S2. KnowiFratios 2, sothi244Pu/239Pu results,the contributionof 239Pu from GF in the sampled core can be calculatedusing a two-end-member linear mixing model, as previously used forthe other Pu isotopic ratios.31 Assumingonly two sources, that is, GF and Studsvik, and considering that no 244Pu released from Studsvik, the model is simplified, andthe 239Pu GF fraction is given byRSample isthe measured 244Pu/239Pu atom ratio in the sample, RGF is the 244Pu/239Pucharacteristic ratio for GF, that is, (7.94 ± 0.31)·10–5, and 239PuGF is the fractionof 239Pu originating from GF. The corresponding GF fractionsfor other Pu isotopes  can becalculated using the 239PuGF fraction and thecharacteristic Pu isotopic ratios for the GF , with a total contribution ranging fromabout 50% in the case of 240Pu to about 97% for 238Pu.From the r the GF 2. The re4.2244Pu/239Pu atomicratios in the three deepest sediment layers dated between 1953 and1957. Remarkably, high 244Pu/239Pu atom ratios(1.51 ± 0.11)·10–4 were measured duringthis period, clearly deviating from the measured ratio of (7.94 ±0.31)·10–5 observed in sediment layers depositedbetween 1959 and 1964, that is, when GF showed a maximum deposition,as discussed in 244Pu concentration is characterizedby a different depth distribution from that of the other Pu isotopesin layers dated from 1953 to 1958 (S2), whichsuggests an enhanced production of 244Pu during this nucleartest period. 244Pu/239Pu and the other Pu atom ratios with a color code indicating thesediment layer’s computed age. In addition, we have superimposedcharacteristic atom ratios reported for other Pu sources  is (4.81 ±0.20)·10–3, a value slightly above the averageGF ratio computed for sediment layers dated 1959–1964 . This different patternis confirmed by the 244Pu/239Pu isotopic ratios,providing a more substantial indication of the influence of the thermonucleartest signal within the three deepest sediment layers dated between1953 and 1958. Within this period, USA’s first thermonucleartests were performed at the Marshall Islands in the Pacific Ocean:Ivy Mike  and Operation Castle. The measured 244Pu/239Pu atom ratio in the three deepest sediment layers (1.51 ± 0.11)·10–4 approaches the reported ratios from samples collectedat the Marshall Islands. 244Pu/239Pu ratiosranging from 2.5×10–4 to 5.7× 10–4 have been reported in sediment and soil samples from the Bikiniatoll ,15 and 244Pu/239Pu ratiosmeasured in soil samples collected at Bikar atoll  are in the(2.1–3.6)·10–4 range.11 The highest 244Pu/239Pu ratio hasbeen reported in airborne debris from Ivy Mike detonation,10 that is, (11.8 ± 0.7)·10–4. Radioactive aerosols are characterized by the stratospheric residencetime between 1 and 5 years, especially when attached to fine aerosolparticles (<0.02 μm diameter).33 The exchangebetween the stratospheric and tropospheric air masses occurs mainlyduring the late spring when the rising hot air and cold stratosphericair masses change place.33 1952’sand 1954’s high-yield thermonuclear detonations were not theonly Pu source in the stratosphere at the time. Multiple low-yieldtests were performed at the Pacific and Semipalatinsk test sites introducing 239,240,241,242Pu isotopes mainly locally and to the troposphere.Still, some of the debris reached the stratosphere in specific detonationconditions.33 Therefore, the obtained 244Pu/239Pu atom ratios in this work are expectedto be a mixture of Pu signatures of the earliest thermonuclear andpure fission tests. As a result, the data for close-in fallout probablydiffer substantially from the worldwide GF signal far from the detonationsite. Likewise, the 240Pu/239Pu ratio obtainedfrom the three deepest samples in this work .34 to 1958 2 and S2, sources 1 in the his work 2 is lowehis work 1. Furthe4.3244Pu, the GF input is unraveledin our sediment core, as discussed in 137Cs has already been discussedin a previous study.18 However, the Chernobylfallout for refractory elements such as Pu was negligible and unevenlydistributed compared to that for 137Cs. The uneven distributionof the Chernobyl-derived 137Cs and Pu in the Baltic Seasediments was shown in a previous study, where multiple cores fromthe Swedish coast of the Baltic Sea were analyzed.26 As seen in –3, and (9.55 ± 0.45)·10–3 for 240Pu/239Pu, 241Pu/239Pu, and 242Pu/239Pu, respectively, and 1.295 ± 0.045for the 238Pu/239+240Pu activity ratio. Theseresults are incompatible with the characteristic isotopic ratios expectedfor the Chernobyl fallout .These 240Pu/239Pu atom ratios prevailed forseveral years in the radioactive effluents from Studsvik. At the beginningof the 1970s, Sweden abandoned the idea of constructing and manufacturingnuclear weapons and using heavy water reactors. The Studsvik researchfacility continued to perform fuel tests with conventional fuel forlight water reactors. One of the international projects completedin Studsvik in the 1970s was the Studsvik Inter Ramp Project, withthe primary objective of investigating failure propensity and characteristicsof 20 unpressurized BWR  fuel rods.37 This paradigmatic political and technical changeis clearly visible in the Pu atom ratio depth profiles, 238Pu activities, and 238Pu/239+240Pu activityratios . During this period, totaldischarges of 15 and 50 MBq were reported for 239+240Puand 238Pu, respectively. It must be mentioned that thedischarge data are not provided with uncertainty estimates, and insome cases, the reported 238Pu/239+240Pu activityratios in liquid discharges were significantly higher than in theC3 sediment core as well as in the other cores collected from thevicinity of the discharge pipeline. These differences may partly beexplained by uncertainties in the methodology used to monitor theliquid effluents by the facility. Only a small fraction  of the discharged volume is analyzed, and homogeneity of thefluid is assumed,18 which may not alwaysbe valid in the case of particle reactive Pu. In addition, the determinationof 238Pu by alpha spectrometry can be biased more easilythan the determination of 239,240Pu due to interferenceof other radionuclides with similar energy to that of 238Pu . We have no information if the Studsvik laboratory applies acorrection for this in their calculations or if the radiochemicalseparation method is applied in their analysis of the discharged water.For that reason, we have only used the information of the 239,240Pu in our evaluation of the Pu discharge history. Based on the availabledata, we can calculate the site-specific transfer fraction FS of the Pu released by Studsvik that is finallyaccumulated in the sediment at the sampling site. It demands comparisonof the Pu inventory after subtracting the GF contribution for thoselayers dated from 2002 to 2016  to the discharge data given by Studsvikfor the same period. We assume that FS has been constant during all the years Studsvik has performed aquaticdischarges of Pu. Using the obtained FS value, that is, (3.5 ± 1.0)·10–6/m2, Pu discharges from Studsvik can be estimated from all Puisotopes as followsxPuSediment is the measured Pu concentration per square meterin the sediment layer and xPuGF is the layer-specific calculated GF fraction  MBq of 239Pu, (35.1 ± 2.6)MBq of 240Pu, (438 ± 30) MBq of 241Pu,and (27.3 ± 2.1) kBq of 242Pu equal to a total aquaticdischarge of Pu of 626 MBq (Table S1).Those estimated values are about 4–7 orders of magnitude lowerthan the reported liquid releases by the main European Nuclear Reprocessingfacilities .39 Thus, Pu Studsvik releases are expected to impact locally, but dueto the Baltic Sea’s semienclosed location, it should not affectthe global marine environment.The earliest reported Studsvik discharges were scarceor even nonexisting for individual Pu isotopes. However, since 2002,some Pu-isotopic-specific data have been published with the annual  to 2016S4. Durin to 2016S4. Durin


Our results support that MPH derivatives may represent a novel class of αSyn clearing agents for PD therapy.Parkinson’s disease (PD) is characterized by dopaminergic nigrostriatal neurons degeneration and Lewy body pathology, mainly composed of α-synuclein (αSyn) fibrillary aggregates. We recently described that the neuronal phosphoprotein Synapsin III (Syn III) participates in αSyn pathology in PD brains and is a permissive factor for αSyn aggregation. Moreover, we reported that the gene silencing of Syn III in a human αSyn transgenic (tg) mouse model of PD at a pathological stage, manifesting marked insoluble αSyn deposits and dopaminergic striatal synaptic dysfunction, could reduce αSyn aggregates, restore synaptic functions and motor activities and exert neuroprotective effects. Interestingly, we also described that the monoamine reuptake inhibitor methylphenidate (MPH) can recover the motor activity of human αSyn tg mice through a dopamine (DA) transporter-independent mechanism, which relies on the re-establishment of the functional interaction between Syn III and α-helical αSyn. These findings support that the pathological αSyn/Syn III interaction may constitute a therapeutic target for PD. Here, we studied MPH and some of its analogues as modulators of the pathological αSyn/Syn III interaction. We identified 4-methyl derivative  The neuronal phosphoprotein Synapsin III (Syn III) plays a key role in the control of dopaminergic striatal neurotransmission . We recethreo-methylphenidate (MPH) in attention deficit hyperactivity disorder (ADHD) patients . For lifetime analysis, fifteen cells were recorded for each condition, included GFP alone.threoI- and threoIV- treatment, SK-N-SH neuroblastoma cell line stably overexpressing αSyn were used as an in vitro model of synucleinopathy as they exhibit αSyn aggregation, as it has been previously reported that three days αSyn overexpression is sufficient to induce