Firmicutes and Proteobacteria were decreased (P ≤ 0.01) with lactating progress and phyla Bacteroidetes were increased (P = 0.008) with lactating progress and dietary transition. At family level, the predominant families were Ruminococcaceae (35.23%), Lachnospiraceae (11.46%), Rikenellaceae (10.44%) and Prevotellaceae (6.89%). A total of 14 genera were different between fecal samples from Fresh1d and Fresh14d, included the predominant genera, such as Ruminococcaceae_UCG-005 (P = 0.008), Rikenellaceae_RC9_gut_group (P = 0.043) and Christensenellaceae_R-7_group (P = 0.008). All fecal bacterial communities shared members of the genera Ruminococcaceae_UCG-005, Bacteroides and Rikenellaceae_RC9_gut_group. These findings help to improve our understanding of the composition and structure of the fecal microbial community in fresh cows and may provide insight into bacterial adaptation time and dietary in lactating cows.The dynamics of the community structure and composition of the dairy cow fecal bacterial communities during early lactation is unclear, therefore this study was conducted to characterize the fecal bacterial communities in dairy cows during early lactation using 16S rRNA gene sequencing. Feces were sampled from 20 healthy fresh Holstein dairy cows on day 1 (Fresh1d group) and day 14 (Fresh14d group) after calving. After calving, cows were fed the same fresh diet. The dominant phyla  The bovine gastrointestinal tract microbiota harbors lots of microbial species that play important roles in the health and productivity of ruminant Bergmann . These mIn addition to studies on the rumen, reports on the microbial composition of the feces in dairy calves are abundant. Previous studies have found an association between fecal microbiota and age  Holstein dairy cows were housed in a free-stall barn at a commercial dairy farm . The dietary and nutritional composition of the feed given to fresh cows is presented in Additional file Forty fecal samples were collected from 20 healthy fresh cows on d1 and d14 after calving, without a history of antibiotic or drug treatment for 3 months prior to collecting samples. Feces were collected by hand from the rectum of cows using sterilized gloves before morning feeding. All samples were immediately transported on liquid nitrogen and later stored at − 80 °C before DNA extraction.2O was used for PCR in triplicate, with the following cycling conditions: 95 °C for 5 min in denaturation, followed by 28 cycles of 95 °C for 45 s in denaturation, 55 °C for 50 s in annealing and 72 °C for 45 s in elongation at with a final extension at 72 °C for 10 min.Total DNA was extracted from all fecal samples using an Omega Stool DNA kit  according to the manufacturer’s instructions. Amplicon library preparation was performed by PCR amplification of the V3 to V4 region of the 16S rRNA gene, using forward  and reverse primers . Then, the amplicon library was sequenced using the Illumina Miseq platform  by Beijing Allwegene Tech. Ltd . A 25 µL reaction mixture containing 12.5 μL of KAPA 2G Robust Hot Start Ready Mix , 1 µL of each primer (5 µM), 30 ng of template DNA and 5.5 µL ddHAmplicons were detected by 1% agarose gel electrophoresis and purified using the Agencourt AM Pure XP Kit . Purified amplicons from samples were quantified using Caliper LabChip GX Touch HT , pooled in equimolar concentrations into the final library, and then 2 × 250 paired-end sequenced on an Illumina MiSeq platform  version 1.8.0  from multiple comparisons in package “ggpurb” of R 3.6.2 . The none-metric multidimensional scaling (NMDS) plot of the microbial profiles was performed based on Bray–Curtis distance  using the “vegan” package in R  were assessed with QIIME 1.8. Differences in community richness and diversity were analyzed with the Kruskal–Wallis test, with P values ≤ 0.05 were considered to indicate significant differences.Comparison of the microbial structure and composition at phylum and genus levels were performed using the Wilcoxon tests with the FDR correlation, as described by Benjamini and Hochberg  for pairAll the raw DNA sequences were deposited in the National Center for Biotechnology Information Sequence Read Archive database and are publicly accessible under the accession number PRJNA628713.P < 0.05) in OTU number and Chao 1 was observed from d1 to d14, indicating significant changes in the diversity and richness of fecal microbiota as lactation progressed .NMDS based on Bray–Curtis distance showed a separation between Fresh1d and Fresh14d groups , Lachnospiraceae (11.46%), Rikenellaceae (10.44%) and Prevotellaceae , Bacteroidaceae (3.85%), Peptostreptococcaceae (3.36%), Family_XIII (2.32%), Bacteroidales_S24-7_group (2.24%) and Bacteroidales_RF16_group (2.21%), and altogether comprised 18.74% of total samples.Among all identified families, the predominant family included aceae 35.3%, LachnRuminococcaceae_UCG-005 (14.90%), Rikenellaceae_RC9_gut_group (7.52%), Ruminococcaceae_UCG-010 (4.94%), Christensenellaceae_R-7_group (4.65%), Eubacterium_coprostanoligenes_group (4.02%), Bacteroides (3.85%), Prevotellaceae_UCG-003 (3.55%), Alistipes (2.50%), Ruminococcaceae_UCG-013 (2.42%), Romboutsia (1.68%), Paeniclostridium (1.68%), Lachnospiraceae_NK3A20_group (1.64%), Clostridium_sensu_stricto_1 (1.54%), Phascolarctobacterium (1.32%), Treponema_2 (1.25%), Family_XIII_AD3011_group (1.06%), and Candidatus_Saccharimonas (1.04%) were the predominant genera  and altogether comprised 59.57% of the fecal community , Lachnospiraceae (6.52%), Rikenellaceae (4.03%), Bacteroidaceae (3.46%), Prevotellaceae (3.41%) and Peptostreptococcaceae (3.36%). In addition, all samples shared two unclassified families belonging to class Clostridiales (0.13%) and phyla Saccharibacteria (0.78%).Among the total number of OTUs, 116 OTUs were shared by all samples, representing a total relative abundance of 49.82% across samples , Christensenellaceae_R-7_group (2.62%), Rikenellaceae_RC9_gut_group (2.78%) and unclassified_Lachnospiraceae (2.66%) each had more than five OTUs shared among all animals. The genera Acetitomaculum (0.14%), Candidatus_Saccharimonas (0.78%), Eubacterium_brachy_group (0.22%), Mogibacterium (0.30%), Prevotellaceae_UCG-001 (0.53%), Prevotellaceae_UCG-003 (1.74%), Ruminococcaceae_UCG-010 (0.29%), Ruminococcaceae_UCG-013 (0.86%) and unclassified_Prevotellaceae (0.95%) each had two shared OTUs. Finally, there have 17 shared OTUs unclassified at the genus level (6.52%), 8 of which were in Lachnospiraceae, 2 in Prevotellaceae, 3 in Ruminococcaceae, 1 in Peptococcaceae,1 in Porphyromonadaceae, 1 in Bacteroidales_RF16_group, 1 in Bacteroidales_S24-7_group and 1 in Clostridiales.Seven OTUs were classified to the genus Firmicutes, Proteobacteria, Saccharibacteria and Cyanobacteria was significantly higher (P < 0.05) in the Fresh1d than in the Fresh14d group (Table Bacteroidetes and Tenericutes had higher (P < 0.01) relative abundance in Fresh14d compared with Fresh1d. Phyla Actinobacteria tended to increase (P = 0.067) and Verrucomicrobia tended to decrease (P = 0.067) as lactation progressed.At the phylum level, the relative abundance of up Table . In contRuminococcaceae_UCG-005, Ruminococcaceae_UCG-013, Prevotellaceae_UCG-003, Alistipes, Ruminococcaceae_UCG-013 and Ruminococcaceae_UCG-014 was lower (P < 0.05) in the Fresh1d group compared with that in the Fresh14d group  in the Fresh1d group than in the Fresh14d group.At the genus level, the relative abundance of P < 0.05) as lactation progressed, these included Bacillus, Clostridium_sensu_stricto_1, Clostridium_sensu_stricto_6 and Escherichia-Shigella  decreased from d1 to d14 after freshening, we cannot say conclusively if these are pathogenic or commensal. Thus, PCR and culture-based methods remain the gold standard for routine detection of these pathogens.Gram-positive rod-shaped Williams . StreptoIn conclusion, we observed significant changes in alpha diversity and relative abundance of fecal microbial taxa at the phylum and genus level using 16S rRNA gene amplicon sequencing analyses, in response to the dietary and lactation period perturbations encountered by dairy cows during early lactation period. Moreover, we found a decrease in genera associated with pathogenic bacteria as lactation progressed during early lactation onward. In accordance with a clear shift in the fecal bacteria after NMDS analyses, we observed significant changes in the relative abundance of the dominant phyla and genera in fecal bacteria as lactation progressed. Further research should focus on the impact of pathogenic and environmental factors on fecal microbial communities and the health of dairy cows.Additional file 1: Table S1. Ingredients and chemical composition of the experimental diets (as dry matter basis). Table S2. Shared bacterial OTUs and taxa among all samples.


