Cellular immune responses, particularly CD8+ T cell responses, probably contribute to protection against severe SARS-CoV-2 infection6. Here we show that cellular immunity induced by current vaccines against SARS-CoV-2 is highly conserved to the SARS-CoV-2 Omicron spike protein. Individuals who received the Ad26.COV2.S or BNT162b2 vaccines demonstrated durable spike-specific CD8+ and CD4+ T cell responses, which showed extensive cross-reactivity against both the Delta and the Omicron variants, including in central and effector memory cellular subpopulations. Median Omicron spike-specific CD8+ T cell responses were 82–84% of the WA1/2020 spike-specific CD8+ T cell responses. These data provide immunological context for the observation that current vaccines still show robust protection against severe disease with the SARS-CoV-2 Omicron variant despite the substantially reduced neutralizing antibody responses8.The highly mutated SARS-CoV-2 Omicron (B.1.1.529) variant has been shown to evade a substantial fraction of neutralizing antibody responses elicited by current vaccines that encode the WA1/2020 spike protein Current vaccines induce broadly cross-reactive cellular immunity against SARS-CoV-2 variants, including Omicron, and provide protection against severe disease despite a substantially reduced neutralizing antibody response. To evaluate the cross-reactivity of vaccine-elicited cellular immune responses against the SARS-CoV-2 Omicron variant, we assessed CD8+ and CD4+ T cell responses in 47 individuals who were vaccinated with the adenovirus vector-based Ad26.COV2.S vaccine9  or the mRNA-based BNT162b2 vaccine10  in Boston, MA, USA  are substantially reduced to the highly mutated SARS-CoV-2 Omicron variantP < 0.0001, two-tailed Mann–Whitney test), as expected12    Fig. . Howeverts) Fig. , consist+ and CD4+ T cell responses by intracellular cytokine staining assays . Spike-specific IFNγ CD4+ T cell responses elicited by Ad26.COV2.S were a median of 0.026%, 0.030% and 0.029% against WA1/2020, Delta and Omicron, respectively, and by BNT162b2 were a median of 0.033% and 0.027% against WA1/2020 and Omicron, respectively, at month 8, indicating that median Omicron-specific CD4+ T cell responses were 82–100% cross-reactive with WA1/2020-specific CD4+ T cell responses   and the BNT162b2 vaccine , although two individuals had undetectable Omicron-specific CD8+ T cell responses following vaccination with BNT162b2  and the BNT162b2 vaccine   Fig. .+ and CD4+ T cell central (CD45RA−CD27+) and effector (CD45RA−CD27−) memory subpopulations elicited by Ad26.COV2.S also showed extensive cross-reactivity to Delta and Omicron variants. At month 8, CD8+ T cell central memory responses were 0.076%, 0.054% and 0.075%, CD8+ T cell effector memory responses were 0.168%, 0.143% and 0.146%, CD4+ T cell central memory responses were 0.030%, 0.035% and 0.038%, and CD4+ T cell effector memory responses were 0.102%, 0.094% and 0.083% against WA1/202, Delta and Omicron, respectively  suggests the generalizability of these findings. The extensive cross-reactivity of Omicron-specific CD8+ T cells contribute to protection against SARS-CoV-2 in rhesus macaques, particularly when antibody responses are suboptimal5. Durable CD8+ and CD4+ T cell responses have also been reported following infection and vaccination16. Given the role of CD8+ T cells in clearance of viral infections, it is likely that cellular immunity contributes substantially to vaccine protection against severe SARS-CoV-2 disease. This may be particularly relevant for Omicron, which dramatically evades neutralizing antibody responses. Recent studies have shown that Ad26.COV2.S and BNT162b2 provided 85% and 70% protection, respectively, against hospitalization due to the Omicron variant in South Africa8. Our data provide immunological context for the observation that current vaccines still provide robust protection against severe disease due to the SARS-CoV-2 Omicron variant despite substantially reduced neutralizing antibody responses.Preclinical studies have shown that CD8Samples from individuals who received the BNT162b2 vaccine were obtained from the Beth Israel Deaconess Medical Center (BIDMC) specimen biorepository. Samples from individuals who received Ad26.COV2.S were obtained from the COV1001 study (NCT04436276). Both studies were approved by the BIDMC Institutional Review Board. All participants provided informed consent. Individuals were excluded from this study if they had a history of SARS-CoV-2 infection, received other COVID-19 vaccines or received immunosuppressive medications.4 cells per well overnight. Threefold serial dilutions of heat-inactivated serum samples were prepared and mixed with 50 μl of pseudovirus. The mixturewas incubated at 37 °C for 1 h before adding to HEK293T-hACE2 cells. After 48 h, cells were lysed in a Steady-Glo Luciferase Assay (Promega) according to the manufacturer’s instructions. SARS-CoV-2 neutralization titres were defined as the sample dilution at which a 50% reduction (NT50) in relative light units was observed relative to the average of the virus control wells.The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were used to measure pseudovirus NAbs. In brief, the packaging construct psPAX2 (AIDS Resource and Reagent Program), the luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene) and the spike protein expressing pcDNA3.1-SARS-CoV-2 SΔCT were co-transfected into HEK293T cells (American Type Culture Collection (ATCC) CRL_3216) with Lipofectamine 2000 (Thermo Fisher Scientific). Pseudoviruses of SARS-CoV-2 variants were generated by using the WA1/2020 strain , the B.1.1.7 variant , the B.1.351 variant , the B.1.617.2 variant  or the B.1.1.529 variant . The supernatants containing the pseudotype viruses were collected 48 h after transfection; pseudotype viruses were purified by filtration with a 0.45-μm filter. To determine the neutralization activity of human serum, HEK293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 1.75 × 10−1 of similarly produced SARS-CoV-2 WA1/2020, B.1.617.2 (Delta), B.1.351 (Beta) or B.1.1.529 (Omicron) RBD protein in 1× Dulbecco’s phosphate-buffered saline (DPBS) and incubated at 4 °C overnight. Assay performance was similar for these four RBD proteins. After incubation, plates were washed once with wash buffer (0.05% Tween 20 in 1× DPBS) and blocked with 350 μl of casein block solution per well for 2–3 h at room temperature. Following incubation, block solution was discarded and plates were blotted dry. Serial dilutions of heat-inactivated serum diluted in casein block were added to wells, and plates were incubated for 1 h at room temperature, before three more washes and a 1-h incubation with a 1:4,000 dilution of anti-human IgG horseradish peroxidase  at room temperature in the dark. Plates were washed three times, and 100 μl of SeraCare KPL TMB SureBlue Start solution was added to each well; plate development was halted by adding 100 μl of SeraCare KPL TMB Stop solution per well. The absorbance at 450 nm, with a reference at 650 nm, was recorded with a VersaMax microplate reader (Molecular Devices). For each sample, the ELISA end point titre was calculated using a four-parameter logistic curve fit to calculate the reciprocal serum dilution that yields a corrected absorbance value (450–650 nm) of 0.2. Interpolated end point titres were reported.SARS-CoV-2 spike receptor-binding domain (RBD)-specific binding antibodies in serum were assessed by ELISA. Ninety-six-well plates were coated with 2 μg mll-glutamine and 0.1% of 55 mM 2-mercaptoethanol) for 2–4 h at 37 °C. SARS-CoV-2 pooled S peptides from SARS-CoV-2 WA1/2020, B.1.617.2 (Delta) or B.1.1.529 (Omicron)  were prepared and plated at a concentration of 2 µg per well, and 100,000 cells per well were added to the plate. The peptides and cells were incubated for 15–20 h at 37 °C. All steps following this incubation were performed at room temperature. The plates were washed with ELISPOT wash buffer and incubated for 2–4 h with biotinylated mouse anti-human IFNγ monoclonal antibody from MabTech (1 µg ml−1). The plates were washed a second time and incubated for 2–3 h with conjugated goat anti-biotin AP from Rockland, Inc. (1.33 µg ml−1). The final wash was followed by the addition of nitro-blue tetrazolium chloride/5-bromo-4-chloro 3 ‘indolyphosphate p-toludine salt (NBT/BCIP chromagen) substrate solution for 7 min. The chromagen was discarded and the plates were washed with water and dried in a dim place for 24 h. Plates were scanned and counted on a Cellular Technologies Limited Immunospot Analyzer.Peptide pools were 16 amino acid peptides overlapping by 11 amino acids spanning the SARS-CoV-2 WA1/2020, B.1.617.2 (Delta) or B.1.1.529  spike proteins . Enzyme-linked immunospot (ELISPOT) plates were coated with mouse anti-human IFNγ monoclonal antibody from MabTech at 1 µg per well and incubated overnight at 4 °C. Plates were washed with DPBS and blocked with R10 media  assays. Peptide pools were 16 amino acid peptides overlapping by 11 amino acids spanning the SARS-CoV-2 WA1/2020, B.1.617.2 (Delta) or B.1.1.529  spike proteins . 106 peripheral blood mononuclear cells were resuspended in 100 µl of R10 media supplemented with CD49d monoclonal antibody (1 µg ml−1) and CD28 monoclonal antibody (1 µg ml−1). Each sample was assessed with mock , peptides (2 µg ml−1) and/or 10 pg ml−1 phorbol myristate acetate (PMA) and 1 µg ml−1 ionomycin (Sigma-Aldrich)  and incubated at 37 °C for 1 h. After incubation, 0.25 µl of GolgiStop (BD) and 0.25 µl of GolgiPlug (BD) in 50 µl of R10 was added to each well and incubated at 37 °C for 8 h and then held at 4 °C overnight. The next day, the cells were washed twice with DPBS, stained with aqua live/dead dye for 10 min and then stained with predetermined titres of monoclonal antibodies ti CD279 , CD4 , CD27 , CD8  and CD45RA  for 30 min. Cells were then washed twice with 2% FBS/DPBS buffer and incubated for 15 min with 200 µl of BD CytoFix/CytoPerm Fixation/Permeabilization solution. Cells were washed twice with 1X Perm Wash buffer (BD Perm/Wash Buffer 10X in the CytoFix/CytoPerm Fixation/ Permeabilization kit diluted with MilliQ water and passed through a 0.22-µm filter) and stained intracellularly with monoclonal antibodies to Ki67 , IL-21 , CD69 , IL-10 , IL-13 , IL-4 , TNF , IL-17 , IFNγ , IL-2 , IL-6  and CD3  for 30 min. Cells were washed twice with 1X Perm Wash buffer and fixed with 250 µl of freshly prepared 1.5% formaldehyde. Fixed cells were transferred to a 96-well round bottom plate and analysed by BD FACSymphony system. Data were analysed using FlowJo v9.9.CD4P < 0.05 were considered significant.Descriptive statistics and logistic regression were performed using GraphPad Prism 8.4.3, (GraphPad Software). Immunological data were generated in duplicate and were compared by Mann–Whitney tests. Correlations were evaluated by linear regression. Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-04465-y.Reporting SummarySupplementary Table 1Peptide sequences for WA1/2020 and Omicron spike.


