Anopheles stephensi mosquito thereby preventing transmission, an essential component of the parasite life cycle. Such vaccines are envisioned as complements to vaccines that target human infection, such as RTS,S as well as drug treatment, and vector control strategies. A number of conserved proteins, including Pfs25, have been identified as promising TBV targets in research or early stage development. Pfs25 is a 25 kDa protein of Plasmodium falciparum expressed on the surface of zygotes and ookinetes. Its complex tertiary structure, including numerous cysteines, has led to difficulties in the expression of a recombinant protein that is homogeneous, with proper conformation, and free of glycosylation, a phenomenon not found in native parasite machinery.Transmission-blocking vaccines (TBVs) have become a focus of strategies to control and eventually eliminate malaria as they target the entry of sexual stage into the While the expression and purification of Pfs25 in various systems, has been previously independently reported, here a parallel analysis of Pfs25 is presented to inform on the biochemical features of Pfs25 and their impact on functionality. Three scalable expression systems were used to express, purify, and evaluate Pfs25 both in vitro and in vivo, including the ability of each protein to produce functional antibodies through the standard membrane feeding assay.Escherichia coli was not achieved, while Pichiapastoris presented Pfs25 as an inhomogeneous product with glycosylation. In comparison, baculovirus produced a pure, monomeric protein free of glycosylation. The glycosylation present for Pichia produced Pfs25, showed no notable decrease in the ability to elicit transmission reducing antibodies in functional evaluation, while a reduced and alkylated Pfs25 (derived from plant and used as a control) was found to have significantly decreased transmission reducing activity, emphasizing the importance of ensuring correct disulfide stabilized conformation during vaccine design and production.Through numerous attempts, soluble, monomeric Pfs25 derived from Pichia showed promise as candidates for vaccine development.In this study, the biochemical features of Pfs25, produced from different expression systems, are described along with their impact on the ability of the protein to elicit functional antibodies. Pfs25 expressed using baculovirus and  Plasmodium falciparum is responsible for nearly a half million deaths annually, based on the estimates from the WHO [Malaria caused by  the WHO . The eme the WHO . Particu the WHO . Such tr the WHO . To advaPlasmodium parasite to the gut of the female Anopheles stephensi mosquito after feeding on an infected human. In the mosquito gut, the Plasmodium parasite undergoes sexual-stage development, replication, and invasion of the mosquito salivary glands leading to infectious sporozoites capable of infecting humans during the mosquito’s next blood meal [Plasmodium parasite [Malaria transmission requires transport of the ood meal . As therparasite . Severalparasite .P. falciparum, which is expressed on the surface of zygotes and ookinetes [Plasmodium parasites lack the N-linked glycosylation machinery, and Pfs25 contains multiple potential glycosylation sites that could then be aberrantly glycosylated when expressed in recombinant eukaryotic systems [One of the primary targets for TBV development is the Pfs25 protein, an approximate 25 kDa sexual stage protein of okinetes –8. Pfs25okinetes , 9. Therokinetes . An addi systems . WhetherEscherichia coli [E. coli, Pichia pastoris, and baculovirus), which have been used for large-scale production of recombinant proteins, were used. Further, the purified proteins were evaluated using in vitro and in vivo tests using a previously reported plant-expressed Pfs25 [Immunogenicity of Pfs25 has been reported in both animals and in human clinical trials , 13. Thehia coli  and algahia coli  along wihia coli . The objed Pfs25  as an adE. coli, Pichia, and baculovirus. The GPI anchor sequence functions poorly in heterologous eukaryotic system and was not included [The Pfs25 sequence from 3D7 clone (ACCESSION P13829) Ala 22 to Thr 193, lacking the native signal sequence and GPI-anchor was used to produce recombinant Pfs25 in included . Codon oErwinia carotovora) and with 5′ NdeI and 3′ XhoI restriction sites was cloned into pET41a (Novagen) via standard cloning procedures. Resulting plasmids denoted as pET41a-peri-Pfs25  and pET41a-Pfs25 , were sequenced and verified.Pfs25 with and without additional N-terminal periplasmic signal sequence  agar plates at 30 °C for 3 days. Approximately 100 clones were screened for the expression of Pfs25 and an additional 2000 clones on G418 plates screened for multi-copy integrants.Briefly, two mutations were introduced (N112Q and N187Q) to avoid N-glycosylation . The resPichia, with an additional N-terminal secretion signal (MKFLVNVALVFMVVYISYIYAD from Honeybee Melittin) was cloned into pFastBac vector (Invitrogen) with BamHI (5′) and EcoRI (3′) sites and the resulting plasmid pFastBac-Pfs25 was sequence verified. The generation of recombinant virus followed Users’ Manual of Bac-to-Bac system (Invitrogen). Briefly, pFastBac-Pfs25 was transformed into E. coli DH10Bac to generate recombinant bacmid and colonies grown at 37 °C for 48 h on LB agar plates containing Tetracycline (10 μg/ml), kanamycin (50 μg/ml), gentamycin (7 μg/ml), IPTG (40 μg/ml) and X-Gal , according to users’ manual. The bacmids from selected colonies were confirmed by PCR and sequencing, then used to transfect Super Sf9 cells (Oxford Expression Technologies) for the generation of recombinant baculovirus stock (P1), using CellFECTIN II (Invitrogen) following Bac-to-Bac manual. P1 virus (approximately 14 ml) was harvested and stored at 4 °C protected from light. Two-milliliters of the P1 virus was used to amplify P2 baculovirus after infecting fresh super Sf9 cells at 27 °C for approximately 90 h and similarly to produce high titer P3 virus; remaining P1 baculovirus supernatant was stored at −80 °C. A P3 virus volume of 400 ml for further expression was harvested and titered using BacPAK titer kit (Clontech).Synthetic pfs25 containing N112Q and N187Q mutations as described in E. coli were used including cell lines BL21 (DE3) and BLR(DE3), co-expression of protein disulfide bond isomerase (DsbC), expression in lower temperature (20 °C), and IPTG concentrations (1 and 0.25 mM). All of which did not yield sufficient quantity of soluble, monomeric Pfs25 for further purification. Briefly, pET41a-Pfs25 (cytoplasmic) and pET41a-peri-Pfs25 (periplasmic) plasmids were transformed in BL21 (DE3) and BLR (DE3), respectively, and clones were selected and verified. Expression induction at 37 and 20 °C was carried out for 3 and 16 h, respectively at an optical density A600 of 0.8. Induced cultures were harvested by centrifugation for 10 min at 4 °C. The cell pellet was collected and lysed using BugBuster Protein Extraction Reagent (Novagen) per the manufacturer’s instructions. Soluble and insoluble fractions were separated via centrifugation.Several methods to express soluble Pfs25 in Pichia pastoris were screened for protein expression in 2 ml deep well plates with 2 ml complex medium containing 2 % dextrose for 2 days. Medium was then removed and replaced with 2 ml of complex medium with 1 % methanol for 3 days, with 1 % methanol feed once a day. Samples were collected on the third day post induction. Three high-yield clones were identified by analyses with reducing SDS-PAGE (Pfs25 expression band at 20 kDa), and evaluated with anti-His (Qiagen) or anti-Pfs25 mAb 4B7 [Approximately 100 clones of pMBL003-Pfs25 in  mAb 4B7 , 22, 23 In parallel, clones were screened on YPD (Yeast peptone dextrose) agar in the presence of G418  in 96-well plates—corresponding to potential increased copies of gene of interest and hence, likely increased protein yield. G418 positive clones, including ten clones by 0.5 mg/ml G418, eight by 1 mg/ml G418, and two by 2 mg/ml G418 were subjected to small-scale expression as described earlier, which led to identification of slightly better protein expressers. A selection of ten clones was then evaluated in 100 ml BMGY (Buffered Glycerol complex medium) and induced with BMMY (Buffered Methanol complex Medium) using 1 % methanol feeding per day for 3 days followed by analysis (reducing SDS-PAGE). Two colonies, C6 and H4, produced similar yield and quality of Pfs25, and C6 was arbitrarily selected for 800 ml expression and further purification.Supernatant from G418 (clone C6) was purified using Ni–NTA resin (Qiagen) using standard protocol. Briefly, 200 ml supernatant was adjusted to 1000 ml with 