Thereby, optimization of the enzymatic hydrolysis and the use of β-glucuronidase from E. coli K12 as well as sulfatase from Aerobacter aerogenes ensures the acquisition of intact analytes without cleavage of ester bonds among parabens. Validation of the method revealed limits of detection between 0.02 and 0.25 µg/L as well as limits of quantification between 0.08 and 0.83 µg/L. Thereby, the use of analyte-free surrogate matrix for calibration and control material influenced the sensitivity of the procedure positively. Furthermore, excellent precision in and between series was observed. Good absolute and relative recoveries additionally proved the robustness of the multimethod. Thus, the procedure can be applied for exploring the exposome to these prominent endocrine disruptors in the general population.Many xenobiotics were identified as possible endocrine disruptors during the last decades. Structural analogy of these substances to natural hormones may lead to agonists or antagonists of hormone receptors. For a comprehensive human biomonitoring of such substances, we developed a simple, reliable, and highly sensitive method for the simultaneous monitoring of the parameters bisphenol A, triclosan, methylparaben, ethylparaben, propylparaben, butylparaben, benzophenone-1, benzophenone-3, 3,5,6-trichloropyridin-2-ol, The online version contains supplementary material available at 10.1007/s00216-022-04438-0. During the last decades, many xenobiotics which modulate the human endocrine system by different mechanisms were identified , 2. A laO-ethyl O-(4-nitrophenyl)phenylphosphonothioate (EPN) [One of the most prominent representative of these substances is bisphenol A propane, BPA), for which effects on and through the endocrine system were demonstrated in vitro as well as in vivo –7. BPA ite (EPN) .Besides xenobiotics, the exposure to naturally occurring endocrine disruptors should be taken into account for a holistic exploration of the EDC exposure , 22. RelIn epidemiological studies, the questions, whether adverse effects are resulted from synergistic or additive effects of several compounds were addresse , 30. TheWe developed a simple, reliable, and highly sensitive method for the simultaneous monitoring of the parameters BPA, TCS, MHB, EHB, PHB, BHB, BP1, BP3, TCPy, PNP, GEN, and DAI , benzophenone-3 , bisphenol A propane, BPA), n-butyl-4-hydroxybenzoate , daidzein (7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzo pyran-4-one, DAI), ethyl-4-hydroxybenzoate , genistein -4H-1-benzopyran-4-one, GEN), methyl-4-hydroxybenzoate , n-propyl-4-hydroxybenzoate , 4-nitrophenol (NP), and triclosan phenol, TCS) were purchased from Sigma-Aldrich . 3,5,6-Trichloropyridin-2-ol  was obtained from Chem Service . Figure 2H5-2,4-dihydroxybenzophenone, BP1-D5, chemical purity 99 %, isotopic purity 99 %), 2,3,5,6-2H4-ethyl-4-hydroxybenzoate , 2,3,5,6-2H4-n-propyl-4-hydroxybenzoate , and 2,3,5,6-2H4-n-butyl-4-hydroxybenzoate  were delivered by Toronto Research Chemicals . Benzophenone-3-D5 , 2,2′,6,6′-2H4-bisphenol A , 2,3,5,6-2H4-methyl-4-hydroxybenzoate , and triclosan-D3 phenol, TCS-D3, chemical purity 98 %, isotopic purity 98 %) were obtained from CDN isotopes . 3′,5′,8-2H3-Daidzein  and 3′,5′,6,8-2H4-genistein  were purchased from Cambridge Isotope Laboratories . 2,3,5,6-2H4-4-nitrophenol  was delivered by Sigma Aldrich . 2,3,4-13C3-2,3,5-trichloro-6-hydroxypyridine  was custom synthesized by the Institute for Organic and Biomolecular Chemistry .Isotope-labeled internal standards were included for every compound in terms of quantitative evaluations. Thereby, benzophenone-1-D5  was acquired for derivatization from Sigma-Aldrich . For hydrolysis, β-glucuronidase from E. coli K12 and β-glucuronidase/arylsulfatase from Helix pomatia were obtained from Roche Diagnostics GmbH . Sulfatase from Aerobacter Aerogenes and β-glucuronidase from Helix pomatia Type H-1  were delivered by Sigma-Aldrich . Acetonitrile , acetic acid , methanol  and toluene  as well as ammonium acetate , sodium acetate , and sodium chloride  were purchased from Merck KgaA . Double distilled water was generated using a milli-Q-system . Polystyrene divinylbenzene copolymer columns for solid-phase extraction (SPE) , pore diameter 100 Å) were supplied from Biotage AB . Artificial urine medium was purchased from Pickering Laboratories, Inc. .13C3, and TCS-D3 in concentrations of 1000 mg/L each. Stock solutions of DAI-D3 and GEN-D4 were received by the supplier in concentrations of 60 µg/mL in ACN-D3 and 100 µg/mL in ACN, respectively. By dilution of the stock solutions with double-distilled water, an internal standard mix was prepared containing 1.5 mg/L GEN and DAI as well as 500 µg/L of the remaining analytes. For the hydrolysis buffer, 19.5 g NH4Ac were weighed in a 250-mL volumetric flask filled up with double-distilled water. The pH value was adjusted to a value of 6.5 through the addition of glacial acetic acid. The buffer was stored in the refrigerator at 4 to 8 °C and prepared freshly every 4 weeks.Stock solutions of BP1, BP3, MHB, EHB, PHB, BHB, NP, TCPy, and TCS were set up by dissolving 1000 mg/L of the reference substances in ACN. In contrast, stock solutions of BPA, DAI, and GEN were prepared in EtOH in a concentration of 1000 mg/L. By dilution of the stock solutions with ACN, an analyte working mix containing 1.5 mg/L DAI and GEN as well as 0.5 mg/L of the remaining analytes was prepared. Stock solutions of the internal standards were set up in ACN by dissolving the reference substances BP1-D5, BP3-D5, BPA-D4, MHB-D4, EHB-D4, PHB-D4, BHB-D4, NP-D4, TCPy-Seven concentration levels were applied for calibration. Therefore, 0.9 % NaCl (w/v) was spiked with different volumes of the analyte working mix to achieve final concentrations of 0.5, 1, 2, 5, 10, 25, and 50 µg/L of BP1, BP3, BPA, MHB, EHB, PHB, BHB, NP, TCPy, and TCS. In contrast, seven calibration levels containing 1.5, 3, 6, 15, 30, 75, and 150 µg/L of DAI and GEN were prepared. The calibration samples were stored frozen as aliquots of 1 mL at −20°C until analysis. Additionally, a blank sample consisting of 0.9 % NaCl (w/v) was integrated in every set of samples. For analysis, the calibration standards were treated according to the standard procedure in the “4Ac hydrolysis buffer (pH 6.5) were added to aliquots of 1 mL urine in 1.8 mL screw glass vials. Afterwards, 10 µL of β-glucuronidase K12 from E. coli and 10 µL of sulfatase from Aerobacter aerogenes were supplemented, and the samples were incubated over night at 37 °C. For solid phase extraction, Isolute® 101 cartridges were preconditioned successively with 500 µL MeOH, 250 µL ACN and twice with 500 µL of 1 M NH4Ac hydrolysis buffer (pH 6.5) and loaded with the hydrolyzed urine samples. A washing step with 500 µL buffer, 750 µL double-distilled water and 500 µL 50% MeOH was included. Afterwards, the SPE cartridges were centrifuged for 10 min at 1900 × g and dried for another 10 min under vacuum. The samples were eluted with 4 × 200 µL of ACN into 1.8 mL screw glass vials containing 200 µL of toluene as a keeper. The eluates were concentrated to a volume of 100 µL using a gentle stream of nitrogen. Afterwards, derivatization was performed  was compared with three further incubations: β-glucuronidase Type H-1 from Helix pomatia pH 5 (B), 10 µL β-glucuronidase from E. coli K 12 pH 5 (C), and 10 µL β-glucuronidase from E. coli K 12 together with 10 µL sulfatase from Aerobacter aerogenes Type VI pH 5 (D). Thereby, 1 mL aliquots of human urine samples containing glucuronide and sulfate conjugates of the investigated analytes were prepared with 500 µL of 0.4 M NaAc buffer (pH 5) (procedures (A), (C), and (D)) and 20 µL of the internal standard mix in 1.8 mL screw vials. Afterwards, hydrolysis enzymes were added according to the respective approach. In case of procedure (B), 40 mg of β-glucuronidase Type H-1 from Helix pomatia were weighed into 10 mL 0.4 M NaAc buffer (pH 5). Afterwards, 500 µL of this mixture and 20 µL of the internal standard mix were applied to 1 mL urine. The mixtures were incubated at 37°C over night and further processed according to the final operating procedure (see the “4Ac buffer (pH 6.5).In a first approach, four different modifications of the enzymatic hydrolysis step were tested. Thereby, deconjugation with 10 µL β-glucuronidase/arylsulfatase from Helix pomatia pH 5 (A) was used as a reference. Furthermore, 10 µL β-glucuronidase from E. coli K 12 together with 10 µL sulfatase from Aerobacter aerogenes Type VI were tested for pH 5 (B) and 6.5 (C). Additionally, 10 µL β-glucuronidase from E. coli K 12 together with 50 µL sulfatase from Aerobacter aerogenes Type VI were prepared at pH 6.5 (D). Thereby, 1 mL aliquots of human urine samples containing glucuronide and sulfate conjugates of the investigated analytes were prepared with 500 µL hydrolysis buffer  and 20 µL of the internal standard mix in 1.8 mL screw vials. Afterwards, hydrolysis enzymes were added according to the respective approach. Again, the mixtures were incubated at 37°C over night and further processed according to the final operating procedure  source installed  were used. Chromeleon Software Version 7.2.8 was applied for device control and data analysis. A volume of 1 μL sample was injected into the system in splitless mode with a purge flow of 3.0 mL/min to vent after 1.2 min. The injector temperature was set to 320°C. A 5%-phenylarylene/95%-dimethyl polysiloxane low-bleed capillary column   was used for gas chromatographic separation at a constant flow of 1.2 mL/min helium carrier gas. The oven temperature was initially held at 120°C for 2 min. Afterwards, the temperature was raised in two steps: the first ramp was heated with a rate of 20°C/min up to 210°C and finally increased with 65°C/ min to 320°C. The final temperature was held for 9 min. A total run time of 17.2 min resulted for the chromatographic run. The transferline was held at 280°C. The ion source was operating at a temperature of 250°C with an electron energy of 50 eV in timed acquisition mode. Argon was used as collision gas. The mass spectrometer was working in multiple reaction monitoring (MRM) mode to detect the eluting analytes. The MS/MS operating conditions were adjusted via Auto SRM using a derivatized mixture of all investigated analytes ) with the investigated analytes in two different concentration levels. Thereby, Q low and Qhigh ten times in one set of samples, precision in series was calculated. After the analysis of one Qlow and one Qhigh sample on three different days, repeatability was determined. For the resulting coefficients of variation, a maximum tolerated value of 15 % was assumed. For the absolute and relative recoveries, four different urine samples with varying creatinine contents were investigated in different approaches. Series A was analyzed as blank sample. Series B was spiked with 10 µg/L of BP1, BP3, BHB, BPA, EHB, 4NP, MHB, PHB, TCPy, and TCS as well as 100 µg/L DAI and GEN before sample preparation and was treated according to the standard procedure. Series C was spiked accordingly after the extraction immediately before derivatization to erase analyte losses during sample preparation.Limits of detection (LOD) and quantification (LOQ) were calculated according to the calibration method as described in guideline DIN 32 645 . Therefocalculated) were