Drosophila for mutants with reduced levels of rhodopsin. We isolated loss-of-function alleles in endoplasmic reticulum membrane protein complex 3 (emc3), emc5, and emc6, each of which exhibited defective phototransduction and photoreceptor cell degeneration. EMC3, EMC5, and EMC6 were essential for rhodopsin synthesis independent of the ER associated degradation (ERAD) pathway, which eliminates misfolded proteins. We generated null mutations for all EMC subunits, and further demonstrated that different EMC subunits play roles in different cellular functions. Conditional knockout of the Emc3 gene in mice led to mislocalization of rhodopsin protein and death of cone and rod photoreceptor cells. These data indicate conserved roles for EMC subunits in maintaining rhodopsin homeostasis and photoreceptor function, and suggest that retinal degeneration may also be caused by defects in early biosynthesis of rhodopsin.Defective rhodopsin homeostasis is one of the major causes of retinal degeneration, including the disease Retinitis pigmentosa. To identify cellular factors required for the biosynthesis of rhodopsin, we performed a genome-wide genetic screen in  Over 120 point mutations in the rhodopsin gene have been associated with ADRP, with most disrupting its trafficking from the endoplasmic reticulum (ER) to the plasma membrane [Retinitis pigmentosa (RP) causes the gradual degeneration of rod and cone photoreceptors and is the most common form of hereditary retinal diseases , 2. Mutamembrane . Proper Drosophila has provided insights into the folding and transport of rhodopsin protein [Drosophila gene involved in rhodopsin biogenesis in the ER that cause retinal degeneration when mutated.Studying photoreceptor cells in  protein , 5. In f protein –8. Witho protein , 9. RanB protein , 10. In emc3 gene, partial optokinetic response b (pob), causes degeneration of long-wavelength photoreceptor cells, and Drosophila EMC3 is required for the stable production of rhodopsins [EMC1 gene has been associated with retinal dystrophy [The ER membrane protein complex (EMC) was first identified as a multi-protein transmembrane complex in a genetic screen for the accumulation of misfolded membrane proteins in yeast. Based on a high-content proteomics strategy, this complex was then found to interact with the ER-associated degradation (ERAD) pathway, suggesting a role in the biosynthesis of transmembrane proteins , 12. Recodopsins , 24. Morystrophy . Howeveremc3, emc5, and emc6 that each reduced levels of the major rhodopsin Rh1, disrupted phototransduction, and caused gradual photoreceptor degeneration. We found that EMC function was independent of ERAD, most likely regulating rhodopsin levels at an earlier step in the biosynthetic process. Furthermore, knocking out the Emc3 gene in mammalian rod photoreceptor cells led to dysregulated rhodopsin trafficking and retinal degeneration, as seen in flies, suggesting that EMC function is conserved in mammals. Our study highlights the different roles played by EMC proteins in rhodopsin biogenesis.In the present study, we conducted an ethyl methanesulfonate (EMS)-based genetic screen to isolate mutations that affect rhodopsin homeostasis. We identified mutations in 3 EMC subunits, ey-flp ninaE-Rh1-GFP;FTR40A flies and the third chromosome of ey-flp ninaE-Rh1-GFP;FTR82B flies were isogenized, and young male flies were fed 25 mM ethyl methanesulfonate (EMS)  in 2% sucrose for 8 h. Mutagenized flies were mated immediately to ey-flp ninaE-Rh1-GFP;GMR-hid CL FRT40A/Cyo hs-hid or ey-flp ninaE-Rh1-GFP; FRT82B GMR-hid CL/TM3 hs-hid flies, and F1 progenies were screened for loss of GFP by performing the fluorescence deep pseudopupil (DPP) assays 1 day following eclosion [emc3G7, emc3G10, and emc5G2 mutants were isolated by screening ey-flp ninaE-Rh1-GFP;FTR40A flies (looking for changes in Rh1 fluorescence); emc6N10 mutant flies were isolated by screening ey-flp ninaE-Rh1-GFP;FTR82B flies.The second chromosome of eclosion . ApproxiCG3678G4215 (emc2B), CG32441MB09773 (emc10), ninaA2, ninaEP332, M(vas-int.Dm)ZH-2A;M(3xP3-RFP.attP)ZH-86Fb, M(vas-int.Dm)ZH-2A;M(3xP3-RFP.attP) ZH-51C, GMR-Gal4 and dmppee02905 lines were obtained from the Bloomington Stock Center. The emc4RNAi (P{TRiP.HMC06642}attP40) flies were obtained from the TsingHua Fly Center. The emc2ARNAi and emc5RNAi flies were generated using short-hairpin (sh) RNA system. The emc1, emc2A, emc4, emc7, emc8/9, and hrd1 knockout lines were generated using Crispr/Cas9 technology. The ey-flp ninaE-Rh1-GFP;FRT40A, ey-flp ninaE-Rh1-GFP;FRT42D, ey-flp ninaE-Rh1-GFP;FRT2A, ey-flp ninaE-Rh1-GFP;FTR82B, ey-flp ninaE-Rh1-GFP;GMR-hid CL FRT40A/Cyo hs-hid, ey-flp ninaE-Rh1-GFP;FRT42D GMR-hid CL/Cyo hs-hid, ey-flp ninaE-Rh1-GFP;GMR-hid CL FRT2A/TM3 hs-hid, and ey-flp ninaE-Rh1-GFP; FRT82B GMR-hid CL/TM3 hs-hid lines were maintained in the laboratory of T. Wang. Flies were raised in a 12h-light-12h-dark cycle at 25 °C on standard corn food.emc1, emc2A, emc4, emc7, emc8/9, and hrd1 mutations were generated using the Cas9/sgRNA system, as described [http://www.flyrnai.org/crispr2/.The escribed . The recemc1, sgRNA1: ACGCGTTCGTTGGTCGCGCG, sgRNA2: CCACCTACAGACACCCAGCG; emc2A, sgRNA1: GTTCCAAATATTCTCAGAAG, sgRNA2: TAGCGACATATTCACTTAGG; emc4, sgRNA: TCTTAAGAGGTCCCAGGGCA; emc7, sgRNA: GATAACCTCGCAGCTGACAA; emc8/9, sgRNA: CGACTACAAGGTCTCGGAGC; hrd1, sgRNA1: TACGGAGACCTGCTTGGCCT, sgRNA2: CTACGACATGCCCAGTACCT). The (nos-Cas9)attP2 flies were used as the Cas9 source, and F1 progeny were screened by PCR and DNA sequencing to identify the mutations. The primers used for genomic PCR were as follows: promoter, the cDNAs were subcloned into the pninaE-attB vector between the NotI and XbaI sites, and the constructs were injected into M(vas-int.Dm)ZH-2A;M(3xP3-RFP.attP)ZH-86Fb embryos. To express emc6 in photoreceptor cells, the cDNA was subcloned into the pninaE-attB vector between the NotI and XbaI sites, followed by injected into M(vas-int.Dm)ZH-2A;M(3xP3-RFP.attP) ZH-51C. The transformants were identified on the basis of eye color, and mini-white marker was eliminated by crossing to a Cre-expressing line.The emc2ARNAi and emc5RNAi flies, the following 21-nt sequences were used to generate shRNA constructs, as described.To generate emc2AshRNA: 5′-GCAAGATTGCCATCCTTAAGG-3′emc5shRNA: 5′-GCAGGAATGGAATAGCCTTCC-3′NheI and EcoRI sites [M(vas-int.Dm)ZH-2A;M(3xP3-RFP.attP)ZH-86Fb embryo, and transformants were identified on the basis of eye color.Annealed oligo pairs were cloned into the VALIUM20 vector at the RI sites . ConstruERG recordings were performed as described . BrieflyPrimary antibodies used for western blotting are listed below: Tubulin , ß-Actin , Rh1 , TRPL  , TRP , Cnx99A , GM130  GFP , RFP , EMC3 , NaK ATPase , M-Opsin opsin , Rho 1D4 monoclonal antibody against rhodopsin was a gift from Dr. Robert Molday, University of British Columbia, Canada. Secondary antibodies were bought from Invitrogen , 1:500 dilution.Retina were dissected and fixed in 4% freshly made paraformaldehyde in phosphate buffer for 2 h on ice, then dehydrated in a series of ethanol dilutions , 30 min each. Samples were then treated in acetone solution, and embedded in LR white resin . Sections (1 μm) were then prepared for immunostaining. Samples were blocked using PBST (0.3% Triton X-100) with 5% goat serum for 1 h, then incubated with primary antibodies at 4 °C overnight. This was followed by incubation with secondary antibodies for 1 h. Samples were examined and images were recorded using a Nikon A1-R confocal microscope, and further processed using Photoshop CC2017 software.Flies were kept in a 12h-light-12h-dark cycle at 25 °C. Approximately 200 flies were collected 5 days after eclosion. The flies were frozen in liquid nitrogen to dissociate the head from the body. Heads were then sieved out and lysed using lysis buffer  at 4 °C overnight. They were then incubated with RFP agarose beads (Chromotek) for 2 h at 4 °C. After several washes, the beads were boiled in SDS loading buffer, and supernatant was loaded onto a gel. Sliver staining (Thermo Fisher Scientific) was performed and bands on the gel were cut out for further mass spectra analysis.Flies were maintained in a 12 h-light/12h-dark cycle at 25 °C. Sixty female flies of each genotype were collected 5 days after eclosion. For each sample, 30 pairs of retinas were dissected in cold phosphate buffer. Two samples were generated for each genotype. Protein extraction was performed as described with several modifications . Briefly6 with a maximum injection time of 50 ms. Mass range was set to 320–1500. The maximum ion injection time for the MS/MS scan was 100 ms with a resolution of 35,000 at m/z 200, and target value for fragment scans was set at 1 × 105. Isolation width was set at 1.2 m/z and normalized collision energy was set at 32. Each fraction was analyzed in twice.About 2 µg of each high-pH fractionated peptides samples were separated on an in-house packed 75-μm ID × 50 cm capillary column with 2.5 μm Venusil C18 beads  using an EASY-nLC 1000 system . The column temperature was maintained at 42 °C using an integrated column oven . The flow rate was set to 200 nL/min, and a total 240 min or 320 min gradient from 2 to 27% acetonitrile was used. Raw data was collected on Q Exactive mass spectrometer  using Thermo Xcalibur (2.0) control software with a data-dependent MS/MS scans (TopN = 15). MS1 spectra was measured at a resolution of 70,000 at m/z 200, and full scan target was 3 × 10Drosophila Melanogaster (Fruit fly) Swiss-Prot database  combined with a common contaminants database (247 entries), using default processing and consensus workflow for MS2 TMT quantification method. The standard searching parameters were used: a 10 ppm MS1 error tolerance was used, with 0.02 Da error used for MS2 product ions on the Q Exactive. Trypsin was set as the enzyme, allowing for 2 missed cleavages. Methionine oxidation was set as a variable modification, and carbamidomethyl on cysteines, TMT6 plex on lysine and peptide N-terminus were set as static modifications for all searches. FDR was set to 0.01 for peptide database search.All raw LC−MS/MS data were submitted to Proteome Discoverer  for TMT quantitation and database analysis using SequestHT. Data were searched against the S2 cells were transfected by adding 5 µg of plasmid mixed with 2 µl VigoFect agent (Vigorous Biotechnology) into the cell media. Twenty-four hours following transfection, cells were suspended and adhered to a poly-lysine treated coverslip for 1 h and then fixed for 0.5 h in freshly made 4% paraformaldehyde in phosphate buffer. Samples were blocked using PBST (0.3% Triton X-100) with 5% goat serum for 1 h, then incubated with primary antibodies for 1 h. After washing with phosphate buffer, cells were incubated with secondary antibodies for 1 h. Samples were then imaged using a Nikon A1-R confocal microscope. Images were further processed using Photoshop CC2017 software.Emc3 conditional knockout allele was generated using the CRISPR/Cas9 system using gRNA sequence CCTTTGTGAAAACCTGTGTGGCA. A DNA template containing two loxp sites flanking exon 2 of Emc3 was used to guide DNA repair: ccttcctcagaaacctttatttgttcagagcccagtcactctggaagactgccttggatccATAACTTCGTATAATGTATGCTATACGAAGTTATggctgtgaaaacctgtgtggcaagttgaatatgtttcattgatttttagggtttttggaaagtttccacacccagcttttgccaaccaggtcctcccttatgtctgagtgggccttctactttacgcttgctttctttgcgtttcacattttaacatctctttgtcttgtgtttttcagTCAGGTCCTAATTCGAAGCAGAGTCCTCAGGGAAAATGGAAAATACATTCCCAAGCAGgtactcactgatattttatttagaggctccaccattcacctgtaagggcagtaaaaacctaaatgtttttttataagaggatgccagagaaaactggaggtggccatccagttattaagctgcagcATAACTTCGTATAATGTATGCTATACGAAGTTATgaattcggtggtggtgtttgctgtcatttgccctcacccttcttaggaaattgtttttaaatgtgactca. Loxp sequences were underlined. Intronic sequences were in lower case letters. Exon 2 was in upper case letters. C57BL/6 J mice were used for embryo injection. Positive founder mice were screened using PCR and DNA sequencing. Primer pair Emc3-screen-F1 and Emc3-screen-R1 was used to identify upstream loxp sequence. Emc3-screen-F1: CCCAGGACGGGGATTATAGTGGTGTG; Emc3-screen-R1: ACTTGCCACACAGGTTTTCACAGCC. A second primer pair Emc3-screen-F1 and Emc3-screen-R2 were used to identify downstream loxp sequence. Emc3-screen-F1: CCCAGGACGGGGATTATAGTGGTGTG; Emc3-screen-R2: CAAATGACAGCAAACACCACCACCG. Cone-specific knockout mice were generated by crossing Emc3loxp/+ to HRGP-Cre mice [rd1 mutation was bred out before the experiment. Emc3loxp/+ mice were crossed to the broadly expressed inducible CRE CAG-Cre-ER mice (stock Tg(CAG-cre/Esr1*) 5Amc/J, https://www.jax.org/strain/004453) to generate inducible adult knockout mice [All mouse study protocols were approved by the Animal Care and Use Committee of Sichuan Provincial People’s Hospital. All experimental procedures were carried out in accordance with the approved study protocols and relevant regulations. Mice were raised in a 12h-light-12h-dark cycle. The Cre mice . The rd1out mice .A total of 100 mg of tamoxifen salt  was dissolved in 10 ml of ethanol as a stock solution. On the day of injection, a 1 mg/ml working solution was prepared by mixing 10 mg/ml stock solution with corn oil  and mixed well. On P20, mutant and control mice were intraperitoneally injected with a daily dosage of 25 mg/kg body weight for 3 days .Genomic DNA extracted from mouse tails was amplified by PCR using the following primer pairs: EMC3-Seq-F1, TGTCTCCCGTCAAATCCAGAAAGG; EMC3-Seq-R1, ATGTGAAACGCAAAGAAAGCAAGC. Amplification was performed using an Invitrogen Platinum SuperFi PCR master mix . The first cycle consisted of 95 °C for 2 min, followed by 33 cycles of 94 °C for 15 s, 58 °C for 20 s, and 72 °C for 30 sec. Cre was genotyped using generic Cre primers: Cre-F, TGCCACGACCAAGTGACAGCAATG, and Cre-R, ACCAGAGACGCAAATCCATCGCTC).For immunohistochemistry, enucleated eyes were removed, marked at the nasal side for orientation, and fixed for 3 h in 4% paraformaldehyde in 100 mM phosphate buffer (PB) (pH 7.4) and then cryoprotected in 30% sucrose. Tissues were embedded in optimal cutting temperature solution (OCT) and frozen on dry ice for sectioning. Sections (10 micron) were blocked and permeabilized with 10% normal goat serum and 0.1% Triton X-100 in phosphate buffer for 60 min. Labelling with various antibodies was performed as previously described . PrimaryQuantification of mislocalized M-Opsin in the cell bodies of cone cells was performed as follows. P30 and P50 cross-sections of retinas were stained using M-Opsin opsin and DAPI, and higher-magnification images were captured on a Leica SP8 confocal microscope. The number of cones in which M-Opsin mislocalized to the inner segment and to the cell body was counted. Slides were photographed on a Zeiss LSM 800 confocal scanning microscope.For H&E staining, eyes from WT and knockout (KO) mice were removed, marked on the nasal side for orientation, and fixed overnight in 1.22% glutaraldehyde and 0.8% paraformaldehyde in 0.08 M phosphate buffer. They were embedded in paraffin, and cut into 5-µm sections. H&E stained sections were used to count the rows of photoreceptors in the outer nuclear layer . Three mRh1::GFP ey-flp/hid system, which utilizes the FLP-FRT recombination system to generate eyes homozygous for mutations, while using GFP-tagged Rh1 to track photoreceptor cell integrity [emc3G7 and emc3G10 mutations are two new emc3 alleles, containing nonsense and missense mutation, respectively. One new (mutant emc5G2) introduces a premature stop codon in the CG15168 gene, which encodes an EMC5 homologue. The emc6N10 allele introduces a nonsense mutation in the CG11781 gene, which encodes an EMC6 homologue  is an extracellular recording that measures the summed light responses of all retinal cells  in emc3G7 and emc5G2 flies, although the Rh1 signal was weak compared to rescued flies. This indicates that Rh1 trafficking was normal in both emc3 and emc5 mutants , EMC6 primarily localized to intracellular vesicles that lacked ER markers Fig. . Howeveremc3, emc5, and emc6. We therefore asked if other EMC components also regulated biosynthesis of rhodopsin. Using the CRISPR/Cas9 system, we generated null mutations for all genes encoding EMC components, namely CG2943 (emc1), CG17556 (emc2A), CG11137 (emc4), CG8397 (emc7), and CG3501 (emc8/9)  and CG32441MB09773 (emc10), which are considered null mutants  Figure . We alsos Figure . We founG2943 emc, CG17556es Table . Howeveremc mutants from different phenotypic categories. Because null emc2A and emc4 mutations completely lacked retinal cells, we generated weak alleles by expressing RNAis of emc2A, emc4, or emc5 in compound eyes using the GMR-Gal4 driver [emc2A, emc4, and emc5 knockdown was >60%   in which Cre is driven by the human red/green pigment gene promoter [HRGP-Cre mice were used as controls (WT). In Emc3 cone-KO retinas, M-opsin levels were not affected on postnatal day 30 (P30)  Figs. . Howeverty Figs. .Fig. 6LoEmc3 in adult mice. We generated Emc3loxp/loxp; CAG-CreER (named Emc3 iKO) mice and treated the mutant and control (CAG-CreER or Emc3loxp/loxp) animals with tamoxifen at P20 and assessed the retina 7 and 11 days later (P27 and P31). Tamoxifen induction efficiently deleted Emc3 in all retinal cells, and EMC3 levels were reduced by 70% at P27 compared with control retinas , Rh1 levels were significantly decreased as well as applying emc5 RNAi, indicating that rhodopsin is a common substrate of EMC complexes. Whereas EMC7 and EMC10 are not required for the EMC complex to participate in rhodopsin biogenesis. Quantitative proteomic analysis to compare changes in protein levels in emc2, emc4, emc5, and emc7 mutants revealed that 13 proteins were downregulated in all 4 mutants, but most (including rhodopsin) were downregulated in ≤3 emc mutants. These data provide additional support that EMC subunits are differentially required for the biogenesis of distinct membrane proteins.In weak emc5G2 and emc3G7 null mutant animals, emc6N10 null mutants exhibited lower levels of rhodopsin and TRP proteins, more disrupted rhabdomere morphology, and more severe retinal degeneration. Moreover, EMC5 and EMC3 localized to the ER, whereas EMC6 was also concentrated in intracellular vesicles. However, EMC6 localized to the ER when co-expressed with EMC5, indicating that EMC6 is a bona fide component of the EMC complex, while also performing independent functions outside of the ER. The independent functions of EMC6 may explain its role in the biosynthesis of acetylcholine receptors and in autophagy [Different EMC subunits have been shown to function together in a number of cellular processes. Compared with utophagy , 47. In emc3, emc5, and emc6 mutant flies only lack the PDA, as seen for ninaE mutants. These are consistent with previous reports that low levels of TRP is enough for maintaining phototransduction, suggesting that loss of rhodopsin is the major cause of the defective visual responses [ninaE alleles results in retinal degeneration [emc mutants could be explained by a disruption in rhodopsin biosynthesis.Both rhodopsin and TRP rely on EMC for their biogenesis. However, the esponses . Rhodopsneration , 55. PhoEmc3 from photoreceptor cells led to their degeneration. Removal of Emc3 from the adult retina resulted in the accumulation of RHO in the inner segment or in cell bodies, and the death of rods. We did not observe mislocalized cone opsin in Emc3 mutants. One potential explanation is that Emc3 is not essential for cone opsin trafficking. To the best of our knowledge, our study provides the first evidence that an ER membrane protein is essential for mammalian photoreceptor structure.The importance of EMC in various human diseases has emerged in recent years. Mutations in EMC1 are associated with global developmental delay, hypotonia, scoliosis, visual impairment and cerebellar atrophy , 57. In Down-regulated proteins identified in emc2A mutantDown-regulated proteins identified in emc4 mutantDown-regulated proteins identified in emc5 mutantDown-regulated proteins identified in emc7 mutantSupplemental figures 1-13Dataset 1Dateset 2


Using a flanker task, inhibitory control was assessed before the intervention (t0), immediately after the session (t1), and then at 30 min (t2), 60 min (t3), and 90 min (t4) after the session. During the flanker task, the response time (RT) for incongruent trials immediately after HIIE was significantly shortened compared to that before exercise. This shortened RT was sustained for 90 min post-exercise during recovery from HIIE. Interference scores of RT were also reduced after HIIE, benefitting inhibitory control, and were maintained for 90 min post-exercise. Reduced accuracy interference scores were recorded following HIIE compared to the control session. Improvements in inhibitory control elicited by HIIE were sustained for at least 90 min post-exercise. In contrast, an improvement in inhibitory control was not observed during the MICE session. HIIE might represent a time-efficient approach for enhancing inhibitory control.This study examined the immediate and sustained effects of high-intensity interval exercise (HIIE) and moderate-intensity continuous exercise (MICE) bouts on inhibitory control in young adults. Participants ( Inhibitory control refers to the ability to resist internal impulses or external lures, and it inhibits inappropriate or prepotent responses. Inhibitory control is necessary for the regulation of behavior and emotions, and is associated with success in daily life . A growiVarious studies have demonstrated that improvement in cognitive performance by acute exercise can be sustained for a certain period of time. Experimental studies have shown that the benefits of acute exercise can last up to 45 min after exercise ,23. Howe0) and at four time points after the intervention, including immediately (t1), 30 min (t2), 60 min (t3), and 90 min (t4). Because previous studies have demonstrated that different exercise intensities and modalities are potential moderators of exercise-induced inhibitory control [The current study examined the immediate and sustained effects of HIIE and MICE on inhibitory control in young adults. Using a within-subjects design, 41 participants engaged in one HIIE session, one MICE session, and one control session on separate days in a counterbalanced order. The flanker task was performed to assess inhibitory control before the intervention  from Qingdao University, China, participated in this study. All participants were right-handed, free of any reported neurological, cardiovascular, or pulmonary diseases, and had normal or corrected-to-normal vision. Participants were asked to refrain from any moderate-to-vigorous physical exercise 24 h before the experiments and to refrain from caffeine and alcohol 12 h before the experiments. The study protocol was approved by the local ethics committee of Qingdao University. All participants provided written informed consent before the experiment. Demographic characteristics and fitness data for all participants are provided in 0) and at four time points after the intervention, including immediately (t1), 30 min (t2), 60 min (t3), and 90 min (t4).The study was conducted using a within-subjects, repeated-measures design. It included one HIIE session, one MICE session, and one control session conducted in a counterbalanced order. These three sessions were separated by at least one week and were completed at approximately the same time of the day. Before the experiment, participants completed the Physical Activity Readiness Questionnaire (PAR-Q) , which imax (208 - 0.7 × age) [max [During the HIIE session, participants completed a 2 min warm-up at a self-determined speed, followed by 10 bouts of repeated 1 min runs on a treadmill at an intensity targeting 85–90% HR7 × age) , intersp7 × age) . After tge) [max , which wge) [max . The HIITo assess inhibitory control, a computer-based version of the flanker task  was admi0, t1, t2, t3, and t4), and congruency (congruent and incongruent) being used as within-subject factors. Interference scores were examined separately for accuracy (congruent‒incongruent) and RT (incongruent‒congruent) using a 3  × 5  repeated-measures ANOVA. Mauchly’s test was used to examine spherical data, and the Greenhouse–Geisser correction was used to analyze non-spherical data. The Shapiro–Wilk normality test was applied to confirm normal distribution of data before the ANOVA. Paired t-tests with Bonferroni corrections for multiple comparisons were used for post hoc analysis if the ANOVA showed significant interactions between different factors. p-Values < 0.05 were considered significant. All statistical analyses were completed using the Statistical Package for Social Sciences software .For the RT analysis, incorrect trials were first removed and then an outlier correction was performed by separately excluding trials with an RT of 3 standard deviations from the mean for each flanker condition (congruent and incongruent). Accuracies and RTs were analyzed using a three-way repeated-measures analysis of variance (ANOVA), with session , time point (tη2 = 0.17) [Sample size was calculated via the statistical power calculation G*power 3.1.9.2) on a medium effect size  = 5.02, p = 0.012, η2 = 0.21). Post hoc analysis showed that accuracy was significantly higher in the HIIE session (94.52 ± 6.36) compared to the control session , while accuracy in the MICE session (94.17 ± 6.12) did not differ from the other two sessions (p > 0.1). A significant main effect of time point was detected  = 3.43, p = 0.017, η2 = 0.27). Post hoc tests showed that accuracy was significantly higher 30 min after the intervention (t2) compared to before the intervention (t0) (p = 0.009). There was a significant main effect of congruency  = 71.91, p < 0.001, η2 = 0.64), with higher accuracy in the congruent trials compared to the incongruent trials.The three-way ANOVA showed a significant main effect of session on accuracy  = 6.69, p = 0.003, 2η = 0.26). Post hoc analysis showed that accuracy was significantly higher in the incongruent trials of the HIIE session (90.88 ± 9.11) compared to the control session , while accuracy in the MICE session (89.93 ± 8.71) did not differ from the other two sessions (p > 0.08 for both). There were no significant effects in the interactions of time point × congruency  = 2.54, p = 0.056, η2 = 0.12), session × time point  = 0.57, p = 0.792, η2 = 0.12), or session × time point × congruency  = 0.21, p > 0.9, η2 = 0.05) (There was a significant interaction between session and congruency ( = 0.05) a,b.F = 6.69, p = 0.003, η2 = 0.26). Post hoc analysis showed that the accuracy interference score was significantly lower in the HIIE session (7.28 ± 9.07) compared to the control session , while the accuracy interference score in the MICE session (8.47 ± 8.16) did not differ from the other two sessions (p > 0.1 for both). There was no significant main effect of time point  = 2.54, p = 0.056, 2η = 0.22), and no significant effect in the interaction of session × time point  = 0.21, p > 0.9, 2η = 0.05)  c. Mean aF = 2.85, p = 0.037, η2 = 0.24). The post hoc test showed that RT was significantly lower before intervention (t0) (420.99 ± 36.00) compared to 60 min (t3)  and 90 min (t4)  after intervention. A significant main effect of congruency was documented  = 720.17, p < 0.001, η2 = 0.95), with longer RTs in the incongruent trials (448.61 ± 37.65) compared to the congruent trials (382.61 ± 32.72).The three-way ANOVA on RT revealed a significant main effect of time point  = 5.65, p = 0.001, η2 = 0.38). The post hoc test showed that, for incongruent trials, RT was significantly slower before intervention (t0) compared to immediately after intervention (t1) (p = 0.02) and at 30 min (t2) (p = 0.004), 60 min (t3) (p = 0.006), and 90 min (t4) (p = 0.003) after intervention. No significant differences were observed between the time points for congruent trials . There was a significant interaction effect for session × time point × congruency  = 2.51, p = 0.03, η2 = 0.38). Contrasts in the interactions between session, time point, and congruency revealed that RT was significantly slower in incongruent trials before the HIIE intervention (t0) compared to immediately after the HIIE intervention (t1) (p = 0.002) and at 30 min (t2) (p = 0.018), 60 min (t3) (p = 0.003), and 90 min (t4) (p = 0.045) after the HIIE intervention. No significant differences were detected between time points for incongruent trials in the MICE and control sessions . For the RTs of congruent trials, no significant differences were detected between the time points in any of the sessions . There was no significant main effect for session  = 1.76, p = 0.186, η2 = 0.08) or significant effects in the interactions of session × congruency  = 2.69 p = 0.081, 2η = 0.12) or session × time point  = 1.24, p = 0.309, η2 = 0.23) (There was a significant interaction between time point and congruency ( = 0.23) a,b.F = 5.65, p = 0.001, η2 = 0.38). The post hoc test showed that the RT interference score was significantly higher before the intervention (t0) compared to immediately after the intervention (t1) (p = 0.001) and at 30 min (t2) (p = 0.002), 60 min (t3) (p = 0.046), and 90 min (t4) (p = 0.008) after the intervention. There was a significant interaction between session and time point  = 2.89, p = 0.03, η2 = 0.38). The interaction contrasts revealed that RT interference score was significantly lower immediately after the HIIE intervention (t1) compared to before the HIIE intervention (t0) (p < 0.001). This shortened RT was sustained for 90 min . No significant differences were detected in the RT interference scores between the time points in the MICE and control sessions . There was no significant main effect of session  = 2.69, p = 0.081, η2 = 0.12)  c.This study assessed the sustainability of the effect of acute treadmill-based HIIE and MICE on inhibitory control in young adults. After the HIIE intervention, participants performed better in an inhibitory-control task, with performance being sustained for at least 90 min after the intervention. This improvement in inhibitory control was not detected in the MICE session. Thus, HIIE could represent an effective strategy for improving inhibitory control.As a stressor, exercise can activate the autonomic nervous system and promote physiological and psychological arousal . Physiolmax, with a mean RPE of 10.85; however, moderate-intensity exercise represents an RPE of 12–13 according to the American College of Sports Medicine (ACSM) guidelines [The present study failed to show an enhancement of inhibitory control after the MICE session compared to the control session, which was inconsistent with studies reporting improvements in inhibitory control after MICE interventions ,41. Firsidelines . This phidelines  showed tidelines ,46.The present study demonstrated that inhibitory control improved immediately after HIIE and was sustained for at least 90 min. This finding supports several recent studies reporting that cognitive improvement could be maintained for a period of time after exercise ,23,24. FExercise-induced enhancement of inhibitory control is associated with brain oxygenation . Particimax in this study was estimated from an equation (208 - 0.7 × age) [The results of the present study have several limitations. First, cardiorespiratory fitness can modulate the effect of acute exercise on cognitive performance ,26. Howe7 × age) , which mThe present study demonstrated how treadmill-based acute HIIE and MICE impact the sustainability of inhibitory control in young adults. Specifically, HIIE enhanced inhibitory control for up to 90 min following the cessation of exercise. However, this improvement in inhibitory control was not detected in the MICE session. Thus, HIIE represents a time-efficient approach for enhancing cognitive performance, with it being recommended that the public be made aware of this. For example, acute HIIE would be recommended for students before implementing a study, to achieve higher levels of concentration and efficiency. This temporary change in executive control is suggested to have an impact on academic performance and success in daily life ,53.


Glechoma curviflora (Boiss.) Kuntze (Nepeta curviflora) is a medicinal herb growing in the eastern parts of the Mediterranean Sea Basin and is widely consumed as a tea. The leaves of this plant have been traditionally used for the treatment of various infectious diseases. The current research was designed to identify the chemical composition of Glechoma curviflora (Boiss.) essential oil (EO) and to assess its antibacterial, antifungal, and cyclooxygenase inhibitory activities and the biophysical gating effect on AMPA receptors. Twenty phytochemicals were identified from G. curviflora leaves and flowers EO amounting to almost 100% of the total constituents using GC-MS technique, of which 1,6-dimethylspiro[4.5]decane (27.51%) 1, caryophyllene oxide (20.08%) 2, and β-caryophyllene (18.28%) 3 were the main constituents. The biophysical properties' effect from the plant extract on various AMPA-type receptors expressed in Human Embryonic Kidney (HEK293) cells was assessed by exploiting the whole-cell patch-clamp technique. Microdilution assay was adopted for assessing the antimicrobial property against eight virulent microbial strains whilst the cyclooxygenase inhibition effect was accomplished utilizing COX inhibitory screening colorimetric assay G. curviflora EO displayed potent activity against P. aeruginosa (MIC = 1.25 μg/mL), S. sonnei (MIC = 3.12 μg/mL), and E. coli (MIC = 1.25 μg/mL), compared with ciprofloxacin (positive control) and potent antibacterial activity against S. aureus, MRSA, S. sonnei, E. coli, and P. aeruginosa compared to Ampicillin (2nd positive control). It also showed anti-Candida (MIC = 6.25 μg/mL) and antimold (MIC = 3.125 μg/mL) activities compared with fluconazole . Likewise, our results showed an inhibition and biophysical impact of G. curviflora on all AMPARs subunits.The rise of the emergence of microbial resistance of antibiotics, the dangerous side effects of nonsteroidal anti-inflammatory drugs, and noncompetent medications of Alzheimer's, Parkinson's, and other neurodegenerative diseases prompt scientists to search for phytochemicals that could be utilized in the remedy of lethal diseases.  The search for medications and dietary supplements derived from plants has accelerated in the last two decades. Botanists, phytochemists, microbiologists, and pharmacologists are combing the Earth searching for natural products that have therapeutic potentials . EssentiPostsynaptic membranes expressing cation glutamatergic AMPA receptors (AMPARs) are implicated in various plasticity and synaptic transmissions. In the mammalian brain, the postsynaptic AMPARs are fundamentally essential in facilitating the predominantly fast excitatory transmission. Overactivated AMPARs trigger excitotoxicity, involved in numerous neurodegenerative severe illnesses such as AIDS encephalitis, Amyotrophic Lateral Sclerosis (ALS), Parkinson's, epilepsy, Huntington's, and other neurological disorders .Microbial infections due to drug-resistant strains turn out to be one of the most serious intimidations to public health. Numerous health care systems and research centers, especially in developed countries, are relentlessly searching for alternative therapies to cure such infections. Herbal medicines are acknowledged to prevent infections as they are considered a better alternative to artificial drugs due to drug-resistant pathogens .Cyclooxygenase (COX-1 or COX-2) inhibitors, classified as NSAIDs-nonsteroidal anti-inflammatory drugs, are considered to be the most common medications for inflammatory diseases due to their effectiveness in the treatment of fever, redness, pain, and edema. Most of the used NSAIDs are associated with harmful side effects, including gastrointestinal ulcers, renal failure, and hepatic dysfunction , 6.Glechoma curviflora (Boiss.) Kuntze (Nepeta curviflora) is a perennial aromatic herb growing in the eastern parts of the Mediterranean Sea Basin, which is widely consumed as a tea in Lebanon, Syria, Jordan, Turkey, and Palestine decane 1 (27.51%), caryophyllene oxide 2 (20.08%), and β-caryophyllene 3 (18.28%) were the major constituents  was then compared to the induced current obtained from glutamate plus EO amplitude (AI) for calculating any significant inhibition solely from the extract as shown in via 500 ms intervals of agonist; the initially acquired amplitude of GluA1, GluA2, GluA1/2, and GluA2/3 was recorded at 964 ± 55 pA, 1293 ± 63 pA, 490 ± 31 pA, and 452 ± 39 pA, respectively. No significant inhibition was observed by treating the cell with the EO as the amplitude slightly dropped to 812 ± 26 pA, 1095 ± 38 pA, 408 ± 33 pA, and 410 ± 28 pA following the previous order. Nonetheless, the impact of G. curviflora EO on AMPAR desensitization and deactivation was tested. The fast amplitude (τf), the slow amplitude (τs), and the relative amplitude of both (τf) and (τs) represented by (af) were used to attain the weighted tau (τw) for AMPAR-current deactivation and desensitization via fitting exponentials. The desensitization before G. curviflora EO treatment for GluA1, GluA2, GuA1/2, and GluA2/3 was 2.8 ± 0.1 ms, 2.3 ± 0.1 ms, 5.3 ± 0.2 ms, and 2.5 ± 0.3 ms, respectively. After treatment, the desensitization significantly increased to 3.7 ± 0.2 ms, 3.9 ± 0.2 ms, 6.8 ± 0.4 ms, and 3.5 ± 0.3 ms, respectively. The desensitization rate was 0.27, 0.26, 0.15, and 0.28, respectively. Likewise, the same significant effect of oil on AMPAR deactivation rate was observed, whereby the rate dropped from 0.43 ± 0.1, 0.45 ± 0.1, 0.42 ± 0.3, and 0.38 ± 0.2 to 2.6 ± 0.3, 0.24 ± 0.2, 0.21 ± 0.4, and 0.26 ± 0.3 for GluA1, GluA2, GluA1/2, and GluA2/3, respectively.The G. curviflora EO ability to inhibit the growth of S. aureus, MRSA, S. sonnei, E. faecium, E. coli, P. aeruginosa, C. albicans, and E. floccosum. The range of concentration of the prepared G. curviflora EO used ranged from 0.025 to 25 μg/mL. The attained results revealed that EO displayed significant antibacterial and antifungal effects against all the tested strains as indicated in μL/mL. It inhibited the growth of tested mold (E. floccosum) at half concertation required for the inhibition of the yeast Candida spp.The well diffusion procedure was employed in order to assess G. curviflora EO was accomplished using Cayman Chemical ELISA kit no. 560131. The calculated inhibition percentage of COX-2 for the tested EO revealed inhibition activity of 98.4% and 99.5% at concentrations of 0.25 μg/mL and 0.5 μg/mL, respectively. Similar results were obtained for the inhibition of COX-1, where the inhibition was 98% and 99% for the two tested concentrations, respectively. The results revealed that the G. curviflora EO demonstrated a potent but nonselective inhibitory activity towards both COX-2 and COX-1 enzymes.The COX enzymatic inhibition activity of There are rising interests towards the use of natural biological sources, which are known in general as phytotherapeutic active compounds. The necessity for the search for potential drug candidate from plants is incited by different reasons including the growth of chronic and degenerative diseases, inadequate access to medications, incidents of epidemic antibiotic resistances, and the emergence of new diseases –24.1, caryophyllene oxide 2, and β-caryophyllene 3 were the most abundant components representing 27.51%, 20.08%, and 18.28%, respectively. The twenty components could be classified into eight phytochemical groups, of which sesquiterpene hydrocarbon was the most abundant group (38.49%), followed by the oxygenated sesquiterpene (caryophyllene oxide) group (20.08%) and spiroalkane group (29.14%). The major components of sesquiterpene hydrocarbon were β-caryophyllene (18.28%), β-farnesene (6.2%), and γ-caryophyllene (4.95%). The comical compositions of EO of G. curviflora growing in Lebanese and Jordanian mountains were reported. Musso et al. reported the identification of 49 compounds in the EO extracted from G. curviflora growing in Lebanon, of which 2-isopropyl-5-methyl-3-cyclohexen-1-one (12.51%), (-)-spathulenol (11.73%), cis-Z-α-bisabolene epoxide (8.07%), widdrol (7.0%), -5, 7-dodecadiene (6.93%), dihydronepetalactone (5.57%), and 4-propyl-cyclohexene (5.43%) were the major constituents decane tituents . A study(10.47%) .The qualitative and quantitative variations in the findings could be attributed to the location and collection time and extraction techniques used. In contrast to the previously reported investigation, the mild microwave ultrasonic apparatus for the extraction of the EO was used in the current study. The previously reported studies used the hydrodistillation procedure, which requires reflux for a long time (≥3 h), which can have an impact on the chemical composition of the EO. The plant material used in the current study was collected on the 19th of May, where the previously reported studies worked on a plant collected in April.G. curviflora EO did not affect the activation of AMPARs, it displayed neuroprotective properties by targeting the receptor's desensitization and deactivation rate. The EO drastically prolonged the state at which the receptor remains in desensitization and deactivation by lowering the rates and consequently reaches a receptor in a nonactive state or a slow current-induced state. The oil showed activity on all tested AMPAR subtypes, yet significantly enhance the desensitization rate in addition to the deactivation rate of the GluA2 subtype comparable to other subtypes. To confirm this finding, we assessed the open state receptors by using 10 mM ligand concentration decane 1, caryophyllene oxide 2, and β-caryophyllene 3 were the major compounds. The G. curviflora VO displayed potent antimicrobial as compared with ciprofloxacin, Ampicillin, and fluconazole. Moreover, it showed potential cyclooxygenase inhibitory activity compared with celecoxib and has potential neuroprotective activity effect. However, further studies in vivo and clinical trial investigations are required to approve these findings and to establish suitable pharmaceutical formulations to be used as formal drugs or food supplements based on the G. curviflora VO.The current investigation explored the chemical ingredients of


Aizoon canariense L., a wild neglected plant traditionally used in gastrointestinal ailments, has been the subject of a limited number of phytochemical and biological studies. Therefore, herein, we investigated the in vitro cytotoxic, antimicrobial, and anticholinesteraseactivity of the aerial parts of A. canariense L. and analyzed the phytochemical compositions of the lipoidal and alkaloidal fractions. Petroleum ether extract showed the presence of behenic and tricosylic acid, while an in-depth investigation of the alkaloidal fraction revealed the identification of new adenine based alkaloids (1–5), which were isolated and identified for the first time from Aizoon canariense L. Their structures were elucidated based on extensive spectroscopic analyses. The alkaloidal extract showed a powerful cytotoxic effect (IC50 14–28 μg/mL), with the best effect against colon carcinoma, followed by liver and breast carcinomas. The alkaloidal extract also had a potent effect against Candida albicans and Escherichia coli, with minimum inhibitory concentrations (MIC) values of 312.5 and 625 µg/mL. The in vitro anticholinesterase activity was potent, with IC50 < 200 ng/mL for the tested extracts compared with 27.29 ± 0.49 ng/mL for tacrine.Aizoaceae is a large succulent family characterized by many psychoactive species.  African plants have been proven to have encouraging healing powers but are scarce in scientific investigations. Many studies have shown promising antimicrobial, cytotoxic, and enzyme inhibitory effects, correlated with the diversity of their phytoconstituents ,2Aptenia + peak at m/z 394, while the negative MS spectrum revealed an [M − H]− peak at m/z 392, suggesting a molecular formula of C20H35N5O3  were noticed as multiplet signal at δH 0.83–0.89 ppm. H-10′′′ was detected at δH 1.84 ppm while H2′′′-6′′′, 8′′′ and 9′′′ were noticed at δH 1.16–1.28 ppm and corresponding to 14Hs. H-14′′′ and 15′′′ were identified downfield as doublet signal at δH 2.35–2.37 ppm counting for 4 Hs. The most deshielded protons were spotted at δH 2.47 ppm (H-7′′′ and 13′′′) and 3.17 ppm (H-1′′′). In HSQC, H at δH 8.50 was correlated to C at 143.07, multiple signals at δH 0.83–0.89 ppm was connected to Cs at δc 12.24, 15.64, 22.27, and 23.25 ppm. Protons at δH 1.16–1.28 ppm were coupled with Cs at δc 21.14–31.18 ppm. Signals at δH 1.84, 2.35–2.37, and 2.47 ppm were associated with Cs at δc 22.76, 30.53, and 21.30 ppm respectively (5 was identified as 2-((11-(6-amino-9H-purin-9-yl)-2-methylundecan-5-yl)oxy)propane-1,3-diol.For compound 0H35N5O3 . Moreoveectively . Compoun1 was also recognized as a purine derivative. The characteristic two protons of purine in the aromatic region corresponding to H8 and H2 were detected at δH 7.67–7.73 ppm. Signals of monoacetylated ribose, methylated rhamnopyranoside, and the alkyl side chain were noticeable in the same range as in compounds B, C, and D, as per H 4.13–4.15 ppm . The positive HR-ESI-MS spectrum -9H-purin-9-yl-3′-monoacetate-α-ribofuranoyl-2′′,3′′,4′′-tri-O-methyl-α-rhamnopyranoside.Compound spectrum  revealedA. canariense L. petroleum ether extract allowed the identification of 91.7% of its fatty acid content, characterized by being long (C14–C22) and very long-chain fatty acids (more than 22 carbons)  . SaturatA. canariense L. extracts were screened for their cytotoxic, antimicrobial, and acetylcholinesterase inhibitory activities , hepatocellular carcinoma (HepG-2), and colon carcinoma (HCT-116), using sample concentrations ranging from 0 to 500 μg/mL. The results of the cytotoxic activity of A. canariense L. extracts revealed that the alkaloidal extract had the most powerful effect (IC50 14–28 μg/mL), with the best effect against HCT-116, followed by HepG-2 then MCF-7. The methanolic extract showed comparable results with higher IC50 . The results are shown in 50 for the different extracts. The results demonstrated that the methanolic extract showed significant activity with IC50 = 112.24 ± 7.73 ng/mL, along with the aqueous alkaloid extract with IC50 =139.27 ± 21.40 ng/mL. Moreover, the dichloromethane extract showed the highest (very potent) and most promising anti-acetylcholinesterase activity with IC50 = 62.48 ± 1.31 ng/mL compared to the standard drug (AChE inhibitor) tacrine, with IC50 = 27.29 ± 0.49 ng/mL  activity is the most characteristic change that occurs in Alzheimer’s disease. AChE is the enzyme responsible for acetylcholine hydrolysis, from both cholinergic and non-cholinergic neurons of the brain. The increase in acetylcholine level can be achieved by inhibition of AChE, which helps in the treatment of Alzheimer’s disease. The inhibitory activity of acetylcholinesterase was assessed using BioAssay Systems’ QuantiChromTM Screening kit based on an improved Ellman method. Through this work, the anti-Alzheimer’s activities (AChE enzyme inhibition activity) of the crude methanolic, dichloromethane, alkaloidal, as well as aqueous alkaloidal extracts of 49 ng/mL . Taken trds AChE , while abo group .Aizoon canariense L. (F. Aizoaceae) were collected from Cairo-Ismailia Road in February 2017 and kindly identified by Prof. Dr. A.A. Fayed, Professor of Plant Taxonomy, Faculty of Science, Assiut University, Assiut, Egypt. A voucher specimen is placed at the Herbarium of the Faculty of Science, Assiut University, Assiut, Egypt.Fresh whole-plant samples of 1H, 13C, COSY, and HSQC) were performed on a Bruker instrument  using DMSO-6d as a solvent and with chemical shift values given in δ (ppm) and referenced to the TMS signal as an internal reference. All samples were prepared in suitable deuterated solvents. An ultra-mass spectrometer was used , equipped with a Nanomate electrospray ionization (ESI) interface (Advion). An electrospray voltage of 1.7 kV (+/−) and a transfer capillary temperature of 200 °C were applied. Chromatographic analysis was carried out on TLC plates  using CH2Cl2–MeOH at different ratios, while column chromatographic separation was performed using a silica gel column with CH2Cl2 and gradient increase of MeOH. The analysis of fatty acid methyl esters was performed on an Agilent 19091J-413 gas chromatography instrument equipped with a flame ionization detector (FID), for which an HP-5 5% phenyl methyl siloxane capillary column (30 m × 320 µm × 0.25 µm) was used. The injector temperature was 250 °C, with an average velocity of 27 cm/s. H2 was the carrier gas, with a flow rate of 30 mL/min. The detector operated at a temperature of 280 °C. NMR analyses  eluted with CH2Cl2: MeOH (90:10) was neglected, as it consisted of traces of many compounds. Fraction 2 CH2Cl2: MeOH (80:20) consisted of a mixture of 2 major spots, with some other impurities. Therefore, it was separated and purified using preparative high-performance liquid chromatography (HPLC) equipped with an RP-C18 column , using a mobile phase consisting of 0.1% trifluroacetic acid in water (A)–MeOH (B) (HPLC grade) in the following sequence: 95% in 2 min, 95% to 50% in 23 min, 50% to 30% in 5 min. A photodiode array detector  was used. Samples were injected using a 100 µL glass syringe  yielding compounds 1 and 2 . Fraction 3 (CH2Cl2: MeOH (70:30)) was further purified on another silica gel sub-column  using a gradient elution of the CH2Cl2–MeOH mixture as a mobile phase to yield compounds 3 (15 mg), 4 (20 mg), and 5 (10 mg). The dried and powdered plant material (0.5 kg) was defatted with petroleum ether until exhaustion, then plant material was exhaustively extracted with 95% methanol. Acid–base extraction was applied as previously reported , allowin2SO4–MeOH at a ratio of 3:50 for three hours. The ethereal layer was then collected and the residue was kept for GLC analysis. The identification of the fatty acid methyl esters was carried out by comparing retention times with the applied authentic sample. The quantitative estimation of each peak was achieved by using a computer integrator, adopting the internal normalization procedures [Here, 1 g of petroleum ether extract was refluxed with KOH for three hours, then the mixture was partitioned with diethyl ether. The aqueous layer was acidified with HCl, extracted with diethyl ether, and then the ethereal extract was esterified by refluxing with Hocedures . v/v) CO2 atmosphere at 37 °C.All cell lines used in this study were obtained from Nawah Scientific, Inc. . Cells were maintained in DMEM media supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% heat-inactivated fetal bovine serum in a humidified 5% (50), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose–response curve for each concentration using GraphPad Prism v.8.4.2. , as indicated. Here, p values < 0.05 were considered statistically significant.The cytotoxic activity of both methanolic and alkaloidal extracts was estimated using 3--2–5-diphenyltetrazolium bromide (MTT) assay against human breast cancer (MCF-7), liver cancer (HEPG2), and colon cancer (HCT-116) cell lines ,41. PrinA. canariense L. extracts using the broth microdilution method against Gram-positive (Staphylococcus aureus (RCMB 010010), Bacillus subtilis ) and Gram-negative bacteria , Escherichia coli , in addition to fungi (Aspergillus flavus (RCMB 002002), Candida albicans ), using 96-well microplates at the Regional Centre for Mycology and Biotechnology (RCMB) at AL-Azhar University, Nasr City, Egypt [The minimum inhibitory concentration (MIC) values were determined for the different y, Egypt . MicrobiA. canariense L. extracts were estimated using a QuantiChromTM kit, IACE-100, . The acetylcholinesterase inhibitor screening kit is dependent on an enzyme-catalyzed kinetic reaction [E. electricus. The enzyme hydrolyzes the substrate acetylthiocholine, resulting in the production of thiocholine, which reacts with 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) to form 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate, which can be detected at 412 nm [test/ΔOD control) ×100 [The most important enzyme controlling acetylcholine (ACh) levels in healthy brains is acetylcholinesterase (AChE), while butyrylcholinesterase (BChE) is involved to a lesser extent . The antreaction . The enzt 412 nm ,47. Brieol) ×100 . p value significance levels are represented as asterisks (*) for p < 0.05, (**) p < 0.01, and (***) p < 0.001.Statistical analysis of the data was performed using one-way ANOVA, followed by Tukey’s multiple range test for post-hoc comparisons . All the data are presented as the means of 3 determinations ± SE. The A. canariense L. The alkaloidal fraction of A. canariense L. showed a promising cytotoxic effect against HCT-116, MCF-7, and, HepG-2, in addition to significant antimicrobial effects. Furthermore, the alkaloid fraction, as well as dichloromethane (flavonoid containing fraction), showed a significant effect against Alzheimer’s disease, which requires further in vivo studies. The predominance of behenic and tricosanoic acids in the non-polar fraction, as well as the adenine-based alkaloids, may correlate to the potential effects in cerebral disorders. Our work revealed A. canariense L. a potential candidate for the treatment of many ailments.This study provides the first report for the isolation and characterization of five adenine-based alkaloids from the polar alkaloidal fraction after acid–base extraction of the aerial parts of


Results: In 970 subjects, albuminuria (≥30 mg/g) was detected in 12.7% (95% CI 85.6–96.3%) with the ACR. Sensitivity was 82.9% (95% CI 75.1–89.1%) and 91.9% (95% CI 88.7–96.9%) and specificity 71.5% (95% CI 68.4–74.6%) and 17.5% (95% CI 15.0–20.2%) for the Combur9-Test® and Micral-Test®, respectively. Correct classification to KDIGO albuminuria stages A2/A3 with the Combur9-Test® was 15.4%, 51.4%, and 87.9% at cut-offs of 30, 100, and ≥300 mg/dL, and with the Micral-Test® it was 1.8%, 10.5%, and 53.6% at cut-offs of 2, 5, and 10 mg/dL, respectively. Overall, disagreement to KDIGO albuminuria was seen in 27% and 73% with the Combur9-Test® and Micral-Test®, respectively. From the total population, 62.5% and 15.3% were correctly ruled out and 2.2% and 1% were missed as false-negatives by the Combur9-Test® and Micral-Test®, respectively. Conclusion: Compared to the Combur9-Test®, the utility of the Micral-Test® is limited, because the fraction of correctly ruled out patients is small and a large proportion with a positive Micral-Test® require a subsequent ACR conformation test.Background: Semiquantitative dipstick tests are utilized for albuminuria screening. Methods: In a prospective cross-sectional survey, we analyzed the diagnostic test validity of the semiquantitative colorimetric indicator-dye-based Combur9-Test The burden of chronic kidney disease (CKD) is increasingly recognized as a global public health problem, with major negative implications on quality of life, premature death, and enormous costs on healthcare systems ,2. It is®) and an albumin-specific immunochromatographic assay  compared with the quantitative ACR reference diagnostic test [The aim of this study was to assess the validity of a semiquantitative standard colorimetric indicator-dye-based multi dipstick  as part of the RenalOne study as previously described . BrieflyWe followed the STARD 2015 guidelines for reporting diagnostic accuracy studies, as described by Cohen and Korevaar et al. .All data were collected in a case report form (CRF), translated from English to Swahili. In all participants, past medical histories and smoking status were assessed through interviews, and body weight and height, and blood pressure (BP) were measured and recorded. Glycated hemoglobin A1c (HbA1c) was measured from capillary blood by using a bedside DCA 2000+ Analyzer . A random clean urine specimen was collected in all patients. Within 30 min after returning the urine specimen to the study team, two different types of semiquantitative dipstick tests and an ACR test were carried out, in order to detect and to determine the degree of albuminuria.®  and the immunochromatographic dipstick Micral-Test® . Both urine dipstick tests were carried out manually according to the manufacturer’s instructions, read, interpreted, and recorded by the onsite investigator and full-time technician (N.H.), and supervised by a senior nephrologist (M.M.) [® is based on the ability of the protein to change the dye color of an acid–base indicator, forming the anion of 3′,3″,5′,5″-tetrachlorphenol-3,4,5,6-tetrabromsulfophthalein in presence of protein in the urine, resulting in a gradational change of the indicator dye [® aims for total protein in the urine, the sensitivity to albumin is higher than to other proteins found in the urine; this is explained by the increased affinity of albumin to protons [® value of ≥15–30 mg/dL (1+) as positive is based on the statement in the KDIGO 2012 guidelines that a reagent strip with (1+) protein positivity can substitute a quantitative albuminuria measurement where it not available [® is a chromatographic immunological procedure based on a gold-labelled monoclonal antibody with high specificity to human albumin. The correct handling is of significant importance for the test accuracy. With the correct amounts of fluid (urine) absorbed, the antibody–albumin conjugate is transported to a reactive detection pad. For the Micral-Test®, the detection limit is 20 mg/L (2 mg/dL) [As index test, two semiquantitative urine dipstick tests were performed and recorded during the time of the ACR measurement: the colorimetric dipstick Combour9-Testt (M.M.) . The semator dye . Althoug protons ,25,26. Tvailable , cardiovvailable ,28. The 2 mg/dL) ; cut-off2 mg/dL) ,30. Furt2 mg/dL) . Two indThe reference test was a POC quantitative ACR measurement in a spot urine sample. According to the KDIGO guidelines, the urine ACR measurement is the “first priority test” for the evaluation of albuminuria for a CKD classification . The ACRIn the “Assessment of Performance” section of the United States (U.S.) Food and Drug Administration (FDA) “510(k) Safety and Effectiveness Summary (K963142)”, it is declared that the DCA 2000+ Analyzer and the albumin/creatinine assay were studied in clinical settings and the results were substantially equivalent to results from methods used in clinical laboratory practice . The lin® was read as follows: negative (neg.), (1+/30 mg/dL), (2+/100 mg/dL), (3+/300 mg/dL), and (4+/≥2000 mg/dL) for total protein. The Micral-Test® was read as (neg.), (20 mg/L (2 mg/dL)), (50 mg/L (5 mg/dL)), and (100 mg/L (10 mg/dL)) for albumin. For the reference test, albuminuria was categorized according to KDIGO stages A1 with ACR (1–29 mg/g) normally to mildly increased, A2 (30–299 mg/g) moderately increased, and A3 (≥300 mg/g) severely increased albuminuria [The Combur9-Testuminuria ,33.®) and an albumin-sensitive immunological assay , for the detection of albuminuria, and the distribution of the semiquantitative urine protein evaluation within the albuminuria stages according to KDIGO guidelines [The primary outcome of this study was the assessment of the diagnostic test validity measures of an indicator-dye-based reagent strip . Discrete variables were expressed as counts (percentage), and comparison between groups was done with Pearson’s chi-square test or Fisher’s exact test. Continuous variables were expressed as mean ± standard deviation (SD) if normally distributed or as median and range if not normally distributed, and ® (≥1+) or Micral-Test® (≥2 mg/dL) and ACR ≥ 30 mg/g), true negatives (Combour9-Test® (<1+) or Micral-Test® (<2 mg/dL) and ACR < 30 mg/g), false positives (Combour9-Test® (≥1+) or Micral-Test® (≥2 mg/dL) and ACR < 30 mg/g), and false negatives (Combour9-Test® (<1+) or Micral-Test® (<2 mg/dL) and ACR ≥ 30 mg/g) cases. For a diagnostic test validity with 95% accuracy, a sample size of n = 945 was estimated, in a population with an assumed albuminuria prevalence of 10% [® ≥ 1+ (30 mg/dL) and Micral-Test® ≥ 2 mg/dL, and for scenario B, Combur9-Test® ≥ 2+ (100 mg/dL) and Micral-Test® ≥ 5 mg/dL. For scatterplots, data were logarithmically transformed. p-values of <0.05 were considered as statistically significant.For the test validity analysis, the sensitivity, specificity, and predictive values were calculated, with numbers extracted from the measures of true positives  patients had albuminuria with ACR ≥ 30 mg/g. The median age was 37 years (range 18–91 years), median body mass index (BMI) was 24 kg/m2 (range 14–53 kg/m2), median HbA1c was 5.4% (range 3.9–14%), median hemoglobin was 12.8 g/dL (range 4.1–22 g/dL), and median systolic and diastolic BP were 124 mmHg (range 70–286 mmHg) and 80 mmHg (range 36–150 mmHg), respectively. Patients with albuminuria were older (45 years (range 18–84 years) versus 36 years (range 18–91 years)), had higher HbA1c (5.6% (range 4.2–14%) versus 5.4% (range 3.9–14%), and more often had diabetes (16% versus 5.5%), higher systolic BP 144 mmHg (range 72–286 mmHg) versus 129 mmHg (70–222 mmHg), and higher diastolic BP 90 mmHg (range 42–150 mmHg) versus 80 mmHg (range 36–140 mmHg) than patients without albuminuria . Patients with albuminuria had more often stage I and stage II hypertension (46% versus 24% and 28% versus 9% (p < 0.001)), were more often HIV-positive (11% versus 5.9% (p = 0.032)), had more often a history of tuberculosis (11% versus 3.8% (p = 0.001)), and had more acute infections (14% versus 7.4% (p = 0.013)) than patients without albuminuria.The study population consisted of 301 (30%) males and 673 (70%) females . Overalln = 102 true positives, with a positive Combur9-Test® and a positive ACR reference test, (b) shows n = 241 false positives, with a positive Combur9-Test® and a negative ACR reference test, (c) shows n = 21 false negatives, with a negative Combur9-Test® and a positive ACR reference test, and (d) shows n = 606 true negatives, with a negative Combur9-Test® and a negative ACR reference test. For the Micral-Test®  shows n = 699 false positives, with a positive Micral-Test® and a negative ACR reference test, (c) shows n = 10 false negatives, with a negative Micral-Test® and a positive ACR reference test, and (d) shows n = 148 true negatives, with a negative Micral-Test® and a negative ACR reference test.In ® and the Micral-Test® at different cut-off values compared with the ACR reference test. Overall, albuminuria prevalence was 12.7% (n = 123/970) with the ACR reference test at a cut-off of ≥30 mg/g. At a Combur9-Test® cut-off level of ≥30 mg/dL (≥1+), the prevalence of Combur9-Test® positive patients was 35.4% (n = 343/970). Of those, 10.5% (n = 102) also tested positive with the ACR reference test (≥30 mg/g). At this cut-off, the sensitivity of the Combur9-Test® was 82.9% (95% CI: 75.1–89.1%), the specificity 71.5% (95% CI: 68.4–74.6%), the PPV 29.7% (95% CI: 24.9–34.9%), and the NPV 96.7% (95% CI 94.9–97.9%). At a Micral-Test® cut-off level of ≥20 mg/L (≥2 mg/dL), the prevalence of Micral-Test® test positive patients was 83.7% (n = 812/970). Of those, 11.6% (n = 113) also tested positive with the ACR reference test at a cut-off of ≥30 mg/g. Compared to an ACR reference cut-off of ≥30 mg/g, the sensitivity of the Micral-Test® was 91.9% (95% CI: 85.6–96.3%), the specificity 17.5% (95% CI: 15.0–20.2%), the PPV 13.9% (95% CI: 11.6–16.5%), and the NPV 93.7% (95% CI: 88.7–96.9%). With increasing cut-off level, Combur9-Test® and Micral-Test® have increasing specificity and decreasing sensitivity  of the patients (n = 606) corresponded to ACR category A1 and 3.4% (n = 21) to A2/A3. The Combur9-Test® was trace-positive (1+) (30 mg/dL) in 24.7% (n = 240) of patients. Of those, 84.6% (n = 203) corresponded to ACR category A1 and 15.4% (n = 37) to A2/A3. The Combur9-Test® was 2+ (100 mg/dL) in 7.2% (n = 70) of patients. Of those, 48.6% (n = 34) corresponded to ACR category A1 and 51.4% (n = 36) to A2/A3. From the 3.4% (n = 33) of patients with Combur9-Test® ≥ 300 mg/dL, 12.1% (n = 4) corresponded to ACR category A1 and 87.9% (n = 29) to A2/A3. Correct classification to KDIGO albuminuria stages A2/A3 with the Combur9-Test® was 15.4%, 51.4%, and 87.9% at cut-offs of 1+ (30 mg/dL), 2+ (100 mg/dL), and ≥3+ (≥300 mg/dL), respectively. Overall, disagreement to KDIGO albuminuria was seen in 27% (n = 262) with the Combur9-Test®.Summarized in patients A,C. Of t® identified 16.3% (n = 158) of the patients as albuminuria-negative (n = 148) corresponded to ACR category A1 and 6.3% (n = 10) to A2/A3. The Micral-Test® dipstick identified 46.2% (n= 448) of the patients having albuminuria of 2 mg/dL. Of those, 98.2% (n = 440) corresponded to ACR category A1 and 1.8% (n = 8) to A2/A3. The Micral-Test® was positive at a cut-off level of 5 mg/dL in 21.5% (n = 209) of patients. Of those, 89.5% (n = 187) corresponded to ACR category A1 and 10.5% (n = 22) to A2/A3. The Micral-Test® was positive at the cut-off level 10 mg/dL in 16% (n = 155) of patients. Of these, 46.4% (n = 72) corresponded to A1 and 53.6% (n = 83) to A2/A3. Correct classification with the Micral-Test® was 1.8%, 10.5%, and 53.6% at cut-offs of 2, 5, and 10 mg/dL respectively. Overall, disagreement to KDIGO albuminuria was seen in 73% (n = 709) with the Micral-Test®.The semiquantitative Micral-Testnegative B,D. Of t® with the ACR reference test and the Micral-Test®. From the 627 (100%) patients with a negative Combur9-Test®, 96.6% (n = 606) had a negative and 3.4% (n = 21) a positive ACR and 22.8% (n = 143) a negative and 77.2% (n = 481) a positive Micral-Test®. From the 240 (100%) patients with a 1+ (30 mg/dL) Combur9-Test®, 84.6% (n = 203) had a negative and 15.4% (n = 37) a positive ACR and 5.5% (n = 13) a negative and 94.5% (n = 227) a positive Micral-Test®. From the 70 (100%) patients with 2+ (100 mg/dL) positive Combur9-Test®, 48.6% (n = 34) had a negative and 51.4% (n = 36) a positive ACR and 4.3% (n = 3) a negative and 95.7% (n = 67) a positive Micral-Test®. From the n = 33 (100%) patients with a ≥3+ (≥300 mg/dL) positive Combur9-Test®, 12.1% (n = 4) had a negative and 87.9% (n = 29) a positive ACR and 100% (n = 33) a positive Micral-Test®.Summarized in ® with the ACR reference test and the Combur9-Test®. From the 158 (100%) patients with a negative Micral-Test®, 93.7% (n = 148) had a negative and 6.3% (n = 10) a positive ACR and 91% (n = 143) a negative and 9% (n = 15) a positive Combur9-Test®. From the 448 (100%) patients with a 2 mg/dL positive Micral-Test®, 98.2% (n = 440) had a negative and 1.8% (n = 8) a positive ACR and 87.3% (n = 391) a negative and 12.7% (n = 57) a positive Combur9-Test®. From the 209 (100%) patients with a 5 mg/dL positive Micral-Test®, 89.5% (n = 187) had a negative and 11.5% (n = 22) a positive ACR and 31.6% (n = 66) a negative and 68.4% (n = 143) a positive Combur9-Test®. From the n = 155 (100%) patients with a 10 mg/dL positive Micral-Test®, 46.4% (n = 72) had a negative and 53.6% (n = 83) a positive ACR and 11% (n = 17) a negative and 89% (n = 138) a positive Combur9-Test®.Summarized in ® positivity was set at (1+) ≥30 mg/dL. A negative test had a high NPV of 96% and included 64.6% (n = 627/970) of the screened population (n = 21/970) and 17% (n = 21/123) cases with a relevant albuminuria of the whole population and of all albuminuria-positives would be missed, respectively. Correctly ruled out would be 62.5% (n = 606/970) and 71.5% (n= 606/847) of the whole population and all albuminuria-negatives, respectively. Of the 343 patients with a positive dipstick Combur9-Test® and a subsequent ACR conformation test, 10.5% (n = 102/970) and 83% (n = 102/123) of the whole population and of all albuminuria-positives would be confirmed, and 24.8% (n = 241/970) and 70.2% (n = 241/343) of the whole population and of all Combur9-Test® positives would be over tested, respectively. In summary, the proposed algorithm would mean that 17% of the true positive results would be missed  and that an unnecessary additional test would have to be carried out in 70.2% of patients with a positive screening dipstick test .In scenario A, the cut-off for Combur9-Testpulation A. Withou® positivity, the cut-off was set at ≥2 mg/dL. A negative test had an NPV of 94% and included 16.3% (n = 158/970) of the screened population. Without further confirmation of a negative test result, 1% (n = 10/970) and 8% (10/123) of cases with a relevant albuminuria of the whole population and of all albuminuria-positives would be missed, respectively. Correctly ruled-out would be 15.3% (n = 148/970) of the population and 17.5% (n = 148/847) of all albuminuria-negatives, respectively. Of the 83% (n = 812/970) with a positive dipstick Micral-Test® and a subsequent ACR conformation test, 11.6% (113/970) of the population and 92% (n = 113/123) of all albuminuria-positives would be confirmed, and 72.1% (n = 699/970) of the population and 86% (n = 699/812) of all Micral-Test® positives would be over tested, respectively. In summary, the proposed algorithm would mean that 8% of the true positive results would be missed  and that an unnecessary additional test would have to be carried out in 86% of the patients with a positive screening dipstick test   A.® positivity was set at (≥2+) ≥100 mg/dL. A negative test had an NPV of 85.4% and included 89.4% (n = 867/970) of the screened population (n = 58/970) and 47% (n = 48/123) of cases with a relevant albuminuria of the whole population and of all albuminuria-positives would be missed, respectively. Correctly ruled out would be 83.4% (n = 809/970) and 95.5% (n= 809/847) of the population and all albuminuria-negatives, respectively. Of the n = 103 patients with a positive dipstick Combur9-Test® and a subsequent ACR conformation test, 6.7% (n = 65/970) and 53% (n = 65/123) of the whole population and of all albuminuria-positives would be confirmed, and 3.9% (n = 38/970) and 37% (n = 38/103) of the whole population and of all Combur9-Test® positives would be over tested, respectively. In summary, the proposed algorithm would mean that 47% of the true positive results would be missed  and that an unnecessary additional test would have to be carried out in 37% of patients with a positive screening dipstick test .In scenario B, the cut-off for Combur9-Testpulation B. Withou® positivity, the cut-off was set at ≥5 mg/dL. A negative test had an NPV of 98% and included 62.4% (n = 606/970) of the screened population. Without further confirmation of a negative test result, 1.9% (n = 18/970) and 15% (18/123) of cases with a relevant albuminuria of the whole population and of all albuminuria-positives would be missed, respectively. Correctly ruled out would be 60.6% (n = 588/970) of the population and 69.4% (n = 588/847) of all albuminuria-negatives, respectively. Of the 37.5% (n = 364/970) with a positive dipstick Micral-Test® and a subsequent ACR conformation test, 10.8% (105/970) of the population and 85% (n = 105/123) of all albuminuria-positives would be confirmed, and 26.7% (n = 259/970) of the population and 71% (n = 259/812) of all Micral-Test® positives would be over tested, respectively. In summary, the proposed algorithm would mean that 15% of the true positive results would be missed  and that an unnecessary additional test would have to be carried out in 71% of the patients with a positive screening dipstick test   A.® showed weak allocation to all albuminuria stages when positive and was too sensitive to classify the large fraction of subjects without albuminuria to stage A1 . The Combur9-Test® has its limitations but was accurately classifying patients with strong positive (≥3+) results to the corresponding albuminuria stages A2/A3 (moderately/severely increased), and a majority of those subjects without albuminuria correctly to stage A1. Our data illustrate a dose-dependency between the two urine dipstick tests, which leads to a clear concordance with increasing albumin amounts in urine  and therefore was correctly ruling out only 15% (n = 148/970) of the population. While the colorimetric indicator-dye reagent strip Combur9-Test® with a specificity of 71.5%  was four times more (62% of the population (n = 606/970)) efficient in correctly ruling out albuminuria-negatives from the population screened. The consequence of the high proportion already ruled out with the reagent strip Combur9-Test® was resulting in a relevant smaller fraction (35% (n = 343/970)) of subsequent ACR testing compared with the Micral-Test®, where 83% (n = 812/970) of the entire population would require an ACR conformation test.In the models for albuminuria screening algorithms, we clearly illustrate that both tests have a high accuracy in correctly ruling out patients with no further need for ACR conformation A,B. Howe® did not exceed a PPV of over 54%. Therefore, the chance for a correct detection of a clinically relevant albuminuria according to the KDIGO guidelines with the albumin-specific dipstick test was nearly fifty-fifty, even at its highest test positivity level (≥10 mg/dL) [n = 699/970) of Micral-Test® positive patients were not confirmed to have albuminuria in a ACR test, and therefore were over tested. Similarly, for the Combur9-Test®, more than two-thirds  of the patients with a positive dipstick test result were not confirmed to have albuminuria in an ACR conformation test. However, the decisive difference is that the overall number for a required ACR conformation test was significantly smaller with the Combur9-Test®  compared with the Micral-Test® . The indicator-dye-based Combur9-Test® strip is of good value to correctly rule out patients with no albuminuria, but the semiquantitative detection device has its limitations, with weak test validity measures for ≥1+ (30 mg/dL) and ≥2+ (100 mg/dL) cut-offs, and therefore these quantifications are of little value for albuminuria classification according to KDIGO [Although the specificity improved with increasing cut-off level , the Micral-Test0 mg/dL) . The vasto KDIGO .® ≥2+ (100 mg/dL), in regard to correctly ruling out patients, further increased to 83.4%, but at the cost of a relevant number of patients that were missed and judged falsely as negatives . If the cut-off level for the Micral-Test® was increased to ≥5 mg/dL, the overall distribution of correctly detected, ruled out, missed, and over tested patients was nearly equal to the Combur9-Test® with its regular cut-off levels at ≥1+ (30 mg/dL) If the cut-off level was increased by one level for each index test, the performance of the Combur9-Test® to correctly rule out patients is only partially correct, because the fraction of correctly ruled out healthy patients was comparatively small, and a large proportion with a positive Micral-Test® would require a subsequent ACR conformation test according to guideline-orientated clinical practice [With these results from the colorimetric indicator-dye-based urine test strip, we confirm the findings of large population-based studies from across the globe, and several smaller studies in at-risk cohorts, including some from SSA ,38,39,40practice .In our study, we face some limitations. A change in prevalence may lead to different test validity measures and must be considered also for event  probabilities . There a®, the more expensive and more specific Micral-Test® does not add any significant benefit to clinical practice and would require a much higher rate of unnecessary subsequent ACR clarification testing, in order to diagnose albuminuria according to guideline recommendations [® with the ability to rule out albuminuria accurately, without a relevant loss of missed albuminuria cases, is therefore a valid initial diagnostic tool to screen for albuminuria and CKD in a walk-in population with an assumed albuminuria prevalence of 10%. These findings are of importance considering the distribution of resources in clinical practice and research settings, where upon a positive dipstick test result a subsequent ACR conformation test should be executed.The two semiquantitative index tests differ strongly in specificity, especially at their lowest positivity levels. Compared to the Combur9-Testndations . The Com