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, applied to column and washed  with 5 mM Imidazole in 50 mM Tris–HCl (pH 8.0), 150 mM NaCl. Proteins were eluted with 300 mM Imidazole in 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, analysed, pooled, and dialyzed into PBS buffer, pH 7.4. The pool was concentrated and further purified by gel filtration (Superdex 75).Using Super Sf9 cells, three different MOI’s  were screened at a 30 ml scale as well as uninfected cells (negative control). Post infection (48 and 72 h), 1 ml of culture was collected and centrifuged for 5 min. The supernatant was analysed via reducing and non-reducing SDS-PAGE and Western blot. Based on analysis of Pfs25 post infection of 72 h, no significant difference between the three MOI’s tested was seen and further expression proceeded with MOI = 1 at 100 ml and 72 h.Supernatant was used for batch binding overnight with ~2 ml of Ni–NTA resin  at 4 °C on table top rocker platform. Two washing steps were used: Wash buffer (1): 1x PBS with 10 mM Imidazole, pH 7.2 (10 CV) and wash buffer (2): 1x PBS with 20 mM Imidazole, pH 7.2 (10 CV). Protein was eluted via a step gradient with elution buffers (1): 1x PBS with 50 mM Imidazole, pH 7.2 and Elution buffer and (2): 1x PBS with 500 mM Imidazole, pH 7.2. Elution fractions were then analysed and those containing Pfs25 were pooled, concentrated, and further purified on Superdex 75 column.Nicotiana benthamiana was provided by Fraunhofer CMB  and utilized as a control antigen. The soluble protein, termed Pfs25MF1E, and encompassing amino acid 23–193 of the Pfs25 sequence, with a theoretical mass of 20,021 daltons is fully described in [Pfs25 derived from ribed in . The proribed in .The samples were used with either (1) 4X non-reducing NuPAGE LDS (Lithium dodecyl sulfate) sample buffer (non-reducing and non-boiled) or with reducing agent and heated to 95 °C for 5 min or (2) 4X SDS sample buffer and boiled for 10 min at 99 °C (reducing and boiled). SDS-PAGE gels (4–12 % NuPAGE Bis–Tris or 15 % Tris–glycine) were loaded in a final volume of 20 μl/well and run at 150–200 V for 35–50 min in 1X MES or 1X SDS running buffer.Following SDS-PAGE, proteins were transferred onto PVDF membrane and blocked in 5 % skim milk  at room temperature for 1 h. Primary antibodies were prepared at 1:5000 dilutions of Penta His antibody (Qiagen Cat No:- 34460), 1:2000 dilution of Anti-Pfs25 mAb 4B7 (BEI), or 1:1000 dilutions of 1G2  in 1 % skim milk in 1X TBS buffer containing 0.05 % Tween-20 (TBS-T). Blots were incubated in the primary antibody solution for 1 h at room temperature or overnight at 2–8 °C. Membranes were washed with 1X TBS-T buffer (3X for 10 min) and secondary antibody 1:1000 dilution of goat anti mouse IgG-HRP (Santa Cruz) or 1:4000 goat anti mouse IgG Alkaline Phosphatase (BioRad) in 1 % skim milk (1X TBS-T buffer) was added and incubated at room temperature for 1 h. Membranes were then again washed with 1X TBS-T buffer (3X for 10 min). HRP labeled blots were developed with TMB  substrate. Alkaline Phosphatase labeled blots were developed using Bio-Rad Alkaline Phosphatase Conjugate Substrate Kit.Spectramax plus spectrophotometer (kinetic endotoxin assay) was used to quantify endotoxin content of purified Pfs25, with EndoSafe Endotoxin standard (Charles River), EndoSafe Lysate (Charles River), and EndoSafe LAL Reagent Water (Charles River).P. falciparum NF54 gametocyte/zygote/ookinete parasites. The human serum and red blood cells used for the gametocyte cultures (extract preparation and SMFA) were purchased from Interstate Blood Bank .Mature gametocyte cultures were prepared as reported previously . From thPichia and baculovirus was performed on a BioAssist G3SWxl column  with a Shimadzu Prominence UFLC HPLC system at a flow rate of 0.7 ml/min in 0.2 M sodium phosphate pH 6.8. A gel filtration standard  was used in column calibration.SE-HPLC analysis for purified Pfs25 proteins from Pichia protein).Samples of Pfs25 were analysed using an Ultimate 3000 UHPLC system (Thermo Scientific) with a 2.6 µm, 2.1 × 150 mm C-18 column (Thermo Scientific) at a flow rate of 0.2 ml/min. C-18 column and auto-sampler temperatures were set at 60 and 5 °C, respectively. The elution of Pfs25 was monitored using absorbance (214 nm). Mobile phase (A) consisted of water with 0.1 % trifluoroacetic acid (TFA) and mobile phase (B) consisted of acetonitrile with 0.1 % TFA with a gradient of: 1 % B (5 min), 1–20 % B (2 min), 20–50 % B (30 min), 50–99 % B (2 min), 99 % B (3 min), 99–1 % B (2 min), and 1 % B (5 min). The Pfs25 samples were also run without column to determine sample recovery (82 ± 2 % for baculovirus protein and 70 ± 3 % recovery for 2 = 0.983) was constructed using known concentrations of reduced glutathione. Fluorescence was measured using a SpectraMax M5 plate reader .Free thiol (number of free cysteine residues) in each protein sample was measured using Measure iT free thiol assay kit  following manufacturer’s instructions. Samples were diluted in either ultrapure water or 2 M guanidine-HCl and 50 μg of each Pfs25 sample was used for non-denaturing conditions and 25 μg of each Pfs25 sample was used for denaturing conditions. A standard curve  were measured using a SYNAPT G2 hybrid quadrupole/ion mobility/TOF mass spectrometer  with the assistance of the Mass Spectrometry and Analytical Proteomics Laboratory at the University of Kansas. The instrument was operated in a sensitivity mode with all lenses optimized on the MH + ion from the control Leucine Enkephalin and sample cone voltage of 40 eV. Argon was admitted to the trap cell operated at 4 eV for maximum transmission. Spectra were acquired at 9091 Hz pusher frequency covering the mass range from 100 to 3000 unified atomic mass unit and accumulating data for two seconds per cycle. Time to mass calibration was made with NaI cluster ions acquired under the same conditions. Mass spectra of [Glu2O, 0.1 % formic acid) and B  over a short gradient from 1 to 70 % B in 4 min with a flow rate of 20 µl/min. MassLynx 4.1 software (Waters Corporation) was used to collect data and deconvolute the protein spectra for molecular weight determination.Samples were desalted via reversed phase PRP-1 column, 1 cm, 1 mm I.D.  using a NanoAcquity chromatographic system (Waters Corporation) and solvents A  and 9-fluorenylmethyl chloroformate (FMOC).Pfs25 protein derived from plant at 1 mg/ml, pH 8.0 was reduced with 20 mM DTT at 60 °C for 30 min and alkylated with 40 mM iodoacetamide at 37 °C for 30 min in the dark; alkylation was quenched with large excess of β-mercaptoethanol. The extent of modified cysteines was analysed by amino acid analysis (AAA) at AI BioTech  with pre-column derivation using Pichia produced Pfs25; Group 3, positive control (plant produced Pfs25 [intramuscularly) on day 0 and 21. On day 42 sera were collected and individual sera were tested by ELISA. In addition, sera were pooled, IgG affinity purified and tested at 0.75 mg/ml as well as three-fold dilutions  for SMFA activity [P. falciparum NF 54 gametocytes and purified IgG from mice immunized with Pfs25 were fed to female Anopheles stephensi mosquitoes. Mosquitoes were then dissected 8 days after the feeding and the number of oocysts counted to measure transmission reduction activity.To produce anti-Pfs25 sera in CD-1 mice for SMFA, 10 µg of Pfs25 was formulated with ISA720 (Montanide) for each of the following groups: Group 1, baculovirus produced Pfs25; Group 2, ed Pfs25 ); Group activity . BrieflyBasic methodology of ELISA has been described by Miura et al. . All ELIE. coli, and as secreted proteins in Pichia and baculovirus (super Sf9) at small scale and purified by Ni–NTA chromatography and gel filtration for subsequent evaluation. In E. coli, the majority of Pfs25 expressed was present in inclusion bodies and, therefore, sufficient soluble monomeric Pfs25 from E. coli lysates for analysis was not achievable.Recombinant His-tagged Pfs25 was produced as an intracellular protein in E. coli protein. Hereafter, Pfs25 solely from the Pichia and baculovirus expression systems were used for further biochemical in vitro and functional in vivo analysis. A well-characterized Pfs25, derived from plant [It has been reported that it is possible to conduct refolding of Pfs25  however om plant , was proPichia gave rise to a major protein migrating at ~20 kDa and a minor band at ~21 kDa present at approximately 50 % of the intensity of 20 kDa band , which implicated the presence of 11 pairs of disulfides [As shown in the same intact mass analysis Fig.  the Pichsulfides . The intsulfides . This trsulfides .Table 1PPichia and baculovirus) contained <0.1 % free thiol, as no fluorescence signal was detected above the buffer control, supporting the observations that all 22 cysteines were oxidized and disulfide paired as suggested by the intact mass observations described above  assay was utilized to further quantify the inhomogeneity Fig.  of the PPichia-expressed Pfs25, whether this modification would affect the biological activity of Pfs25, was investigated. While a previous study utilizing DNA vaccination showed limited detrimental effects of glycosylation on transmission reducing activity [After observing glycosylation in activity , data onactivity . Before For the immunizations, the plant protein was used as a positive control and a reduced alkylated form of the plant protein was used as a negative control to confirm the importance of disulfide bond formation of Pfs25. Further analysis of the reduced alkylated Pfs25 preparation via amino acid composition analysis recovered 13 carboxymethylated cysteine, indicating at least 13 out of 22 cysteines were modified by alkylation.Pichia expressed Pfs25 (Group 2), glycosylation did not apparently affect the transmission reducing activity as detected here, with results comparable to Pfs25 produced from baculovirus (Group 1) or plant (Group 3) that each exhibited little or no glycosylation  were not statistically different between the Pfs25 groups as determined through a non-parametric Kruskal–Wallis test and Dunn’s multiple comparisons test Fig. . Statiston Table  and yielPichia and baculovirus systems. In the E. coli system, in spite of numerous attempts, soluble, monomeric Pfs25 was not achieved. While it may be possible to generate native configuration Pfs25 using refolding methods from the generated inclusion bodies [Pichia and baculovirus expression resulted in proteins with many characteristics consistent with the expected structure of the native Pfs25. In Pichia, the expressed Pfs25 was likely glycosylated, despite mutations of probable N-linked sites, and consequently resulted in multiple molecular weight forms. The baculovirus expressed Pfs25 presented a non-glycosylated and homogeneous recombinant protein. Through multiple methods, including the free thiol assay and intact mass analysis, both Pichia and baculovirus produced Pfs25 proteins contained 11 pairs of disulfides as expected. Two transmission blocking monoclonal antibodies 4B7 [Pichia produced Pfs25 protein suggesting proper folding of the purified proteins.In the search for a transmission blocking vaccine based on Pfs25, the malaria community has been guided by the concept that an immunogen should mimic as much as possible the Pfs25 found on the surface of the parasite. In the current studies, Pfs25 was expressed, purified, and characterized in the n bodies , this wadies 4B7 , 23 and dies 4B7 , also biP. falciparum apparently lacks such modifications, glycosylation has been a concern in the development of recombinant antigens for use as TBV and other malaria vaccine candidates. In this study, the evidence of limited glycosylation present in Pichia produced Pfs25 did not appear to diminish the ability to elicit transmission blocking antibodies and was consistent with findings carried out with Pfs25 DNA vaccines evaluating glycosylation of Pfs25 [The ability to elicit functional transmission blocking antibodies is the most important characteristic of any protein preparation to be considered for the generation of a vaccine. Given that of Pfs25 . It is iPichia, baculorvirus as well as the control plant produced protein showed evidence of no free cysteines by two methods, free thiol and mass spectrometric analysis. When these disulfide bonds are disrupted by reduction and alkylation the elicitation of functional antibodies (as measured by SMFA) was diminished, demonstrating that at least some of the disulfide bonds are essential for the function of the Pfs25 protein as an immunogen. Taken together, these results indicate that both Pichiapastoris and baculovirus expression systems have potential for the production of Pfs25 based transmission-blocking vaccines.Lastly, the status of the disulfide bonds formed in the recombinant proteins was investigated. Proteins produced in


PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize γ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates of γ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the γ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing the γ-PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications.To clone and express the  It is an anionic polypeptide formed by the condensation of amide linkages between α-amino and γ-carboxylic acid groups of the D- and/or L-glutamate in microorganisms. It has nontoxic, edible, adhesive, film-forming, and moisture retention properties [γ-PGA and its derivatives can be used as drug carriers and bioadhesive materials that have been widely used in pharmaceutical, cosmetics, food, agriculture, and sewage treatment industries and have become one of the most interesting topics in biopolymer research [Gamma-polyglutamic acid  and are classified as glutamate-dependent or glutamate nondependent types based on their needs for glutamate [γ-PGA-producing strains have unstable heritability, easily leading to a reduction or loss in the ability to synthesize γ-PGA during fermentation, undergo γ-PGA degradation, and produce extracellular polysaccharide by-products, thereby lowering product yield. Compared to traditional mutation breeding, genetic engineering technologies have been expected to become an effective method to create γ-PGA high-yield strains. Ashiuchi et al. [ pgsB, pgsC, and pgsA are three essential genes involved in γ-PGA synthesis in glutamate-dependent strains. Urushibata et al. [ pgsBCA gene through different methods of fusion expression and further transformed the plasmids into Escherichia coli to obtain positive clones that were capable of producing γ-PGA. E. coli, a gram-negative bacterium, has been reported as the primary host strain for transforming the recombinant vector of the γ-PGA synthase gene. However, its synthase gene is mainly derived from Bacillus subtilis (gram-positive bacteria). The membrane structures and protein secretion systems of both types of bacteria vary, which in turn may result in poor positioning of the recombinant expressed γ-PGA synthase system on the bacterial cell membrane [γ-PGA in the host strain is lower; and the amount of γ-PGA obtained from positive clones is only within the range of 0.024–0.134 g/L [ B. subtilis, as a prokaryotic expression host for food safety, carries some excellent features in expressing γ-PGA that E. coli does not possess. For example, B. subtilis is capable of expressing the soluble and nonfusion proteins, as well as preferentially expressing the nonpathogenic and nonapparent codons [ pgsBCA in B. subtilis was comparatively scarce. To date, the expression of the γ-PGA synthase gene pgsBCA still need D-xylose and L-arabinose induced, generally with poor expression yield and low molecular weight (only 200–500 kDa) [ pgsBCA gene was reconstructed and highly expressed in B. subtilis as to improve the yield and molecular weight of γ-PGA. B. subtilis 168 has been widely used in the study of γ-PGA regulation. It is one of the few bacterial strains that has a complete set of γ-PGA synthase genes but does not produce γ-PGA [ B. subtilis 168 as DNA template to amplify the γ-PGA synthase gene pgsBCA and to further clone the pgsBCA gene into the B. subtilis expression vector pWB980 to transform into type strain B. subtilis WB600. We constructed a recombinant B. subtilis expression system for γ-PGA synthesis, which may serve as a foundation for the high-yield industrial production of γ-PGA based on an engineered B. subtilis expression system.Traditionally, entation . Bacterilutamate . Wild-tyi et al.  and Tarui et al.  confirmea et al.  and Jiana et al.  construcmembrane . Therefo.134 g/L . B. subtt codons . In addi500 kDa) , indicatB. subtilis 168 and B. subtilis WB600 were purchased from Shanghai Genemy BioTech Co., Ltd. ; E. coli JM109 was prepared and preserved at our laboratory and described in a previous study. pMD19-T vector and B. subtilis expression vector pWB980 were purchased from TakaRa Biotechnology  Co., Ltd. . Taq DNA polymerase, dNTPs, DNA ladder marker, and protein molecular weight markers, were purchased from TakaRa Biotechnology  Co., Ltd. Plasmid extraction and agarose DNA extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. . Bacterial genomic DNA extraction kits were purchased from and primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. . Silica gel plates for thin layer chromatography (TLC) were purchased from Qingdao Jiyida Silica Reagent Factory .All restriction endonucleases, T4 DNA ligase, E. coli and B. subtilis transformants were