Its novel chemistry with a synthetically useful second nitrogen atom on the N-tert-butyl piperazine substructure generates a pharmacologically useful core.A sterically congested piperazine derivative,  20H27FN2O2, was prepared using a modified Bruylants approach. A search of the Cambridge Structural Database identified 51 compounds possessing an N-tert-butyl piperazine substructure. Of these only 14 were asymmetrically substituted on the piperazine ring and none with a synthetically useful second nitrogen. Given the novel chemistry generating a pharmacologically useful core, determination of the crystal structure for this compound was necessary. The piperazine ring is present in a chair conformation with di-equatorial substitution. Of the two N atoms, one is sp3 hybridized while the other is sp2 hybridized. Intermolecular interactions resulting from the crystal packing patterns were investigated using Hirshfeld surface analysis and fingerprint analysis. Directional weak hydrogen-bond-like interactions (C—H⋯O) and C—H⋯π interactions with the dispersion interactions as the major source of attraction are present in the crystal packing.The title sterically congested piperazine derivative, C Nucleophilic attack by an organometallic reagent into a transient 1-N-ethylidenepiperazinium has a literature precedent, but nucleophilic attack into the more sterically congested 1-N-propylidenepiperazinium intermediate by an alkynyl Grignard reagent is presented here for the first time. Four potential synthetic routes were identified including Katritzky benzotriazole trapping of an iminium piperazines. These could be prepared in several steps from the corresponding alkyne P21 with one molecule in the asymmetric unit as shown in the Scheme and Fig. 2et al., 2013et al., 2015et al., 2004et al., 1999sp3 hybridization, while the sum of the bond angles around N2 (360°) indicates sp2 hybridization. This is also evidenced by the tetragonal molecular geometry of C12—N1—C9 [113.89 (18)°], C14—N1—C9 [113.48 (16)°], and C12—N1—C14 [108.36 (16)°] and the trigonal planar molecular geometry of C16—N2—C15 [126.30 (19)°], C16—N2—C13 [120.9 (2)°], and C15—N2—C13 [112.8 (2)°]. The delocalization of the lone pair of N2 into the π bond of carbonyl group causes sp2 hybridization of N2.The title compound, prepared from achiral reagents as a racemic mixture, crystallizes in the chiral monoclinic space group et al., 2007a-axis  = 2.595 Å] present in the crystal packing could be weak C—H⋯O hydrogen-bond-like interactions  = 2.804 Å] interactions could be C—H⋯π interactions with dispersion interactions as the major source of attraction. Fingerprint analysis .Hirshfeld surface analysis and fingerprint analysis were performed using CrystalExplorer piperazine-1-carboxylate (3): Ethereal HCl  was added dropwise to a stirred solution of tert-butyl piperazine-1-carboxylate 2  in MeOH (60 mL) and CH2Cl2 (60 mL) at 273 K under Argon. The resulting mixture was stirred at 273 K for 1 h, after which the solvent and excess HCl were removed under reduced pressure and the white residual solid was dissolved in water (150 mL). In a well-ventilated fume hood, solid NaCN  and then a solution of acetone  in water (20 mL) were added sequentially at room temperature (296 K). The resulting mixture was stirred at room temperature under air for an additional 48 h. Water (100 mL) was added and the mixture was extracted with EtOAc (3 × 100 mL) then NaCl . The combined organic extracts were dried (MgSO4) and the solvent was removed under reduced pressure to give tert-butyl 4-(2-cyanopropan-2-yl)piperazine-1-carboxylate 3 as a white crystalline solid, 11 g (64%). MP: 381.2 K (reported 381–383 K) matching the literature , 2.62 , 1.54 , 1.49  matches literature  were collected and the residual cyanide was oxidized to cyanate with sodium hypochlorite -2-methylbut-3-yn-2-yl]piperazine-1-carboxylate (1):4  in 50 mL of anhydrous THF. This solution was cooled with an external ice-bath. A commercial solution of methyl magnesium bromide   was added with slow dropwise addition over 10 minutes. The internal temperature was maintained between 274–275 K. This mixture was stirred at ice-bath temperature for an additional 20 minutes, which resulted in a pale-yellow solution. A solution of tert-butyl 4-(2-cyanopropan-2-yl)piperazine-1-carboxylate 3 . Following stirring for 12 h at 296 K, the crude reaction mixture was cooled to ice-bath temperature and the reaction was quenched with the addition of 10 mL of ice-cold water at a rate of addition that maintained the internal temperature below 278 K. After quenching the organo-base, an additional 50 mL of water were added. Small aliquots of brine and ethanol were used, as required, to break the emulsion in the following extraction. This mixture was extracted with 3 × 20 mL of ethyl acetate, washed  dried (Na2SO4), decanted, and the solvent removed under reduced pressure to afford 30.6 g of a yellow solid. This was separated on 50 g of SiO2 with hexane/ethyl acetate (1/1) as the eluent to yield tert-butyl 4-[4-(4-fluorophenyl)-2-methylbut-3-yn-2-yl]piperazine-1-carboxylate 1 as a white powder, 2.74 g (86.3%). This compound was recrystallized from ethyl acetate as colorless plates, having a melting point of 388.1 K. 1H NMR  δ 7.36 , 6.96 , 3.46 , 2.63 , 1.45 . HRMS: (C20H27FN2O2) calculated for [M + H]+ 347.2129, found 347.2127.A 250 mL flame-dried, round-bottom flask was cooled under argon and then charged with 1-ethynyl-4-fluorobenzene Uiso(H) = 1.2Ueq(C) for aromatic and 1.5Ueq(C) for methyl groups.Crystal data, data collection, and structure refinement details are summarized in Table 310.1107/S2056989021002346/zl5007sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021002346/zl5007Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021002346/zl5007Isup3.cmlSupporting information file. DOI: 2067318CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The reaction of dichlorodimethylsilane with a polydentate Schiff base ligand derived from pyridoxal and 2-ethanolamine yielded a macrocyclic silicon compound. E,22E)-4,4,12,18,18,26-hexamethyl-3,5,17,19-tetraoxa-8,13,22,27-tetraaza-4,18-disilatricyclooctacosa-1(24),8,10,12,14,22,25,27-octaene-11,25-diol, C24H36N4O6Si2. The asymmetric unit contains the half macrocycle with an intramolecular O—H⋯N hydrogen bond between the imine nitrogen atom and a neighbouring oxygen atom. The crystal structure is dominated by C—H⋯O and C—H⋯π interactions, which form a high ordered molecular network.The reaction of dichlorodimethylsilane with a polydentate Schiff base ligand derived from pyridoxal and 2-ethanolamine yielded the macrocyclic silicon compound (8 This vitamin is an essential cofactor to a large number of enzymes that catalyze many reactions of amino acids iminomethyl]-5- hydroxymethyl-2-methylpyridine-3-ol (I)bThe heterocyclic aldehyde pyridoxal is one of the active forms of vitamin B2SiCl2. The reaction was performed in tetrahydrofuran in presence of triethylamine as supporting base to remove the hydrogen chloride, which is formed during the reaction. Recrystallization of the raw product from 1,2-dimethoxyethane and diethyl ether gave yellow crystals suitable for structure analysis.There are several potential coordination sites at the ligand molecule (I)I2/c with the half macrocycle in the asymmetric unit. Fig. 1C2 axis through the centre of the macrocycle  crystallizes in the monoclinic space group le Fig. 2. The sille Fig. 2. A quite al. 1990, these hds Fig. 2.A⋯O2 and C5—H5⋯O1 results in a chain along the crystallographic b-axis. The C—H⋯O interaction of C9—H9B with O2 connects adjacent chains  are observed. First, a bifurcation at H9B (d = 3.31 Å) shows up within the C—H⋯O chains along the c axis. Furthermore, C11—H11A⋯Cg1 (d = 2.85 Å) supports the C—H⋯O interactions of H5 and H6A.Apart from the relevant C—H⋯O interaction, two C—H⋯π contacts with the pyridine moiety  a suitable candidate for supramolecular recognition processes. The available pyridine N, azomethine N, and OH groups could be useful for the generation of nanostructures ConQuest  presented here and the former investigation  was performed in Schlenk tubes under argon with dry and air-free solvents.in vacuo from the resulting clear yellow solution. The remaining solid was extracted with 1,2-dimethoxyethane. Addition of diethyl ether and cooling to 278 K yielded yellow crystals of (III) .Compound (III) was prepared by reaction of 4-[(2-hydroxyethyl)iminomethyl]-5-hydroxymethyl-2-methylpyridine-3-ol (I)3, 300 K, TMS, in p.p.m.): 29Si: −0.1. 1H: δ = 0.14 , 2.50 , 3.71, 3.90 , 4.78 , 7.89 , 8.84 , 14.05 . 13C: 3.0 (Me2Si), 22.0 , 63.3, 64.6 (N—CH2—CH2—O), 66.4 , 122.6, 133.4, 140.8, 153.8, 157.8 , 167.5 (HC=N).NMR . The Uiso(H) values were fixed at 1.5 (for the methyl H) or 1.2 times the equivalent Ueq value of the parent carbon atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021010185/zq2266sup1.cifCrystal structure: contains datablock(s) I, I_7. DOI: 10.1107/S2056989021010185/zq2266Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021010185/zq2266Isup3.cmlSupporting information file. DOI: 2113407CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Globodera pallida, which assimilates intact peptides from the external environment, was investigated as a new potential non-transgenic model system of AD. Methods: Fresh second-stage juveniles of G. pallida were used to measure their chemosensory, perform immunocytochemistry on their neurological structures, evaluate their survival rate, measure reactive oxygen species, and determine total oxidized glutathione to reduced glutathione ratio (GSSG/GSH) levels, before and after treatment with 100 µM of various amyloid beta (Aβ) peptides . Wild-type N2 C. elegans (strain N2) was cultured on Nematode Growth Medium and directly used, as control, for chemosensory assays. Results: We demonstrated that: (i) G. pallida (unlike Caenorhabditis elegans) assimilates amyloid-β (Aβ) peptides which co-localise with its neurological structures; (ii) pre-treatment with various Aβ isoforms  impairs G. pallida’s chemotaxis to differing extents; (iii) Aβ peptides reduced survival, increased the production of ROS, and increased GSSG/GSH levels in this model; (iv) this unique model can distinguish differences between different treatment concentrations, durations, and modalities, displaying good sensitivity; (v) clinically approved neuroprotective agents were effective in protecting G. pallida from Aβ (1–42) exposure. Taken together, the data indicate that G. pallida is an interesting in vivo model with strong potential for discovery of novel bioactive compounds with anti-AD activity.Background: Whole transgenic or non-transgenic organism model systems allow the screening of pharmacological compounds for protective actions in Alzheimer’s disease (AD). Aim: In this study, a plant parasitic nematode,  G. pallida, a plant parasitic nematode, can be used as a non-transgenic model of AD.G. pallida appears to be a reliable non-transgenic nematode compared to C. elegans transgenic strains, for studying AD experimentally.G. pallida can assimilate amyloid beta (Aβ) peptides, which co-localize with its neurological structures mimicking AD physiopathology.G. pallida’s chemotaxis, survival, production of ROS, and GSSG/GSH levels.Treatment with various Aβ isoforms 1–40, 1–42, 17–42, 17–40, 1–28, or 1–16) impaired G. pallida represents a unique model that can sensitively distinguish differences between different treatment concentrations, durations, and other modalities.G. pallida from Aβ (1–42) exposure.Clinically approved neuroprotective agents were effective in protecting G. pallida is an interesting new in vivo model with strong potential for discovery of novel bioactive compounds with anti-AD activity.Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of age-related dementia affecting an estimated 50 million people worldwide ,2. The ADrosophila melanogaster and the nematode Caenorhabditis elegans have been used extensively in studying AD pathology, including the effects of Aβ [C. elegans has been particularly used to study genetic and biological mechanisms of AD, among other neurodegenerative diseases [Various Aβ isoforms of differing length exist, the longest of which, Aβ (1–42), is the most toxic ,7. Animats of Aβ . The extdiseases ,11,12.C. elegans may exhibit transgenesis leading to extrachromosomal arrays containing hundreds of copies of the transforming DNA [C. elegans AD models are muscle-expressed systems [C. elegans [C. elegans models of AD.Nevertheless, such transgenic models are not completely without problems. Thereby, a common criticism of invertebrate transgenic AD models is that non-transgenic organisms are not appropriate to be used as controls. Indeed, it is difficult to ascertain whether transgenic expression of any aggregating proteins (instead of Aβ) would have had the same behavioral effects compared to that of the corresponding non-transgenic aggregating proteins or would reflect the natural behavior effects . Transgeming DNA , which s systems , which m elegans , there iG. pallida, a plant parasitic nematode. The proposed G. pallida model would have potential advantages over C. elegans. First, G. pallida is capable of assimilating peptides from the external environment, an innate trait lacking in C. elegans [G. pallida takes up peptides through the neuronal amphids of its nervous system, enabling the direct observation of neurotoxicity through the measurement of behavioral and biochemical changes [G. pallida. Third, as Aβ exists in many forms in the human brain, it becomes feasible to isolate the effects of each individual Aβ species and examine their combined effects in G. pallida. Fourth, in terms of HTS of drugs, G. pallida model would offer greater speed and convenience over C. elegans. Fifth, G. pallida is extremely sensitive to neuronal RNAi, which is a powerful tool for studying and manipulating the mechanisms that underlie Aβ toxicity [As a step in this direction, we investigate a novel non-transgenic model, namely  elegans ,18. Inde changes . Second,toxicity ,20.G. pallida as a novel in vivo model for studying AD. Specifically, we checked if (i) G. pallida can assimilate specifically Aβ peptides, (ii) these peptides co-localise with neurological structures , and (iii) the behaviour of this nematode is consequently impaired.Herein, we then decided to explore All Aβ peptides ((1–40), (1–42), (17–42), (17–40), (1–28) and (1–16)) as well as the cocoa peptide 13L (DNYDNSAGKWWVT) were obtained from GL Biochem Ltd. .C. elegans were obtained from Sigma-Aldrich Co Ltd. . Alamar Blue reagent, agar base and agar slurry were purchased from Fisher Scientific Ltd. .Mannitol, galantamine, memantine, caffeine, 2,7-dichlorofluorescein diacetate and phosphate-buffered saline (PBS), and all reagents used in chemotaxis assays for G. pallida) were maintained in potato plants at the Agri-Food and Bioscience Institute (AFBI), Belfast, Northern Ireland. The cysts were hatched in fresh potato root diffusate (PRD) at 16 °C in darkness over ten days. Second-stage juveniles (J2s) were first washed in spring water then used directly in assays.Potato cyst nematodes , the Chemotaxis Index (CI) [Chemosensory assays were carried out using a modified protocol of a previously published method . Brieflydex (CI)  describiG. pallida J2 organisms were washed in spring water to remove PRD liquid and then centrifuged . Pellets were reconstituted in 196 µL of spring water and transferred to a 24-well plate; 4 µL of either Aβ  in double-distilled water (ddH2O)) or the vehicle control (1% DMSO in ddH2O) was then added to achieve a final peptide concentration of 100 µM of Aβ (1–42), Aβ (1–40), Aβ (17–42), Aβ (17–40), Aβ (1–28) or Aβ (1–16). Studies with Aβ (1–42) were conducted with a range of pre-treatment temperatures , periods (3 h to 24 h), and concentrations (1–200 µM). Drug treatments, at the concentrations indicated in Figures 1–5 , caffeine (3.6 mM), memantine (10 mM), or 13L cocoa peptide (200 µg/mL), were co-incubated with Aβ (1–42) .For Aβ pre-treatments , approximately 100 G. pallida J2s were immuno-stained using indirect immunofluorescence technique [Approximately 1000 freshly hatched echnique . Organis® 488 conjugate  with fixed worms [v/v), Triton X-100, 0.1% (w/v), sodium azide (NaN3) and 0.1% (w/v) bovine serum albumin (BSA)), the specimens were incubated at 4 °C in the primary antibody (1:700 dilution in AbD) for three days. Then, a wash in AbD for 24 h preceded the incubation of the specimens with the secondary antibody conjugated to the fluorophore goat anti-mouse Alexa Fluor 488 (Molecular Probes) for 72 h at 4 °C. Subsequently, an additional wash in AbD for 24 h was followed by incubation in tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin for 24 h at 4 °C to visualise endogenous muscle systems . After a final wash in AbD for 24 h at 4 °C, the specimens were mounted on glass slides with glycerol/PBS and viewed under the Leica TCS SP5 confocal scanning laser microscope.Immunocytochemical staining employed purified monoclonal anti-Aβ (1–16) primary antibody 6E10, Isotype: Mouse IgG1 , goat anti-mouse Alexa Fluor 488 (Molecular Probes) pre-adsorbed IgG (H + L) Secondary Antibody, and Alexa Fluored worms . TherebyG. pallida J2 organisms was manually counted [The number of live and dead  counted , based oG. pallida J2 organisms. The Alamar Blue assay is based on the ability of viable cells to produce formazan from the cleavage of tetrazolium salt by functional mitochondria. The Alamar Blue assay was carried out according to manufacturer’s instructions. Briefly, a 96-well plate containing G. pallida and the Aβ peptides to be tested was prepared using standard methods, and Alamar Blue was added directly to each well. The plates were then incubated at two different temperatures points, 18 °C (ambient/RT) and 37 °C , to allow cells to convert resazurin to resorufin, and the signal was measured. Fluorescence was quantified every 30 min for 24 h at Ex550 nm and Em590 nm using a FLUOstar Omega microplate reader, and a mean value was obtained for three independent experiments.Alamar Blue reagent  was used to assess the cell viability of G. pallida in response to peptides Aβ (1–42), Aβ (1–40), Aβ (17–42), Aβ (17–40), Aβ (1–28), Aβ (1–16), or to the vehicle control. The method used a modified protocol based on previous studies with C. elegans [The dichlorofluorescein-diacetate (DCF-DA) assay was used to measure the amount of ROS production in  elegans ,27,28. RG. pallida was determined using an OxiSelectTM Total Glutathione Assay Kit  in accordance with the manufacturer’s instructions.The content of reduced glutathione (GSH) and oxidized glutathione (GSSG) in C. elegans (strain N2) was obtained from the Caenorhabditis Genetics 91 Centre, University of Minnesota, Minneapolis, USA, and was cultured on Nematode Growth Medium. Chemosensory assays were performed as described by [Wild-type N2 ribed by  with somn = 3). Data were statistically analysed by a one-way ANOVA with Tukey’s multiple comparison test to compare differences between groups: ns (not significant), * p < 0.05, ** p < 0.01, *** p < 0.001. All statistical analyses were performed using Graphpad Prism 5.0 software .All data are expressed as mean ± standard error mean (SEM)  of As shown in G. pallida was highest at 2 h (CI: 0.52 ± 0.06) and gradually diminished with increasing incubation time.Next, the optimal time for chemosensory assays was determined. As shown in G. pallida was evaluated using the optimized concentration mannitol of 50 mM.The effect of Aβ exposure on the chemotaxis of p < 0.001), with the most pronounced effects observed at 21 h  and 24 h  compared with the vehicle control group (0.47 ± 0.03).G. pallida with different Aβ fragment peptides affected its chemoattraction to mannitol . At a concentration of 100 μM, the CI value was significantly altered for Aβ (1–42), Aβ (1–40), Aβ (17–42), Aβ (17–40), Aβ (1–28), and Aβ (1–16) compared with the vehicle control (CI: 0.47 ± 0.06). The greatest effect on the CI was observed for Aβ (1–42) peptides , followed by Aβ (17–42) (CI: −0.23 ± 0.07), Aβ (1–40) (CI: −0.09 ± 0.04), Aβ (17–40) (CI: −0.02 ± 0.04), Aβ (1–28) (CI: 0.18 ± 0.02) and Aβ (1–16) (CI: 0.18 ± 0.06).A pre-incubation time of 24 h was deemed optimal where the response was maximized. G. pallida were found to be dependent on the peptide concentration  compared with the vehicle control (CI: 0.52 ± 0.02). Further studies involving 10-fold dilutions of Aβ (1–42) (p < 0.001), followed by 10 µM , whereas 1 µM had no significant effect on CI compared with the vehicle control .The effects of Aβ (1–42) on the chemotaxis response of ntration A. Concenβ (1–42) B demonstG. pallida was not evident in C. elegans. Chemosensory assays were carried out for C. elegans wild-type N2 using 0.1% diacetyl as an attractant. Organisms were exposed to Aβ (1–42) for 24 h. C. elegans. No significant differences between Aβ (1–42)-treated organisms and the vehicle control could be detected at any time point over a 120-min period.Additional studies were carried out to demonstrate that Aβ-induced impairment of chemosensing by G. pallida to assimilate Aβ (1–42), and to establish in which physiological structures of G. pallida the peptides are localised.Immunocytochemistry studies were conducted to confirm the capacity of G. pallida exposed to the vehicle control (left) or Aβ (1–42) (right). Aβ (1–42)-treatment (100 µM) produced clear Aβ-specific fluorescence staining (green) which was not co-localised with muscle staining (red). Aβ (1–42) was extensively located throughout the nervous system of G. pallida J2s. It was particularly abundant in the amphid and phasmid neurons and in the ‘brain’ i.e., the longitudinal nerve cords of the circumpharyngeal nerve ring (CNR) of G. pallida J2s. This confirms the hypothesis that these nematodes can assimilate peptides from the external environment. Furthermore, it is evident that the chemotaxis dysfunctions exhibited in pre-incubation of Aβ (1–42) with G. pallida were mediated by the nervous system and not by muscle action. It appears that Aβ (1–42) is localised in both the paired neurons within the central nerve ring and the sensory amphid neurons of G. pallida J2s.G. pallida, at 18 °C and 37 °C, respectively, as measured by mitochondrial reductase activity (Alamar Blue). 