calculated using the applied external calibration. Afterwards, the resulting concentrations were divided through the known, actual spiked concentration (cspiked) and multiplied by 100:For the determination of relative recoveries, the analyte concentrations in the samples spiked before sample preparation (ccB) and of the samples spiked after extraction directly before derivatization  were determined for the calculation of the absolute recoveries. The analyte concentrations in the urine blanks  were subtracted from both series. Afterwards, the ratio was calculated and multiplied by 100:In contrast, the analyte concentrations of the samples spiked before sample preparation , nail polishes, sun protection products or perfumes , and biocides (TCS) as well as food , humans are exposed to these chemicals through their daily routines , 39–41. Comparison of the averaged calibration curves showed similar slopes for all matrices. Using the slope of the averaged calibration curve in pooled urine as a reference equal to 100%, acceptable values between 80 and 120% were obtained for all analytes in both alternative matrices which was in line with generally accepted criteria for matrix effects . Figure The original sample preparation procedure developed by Schmidt  was adjutert-butyldimethylsilyl derivate of the considered analyte. For some analytes, the parent ions were detected, and others were measured as fragments of the original tert-butyldimethylsilyl derivates after the loss of methyl moieties (data not shown). As a result, MtBSTFA was considered as suitable for the derivatization of the recently added analytes BP1, BP3, MHB, EHB, PHB, BHB, and TCS. In total, the derivatization procedure was only slightly modified, as the SPE eluates were concentrated to 100 µL volume and directly derivatized without further concentration  and its metabolites [Helix pomatia. Thereby, they did not investigate the influence of possible ester cleavage reactions of parabens by lipases. If such analyte losses would have to be considered for these hydrolysis conditions, the sulfate shares would have been overestimated in the calculation, since the glucuronide components were determined without any possible lipase influence, in contrast to the total quantity. This could explain the discrepancies to the calculated sulfate proportions of the present method, for which only lipase-free enzymes were used.Enzymatic β‑glucuronidase/aryl sulfatase preparations from Helix Pomatia are known to cleave ester moieties of certain metabolites, such as phthalate and terephthalate metabolites –47. Therabolites . As onlyabolites , 48, 49.abolites , 51. In Helix pomatia pH 5 (A) was compared to three further incubations, namely β-glucuronidase Type H-1 from Helix pomatia pH 5 (B), 10 µL β-glucuronidase from E. coli K12 pH 5 (C), and 10 µL β-glucuronidase from E. coli K12 together with 10 µL sulfatase from Aerobacter aerogenes Type VI pH 5 (D). Figure E. coli K12 combined with sulfatase from Aerobacter aerogenes Type VI delivered comparable results to the application of hydrolysis enzymes from Helix pomatia. However, the situation is different for the group of investigated benzoic acid esters. As both analytes and internal standards contain ester moieties, their responses were illustrated separately. Figure Helix pomatia were used. This indicates cleavage of ester bonds of hydroxybenzoates by non-specific esterases, as previously observed for phthalates, therephthalates and EHS [E. coli K12, especially in combination with sulfatase from Aerobacter aerogenes Type VI, offers a good alternative for enzymatic hydrolysis, which was further investigated in a second experiment.In the first approach, deconjugation with 10 µL β-glucuronidase/arylsulfatase from  and EHS –48. HoweHelix pomatia pH 5 (A) was used as a reference. Furthermore, 10 µL β-glucuronidase from E. coli K12 together with 10 µL sulfatase from Aerobacter aerogenes Type VI were tested for pH 5 (B) and 6.5 (C). Additionally, 10 µL β-glucuronidase from E. coli K 12 together with 50 µL sulfatase from Aerobacter aerogenes Type VI were prepared at pH 6.5 (D). Figure E. coli K12 by the manufacturer , did not negatively affect the determination of TCPy, NP, TCS, BPA, BP1, DAI, GEN, and BP3. Furthermore, the increase in enzyme volume from 10 to 50 µL sulfatase from Aerobacter aerogenes Type VI did not further enhance the analyte responses which indicates that a quantity of 10 µL is already sufficient for the cleavage of sulfate conjugates. A similar pattern was observed for the group of investigated benzoic acid esters  was defined as the optimized standard procedure for the analysis of the investigated endocrine disruptive analytes.In the next step, another four different modifications were tested for the enzymatic hydrolysis. Again, deconjugation with 10 µL β-glucuronidase/arylsulfatase from see Fig. . TherebyThe derivatized analytes showed molecular weights ranging from 253 g/mol for NP to 612 g/mol for GEN , coefficients of variation between 0.4 and 11.4 % for Qlow and between 0.4 and 9.8 % for Qhigh were calculated. Thus, very good intra- and inter-day precision of the procedure was proven.The variation coefficients for precision in series ranged from 1.8 to 7.9 % for QCalculation of the relative recoveries revealed values between 83 and 105 % for all analytes. Thus, the relative recovery rates were in an acceptable range close to 100 %, proving good accuracy of the method. In contrast, absolute recoveries ranged from 73 to 100 %. Thereby, the lowest values were calculated for TCS and BP1. Still, the losses during sample preparation were acceptable as the relative recovery rates were close to 100 % for all analytes by use of structural-identical isotope-labeled internal standards. Additionally, good sensitivity of the analytical procedure was already proven through low LOD and LOQ values. The validation data of the optimized method is summarized in Table The analysis of urine samples of environmentally exposed humans revealed different amounts of the considered substances, which is exemplary illustrated in Fig. Table Helix pomatia was applied for enzymatic hydrolysis at 37°C over night. Afterwards, the analytes were extracted by solid phase extraction, derivatized by silylation with MtBSTFA and measured via GC-MS/MS. However, the use of a crude solution of β-glucuronidase/arylsulfatase from Helix pomatia may lead to sample contamination with GEN and DAI, since the gastric juice of these snails can contain isoflavones [E. coli K12, especially in combination with sulfatase from Aerobacter aerogenes Type VI in the present method, no contaminants of isoflavones were introduced into the investigated samples. Moors et al. [E. coli were added for enzymatic hydrolysis at 37 °C over night. Consequently, they only used lipase-free and contaminant-free enzymes. Nevertheless, the presented method encompasses a much broader analytical spectrum than BPA, GEN, and DAI. Moos et al. [Helix pomatia and an incubation duration of 3.5 h at 37 °C for hydrolysis prior to the analysis of MHB and BHB. Likewise, Dewalque et al. [Helix pomatia but with an overnight incubation at 37 °C. Thus, lipase activity was not excluded in both cases completely. However, shortening the hydrolysis time to 3.5 h may result in the cleavage of glucuronides and sulfates occurring preferentially first, while ester cleavage might be less apparently within this smaller period. Nevertheless, the analytical losses of the parabens during enzymatic hydrolysis were not investigated in both studies. Ye et al. [Helix pomatia. In the present study, the hydrolysis step was investigated using this enzyme for an overnight incubation Below is the link to the electronic supplementary material.


There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen  with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines. These designs also included both of the mutations to the hydrophobic patch in the RBM from our previous work 2.2We compared the biophysical properties of RBD-J and RBD-J6. First, we performed far-UV circular dichroism (CD) spectroscopy as a function of temperature and observed that the thermal melting temperature (Tm) value of RBD-J6 (63.0 ± 0.4 °C) was ∼ 1.5 °C higher than that of RBD-J (61.6 ± 3.5 °C). This result suggested that the overall secondary structure of RBD-J6 was modestly more stable when compared to RBD-J d. Next, 2.3Modification of the RBD to improve its physical qualities and stability as a vaccine candidate could potentially alter the antigenicity of the RBD. Unlike our previous engineering of the RBD, the two mutations introduced in RBD-J6  are not native to the sarbecoviruses, and could affect the antigenicity of the RBDWe then evaluated how polyclonal antibodies raised in mice immunized with RBD-J bound to RBD-J6. In our previous study, we formulated RBD-J with different adjuvant combinations including alum alone, CpG1826 alone, alum + CpG1826, or a saponin-based adjuvant SMNPt-test). These data, together, suggest that the S383D and L518D RBD mutations do not impact binding to a breadth of human antibodies raised in response to infection or vaccination where native S protein is present.Finally, we evaluated the binding to both engineered RBDs of antibodies from convalescent sera obtained from patients infected with SARS-CoV-2 Delta (B.1.617.2), including ones who had been vaccinated with approved mRNA COVID-19 vaccines before infection. The binding of these antibodies to either RBD-J or RBD-J6 was not significantly different, regardless of vaccination status (2.4Emerging variants of concern (VOCs) of SARS-CoV-2 have spurred the development of updated vaccine candidates that incorporate new viral mutations. In this study, we focused on two SARS-CoV-2 variants Alpha (B.1.1.7) and Beta (B.1.351). Several natural RBD mutations, including N501Y (Alpha and Beta), K417N (Beta), and E484K (Beta), reportedly increase virus transmissibilityWe added the three mutations found in the RBD of the Beta variant to RBD-J6 . We first evaluated the secreted expression of RBD-J6 β in yeast and observed 30% lower titers compared to RBD-J6. This reduced titer of the β version of RBD-J6 agrees with our previous report on the expression of the Wuhan RBD and its B.1.351 variant in yeastt-test).We evaluated binding of RBD-J6 β to ACE2 and observed similar binding as RBD-J6 . Previou2.5We next sought to compare the immunogenicity and protectiveness of the RBD-J β and RBD-J6 β. We previously reported a vaccine design in which an RBD antigen was displayed on the surface of Hepatitis B surface antigen virus-like particles (HBsAg VLPs)We previously evaluated both monomeric RBD and multimeric VLP – RBD vaccine candidates with different adjuvantsNext, at week 7, we challenged K18-hACE2 mice that received RBD-J β and RBD-J6 β vaccines with SARS-CoV-2 Alpha or Beta. We monitored weight loss and temperature for 11 days following challenge . Four ouFinally, we evaluated neutralizing activity of sera from mice challenged with Alpha and Beta variants against RBDs from SARS-CoV-2 VOC  of a dose–response curve between serum dilutions and electrochemiluminescence). No significant differences were observed in the sera neutralizing activity from mice immunized with either the VLP – RBD-J β or VLP – RBD-J6 β conjugates, and Comirnaty®, after challenge with the Alpha variant f. We als3in