selected with 50 μg/mL ampicillin (Ampr) and 30 μg/mL kanamycin (Kmr), respectively. Fermentation broth for the genetically engineered recombinant bacteria contained 40 g/L glucose, 0–100 g/L sodium glutamate, 6 g/L (NH4)2SO4, 2 g/L K2HPO4, and 0.2 g/L MgSO4 (pH 7.5).Lysogeny broth (LB) was prepared using 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl (pH 7.0) and 2.0% (W/V) agar powder to solidify the medium. pgsB, pgsC, and pgsA coding gene sequences of B. subtilis 168 were designed as follows: BAC1: 5′-CGCGGATCCATGTGGTTACTCATFATAGCC-3′ (restriction site of BamHI endonuclease is underlined); BAC2: 5′-CCCA AGCTTTTATTTAGATTTTAGTTTGTCA C-3′ (restriction site of HindIII endonuclease is underlined).With reference to the NCBI database, the upstream and downstreamB. subtilis 168 genomic DNA was used as template. BAC1 and BAC2 primers were used to amplify the gene. The PCR reaction system included 2 μL of DNA template, 10 μL of 5x buffer, 2 μL of dNTPs, 2 μL of individual primers of BAC1 and BAC2, 0.5 μL of 5x Primer STAR, and sterile double-distilled water to prepare a final volume of 50 μL. Reaction conditions were as follows: 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 55°C for 15 s, and 72°C for 3 min, and a final 72°C extension for 10 min. One percent agarose gel electrophoresis was used to identify the PCR reaction products. PCR products were recovered using a DNA rapid recovery reagent and ligated into the pMD19-T vector, which was followed by transformation into E. coli JM109 competent cells using CaCl2 methods. The selected single colonies were inoculated into liquid LB to expand the plasmid. Intermediate vectors pMD-pgsBCA were then obtained and identified using BamHI and HindIII double digestion as well as sequencing.BamHI and HindIII double digestion was performed to cut the intermediate vector pMD-T-pgsBCA and pWB980 plasmid, followed by ligating these into the recombinant expression vector, pWB980-pgsBCA  [γ-PGA samples underwent infrared spectroscopy using Shimadzu's IR Prestige-21 infrared spectrometer, Shimadzu (China) Co., Ltd. . Potassium bromide (KBr) was used as reference material [γ-PGA was determined by SDS-PAGE [y (HPLC) . The purmaterial . The molSDS-PAGE . pgsBCA fragment, 2.8 kb, was in agreement with our expected results. An agarose DNA extraction kit was used to recover and purify the PCR products. After confirming with DNA sequencing, the DNA sequence of the PCR products was determined to be 100% identical with the sequence of the reported gene of B. subtilis 168.The target gene was amplified by PCR. pgsBCA, into competent cells, the plasmids were collected and identified using BamHI and HindIII restriction enzyme digestions.  pgsBCA PCR products, thereby initially confirming the successful construction of the recombinant expression vector, pWB980-pgsBCA.After transforming the constructed recombinant expression vectors, pWB980-γ-PGA was enhanced. However, when glutamate concentration was >50 g/L, the synthetic yield of γ-PGA declined. This result suggested that pgsBCA was secreted by B. subtilis WB600. The expressed product, γ-PGA, could be secreted into extracellular fermentation broth. Using a lower substrate concentration, we observed that the recombinant bacteria did not synthesize γ-PGA, indicating that an excess amount of substrate was necessary for the recombinant bacteria to synthesize γ-PGA. Therefore, from the perspective of economic efficiency, we identified that a substrate concentration of 50 g/L was optimal to synthesize the highest possible amount of γ-PGA (1.74 g/L).γ-PGA, no other band was observed on the silica gel plates but only single spots of uniform color intensity. Its retention (Rf) value was consistent with that of the standard, glutamate spots, indicating that the hydrolysates had no other amino acids and other protein impurities. These hydrolysates were in single form, solely consisting of pure glutamic acid. γ-PGA. The absorption peak at 3,421 cm−1 was the symmetric stretching vibration band of N-H; and the absorption peak at 1,649 cm−1 was the asymmetric stretching vibration band of an amide group, -CONHR. Both peaks were the main indicators used in the identification of amides and for the presence of amide groups in γ-PGA molecules. The absorption peak at 1,408 cm−1 was the symmetric stretching vibration band of COOH; the absorption peak at 1,076 cm−1 was the hallmark peak representing the presence of aliphatic hydrocarbons, -CH2 or -CH3 , in the molecular structure; and the absorption peaks within the range of 1,000 cm−1–500 cm−1 were caused by the (CH2)n (n > 4) planar rocking vibration, as well as in-plane bending vibration. The spectral characteristics of recombinant γ-PGA in fermentation broth was consistent with those of the standard γ-PGA's IR spectroscopy, indicating that the sample obtained in the present study contained the N-H and C=O functional groups, as well as the aliphatic hydrocarbon structure (CH2)4 of the γ-PGA [γ-PGA. The molecular weight of the γ-PGA sample obtained after the fermentation, isolation, and separation of recombinant strain Bacillus WB600-pgsBCA was determined using SDS-PAGE. γ-PGA was between 500 and 600 kDa and occurred as aggregates of a multimolecular mass, but not of a single molecular composition.he γ-PGA , therebyγ-PGA synthase gene pgsBCA in B. subtilis and used plasmid pWB980 to construct the recombinant expression vector, pWB980-pgsBCA, and to further transfer the recombinant expression vector into B. subtilis WB600. The P43 promoter of pWB980 induced the expression of pgsBCA; then the host cells of this expression vector showed a capacity to synthesize γ-PGA, and the product yield of γ-PGA reached ≥1.74 g/L. The isolated and purified γ-PGA sample from the fermentation broth was confirmed to have a single form of hydrolysates that solely consisted of pure glutamic acid. This result matched the characteristics of the standard γ-PGA's IR spectroscopy and showed the aggregates of a multimolecular mass, with a molecular weight ranging between 500 and 600 kDa.The present study evaluated the cloning and expression of  B. subtilis as the expression host, and the pgsBCA gene originated and was expressed in B. subtilis. The γ-PGA synthase system is better positioned in the cell membrane , D-xylose, and L-arabinose as an inducer to secrete the pgsBCA into the extracellular fermentation broth is circumvented using the methodology developed in the present study. This technique may also be potentially used in industrial production as it can increase the stability of products, simplify the purification work, and have more obvious application potential advantage.The present study usedficiency –20. The g report –24. The  Bacillus WB600-pgsBCA showed the capacity to synthesize γ-PGA, our results still could not match the highest synthetic yield of γ-PGA (40–50 g/L) that is induced by the fermentation of mutated bacteria [γ-PGA and to increase the bacterial concentration, oxygen uptake, or endogenous synthase expression, thereby ultimately increasing γ-PGA yield [γ-PGA-producing strains to reduce γ-PGA degradation, thereby increasing γ-PGA yield [γ-PGA yield, thereby laying the foundation for the industrial production of high-yielding γ-PGA engineered bacteria based on the B. subtilis expression system.Although the constructed recombinant bacteriabacteria , 26. TheGA yield , 28. AltGA yield . Therefo


Mental disorders are always remained a neglected public health problems in low and middle-income countries (LMICs), most people with mental disorders never receive effective care and there is a large treatment gap. In order to solve the problem integration of mental health into primary health care is recommended and in Ethiopia implementation of the scale of mental health services at primary health care level was started in 2014. For the success of the integration of mental health into primary health care, primary care health professionals are the key personnel who are responsible for the management of mental, neurologic and substance use disorders. However, proper training and education of primary care health professionals is mandatory for an optimal performance and success of integration. This interventional study was conducted to assess the effectiveness of mental health training course for scale up of mental health services at primary health care level in Ethiopia.t test with p values was performed to test the differences between the pre- and post-test. In additions mean and standard deviation of the responses were calculated. Overall the response rate was 100% at the end of the intervention.This quasi-experimental pre- and post-study design was conducted in Ethiopia from October to December 2016 using quantitative