100 µM of Aβ peptide fragments was used. These effects were compared to the vehicle control.Aβ (1–42) increased mitochondrial reductase activity by 26.33% at 18 °C and by 255% at 37 °C. Aβ (1–40) caused a negligible change in this activity (1.51%) at 18 °C but increased it drastically by 53% at 37 °C. Aβ (17–42) increased this activity by 12.95% and 120% at 18 °C and 37 °C, respectively. Aβ (17–40) reduced this activity by 4.48% at 18 °C but increased it by 23% at 37 °C. Aβ (1–28) increased this activity by 10.15% and 71% at 18 °C and 37 °C, respectively. Eventually, Aβ (1–16) slightly increased the mitochondrial reductase activity by 6.74% at 18 °C, it is worth noting that this activity increased drastically by 99% at 37 °C.G. pallida based on counting using visual observation. Specifically, 24 h exposure of G. pallida to Aβ (1–42) significantly decreased the survival rate of G. pallida (74.3 ± 0.66%) compared with the vehicle control (97.66 ± 0.88%).100 µM of Aβ peptide fragments was used. G. pallida J2s following 24 h exposure to amyloid-β Aβ (1–42). G. pallida compared with the vehicle control. Aβ (1–42) increased ROS production to the greatest extent with 329%, followed by Aβ (17–42) with 251%, Aβ (1–28) with 194%, and Aβ (1–16) with 188%. Aβ (1–40) and Aβ (17–40) affected ROS production the least, by 99% and 62%, respectively. When G. pallida were not subject to any incubations, there was significantly less ROS production, indicating that these organisms maintain a basal level of ROS.The ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH) within cells is believed to be a measure of cellular oxidative stress.G. pallida treated with 100 µM Aβ (1–42) for 24 h were measured .Therefore, glutathione levels in measured B. BrieflG. pallida is used as a novel in vivo invertebrate model of AD -induced impairment of the CI when el of AD .G. pallida J2s is shown in G. pallida J2s were pre-incubated (24 h) with either the vehicle control, galantamine (100 μM), Aβ (1–42), or Aβ (1–42) in combination with 100 μM of galantamine. Insignificant difference was noticed between the vehicle control group (CI: 0.50 ± 0.02) and the group treated with just galantamine at 100 μM (CI: 0.53 ± 0.03). However, when galantamine is combined to Aβ (1–42), significant improvements in CI values  were found compared to that of the Aβ (1–42)-treated group (CI: −0.3 ± 0.01).Among anti-AD drugs, galantamine is an acetylcholinesterase inhibitor (AChEI) commonly used clinically to treat mild-to-moderate AD . The impG. pallida J2s  with either the vehicle control, Aβ (1–42) at 100 µM or Aβ (1–42) at 100 µM in combination with caffeine (3.6 mM), 13L cocoa peptide (200 µg/mL), or memantine (10 mM). When tested alone in G. pallida, none of these agents significantly affected the CI  compared to the vehicle control group (CI: 0.5 ± 0.04). However, all of them did ameliorate Aβ (1–42)-induced impairments in CI (p < 0.001). Indeed, all these agents in combination with Aβ (1–42), a significant attenuation in the chemotaxis responses was noticed  compared to the Aβ (1–42)-treated group .C. elegans models of AD. For this purpose, the innate ability of the plant parasitic nematode G. pallida, to assimilate intact peptides from the external environment into neuronal structures [The principal aim of this investigation was to develop a novel non-transgenic model as a less complicated alternative to ructures ,18,31, wG. pallida, in anticipation that it could provide new insights into AD mechanisms or assist with the identification of new therapeutic targets. There are several potential advantages to this approach, not least that it allows the direct observation of Aβ toxicity through the measurement of behavioral changes on sensory neurons of this nematode [Aβ-induced toxicity is one of the central mechanisms thought to be involved in the induction of AD pathology . It has nematode ,18.G. pallida J2s. We determined that a 2 h period of chemotaxis towards mannitol as a chemoattractant was optimal. The investigation then turned to how G. pallida responded to a range of human Aβ fragments. All Aβ peptide fragments (Aβ (1–42), Aβ (1–40), Aβ (17–42), Aβ (17–40), Aβ (1–28) and Aβ (1–16)) tested in this study significantly affected the chemotaxis index (CI) of G. pallida. However, Aβ (1–42) had the most marked effect on CI compared with the vehicle control. In contrast, Aβ (1–28) and Aβ (1–16) exerted the least effect on the CI. These findings corroborate earlier studies showing that Aβ (1–42) is the most toxic in human AD [We applied conventional chemosensory assays to objectively assess Aβ-induced effects on the behavior of human AD ,35, and human AD .G. pallida. These data confirmed that this nematode can assimilate peptides through chemosensing amphid neurons within cells of the central nerve ring of its nervous system [G. pallida [The Aβ peptide is derived from a single transmembrane protein, known as the amyloid precursor protein (APP), and production of Aβ is completed by the sequential actions of two enzymes, β-secretase (BACE) and γ-secretase. The Aβ (1–40) and Aβ (1–42) species are components of the plaques implicated in AD progression. With Aβ (1–42) established as the most potent peptide in our model system, we examined how the duration of Aβ (1–42) treatment affected the CI. We found that incubations of as little as 6 h significantly affected the CI, with the most pronounced effects were observed at 18 h and 24 h. These time-related changes may reflect the typical transit time required for assimilated peptides to reach the nervous system of s system ,18,21. I pallida ,18,31. TG. pallida are not recapitulated in C. elegans. Wild-type C. elegans was not affected by exposure to Aβ (1–42), further demonstrating that G. pallida’s known ability to assimilate peptides was critical for sensory impairment to occur. It should be noted that G. pallida and C. elegans respond to different chemoattractants and therefore a direct like-for-like comparison is not possible.Importantly, we then confirmed that Aβ-induced impairments of chemosensing of G. pallida, as an original in vivo non-transgenic model system for AD studies, was assessed by gauging whether Aβ (1–42) affected chemotaxis in a concentration-dependent manner. Concentrations of between 50 and 200 µM of Aβ (1–42) profoundly affected the CI of G. pallida compared with the vehicle control. Even Aβ (1–42) concentrations as low as 10 µM had a significant impact on the CI. However, very low Aβ (1–42) concentrations (1 µM) were completely ineffective, showing the relative sensitivity of our method.Next, the sensitivity of G. pallida in response to each of the tested Aβ peptide fragments. There is growing evidence that Aβ peptides play a role in increasing ROS production and oxidative stress in AD. Moreover, Aβ induces oxidative damage to the vital cellular biomolecules, DNA, proteins, and lipids oxidation, which are associated with AD pathology [G. pallida which are acting against the increased oxidative stress [We then probed putative mechanisms through which Aβ peptides could be impairing chemotaxis. Mitochondrial dysfunction and oxidative stress contribute to ageing and to the occurrence and progression of AD . Oxidatiathology ,41,42,43athology . Indeed,athology ,41,45. Lathology . Aβ pepte stress ,48,49.G. pallida, and this added heat stress probably accelerated the mitochondrial responses to Aβ. A similar temperature (35 °C) has been applied to C. elegans. Periods of heat stress in C. elegans can increase life span, increase mitochondrial ROS, and enhance maintenance and repair (hormesis) [Mitochondrial reductase activity was assessed at two different temperature conditions: ambient (18 °C) and human physiological (37 °C). All Aβ-treated groups had significantly higher mitochondrial reductase activity at 37 °C than at 18 °C. A temperature of 37 °C is considered high for ormesis) ,51,52. TAlthough Aβ (1–16) and Aβ (1–28) are not cytotoxic and usually do not aggregate, our results suggest that they nevertheless induce some ROS production. This is consistent with ,54, who C. elegans [Increased ROS production causes severe damages to mitochondrial DNA and cellular injury; moreover, ROS are linked to systemic necrosis and lethality in  elegans . Some st elegans ,58,59.G. pallida or in G. pallida incubated in Aβ (1–42) for 24 h. The results show that Aβ (1–42) led to significantly higher GSSG/GSH compared with the control in different numbers of worms.High rates of oxidative stress increase the levels of antioxidants such as glutathione, which is an anti-oxidative defence system common in all biological processes ,60,61,62G. pallida from impairment by Aβ (1–42). Firstly, we examined if inhibition of acetylcholinesterase (AChE) was effective in achieving this. AChE is the key enzyme that hydrolyses the neurotransmitter acetylcholine, which plays a vital role in maintaining effective synaptic communication. Reduced levels of acetylcholine can affect the communication between neurons and ultimately can contribute to cognitive decline [In a final phase of the study, we evaluated whether a range of reported neuroprotective compounds could protect  decline ,68,69. TGalanthus caucasicus, Galanthus woronowii, Narcissus (daffodil), Leucojum aestivum, and Lycoris radiata. Research indicates that there is a high level of AChE in the brain cells of AD patients [G. pallida. It was demonstrated that galantamine significantly ameliorated Aβ (1–42)-induced impairment of chemosensing.Galantamine is a known AChE inhibitor which is clinically approved for treatment of mild-to-moderate AD ,70,71,72patients . TherefoC. elegans [C. elegans [Similarly, memantine is a known attenuator of glutamatergic (N-methyl-D-aspartate (NMDA), serotonergic (5-HT3) and cholinergic (nicotinic acetylcholine) receptors . Memanti elegans . A conce elegans . Further elegans .G. pallida. Neuroprotective agents were selected based on their reported protective effects in studies of transgenic C. elegans AD models. First, we examined caffeine, since [C. elegans [C. elegans AD model; it was suggested that coffee extracts significantly reduced the paralysis that was induced by Aβ (1–42) expression and subsequently protected against Aβ (1–42) toxicity. Second, we examined a Cocoa peptide known as 13L, which reduces paralysis resulting from Aβ (1–42) peptide expression in transgenic C. elegans [Non-clinical neuroprotective agents were also examined for their ability to ameliorate Aβ (1–42)-induced impairment of chemotaxis in e, since  have sho elegans . Another elegans  examined elegans . Along w elegans , which p elegans .G. pallida treated with Aβ (1–42). The non-transgenic G. pallida model system provided similar results to the Aβ-expressing transgenic C. elegans model system. Furthermore, we showed that G. pallida represent a promising non-transgenic invertebrate to screen faster, easier, and reliably, for novel therapeutic compounds which protect against Aβ-induced damage.Taken together, we demonstrated that both caffeine and 13L significantly improved the CI responses of G. pallida responses. We evaluated how treatment of G. pallida with Aβ peptides  impaired the chemotaxis response to a chemoattractant and increased mitochondrial reductase activity and ROS production (leading to increased glutathione). Importantly, we have determined that, unlike C. elegans models, G. pallida assimilates Aβ (1–42), which co-localises with specific neurological structures. The ability of this unique model to distinguish between different treatment concentrations, durations, and modalities, shows that it offers remarkable sensitivity and specificity, which could be of benefit to the pharmaceutical industry. The fact that clinically approved neuroprotective agents were effective in protecting G. pallida from Aβ (1–42) exposure outlines its clear potential for the discovery of novel bioactive compounds with anti-AD activity.In this present study, we have optimized a chemosensory assay capable of reliably measuring