situ, this second hydrophobic patch is primarily exposed when the full S protein RBD is in the “up” position  in the engineered variant RBD-Jposition a, and reWe also evaluated the antigenicity of the RBD-J6 β variant and observed that binding to the target receptor ACE2 and several known neutralizing antibodies was not impacted by the addition of mutations in the RBD core hydrophobic patch relative to RBD-J β. Binding was only disrupted, however, for antibodies that targeted the mutated epitope. Notably, these antibodies that target the class IV epitope do not block binding of the RBD to the ACE2 receptor. These results suggest that the overall secondary and tertiary structure of the protein was not measurably impacted by the additional mutations. When we tested the RBD-J6 β antigen in a transgenic mouse model, we observed higher titers of antibodies that bind to several RBD variants compared to the RBD-J β antigen . LikewisWe acknowledge that one limitation of the current study is that there was no direct comparison of the engineered β variants of RBD to their corresponding ancestral sequences (used in current vaccines). While the results here emphasize the influence of the engineering changes in RBD-J6 β to the RBD-J β, this additional comparison to the ancestral sequences may have also informed the potential benefits of breadth afforded from the mutations intrinsic to the beta variant itself.Over two rounds of rational engineering, we have now achieved a >10-fold increase in expression titer over the sequences for the ancestral RBD, and overall improvements to its biophysical stabilityMice challenged with Alpha and Beta variants of SARS-CoV-2 after immunization with protein subunit VLP-RBD conjugates had nearly complete protection, and the neutralizing activity elicited by three doses of the VLP – RBD-J6 β conjugate was comparable to two doses of Comirnaty®. Recent literature has demonstrated that individuals immunized with currently approved mRNA vaccines show a marked decline in anti-RBD IgG titers after several monthsWhen designing RBD-based vaccines, presentation of a variety of pan-sarbecovirus RBDs can elicit cross-neutralizing antibodies that target conserved epitopes, potentially withstanding the continuous mutation of the SARS-CoV-2 virusTo date, the optimal frequency of vaccine boosters for SARS-CoV-2 and, subsequently, the overall yearly global demand for vaccine doses are unknown44.1Komagataella phaffii expression, and synthesized cloned into a custom vector on a BioXP (Codex). Linear DNA was purified, and constructs were transformed into a modified wild-type K. phaffii (NRRL Y-11430) as described previouslyRecombinant genes for RBD variant expression were codon optimized for 4.2600, growth for 24 h in complex media containing 4% glycerol, pelleted, resuspended, and grown for 24 h in complex media containing 40 g/L sorbitol and 1% methanol.Strains for titer evaluation of each RBD mutant were cultivated in 3 mL in 24-deep well plates as described previously4.3Purification of harvested cell culture supernatant containing the recombinant protein was performed using the downstream processing (DSP) module of InSCyT as described previously4.4Purified RBD-SpyTag was conjugated onto Hepatitis B surface antigen (HBsAg) SpyCatcher VLPs overnight at a 1.5:1 RBD to HBsAg molar ratio. Conjugated VLP-RBD was buffer exchanged and concentrated with 100 K molecular weight cutoff centrifugal filters. Materials were formulated with a 10 mM Histidine, 20 mM Sodium phosphate, 5 mM Tris, 37.5 mM NaCl, pH 7.4 buffer. VLP-RBD formulations were diluted to a final concentration of 100ug/mL. Alhydrogel (Invivogen) was added to a final concentration of 400ug/mL.4.5Reverse phase high performance liquid chromatography (HPLC) for supernatant titer measurement was performed on an Agilent 1260 HPLC system  using a PLRP-S column   as described previously4.6Far-UV CD spectroscopy was performed using a Chirascan-plus CD spectrometer  equipped with a 6-cuvette position Peltier temperature controller  and a high-performance solid-state detector as described previously4.7SLS measurements as a function of temperature were made in triplicate using a dual emission PTI QM-40 Spectrofluorometer  equipped with a 4-position cell holder Peltier temperature control device, a high-power continuous 75 W short-arc Xe lamp (Ushio), and a Hamamatsu R1527 photomultiplier tube as described previously4.8DSC was performed in duplicate using an auto-VP capillary differential scanning calorimeter  equipped with Tantalum sample and reference cells pressurized at ∼ 60 psi with nitrogen as described previously4.9Biolayer interferometry was performed using the Octet Red96 with Protein A (ProA) biosensors , which were hydrated for 15 min in kinetics buffer prior to each run. Kinetics buffer comprising 1X PBS pH 7.2, 0.5% BSA, and 0.05% Tween 20 was used for all dilutions, baseline, and disassociation steps. ACE2-Fc was loaded at a concentration of 10 ug/mL. Antibodies CB6, CV30, S309, CR3022, EY6A were loaded at a concentration of 2ug/mL. Samples were loaded in a 96-well black microplate  at starting concentrations of 15 µg/mL for ACE2-Fc binding and 10 µg/mL for antibody binding. Association and dissociation were measured at 1000 rpm for 300 and 600 s, respectively. Binding affinity was calculated using the Octet Data Analysis software v10.0 , using reference subtraction, baseline alignment, inter-step correction, Savitzky-Golay filtering, and a global 1:1 binding model.4.10B6.Cg-Tg(K18-ACE2)2Prlmn/J mouse vaccine and SARS-CoV-2 challenge studies were executed under IACUC protocol number 2009036460. All mice were humanely euthanized based on the disease scoring system4.11Four week old B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were purchased from Jackson Laboratory (stock no: 034860). Mice were first primed with the VLP-RBD vaccine at 9 weeks old, and boosted 3 and 5 weeks after. Mice immunized with Comirnaty® were primed at 9 weeks old and boosted 3 weeks later. Mice were immunized intramuscularly with 50uL of vaccine formulation.4.12Anti-RBD IgG was measured in mice serum on a MSD QuickPlex SQ120 following the SARS-CoV-2 Plate 11 Multi-Spot 96-well, 10 spot plate manufacturer’s protocol. Sera from unvaccinated mice collected at weeks 2 and 7 was diluted 1:1000. Sera from vaccinated mice collected two weeks post prime (week 2) was diluted 1:4000–1:512000, and vaccinated sera collected two weeks post 2nd booster (week 7) was diluted 1:32000–1:4096000.4.13SARS-CoV-2 challenged serum was analyzed using the SARS-CoV-2 Plate 11 Multi-Spot 96-well, 10 spot plate following the manufacturer protocol  on the MSD QuickPlex SQ120. The 10 spots contained the following RBD antigens, common designations, and lineages: 1) Epsilon - L452R  2) Beta - K417N, E484K, N501Y  3) Eta, Iota, Zeta - E484K  4) Gamma - K417T, E484K, N501Y (P.1) 5) New York - S477N 6) Alpha - N501Y (B.1.1.7) 7) UK, Philippines - E484K, N501Y  8) Kappa - L452R, E484Q  9) Delta - L452R, T478K  and 10) Wuhan. Four dilutions of serum, 1:5, 1:50, 1:500, and 1:5000 was analyzed on the MSD neutralization assay for each mouse to perform Area Under the Curve analysis on the electrochemiluminescence using GraphPad Prism.4.14Alpha (NR-54000) and Beta (NR-54008) SARS-CoV-2 variants were obtained from BEI Resources. Alpha and Beta VOC were propagated in Vero E6 cells (ATCC-CRL-1586) and re-sequenced before use in mouse challenge. K18-hACE2 mice were anesthetized using an intraperitoneal injection of ketamine  / xylazine  and were intranasally challenged with 50uL of 104 PFU/dose of Alpha or Beta variant, 25uL per nare. Mice were monitored until awake.4.15Challenged K18-hACE2 mice were evaluated daily through both in-person health assessments in the BSL3 and SwifTAG Systems video monitoring for 11 days. Disease assessments of the mice were scored based on five criteria: 1) weight loss , 2) appearance , 3) activity , 4) eye closure , and 5) respiration 4.16Challenged mice that were assigned a disease score of 5 or above or reached the end of the experiment were euthanized with an IP injection of Euthasol (390 mg/kg)  followed by secondary measure of euthanasia with cardiac puncture. Blood from cardiac puncture was collected in BD Microtainer gold serum separator tubes (BD 365967), centrifuged at 15,000 × g for 5 min and serum was collected for downstream analysis. Lungs were separated into right and left lobes. Right lobe of the lung was homogenized in 1 mL of PBS in gentleMACS C tubes (order number: 130–096-334) using the m_lung_02 program on the gentleMACS Dissociator. 300 μL of lung homogenate was added to 1000 μL of TRI Reagent (Zymo research) for downstream RNA purification and 300 μL of lung homogenate was centrifuged at 15,000 × g for 5 min and the lung supernatant was collected for downstream analyses. Brain was excised from the skull and was homogenized in 1 mL PBS in gentleMACS C tubes using the same setting as lung on the gentleMACS Dissociator. 500 μL of TRI Reagent was added to 1000 μL of brain homogenate for RNA purification.4.17RNA purification of the lung and brain were performed using the Direct-zol RNA miniprep kit (Zymo Research R2053) following the manufacturer protocol. SARS-CoV-2 copy numbers were assessed through qPCR using the Applied Biosystems TaqMan RNA to CT One Step Kit (Ref: 4392938), as described previously4.18Left lobes of lungs were fixed in 10 mL of 10% neutral buffered formalin. Fixed lungs were paraffin embedded into 5 μm sections. Sections were stained with hematoxylin and eosin (H&E) and were analyzed by iHisto. Lungs were scored by a pathologist for chronic and acute inflammation in the lung parenchyma, blood vessels, and airways, as described previously5All data generated or analyzed during this study are included in this published article and its  files.S.R.A., N.C.D., and J.C.L. conceived and planned experiments. N.C.D. and R.S.J. generated and characterized yeast strains. S.R.A. and C.A.N performed HPLC assays. S.R.A. designed and performed protein purifications. S.B., K.K. and O. K. performed biophysical characterization. S.R.A performed antigenic characterization by BLI. H.D.R, M.P.R, R.R.L., U.S.S, S.B., R.C., G.N., and M.R. produced, purified and validated the HBsAg VLP material. N.C.D. and S.R.A. formulated and analyzed samples for animal studies. T.Y.W., B.P.R., K.S.L., H.A.C., and M.B. designed and performed mice immunizations, challenge and monitoring. T.Y.W. performed RBD ELISA assays. S.R.A., N.C.D., T.Y.W. and J.C.L. wrote the manuscript. J.C.L., F.H.D.., D.B.V., S.B.J., J.R.B., S.B, and H.K. designed the experimental strategy and reviewed analyses of data. All authors reviewed the manuscript.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sergio Andre Rodriguez-Aponte, Neil Chandra Dalvie, and J. Christopher Love have filed a patent related to the RBD-L452K-F490W (RBD-J) sequence. J. Christopher Love has interests in Sunflower Therapeutics PBC, Honeycomb Biotechnologies, OneCyte Biotechnologies, QuantumCyte, and Repligen. J.C.L.’s interests are reviewed and managed under MIT’s policies for potential conflicts of interest. Harish D. Rao, Meghraj P. Rajurkar, Rakesh R. Lothe, Umesh S. Shaligram, Saurabh Batwal, Rahul Chandrasekaran, Gaurav Nagar are employees of Serum Institute of India Pvt. Ltd. Sumi Biswas is an employee of SpyBiotech Limited.