data collection methods. A total of 94 primary health care professionals were included in the study. The intervention was conducted by psychiatry professionals using standardized World Health Organization (WHO) Mental Health Gap Action Programme (mhGAP) guide prepared for scaling up of mental health care through integration into primary health care (PHC) and general medical services. Pre- and post intervention assessment was done for knowledge, attitude and practice (KAP); and statistically analyzed. A paired sample The study resulted in a significant improvement in knowledge, attitude and practice (KAP) of PHC workers about all the four mental, neurologic and substance use disorders during the post intervention survey (p < 0.05). Post intervention the knowledge of health professionals increased by 53.19% for psychosis, 42.56% for depression, 19.25% for epilepsy and 54.22% for alcohol use disorders. Similarly, post intervention attitude increased by 55.32% for psychosis, 40.42% for depression, 36.17% for epilepsy and 43.6% for alcohol use disorders. In addition, post intervention case identification rate increased by 62.78% for psychosis, 55.46% for depression, 21.35% for epilepsy and 41.49% for alcohol use disorders with significant p value (p < 0.05).The study results suggest that mental health training could be an effective intervention for improving knowledge, attitudes, and practices among primary health care professionals regarding mental, neurologic and substance use disorders. Training is a prerequisite and vital to enhance the knowledge, attitude, and practice of primary care professionals which plays a significant role for the easy success of integrated care and treatment of mental, neurologic and substance use disorders into the existing general health care services. Mental, neurological and substance use (MNS) disorders constitute a huge global burden of disease, and there is a large treatment gap, particularly in low- and middle-income countries (LMICs) and they are major contributors to morbidity and premature mortality . MNS disDespite the high morbidity and premature mortality due to mental disorders in LMICs, which are often comorbid with physical diseases, there is a scarcity of mental health specialists , 12, 13.Scaling up of mental health care through integration into PHC and general medical services in Ethiopia was implemented in 2014 , 17. TheOne of the major challenges of successful integration of mental health into PHC is the lack of adequate knowledge, positive attitude, and skills for mental health service of primary health care professionals participating in care and treatment of peoples at primary health care levels . This paQuasi-experimental a single group pre-test/post-test design was used in order to evaluate the effect of training on knowledge, attitude and practice of health professionals working at primary health care level in Ethiopia from October to December 2016. We carried pre-intervention measurements of knowledge, attitude and practice among participates of the total sample and we assessed post training knowledge and attitude of the same participants after 5 days of training. Post training practice was assessed after 3-month work experience at the primary health care level by reviewing charts and records.The study is conducted in Ethiopia. In Ethiopia mhGAP scale upfor people suffering from MNS disorders was implemented in different regions in 2014. The main aim of mhGAP program is implementation and Scaling up care for MNS disorders in PHC facilities  by non-specialized professionals (working at first- and second-level facilities) , 19. SelThe training programme is the result of cooperation between the Federal ministry of health of Ethiopia, Addis Ababa Health Bureau, and Amanuel Mental specialized Hospital. Training was given on four selected WHO mhGAP priority MNS disorders for scale up service in Ethiopia , 19. TraAlcohol use disordersDepressionPsychosisEpilepsyHere are the four selected WHO mhGAP priority disorders:The training was given in four rounds and 23, 24, 24 and 23 health professionals participated in the first, second, third and fourth round training respectively.We used non-probability sampling technique and all participant who came for the training were included in the study. A total of 94 primary health care professionals  selected from different primary health care levels were included in the study.Data were collected using a self-completed questionnaire that contained three sections.Questionnaires were asked about vignette case identification after Vignette descriptions of common priority MNS disorders such as psychosis, depression, epilepsy and alcohol use disorders both pre- and post training. WHO study design with case vignettes has also been used in a study Butajira to assess attitude about mental disorders , on EthiSelf-report of years of clinical experience and whether or not the respondent had received pre-service training in mental health care and whether or not the patient had experience in diagnosis and treatment of common mental, neurologic and substance use disorders. In addition, cases identified and treated post training at primary health care level by trained primary health care professionals were collected by the chart review after 3 months of the training.A structured questionnaire to investigate knowledge and attitudes of PHC workers towards mental illness was developed for the purpose of the study. The questionnaire drew on previous research in this area from LMIC settings –27. KnowGenetic exposure causes the disorderUse of psychoactive substance causes the disorderNeurochemical imbalance causes the disorderLoss of loved one causes the disorderConflict in marriage causes the disorderAcademic failure causes the disorderDivorce causes the disorderPhysical or sexual abuse causes the disorderUnemployment causes the disorderWork overload causes the disorderFinancial constraints cause the disorderEvil eye causes the disorderEvil spirit causes the disorderCurse causes the disorderDue to sins committed causes the disorderWill of God causes the disorderMagic causes the disorderThe condition is treatableThe condition has good outcome in most patientsThe person treated in the same health center with the general patient. What about you?Traditional healers are better in effectiveness than our medical care in treating this condition? What about you?The person can marry and may bring children’s?The person can be employed and able to work effectively.The person can leave with others in society?The data were cleaned before final analyses by looking at the distribution of the data, identifying outliers and checking back against the original data. Data analysis was carried out using SPSS version 23 for Windows. Most of the responses to the structured questionnaire were analyzed descriptively, as percentages or summary measures of central tendency. Sociodemographic and other factors were analyzed and reported by using words and table. A paired sample t test was performed to test the differences between the pre- and post-test. In additions mean and standard deviation of the responses were calculated.Ethical clearance was obtained from the research ethics committee of Amanuel mental specialized hospital research and training department. Informed, written consent was obtained from each study participant. The right to withdraw from the research process at any point in time was respected. Privacy and strict confidentiality were maintained during the interview process.A total of 94 participants were included in the study which makes the response rate 100%. The mean age of the respondents was 27.80 (± standard deviation = 11.70) years.Among total participants, more than two-third of them 66 (70.21%) were females, 34 (36.14%) were married, 44 (46.81%) were between the ages of 25–30 years.More than one-third of the study participants 44 46.81%) were health officers, the majority of the participants, 70 (74.47%) were Orthodox Christians and around forty-two participants 44.68% were Oromo by ethnicity. Majority of participants, 72 (76.60%) had taken psychiatry courses during under graduate training and more than half of them had 4–7 years of experience  for psychosis, 95.74% (p = 0.001), for depression 89.36% (p = 0.002) for epilepsy and 90.42% (p = 0.001) for alcohol use disorders showed favorable attitude  disorders, post- intervention the participant’s case detection and treatment showed significant improvement, which was assessed after three-month practice at primary health care level. Post intervention case identification of the participants increased from 9.53 to 20.84% (p = 001) for psychosis, and 15.87–18.75% (p = 0.001) for depression. Similarly, post- intervention case identification of the participants showed significant improvement for epilepsy and alcohol use disorders  countries due to lack of attention, problems related to of awareness and negative attitude by health care professionals , 17, 18.Consistent with previous research , 18, priAccording to the current study the participants showed significant increase post- intervention in proportion of knowledge about mental, neurologic and substance use. Pre- and post-training evaluation indicated that post-intervention proportion of the participant’s knowledge about mental, neurologic and substance use disorders showed significant improvement. Post intervention knowledge of the participants about psychosis increased from 34.04 to 87.23% (p = 000). Similarly, greatest improvement was observed after training on participant’s knowledge about depression, epilepsy and alcohol use disorders. This result indicates that training has significant effect on knowledge primary health care workers related to mental, neurologic and substance use disorders which is vital for success of integrated services. This findings are in line with other studies done in Nigeria  and otheThis study demonstrated that training has significant effect on attitude of primary health care workers about mental, neurologic and substance use disorders. The effect is higher for psychosis followed depression and alcohol use disorders. Post-intervention the majority of the participants, 89.36% (p = 0.000) for psychosis, 95.74% (p = 0.001), for depression 89.36% (p = 0.002) for epilepsy and 90.42% (p = 0.001) for alcohol use disorders showed favorable attitude. This findings are in agreement with other studies done in Nigeria  and otheWe found a change in the practice of primary health care professionals about mental, neurologic and substance use disorders after the training. This findings suggests that effectiveness of mental health training on practice of primary health care workers and continues training and education is vital for success of mental health integration into general health care services. Post- intervention the participants case detection and treatment showed significant improvement, which was assessed after three-month practice at primary health care level. Post intervention case identification of the participants increased from 9.53% to 20.84% (p = 001) for psychosis, and 15.87 to 18.75% (p = 0.001) for depression. Similarly, post- intervention case identification of the participants showed significant improvement for epilepsy and alcohol use disorders. This findings are in agreement with other studies done in Nigeria , 29.This study also indicated that there is statically significant difference in vignette case identification of primary health care workers pre- and post-training. The intervention had a large impact on case identification of primary health care workers, which is essential for the success of the integration of mental health into primary health care services. The vignette case identification of trainees increased from 31.92 to 94.68% (p = 000) for psychosis, 34.04 to 90% (p = 001) for depression, 75.45 to 96.80% (p = 003), for epilepsy and 53.19 to 94.68% (p = 002) for alcohol use disorder. This findings are in agreement with other studies done in Nigeria  and otheThe study resulted in a significant improvement in KAP of PHC workers about all the four mental, neurologic and substance use disorders during the post intervention survey. PHC workers showed significant increase post intervention in proportion of knowledge, attitude and practice related to common mental, neurologic and substance use disorders selected and implemented for scale and integration of mental health services into primary health care in Ethiopia. Training is a prerequisite and vital to enhance the knowledge, attitude, and practice of primary care professionals which plays a significant role for the easy success of integrated care and treatment of mental, neurologic and substance use disorders into the existing general health care services.The mail limitations of this study was failure discuss and compare the results with other previous studies due to lack adequate studies in the area.


A high‐fibre diet and one rich in fruit and vegetables have long been associated with lower risk of chronic disease. There are several possible mechanisms underpinning these associations, but one likely important factor is the production of bioactive molecules from plant‐based foods by the bacteria in the colon. This links to our growing understanding of the role of the gut microbiome in promoting health. Polyphenolic‐rich plant foods have been associated with potential health effects in many studies, but the bioavailability of polyphenol compounds, as eaten, is often very low. Most of the ingested molecules enter the large intestine where they are catabolised to smaller phenolic acids that may be the key bioactive effectors. Dietary fibres, present in plant foods, are also fermented by the bacteria to short‐chain fatty acids, compounds associated with several beneficial effects on cell turnover, metabolism and eating behaviour. Polyphenols and fibre are often eaten together, but there is a lack of research investigating the interaction between these two groups of key substrates for the colonic bacteria. In a project funded by the Biotechnology and Biological Sciences Research Council Diet and Health Research Industry Club, we are investigating whether combining different fibres and polyphenol sources can enhance the production of bioactive phenolic acids to promote health. This could lead to improved dietary recommendations and to new products with enhanced potential health‐promoting actions. There has been intense interest in the role of the gut microbiome in human health over the last decade. The ability to describe the genetic repertoire of bacterial populations without the need to isolate, culture and characterise each organism has revolutionised our ability to understand the complexity of this important ecosystem. It is becoming increasingly evident that our gut bacteria have important influences on several functions of the human body. The diversity and composition of the gut microbiota has been associated with a wide variety of disorders and pathologies including obesity  study. In Mediterranean countries, flavonoids were the main polyphenolic contributors but were only the second contributor in non‐Mediterranean Europe where phenolic acids were the main contributor. In Mediterranean countries, the main food sources were coffee, fruit and then wine. In non‐Mediterranean countries, the order of contribution was coffee, tea and then fruit. Fruit was the main source of flavonoids in Mediterranean countries, whereas tea was the main source in the non‐Mediterranean countries. Flavonoids include flavonols, flavones, flavanones, isoflavones, flavanols and anthocyanins. However, most of these parent polyphenols are not well absorbed in the small intestine  thus complicating the interpretation of plasma and urine phenolic acid measurements. One of the main intermediate metabolites of rutin catabolism, 3,4–dihydroxy phenyl acetic acid , exhibited greater inhibition of anti‐platelet aggregation , which are expressed on a wide array of cell types . In previous pilot studies, we have shown that combining fermentable carbohydrates and polyphenols in an in vitro model of colonic fermentation speeded up the breakdown of the polyphenol rutin  Diet and Health Research Industry Club (DRINC) initiative, we are exploring the interactions between dietary fibres and colonic polyphenol catabolism in a systematic fashion. Starting with in vitro fermentation models, using human faecal bacteria, a range of fibres has been combined at different doses with a range of polyphenols, so that the relative effects of (1) fibre on individual polyphenol catabolism and (2) the polyphenols on SCFA production can be estimated. The results of these fermentations will then inform the choice of two fibre‐polyphenol mixtures to be studied in acute bioavailability studies in humans. Phenolic acid production will be measured in urine over 24 hours after a test meal. Finally, a 6‐week feeding study will explore the longer term interactive effects of the fibre and polyphenol mixture on phenolic acids and also biomarkers of inflammation and health. We are using stable isotope‐labelled polyphenols and foods to confirm the source of phenolic acids in these studies. Thus, the results of this project should inform clearer dietary recommendations and may lead to new product designs for enhancing the positive actions of the dietary polyphenols.In our project We have no conflict of interest to declare.


Cognitive impairment (CI) has been described in 3–80% of Systemic lupus erythematosus (SLE) patients but only short-term studies evaluated its over-time changes, suggesting that CI is usually a stable finding. We aimed at evaluating the changes of SLE-related CI in a 10-years prospective single center cohort study.We evaluated 43 patients  at baseline (T0) and after 10 years (T1). A test battery designed to detect fronto-subcortical dysfunction across five domains  was administered. A global cognitive dysfunction score (GCD) was obtained and associated with clinical and laboratory features.Prevalence of CI was 20.9% at T0 and 13.9% at T1 (P = NS). This impairment was prevalently mild at T0 (55.5%) and mild or moderate at T1 (36.3% for both degrees). After 10 years, CI improved in 50% of patients, while 10% worsened. Impaired memory (P = 0.02), executive functions (P = 0.02) and abstract reasoning (P = 0.03) were associated with dyslipidemia at T0. Worsening of visuospatial functions was significantly associated with dyslipidemia and Lupus Anticoagulant (P = 0.04 for both parameters). Finally, GCD significantly correlated with chronic damage measured by SLICC/damage index at T0  and T1 .For the first time, we assessed CI changes over 10-years in SLE. CI improved in the majority of the patients. Furthermore, we observed an improvement of the overall cognitive functions. These results could suggest that an appropriate management of the disease during the follow-up could be able to control SLE-related CI. Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by a multifactorial etiology, in which genetic and environmental factors determine disease development . The proAmong the different clinical manifestations, neuropsychiatric involvement can affect up to 90% of SLE patients.–9 A wideCognitive impairment (CI) represents one of the most common neuropsychiatric feature in SLE patients, with a prevalence ranging from 3% to 80%. –15 This From a pathogenic point of view, NPSLE development has been related to the presence of autoantibodies and cytokine-mediated neuronal dysfunctions, vasculopathy, and coagulopathy. Several With regard to the assessment of SLE-related CI, the ACR Ad Hoc Committee on Neuropsychiatric Lupus nomenclature proposed in 1999 a brief research battery able to quantify these dysfunctions. So far, the studies assessing cognitive impairment in SLE patients are mostly cross‐sectional, without providing information about over-time changes.To the best of our knowledge, only four longitudinal studies have been conducted, with a maximum follow-up of 5 years. –25 TakenThus, in the present 10-year prospective study, we aimed at evaluating the changes of CI in a single center SLE cohort. Secondly, we evaluated the correlations between CI and clinical and laboratory SLE-related features.Fifty-eight adult patients affected by SLE according to the ACR revised criteria, were enrolled consecutively in this longitudinal study at the Lupus Clinic, Sapienza University of Rome. Written informed consent was obtained from each patient and the local ethic committee approved the study design. The baseline features of this cohort were described in a previous study. According to the study protocol, the patients were evaluated at baseline (T0) and after 10 years (T1).Study protocol included complete physical examination and blood drawing. The clinical and laboratory data were collected in a standardized computerized electronically filled form including demographics, past medical history with date of diagnosis, co-morbidities, and previous and concomitant treatments.Crithidia Luciliae (titer ≥1: 10), ENA  by ELISA assay considering titers above the cut-off of the reference laboratory, anti-cardiolipin (anti-CL) (IgG/IgM isotype) by ELISA, in serum or plasma, at medium or high titers , anti-β2 Glycoprotein-I (anti-β2GPI) (IgG/IgM isotype) by ELISA, in serum (above the 99th percentile), and lupus anticoagulant (LA) according to the guidelines of the International Society on Thrombosis and Hemostasis. Finally, C3 and C4 serum concentrations were determined by means of radial immunodiffusion.Each subject underwent peripheral blood sample collection. The study protocol included the determination of autoantibodies and the evaluation of C3 and C4 serum levels. Specifically, ANA has been determined by means of indirect immunofluorescence (IIF) on HEp-2 , anti-dsDNA with IIF on Disease activity was assessed by using the SLE Disease Activity Index 2000 (SLEDAI-2K).  AccordinThe Systemic Lupus International Collaborative Clinics/American College of Rheumatology (SLICC/ACR) Damage Index (SDI) was applied to evaluate the chronic damage. All patients underwent a comprehensive cognitive-behavioral neuropsychological assessment, performed by the same neurologist (CM) at baseline and after 10 years of follow-up.Neurocognitive assessment was performed during a 1-hour interview and included standardized testing for five domains: memory, attention, abstract reasoning, executive and visuospatial functions. This assessment included those tests from the ACR and the CSI standardized in an Italian population, and was specifically designed to detect the fronto-subcortical dysfunction typical of SLE.A Minnesota Multiphasic Personality Inventory (MMPI) was administered to all the patients in order to exclude the influence of behavioral abnormalities on cognitive dysfunction.  The follMini Mental State Examination (MMSE) for general cognitive status , 33, 34.Rey Auditory Verbal Learning Test and Digit Span forward, two efficient neuropsychological instruments for testing verbal memory;Immediate Visual Memory Test  and Corsi Block-Tapping Test forward, used to measure visuospatial memory;Copying of Drawings with and without elements of programming, two common tools to evaluate visuospatial abilities;Attentive Matrices for both selective and sustained attention;Raven’s Progressive Matrices, a widely used non-verbal intelligence test for abstract reasoning;Digit Span backward, Corsi Block-Tapping Test backward, Phonological Verbal Fluency Test, Trail Making Test A, Trail Making Test B, Wisconsin Card Sorting Test, Analogies Test and Time and Weight Estimation Test, STEP, to investigate deeply the presence of executive dysfunctions. Unadjusted analysis was performed as previously described. , 35Briefly, for each patient, the raw scores from each test were compared with published norms  and transformed into Z scores to express the deviation from the normal mean [Z = (raw data2test mean)/test standard deviation]. Mean domain Z scores (MDZs) were defined as the average of the Z scores from the tests comprising each domain. To indicate cognitive function as a composite score, the Z score for each domain was transformed into a Domain Cognitive Dysfunction score (DCDs), with higher values representing more impairment in a particular domain. The sum of all DCDs across the five domains resulted in the Global Cognitive Dysfunction score (GCDs), which was transformed into a Global Cognitive Dysfunction category (GCDc).This method was summarized in 2) test or Fisher’s exact test where appropriate. Two-tailed P values were reported, P values less than or equal to 0.05 were considered significant. GCDs were compared in patients grouped by antibody level. The binary outcomes variable for the antibody testing were serum autoantibody status, defined either as present versus absent or low/absent versus high. The results were verified through analysis of the domain Z scores and single-test Z scores. Descriptive statistics were computed for all study variables. Multivariable logistic regression analysis was performed including only variables that achieved P value <0.100 in the univariate analysis were included for calculation.The statistical calculations were performed using Statistical Package for Social Sciences 13.0  and GraphPad 5.0 . Normally distributed variables were summarized using the mean±SD, and non-normally distributed variables by the median and interquartile range. Wilcoxon’s matched pairs test and paired t-test were performed. Univariate comparisons between nominal variables were calculated using chi-square  were re-evaluated: at baseline, these patients showed a mean age of 36.7±10.0 years (range 19–58 years), a mean disease duration of 110.9±73.6 months, a mean ±SD duration of scholar education of 12.2±3.5 years.Considering the 15 SLE patients lost to follow-up, 13 refused to participate to the second evaluation, and two patients died (one for complicated infection and one for cardiovascular event). No significant differences between re-evaluated and missing patients were observed as regards demographic, clinical and laboratory features. In myasthenia gravis. In With regard to other autoimmune diseases, eleven patients (25.6%) were affected by anti-phospholipid syndrome (APS), six (13.9%) by Sjögren’s Syndrome. Furthermore, the presence of comorbidity was registered: arterial hypertension was identified in 21 patients (48.8%), thyroid pathology in 15 (34.9%), dyslipidemia, defined as raised plasma triglycerides (≥ 150 mg/dl) and/or low HDL-C (<40 mg/dl in men and <50 mg/dl in women) , in 11  at T0 and in 13.9% (6 patients) at T1 (P = NS). This impairment was prevalently mild at T0 (55.5%) and mild or moderate at T1 (36.3% for both degrees).Considering patients with CI at baseline, 55.5% experienced an improvement, while the other patients remained stable. Only one patient among the 34 without CI at baseline experienced a neurocognitive dysfunction with mild impairment.The mean domain Z scores were graphically represented in All the domains showed an improvement over-time, with a significant difference from baseline for executive function (P = 0.04). After transforming the MDZs into DCDs, the percentage of patients with impairment in the different domains was calculated as shown in The domain referring to executive functions was the most frequently involved at baseline ; this frequency was significantly reduced at the follow-up, when the domain was compromised in six patients . Of note, after 10 years the frequency of impairment was reduced in all the domains evaluated even if without reaching significant difference.Moreover, the presence of dyslipidemia, considered in our statistical analysis as a categorical variable, resulted significantly associated with memory impairment (P = 0.02), executive functions (P = 0.02) and abstract reasoning (P = 0.03) at the baseline, and with visuospatial functions (P = 0.009) and abstract reasoning (P = 0.004) at T1. Of note, worsening of visuospatial functions was significantly associated with dyslipidemia and positivity for LA (P = 0.04 for both parameters). Finally, the GCDs significantly correlated with SDI value at the baseline  and at T1 .No correlations between CI and demographical characteristics (including age and education level), SLE clinical features, ongoing and past treatments , comorbidities, activity and damage indices were observed in the present SLE cohort at baseline and follow-up.Moreover, all the lupus features  and ongoing and past medications  were considered in the multivariate logistic regression analysis, without identifying significant association.Moreover no significant associations were identified between CI and behavioral abnormalities evaluated by MMPI scales.In the present longitudinal study, the CI changes after 10 years of follow-up were evaluated in a cohort of Caucasian SLE patients. We observed an improvement of this manifestation in the majority of patients evaluated, with a worsening in only 10% of subjects. Furthermore, all cognitive functions improved and a statistically significant difference was achieved for the executive functions.Interestingly, the same operator performed the neurocognitive assessment at baseline and after 10 years, safeguarding the reliability of the results and reducing the risk of performance bias. At the same time, the patients were followed constantly in our Lupus Clinic during the 10 years follow-up, suggesting that clinical decisions among this time lapse were homogenous.Despite the high number of studies evaluating SLE-related CI, few data have analyzed its over-time changes, with a maximum follow-up of 5 years. To sum up, in most of the studies a fluctuating or stable trend was identified more frequently than worsening. –25The one-year follow-up study of Carlomagno and colleagues demonstrated a stable trend in more than 90% of SLE-patients. The 5-yeaWe previously evaluated a 58-patients SLE cohort in order to assess the CI prevalence and the possible association with other clinical and laboratory SLE-related features. We observed a mild GCDs impairment in 19% of patients, moderate (GCDs 4–5) in 7% and severe in 5%.15) In this cohort, the impairment of visuospatial domain resulted the most compromised and significantly associated with aCL IgM levels. Moreover, disease activity and chronic damage correlated with different domains. [ In this In the present study, we re-evaluated 43 out of 58 patients (74.1%), observing a reduction of the CI prevalence from 20.9to 13.9% after 10-yearfollow-up. Specifically, half of patients with CI at the baseline showed an improvement and only 10% a worsening. These results reinforce those obtained in above-described studies: in our cohort, even the majority of patients experienced an improvement of cognitive functions, differently from the stable trend previously described. We could hypothesize that the improved management of SLE patients and new therapeutic strategies allowed these results. In fact, previous studies were conducted more than 10 years ago and a wider knowledge on the SLE management as well as of therapeutic arrows can be expected in this time interval. , 38This suggestion was confirmed indirectly by the comparison of treatments at baseline and after 10 years. Of note, a significantly lower mean weekly dosage of GC was documented at the follow-up, with a significant increase of percentage of patients assuming dosage lower than 35mg/weekly of prednisone equivalents. Moreover, in our cohort we observed a significant increase of immunosuppressant drugs administration  and the appearance of biological drugs usage. Of note, in the last years a growing use of Mycophenolate was registered in Lupus cohorts, demonstrating the efficacy of this drug in other than renal involvement features, such as neurological manifestations. Furthermore, all patients were treated by antimalarial drugs and/or immunosuppressant and, in case of positivity for aPL antibodies, by anti-thrombotic therapy. Taken together, these treatments could influence the different inflammatory and thrombotic pathogenic mechanisms potentially determining CI. Nonetheless, it should be considered that the low mean age at the baseline (lower than 40 years) and the high scholar level of our cohort could influence the ability to perform neurocognitive assessment.Furthermore, we evaluated the association between CI and the different clinical/laboratory SLE-related features, as well as cardiovascular comorbidities. We observed a significant association between dyslipidemia and CI in all the domains, except for the attention, at baseline and after 10 years. Moreover, this comorbidity significantly correlated with the worsening of visuospatial functions. These results are in agreement with previous evidences: in particular, a multicenter Italian study including about 1.000 SLE patients identified dyslipidemia as a risk factor for the presence of CI. We could hypothesize, as a possible pathogenetic explanation, a concurrent subclinical vascular injury in both dyslipidemic and cognitive impaired patients which may justify this association.In addition, our analysis confirms the possible pathogenic role of aPL in the CI development: a significant correlation between LA positivity and the worsening in visuospatial functions was identified in our cohort. This finding suggests that aPL may act determining not only a focal damage (at level of thrombotic event), but also a more diffuse damage, with a direct mechanism on neural cells. , 42 MoreWe would highlight that generally MMPI is not an optimal battery to evaluate mood disorders. However, since at baseline evaluation, performed about 10 years ago, MMPI has been chosen to exclude the influence of behavioral abnormalities on cognitive dysfunction, we decided to keep the same protocol in order to maintain continuity and consistency of our longitudinal study. Nonetheless we observed that the assessment by MMPI depression scale indeed confirmed the presence of this mood disorder in the same patients previously diagnosed by a neuropsychiatrist specialist. Thus, all MMPI scales were included in the multivariate analysis but did not correlate with CI identified.In conclusion, the present study provides data concerning the changes over-time of SLE-related CI, by considering for the first time a follow-up of 10 years. Our data demonstrated a stability of cognitive functions, with a trend to the improvement in all the evaluated domains. The risk factors for a worse prognosis resulted the positivity for aPL, in particular LA, and the presence of a concomitant dyslipidemia. Moreover, the prevention of disease relapse and chronic damage development is mandatory in order to prevent CI worsening.