Ochyrocera Simon, 1892, are described for caves located in the iron formation of Floresta Nacional (FLONA) de Carajás in southeastern Pará State, Brazil. The caves are located in the municipalities of Parauapebas and Canaã dos Carajás, in the eastern portion of the Amazon Forest domain. Ochyroceraritxocosp. nov. and O.ritxoosp. nov. are described based on males and females. The species have similar body characteristics with the total absence of eyes and complete depigmentation, characteristics that indicate possible evolution in subterranean environments , and thus are classified as troglobites. Each species is associated with a single geomorphological unit (mountain range), with Ochyroceraritxocosp. nov. being restricted to caves of Serra Norte (North Mountain) and O.ritxoosp. nov. to caves of Serra Sul (South Mountain). Both species were collected in aphotic zones of the caves. Small and tangled webs of O.ritxocosp. nov. were observed under blocks of stone in the soil or in cracks of the walls.The first two anophthalmic species of spiders of the genus  Ochyroceratidae currently contains 10 genera and 166 species , while the others are characterized by reduced or small size of the eyes, depigmented body and long thin legs , state of Pará, northern Brazil and expand the diversity of spiders known for this ferruginous region  and Museu Paraense Emílio Goeldi, Belém .Specimens are deposited in the following collections (abbreviation and curator in parentheses): SEM), body parts were dehydrated in a series of graded ethanol washes (80% to 100%), critical point dried, mounted on metal stubs using adhesive copper tape and nail polish for fixation and covered with gold. SEM images were taken with FEI Quanta 250 and LEO 1450VP scanning electron microscopes, at Laboratório de Biologia Celular of Instituto Butantan, São Paulo and Museu Paraense Emílio Goeldi, Belém, respectively.Morphological terms follow FLONA de Carajás (Brazilian System of Conservation Units), which encompasses approximately 411 thousand hectares and includes parts of the municipalities of Parauapebas, Canaã dos Carajás and Água Azul do Norte. In the region of the park there is a mosaic of protected areas forming a continuous area of 1.31 million hectares of preserved forest , FLONA de Carajás, Parauapebas, Pará, Brazil, 28/XI-03/X/2007, R. Andrade et al. col. (IBSP 115497). Paratype female from Cave N1_0075 , FLONA de Carajás, Parauapebas, Pará, Brazil, 28/XI–03/X/2007, R. Andrade et al. col. (IBSP 115499).FLONA de Carajás, Cave N1_0015 (GEM-1211) , 11/VI–02/VII/2014, 1♀ (IBSP 186123); 1♀ (IBSP 186124); 1♂ (IBSP 186125); 2♀ (IBSP 186126); Cave N1_0016 (GEM-1212) , 04/IX–06/X/2014, 1♀ (IBSP 186130); 02–29/IV/2015, 2♀ (IBSP 186153); 3♀ (IBSP 186154); 1♀ (IBSP 186155); Cave N1_0055 (GEM-1253) , 07–28/I/2015, 1♀ ; Cave N1_0056 (GEM-1254) , 07–28/I/2015, 1♂ ; 1♂ 1♀ (IBSP 186149); Cave N1_0060 (GEM-1258) , 11/VI–02/VII/2014, 1♀ (IBSP 186127); 1♀ (IBSP 186128); 07–28/I/2015, 1♀ (IBSP 186150); 1♀ (IBSP 186151); 1♂ 1♀ ; Cave N1_0062 (GEM-1260) , 04/IX–06/X/2014, 1♀ (IBSP 186131); 1♀ (IBSP 186132); 2♀ (IBSP 186133); 1♀ (IBSP 186134); 1♂ 2♀ (IBSP 186135); 02–29/IV/2015, 2♀ (IBSP 186157); 3♂ 1♀ (IBSP 186156); 1♀ (IBSP 186158); Cave N1_0073 (GEM-1271) , 02–29/IV/2015, 1♀ (IBSP 186159); 1♂ (IBSP 186160); 1♀ (IBSP 186161); 1♀ (IBSP 186162); Cave N1_0084 (GEM-1282) , 11/VI–02/VII/2014, 1♀ (IBSP 186129); Cave N1_0101 (GEM-1299) , 04/IX–06/X/2014, 1♂ (IBSP 186136); 1♀ (IBSP 186137); 1♀ (IBSP 186138); 1♂ (IBSP 186139); 2♀ (IBSP 186140); 1♂ (IBSP 186141); 1♂ (IBSP 186142); Cave N1_0240 , 04/IX–06/X/2014, 1♂ (IBSP 186143); 1♀ (IBSP 186144); 1♂ (IBSP 186145); 1♀ (IBSP 186146); 02–29/IV/2015, 1♀ (IBSP 186163); 1♂ 1♀ (IBSP 186164); all collected by Equipe Carste; Cave N4WS_0067 (GEM-1846) , 18/XI–01/XII/2010, F.P. Franco & C.A.R. Souza et al. col., 1♂ (IBSP 174069); 2♀ (IBSP 174070); Piranha, Cave N1-75 , 28/IX–03/X/2007, 1♂ 2♀ ; all collected by R. Andrade et al.Brazil. Pará: Parauapebas, Ochyroceraritxoco sp. nov. is distinguished from O.ritxoo sp. nov. by having an elongated embolus, which is two times longer than the bulb in the male palp . Total length 1.1. Carapace length 0.45; ovoid, narrowing gradually anteriorly, yellowish and bright, pars cephalic flat, fovea absent . Total length 1.15. Carapace length 0.55 as in male with light yellowish pattern . Ochyroceraritxoco sp. nov. was generally found in large cavities with horizontal projections varying from 9.5 to 216 meters . All caves where the species was found have only one entrance and are located only in the middle and high slopes of Serra Norte. Most caves have aphotic zones or twilight zones (except for cavities N1_0103 and N1_0084) and high humidity, thus explaining the observed presence of small bodies of water in almost all cavities, especially during the wet season. The number of troglobitic species in these caves varied from one to ten (average 5.3 per cave), with species of the following taxa: spiders – Oonopidae (many species), Caponiidae  and Ochyroceratidae (Speocera spp.); pseudoscorpions – Bochicidae, Chthoniidae and Ideoroncidae; springtails – Paronellidae , Entomobryidae (Pseudosinella sp.) and Sminthuridae ; beetles – Carabidae (Coarazuphium spp.), Dytiscidae ; isopods – Scleropactidae (Circoniscus spp.), Calabozoidae; amphipods – Bogidiellidae (Bogidiella sp.); planarian – Prorhynchidae (Geocentrophora sp.); and Harvestmen – Escadabiidae.its Fig. . The obsFLONA de Carajás, Parauapebas, state of Pará, northern Brazil , zil Fig. .Taxon classificationAnimaliaAraneaeOchyroceratidae968D35C3-CEA4-523D-A92B-B62CC63F4ED0http://zoobank.org/F161E5C9-B893-46D7-B724-737B01AF5705Holotype male from Cave S11C_0201 , FLONA de Carajás, Canaã dos Carajás, Pará, Brazil, 27/VII/2015, BioEspeleo Consultoria Ambiental col. (IBSP 193194). Paratype female from Cave S11C_0052 , FLONA de Carajás, Canaã dos Carajás, Pará, Brazil, 09/III/2016, BioEspeleo Consultoria Ambiental col. (IBSP 193196),FLONA de Carajás, Cave S11C_0194 , 12/III/2016, 1♂ (IBSP 193078); Cave S11C_0046 , 19/IV/2016, 1♀ imm. (IBSP 193083), all collected by BioEspeleo Consultoria Ambiental; Cave S11D_0064 (710) , 13–30/I/2010, R. Andrade & I. Cizauskas et al. col., 1♂ 1♀ 3 imm. (IBSP 174071); 10–19/V/2011, D. Bebiano col., 1♀ (IBSP 196512); Cave S11D_0064 (710) , 13–30/I/2010, 1♀ (IBSP 196513); 01–14/VII/2010, R. Andrade & I. Cizauskas et al. col., 2♀ (IBSP 196514); 2♂ ; Cave S11D_0096 (742) , 13–30/I/2010, 1♂ (IBSP 196481); Cave S11D_0064 (710) , 13–30/I/2010, 1♀ (IBSP 196482); Cave S11D_0064 (710) , 1–14/VII/2010, 1♂ 1♀ (IBSP 196483) all collected by R. Andrade & I. Cizauskas et al.; Cave S11B_078 , 28/IX/2018, Ativo Ambiental col. 1♂ (IBSP 260308).Brazil. Pará: Canaã dos Carajás, Ochyroceraritxoo sp. nov. is distinguished from O.ritxoco sp. nov. by having the embolus as long as the bulb of the male palp  Total length 0.90. Carapace length 0.40, ovoid, narrowing gradually anteriorly, cream-colored and bright, pars cephalic flat, fovea absent . Total length 1.0. Carapace length 0.35, as in male . Ochyroceraritxoo sp. nov. was generally found in large cavities with horizontal projections varying from 26 to 245 meters . These caves have one to three entrances and are located in all compartments of the Serra Sul landscape . All caves have aphotic zones and other troglobitic species were found in most caves, with the richness of troglobitic species per cave varying between one and six (average of four). The following taxa were found: spiders – Gnaphosidae , Caponiidae  and Tetrablemmidae (Matta sp.); tailless whip scorpions – Charinidae ; pseudoscorpions, Chthoniidae; diplopods – Glomeridesmidae (Glomeridesmus sp.), Pyrgodesmidae, Pseudonannolenidae (Pseudonannolene spp.); springtails – Sminthuridae, Paronellidae; and beetles – Staphylinidae (Pselaphinae).FLONA de Carajás, Canaã dos Carajás, state of Pará, northern Brazil , Ochyroceratidae located in caves in Brazil is represented mainly by specimens of the genera Ochyrocera Simon, 1892, Speocera Berland, 1914, and Theotima Simon, 1893. The first two genera have troglomorphic spiders among their representatives and are a significant part of the yet unknown diversity of subterranean spiders in these environments  and, together with Carajasparaua Brescovit & Sánchez-Ruiz, 2016 (Caponiidae) and Paracymbiommacaecus and P.bocaina , is evidence of a diversified subterranean araneofauna in the Carajás ferruginous system which must be considered in projects aimed at conservation and sustainable use of its natural resources.Both


Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells, and its mRNA destabilization activity is regulated by protein phosphorylation.We generated an antibody against phospho-Serine316 located at the C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP was created in RAW264.7 cells using CRISPR/Cas9 gene editing to explore TTP functions.TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1.We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2) and dephosphorylated by Protein Phosphatase 2A (PP2A). A phosphorylation-mimic mutant of S316D resulted in dissociation with the CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to the CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced, and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation via regulating the TTP interaction with the CCR4-NOT deadenylase complex.The online version contains supplementary material available at 10.1186/s12950-021-00288-2. Regulation of mRNA stability plays a central role in controlling gene expression in the vast majority of eukaryotic cells –4. The m factors , 6. The ved mRNA . Severalved mRNA , which ived mRNA . CCR4-NOved mRNA , 11. Theved mRNA , 13.Saccharomyces cerevisiae, and Zfs1p in S. pombe, are no containing NOT1-binding domain [TTP is an extensively studied ARE-binding protein. There are four TTP members in rodents, including TTP, Zfp36l1, Zfp36l2, and Zfp36l3, and they all contain conserved tandem CCCH zinc finger RNA–binding domains and a conserved C-terminal NOT1-binding domain . TTP cong domain . TTP wasg domain . We demog domain . Zfp36l3g domain . TTP famg domain , 22. TTPg domain , 24. In g domain , 26. Theg domain . One molg domain , 29. TTPg domain , 31. In g domain , 33.Previously, Ser316 phosphorylation was identified by mass spectrometry analysis in both in vitro MK2 phosphorylation and LPS-stimulated RAW264.7 macrophages . Ser316 2 humidified atmosphere (37 °C). Mouse BALB/c macrophage RAW264.7 cells (TIB-71) were purchased from ATCC and were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% FBS (Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco) in a 5% CO2 humidified atmosphere (37 °C). The RAW264.7 cells were treated with 100 ng/ml of LPS from E. coli O111:B4 (Sigma-Aldrich) for different time intervals, combined with the 2 or 5 μM of MK2 inhibitor PF3644022 (Sigma-Aldrich) and 25 or 50 μM of RSK1 inhibitor BI-D1870 (Abcam).Human embryonic kidney (HEK) 293T cells (CRL-3216) and mouse NIH3T3 cells (CRL-1658) were purchased from American Type Culture Collection (ATCC) and were cultured in DMEM containing 3.7 g/l sodium bicarbonate and supplemented with 10% FBS (Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco) in a 5% COThe plasmids for TTP, Zfp36l1, Zfp36l2 and 14–3-3ζ expression were constructed as described , 22, 36.