protein aggregation [2 and 95% O2 in Dulbecco’s modified Eagle medium with 1000 mg glucose/L , 10% heat-inactivated fetal bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin and 0.01 μM non-essential amino acids . When the cells reached 80% confluence, these were treated with threoI- 0.05, 0.1, 10 μM or with threoIV- 10 μM for 24 h, to assess the effect of the compounds on αSyn aggregation. Cells were then fixed with 4% PFA  for 15 min, at 4 °C (n = 3 replicates).For regation . Briefly2 and 95% O2 in Neurobasal medium , and after 7 days in vitro (DIV), they were exposed to GD in order to induce αSyn aggregation [threoI- and threoIV- for 24 h and fixed in 4% PFA  for 15 min, at 4 °C (n = 3 replicates).Primary ventral mesencephalic cells from C57 BL/6 J 13.5 days embryos were isolated and cultured as previously described [2 and 95% O2 for at least 10 days in vitro prior to their use to allow their maturation. At DIV 10, cells were treated with increasing concentrations  of the different MPH analogues. Twenty-four hours after the treatments, culture medium was removed, and 300 μL of MTT 0.5 mg/mL diluted in culture medium was added to each well. After 90′ incubation at 37 °C, the medium was removed, and 250 μL aliquots of DMSO were added to each well to solubilize the formazan crystals. Absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed as absorbance value (n = 3 replicates).The cytotoxic effect of compounds treatment was measured by evaluating mitochondrial dehydrogenase activity using the MTT salt assay. Primary neuronal cultures from mesencephalic tissues were dissected from C57 BL/6 J 13.5-day-old embryos. Briefly, after mechanical dissociation, the single cells were re-suspended in Neurobasal medium  containing 100 μg/mL penicillin, 100 μg/mL streptomycin, 0.5 mM glutamine and 1% B27 supplement. Cells were then centrifuged and seeded onto poly-D-lysine-coated 24-well plates . Cells were maintained at 37 °C under a humidified atmosphere of 5% COw/v bovine serum albumin  plus 3% v/v normal goat serum  in PBS, then overnight, at 4 °C, with αSyn antibody  in the case of SK-N-SH cells, and with Syn III antibody  in the case of mesencephalic neurons. On the following day, SK-N-SH cells overexpressing αSyn were incubated for 1 h, at room temperature, with the Cy3 fluorochrome-conjugated secondary antibody  diluted in 0.1% Triton X−100 PBS plus BSA 1 mg/mL. The following day, mesencephalic neurons were incubated for 1 h, at room temperature, with the Alexa 488 fluorochrome-conjugated secondary antibody  diluted in 0.1% Triton X−100 PBS plus BSA 1 mg/mL. After that, primary neurons were incubated with the second primary antibody for αSyn antibody  for 2 h at room temperature and then with Cy3 fluorochrome-conjugated secondary antibody , diluted as mentioned above. After three washes with 0.1% Triton X−100 PBS, cell nuclei were counterstained with ToProTM−3 dye , and the coverslips were mounted onto glass slides using Vectashield .After 24 h of treatment with the MPH analogues, SK-N-SH cells and primary neurons were fixed by incubating for 15 min in 4% PFA (Bio-Optica), at 4 °C. Coverslips were incubated for 1 h, at room temperature, in blocking solution , as described before. Total distance travelled was recorded in the open field arena (50 × 50 cm2), data were analyzed in 14 trials of 2.5 min each. First, mice were placed in the arena to explore the environment for 5 min before starting the test. After that, 10 min of registration in basal condition were recorded, and then mice were injected with MPH or threoI-  10 mg/kg, dissolved in normal saline 0.9%), while control mice were treated with vehicle , placed again in the open field arena, and recorded for the following 25 min. All the experiments were conducted during daylight hours. n = 3–4 animals per group were analyzed.Acute locomotor activity of twelve-month-old SYN120 tg treated with vehicle or MPH or Time spent to traverse the beam in the beam walking test was assessed as previously described by our group . BrieflyThe acquisition of images from primary neuronal cells was performed by using a LSM 880 Zeiss confocal laser equipped with Airyscan detector (Carl Zeiss) with λ = 405–488–543 nm lasers. In particular, z-stack images were acquired with the height of the sections scanning = 1 μm. Images (512 × 512 pixels) were processed by Airyscan processing and maximum intensity projection by using Zen Black 2.3 Software, Carl Zeiss, Germany.threo) were assessed by using two-way ANOVA followed by Bonferroni’s multiple comparisons test . Differences between the time to transverse the beam of vehicle, MPH or threoI--treated SYN120 tg mice in the balance beam test were assessed by using one-way ANOVA followed by Newman–Keuls multiple comparisons test .Differences between the FRET efficiency/Amplitude (FLIM) of GFP-Syn III and RFP-αSyn overexpressing SK-N-SH cells exposed to MPH or MPH-analogues were assessed by one-way ANOVA followed by Tukey multiple comparison test. At least n = 15 cells for each experimental condition were analyzed. In each cell, 3–6 different ROIs were acquired, and the resulting median value for each cell was then plotted and subjected to statistical analysis. Differences between αSyn-positive particle average size treated with the compounds were assessed by one-way ANOVA followed by Newman–Keuls multiple comparison test. At least n = 15 images for each experimental condition were analyzed, and the resulting quantification for each image was then plotted and subjected to statistical analysis. Statistical differences between the total distance travelled in the open field test by the different experimental groups  [For all the studied compounds, physicochemical descriptors relevant for drug-likeness, ADME parameters and pharmacokinetic properties were predicted using the SwissADME tool  ,42. Resuwww.rcsb.org, accessed on 17 November 2021). Protein–protein docking was performed using the HDOCK server  [The files containing the atomic coordinates for the considered proteins were obtained from the RCSB Protein Data Bank  ,45,46,47ong, CN)  was loadong, CN)  as the long, CN) . Out of ong, CN)  was usedThe blind docking experiment of the studied ligands toward the previously obtained model of αSyn /Syn III complex was performed using Autodock Vina  . The numMD simulations were carried out using PlayMolecule  starting from the output models of docking experiments. More in detail, ligand was prepared by running the Parametrize function (playmolecule.org/parameterize), based on GAFF2 force field . The αSyI–IV to modulate the interaction between αSyn and Syn III in a cell model of PD exhibiting αSyn and Syn III co-aggregates by acceptor photobleaching FRET as previously described [threoI-, erythroI-, threoII- and threoIV- to promote αSyn/Syn III interaction were performed at 15 min from the addition of 10 μM of each of the different compounds to the cell culture media as previously described [First, we evaluated the ability of MPH analogues escribed . This teescribed ,35,36,37escribed . The FREescribed .threoI- and, to a lesser extent, threoIV- were found to significantly increase FRET efficiency and thus Syn III and αSyn binding . Compounds threoI- and threoII- significantly reduced cell viability only at 100 μM concentration, which is 10 times higher than the effective concentration tested in the FRET and FLIM experiments  αSyn, an established in vitro model of PD, as three days of human αSyn overexpression is sufficient to induce the formation of aggregates [As compounds gregates .threoI- or threoIV- and stained for αSyn. A negative control was performed by omitting the incubation with the primary antibody  were used to reduce off-target effects on neuronal cells (compared to the SK-N-SH cells treatments). We observed that 0.1 μM of compound threoI- was required to significantly decrease the size of αSyn-positive particles . On the other hand, MD simulations predicted that while threoI- could be retained within the binding site with small fluctuations, MPH did not reach stabilization within the simulation timeframe, showing high RMSD values that are not consistent with a structured ligand-target binding  of compound threoI- has never been determined. In light of the above-described results, we thus evaluated the DAT Ki of threoI- and of MPH as reference control , were both unable to stimulate Syn III/αSyn interaction.Interestingly, even compounds The different behavior of MPH and its analogues towards DAT inhibition and Syn III/αSyn interaction, especially for compounds showing hindered aromatic moieties, suggests that the binding sites for these compounds on the targets are structurally different.Overall, these results support the hypothesis that MPH and MPH analogues, having an αSyn/Syn III complex stabilizing ability, may sequester Syn III from the pathological αSyn aggregates, thus enabling their reduction analogously to Syn III gene silencing. More strikingly, through this mechanism, these compounds restore the functional αSyn/Syn III interaction, which is pivotal for ensuring DA release ,13,16.threoI- as a suitable lead compound for counteracting motor dysfunction and reducing αSyn pathology in PD and could be the starting point for further structural modifications aimed at avoiding unwanted activities as MAT binding, to design novel drug candidates exerting a disease-modifying effect on αSyn aggregation in PD. These findings have significant implications in the field of PD and other synucleinopathies, but may even open insightful therapeutic perspectives in the field of ADHD, which can be associated with Syn III and αSyn polymorphisms [Collectively, our findings highlight orphisms ,58,59 anWO2022/029151 A1: Structural analogues of Methylphenidate as Parkinson’s disease-modifying agents.