However, the mechanisms of inhalative anesthetics on hippocampal dendritic spine plasticity and BACE-dependent APP processing remain unclear. In this study, hippocampal slices were incubated with equipotent isoflurane (iso), sevoflurane (sevo), or xenon (Xe) with/without pretreatment of the BACE inhibitor LY2886721 (LY). Thereafter, CA1 dendritic spine density, APP processing-related molecule expressions, nectin-3 levels, and long-term potentiation (LTP) were tested. The nectin-3 downregulation on LTP and dendritic spines were evaluated. Sevo treatment increased hippocampal mouse Aβ1–42 (mAβ1–42), abolished CA1-LTP, and decreased spine density and nectin-3 expressions in the CA1 region. Furthermore, CA1-nectin-3 knockdown blocked LTP and reduced spine density. Iso treatment decreased spine density and attenuated LTP. Although Xe blocked LTP, it did not affect spine density, mAβ1–42, or nectin-3. Finally, antagonizing BACE activity partly restored sevo-induced deficits. Taken together, our study suggests that sevo partly elevates BACE activity and interferes with synaptic remodeling, whereas iso mildly modulates synaptic changes in the CA1 region of the hippocampus. On the other hand, Xe does not alternate dendritic spine remodeling. Evidence indicates that inhalative anesthetics enhance the β-site amyloid precursor protein (APP)-cleaving enzyme (BACE) activity, increase amyloid beta 1-42 (Aβ The inhalational anesthetics  and sevoflurane (sevo)) are widely used in clinics . HoweverDendritic spines are protrusions that receive excitatory synaptic input and trigger postsynaptic responses , thus pr1–42 has been reported to be one of the solo pathogenic forms related to synaptic loss and memory deficits , n = 4; p = 0.0006; n = 8; iso [15.65 ± 0.52], n = 4; p = 0.0012, post hoc with Tukey’s test of one-way ANOVA). Application of LY did not reverse the iso-induced effects on spine density .As shown in n = 8; sevo [9.37 ± 0.44], n = 4; p = 0.0066; n = 8; sevo [15.38 ± 0.49], n = 4; p = 0.0370; post hoc with Tukey’s test of one-way ANOVA). Moreover, pretreatment with LY partly reversed the sevo effects on spine dynamics .In the presence of sevo, thin and total spines were also reduced (ANOVA).The neuroprotective effects of Xe have been largely investigated previously ,34,35. XTaken together, these results suggest that Xe, in contrast to iso or sevo, does not affect spine dynamics on CA1 pyramidal neurons. Furthermore, sevo reduced spine density in CA1, which was mildly prevented by the inhibition of β-secretase activity.Cell adhesion molecules are critical in neuronal communication. Adhesion disruption may result in structural and functional imbalance leading to neurodegenerative diseases such as AD ,37. Postn = 13; sevo [78.16 ± 4.48], n = 7; p = 0.0205, post hoc with Tukey’s test of one-way ANOVA).We measured the nectin-3 levels in either the whole hippocampus or the subregions by means of Western blot and immunohistochemistry, respectively . Althougn = 6; KD [75.06 ± 8.25], t10 = 2.361, p = 0.0399, two-tailed unpaired t-test), but not in CA3, DG or the whole hippocampus (HC) . Decreased nectin-3 levels in CA1 pyramidal neurons evoked a reduction in total spines , particularly thin spines .For mimicking the sevo-induced nectin-3 downregulation, we used an AAV-induced knockdown (KD) of nectin-3 to examine the effects of a specific nectin-3 reduction in the CA1 region on LTP and dendritic spine dynamics. Two weeks after the microinjection, the nectin-3 protein levels were significantly reduced in CA1 (pus (HC) F–G. Nectn = 6; LY + iso [84.95 ± 5.06], n = 5; p = 0.0238; sevo [117.90 ± 20.09], n = 6; LY + sevo [66.15 ± 8.15], n = 5; p = 0.0225; post hoc with Tukey’s test of one-way ANOVA). Moreover, sAPPβ and BACE levels in the whole hippocampus were comparable between groups . Incubation with LY, together with either iso or sevo, reduced APP expression levels in CA3 and HC significantly in comparison with iso or sevo treatment alone, respectively .Additional immunohistochemistry experiments were performed to detect APP levels in the subregions of the hippocampus. These results showed that, in the CA1 region, APP levels were reduced in LY + sevo groups compared with the sevo group .A trend of elevated APP protein levels was observed in the hippocampus B. LikewiWestern blot and immunohistochemistry procedures were performed to ensure that LY alone did not affect APP levels in the whole or the subregions of the hippocampus .n = 6; LY + sevo [0.15 ± 0.01], n = 6; p = 0.0104, post hoc with Tukey’s test of one-way ANOVA).APP is also a type of cell adhesion molecule supporting the maintenance of dendritic spines and synaptic plasticity ,27. Inte1–42 levels, aberrant spine dynamics and cell adhesions may affect synaptic transmission. Moreover, previous studies have demonstrated a decrease in synaptic transmission as well as LTP in the presence of either iso, sevo, or Xe  vs. [sevo: 99.18 ± 5.12%], p < 0.0001; 7h [ctl: 133.50 ± 4.50%] vs. [Xe: 106.40 ± 4.01%], p = 0.0006; n = 6 mice/group, post hoc with Tukey’s test of one-way ANOVA).Elevated mAβo, or Xe ,38. In tOur results demonstrated that BACE inhibition partly reversed the detrimental effects of iso and sevo. Therefore, we further investigated the effects of β-secretase inhibition on attenuated potentiation by either anesthetic. t10 = 0.6612, p = 0.5234, two-tailed unpaired t-test, n = 6 mice/group). We then incubated slices with the BACE inhibitor LY for 2 h before equipotent concentrations of iso, sevo, or Xe exposure. Analysis of the fEPSP slopes for the last 10 min of LTP revealed that inhibiting β-secretase activity partly restored the negative effects of sevo on LTP, but not that from iso and Xe, as detected by post hoc with Tukey’s test of one-way ANOVA, respectively (n = 6 mice/group): iso [106.40 ± 4.31%] and LY + iso [115.0 ± 3.61%] (p = 0.4282); sevo [99.18 ± 5.12%] and LY + sevo [122.2 ± 2.06%] (p = 0.0005), Xe [106.40 ± 4.01%] and LY + Xe [104.30 ± 4.39%] (p = 0.9823). A two-tailed independent t-test  confirmed a significance only between the control—sevo and LY—LY + sevo groups.To ensure that LY did not affect LTP per se, we found that incubating the slices with LY at 3 µM for 2 h showed no effects on LTP compared with the control conditions . Given  removal .The inhaled anesthetics may affect physiological processes such as autophagy , along wPrevious work has shown that nectin-3 is critical for the maintenance of neuronal morphology and function. Downregulation of nectin-3 protein levels in either CA3, DG, or the whole hippocampus evokes a reduction in spines and impairs learning and memory ,29. In tIn summary, our data indicate that, in the CA1 region, sevo modulates the expression of peptides important for synapse dynamics, neuronal connectivity, and synaptic plasticity, e.g., LTP. BACE is the rate-limiting protease in the Aβ production, so it represents a promising drug target for the treatment of AD. We have shown herein that BACE inhibition was partly able to restore sevo-induced synaptic deficits in the CA1 region. A contribution of sevo in aggravating or even triggering AD is therefore conceivable. The gaseous anesthetic Xe blocked LTP but did not interfere with spine dynamics and synaptic remodeling, indicating a putative neuroprotective mechanism. Our study unraveled the effects of commonly used inhalational anesthetics on the modulation of synaptic plasticity and dendritic spine dynamics and identified BACE as a potential new target for attenuating cognitive deficits as reported after the administration of volatile anesthetics.Adult male C57BL/6J and Thy1-eGFP mice  were used. All animals were single-housed and were fed under standard conditions  with free access to tap water and standard mouse food. Mice were randomly assigned to each group. All animal procedures were performed in accordance with a protocol approved by the Technical University Munich and the Government of Upper Bavaria ,63.The beta-secretase inhibitor LY2886721   at a con1–42  was firstly suspended in hexafluoroisopropanol (HFIP) at 37 °C for 1.5 h. Then the mixture solution was aliquoted into 5–50 µg portions. Thereafter HFIP was removed by evaporation using a Speedvac for 30 min. When it was completely dry, the aliquots were stored at −20 °C. Before we used it, the lyophilized Aβ1–42 was dissolved in DMSO in an ultrasonic water bath. The solution was further dissolved in aCSF to a final concentration of 50 nM.The mAβ2, 5% CO2) [2, 30% O2, 5% CO2) as a control, both co-applied in exchange with carbogen gas (95% O2/5% CO2) to the aCSF. For the sake of comparability and ensuring equipotent MAC-awake [To assure a sufficient oxygen supply and a stable pH level of 7.2–7.4 during Xe application, the maximum Xe concentration used in the slice experiments was limited to 65% (1.9 mM in aCSF) . The Xe  5% CO2) ,41 and aAC-awake ,59 conce2/5% CO2 (carbogen gas) was applied, which led to a pH level of 7.4 during the preparation, incubation, and recording stages [Mice were anesthetized with iso and decapitated. The brains were removed and quickly placed in ice-cold aCSF . The slices were incubated at 35 °C for 0.5 h, then allowed to recover at room temperature (RT) for at least 1 h. A sufficient mixture of 95% Og stages ,35.The fEPSPs were recorded as previously described ,64. RecoThe anesthetics or LY2886721 were applied after HFS1 was triggered. Thereafter, the first LTP was maintained for 1 h; either iso, sevo, or Xewas applied. After 1.5 h, HFS2 was delivered, and the fEPSPs were recorded for an additional 1 h. The fEPSP slopes recorded 20 min before HFS2 were used as the baseline data for the secondary LTP.For the LY2886721 + anesthetics application experiments, LY2886721 was applied and mixed well in the aCSF before the slices were transferred to the recording chamber. After 2 h of incubation, HFS1 was triggered, and fEPSPs were monitored for 1 h. Either iso, sevo, or Xewas applied for 1.5 h before the trigger of HFS2.For the experiments conducted with slices from AAV-treated animals, two electrodes were used for the fEPSP slopes recordings, but LTP was induced in one input only.9 viral genomes/mL) and scrambled control virus  were presented by Applied Biological Materials Inc. .We used AAV9 vectors to suppress nectin-3 expression levels in the CA1 region. Previous work has validated the short hairpin RNA (shRNA) sequence targeting nectin-3 5′-TGTGTCCTGGAGGCGGCAAAGCACAACTT-3′ ,29,30. AStereotaxic microinjection was performed as previously described . BrieflyRight after the extracellular recordings, slices were collected, fixed with 4% paraformaldehyde for 2 days, cryoprotected with 30% sucrose for 3 days, and further cut into 40 µm thickness sections using cryostat  for further staining.For immunohistochemistry experiments, free-floating sections were simultaneously treated with 0.1 M PBS (3 × 10 min), hydrogen peroxide block (10 min), 0.1 M PBS (3 × 10 min), 0.1% normal goat serum (1 h) at RT, and were then labeled with mouse anti-APP (22C11)  or mouse anti-nectin-3 (H-11)  at 4 °C overnight. On the second day, sections were washed with 0.1 M PBS (3 × 10 min), incubated with biotinylated goat anti-mouse polyvalent , washed with 0.1 M PBS (3 × 10 min), and treated with streptavidin peroxidase . After rinsing, sections were stained with 3,3′-Diaminobenzidine Horseradish Peroxidase Color Development Kit DAB (Chromogen and DAB Substrate (1:50)) for 10 min. Thereafter, sections were washed, transferred onto slides, dehydrated with EtOH  and Xylene (3 × 5 min) sequentially, and coverslipped. Images from 4 to 8 sections per animal were acquired with the Zeiss Microscope  to quantity the immunoreactivity of APP and nectin-3 levels. As previously described , we deteFor immunofluorescence experiments, free-floating sections were simultaneously treated with 0.1 M PBS (3 × 10 min) and 0.1% normal goat serum (1 h) at RT and were thereafter labeled with mouse anti-APP (22C11)  or rabbit anti-nectin-3  or rabbit anti-GFP  at 4 °C overnight. On the second day, sections were rinsed with 0.1 M PBS (3 × 10 min) and then labeled with Alexa Fluor 488/594-conjugated goat anti-mouse/rabbit secondary antibody  for 2 h at room temperature. Afterward, sections were washed, transferred onto slides, and coverslipped. For the evaluation of the colocalization patterns of APP and nectin-3, images from 4 to 8 sections per animal were acquired with the Zeiss Microscope  and analyzed with Imaris software .1–42 levels were detected by a sandwich ELISA assay . A sufficient amount of hippocampal tissues was collected and weighted. Tissues were homogenized with guanidine buffer (8× the tissue mass) at RT for 4 h, diluted four-fold with cold 0.1 M PBS including 1× protease inhibitor cocktail, and centrifuged at 12,000× for 30 min at 4 °C. The supernatant was collected and added to the appropriate wells at RT for 4 h. After washing, samples were sequentially treated with mAβ1–42 detection antibody , wash buffer (4 times), and anti-rabbit IgG HRP . After rinsing, stabilized chromogen was added and incubated for 30 min at RT in the dark. Stop solution was applied, and the absorbance at 450 nm was read by an absorbance microplate reader. The results were described as a percentage of the control group.Mouse AβHippocampus was dissected and frozen after LTP experiments. Tissues were homogenized in ice-cold lysis buffer ), 0.1% Pepstatin , 0.1% 100× phenylmethylsulfonyl fluoride  and 97% PIPA buffer . Thereafter, samples were centrifuged at 12000 RPM for 30 min at 4 °C. The supernatant was collected, and the protein concentrations were determined by a pre-diluted protein standard assay. Samples containing 20 μg of protein were separated by sodium dodecyl sulfate-polyacrylamide gels (10%) and transferred onto polyvinylidene difluoride membranes . After rinsing with TBST and blocking with 10% Roti-Block  at RT for 1 h, membranes were labeled with primary antibodies (mouse anti-APP (22C11), 1:2000, #14-9749-82, Thermo Fisher; rabbit anti-BACE, 1:2000, #5606S, Cell Signaling; rabbit anti-GAPDH, 1:5000, PA1-987, Thermo Fisher Scientific; rabbit anti-nectin-1, 1:2000, ab-66985, abcam; mouse anti-nectin-3 (H-11), 1:1000; sc-271611, Santa Cruz; rabbit anti-sAPPβ, 1:2000, #813401, Biolegend) overnight at 4 °C. Following incubation with horseradish peroxidase-conjugated secondary antibodies  for 1 h at RT, bands were visualized using an Enhanced Chemiluminescence detection kit and BioRad Image Lab. The results were normalized as a percentage of the control group. Each assay was repeated 2–4 times.−ΔΔCt). The relative mRNA expressions of APP gene were calculated using the following equations: ΔCt = Ct (APP) − Ct (18S rRNA), ΔΔCt = ΔCt (iso/sevo group) − ΔCt (control group).RNA was extracted from the hippocampus using an RNA extraction kit . RNA (0.5–1 µg) was reversely transcribed using QuantiTect Rev. Transcription Kit (Qiagen). RT-PCR analysis was conducted using QuantiNova SYBR green PCR kit (Qiagen) on a Rotor GeneQ (Qiagen). The APP primer sets contained 10 pM of each sense primer 5′-GACCCGTCAGGGACCAAAAC-3′ and the antisense primer 5′-AACGGTAAGGAATCACGATGTG-3′ . The controlled 18S rRNA primer sets were as follows: sense 5′-GTAACCCGTTGAACCCCATT-3′, antisense 5′-CCATCCAATCGGTAGTAGCG-3′ . The cycle threshold (Ct) values of the APP genes were normalized to the CT values of the 18S rRNA gene for the quantification of the relative APP mRNA expressions. The results were shown as fold change . After washing with PBS, the slices were transferred onto slides and coverslipped.Images containing apical oblique dendritic spines in the CA1 region were obtained using a confocal microscope  at 0.3 µm interval z-stacks with a 60× oil-immersion objective. Dendritic segments  from the stratum radiatum in the CA1 region were analyzed with Leica Application Suite X software . Dendritic spines were categorized as thin, mushroom, and stubby subtypes, based on established criteria as follows ,30: (1) 1–42 levels, hippocampal protein levels, and dendritic spine density between three or four groups, one-way analysis of variance one-way (ANOVA) followed by Tukey’s post-hoc multiple comparisons test were used. A two-tailed unpaired t-test (homogeneity of variance was met) or a two-tailed Mann–Whitney test (homogeneity of variance was not met) was performed for the comparisons of the two groups. Data were reported as mean ± standard error of the mean (SEM). The statistical significance was defined at p < 0.05.Statistics were performed using GraphPad Prism 8 software. For the comparisons of fEPSP slopes, mAβ


We measured toll-like receptor (TLR) 2, TLR4, nuclear factor kβ (NF-kβ), interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α. Hypertensives showed significantly worse metabolic and lipid profiles, and higher values of body mass ass index (BMI), creatinine, and inflammatory parameters, compared to controls. NGT 1-h ≥ 155 had a worse glycometabolic profile and higher values of TLR2 (9.4 ± 4.2 vs. 5.9 ± 2.6 MFI), TLR4 (13.1 ± 3.9 vs. 7.8 ± 2.3 MFI), NF-kβ (0.21 ± 0.07 vs. 0.14 ± 0.04), IL-1β (6.9 ± 3.4 vs. 3.2 ± 2.1 pg/mL), IL-6 (10.8 ± 2.6 vs. 4.1 ± 1.6 pg/mL), IL-8 (27.6 ± 9.3 vs. 13.3 ± 5.6 pg/mL), TNF-α (6.4 ± 2.9 vs. 3.3 ± 1.4 pg/mL), and high-sensitivity C-reactive protein (hs-CRP) (4.8 ± 1.5 vs. 2.7 ± 1.0 mg/dL) in comparison with NGT 1-h < 155. Matsuda-index and 1-h post-load glycemia were retained as major predictors of TLRs and NF-kβ. These results contribute to better characterizing cardiovascular risk in hypertensives.Inflammation plays a key role in the pathogenesis/progression of atherosclerosis, and inflammatory molecules contribute to the progression of cardiovascular disease. Subjects with normal post-load glucose tolerance and 1-h post-load plasma glucose >155 mg/dL have an increased risk of subclinical target organ damage and incident diabetes. We aimed to test possible differences in immune-mediated inflammatory parameters in newly-diagnosed hypertensives with or without 1-h post-load hyperglycemia. We enrolled 25 normotensives (NGT) and 50 hypertensives normotolerant on oral glucose tolerance test, further divided into two groups based on 1-h post-load plasma glucose: NGT 1-h ≥ 155 ( It is well established that inflammatory processes play a key role in the pathogenesis and progression of the atherosclerotic disease and its related clinical complications ,2,3,4. ISimilarly, CV risk factors are associated with an increase in oxidative stress and an elevation of some pro-inflammatory agents indicating the response of the vascular wall to injury as a defense reaction that, mechanistically, promotes both proliferation and expansion of smooth muscle cells in the intimal space . In accoIn the last few years, we and others have contributed to defining a specific phenotype characterized by a post-load normal glucose tolerance (NGT) but with 1-h post-load plasma glucose ≥155 mg/dL (NGT 1-h ≥ 155) ,15,16,17Nevertheless, to our knowledge, at this time, no data exist about a different activation of immune-mediated inflammation in NGT 1-h >155 subjects in comparison with NGT 1-h < 155. Thus, the purpose of this study was to verify possible differences in immune-mediated inflammatory parameters, evaluated by the expression of toll-like receptors (TLRs), and oxidative stress [evaluated by nuclear factor kβ (NF-kβ)] in a group of newly diagnosed hypertensive patients with or without 1-h post-load hyperglycemia.n = 50) were further divided into two groups on the basis of 1-h post-load plasma glucose: hypertensives with  or without  1-h post-load hyperglycemia. In Hypertensive patients  study , we seleThe Ethical Committee approved the protocol , and informed written consent was obtained from all participants. All the investigations were performed in accordance with the principles of the Declaration of Helsinki.Clinic blood pressure (BP), after an initial evaluation at both upper arms, was measured in the arm with higher BP values of the sitting patients, after 5 min of quiet rest, with a validated sphygmomanometer. SBP and DBP were recorded at the first appearance (phase I) and the disappearance (phase V) of Korotkoff sounds. A minimum of three BP readings were taken on three separate occasions at least 2 weeks apart. Baseline BP values were the average of the last two of the three consecutive measurements obtained at intervals of 3 min. Patients with a clinic SBP > 140 mmHg and/or DBP > 90 mmHg were defined as hypertensive .2) were calculated by using the CKD-EPI equation . Total, LDL-, and HDL-cholesterol and triglyceride concentrations were measured by enzymatic methods . Uric acid and creatinine were measured using the Jaffe methodology. Values of estimated glomerular filtration rate (e-GFR)  criteria. Insulin sensitivity was evaluated using the Matsuda index:[insulin sensitivity index (ISI)], calculated as follows: 10,000/square root of [fasting glucose (mmol per liter)×fasting insulin (μU per liter)] × [mean glucose×mean insulin during OGTT] .The Matsuda index is strongly related to the euglycemic hyperinsulinemic clamp that represents the gold standard test for measuring insulin sensitivity.Peripheral blood mononuclear cells (PBMCs) were isolated from fresh peripheral samples by Ficoll gradient according to the manufacturer’s instructions within 2 h after blood collection. Isolated PBMCs were harvested and suspended in phosphate-buffered saline (PBS) for TLRs expression analysis by FACS, or in hypotonic buffer, and further processed to evaluate NF-kβ (p65) activity. Isolated PBMCs were labeled with an anti-CD14 antibody conjugated with phycoerythrin (PE) to identify the monocytes within the isolated population, and with an anti-TLR2 antibody conjugated with fluorescein isothiocyanate (FICT), or with an anti-TLR4 antibody conjugated with allophicocyanine (APC). An ISO CD-14 (PE) non-specific antibody was employed as a control isotype to exclude non-specific bonds. Samples were acquired with a FACSCalibur (BD Biosciences) and analyzed by Flow JO software (BD Biosciences). The monocyte population was identified by both forward (FSC) and side scatter (SSC). TLRs expression was considered as the ratio of mean fluorescence intensity (MFI) of the sample and MFI of the isotype control.2, 420 mM NaCl, and 10% glycerol. NF-kβ activity in nuclear lysates was then measured with NF-kβ specific ELISA assay , according to the manufacturer instructions. A positive control was included in each ELISA plate; samples were assessed in duplicate.To evaluate NF-kβ (p65) activity, PBMCs were lysed with a hypotonic buffer containing 10 mM Hepes, 0.1 mM EDTA, and phosphatase inhibitors. Supernatants containing cytosolic fractions were removed, and pellets were resuspended in a nuclear extraction buffer containing 10 mM Hepes, 0.1 mM EDTA, 1.5 mM MgClTo assess cytokines levels, serum samples were loaded into a biochip array containing specific primary antibodies  and processed according to the user manual. Briefly, biochips were incubated for 1 h at 37 °C, washed and incubated for an additional hour with the HRP-conjugated secondary antibody. Biochips were acquired with a biochip reader and analyzed with dedicated software. The assay measuring range was 0.0–2.4 pg/mL for IL-1β; 0.0–5.6 pg/mL for IL-6; 1.9–17.4 pg/mL for IL-8; 0.0–6.3 pg/mL for IL-10; 0.0–13.3 pg/mL for TNF-α. Intra and inter assay precision values were respectively 8.1% and 9.4% for IL-1β, 7.3% and 12.9% for IL-6; 7.7% and 6.7% for IL-8; 7.8% and 5.2% for IL-10, and 9.9% for TNF-α.t-test when clinical and biological data were expressed as continuous variables, and the χ2 test for categorical variables. Relationships between paired parameters were analyzed by the Pearson product moment correlation coefficient. Linear regression analysis was performed to test the relationship between TLRs and NF-kβ with some independent covariates . Successively, variables reaching statistical significance and gender, as dichotomic value, were inserted in a stepwise multivariate linear regression model to determine the independent predictors of TLRs and NF-kβ in the whole study population and in the two groups of NGT hypertensive patients. Differences were assumed to be significant at two-tailed p values < 0.05. All calculations were done with a standard statistical package .Descriptive data of normally distributed variables are reported as the mean ± SD, while binary data as percent frequency. Distribution normality was assessed with the Shapiro–Wilk test. Differences between groups were compared using an unpaired