–∆∆CT relative quantitation method.Total RNA was extracted from cell cultures using TRIzol reagent (Invitrogen). For mRNA stability analysis, the cells were treated with 10 μg/ml actinomycin D (transcription inhibitor) for various times to inhibit new transcription. After DNase I digestion, 2 μg total RNA was reverse-transcribed to produce cDNA using M-MLV reverse transcriptase and oligo dT primer (Promega). Real-time PCR was performed with the 7300 Real-Time PCR System (Applied Biosystems) in a total volume of 20 μl. Expression of genes encoding TTP, TNFα and actin was assessed using SYBR Green PCR Master Mix (Applied Biosystems) with 50 ng of cDNA and 160 nM of each primer: 5′-GGATCTCTCTGCCATCTACGA-3′ and 5′-CAGTCAGGCGAGAGGTGAC-3′ for TTP; 5′-GACCCTCACACTCAGATCATCTTCT-3′ and 5′-CCTCCACTTGGTGGTTTGCT-3′ for TNFα; 5′-TCCTTCCTGGGCATGGAGTC-3′ and 5′-ACTCATCATACTCCTGCTTG-3′ for β-actin. The PCR amplification conditions were 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Real-time PCR data were analyzed using the 22MoO4, 0.1 mM Na3VO4, pH 10.0, 10 mM NaF) and centrifuged at 15,000×g for 10 min. The supernatants were immunoprecipitated using anti-Flag M2 agarose (Sigma-Aldrich) at 4 °C for 2 h. After the IP mixture was washed three times with NET buffer, bound proteins were eluted by boiling in SDS-PAGE sample buffer. The proteins were separated by SDS-PAGE (10% polyacrylamide) and transferred to a polyvinylidene difluoride membrane (Millipore), and western blotting was performed using anti-Flag (1:2000)(Sigma-Aldrich), anti-HA (1:2000) (Bethyl Laboratories), anti-TTP and anti-p-S316 (1:1000)  (produced in our lab), anti-α-tubulin (1:1000), anti-β-actin (1:1000), anti-phospho-ERK1/2 (1:1000), anti-p38 (1:1000), anti-phosph-p38 (1: 1000), anti-MK2 (1:1000), anti-phospho-MK2 (1:200), anti-phospho-RSK1(1:200), anti-CNOT3 (1:1000) and anti-CNOT6 (1:1000) , anti-GAPDH (1:5000), anti-CNOT1 (1:1000), and anti-CNOT7 (1:500) , anti-ERK1/2 (1:2000) and anti-RSK1(1:2000) (both from Santa Cruz Biotechnology), and anti-DDX6 (1:1000)(Abcam). All experiments were carried out at least three times, and represented results were displayed.HEK293T cells were transfected with the plasmids using Turbofect reagent (Thermo). Cells harvested 24 h after transfection were lysed with NET buffer  Triton X-100) containing a protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors  was synthesized and purified by Kelowna International Scientific Inc. (Taiwan). A specific rabbit antiserum was produced by LTK BioLaboratories (Taiwan). The antiserum was affinity purified using the immunizing peptide (LTK BioLaboratories).2, 0.1 mM EDTA, 0.05% Triton X-100, 0.5 mM DTT, 20 mM β-glycerolphosphate, 0.1 mM Na3VO4, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 100 μg/ml PMSF. The mixture was rotated at 4 °C for 3 h and pelleted by centrifugation at 10,000×g for 20 s. After 4 × 1-ml washes in HEPES binding buffer , the beads were resuspended in 30 μl for kinase assay. The reaction mixtures were incubated at 30 °C for 30 min and stopped by adding one volume of protein sample buffer. Samples were subjected to SDS-PAGE for western blotting with anti-phospho-S316 and ponceau S staining.Each reaction mixture contained 2 μg of recombinant GST-tagged TTP (wild type or mutants) served as substrates, 3 μl of 10X reaction buffer (New England Biolabs), 30 μM of ATP, and kinases including ERK2 (New England Biolabs), p38 alpha , RSK1 , and MK2  in a final volume of 30 μl. The kinases can be from cell extracts. 300 μg of LPS-treated RAW264.7 whole cell extracts were incubated with GSH-Sepharose bound 2 μg of GST-TTP in the buffer containing 20 mM HEPES, pH 7.7, 75 mM NaCl, 0.5 mM MgCls combined with lysates (300 μg protein) of RAW264.7 cells that had undergone various treatments in a final volume of 200 μl of buffer containing 20 mM HEPES, pH 7.9, 100 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% (v/v) NP-40, 1% (v/v) Triton X-100, 1 mM DTT, and 1 mM PMSF. The mixtures were incubated at 4 °C for 2 h on a rotary shaker, and then the Sepharose was washed four times with the same buffer lacking DTT and PMSF but containing 0.2 M NaCl and once with 50 mM Tris, pH 6.8. Bound proteins were eluted by boiling in SDS-PAGE sample buffer and analyzed by western blotting.Glutathione-Sepharose 4B  were incubated with 2 μg of bacterially expressed GST, or GST-Cnot1@800–1015 or GST-14-3-3 in phosphate-buffered saline containing 1% (v/v) Triton X-100 on a rotary shaker for 20 min at room temperature. After washing three times with the same buffer, the Sepharose wa− 1 RNasin (Promega) and 20 μg·μL− 1 yeast tRNA was added to each lysate. To prevent non-specific binding, heparin-agarose (Sigma-Aldrich) was incubated with each lysate for 15 min at 4 °C and then centrifuged for 1 min at 8000 rpm, 4 °C. Each supernatant was further cleaned with streptavidin-Sepharose  for 1 h at 4 °C and then centrifuged for 1 min at 8000 rpm, 4 °C. The biotin-labeled TNFα ARE was added as described [Cytoplasmic extracts from LPS-stimulated RAW264.7 cells were prepared by hypotonic buffer . Potassium acetate was adjusted to 90 mM, and 0.1 U·μLescribed . The pul5) were seeded in each well of a 12-well plastic culture plate. For gene knockdown, the cells were transfected with 5 nM of a small interfering RNA (siRNA) for CNOT1, CNOT6, CNOT7, RSK1, or MK2 (Invitrogen) using Lipofectamine 3000 (Invitrogen). After 24 h, the cells were transferred into a fresh medium and transfected with 1 μg of Flag-TTP wild-type, −TTP S52,178A or –TTP S316A expression plasmids using Turbofect reagent (Thermo). After another 24 h, cells were harvested, and whole cell extracts were isolated for western blotting or immunoprecipitation.HEK293T cells (1 × 10https://benchling.com) and CHOPCHOP (https://chopchop.cbu.uib.no/) to search the specific target-sequences of Cas9 RNP complexes on mouse Ttp gene. Based on the description of the target score [https://crispr.mit.edu/about), we designed four sgRNAs, which contain a relatively higher on-target with a lower off-target score  at 60 °C for 5 min. The RNA was purified by electrophoresis in 10% polyacrylamide gel containing 6 M urea. The RNA band was excised from the gel, ground up in a 15-ml tube, and eluted with 5 vol. of 300 mM sodium acetate (pH 5.0) overnight at 4 °C. One equivalent of isopropanol was added to precipitate the RNA at − 20 °C. The RNA pellet was centrifuged and washed three times with 70% ethanol and dried by vacuum. To refold the sgRNA, the RNA pellet was first dissolved in 20 mM HEPES (pH 7.5), 150 mM KCl, 10% glycerol, and 1 mM TCEP. The sgRNA was heated to 70 °C for 5 min and cooled to room temperature. MgCl2 was added to a final concentration of 1 mM. The sgRNA was again heated to 50 °C for 5 min, cooled to room temperature and kept on ice. The sgRNA concentration was determined by OD260nm and adjusted to 100 μM using 20 mM HEPES (pH 7.5), 150 mM KCl, 10% glycerol, 1 mM TCEP, and 1 mM MgCl2. The sgRNA was store at − 80 °C [The assembled PCR products were extracted once with phenol:chloroform:isoamyl alcohol and then once with chloroform, before isopropanol precipitation overnight at − 20 °C. The DNA pellet was washed three times with 70% ethanol and dissolved in DEPC-treated water. The T7 in vitro transcription reaction consisted of 30 mM Tris–HCl (pH 7.9), 20 mM MgCl − 80 °C .Transfection of RAW264.7 cells was performed according to the instructions of Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (Invitrogen). The Cas9 RNP were prepared by incubating the purified Cas9 protein with sgRNA at 1:4 molar ratios. After transfection, the cells were incubated at 37 °C for 48 h, and followed by single-cell sorting using BD, FACSJazz automated cell sorter to perform the single isolation (TechComm at NTU).Ttp gene in RAW264.7 cells. Therefore, the size of PCR product generated by primer-1 and primer-4 will be reduced from 2001 nt to 1208 nt. Besides, primer-2 and primer-3 locate on the predicted cutting fragment. If the TTP knock-out happened, it would not have PCR products between primer-1 and primer-2 or primer-3 and primer-4 theoretically (Table Genomic DNA was extracted by QuickExtract™ DNA Extraction Solution (Epicentre). Cells were mixed by 200 μl of QuickExtract Solution and vortex for 15 s. The tubes were transferred to 65 °C and incubate for 6 min. After 15 s vortex, samples were incubated at 98 °C for 2 min. Store the DNA at − 20 °C or − 80 °C for long-term storage according to the manufacturer’s protocol. Genomic DNA was PCR amplified using primer-1 and primer-4 Table . Theoretly Table .NIH3T3 cells were typically seeded at 50% on sterile glass coverslips before 16–18 h. After transfected with GFP-fused TTP expression vector using Lipofectamine 2000 (Thermo) for 24 h and treated with 20 ng/ml of leptomycin B (LMB) for 6 h, cells were washed briefly with PBS and then fixed in 4% paraformaldehyde/PBS for 30 min at room temperature. After gently washed twice with PBS (5 min for each), cells were permeabilized with 0.1% Triton X-100/PBS for 10 min at room temperature. Following another two times washed with PBS (5 min for each), blocked the cells for 30 min with PBS containing 1% BSA. Then the cells were incubated with appropriately diluted DDX6 antibody overnight at 4 °C, washed with PBS three times (10 min for each), further incubated with secondary antibody  and DAPI (Sigma-Aldrich) for 1.5 h in the dark, and washed with PBS three times (10 min for each) in the dark. Mount in mounting fluid and store at − 20 °C until detected by Leica SP5 confocal Microscopy system.P < 0.05, **P < 0.01 or ***P < 0.001) was determined by one-tailed Student’s t-test.All data are presented as the mean ± SD of at least three independent experiments. Statistical significance  was generated, and its specificity was established using wild-type or mutant TTP Fig. A. The seThe NOT1-binding domain contains two serines, Ser316 and Ser318, which might be phosphorylated to affect CNOT1-binding. The phosphomimetic mutant  and non-phosphorylated mutant  were created for co-immunoprecipitation analysis. Compared to wild-type and non-phosphorylated S316,318A mutant, the S316,318D phosphomimetic mutant weakened associations with CNOT1, CNOT3, CNOT6 and CNOT7  to recognize the TTP sequence on mouse genome and were in vitro transcribed  or GFP-TTP(S316D) or GFP vector control. After transfection, the cells were treated with LPS to induce TNFα expression and RNA was isolated for qPCR analysis  ERK and p38 signaling pathways result in S316 phosphorylation. Two hundred ninety three T cells were transfected with constitutive-active (CA) or dominant-negative (DN) MKK1 or MKK3 expression plasmids. After treated with RSK1 inhibitor (RSKi: 50 μM of BD-I1870) or MK2 inhibitor (MK2i: 5 μM of PF364402) for 2 h, the whole cell extracts were isolated for western blotting