High values of V̇O2max, maximal aerobic power (MAP), and power output at anaerobic thresholds are key variables in elite rowers. Endurance athletes often use altitude training as a strategy to improve performance. However, no clear evidence exists that training at natural altitude enhances sea-level performance in elite rowers. This study aimed to evaluate the effect of altitude training on rowing-performance parameters at sea level. The study was conducted on eleven rowers  from the Chilean National Team during a 3-week moderate altitude training  under the live high-train high (LHTH) model. It included a rowing ergometer maximal incremental test and blood analysis . Gas exchange analysis was performed to measure V̇O2max, ventilatory thresholds (VTs) and rowing economy/efficiency (ECR/GE%). LHTL training improves performance-related variables at sea level ; hemoglobin concentration ([Hb]): 4.3% ; hematocrit (%): 4.5% ; RBC (red blood cells) count: 5.3% ; power at VT2: 6.9% , V̇EVT2: 6.4% ; power at VT1: 7.3% , V̇EVT1: 8.7% ) and economy/efficiency-related variables ; GE(%): 5.8% ). The LHTH training decreased breathing economy at MAP ), pVT2 ), and pVT1 ). Non-significant changes were found for V̇O2max and MAP. This study describes the effects of a 3-week moderate altitude (LHTH training) on performance and economy/efficiency-related variables in elite rowers, suggesting that it is an excellent option to induce positive adaptations related to endurance performance.Maximal oxygen consumption (V̇O However economy .2max is mandatory for elite endurance athletes   of endurance performance , where cathletes , represeathletes . In rowifemales)  maximal females) .2-carrying capacity  model, which consists of living and training for weeks at moderate-to-high natural altitude. In addition to this model, other protocols of natural or artificial hypoxia as live-high train-low (LHTL), artificial LHTL with daily exposure to long (8–18 h) continuous, brief (1.5 to 5 h) continuous or brief (<1.5 h) intermittent periods of hypoxia, and artificial live-low train-high (LLTH), among others , has beerotocols . Regardirotocols . Few sturotocols . However2max value lost for every 1,000 m, starting at about 1,500 m; however, at any given altitude, the V̇O2max decrement varies significantly between individuals, with the majority of the variance explained by the sea-level value  on parameters related to rowing performance at sea level. It is hypothesized that altitude training will improve physiological thresholds and economy/efficiency-related variables in elite rowers.2max: 3.9 ± 0.31 L/min; 58.2 ± 4.5 ml/kg/min), and five males  participated in the study. The subject’s characteristics are presented in i.e. injuries in bone tissues and joints): or muscle lesions during the study; (ii) could not complete the LHTL sessions and tests proposed by this research (iii) use of ergogenic substances ; which could enhance physical performance or change V̇O2max values , and; (iv) having participated in LHTH camp or hypobaric environment before the experiment. To our knowledge, the participants did not consume any dietary supplements or medication at the moment of the study.Eleven  heavyweight scull rowers, six females . In addition, the present research complies with the requirements of the international STROBE checklist .Subjects arrived at the laboratory between 10:00 a.m. and 12:00 p.m., having fasted for at least 2 h and refrained from training for 48 h. All tests were performed in a temperature-controlled laboratory . Anthropometric, hematological measurements, and a rowing ergometer maximal incremental testing were conducted 1 week before and 14 days after the moderate altitude training  Figure . To adju2), expired carbon dioxide (V̇CO2), and respiratory exchange ratio (RER) were averaged over 10 s in the mixing chamber mode, with the highest 15 s value used in the analysis. V̇O2max was determined according to previously established criteria , (ii) RER >1.1, (iii) ≥90% of theoretical maximal heart rate, and (iv) blood lactate levels >8.0 mmol/L. The V̇O2max was expressed as absolute values (L/min) and relative to body mass (ml/kg/min). Ventilatory threshold 1 (VT1) and ventilatory threshold 2 (VT2) were identified according to the following criteria : the intensity that causes the first rise in the ventilatory equivalent of oxygen (V̇E/VO2) without a concurrent rise in the ventilatory equivalent of carbon dioxide (V̇E/V̇CO2).- VT1 : the intensity that causes a rise in V̇E/V̇O2 and V̇E/V̇CO2 and a fall in end-tidal CO2 (PETCO2).- VT2  Concept 2, Model D, Nottingham, United Kingdom) according to the criteria previously described . An initcriteria : (i) placriteria :- VT1  divided by the power generated (W) at ventilatory thresholds intensities:Mean RER values at pVT1 and pVT2 were 0.89 ± 0.018 and 0.96 ± 0.016, respectively. Rowing economy, calculated as the energy cost of rowing (ECR), was determined as the total V̇OAs a complement, gross rowing efficiency (GE%) was calculated at pVT2 as previously described .An automated hematology analyzer Sysmex XN-1000 , performed a complete blood count using whole venous blood according to the manufacturer’s instructions. Hematocrit (%), the concentration of hemoglobin , and red blood cell  count were measured.The 3-week moderate  altitudei.e., greater than the smallest worthwhile change, SWC), similar to or smaller than another time point was calculated. SWC was calculated as 0.2 multiplied by the between-subject standard deviation, based on Cohen’s d principle (p < 0.05.All data were expressed as mean ± standard deviation (SD) or 95% confidence interval (CI). Data normality was initially confirmed by Shapiro–Wilk tests. Paired-sample t tests were used to test the null hypothesis stating no difference between pre and post values. The chances that change in performance or physiological variables were substantial , affecting the relative values of performance and economy-related variables. Basal values, the difference between pre and post, and effect size are shown in Seventy-two hours after arriving at the moderate altitude training camp MAP, pVT2 and pVT1 decreased -14.6% , −9.5% , and −14.6% , respectively . Also, LHTH training period induced a substantial decrease in body weight  p = 0.014)], on power output at VT2 as a percentage of MAP , SWC-Factor = 4.3, 90% CL  and V̇E at VT2 as a percentage of V̇Emax , SWC-Factor = 3.6, 95% CL  ], SWC-Factor = 0.9, 95% CL .The LHTH induced a moderate increase on power output at VT2 , on power output at VT1 as a percentage of MAP , and V̇E at VT1 as a percentage of V̇Emax . No changes were observed on V̇O2 at VT1 .Regarding variables associated to VT1 , there w2) at MAP . Regarding submaximal values, there was a decrease in breathing economy observed through a large increase in the ventilatory equivalents for oxygen (V̇E/V̇O2) at VT2 , and a large increase in the ventilatory equivalents for oxygen (V̇E/V̇O2) at VT1 , and ], and .2/W, 95% CL , SWC-Factor = −0.3, 95% CL ], a moderate decrease at VT2 , and an unclear effect at VT1  ] .To our knowledge, this study is the first to describe the effect of a 3-week moderate altitude  camp under the LHTH model on (i) performance-related variables and (ii) economy/efficiency-related variables in elite rowers. The main findings are as follows:max: 3.3%  [Hb]: 4.3% ; hematocrit: 4.5% ; RBC count: 5.3% ; pVT2: 6.9% , V̇EVT2: 6.4% ; pVT1: 7.3% , V̇EVT1: 8.7% ; and economy/efficiency-related variables ; GE (%): 5.8% ). Nevertheless, LHTH training also induces a decrease in breathing economy at MAP ), VT2 ), and VT1 ).LHTH training improves performance-related variables  camp under the LHTH model on rowing performance-related variables after returning to sea level. According to Millet et al.  the retu2max and lactate/ventilatory thresholds interact to determine how long an athlete can sustain a given rate of aerobic and anaerobic metabolism, whereas efficiency determines the velocity or power that can be achieved with a given amount of energy consumption  are related to endurance performance. Recently, Breda et al. demonstrated an integrated LHTH training model of complex networks and confirmed the prominence of hematological factors, particularly hematocrit and hemoglobin, as central nodes in this adaptive process, suggesting that the LHTH training followed by a period of 15–16 days of re-adaptation to low altitude, is effective in improving the physical performance of paralympic-runners , responsible for neovascularization by activating angiogenic mechanisms that mediate skeletal muscle adaptations , pH regulation, and muscle buffer capacity . Recently, a study showed that the LHTH training model at moderate altitude, followed by a period of 15–16 days in the third phase, is effective in improving the performance of long- and medium-distance paralympic runners  are a good indicator of rowing performance  during incremental tests are commonly used variables in rowing, are highly correlated to ergometer