The glycine was utilized as 0 mM, 5 mM, 10 mM, 15 mM, and 20 mM along with vitamin E @ 2.3 mM added constantly in each concentration. The control group contained all extenders except glycine. The results revealed that post-thawed spermatozoa motility was found significantly higher (P < 0.05) at 10 mM as compared to 5 mM, 15 mM, and 20 mM. Compared with control group, glycine concentration at 10 mM and other concentrations increased progressive and fast motility (%), curvilinear, straight line, and average path velocity (μm/s). Moreover, beat cross frequency (Hz) was higher (P < 0.05), and post-thaw viability (%), plasma membrane integrity, and mitochondrial membrane potential were significantly higher (P < 0.05) at 10 mM of glycine concentration in comparison to control and other glycine concentrations. Besides, acrosome integrity (%) and DNA integrity (%) as well as post-thawed T-AOC were also significantly higher (P < 0.05) at 10 mM of glycine concentration as compared to other glycine concentrations and control group. It is concluded that 10 mM of glycine along with vitamin E @ 2.3 mM improved cryopreserved semen quality of Achai bull.Achai is a small size cattle breed, resilient to harsh and cold environment. Cryopreservation of Achai bull semen may help to improve its genetics and preserve the germplasm. Reactive oxygen species (ROS) affects the structural and functional integrity of the spermatozoa. During freezing and thawing processes, the ROS make changes in the spermatozoa quality parameters and reduce total antioxidant capacity (T-AOC) of semen that is considered as marker of oxidative stress. This study was designed to determine the effect of glycine along with vitamin E on post-thawed spermatozoa quality and total antioxidant capacity in Achai cattle. The semen collection was done twice a week from four mature fertile Achai cattle bulls ( Livestock is the main subsector of agriculture which contributes 58.92% to agriculture and 11.11% to the overall GDP. The milk, meat, and hides are the demands for sustainable livelihood. Pakistan is endowed with best breeds of the buffalo and cattle, and approximately, 96% milk is produced from these animals .Frozen semen helps to improve genetics, preserve endangered species, and easy transport of genetic material. Cryopreservation during semen preservation subjects the spermatozoa to acrosomal damage and other spermatozoa abnormalities and thus is associated with reduced post-thawed spermatozoa quality parameters. It has been estimated that 50% spermatozoa livability is dropped during semen cryopreservation due to different stressors, i.e., osmotic stress, oxidative stress, and cold shock. Semen extenders are being used since long to minimize these adverse effects on spermatozoa , 5.Oxidant level is another factor that can affect semen quality. A limited concentration of oxidants is advantageous for spermatozoa's physiological functions. The production of free radicals is associated with the physiology of spermatozoa hyper-activation, capacitation, acrosomal reaction, and fertilization. Similarly, the production of reactive nitrogen species (RNS) is also formed resulting in increased level of these ROS and RNS that damage the spermatozoa due to oxidative stress , 7. A 20Inclusion of antioxidants results in improved post-thawed total motility of the spermatozoa and avoids damage using suitable level of semen extender for cattle . DiffereGlycine is an amino acid that plays a crucial role in preventing spermatozoa cell damage during cryofreezing and has been reported in goats and rams , 13. Addad-libitum fresh water. Green fodder was made available to all animals equally.The experimental approval was granted by the ethical committee of the Faculty of Animal Husbandry and Veterinary Sciences and semen processing unit, the University of Agriculture, Peshawar, Pakistan. Four fertile Achai cattle bulls, aged 4 to 6 years, having weight of 300-350 Kg, body condition score (BCS) 3-4, were selected with no provision of disease history. The semen was performed during summer season for a period of approximately six weeks. During that period, the temperature was around 35 to 40°C. The bulls were provided standard ration and Semen samples were collected with artificial vagina having temperature 42°C twice a week. After collection, the ejaculates were incubated in water bath at 37°C until the volume, pH, concentration, and motility parameters were measured. Each ejaculate was examined with naked eyes in glass tube. Ejaculate having no contamination like blood or dirt was recommended for further processing. Each sample volume was recorded by using graduated collection tube. Mass activity was observed by putting an undiluted drop of semen on glass slide under warm stage of microscope and recorded as follows: 0 marked no mass motility, + marked as >20 percent of spermatozoa showing progressive motility, ++ marked as 40-60 percent showing progressive motility with slow wave, +++ marked as 60-80 percent showing progressive movement with wave more intense, and ++++ marked as 80 to 100 percent showing progressive movement with rapid wave waking eddies.μg/mL, glycerol 5%, and egg yolk 25%, and doubled-distilled water was added up to 100 mL. The spermatozoa concentration was kept twenty million (approximately) in 0.5-mL straw. The samples after dilution were incubated in cold cabinet at 4-5°C for 5-6 hours for equilibration. The straws were marked according to experimental design and filled by using automatic filling machine . Later on, the semen straws were kept for 10 minutes at 4 cm above the liquid nitrogen vapors. Thereafter, semen straws were dipped into liquid nitrogen (LN2) and were stored in LN2 container until further analysis. All the experiments were performed in triplicates. For motility, viability, plasma membrane integrity, acrosome integrity, DNA integrity, mitochondrial membrane potential, and T-AOC evaluation, five semen-filled straws were thawed for 20-30 seconds in water bath at 37°C. Mass motility was determined using a 20 μL of undiluted semen on prewarmed slide at 37°C without using a coverslip and examined through (40×) phase contrast microscope . Semen extender was prepared with little modification as described by (PARC/JICA) [The samples met the minimum requirement (motility >65%) after initial evaluation of semen and then were pooled. The same spermatozoa motility was reported by others in previous studies using Achai cattle , 22. TheRC/JICA) .Thawing of frozen semen was done at 37°C for 20-30 seconds in a water bath and subjected to post-thaw evaluation following semen characteristics.μL of frozen-thawed semen from all concentrations of glycine were examined for total motility, motility kinematics, and progressive motility by a preheated glass slide and coverslip using 100× magnification. A minimum 400 spermatozoa from each sample were calculated under microscopic. Spermatozoa motility kinematics such as curvilinear velocity (VCL μm/s), straight line velocity (VSL μm/s), average-path velocity (VAP μm/s), distance average path (DAP μm), linear distance (DSL μm), linearity (VAP/VCL) and straightness (VSL/VCL), and beat cross frequency (BCF Hz) were analyzed. The machine setting for selected CASA parameters was made using previous procedure [μm/s. By contrast, if spermatozoa had >65% progressive motility, they were referred as highly motile sperms with a forward progression/speed of 25 μm/s. But in our study, the post-thaw sperm motility was obtained from 33.46 to 49.41 which were considered as progressive motile sperms enabling to impregnate the ovum which falls in the range of previous studies [Spermatozoa kinematics was examined using computer-assisted semen analysis . A total of 10 rocedure . The spe studies , 29.DNA integrity was evaluated by using the acridine orange (AO)  solution of stain which was made from 10 mL stock and then added to one-liter distilled water and mixed and stored at 4°C temp in dark. A 40 mL of 0.1 M citric acid solution, 2.5 mL of 0.3 molar disodium phosphate. The pH of the solution was adjusted up to 2.5. A single drop of semen was put on standard glass slide, and then, smear was made and air dried, and the slide was fixed for 2 hrs in Carnoy's solution. A slide was stained with AO for three minutes and finally examined under phase contrast microscope . A total of 250 spermatozoa per smear were estimated for DNA abnormalities .μL of frozen-thawed semen sample was diluted with 50 μL tris-citric acid-fructose (TCF) and centrifuged at 300 g for five minutes. Spermatozoa pellet was added with TCF to make a volume up to 245 μL, plus added with 5 mL Rhodamine , and incubated at room temperature for twenty minutes. A 50 μL drop was placed on glass slide, and a coverslip was applied and examined under fluorescence microscope . At least 200 spermatozoa were observed for the presence of green fluorescence at midpiece and were considered to have normal intact mitochondrial membrane [A 50 membrane .μL tris-citric acid-fructose (TCF) was added to the 50 μL of semen from each group concentration of glycine and centrifuged (300 g for five minutes) resulting in spermatozoa pellet. A volume of 2.5 μL of PI was mixed to spermatozoa pellet after resuspending TCF to a volume of 47.5 μL. Later on, the spermatozoa were stored at 37°C for five minutes. A total of at least 200 spermatozoa were calculated under the microscope to evaluate spermatozoa viability. Nonviable spermatozoa were noted in red color under the fluorescence microscope  [The viability of spermatozoa was examined by using propidium iodide (PI) under florescent microscope. A 50 , Japan) .μL frozen-thawed semen was added with 50 μL of 1% formal citrate formaldehyde and observed under phase contrast microscope (100×), and spermatozoa with viable intact acrosome had a sharp black crescent on their apical ridges [Normal apical-ridge (NAR) solution consisting of 1% formal citrate  was used for assessment of acrosomal integrity. A 500 l ridges .μL of semen sample was mixed with 500 μL solution of HOS and placed for 30 min at 37°C in incubator. After incubation, 5 μL semen droplets were examined under advance microscope (400×). A total of 100 spermatozoa were calculated for swelling in HOS solution. Spermatozoa swollen were recognized by coiling of spermatozoa tail impaired with plasma membrane [The plasma membrane integrity (PMI) of spermatozoa was examined by using hypo-osmotic swelling test (HOST). A 50 membrane .+ to Fe2+, and Fe2+ can form stable complexes with phenanthroline substances. The antioxidant capability (T-AOC) can be premeditated by measuring the absorbance at 520 nm. The data was computed through the following company protocol after obtaining the OD value .A total antioxidant capacity (TAC) colorimetric assay kits were used for measurement of a range of antioxidant macromolecules or protein antioxidants, and enzymes in a system can eradicate all types of reactive oxygen species and inhibit oxidative stress . The entP < 0.05.Statistical analysis by one-way analysis of variance (ANOVA) and differences among treatment groups were determined by post hoc Duncan's multiple range test using SAS (version 9.1). Data was presented as mean ± SE. Differences were considered significant at P < 0.05) in 10 mM glycine concentration (56.77 ± 2.59) as compared to other concentrations. At 15 mM concentration, the total spermatozoa motility was (46.97 ± 1.75) but lower than 10 mM followed by 5 mM (45.81 ± 0.80) and 20 mM (42.35 ± 1.09) concentration of glycine, respectively. However, the lowest total spermatozoa motility was recorded in control group (41.54 ± 0.68). The progressive linear motility (%), the progressive linear fast motility (%), and the progressive slow motility (%) were significantly higher (P < 0.05) in semen extender group containing 10 mM glycine concentration in comparison to control and other remaining glycine concentrations. Progressive motility (%) at 10 mM was 49.41 ± 2.12 followed by 5 mM (39.33 ± 1.08), 15 mM (38.30 ± 1.44), 20 mM (34.80 ± 1.36), and control (33.46 ± 1.78). The progressive fast motility (%) was recorded higher 27.80 ± 1.75 at 10 mM followed by 20.41 ± 1.71, 17.35 ± 1.62, and 16.67 ± 1.22 at 5 mM, 15 mM, and 20 mM glycine concentrations.The progressive fast motility post-thawed CASA parameters like total spermatozoa motility, progressive slow motility (%), and progressive fast motility (%) are shown in μm/s) was higher at (P < 0.05) 10 mM that is 82.27 ± 3.27 followed by 69.6 ± 4.25, 64.83 ± 2.83, and 63.63 ± 2.01 at 20 mM, 15 mM, and 05 mM, respectively. The lowest curvilinear velocity (μm/s) (54.48 ± 2.97) was recorded in control. The straight line velocity (μm/s) was significantly high (P < 0.05) at 10 mM (30.40 ± 1.03) glycine concentration (27.66 ± 1.70) at 20 mM of glycine concentration followed by 26.13 ± 1.31 and 25.32 ± 3.00 at 5 mM and 15 mM concentrations, respectively.In current study, the curvilinear velocity (μm/s) followed the same trend as it was higher (P < 0.05) at 10 mM (40.04 ± 1.02) glycine concentrations. The other glycine concentrations had lowered average path velocity (μm/s), i.e., 33.06 ± 1.87, 32.20 ± 2.00, and 32.13 ± 1.51 at 20 mM, 15 mM, and 5 mM glycine concentrations, respectively. The control had lowered value of average path velocity (μm/s) (27.6 ± 1.47) than other glycine concentrations. However, the value for linear distance (μm/s) was recorded higher in 5 mM (9.50 ± 0.82) glycine concentration followed by 8.07 ± 0.85, 8.04 ± 0.38, and 7.43 ± 0.50 at 10 mM, 15 mM, and 20 mM, respectively. The control group has shown the lowest value, i.e., 7.38 ± 0.76 for linear distance (μm/s).The average path velocity (μm/s) was found significantly higher (12.22 ± 0.81) in 5 mM glycine concentration. The other values showed lower distance average path (μm/s) that are 11.80 ± 0.77, 10.40 ± 0.55, and 9.94 ± 0.52 at 10 mM, 15 mM, and 20 mM glycine concentrations, respectively. The control group shows the lowest value (9.93 ± 0.75) for distance average path (μm/s) as shown in P < 0.05) in semen extenders containing 10 mM glycine concentration in comparison to control and other glycine concentrations presented in The post-thawed CASA parameters like total velocity distribution values are shown in P < 0.05) at 10 mM glycine concentration as compared to control and other glycine concentrations. It was followed by 5 mM concentration having 47.58 ± 0.86 viability%. The other glycine concentrations had 44.93 ± 0.46 and 41.65 ± 0.38 viability% for 15 mM and 20 mM, respectively. The control group had lower viability%, i.e., 44.22 ± 1.14, as compared to all other glycine concentrations as shown in P < 0.05) at 10 mM (53.14 ± 0.41) glycine concentration in comparison to control and other glycine concentrations. The PMI% was 45.07 ± 0.61 at 5 mM followed by 43.92 ± 0.49 and 43.35 ± 0.58 at 20 mM and 15 mM, respectively. The control had lower value (43.28 ± 0.44) as compared to other glycine concentrations. The mitochondrial membrane potential was also significantly higher (50.72 ± 0.44), (P < 0.05) at 10 mM glycine concentration in comparison to control and other glycine concentrations. The mitochondrial membrane potential for other concentrations was 46.41 ± 0.94, 45.58 ± 0.61, and 44.93 ± 0.76 for 5 mM, 20 mM, and 15 mM, respectively. Post-thawed semen parameters viability percentage, plasma membrane integrity, and mitochondrial membrane potential of Achai bull semen are shown in Post-thawed viability (%) was higher (51.08 ± 0.87) (P < 0.05) at 10 mM (50.72 ± 0.21), followed by 5 mM (50.15 ± 0.57), 15 mM (50.08 ± 0.55), and 20 mM (48.72 ± 0.58) of glycine concentration as compared to control (48.43 ± 0.44). The DNA integrity was significantly high (P < 0.05) at 10 mM (98.93 ± 0.20) glycine concentration. At different glycine concentrations 5 mM, 15 mM, and 20 mM, the values for DNA integrity percentage were 98.15 ± 0.43, 98.01 ± 0.83, and 95.65 ± 0.76, respectively. Post-thawed DNA and acrosome integrity are presented in However, acrosome (%) was significantly high (P < 0.05) in samples having 10 mM (82.29 ± 1.19) of glycine concentration as compared to control and other concentrations. T-AOC was 67.28 ± 1.63, 75.55 ± 1.98, and 79.29 ± 0.68 at 5 mM, 15 mM, and 20 mM glycine concentrations, respectively. The T-AOC values are lower in control as compared to other glycine concentrations presented in post-thaw semen profile as shown in Post-thaw total antioxidant capacity (T-AOC) was significantly higher , the straight line velocity (μm/s), and the average path velocity (μm/s) were higher at (P < 0.05) 10 mM as compared to other glycine concentrations and control. However, the values of linear distance (μm/s) and distance average path (μm/s) were higher in 5 mM glycine concentrations as compared to other glycine concentrations and control. The current study findings are in agreement with [Semen quality parameters were estimated by CASA which shows that glycine improved spermatozoa quality parameters and antioxidant capacity of cryopreserved semen. Post-thawed total spermatozoa motility was highly significant at 10 mM glycine concentration as compared to other glycine concentrations and control. Although progressive linear motility (%), progressive fast motility (%), and progressive slow motility (%) were higher at significant level in semen extender containing 10 mM glycine concentration in comparison to control and other glycine concentrations, in current study, the curvilinear velocity  at 10 mM glycine concentration in comparison to control and other glycine concentrations. In addition, acrosome integrity (%) was significantly high (P < 0.05) at 10 mM (50.72 ± 0.21), followed by 5 mM, 15 mM, and 20 mM of glycine concentration and as compared to control group. The DNA integrity was found significant (P < 0.05) at 10 mM glycine concentration as compared to other glycine concentrations and control. The findings of this study are in line with Khalili et al. [Post-thawed viability (%), plasma membrane integrity, PMI percentage, and mitochondrial membrane potential (MMP) were significantly higher  in extender having 10 mM of glycine concentration in comparison to control and all other remaining concentrations. The T-AOC values for all other concentrations and control were lower than 10 mM glycine concentrations. These findings are in line with Shannon [Post-thawed total antioxidant capacity (T-AOC) was higher ( Shannon , who des Shannon . The enh Shannon , 48 cond Shannon . In our  Shannon , 48.It is concluded that addition of glycine at low level of 10 mM concentration along with vitamin E improved post-thaw total motility of Achai bull semen. The progressive total and progressive fast motility was also improved by the use of glycine @ 10 mM, and the supplementation of glycine coupled with vitamin E enhanced the total antioxidant capacity of cryopreserved semen, ultimately resulting in better conception rate.


Objective: This study was conducted to investigate the safety, tolerability and pharmacokinetics (PK) of WXFL10203614 after single and multiple oral doses in healthy Chinese subjects.Methods: A single-center, randomized, double-blind, placebo-controlled phase Ⅰ study was performed on healthy Chinese subjects. In the single-dose study, Subjects were randomized into 7 dose levels of WXFL10203614 . In the multiple-dose study, subjects received 5 or 10 mg WXFL10203614 once daily (QD), 5 mg twice daily (BID) or placebo for 7 consecutive days. Safety, tolerability and PK of WXFL10203614 were all assessed.Results: A total of 592 subjects were screened, 50 subjects were enrolled in the single-dose study and 30 in the multiple-dose study. All adverse events (AEs) were mild or moderate and resolved spontaneously. No Serious Adverse Events (SAEs) or deaths were reported during the study. WXFL10203614 was absorbed rapidly after dosing with Tmax of 0.48–0.98 h, Cmax, AUC0-t and AUC0-∞ were all increased in a dose-related manner over the range of 1–33 mg. Renal excretion was the major route of elimination of WXFL10203614. Steady-state PK parameters  were elevated after once-daily administration of 5–10 mg WXFL10203614 and non- and weak drug accumulations were observed, whereas moderate drug accumulation occurred in the 5 mg BID group.Conclusion: WXFL10203614 exhibited good safety, tolerability and favorable PK profiles in healthy Chinese subjects, supporting further clinical development in patients with rheumatoid arthritis.Clinical Trials Registration Number:http://www.chinadrugtrials.org.cn/index.html, #CTR20190069 and CTR20200143. Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. It is characterized by joint synovitis, bone erosion, progressive cartilage destruction and disability, which seriously influence life quality and even increase mortality . So far,Although significant progress has been made in non-steroidal anti-inflammatory drugs (NSAIDs), conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) and biological therapeutics for the treatment of RA, some shortcomings were still inevitable during the treatments, such as inadequate or no response to traditional therapies, serious complications of infection, inconvenient injection and high cost of biological therapy, which limited their long-term use . A more via inhibiting the JAK-mediated signal pathways. A series of small molecule inhibitors of JAK isoforms have been approved for treating RA since 2009 , mediating the signal pathways of numerous cytokines and growth factors which participate in the regulation of immune function, inflammation and hematopoiesis . Howevernce 2009 , includince 2009 . TherefoWXFL10203614, a potential selective JAK1 inhibitor, was under development as an oral DMARD for RA treatment. The preclinical studies confirmed that WXFL10203614 was effective for RA with acceptable safety and tolerability profiles and superior to tofacitinib (data not shown). Based on these promising preclinical data, the clinical trials of WXFL10203614 were eventually approved by National Medical Products Administration (NMPA) in 2018 .This single- and multiple-dose study aimed to evaluate the safety, tolerability and pharmacokinetics (PK) of WXFL10203614 in healthy Chinese subjects. Results supported the dose selection and design of phaseⅡclinical trials of WXFL10203614 in RA patients.2 and body weight ≥50 kg  or ≥45 kg , were enrolled in this study. Subjects were included if they were evaluated to be healthy by vital signs, physical examination, medical history, laboratory tests, virological examinations, electrocardiograph (ECG), chest X-ray, abdominal ultrasound, urine nicotine test, alcohol breath analysis and urine drug screening test within 2 weeks before dosing. Subjects were excluded if they had any of the following: allergic to any medication, a history of corrected Q-T (QTc) interval prolongation, QTc interval ≥450 milliseconds, PR interval ≥210 milliseconds, QRS ≥120 milliseconds, positive for T-SPOT® TB test, took any prescription or nonprescription medicine  within 1 month before the screening, or participated in any clinical trial within the past 3 months. Besides, pregnant or lactating women were also excluded.Healthy Chinese subjects aged 18–45 years, with a body mass index of 19.0–26.0 kg/mThis study was conducted according to Good Clinical Practice guidelines and the ethical principle of the Declaration of Helsinki. The study protocol and informed consent form were approved by the independent Ethics Committee of Affiliated Wuxi People’s Hospital of Nanjing Medical University . The written informed consent was signed by each subject freely and voluntarily before the screening.http://www.chinadrugtrials.org.cn/index.html). The WXFL10203614 tablet  and the placebo tablet (Lot Number: XS180901A) were manufactured and supplied by Wuxi Fuxin Pharmaceutical Research and Development Co., Ltd.A single-center, randomized, double-blind, placebo-controlled, single- and multiple-dose phase Ⅰ study of WXFL10203614 was conducted in the phase Ⅰ center of Wuxi people’s hospital from 7 March 2019 to 13 December 2019 (the single-dose study) and from 31 March 2020 to 10 July 2020 (the multiple-dose study)  or placebo at a 3:1 ratio in a dose-escalation manner. All subjects fasted overnight for at least 10 h before dosing and received a single oral dose of WXFL10203614 with 240 ml water. Moreover, none of the subjects was permitted to drink water within 1 h before and after dosing or eat anything within 4 h after dosing. Subjects remained in the center for 4 days and completed follow-up on day 7 after discharge. Safety, tolerability and PK evaluations were performed before escalation to the next dose level.In the multiple-dose study, three doses of WXFL10203614 (5 or 10 mg once daily [QD] or 5 mg twice daily [BID]) were selected on the basis of the single-dose study. 30 subjects were enrolled, 10 subjects per cohort were randomly assigned to receive WXFL10203614 or placebo at a 4:1 ratio for 7 consecutive days . Subjects fasted overnight before the first and last dosing and orally received WXFL10203614 with 240 ml water at each dosing time. Subjects remained in the center until day 10 and completed follow-up on day 7 after discharge. Safety, tolerability and PK were also evaluated at the end of the study. Dose escalation was only allowed when the previous dose was safe and well-tolerated in subjects.Safety and tolerability were assessed on the basis of Adverse Events (AEs), the clinically significant changes in physical examination, vital signs, laboratory tests and ECG. The severity of AEs was referenced in terms of the National Cancer Institute- Common Terminology Criteria for Adverse Events (NCI-CTCAE) (version 5.03). Furthermore, the relationship of AEs to WXFL10203614 was evaluated by the investigator.In the single-dose study, blood samples (4 ml) were collected in Ethylene Diamine Tetraacetie Acid (EDTA)-2K vacuum tubes at 0 (pre-dose), 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48 and 72 h. Urine samples were collected at -2-0 h (pre-dose), 0–4, 4–8, 8–12, 12–24, 24–36, 36–48 and 48–72 h. In the multiple-dose study, blood samples (4.0 ml) were collected on day 1 at the same points as in the single-dose study until 24 h (before the second dosing) and on day 5 and 6 before dosing. Moreover, the sampling time points were 0 (pre-dose), 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 8, 12, 24, 36 and 48 h after the last dosing on day 7. Blood samples were gently mixed 6–8 times after collecting and centrifuged at 1500 g for 10 min at 4°C. All plasma and urine samples were stored at −80°C until analyzed.13CD3-Target as internal standard (IS). Briefly, 50 μL of IS working solution (50 ng/ml for plasma or 200 ng/ml for urine) and 300 μl of acetonitrile were added to 50 μl of the sample. The mixture was vortexed for 15 min, followed by centrifugation at 2,510 g for 15 min. The supernates were assayed for the WXFL10203614 concentration in plasma. For the urine sample, the supernate should be diluted 10-fold before analysis.Samples were prepared by simple protein precipitation with acetonitrile containing The concentrations of WXFL10203614 in plasma and urine were analyzed using a validated liquid chromatography-tandem mass spectrometry (LS-MS/MS) method. WXFL10203614 and IS were chromatographed by injecting 1 μl sample into a BEH C18 column  with a column temperature at 40°C. The mobile phase consisted of solvent A (0.05% formic acid in a 2 mM aqueous ammonium acetate solution) and solvent B (acetonitrile). The gradient elution was programmed as follows: 90% solvent A for 0.3 min, and then gradually decreased to 5% for 1.4 min, followed by re-equilibration at 90% for 0.8 min. The total analytical run time was 2.5 min and the flow rate was 0.55 ml/min. The mass transitions of WXFL10203614 and IS monitored in multiple reaction monitoring (MRM) were m/z 294.1→146.0 and 298.2→146.1. The retention times of WXFL10203614 and IS were 1.06 and 1.05 min, and no apparent interference affected the detection. The linearity ranges of WXFL10203614 were 0.4–500 ng/ml and 5–8,000 ng/ml, the lower limit of quantitation (LLOQ) was 0.4 and 5 ng/ml, and the recoveries were 90.9–94.2% and 92.0–94.0% in plasma and urine, respectively. The intra-and inter-day precision did not exceed 6.1% with an accuracy of −4.2-2.9% in plasma . The plasma samples were stable at room temperature for 18 h, and −20°C and −80°C for 105 days. The intra-and inter-day precision did not exceed 10.7% and the accuracy was within ±7.2% in urine . The urine samples were stable at room temperature for 24 h, and −20°C and −80°C for 69 days. The concentrations of WXFL10203614 in plasma or urine that exceed the upper limit of quantification (ULOQ) should be diluted, and 10-fold dilutions did not affect the accuracy and precision.max), time to maximum concentration (Tmax), area under the plasma concentration-time curve from time 0 to the last measurable concentration (AUC0-t), area under the plasma concentration-time curve from time 0 to infinity (AUC0-∞), terminal elimination half-life (t1/2), mean residence time of 0 to the last measurable concentration (MRT0-t), apparent clearance rate (CL/F), apparent volume of distribution (Vd) and the cumulative urinary excretion in 72 h (Ae0-72). In the multiple-dose study, the primary PK parameters were Cmax at steady state , Tmax at steady state , AUC0-t at steady state , AUC0-∞ at steady state , CL/F at steady state (CLss/F) and the accumulation index (Ra). Ra (AUC) was calculated as the ratio of AUC0-t,ss to AUC0-t and Ra (Cmax) was the ratio of Cmax,ss to Cmax.PK analysis was performed by a non-compartmental method using Phoenix WinNonlin software (version 8.2) to calculate PK parameters. In the single-dose study, the primary PK parameters included maximum concentration in plasma  and Safety Set (SS) included all randomized subjects receiving at least one dose of WXFL10203614 or placebo. The pharmacokinetic concentration set (PKCS) included all subjects with at least one drug concentration result. The pharmacokinetic parameter set (PKPS) included all subjects with at least one viable PK parameter.max, AUC0-t and AUC0-∞) proportionality was assessed with linear regression by using natural logarithm value based on the power model. The corresponding 90% confidence intervals (CIs) for the slopes of the log-transformed Cmax, AUC0-t and AUC0-∞ were compared with the modified acceptance range , it was inconclusive that PK parameters were proportional to dose within the dose range  on day 1 following 5–10 mg QD WXFL10203614, the median Tmax, ss and t1/2 was 0.73–1.23 h and 9.2–12.0 h on day 7, while CLss/F remained constant (3.6 L/h) regardless of the dose. Steady-state trough concentrations were 26.2 ± 11.8, 25.5 ± 13.4 and 25.6 ± 13.4 ng/ml for 5 mg QD WXFL10203614, as well as 59.3 ± 15.5, 58.8 ± 17.9 and 58.9 ± 19.4 ng/ml for 10 mg QD WXFL10203614 in day 5, 6 and 7 mornings, indicating that the steady state of WXFL10203614 in plasma has been reached after multiple doses. AUC0–t,ss and Cmax,ss were increased with the dose ranging from 5 to 10 mg. Ra (AUC) and Ra (Cmax) were 1.17–1.34 and 1.14–1.37, respectively. In the 5 mg BID group, Steady-state trough concentrations of WXFL10203614 in 3 days were 97.5 ± 12.6, 96.5 ± 13.9 and 95.8 ± 14.9 ng/ml, which confirmed the steady-state trough concentration was stable. The median Tmax, ss was 0.48 h and t1/2 was 11.1 h, Ra (AUC) and Ra (Cmax) were 2.17 ± 0.15 and 1.85 ± 0.21, indicating that the obvious accumulation might occur following multiple doses of 5 mg BID WXFL10203614.The numbers and percentages of subjects who experienced AEs are summarized in In the single-dose study, 58 AEs occurred in 28 (73.7%) of the 38 subjects who received 1–33 mg WXFL10203614. The most common AEs included polyuria (11/28.9%), the proportion of neutrophils decreased (9/23.7%), fecal occult blood (positive for transferrin) (7/18.4%), neutrophil counts decreased (6/15.8%), white blood cell count decreased (3/8.0%) and creatinine increased (3/8.0%), all deemed related to WXFL10203614 before unblinding. 20 AEs were reported in 8 (66.7%) of the 12 subjects who received placebo, mainly including white blood cell count decreased (3/25.0%), polyuria (3/25.0%) and fecal occult blood (positive for transferrin) (2/16.7%). All AEs were mild or moderate and resolved spontaneously.In the multiple-dose study, 28 AEs occurred in 11 (68.8%) of the 16 subjects in the 5 and 10 mg QD groups. The most frequency AEs were polyuria (8/50.0%), fecal occult blood (positive for transferrin) (5/31.3%), creatinine increased (3/18.8%), mucositis oral (3/18.8%) and the proportion of neutrophils decreased (3/18.8%), respectively. Meanwhile, 21 AEs occurred in all subjects (8/100%) who received 5 mg BID WXFL10203614, mainly including polyuria (8/100%), fecal occult blood (positive for transferrin) (2/25.0%) and constipation (2/25.0%). All AEs were considered to be related to WXFL10203614 before unblinding. However, 15 AEs occurred in the placebo group (6/100%), which was similar to WXFL10203614 groups, such as polyuria (4/66.7%), mucositis oral (3/50.0%), fecal occult blood (positive for transferrin) (2/33.3%). All AEs were mild and subsided without treatment.There were no AEs, Serious Adverse Events (SAEs) or deaths leading to withdrawal from the single- and multiple-dose study. All AEs were reported to the Ethics Committee of Wuxi people’s hospital.As far as we know, none of the domestically produced JAK1 inhibitors are marketed in China. It is the first-in-human clinical trial of WXFL10203614, a potent and selective JAK1 inhibitor, conducted in healthy Chinese subjects. The study showed that WXFL10203614 was well-tolerated after a single dose and multiple doses. The most frequent AEs were polyuria, the proportion of neutrophils decreased, fecal occult blood (positive for transferrin), neutrophil count decreased, white blood cell count decreased and creatinine increased, most of which were more common with the higher doses, and have previously been reported in healthy subjects or patients treated with JAK inhibitors . As for max of 0.48–0.98 h, which was in accordance with the PK profiles of JAK1 inhibitors  were elevated in a dose-related manner, the conclusion of an obvious linear correlation could not be made yet. Renal excretion was proved to be the main excretion route of WXFL10203614. The slow elimination of WXFL10203614 might increase AEs caused by drug accumulation when multiple doses were given consecutively. The kidney was the main route of WXFL10203614 excretion, and chronic kidney disease was reported in 10.4% of RA patients before and 89.6% of RA patients after treatment  originating from the long-term toxicological studies of WXFL10203614 conducted in Sprague Dawley rats and beagle dogs (2 and 6 mg/kg). The NOAELs were finally converted to human equivalent doses (HEDs) according to the guidelines issued by US Food and Drug Administration . The safOnce-daily and twice-daily treatment regimens are two options for treating RA. According to the results of the preclinical pharmacodynamics studies of WXFL10203614, the efficacy of 3 mg/kg WXFL10203614 was equal to 5 mg/kg tofacitinib in the rat model of RA, and the steady plasma concentration of tofacitinib with the therapeutic dose of 5 mg BID. It is speculated that when WXFL10203614 achieved the same clinical efficacy as tofacitinib, the steady-state peak concentration and steady-state exposure should reach 39.23 ng/ml and 287.90 ng*h/mL, respectively. PK model of WXFL10203614 after multiple dosing developed by WinNonlin software which predicted that administration of 5 mg QD, 10 mg QD, 5 mg BID or 10 mg BID WXFL10203614 for 7 consecutive days could achieve this PK profile. Thus, 5 mg QD, 10 mg QD, 5 mg BID and 10 mg BID were chosen as the administration dosages in the multiple-dose study. However, due to the drug accumulation (Ra: 2.17 ± 0.15) of the 5 mg twice-daily regimen, the 10 mg BID study was not performed.The present study will help to optimize the WXFL10203614 dosing regimen for the phaseⅡstudy of WXFL10203614 in patients with moderate to severe active RA , and the drug-drug interaction (DDI) of WXFL10203614 with methotrexate in patients with RA  to evaluate the efficacy, safety and DDI of WXFL10203614 better.WXFL10203614, the potential selective JAK1 inhibitor, was well-tolerated and safe in healthy Chinese subjects, and plasma exposure increased in a dose-related manner after single-dose and once-daily administration, supporting further evaluation of WXFL10203614 in patients with RA.