analysis with indicating antibodies. (B) PP2A decreases Ser316 phosphorylation. HEK293T cells were coexpressed with wild type or S316A or S52, 178A TTP, and the wild type or non-activity mutant H59Q of the catalytic subunit of PP2Ac or the regulatory subunit PR55. The western blotting was performed with indicated antibodies. Figure S2. (A) S316 is critical for TTP binding to CNOT1. HEK293T cells were transfected with 4 μg of Flag-tagged TTP expression plasmids as indicated. IP was performed with anti-Flag M2 agarose, and the precipitated protein complexes were analyzed by western blotting with indicated antibodies (B) Luciferase reporter assay of TTP phosphomimetic mutants. Schematic diagram of MKP-1-3’UTR (three AREs) containing luciferase reporter. The TTP S316D mutant resulted in higher luciferase activity than wild-type. * P < 0.05. (C) Knockdown of CCR4-NOT complex reduces TTP mRNA-destabilizing ability. HEK293T cells were seeded in 12-well culture plates and transfected with 5 nM of siRNA targeting CNOT1, CNOT6, CNOT7, and negative control (NC). After 24 h, cells were transfected again with 0.2 μg of Flag-tagged TTP, 0.5 μg of luciferase reporter carrying MKP-1-3’UTR or reporter alone, and 0.5 μg of Renilla luciferase reporter . Dual-luciferase reporter assays were performed after 24 h post-transfection. The relative MKP-1-3’UTR-mediated luciferase activities were normalized to the Renilla luciferase activities and to that of the reporter alone. Each treatment group contained two duplicates, and experiments were repeated three times. Data are presented as means ± SD. The protein expression level and knockdown efficiency were examined by western blot analysis (lower panel) using indicated antibodies. The asterisk indicates Flag-tagged firefly luciferase. (D) Another IP result of Fig. Figure S3TNFα mRNA stability analysis. RAW264.7 cells were pre-treated with RSK1 inhibitor (RSKi: 50 μM of BD-I1870) or together with MK2 inhibitor (MK2i: 5 μM of PF364402) for 30 min, and then treated with 100 ng/ml of LPS for 1 h. The cells were added transcription inhibitor actinomycin D  for 15 min, 30 min, and 45 min. The cells were harvested for RNA isolation and RT-qPCR analysis. Figure S4. The generation of TTP KO RAW264.7 cells. (A) The genomic TTP sequence is located on chromatin7:28376784–28,379,700 which has two exons and one intron. The four sgRNAs were designed to recognize the specific unique sequence positioned on TTP exons that contain NGG, which were assembled with T7 promoter and generated by in vitro transcription. (B) Different combinations of sgRNAs were co-transfected with Cas9 protein in RAW264.7 cells and checked by genomic PCR  in LPS-stimulated RAW264.7 cells with anti-TTP. To prepare cytosolic extract, 5 × 106 cells were resuspended in 400 μl of hypotonic buffer . The cell suspension was on ice for 15 min, and then 25 μl of 10% NP-40 was added followed by vortexing for 10 s. After centrifugation at 10,000×g for 30 s, the supernatant was collected as cytoplasmic extract. 1 mg cytoplasmic extracts from RAW264.7 cells were adjusted to 25 mM HEPES, pH 7.5, 150 mM 5NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM DTT and 1u/μl of RNasin and were pre-cleaned by protein-A Sepharose (Amershan Pharmacia) for 1 h. After centrifugation, the supernatants were added 1 μg of normal IgG or anti-TTP antibody and protein A-Sepharose at 4 °C rotated for 2 h. Beads were washed using NT2 buffer  for three times. For co-IP, the precipitated protein complexes were added with SDS-PAGE sample buffer, boiling for 10 min, and analyzed by western blotting with anti-Cnot1 and anti-TTP (A). For RNA-IP, the beads were incubated with 100 μl NT2 buffer containing 5 U RNase-free DNase I (Ambion) for 15 min at 30 °C, washed with NT2 buffer, and further incubated in 100 μl NT2 buffer containing 0.1% SDS and 0.5 mg/ml proteinase K at 55 °C for 15 min. RNA was extracted with TRIzol reagent and reverse transcribed in cDNAs as mentioned above for semi-quantitative PCR analysis. The specific primers of Gapdh and TNFα was amplified using 5% of the cDNAs from IP and 2% from input in 20 μl containing 10 pmol of forward and reverse primer as shown in Table S1, and lypholized Taq DNA polymerase, buffer and dNTPs . PCR was performed in a Robocycler gradient 96 PCR thermal machine (Stratagene) using the following conditions: 95 °C (3 min) for one cycle, 95 °C (30 s), 55 °C (30 s), 72 °C (20 s) for 35 cycles, and a final incubation at 72 °C for 3 min. One-third of PCR products were separated in 2% agarose gel (B). Table S1. Primers for generating murine TTP mutants. Table S2. Sequences for TTP knock-out in CRISPR/Cas9 system.


PLoS Medicine volume 2, issue 11: DOI:10.1371/journal.pmed.0020298InTable 4: Under “Religion,” the values for “HIV cases,” “Follow-up,” and “HIV incidence rates” for the categories “Catholic or Protestant” and “Others” were reversed in error. The correct values are as follows.Catholic or Protestant: HIV cases, 5; Follow-up (py), 576; HIV Incidence Rates (95% CI per 100 py), 0.87 (0.33–2.09).Others: HIV cases, 25; Follow-up (py), 1,845; HIV Incidence Rates (95% CI per 100 py), 1.36 (0.92–2.02).


PLoS Biology, volume 4, issue 2: DOI: 10.1371/journal.pbio.0040031In In the first paragraph of the Materials and Methods subsection “Retroviral infection of INS1 and MIN6 cells,” “pSUPERretro SiRNA-T1 (5′-GCTGCATCCAAGGGCCATG-3′)” should be “pSUPERretro SiRNA-T1 (5′-gatgaagttgacctcctca-3′)”.


CreIL-4Rα−/lox) were generated and characterized to uncover any role for IL-4/IL-13 in this non–immune cell type in response to Nippostrongylus brasiliensis infection. IL-4Rα was absent from α-actin–positive smooth muscle cells, while other cell types showed normal IL-4Rα expression, thus demonstrating efficient cell-type–specific deletion of the IL-4Rα gene. N. brasiliensis–infected SM-MHCCreIL-4Rα−/lox mice showed delayed ability to resolve infection with significantly prolonged fecal egg recovery and delayed worm expulsion. The delayed expulsion was related to a delayed intestinal goblet cell hyperplasia, reduced T helper 2 cytokine production in the mesenteric lymph node, and reduced M3 muscarinic receptor expression during infection. Together, these results demonstrate that in vivo IL-4Rα–responsive smooth muscle cells are beneficial for N. brasiliensis expulsion by coordinating T helper 2 cytokine responses, goblet hyperplasia, and acetylcholine responsiveness, which drive smooth muscle cell contractions.Interleukin 4 receptor α (IL-4Rα) is essential for effective clearance of gastrointestinal nematode infections. Smooth muscle cells are considered to play a role in the type 2 immune response–driven expulsion of gastrointestinal nematodes. Previous studies have shown in vitro that signal transducer and activator of transcription 6 signaling in response to parasitic nematode infection significantly increases smooth muscle cell contractility. Inhibition of the IL-4Rα pathway inhibits this response. How this response manifests itself in vivo is unknown. In this study, smooth muscle cell IL-4Rα–deficient mice  responsiveness for worm expulsion in global IL-4Rα–deficient mice. In this study, we specifically addressed the role of IL-4Rα responsiveness in a novel smooth muscle cell–specific IL-4Rα–deficient mouse model. These mice showed decreased ability to control the worm infection, with delayed expulsion and reduced protective immune responses. These data provide compelling evidence for smooth muscle cell IL-4Rα being an important coordinator of both the immune and physiological responses to intestinal worm infections. A proposed model is suggested with IL-4Rα responsiveness on smooth muscle cells coordinating T helper 2 cytokine responses, goblet hyperplasia, and acetylcholine-driven smooth muscle contractions for optimal worm expulsion. Nippostrongylus brasiliensis, while mice demonstrated an increased susceptibility to infection by the trematode Schistosoma mansoni. The work presented here describes the effect of a smooth muscle–specific disruption of IL-4Rα expression on the immune response to N. brasiliensis.The interleukin (IL)-13/IL-4 receptor α (IL-4Rα)/signal transducer and activator of transcription 6 (STAT-6) signaling pathway is essential in the control of a number of infectious diseases as well as being a key factor in the induction of allergic responses. Signaling through this pathway can either confer protective immunity or mediate tissue damage depending on the antigenic stimuli and the cell-specific response . It was N. brasiliensis induces a strong protective host T helper 2 (TH2) response for which IL-13 production and signaling through IL-4Rα are essential for successful clearance of infection [N. brasiliensis larva penetrate the skin and migrate via the blood system, to the lungs. Larva emerge from blood vessels and enter the airways, from which they are coughed up and swallowed. Upon reaching the intestine, larva develop into egg-producing adult worms that attach to the small intestine epithelium. BALB/c mice clear N. brasiliensis infection after approximately 9 d [Murine infection with tely 9 d .N. brasiliensis from the intestine, the precise role of IL-4Rα in coordinating the immune and physiological response remains unclear [N. brasiliensis infection. Additionally, acetylcholine-driven contractions of longitudinal smooth muscle in the intestine are also implicated in playing a role in worm expulsion [−/− mice. Therefore, the IL-13/IL-4Rα/STAT-6 pathway is necessary for elevated smooth muscle cell contractility required to aid worm expulsion [Although essential for expulsion of  unclear . IL-13/I unclear . Disruptxpulsion . A numbexpulsion ,10. Addixpulsion , as wellxpulsion , in ordeCreIL-4Rα−/lox mice, we demonstrate that disrupted IL-4Rα expression in smooth muscle cells influences host immunity to an intestinal nematode infection. The absence of smooth muscle IL-4Rα delays worm expulsion and goblet cell hyperplasia. Furthermore, induction of TH2 cytokines is delayed and/or reduced, as is intestinal expression of the M3 acetylcholine receptor, in response to infection with N. brasiliensis.To date, no studies have been able to demonstrate in vivo the effect of a cell-specific inhibition of the IL-13/IL-4Rα/STAT-6 pathway in smooth muscle cells. Using smooth muscle myosin heavy chain (SM-MHC)Cre), were backcrossed to the BALB/c genetic background for nine generations and then intercrossed with IL-4Rα−/− and “floxed” IL-4Rαlox/lox BALB/c mice to establish smooth muscle cell–specific IL-4Rα–deficient BALB/c mice (SM-MHCCreIL-4Rα−/lox), with one deleted and one floxed IL-4Rα allele (SM-MHCCreIL-4Rα−/lox) to increase the efficiency of Cre-mediated site-specific recombination. Mutant mouse strains were identified by PCR genotyping  and global IL-4Rα−/− (GMF: 11.2) mice (−/lox mice (GMF: 18.37). IL-4Rα expression on α-actin–positive smooth muscle cells isolated from small intestine and lung was too low to detect using FACS analysis (unpublished data). However, Cre mRNA was highly expressed in tracheal and intestinal tissue in the SM-MHCCreIL-4Rα−/lox mice. As expected, IL-4Rα−/lox mice demonstrated no Cre expression. In agreement with the smooth muscle specificity of the deletion, IL-4Rα mRNA expression was substantially depressed in both tracheal and intestinal