performance  is sensitive to training , which is a good indication of the economy of respiration. Ventilation of a larger air volume requires a greater activity of the respiratory muscles, which requires a greater percentage of V̇O2. This means less oxygen will be available to the skeletal muscles involved during exercise. The more economical the respiratory effort is during exercise, the lower the V̇E/V̇O2 ratio . These results are concordant with an increase in V̇E at physiological thresholds (8.7% at pVT1 (as % V̇Emax) and 6.4% at pVT2 (as % V̇Emax)), despite an improvement in V̇Emax (3.3%), suggesting a performance improvement in a breathing-economy independent manner in highly-trained athletes. A possible explanation could be the increases in hematological parameters contributing to buffering changes in the blood  the fraction of maximal uptake that can be sustained during the exercise; and (iii) economy or efficiency of conversion of oxygen consumption into power output  under the LHTH model on performance and economy/efficiency-related variables in elite rowers. The main findings are a rightward shift of the power at ventilatory thresholds, increments in hematological parameters, and improvements in rowing economy and gross efficiency, primarily related to the anaerobic threshold, which is key to improving performance parameters in highly-trained rowers see . The res


Switching dual therapy with dolutegravir (DTG) plus rilpivirine (RPV) was assessed in the SWORD-1 and SWORD-2 studies. Real-life data regarding the immunological impact of this approach on CD4+ and CD8+ T lymphocyte counts and the CD4/CD8 ratio are scarce. We evaluated this strategy on the basis of clinical practice data.A multicentric retrospective cohort study.Treatment-experienced virologically suppressed HIV-1-infected patients who were switched to DTG plus RPV were included. Using different models for paired data, we evaluated the efficacy and immune status in terms of CD4+ and CD8+ T-cell counts and CD4/CD8 ratio at 24 and 48 weeks of treatment.P = .0038), but no significant changes in the CD8+ T-cell count . No differences were found in the CD4/CD8 ratio between the acquired immunodeficiency syndrome subgroup and sex or age.The study population comprised of 524 patients from 34 centers in Spain. Men accounted for 76.9% of patients, with a median age of 53 years. Patients receiving DTG plus RPV reached weeks 24 and 48 in 99.4% and 83.8% of cases, respectively, with only three (0.57%) virological failures. We found a significant decrease in CD8+ T-cell count (log OR –40) at week 24 and an increase in CD4+ T-cell count at week 48 (log OR +22.8). In acquired immunodeficiency syndrome-diagnosed patients, we found a significant increase in the CD4+ T-cell count at week 48 (log OR = 41.7, In patients with controlled treatment, dual therapy with DTG plus RPV slightly improved the immune status during the first 48 weeks after switching, not only in terms of CD4+ T-cell count but also in terms of CD8+ T-cell count, with persistently high rates of viral control. Restoration of the immune system was defined as an increase in the CD4+ T-cell count to normal values and an improved CD4/CD8 ratio >0.9.Antiretroviral therapy with three active drugs has been recognized as the standard of care for the treatment of human immunodeficiency virus type 1 (HIV-1) infection for the last 25 years. This therapeutic strategy, based on the combination of a backbone of 2 nucleos(t)ide reverse transcriptase inhibitors (NRTIs) and a third agent that could be a non-nucleoside reverse transcriptase inhibitor (NNRTI), a boosted protease inhibitor (PI), or an integrase strand transfer inhibitor (INSTI), and better tolerability and safety, the currently used NRTI backbones are not entirely free of potential tolerability and toxicity issues.,6 The increased potency of new third drugs, mainly INSTIs, together with the persistent toxicity of NRTIs, has led to the study and approval of therapeutic strategies based on 2 drugs , which makes it possible to avoid classic NRTIs. These combinations have been approved for use in both naïve and experienced patients. They guarantee not only undetectability, but also better tolerability and safety profiles, with less risk of developing potential short or long-term toxicities, especially in patients with comorbidities. Moreover, adherence may improve in some patients, and the costs are reduced.Despite improvements in triple therapy in terms of potency, efficacy, ease of dosing with single-tablet-a-day regimens,–11 Dual therapy with DTG and lamivudine (3TC) or rilpivirine (RPV) has been approved for treatment-naïve (DTG/3TC) and experienced patients (DTG/3TC and DTG/RPV). The combination of DTG plus RPV was studied as a switching strategy in SWORD-1 and SWORD-2 studies and small real-world cohorts, which showed very favorable efficacy, safety, and tolerability.–16 Data regarding the impact of dual therapy on CD4+ and CD8+ T lymphocyte counts and CD4/CD8 ratio in treatment-experienced patients are scarce. CD8+ T-cell count and CD4/CD8 ratio are indirect markers of immune activation and inflammation.–20 Improvement in these laboratory markers with dual therapy is controversial and uncertain because data are scarce, and some clinicians doubt that dual therapy is as potent as triple therapy in restoring the immune system and reducing immune activation.Dolutegravir (DTG) is a highly potent and effective INSTI that has become the gold standard treatment for naïve patients.This study aimed to provide data on the impact of this strategy in real-life patients regarding changes in immune status. We explored efficacy and immune status based on CD4+ and CD8+ T lymphocyte counts in a cohort of 524 patients from 34 hospitals in Spain.22.1  HIV-1 infection with age ≥18 years, b) switching from three-drug combination antiretroviral treatment (cART) to dual therapy with DTG+RPV, c) HIV RNA viral load <50 copies/ml in the previous 24 weeks before switching, and d) switching to DTG plus RPV at least 48 weeks before the start of the study in May 2020. Data were collected from medical records, anonymized, and entered into an online electronic database, REDCapThe study protocol was approved by the Ethics Committee of Complejo Hospitalario de Avila in August 2020 (code 3/20) and was re-approved or registered by other local ethics committees. Given the retrospective nature of the study and the fact that data were obtained from routine clinical records, written informed consent was not necessary.2.23) and CD4+/CD8+ ratio, b) safety profiles at weeks 24 and 48, and c) reasons for switching to DTG+RPV.The primary outcome was efficacy analysis to determine the proportion of patients with undetectable viral loads (<50 copies/ml) at weeks 24 and 48. Secondary outcomes included the following: a) changes in the immune status of patients in terms of CD4+ and CD8+ T lymphocyte counts  and three different third agents ;2.at 24 weeks after switching to dual therapy with DTG+RPV; and3.at 48 weeks after switching to dual therapy with DTG plus RPV Fig. .3.Given that the experimental design requires a study to compare CD4+ and CD8+ T lymphocyte counts in individuals at different stages, these data points must be considered paired. A series of variables that can interact with lymphocyte count was also obtained, including sex, age, and previous antiretroviral treatment. We also recorded a diagnosis of AIDS and a CD4 nadir below 200 cells/mm2.3t-test or paired t-test with n/2–1 degrees of freedom. Using this approach, we tested the differences in lymphocyte counts between the groups  without correcting for covariates or considering the effect of the backbone and third agents.The statistical methods applied in this study were specific to the paired data. Our main objective was to assess differences in the lymphocyte counts of individuals receiving cART at baseline versus 24 and 48 weeks after the administration of dual therapy. Thus, we started by applying a simple paired sample To include covariates and address the effect of the drugs used at baseline, we applied a multiple generalized linear mixed model. By considering the individual as a random effect, this model enabled us to address the paired nature of the data:p,0p is the random effect of each individual, β1Xi is the fixed effect of the different treatments to be tested , β2Xj is the fixed effect of the backbone drug, β3Xk is the fixed effect of the third agent, β4Xl is the fixed effect of sex, and β4Xm is the fixed effect of age.where b3The study population was comprised of 524 patients from 34 centers in Spain. The baseline characteristics are summarized in Table P = .1803 or Fisher exact test: OR = 0.6301 (0.28–1.5); P = .279. At week 48, discontinuations due to reasons other than virologic accounted for 16.2% of cases, with 2.3% due to toxicity issues.The percentages of patients with undetectable HIV viral load who reached weeks 24 and 48 with this switching strategy were 99.4% and 83.8%, respectively. Virological failure was recorded in only 3 patients (0.57%). Resistance mutations were not detected. No differences were found in terms of efficacy between the AIDS and non-AIDS subgroups using the general linear model (GLM): –0.536, P = .6177).Immune status, assessed through the median baseline CD4+ and CD8+ T lymphocyte counts, was shown to change in the complete dataset. A simple analysis of paired data showed an increase in CD4+ T-cells  at week 48 after switching treatment to dual therapy, and a decrease in CD8+ T-cells  at week 24 after switching . Patients diagnosed with AIDS also showed a statistically significant increase in CD4+ T-cell counts. The mean difference between baseline cART and 48 weeks after switching was 46.34 (95% CI = 90.5–2.12) . This effect was also significant in the multiple mixed models at 48 weeks (log OR = 41.78). No differences were found in the CD8+ T-cell count, effect of the baseline antiretroviral backbone, or third drug.Adverse events (AEs) were reported in 20 patients (3.8%), including renal toxicity in 6 patients (35%), central nervous system toxicity in 6 (30%), and gastrointestinal issues in 4 (20%). No severe AEs were observed. Twelve patients (2.3%) discontinued treatment because of mild-to-moderate toxicity issues.P = 6.47E-06; eGFR log OR = –4.37, P = 1.17E-10. Week 48: creatinine: log OR = 0.069, P = 1.42E-04 and eGFR log OR = –3.79; P = 1.85E-07). We did not find significant differences in the subgroup of 395 patients who used tenofovir disoproxil fumarate (TDF) as part of their previous treatment, apart from those who also used boosted PIs .Changes in laboratory values included an increase in creatinine and estimated glomerular filtration rate (eGFR) at weeks 24 and 48  and toxicity of baseline cART (24.6%). Clinicians indicated this dual therapy with DTG plus RPV as a transition to future injectable treatments with RPV in 4.5% of the patients.4–16 In our cohort, these rates align with published data, around 100% at week 24, with only three virological failures due to poor adherence during the first 48 weeks, even in patients diagnosed with AIDS and those treated with multiple regimens. Similar results have been reported in previous real-life cohorts of patients undergoing dual therapy.,22 These high rates of virological response make this regimen a valid alternative to triple therapy in virologically controlled HIV-1-infected patients without NNRTI resistance mutations, even in those with long-term disease, multiple previous treatments, and a prior diagnosis of AIDS.The efficacy rates of the DTG plus RPV strategy are high in clinical trials and real-life cohort studies.,18,19 and CD8+ T-cell count and, more specifically, the CD4/CD8 ratio, are indirect markers of immune activation and inflammation, both of which affect prognosis. Some retrospective and observational cohort studies have suggested that dual therapy could lead to less powerful control of HIV replication, with a subsequent negative impact on immune status in the form of increased activation, inflammation, and lower CD4/CD8 ratios.,19 In our cohort, no significant changes were observed in the CD4/CD8 ratio (0.85 at baseline) during the first 48 weeks after switching to DTG plus RPV. Similar findings have been reported in patients diagnosed with AIDS. Our data contrast with other published real-life cohort studies, which reported a slight increase in the CD4/CD8 ratio, possibly because of the lower baseline CD4/CD8 ratio (0.71) and smaller study population (91 patients in the DTG/RPV group).Immune activation and biomarkers of inflammation improve within the first year after cART-induced HIV-1 suppression; however, some residual immune activation is present, In our cohort of 524 patients, we observed a significant decrease in the CD8+ T-cell count at week 24 and an increase in the CD4+ T-cell count at week 48, with a switching baseline count >700 cells/mm3. It is mandatory to note this aspect in our cohort, because, despite a general low nadir of CD4+ T-cell count or a long-term HIV infection, the CD4+ T-cell count at the switching time was over 500 cells/mm3, and the CD4/CD8 ratio was 0.85, in contrast with published data. This suggests that most patients fail to normalize their ratio if they start treatment during chronic HIV infection, even after a decade of viral suppression.Some authors reported an increase in the CD8+ T-cell count after switching 104 patients to dual therapy with boosted PIs. Moreover, the impact of dual therapy on the restoration of the immune system could differ depending on the antiretrovirals used, including INSTIs, boosted PIs, and NNRTIs. Nevertheless, long-term follow-up would probably be necessary to account for changes in immune status with DTG plus RPV.Our findings suggest that in treatment-experienced patients with virologically controlled HIV infection and a high CD4+ T-cell count, especially those with a long history of HIV infection, more than 48 weeks are needed to evaluate the significant changes in the CD4/CD8 ratio after switching.Co-infection with other viruses may play an important role in immune activation. In our cohort, we found no difference in patients with active HCV infection, but we were unable to determine the potential impact of other viruses, such as cytomegalovirus due to the retrospective nature of the study.,16 This observation is of great interest because toxicity continues to be one of the main reasons for switching antiretrovirals, although only 2.3% of patients in our cohort discontinued treatment due to mild or moderate issues. The main reasons for withdrawal were physicians’ decisions or clinical conditions other than HIV infection, which prevented continuation of DTG plus RPV. Good safety and tolerability profiles make this regimen a simplified option for patients with significant comorbidities who need drugs that are readily metabolized and for patients who have or are predisposed to toxicities.The DTG plus RPV strategy was well-tolerated and safe, with AEs registered in only 3.8% of the patients. These results correlate with published data from clinical trials and cohort studies. This result contrasts with data from clinical trials and real-life cohort studies,,16,22 which found significant changes in renal function after discontinuing TDF. This may be explained by chronic damage to renal function in patients with long-term exposure to TDF. DTG plus RPV have a good renal profile and constitute a favorable option for this group of patients.In our cohort, no improvement in renal function (creatinine or eGFR) was observed in patients whose previous regimen included TDF except when boosted PIs were present. Remarkably, the combination of boosted PIs and TDF is associated with renal problems.Our study was limited by its retrospective design and the absence of a control group. In addition, the clinical protocols and visit timetables differed between the participating hospitals. Nevertheless, the strength of this study is its large sample size (>500 patients), which is greater than that of other real-life cohorts (approximately ≤100 patients). Further investigations are needed to elucidate this aspect in clinical trials and prospective observational studies.In conclusion, administration of dual therapy with DTG plus RPV to treatment-experienced and virologically controlled patients was highly effective and slightly improved the immune status during the first 48 weeks after switching, in terms of not only the CD4+ T-cell count but also the CD8+ T-cell count. The low tolerability and safety issues, together with the low number of discontinuations owing to these toxicity issues, make this regimen a good switching strategy for aging patients, those with several comorbidities, and those with previous or potential long-term toxicities.We thank the patients, investigators, and participating institutions for enabling this study possible.Conceptualization: Carlos Dueñas, Jesús Troya, Luis Buzón.Data curation: Carlos Dueñas, Jesús Troya, Luis Buzón.Formal analysis: Francisco Ceballos, Jesús Troya.Investigation: Ana Pelaez, Elisabeth Delgado, Jesús Troya, Luis Buzón, María Garcinuño, María José Galindo, Marta Montero, Miguel Ángel Morán, Miguel Cervero, Sara de la Fuente, Teresa Aldamiz, Carlos Dueñas, Guillermo Posada, Idoia Irazola, Ignacio Santos, Javier Fuente, Josean Uribaren, Josefa Galindo, Julia Gomez.Methodology: Carlos Dueñas, Jesús Troya, Luis Buzón.Project administration: Jesús Troya.Supervision: Jesús Troya.Writing – original draft: Carlos Dueñas, Luis Buzón, Jesús Troya.Writing – review & editing: Ana Pelaez, Luis Buzón, Carlos Dueñas, Carlos Galera, Cristina Hernández, Desiré Gil, Elisabeth Delgado, Estela Moreno-García, Francisco Ceballos, Guillermo Posada, Idoia Irazola, Ignacio Santos, Javier Fuente, Jesús Troya, Jose Antonio Iribarren, Jose Manuel Guerra, Josean Uribaren, Josefa Galindo, Julia Gomez, María Garcinuño, María José Galindo, Marta Montero, Miguel Ángel Morán, Miguel Cervero, Sara de la Fuente, Teresa Aldamiz.


Our synthesized NMO was characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), field emission scanning electron spectroscopy (FE-SEM), and energy dispersive X-ray spectroscopy (EDX). The cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to detect the FZE. Under optimized conditions, the obtained results showed that the NMO had an excellent electrocatalytic property towards FZE. As a result, NMO/GCE showed a good linear range of 0.001–1765 µM, an excellent detection limit (LOD) of 0.02 µM, and sensitivity of 0.2042 µA µM Deionized (DI) water and ethanol were used for solution making and other washing purposes throughout the experiments. All experiments were carried out in N2 gas saturated with 0.1 M phosphate buffer solution (PBS).Nickel (II) chloride hexahydrate (NiCl2) as a working electrode, platinum wire as a counter electrode, and Ag/AgCl as a reference electrode. The electrochemical experiments were carried out in an electrochemical workstation with cyclic voltammetry (CV CH1205C) and differential pulse voltammetry (DPV CHI900) in the potential range of +0.5 to +1.2 (Ag/AgCl vs. V) at a scan rate of 50 mVs−1. A Nyquist plot was obtained by electron impedance spectroscopy (EIS).The morphology and the structure of the as-synthesized material were analyzed by using field emission electron microscopy . The XRD pattern of the material was recorded . The composition and chemical state of the catalyst was analyzed by X-ray photoelectron spectroscopy . The Raman spectrum data were obtained from a Micro-Raman spectrometer . FT-IR spectroscopy (Perkin-Elmer IR spectrometer) was used to detect the functional groups. The electrochemical measurements were verified by using a three-electrode system consisting of a glassy carbon electrode (surface area 0.071 cm2.6H2O and 0.5 g of (NH4)6Mo7O24·4H2O were stirred for about 30 min in 60 mL of DI water. The mixture was transferred to a 80 mL Teflon-lined stainless steel autoclave, and kept for a period of 6 h at a temperature of 150 °C in a muffle furnace 3−/4− with 0.1 M KCl solution. At high frequencies, the surface electron transfer resistance equals the semicircle formed partially. The charge transfer resistance (Rct) values were calculated to be 444.4 and 299 Ω for bare GCE and NMO/GCE, respectively. The observed low Rct value indicates the high electrical conductivity of NMO/GCE compared to bare GCE, which confirms that the NMO modified GCE has more active sites compared to bare GCE.The interfacial properties of the electrode were investigated by an electrochemical impedance study. The surface of the bare GCE and NMO/GCE was investigated by the EIS technique. The Nyquist plots of bare GCE and NMO/GCE are shown in −1 shown in The CV technique was used to examine the catalytic activity of NMO/GCE in a ferric/ferrous system containing 0.1 M KCl solution at a scan rate of 50 mVs−1 in a [Fe (CN)6]3−/4− system containing 0.1 M KCl solution. With increasing scan rate, the redox peak current increased, which reveals the fast kinetics of NMO/GCE. pa = 0.0003x + 7 × 10−6, R2 = 0.9994 and Ipc = −0.0002x − 2 × 10−5, R2 = 0.9965, respectively. The Randles–Sevcik equation was used to calculate the electroactive surface area of NMO/GCE,pa is peak current value, n is number of electrons involved in the electrochemical reaction, Dis the diffusion coefficient, v is scan rate, and C is concentration of the Ferro ferricyanide solution. The calculated EASA of bare GCE and NMO/GCE was 0.72, and 0.117 cm2, respectively. The results revealed that NMO/GCE had a higher EASA value, which will be more advantageous for FZE detection.The electroactive surface area of bare GCE and NMO/GCE was calculated by different scan rate investigations by CV analysis. −1, depicted in For efficient electrochemical determination, the loading of the catalyst is a key parameter. The surface of the electrode was coated with different loading concentrations with FZE (100 µM) at a scan rate of 50 mVs−1 containing 100 µM of FZE. The electrochemical behavior of bare GCE and NMO/GCE was investigated by CV analysis for the detection of FZE, shown in −1 containing 100 µM of FZE (pH-7), shown in pc = −0.0395x − 0.1355 and R2 = 0.9989, respectively. According to the results, pH 7 was chosen as a supporting electrolyte for the electrochemical detection of FZE. The reduction of a nitro group (–NO2) in FZE to hydroxylamine (–NHOH) is shown in The pH range of electrolytes plays a major role in the electrochemical response of the NMO/GCE. The effect of pH on NMO/GCE was evaluated by CV analysis at a scan rate of 50 mVs−1, shown in pc = −0.3195x − 2.75 and R2 = 0.9941, respectively. According to the results, NMO/GCE has great electrocatalytic activity and fast electron transfer for the detection of FZE.The effect of concentrations of FZE at NMO/GCE was observed by the CV analysis, and the CV current response of FZE (20–100 µM) at NMO/GCE was performed at a scan rate of 50 mVs−1, shown in pc = −107.9x − 14.411 and R2 = 0.9945, respectively.The kinetic electrochemical performance of NMO/GCE was examined at various scan rates with 100 µM of FZE at a scan rate of 20−100 mVspc = −0.1324x − 5.2921, R2 = 0.9976, and Ipc = −0.0145x − 17.07, R2 = 0.9989, respectively. The LOD was determined to be 0.022 µM, calculated using the standard equation formula LOD = 3 s/q, where “s” is the standard deviation of the blank line and “q” is the slope of the calibration plot  Na+, (9) Mg+, (10) Cl−, and (11) Co2+, which were added, and the DPV response is displayed in The DPV method is used for evaluating the selectivity of the NMO/GCE sensor, which was performed in the presence of FZE (100 µM) along with some interfering molecules and ions such as (2) glucose, (3) ascorbic acid, (4) dopamine, (5) 4-nitrophenol, (6) urea, (7) KThe repeatability of FZE (100 µM) on the same NMO/GCE was recorded in five repeatable measurements. The feasibility of NMO/GCE was evaluated in human serum and urine by DPV analysis. The human serum and urine were collected from healthy volunteers. The collected biological samples were filtered by centrifuging for 20 min at 6000 rpm. The collected filtrates were diluted with 0.1 M PB. Moreover, a known concentration of FZE  was spiked into the human serum and urine samples, and calculated using the standard addition method. The recovery results are shown in In summary, we synthesized NMO/GCE via the hydrothermal method as an electrochemical sensor to detect FZE. Experimental results showed that NMO/GCE had excellent electrocatalytic activity towards FZE, possessing a broad linear range of 0.001–1765 µM, narrow LOD of 0.02 µM, selectivity, reproducibility, repeatability, and stability. The modified GCE was employed for the electrochemical detection of FZE. Due to rapidity, high sensitivity, and low cost, the proposed electrochemical sensor NMO/GCE provides a promising future for sensitive and simple detection of FZE in biological samples.


The efficacy of the one-step crosslinking reaction was assessed using swelling and compression testing, SEM and FTIR analysis, and biodegradation studies in simulated body fluid. Results indicate that the scaffolds had comparable mechanical properties, which were: 13.69 ± 1.06 (CS/HAp), 12.82 ± 4.10 (CS/TCP-α), 13.87 ± 2.9 (CS/HAp/TCP-α), and 15.55 ± 0.56 (CS/FAp). Consequently, various benzophenone concentrations were added to CS/HAp formulations to determine their effect on the degradation rate. Based on the mechanical properties and degradation profile of CS/HAp, it was found that 5 µL of 0.1% (w/v) benzophenone resulted in the highest degradation rate at eight weeks (54.48% degraded), while maintaining compressive strength between (4.04 ± 1.49 to 10.17 ± 4.78 MPa) during degradation testing. These results indicate that incorporating bioceramics with a suitable photoinitiator concentration can tailor the biodegradability and load-bearing capacity of the scaffolds.Bone tissue engineering (BTE) is an ongoing field of research based on clinical needs to treat delayed and non-union long bone fractures. An ideal tissue engineering scaffold should have a biodegradability property matching the rate of new bone turnover, be non-toxic, have good mechanical properties, and mimic the natural extracellular matrix to induce bone regeneration. In this study, biodegradable chitosan (CS) scaffolds were prepared with combinations of bioactive ceramics, namely hydroxyapatite (HAp), tricalcium phosphate-α (TCP- α), and fluorapatite (FAp), with a fixed concentration of benzophenone photoinitiator (50 µL of 0.1% ( Bone tissue defects are a rising global concern and are one of the leading causes of morbidity and disability, especially in elderly patients. Normal fractures typically heal within 6–8 weeks in healthy adults, but in 5–13% of cases, the bone does not heal properly and can lead to delayed or non-union, which is classified as the absence of bone healing signs three months post-trauma ,3,4,5,6.Autografts harvested from the patient’s own non-essential bone stock are considered the gold standard in treating fractures. Autografts account for 2.2 million transplantations in orthopaedic and dentistry repair worldwide due to their high success rate of 80–90% ,15,16,17®, which incorporates recombinant bone morphogenetic protein-2 (rhBMP-2) in a collagen sponge carrier  carbodiimide hydrochloride (EDC) as their crosslinker . As chitMoreover, it was evident that the CS/FAp-containing scaffolds recorded the highest values in EWC, WU, and swelling percentage showing the weak crosslinking degree of the scaffolds. EWC of the scaffolds were shown to be 59.29 ± 0.95 for CS/TCP-α, 65.74 ± 0.79 for CS/HAp and 87.71 ± 1.92 for CS/FAp scaffolds. Meanwhile, the EWC for the scaffolds with different BP contents were 63.61 ± 0.43 (CS/HAp/5 µL BP), 64.60 ± 0.5 (CS/HAp/10 µL BP) and 64.47 ± 0.08 (CS/HAp/20 µL BP), indicating that their swelling ability was around a similar level despite the varied photoinitiator amount. While the EWC of human bone is about 15–25%, this shows the importance of water in bones since it is the main factor affecting the mechanical behaviour of the bones ,56. As sIn addition, the swelling test recorded the water uptake (WU) capacity of 145.74 ± 5.68, 191.97 ± 6.84, and 726.65 ± 127.2 for CS/TCP-α CS/HAp and CS/FAp scaffolds, respectively, while CS/HAp/5 µL BP, CS/HAp/10 µL BP and CS/HAp/20 µL BP recorded WU values of 174.85 ± 3.28, 182.51 ± 4.04 and 181.43 ± 0.63, respectively, showing a reduced WU for CS/HAp/5 µL BP. Although water uptake is essential for nutrient transport and gas interchange, swelling under physiological conditions should be controlled to avoid excessive degradation from the diffusion of water into weakly crosslinked scaffolds and thus further causing the loss of mechanical integrity and compressive stresses to the cellular environment ,57,58,59−1, asymmetrical C-H stretch of -CH2 at 2869–2921 cm−1, C=O stretching of amide I at 1638–1657 cm−1, the N–H deformation of amide II at 1540–1559 cm−1, C–CH3 band at 1372 cm−1 and the saccharide C–O–C stretching at 1149–1153 and 1024–1027 cm−1, which correspond to reported values in the literature , orthophosphate asymmetrical stretching , carbonate groups  as well as hydroxyl group stretching bands  [HAp powder presented characteristic peaks corresponding to PO75 cm−1) ,67,68,69−1 and the presence of TCP-α scaffolds was indicated by PO43− peaks at 944, 1024, 1063 and 1149 cm−1 and CO32−-region at 895 cm−1, validating the structural characteristics of the calcium phosphate ceramics. TCP-α is characterised by its main PO43− bands of v3 anti-symmetric P-O stretching at 1107, 1058, 1039, 1013, 1022 and 984 cm−1, as well as v1 symmetric P-O stretching at 935–938, 954–959 cm−1 [3 phosphate ions at 960, 1020–1026, and 562 cm−1, where scaffolds produced in this work which contained FAp demonstrated the presence of PO43− peak at 1024 cm−1, which is in agreement with the values reported in the literature [TCP-α presented phosphate peak at 1009 cm959 cm−1 ,72,73,74terature ,76,77.−1 for C=C and 1720–1760 cm−1 for C=O [−1 for C=C and 1717 cm−1 for C=O. Following the UV reaction, these peaks were observed at 1638–1657 cm−1 for C=C and 1715 cm−1 for C=O with reduced intensity.PEG600DMA contains two different unsaturated bonds at both ends of its repeated unit. These peaks are represented on FTIR spectra at 1637–1650 cm for C=O ,79,80,81w/v benzophenone) in The FTIR spectrum of the CS/HAp scaffolds with different BP contents . These values are higher than values reported in the literature for bone tissue engineering scaffolds, where the compressive modulus of chitosan/HAp-based composites and also aneroin/HAp-3D complex construct were between 4–6 MPa at 60% stain and 6.42 MPa at 40% strain, respectively [Alterations in the photoinitiator concentration in the scaffold formulations did not have a significant effect on the compressive strength of the CS/HAp scaffolds , where vectively . AdditioThe mechanical strength of the scaffolds is important for the recovery of two primary factors in bone healing: the load-bearing capacity and bone strength. Mechanical stimulation on the cellular level in the healing area will contribute to normal bone repair and regeneration in three stages of healing: mesenchymal stem cells proliferation in the early inflammatory phase, soft callus/non-mineralised cartilage in the reparative phase and the hard callus reconstitution in the remodelling phase ,88,89,90The biodegradability of a scaffold is another vital characteristic of an engineered bone scaffold. The ideal scaffold is postulated to be able to have a degradation rate similar to the rate of the new bone formation to promote ideal bone healing ,83,97,98After one week in the SBF, it was shown that the non-crosslinked chitosan scaffolds had lost 7.69%, which was higher than the other scaffolds tested . This wew/v BP scaffold profile, where 54.48 (±10.89)% weight degraded after eight weeks in SBF. The scaffolds were then left to continue degrading until week 12 and were observed to lose their integrity during handling. As such no measurement was possible at this stage, thus establishing that this representative high-strength scaffold can induce osteotransduction within the desired timeframe [Additionally, BP loading also affected the degradation rate of the scaffold . Degradaimeframe ,39,107. imeframe . The losimeframe . In geneSince scaffolds degraded slowly over the increasing incubation period, the scaffolds’ strength was also presupposed to decrease over time . The comp < 0.05, for both comparisons). The decreasing trend of Young’s modulus values obtained in this degradation procedure could indicate that the bonds and linkage between the materials were broken gradually, thus validating that biodegradation of the materials had occurred [The compression test result  shows thoccurred . w/v benzophenone showed a reduction in strength from the initial 11.20 MPa to 7.85 MPa after week 2 before increasing to 10.15 MPa in week 4. Interestingly, the compressive strength of CS/HAp/5 µL BP was observed to increase over the four weeks in the SBF. However, no significant difference was observed, illustrating that the scaffolds had maintained their strength for four weeks prior to complete disintegration in week 8 . An increase in Young’s modulus values after the initial reduction observed could be explained by the polymeric structural breakdown process. In the beginning, the shorter polymer chain in the scaffold composite has broken down, leaving the longer chains behind. Thus, this could result in the greater strength of the scaffold before the breaking down of the longer chain takes place. This result was highly preferable as it was aimed to maintain the strength of the scaffold following the application in vivo [The compressive strength of the scaffolds fabricated with various BP content while degrading was also tested . It was  in vivo .Surface analysis was performed using SEM and the elemental compositions were confirmed using EDX. The main elements recorded for all scaffolds were Carbon (C), Oxygen (O) and Sodium (Na), which originated from the neutralised chitosan, while Calcium (Ca) and Phosphorus (P) represented the incorporated ceramics. In addition, the unique Fluorine (F) component present in CS/FAp scaffolds (0.9%) validated the fluorapatite prepared, although this value was less than that of the prepared FAp powder 2.5%). Chen et al. (2006) and Kimoto et al. (2011) previously validated the synthesised FAp through the presence of a fluorine peak in the EDX analysis compared to the absence of this peak in the HAp sample, thus showing the importance of fluorine in characterising FAp ,114. CS/%. Chen eIn addition, the SEM photomicrographs also revealed the porosity of the fabricated scaffold composites, which was analysed by using ImageJ software. CS/HAp presented 3.68 ± 0.2% porosity, followed by CS/FAp (3.06 ± 0.2%), and CS/TCP-α (3.02 ± 0.1%), where these values are close to the reported porosity of the cortical bone (5–30%). While it was proposed that a scaffold should possess a porosity of >90% to facilitate optimum nutrients diffusion, this will compromise the mechanical strength and thus leading to failure of load-bearing support . The surface and elemental analyses were also conducted on the degrading CS/HAp scaffolds to observe the Ca/P ratio representing the apatite formation in SBF . The Ca/Developing a biodegradable bone scaffold with sufficient mechanical properties and can provide osteoconductive features is crucial in this tissue engineering field. The present study demonstrated an improved chemical crosslinking method of a chitosan-based scaffold with various bioceramics and photoinitiator alterations. The swelling test in acidic conditions presented a gel fraction of more than 50%, demonstrating crosslinking was achieved. The FTIR showed the scaffold characteristics for both chitosan and ceramics following crosslinking, where a reduction in C=C peaks were recorded with decreasing BP content. Moreover, the surface morphology was confirmed through SEM-EDX analysis. The compression testing validated the mechanical performance of the scaffolds achieving 12–15 MPa, which was sustained to at least 10 MPa during degradation recorded over eight weeks in SBF. The scaffold mineralisation in SBF was monitored semi-quantitatively using SEM-EDX, where an increase in Ca/P ratio was recorded from week 1 to week 2 before gradually reduced from week 2 to week 8. w/v benzophenone scaffold formulation is proposed for future investigation as a biomimetic bone scaffold candidate since it was found promising mechanical properties while degrading in addition to favourable swelling and gel fraction characteristics. Ultimately, the scaffold fabrication method presented is hypothesised to provide better control of the covalent grafting of active ingredients between the scaffold structure and its release in vitro and later in vivo.CS/HAp with 5 µL of 0.1% Chitosan (high MW), hydroxyapatite, ethanol ≥ 99.8%, and sodium fluoride ≥ 99% were obtained from Sigma Aldrich. Sodium bicarbonate 99.5% was purchased from Acros Organics , poly(ethylene glycol) (600) dimethacrylate was obtained from Polysciences Inc.  and benzophenone, 99% (A10739.30) was purchased from Alfa Aesar (Thermo Fisher (Kandel) GmbH). All materials were used as received.v/v) acetic acid, yielding a 12% (w/v) paste. The paste was left on the bench for an hour, allowing a protonation reaction to occur before being neutralised in 0.1 M sodium bicarbonate solution for 10 min. It was then pressed between filter papers to remove excess sodium bicarbonate solution. Subsequently, 100 µL of PEGDMA600 and 50 µL of 0.1% (w/v) benzophenone in ethanol were consecutively added to the paste and mixed well.A chitosan-based scaffold was prepared by dissolving 1.5 g of high MW chitosan powder in 12.5 mL of 1%  crosslinking process using a UV curing system  under 20 UV lamps with a spectral range between 315–400 nm and at the average intensity of 10–13.5 mW cmmposites ,131.Chitosan paste was prepared as per 3PO4) was prepared by making up 6.85 mL H3PO4 with water in a 10 mL volumetric flask. Subsequently, 3.2 mL of the acid solution prepared was added to 4.6 g of sodium fluoride (NaF). The mixture was subjected to magnetic stirring until all the NaF was dissolved. Once all the NaF was dissolved, the solution was added gradually to 5 g of hydroxyapatite powder until mixed thoroughly. The fluorapatite powder was finally obtained at the end of the process and was assessed using scanning electron microscopy and energy dispersion X-ray (SEM-EDX) to confirm the elements present. This FAp powder was then added into chitosan paste similar to the HAp and TCP-α mixing method, making CS/FAp composite (1:1).Subsequently, fluorapatite was prepared from the chemical substitution of hydroxyapatite and sodium fluoride before incorporating into the chitosan-based scaffolds. A solution of 10 mol/L phosphoric acid (Hw/v) benzophenone    and follv/v) acetic acid for five minutes, 1.5 h and 24 h.The crosslinkage between the materials post-UV curing process was qualitatively assessed by submerging the scaffolds in 1% (n = 3) with different bioceramic combinations  and also the reduced photoinitiator .The scaffold fluid uptake under physiological conditions was evaluated through swelling studies on the crosslinked scaffolds  by using the following formula:The scaffolds were dried in the vacuum oven at 37 °C and 70 mbar for 72 h and recorded as the dry weight, ws. Subsequently, the scaffolds were dried again in the vacuum oven for 72 h until they reached equilibrium dry weight, we. The swelling percentage, equilibrium water content (EWC), water uptake (WU) and gel fraction in PBS (GFPBS) of the scaffolds were calculated by using the formulas:Subsequently, a new set of scaffolds as detailed above were further submerged in pH 7.4 phosphate-buffered saline (PBS) at ambient temperature for 48 h until the samples had reached swelling equilibrium. The samples were pat dried using filter papers, and the weight was recorded as w−1. Four scans per sample cycle were utilised with a resolution of 0.5 cm−1 at room temperature. Following the tests, the spectra obtained were analysed using Origin software.The linkage and structural properties of the scaffolds were analysed using FTIR spectroscopy on a Perkin-Elmer Spectrum One FTIR spectrometer fitted with a universal ATR sampling accessory. All samples were dried in a vacuum oven at 37 °C and 70 mbar prior to the tests to avoid the broad water peak from shadowing the significant signature peaks of the materials. All the tests were run using a spectral range of 4000–650 cm™ software. The samples were dried using a vacuum oven  at 37 °C and 70 mbar for 24 h. Then, they were submerged in phosphate-buffered saline (PBS) solution for an hour before testing. Subsequently, the compression test was carried out at a rate of 0.5 mm/min and set to stop at 60% strain. The Young’s modulus values obtained from the software were analysed.The strength of the scaffolds was evaluated through compression testing using a Lloyd LRX tensometer  with a 2.5 KN load cell in compression mode, and the results were analysed in NEXYGEN0. The samples were then placed into small containers with a lid, and 5 mL of SBF were added to each container and kept in the oven  at 37 °C under static conditions. The SBF solution for all samples was refreshed twice a week.Simulated body fluid (SBF) was prepared using a previously documented method . All chi1. The degraded weights of all formulations were calculated using the following formula, where w0 represents the initial dry weight and w1 represents the final dry weight.All samples were collected at weeks 0, 1, 2, 4 and 8 or until the samples were disintegrated during handling. The integrity was assessed during handling before being subjected to compression testing to evaluate their degradation profile and strength while degrading. Following the compression test, scaffolds were dried in the vacuum oven, and the weights were recorded as weight after degradation, wThe scaffold surface morphology following the fabrication and biodegradation was assessed using a Mira SEM (TESCAN Performance in Nanospace) in backscattered electron (BSE) mode using magnifications ranging from 200–250×. Dried scaffolds were snapped to expose the cross-sectional regions before placing them on the stub giving both scaffold surface and cross-sectional view. Prior to imaging, the samples were gold sputter-coated using a Baltec SCD 005 for 110 s at 0.1 mBar vacuum. Energy Dispersive X-ray (EDX) scanning was conducted using an Oxford instruments detector to confirm the elemental composition of the composite components. t-test. Subsequently, a one-way ANOVA was utilised to compare three or more parametric data groups, followed by a Tukey’s test to assess the significant changes in the data. Significance was achieved with data exhibiting a p-value less than 0.05 (p < 0.05).All statistical analysis in this document was performed using the Minitab software. Firstly, a descriptive statistic was performed to evaluate the mean and standard deviation between the samples. The significance between the two data groups was then tested using a student