tissue in SM-MHCCreIL-4Rα−/lox mice compared to IL-4Rα−/lox mice . Together, these results provide convincing support for the specificity of smooth muscle cell disruption of IL-4Rα in SM-MHCCreIL-4Rα−/lox mice, in agreement with previously published data on the characterization of SM-MHCCre transgenic mice [IL-4Rα expression was analyzed on α-actin–positive cells derived from aortic cells B. Surfac.2) mice B. Low lelox mice C. Import B cells D, and manic mice .N. brasiliensis were performed. Worm fecundity in the host was followed by determination of egg production in a time kinetic . As expected, IL-4Rα−/− mice demonstrated a chronic infection with eggs present in feces at day 14 PI. Determining the number of worms in the intestine at various time points following infection with N. brasiliensis resulted in comparable worm burdens between IL-4Rα−/lox, IL-4Rα−/−, and SM-MHCCreIL-4Rα−/lox mice at days 4 and 7 PI. However, at day 10 PI, IL-4Rα−/lox control mice, but not SM-MHCCreIL-4Rα−/lox or IL-4Rα−/− mice, had cleared the worm  was analyzed at days 4, 7, and 10 PI. A reduction (p < 0.05) of TH2 cytokine responses was observed from CD4+ T cells of SM-MHCCreIL-4Rα−/lox mice at all time points compared to IL-4Rα−/lox control mice, including IL-4, IL-5, IL-9, and IL-13 . By day 10 PI, intestinal IL-13 levels were reduced in IL-4Rα−/lox mice but still significantly higher than those in IL-4Rα−/− mice (p < 0.05). SM-MHCCreIL-4Rα−/lox mice, however, also showed significantly higher levels of IL-13 than did IL-4Rα−/− mice at day 10 PI (p < 0.05) in accordance with the delayed worm expulsion.Tnfection . Therefond IL-13 . Impairmnd 10 PI . At daysH2 responses in the MLNs had no influence on systemic type 2 antibody responses, as there were similar total serum IgG1 (unpublished data) and IgE . As expected [−/lox–treated mice were unable to clear infection, and CD4+ T cells were also essential for clearance in SM-MHCCreIL-4Rα−/lox mice, as depletion resulted in increased adult worm burdens in SM-MHCCreIL-4Rα−/lox mice , IL-4Rα−/lox mice had significantly higher (p < 0.05) expression levels of M3 than both IL-4Rα−/− and SM-MHCCreIL-4Rα−/lox mice. By day 10 PI, IL-4Rα−/− mice still showed a significantly lower level of M3 mRNA expression compared to IL-4Rα−/lox mice. However, SM-MHCCreIL-4Rα−/lox mice showed increased M3 expression compared to that on day 7 PI. This important result is the first report of IL-4Rα expression having an effect on the expression of acetylcholine receptors in vivo.In addition to goblet cell hyperplasia, another proposed mechanism of expulsion of le cells . Inductile cells ,16,17. WCreIL-4Rα−/lox mice results in delayed expulsion of the parasites.Together, these results show smooth muscle IL-4Rα plays an important role in the regulation of both draining lymph and intestinal cytokine production, goblet cell hyperplasia, and acetylcholine responsiveness. Disruption of these responses in the SM-MHCThis work provides the first description of the generation, characterization, and functional analysis of a smooth muscle cell–specific IL-4Rα–deficient mouse model. Disruption of IL-4Rα expression in smooth muscle cells was applied to a disease model where smooth muscle cells are proposed to play an important role in the resolution of infection, namely, a gastrointestinal nematode infection .H2-associated antibodies and cytokines [N. brasiliensis cleared the infection at day 9 PI, while SM-MHCCreIL-4Rα−/lox mice had an impaired ability to clear the nematode until day 12 PI. We demonstrated this impairment to be associated with a delay in goblet cell hyperplasia and the subsequent influx of mucus into the lumen of the host intestine. These physiological disruptions were related to an inability of the host to amplify appropriate cytokine production both locally and by CD4+ T cells from the draining MLNs.Clearance of nematode pathogens from the intestine is considered to require a number of physiological and immunological responses by the host. Increased intestinal contractions , increasytokines  are all N. brasiliensis–infected STAT6−/− mice have a significantly decreased tensile potential in vitro [2a is a potent inducer of IL-13– and N. brasiliensis–dependent intestinal contractions. However, specific inhibition of 5-HT2a failed to affect the ability of the host to resolve infection [CreIL-4Rα−/lox and IL-4Rα−/− mice following N. brasiliensis infection. The M3 expression data we present here are similar to those of 5-HT2a in response to N. brasiliensis infection. However, the potential role of M3 in mediating expulsion of intestinal parasites is more compelling. M3−/− mice are incapable of eliciting smooth muscle contractions [2a−/− mice [N. brasiliensis–infected mice [A number of authors have demonstrated nematode-induced amplification of intestinal smooth muscle contractions to be dependent on IL-13/IL-4Rα/STAT-6 signaling. Isolated strips of smooth muscle from the small intestine of in vitro . The signfection . We demoractions ; this is−/− mice . Previouted mice . As suchted mice .Trichuris muris, expulsion is driven by epithelial cell turnover [N. brasiliensis is induction of goblet cell–driven mucus production. Goblet cell–derived mucus is essential for clearance of N. brasiliensis infection [H2 polarization severely impairs the ability of goblet cells to secrete mucus and expel N. brasiliensis [N. brasiliensis infection [In addition to contractile responses, host epithelial responses constitute a second major physiological response to the parasite. This response varies according to parasite; in the case of the intraepithelial nematode turnover . The prinfection ,23. Secrnfection ,25 and anfection . Pathogeiliensis . Mucus piliensis  and gobliliensis . Innervanfection ,30. PrevN. brasiliensis infection, due to a deficiency in smooth muscle cell expression of IL-4Rα, may therefore be a result of the host's inability to mount an effective mucus response. Delayed mucus responses to N. brasiliensis infection are associated with an impaired TH2 response [+ TH2 response . These mice were then mated with IL-4Rα−/lox mice (n = 2) to generate SM-MHCCreIL-4Rα−/lox mice. Transgene negative littermates (IL-4Rα−/lox) were used as controls in all experiments. Mice were housed under specific pathogen-free barrier conditions in the University of Cape Town animal facility. All work was approved by the University of Cape Town animal ethics board.SM-MHCd States ,32. SM-Mhttp://www.biozym.com).Specific PCR primer pairs were for the IL-4Rα, 5′-GTACAGCGCACATTGTTTTT-3′ and 5′-CTCGGCGCACTGACCCATCT-3′; deletion, 5′-GGCTGCCCTGGAATAACC-3′ and 5′-CCTTTGAGAACTGCGGGCT-3′; LoxP, 5′-CCCTTCCTGGCCCTGAATTT-3′ and 5′-GTTTCCTCCTACCGCTGATT-3′; and Cre, 5′-ATGCCCAAGAAGAAGAGGAAGGT-3′ and 5′-GAAATCAGTGCGTTCGAACGCTAGA-3′. PCR conditions were as follows: 94 °C for 1 min, 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min for 40 cycles on an MJ thermocycler . Stained cells were then washed, fixed in 2% paraformaldehyde, permeabolized with saponin, preblocked with 2% NRS and Fc block (2.4G2), and stained with anti–α-actin FITC  or isotype control IgG2a-FITC (BD Biosciences). For lymphocyte staining, anti–CD3-FITC, anti–CD19-PE, and anti–IL-4Rα biotin in combination with SA-APC were used to identify lymphocyte subsets . Nonviable cells were stained with 7-AAD and excluded from analysis . Acquisition was performed using FACSCalibur and cells analyzed using Cellquest .A single cell suspension of smooth muscle cells was prepared as previously described  along wiN. brasiliensis L3 larva . Analysis of parasite eggs in feces was carried out using the modified McMaster technique [Mice were injected subcutaneously with 750 echnique . Adult w+ T cells were depleted from mice by intraperitoneal injection of 0.5 mg of anti-CD4+ monoclonal antibody (clone GK1.5) 3 d prior to infection. Mice received booster injections every 3 d to maintain depletion. Effective depletion was confirmed by FACS analysis.CD4+ T cells were purified from pooled MLNs at days 7 and 10 PI. Enrichment was carried with a negative selection. Briefly, cells in single cell suspension were stained with anti-CD8, CD11b, GR-1, B220, and CD16/32. Stained cells were depleted using goat anti-rat IgG–coated magnetic beads . Cell purity was approximately greater than 98%. CD4+ T cells were restimulated for 72 h with anti-CD3 . Supernatants were then collected and stored at −80 °C until analysis.CD4Cytokines in supernatants and serum antibody isotype levels from infected animals were determined as previously described .Tissue samples were fixed in a neutral buffered formalin solution. Following embedding in paraffin, samples were cut into 5-μm sections. Sections were stained with hematoxylin and eosin or periodic−acid Schiff reagent. The number of positively stained cells per five villi were counted by light microscopy. All samples were randomized and counted in a blinded fashion.http://www.promega.com). M3 cDNA was amplified using the following primers: 5′-CGG AAA AGG ATG TCG-3′ and 5′-GGC ACT CGC TTG TGA A-3′. Data were normalized using the α-actin housekeeping gene.RNA was extracted from the intestine of infected mice with the use of Tri-reagent (Sigma), and cDNA was synthesized using the ImProm-II Reverse Transcription System  accession numbers for the genes and gene products discussed in this paper are IL-13 (16163), IL-4Rα (16190), STAT-6 (20852), M3 (12671), smooth muscle myosin heavy chain (17880), and α-actin (11475).The GenBank (


Present: In the title and abstract, the ‘β’ symbol was mistakenly inserted instead of ‘α’.p63β modulates c-Myc activity via direct interaction and regulation of MM1 protein stability1, Juan Li1,*, Yimin Li1,*, Yang Wang1, Johann Bergholz1, Yujun Zhang1, Chenghua Li1 and Zhi-Xiong Xiao1Anning HanThis article corrects: p63β modulates c-Myc activity via direct interaction and regulation of MM1 protein stability, Volume 7, Issue 28, 44277–44287. Article first published online: 2016 Jun 20.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5190095/Link of original article: doi: 10.18632/oncotarget.10187Correct: The proper title and abstract appear below.The title should readp63α modulates c-Myc activity via direct interaction and regulation of MM1 protein stabilityAll symbols of “β” in the ABSTRACT should be changed into “α”.ABSTRACTBoth p53-related p63 and c-Myc are transcription factors playing key roles in cell proliferation, survival, development and tumorigenesis. In the present study, we identified that MM1, a c-Myc inhibitor, specifically binds to C-termini of p63α (including ΔNp63α and TAp63α). Further study demonstrates that p63α facilitates MM1 protein degradation via proteasomal pathway, resulting in elevation of c-Myc transactivation activity. Knockdown of ΔNp63α leads to decrease in c-Myc protein levels, concomitant with reduced expression of CDK4 and Cyclin D1, and impaired cell cycle progression, both of which are effectively reversed by simultaneous knockdown of MM1. Moreover, expression of p63 and CDK4 is concomitantly up-regulated in B-cell acute lymphoblastic leukemia. Together, this study reveals a novel crosstalk between p63 and c-Myc that may play an important role in cell cycle progression and tumorigenesis.44277-44287. doi: 10.18632/oncotarget.10187Original article: Oncotarget. 2016; 7: