To gain a better understanding of the role of Pcgf1 in early development, we analyzed mRNA profiles from Pcgf1-deficient P19 cells using RNA-seq. We found that the differentially expressed genes were enriched in many functional categories, which related to the development phenotype, and knockdown of Pcgf1 increased the expression of histone demethylases. Finally, our results showed that Pcgf1 loss-of-function decreased the levels of transcriptional repression mark H3K27me3 at the promoters of Ngn1 and Otx2, and the levels of transcriptional activation mark H3K4me3 at the promoters of Pou5f3 and Nanog. Together, our findings reveal that Pcgf1 might function as both a facilitator for pluripotent maintenance and a repressor for neural induction.The neural induction constitutes the initial step in the generation of the neural tube. Pcgf1, as one of six Pcgf paralogs, is a maternally expressed gene, but its role and mechanism in early neural induction during neural tube development have not yet been explored. In this study, we found that zebrafish embryos exhibited a small head and reduced or even absence of telencephalon after inhibiting the expression of Pcgf1. Moreover, the neural induction process of zebrafish embryos was abnormally activated, and the subsequent NSC self-renewal was inhibited after injecting the Pcgf1 MO. The results of  Early neural tube development in the embryo includes two important processes: neural induction and self-renewal of neural stem cells (NSCs). Neural induction refers to the process in which the mesoderm induces the ectoderm to develop into a neural plate, and then closes to form a neural tube in the early stage of embryonic development. Later in development, the self-renewal of NSCs is the cytological basis of early neural tube development  and were cultured in a constant temperature incubator at 28.5°C. The hatched eggs were fed twice a day, in the morning and evening. Paramecium was used as a first food for zebrafish at 3 dpf when some embryos started to hatch and swim freely to foraging, and then hatched artemia cysts became the only food at 2 weeks post-fertilization. The development of embryo was observed with a stereomicroscope.Zebrafish maintenance was performed as described previously  that was supplemented with 3% fetal bovine serum , 7% calf bovine serum, 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, and 0.25 μg/ml of amphotericin B. Cells were incubated at 37°C in a humidified atmosphere containing 5% COFor RA-induced differentiation, P19 cells were digested with 0.25% trypsin and resuspended with α-MEM medium that was supplemented with 0.5 μM all-trans-RA , then replaced with N2 medium after 3–4 days  was designed by GENECHEM . Pcgf1 cDNA was PCR amplified and cloned into the pLVX-EGFP-Puro vector, and lentiviruses were packaged using 293T cells. P19 cells were infected with LV-shPcgf1, LV-Pcgf1, or LV-vector lentivirus for 3 days. The stable cell lines expressing Pcgf1 or Pcgf1-shRNA were selected with puromycin . Synthetic mRNA was injected into a one-cell stage combined with MO for rescue experiments  was five mismatches with regard to Pcgf1 MO. The defects in morphogenic events were dose dependent. In the following study, we chose 5 ng/embryo as the experimental concentration, which resulted in the highest proportion of mild phenotype and the lowest death rate . Human P7 P19 cells are needed . The reverse transcription system is as follows: 1 μg of total RNA, 1 μl of oligo (dT) primer (100 μM), and nuclease-free water  were mixed and incubated at 65°C for 5 min. Then the following components were added and incubated for 60 min at 42°C: 4 μl of 5× reaction buffer, 1 μl of RiboLock RNase inhibitor (20 U/μl), 2 μl of 10 mM dNTP mix, and 1 μl of RevertAid RT (200 U/μl). This was followed by heating at 70°C for 5 min to terminate the reaction. Real-time PCR was performed with SYBR Green Realtime PCR Master Mix  according to the manufacturer's instructions. The qPCR reaction system is as follows: 10 μl of SYBR Green Realtime PCR Master Mix, 0.8 μl of PCR forward primer (10 μM), 0.8 μl of PCR reverse primer (10 μM), 2 μl of template cDNA, and 6.4 μl of ddH2O. The qPCR reaction conditions were set as follows: initial denaturation at 95°C for 5 min, 40 PCR cycles including denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extending at 72°C for 30 s. Melting curve analysis was performed at the end of each reaction. The expression of β-actin gene represented internal controls, and the relative expression of genes was calculated with the 2−ΔΔCT method. The primer sequences were as follows: zebrafish: actin , Pcgf1 , Sox2 , Sox3 , Otx2 , Ngn1 , P21 , P57 , Smad1 , Smad4 , Smad5 , Wnt3a , Wnt8a , β-catenin . Mouse: Actin , Pcgf1 , Pou3f1 , Nanog , Zpf521 , Oct4 , Hes1 , Pax6 .Total RNA was extracted from different stages of embryos or P19 cells following the standard protocols using TRIzol reagent (TRANS). Thirty embryos of every stage or about 1–5 × 10in situ hybridization was carried out as described below using the antisense RNA probes prepared . Then the pEASY-T3-derived plasmids were linearized. Antisense RNA probes were transcribed from linearized templates by T7 or SP6 polymerases (Thermo) in the presence of DIG-labeled nucleotides , and Zebrafish embryos were fixed with 4% paraformaldehyde in PBS at 4°C overnight at different developmental stages . The fixed embryos were pretreated with proteinase K (10 μg/ml) for 10 min, washed by PBST, and then the egg chorion was removed. The embryos were subjected to prehybridization in Hyb– (50% deionizing formamide buffer + 25% 20x SSC + 0.1% Tween) for 5 min and Hyb+  for 4 h at 65°C, then they were incubated in 1 ng/μl antisense RNA probes for hybridization overnight at 65°C. The next day, the embryos were washed with Liquid I (50% deionizing formamide buffer + 10% 20x SSC + 0.1% Tween), Liquid II (10% 20x SSC + 0.1% Tween), Liquid III (10% Liquid II + 0.1% Tween), and blocked in blocking solution [1% blocking powder  and 10% goat serum  dissolved in Maleic Acid Buffer] to block the non-specific binding sites at room temperature for 4 h. The embryos were then incubated with anti-DIG-AP  at 4°C overnight. On the third day, NBT/BCIP  was used for color detection, and images were acquired with a stereomicroscope   and zebrafish embryos (25–30 embryos) were lysed in RIPA buffer with protease inhibitors on ice. The supernatants were collected by centrifuging at 12,000 rpm at 4°C for 15 min. The supernatants were then quantified using the bicinchonininic acid (BCA) assay. The samples were mixed with loading buffer, and equal amounts of proteins (20–30 μg) were separated by SDS-polyacrylamide gel and transferred to PVDF membranes. The membranes were incubated with 5% skimmed milk for 2 h and probed overnight at 4°C with the indicated primary antibodies. The primary antibodies used were as follows: Pcgf1 , Pax6 , Oct4 , H3K4me3 , and H3K27me3  were from Abcam; GFP  and H3  were from Cell Signaling Technology, and β-actin  was from TransGen Biotech. Then secondary antibodies were incubated for 1 h at room temperature after washing. Secondary antibodies were HRP conjugated to either goat anti-mouse or anti-rabbit IgG antibodies. Protein bands were visualized by Immobilon™ Western Chemiluminescent HRP Substrate (MLLIPORE), and immunoreactive protein bands were analyzed by densitometry using Quantity one.Total protein of P19 cells . The significance of the differential expression of genes was confirmed by the bioinformatics service of BGI to the combination of absolute value of log2-ratio ≥ 1 and FDR ≤ 0.001. The original sequence data were submitted to the database of NCBI Sequence Read Archive.To perform RNA sequencing (RNA-seq), wild-type P19 cells and Pcgf1-KD P19 cells were harvested and lysed by TRIzol, and total RNA was collected according to the manufacturer's instructions. We mixed RNA samples from three independent experiments, and RNA-seq was carried out on a BGISEQ-500  antisense oligonucleotides. The efficiency of MO treatment was assessed by Western blot after coinjection with Pcgf1 MO or control MO with GFP-tagged Pcgf1 mRNA. The results confirmed that the MO oligonucleotides inhibit the translation of Pcgf1 mRNA . The phein situ hybridization showed that the expression of neural markers Sox2, Otx2, and Ngn1 was activated abnormally compared with the control group, and the expression of Sox3 increased at shield phase and decreased from 75% epiboly phase  of zebrafish embryonic development. The results of ly phase . In ordedization . These rin situ hybridization and qPCR showed that the expression of all the neural markers Sox2, Otx2, Ngn1, and Sox3 were decreased significantly during NSC self-renewal phase  staining was used to detect the effect of Pcgf1 on the apoptosis of NSCs. The results showed that there was no significant change in apoptosis  of zebrafish embryonic development. The results of al phase . Moreovepoptosis . Cyclin-poptosis . Taken tP19 cells stimulated by retinoic acid (RA) can differentiate into neural ectoderm and endoderm-derived cells, which was used to study the process of neural induction  was decreased, especially Smad4, but the activity of key molecules  in the Wnt signaling pathway did not change observably after knocking down Pcgf1 . HoweverPcG genes are identified as essential factors in epigenetic developmental processes. Epigenetic modification of chromatin structure results in the activation or silencing of specific genes, which has been proven to be an important molecular mechanism in development and disease. In our study, we found that the expression levels of H3K4me3 and H3K27me3 were both decreased after Pcgf1 knockdown . ChIP-qPNeural induction is defined as the process of specification of ectodermal cells into neural stem or precursor cells, which play essential roles in early neural tube development. To study the molecular regulation of neural induction is the key to reveal the pathogenesis of the abnormal neural tube development. Here, we showed that Pcgf1 is maternally expressed during the embryonic development of zebrafish. However, the biological function of Pcgf1 in the early neural tube development is still unclear. In order to study the effect of Pcgf1 on the early neural tube development, we constructed Pcgf1 knockdown zebrafish as an animal model. We found that loss of Pcgf1 resulted in telencephalon malformations  in zebrafish embryos. Furthermore, knock down of Pcgf1 resulted in the abnormal neural induction process, which led to the decrease in NSC proliferation in the zebrafish embryo. Finally, our results showed that Pcgf1 regulated the levels of H3K27me3 in the Ngn1 and Otx2 promoter regions, and the levels of H3K4me3 at the promoters of Pou5f3 and Nanog, which explained the role of Pcgf1 in neural induction from the perspective of epigenetics .in vivo is still unclear. To investigate the function of Pcgf1, we inhibited the expression of Pcgf1 in zebrafish embryos using MO oligonucleotides. We found that after the injection of Pcgf1 MO, embryos exhibited obvious morphogenesis defects with a small head, reduced or even disappeared telencephalon, and shortening of the body axis by 24 hpf. In fact, a previous study has reported that Pcgf1 knockout leads to retarded growth and abnormal craniofacial development in zebrafish, which proves that Pcgf1 plays an important role in the early developmental stages and also supports our conclusion to a certain extent (Dupret et al., in vivo, which will be a breakthrough point to study the role of Pcgf1 in early neurodevelopment. In addition, disruption of Ring 1b/Rnf2, the E3 ubiquitin ligase in PRC1 in mice, leads to an arrest at gastrulation, and Ring 1b mutants in zebrafish displayed several defects, including jaw malformations, and they died at around 4–5 dpf (Voncken et al., The PRC1 complex functions as a transcriptional repressor in stem cell biology and development. They are divided into six groups based on the critical factors Pcgf genes. Although it had been reported that Pcgf1-6 play important roles in the self-renewal, proliferation, and differentiation of ESC (Yadirgi et al., in vitro, and the role of Pcgf1 in neural development in vivo has not been fully revealed. In the neural differentiation process of RA-induced P19 cells, Pcgf1 can positively regulate the Oct4–Nanog–Sox2 axis, thus maintaining the pluripotency of P19 cells (Li et al., in vivo.The neural induction process and NSC self-renewal are two most important processes of neural tube development. However, most of the studies on Pcgf1 are carried out P19 cells are derived from an embryo-derived teratocarcinoma, and these cells can differentiate into each of the three germ layers (Boheler et al., in vivo and in vitro, and knockdown of Pcgf1 reduces H2A ubiquitinylation in Hela cells (Wu et al., in vivo further confirmed that the levels of H3K27me3 at the promoters of Ngn1/Otx2, and the levels of H3K4me3 at the promoters of Pou5f3/Nanog, were decreased after injecting Pcgf1 MO to the embryos. There are a variety of evidences supporting the role of PcG in transcriptional activation (Morey et al., −/− mutants with reduced H3K27me3 did not reveal severe brain phenotypes, suggesting that the role of Pcgf1 may be involved in other related mechanisms, which needs further study (San et al., The PcG protein family has always been considered as a class of proteins that inhibit the transcription of target genes at the level of chromatin. A previous study showed that Pcgf1 promotes monoubiquitinylation of histone H2A Taken together, we first discovered the crucial role of Pcgf1 in early neural tube development especially in neural induction phase in the embryo and established the regulatory network of transcription factors and epigenetic factors to neural induction, which laid a certain foundation for further exploring the function of other members during early neural tube development. From the perspective of disease treatment, this study provides a good basis for further revealing the role of the Pcgf family in the abnormal development of the neural tube and provides new ideas and new clues for the clinical treatment of abnormal neural tube closure.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the National Institutes of Health Guide for the Care and Use of Laboratory, and the Institutional Animal Care and Use Committees of Shandong University. Written informed consent was obtained from the owners for the participation of their animals in this study.in vivo experiments and assembly of data. JD, XiaL, WS, and YH contributed to the cell culture experiments and data analysis. WZ interpreted the data and co-wrote the manuscript with AH. AH provided critical input to the overall research design and final approval of the manuscript. All authors contributed to the article and approved the submitted version.XinL performed most of the experiments and interpreted the data. GJ and JZ were involved in the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


S-glutathionylation, the post-translational modification forming mixed disulfides between protein reactive thiols and glutathione, regulates redox-based signaling events in the cell and serves as a protective mechanism against oxidative damage. S-glutathionylation alters protein function, interactions, and localization across physiological processes, and its aberrant function is implicated in various human diseases. In this review, we discuss the current understanding of the molecular mechanisms of S-glutathionylation and describe the changing levels of expression of S-glutathionylation in the context of aging, cancer, cardiovascular, and liver diseases. Reactive oxygen species (ROS) are derivatives of molecular oxygen produced in a wide range of cellular processes . MitochoAlthough historically viewed as harmful byproducts of metabolism, ROS may also function as important second messengers within intracellular redox signaling pathways ,5. The mIn mammalian cells, S-glutathionylation can function as a regulatory mechanism or protect proteins against irreversible oxidation. By introducing the tripeptide glutathione with additional ionic charges into a protein, S-glutathionylation resembles the well-characterized mechanism of protein phosphorylation. The dynamic and reversible nature of S-glutathionylation highlights its ability to function as a redox “switch”, regulating protein function, interactions, and localization . In thisCysteine is one of the least abundant, but perhaps the most functionally diverse, of all twenty standard amino acids . Despitea) of the thiol group. The pKa of the unperturbed cysteine thiol is approximately 8.5 [a values [a means higher availability of thiolates at neutral pH, the nucleophilicity of thiolates is either decreased or unaffected at lower pKa depending on the substrate tested [The human genome encodes about 214,000 cysteines . In aquetely 8.5 , indicata values . Additioe tested .•). Free radical species that oxidize thiols to thiyl radicals in vivo include hydroxyl (•OH), carbonate (CO3•–), nitrogen dioxide (•NO2), superoxide (O2•–), and peroxyl and phenoxyl radicals [Under oxidative stress conditions, the one-electron oxidation of a cysteine thiol leads to the generation of the thiyl radical  . This mel nature . Enzymesl nature .2H) and sulfonic acid (RSO3H), which are more stable, irreversible type modifications generally associated with protein misfolding, degradation, and pathology [Sulfenic acids are transient species that can be subject to several alternative fates. Depending on the microenvironment, a sulfenic acid may react with an adjacent thiol to form a disulfide (RSSR’) or it may react with a backbone amide to form a sulfenyl-amide (RSNHR’) . In the athology . The onlathology . Figure a [2O2), react exclusively with the thiolate form of cysteine [2O2 at pH 7.4 increase with decreasing thiol pKa [a of a thiol is not the sole determinant of its oxidation susceptibility. This fact is highlighted by comparing protein tyrosine phosphatase 1B (PTP1B) (pKa = 5.4) and peroxiredoxin (pKa = 5.6). Although the pKa of PTP1B and peroxiredoxin are similar, there is a 107-fold difference in the reaction rate constants of H2O2 with their active sites [It is well established that only particular cysteine residues in proteins are susceptible to oxidative modifications ,25. Aboua . Many bicysteine . Winterbhiol pKa . Howeverve sites .a and increasing the reactivity of cysteine [a and higher reactivity than cysteines near the C-terminal [Chemical features of the local microenvironment may also affect the reactivity of cysteine. For instance, vicinal polar or positively charged amino acids stabilize the thiolate anion, thereby lowering its pKcysteine . Likewisterminal . Other fIn the reducing environment of the cytoplasm, low-molecular-weight (LMW) thiols are produced in high concentrations to serve as cofactors, reduce existing oxidative modifications, and form covalent linkages with protein thiols ,32,33. OGlutathione is a ubiquitously distributed tripeptide consisting of three amino acids: glycine, cysteine, and glutamic acid. The thiol group of cysteine is the key functional component of the molecule responsible for its biological activity. The structure of glutathione is unique such that the N-terminal glutamate and cysteine residues are bonded by the γ-carboxyl group of glutamate rather than the conventional peptide bond formation in proteins utilizing the α-carboxyl group . ConsequIn most cells, glutathione is present at concentrations of 1–10 mM in the cytosol, and at lower concentrations in subcellular organelles, including the nucleus, mitochondria, and endoplasmic reticulum . The tria of the thiol group of GSH, shown to be around 9.6, confers low reactivity to the molecule [a of the thiol group of GSH to catalyze the nucleophilic addition of the tripeptide to xenobiotics [2O2 to H2O by the coupled oxidation of GSH to GSSG. Subsequently, GSSG is reduced back to GSH by the action of glutathione reductase and NADPH. Other enzymes such as peroxiredoxins and glutaredoxin that require thiol-reducing equivalents also utilize glutathione as a reductant to restore enzymatic function by keeping their redox-sensitive active sites in the reduced state [Glutathione serves vital functions inside the cell, including but not limited to antioxidant defense, maintenance of redox potential, redox signaling, and regulation of cell growth and death. The pKmolecule . Thus, amolecule . Glutathobiotics . Similarobiotics . These eed state .The modification of protein-reactive thiols with glutathione was initially thought to be a non-specific reaction induced in pathological, highly oxidative situations or upon exposure to strong oxidants, particularly under in vitro conditions . Recent mix, Equation (2)) represents the specific oxidation potential for the formation of the mixed disulfide.One of the most intensively studied mechanisms for protein S-glutathionylation involves a thiol-disulfide exchange reaction . The thimix is approximately 1, indicating that the GSH:GSSG ratio would have to decline dramatically to drive S-glutathionylation [mix~13) [For most protein cysteines, Knylation . The ratnylation . Thus, anylation . Therefo[mix~13) , the thiThe mechanisms that are more likely to mediate S-glutathionylation reactions in vivo involve reactive thiol derivates such as sulfenic acids, sulfenyl-amides, thiyl radicals, S-nitrosylated thiols, and thiosulfinates.a = 9.6) is likely to limit this reaction in vivo [The two-electron oxidation of a cysteine thiolate generates a sulfenic acid, the simplest oxyacid of sulfur. The oxidants most commonly implicated in the conversion of protein thiolates to their corresponding sulfenic acids include hydrogen peroxide, alkyl hydroperoxides, peroxynitrite, hypochlorous acid, and chloramines . Under p in vivo .2O2 to form a protein sulfenic acid [2O2, thereby escaping further oxidative modification [Many proteins have been identified as candidates for regulation by sulfenic acid formation. However, most of these studies were performed in vitro and in the absence of GSH. Recent literature suggests that although sulfenic acid formation may be the initial oxidative modification for these proteins, S-glutathionylation serves as the more stable intermediate in redox signaling . This wanic acid ,57, havenic acid . More renic acid . In agrefication .2O2 to sulfenic acid, followed by the rapid elimination of oxygen to generate a more stable derivative, the sulfenyl-amide species [The formation of the sulfenyl-amide species was first demonstrated in vitro by Salmeen et al., as an alternative protective modification for the active site cysteine of protein tyrosine phosphatase 1B (PTP1B) . The pro species . X-ray c species . The sul species , the sul species . At pres•OH), carbonate (CO3•–), nitrogen dioxide (•NO2), superoxide (O2•–), and peroxyl and phenoxyl radicals [Thiyl radicals are highly reactive species that can be generated via electron transfer or hydrogen atom abstraction from a thiol ,63,64. Fradicals .2 [2+/ADP/H2O2 + GSH or horseradish peroxidase/H2O2 + GSH), several proteins have been shown to undergo S-glutathionylation in vitro [The formation of protein thiyl radicals or glutathione thiyl radicals may lead to protein S-glutathionylation reactions through radical recombination or reaction of a radical with a thiolate followed by reaction with O2 . In the in vitro . A recenin vitro ,67. Othein vitro ,69. Thusin vitro .2O3) is considered to be the primary nitrosylating agent [S-nitrosylation is a type of post-translational modification that can occur under normal and pathological cellular conditions, analogous to S-glutathionylation . It is cng agent . During ng agent ,75. AlteIn the presence of physiologically relevant concentrations of GSH, one study showed that NO inhibits the DNA binding activity of the transcription factor c-Jun by specifically inducing S-glutathionylation in its DNA binding site . Another2•–/H2O2-producing system xanthine/xanthine oxidase [Glutathione thiosulfinate SG) is the anhydride of glutathione sulfenic acid . Li et a oxidase ,81.Glutathione thiosulfinate is highly reactive with thiols and has been suggested to mediate S-glutathionylation of several proteins. In the rat brain, glutathione thiosulfinate was shown to be one of the most potent S-glutathionylating agent among the various glutathione derivatives tested, including GSNO and GSSG . As glutAlthough protein S-glutathionylation in cells may involve non-enzymatic chemical reactions, several enzymes have been proposed to catalyze the transfer of glutathione to the target protein.Glutathione S-transferases (GSTs) are an abundant family of detoxification enzymes that catalyze the conjugation of glutathione to chemically reactive electrophilic compounds . The Pi A critical step in the detoxification reaction catalyzed by peroxiredoxin VI is the formation of the enzyme sulfenic acid on Cys47, the single conserved cysteine residue of peroxiredoxin VI . Cys47 iGSTπ has been described to play a key role in the S-glutathionylation mechanism of several proteins, especially during oxidative stress conditions in vivo and in vitro ,88. TownRecent studies have identified additional proteins that may also undergo selective S-glutathionylation by GSTπ, including Nrf2 protein inhibitor Keap1, , estroge•, Grx1 has been proposed to catalyze the forward S-glutathionylation reaction [a of the active site Cys22 of Grx1 and the stability of disulfide-anion radicals  as opposed to the glutathionyl radical (GS•), Grx1 may catalyze the S-glutathionylation of proteins through a radical intermediate [2 with Grx1- SSG•−, confirming that the redox environment likely determines the mode of catalysis by Grx1 [Under normal physiological conditions with high levels of GSH, glutaredoxin (Grx) primarily functions in catalyzing the deglutathionylation reaction , which is discussed in more detail below. However, during increased levels of oxidative stress, when glutathione is present mainly in its oxidized forms including GSSG, GSNO, and GSreaction . Mieyal rmediate . Grx1 harmediate . Besides by Grx1 .Recently, a possible role for the glyoxalase II enzyme in the S-glutathionylation of specific proteins has been reported ,99. The Protein S-glutathionylation is a dynamic and reversible process, dependent on both the rate of formation and the rate of deglutathionylation. The reversibility of protein S-glutathionylation provides an additional layer of regulation for controlling cellular processes . DeglutaIsoforms of glutaredoxin are found in prokaryotes and the various cellular compartments of eukaryotes. Based on their active site sequences, mammalian glutaredoxins can be broadly categorized into two major subfamilies: class I and class II glutaredoxins . Class IThe active site of the vast majority of glutaredoxin isoforms contain variations of the redox-active Cys-X-X-Cys motif . The maiAnother enzyme that has been proposed to have deglutathionylating activity is sulfiredoxin (Srx), a small oxidoreductase that catalyzes the reduction of sulfinic acid derivatives of 2-Cys peroxiredoxins . FindlayS-glutathionylation can directly regulate the structure/function of a wide range of proteins, and thereby transduce redox signals in cellular pathways. S-glutathionylation has been shown to either increase or decrease the activity of several classes of proteins including mitochondrial enzymes, heat shock proteins, transcription factors and cytoskeletal proteins . In contOne study demonstrates how S-glutathionylation can alter the structure and regulate the function of hHsp70, a cytosolic stress-induced form of heat shock protein 70 (Hsp70), in HeLa cells . Hsp70 pAnother study shows how S-glutathionylation of the binding immunoglobulin protein (BiP) regulates the balance between its foldase and ATPase activities by altering protein structure . BiP is A large body of evidence has accumulated over the past few decades suggesting that oxidative stress may be involved, with varying degrees of importance, in the pathogenesis of diseases . In manyHere, we discuss recent studies that have associated a number of conditions such as aging, cardiovascular disease, cancer, and liver disease with changing levels of S-glutathionylation.The aging process is a complex phenomenon broadly defined as the progressive decline in physiological functions accompanied by a wide variety of pathological conditions . SeveralAging is considered to be the biggest risk factor for most neurodegenerative diseases. Neurodegenerative diseases involve loss of neurons, aggregation of misfolded proteins, mitochondrial dysfunction, and overexposure to ROS . Due to The major neurodegenerative disease known to affect the aging population is Alzheimer’s disease (AD). Although the exact causes of AD are not fully understood, the loss of synapses and the accumulation of plaques formed by the protein amyloid beta, and neurofibrillary tangles formed by the protein tau are widely studied . ImplicaMitochondria are one of the most important sources of ROS in the cell. Mitochondria are abundant in cardiac cells due to the increased energy demands of producing ATP through coupled electron transport and oxidative phosphorylation . TherefoMyocardial infarction, also known as a heart attack, occurs due to the formation of plaques in the interior walls of the arteries, which block blood flow and reduce oxygen supply to the cardiac muscle . MyocardA more recent study shows an increase in the S-glutathionylation of a high-molecular-weight protein (most likely titin) in the early postmyocardial infarction period in mouse hearts . Titin iAnother protein involved in the regulation of cardiac dynamics is cardiac myosin binding protein C (cMyBP-C). One study showed that a decrease in the GSH/GSSG ratio in isolated perfused rat hearts led to increased S-glutathionylation and reduced protein kinase A (PKA)-dependent phosphorylation of cMyBP-C . The phy2+ out of the sarcoplasmic reticulum in cardiac muscle [2+ release and decreasing diastolic Ca2+ leak [Ischemic preconditioning refers to brief, repetitive periods of ischemia that make the myocardium more resistant to larger ischemic insult and thus confer protection against subsequent myocardial infarction . Precondc muscle . Severalc muscle . Sanchezc muscle . Subsequc muscle . Althouga2+ leak ,153.2+-handling proteins, as well as mitochondrial dysfunction, and eventually progresses to heart failure [Cardiac hypertrophy refers to the enlargement and thickening of cardiomyocytes. It is classified as physiological when associated with normal cardiac function or pathological when associated with cardiac dysfunction . Patholo failure . Among t failure ,157. Pim failure . The mec failure . Likewis failure . Collect failure  via actiCellular antioxidant defense mechanisms minimize the harmful effects of oxidative stress and maintain the optimal redox environment under normal physiological conditions. However, cancer cells differ from normal cells as they exhibit high ROS levels due to genetic, metabolic, and microenvironment-associated alterations and mitochondrial dysfunction . To prevNumerous studies have reported that S-glutathionylation can modify proteins involved in the signaling mechanisms of cancer cells. In particular, protein kinase C (PKC) isozymes, which link multiple cellular processes to cancer, contain cysteine-rich regions that are highly susceptible to oxidants ,163. PKCMany transcription factors have conserved cysteine residues that are susceptible to thiol modifications. S-glutathionylation of these transcription factors may interfere with their ability to bind to DNA, thereby dysregulating gene expression. For instance, the tumor suppressor p53, known to play an essential role in cell cycle control and apoptosis, is inhibited by the S-glutathionylation of cysteines present within its proximal DNA-binding domain . UntreatCancer is a disease of uncontrolled cell growth that occurs by the overactivation of proliferation and corresponding inhibition of apoptosis. Thus, reinstating apoptosis in malignant cells can lead to the selective elimination of cancer cells . CaspaseNonalcoholic fatty liver disease (NAFLD) is a common hepatic condition that encompasses a wide variety of liver diseases characterized by the accumulation of excess fat stored in liver cells . Althoug+-dependent histone deacetylase, has been identified as a target protein for increased S-glutathionylation [Recent studies have shown that mice fed with a high-fat diet develop NAFLD and show increased S-glutathionylation ,182. In nylation . S-glutanylation . The actnylation . Under hnylation . The ovenylation . In agrenylation  and convnylation .In another study, S-glutathionylation was associated with TNF α-mediated cytotoxicity . The proOver the past few decades, the oxidative post-translational modifications of protein cysteine residues have emerged as an attractive area of research in redox biochemistry. In this regard, protein S-glutathionylation appears to be the predominant modification due to the abundance of the endogenous glutathione pool. S-glutathionylation serves both to protect proteins from oxidative damage and modify structure/function relationships. Increasing evidence shows that redox regulation by S-glutathionylation contributes to physiological processes, and aberrant S-glutathionylation is associated with various health conditions, from cancer to cardiovascular disease.Despite the vast body of accumulated research in this field, much work is still needed to further our understanding of this unique protein modification. As we have discussed in this review, multiple cellular mechanisms of S-glutathionylation have been proposed. However, the contributions of these mechanisms in experimental models remain uncertain, and the functional implications of the enzyme-mediated S-glutathionylation in physiological settings are unclear. Further investigation into the mechanisms of protein S-glutathionylation and the identification of the changes in S-glutathionylation patterns in disease contexts will provide more insight into the significance of S-glutathionylation as a fundamental post-translational modification.


Participants were followed for 3 to 16 years (median of 11 years). The PVL, IFN-γ, TNF, and IL-10 levels were quantified at entry and at the end of the follow-up. Among the self-reported symptoms in the initial evaluation, only the presence of paresthesia on the hands was more frequent in the group with high PVL (p < 0.04). The production of IFN-γ was higher in the group with high PVL group  when compared with the control group in the first assessment. There was no difference in the occurrence of urinary symptoms or erectile dysfunction, periodontal disease, Sicca syndrome, and neurologic signs between the two groups during the follow-up. The observation that none of the HTLV-1 carriers with high PVL and with exaggerated inflammatory response progressed to HAM/TSP indicates that other factors in addition to the PVL and an exaggerated immune response are involved in the pathogenesis of HAM/TSP.A high proviral load (PVL) is recognized as a risk factor for human T cell leukemia virus-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), but there is a lack of prospective studies evaluating whether or not HTLV-1 carriers with high PVL are at risk of developing HAM/TSP or other HTLV-1-related diseases. Here, we compare the incidence of clinical manifestations and the cytokine levels in 30 HTLV-1 carriers with high (> 50,000 copies/10 The exposed group had PVL greater than 50,000 copies/10The HTLV-1 infection was diagnosed by the enzyme-linked immunosorbent assay (ELISA)— and confirmed by Western blot test, HTLV blot . Initially, 92 HTLV-1 carriers were identified, and 31 had high PVL. As one of these patients had neurosyphilis, he was excluded. Thus, 30 subjects with a high PVL and 30 with a low PVL matched for age (± 5 years) and gender participated in the study. None of the subjects was taking immunosuppressive drugs or was using phosphodiesterase inhibitors.The subjects answered a standard questionnaire and had a clinical and neurological physical examination performed once a year. Information about how they were referred to the clinic, previous history of blood transfusions, breast feeding, and sexual behavior were recorded. Patients were evaluated by neurologists, urologists, rheumatologists, dentists, and psychologists. The outcomes determined were Sicca syndrome, periodontitis, urinary dysfunction, erectile dysfunction, walking difficulty, running difficulty, hyperreflexia (grade 3 or 4), and Babinski reflexes. Sicca syndrome was defined by xerophthalmia, and evidence of dry mouth and chronic periodontal diseases by the criteria was established by the International Association of Periodontal Disease. Low urinary tract symptoms such as nocturia, frequency, urgency, and incontinence were recorded, as well as void dysfunction. The erectile dysfunction was determined by the international index of erectile function (IIEF-5).6 cells/ml in RPMI 1640  supplemented with 10% of fetal bovine serum , glutamine, HEPES, and antibiotics. Unstimulated cells, 3 × 106 cells/ml, were incubated for 72 h at 37 °C 5% CO2, and the supernatants were harvested. Cytokines were determined in supernatants of unstimulated PBMC cultures by sandwich ELISA using reagents from BD Biosciences Pharmingen, San Jose, CA, USA, and the results are expressed in pg/ml.Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples by density gradient centrifugation with Ficoll-Hypaque . The cells were cultured in RPMI 1640 , 10% human AB serum , glutamine, HEPES, and antibiotics (complete RPMI). Cytokines were determined in supernatants of unstimulated PBMC cultures as previously described  . In the group of carriers with high PVL, the HTLV-1 carriers were followed a median of 11 years versus 11.5 years in the non-exposed group (p = 0.57).A total of 60 individuals infected by HTLV-1 were eligible for this study and included in the analysis. The demographic, epidemiological, and clinical features of the participants are shown in Table The comparative analysis of HTLV-1 carriers at study entry and in the last evaluation is shown in Table p = 0.011) when compared with the control group in the first evaluation, as well as the concentration of IL-10 . There were no differences in the levels of TNF and IL-10 at baseline, and in the last evaluation, there was a decrease in TNF concentration in both groups, with a significant difference in the group with low proviral load with median of 105 pg/ml at admission versus 22 pg/ml in the last evaluation, p = 0.01).The cytokine production at baseline and in the last evaluation is shown on Fig. r = 0.55) and in the last (r = 0.65) evaluation and between TNF and PVL in the same group in the last evaluation (r = 0.43). Regarding the correlation between proinflammatory versus anti-inflammatory cytokines, it is important that the only strong correlation observed was a direct correlation between IFN-γ and IL-10 in the high PVL (r = 0.76) in the last evaluation. There was a direct correlation between TNF and IL-10 in all evaluations except in the first evaluation of the group with low PVL. Regarding the proinflammatory cytokines, there was a direct correlation between IFN-γ and TNF in all evaluations in both groups.The correlations between the production of cytokines in supernatants of PBMC as well as between cytokines and PVL are shown in Table The HAM/TSP only occurs in less than 5% of HTLV-1-infected subjects (Gessain and Mahieux The two groups of HLV-1 carriers of the present study had similar demographic and epidemiologic profiles at admission, but they differ regarding the proviral load and the production of proinflammatory cytokines. Additionally, carriers from both groups had no difference in the clinical or neurological examination at admission. The only exception was the presence of paresthesia on the hands, which was more frequent in the group with a high PVL. It is relevant that two previous studies aimed to identify clinical and neurological manifestations related to HTLV-1 found higher frequencies of Sicca syndrome, periodontal disease, urinary complaints of neurogenic bladder, hyperreflexia, and even difficulty walking in HTLV-1 carriers than in the no infected subjects (Caskey et al. 6/lymphocytes is a good predictor for the diagnosis of HAM/TSP (Grassi et al. The enhanced T cell activation contributes to the passage of cells through the brain-blood barrier and persistent secretion of proinflammatory cytokines Bangham . A previPrevious studies have shown that proinflammatory cytokine levels in serum and supernatants of PBMC are higher in patients with HAM/TSP than in HTLV-1 carriers (Grassi et al. A correlation between proinflammatory and anti-inflammatory cytokines has been observed in subjects with HTLV infection, and there is a direct correlation between IFN-γ and IL-10 as well in TNF and IL-10 in HTLV-1 carriers (Carvalho et al. The increase in the frequency of cells expressing TNF is associated with pathology in HTLV-1 infection (Santos et al. An analysis of the correlation between PVL and cytokines in the two groups studied showed different profiles. While low PVL carriers showed a direct correlation between IFN-γ and TNF production and PVL in the last evaluation, in the group of subjects with a high PVL, this correlation was not observed. The existence of a correlation between proviral load and cytokine production was observed in other studies (Lima et al. + T cells. The presence of HLA-A * 02 or HLA-Cw * 08 was associated with a lower proviral burden and decreased prevalence of HAM/TSP (Assone et al. We recognized that this study has some limitations including the identification of other factors associated with decreased susceptibility to the development of HAM/TSP, the limited follow-up of the carriers, and the lack of evaluation TAX-specific CD8Our data indicate that a large number of HTLV-1 infected subjects with a high PVL and an exaggerated inflammatory response do not develop neurological manifestations, urinary and erectile dysfunction, nor other diseases associated with HTLV-1. These findings suggest that other factors, in addition to the PVL and the inflammatory response observed in the peripheral blood, are necessary for carriers to develop HTLV-1-associated diseases.


Background: This meta-analysis aimed to evaluate the relationship between serum uric acid (UA) and the risk of dementia and its subtypes.Methods: Embase, PubMed, and Web of Science were searched from inception to July 2020. Random-effect models were employed to analyze the standard mean difference (SMD) with the corresponding 95% confidence intervals (CI).Results: Twenty-three eligible studies involving 5,575 participants were identified. The overall results showed lower levels of UA in dementia relative to non-dementia controls [SMD = −0.32  p = 0.04]. The subgroup analysis of the type of dementia demonstrated a significant association of UA with Alzheimer's disease (AD) [SMD = −0.58  p = 0.009] and Parkinson's disease with dementia (PDD) [SMD = −0.33  p = 0.001] but not with vascular dementia (VaD). The stratification analysis of the concentrations of UA revealed that the UA quartile 1–2 was negatively correlated with dementia and neurodegenerative subtypes (p < 0.05), whereas a positive correlation of UA quartile 4 with dementia was noted (p = 0.028). Additionally, the meta-regression analysis on confounders showed that not age, body mass index, diabetes mellitus, hypertension, or smoking but education (p = 0.003) exerted an influence of the UA in the risk estimate of dementia.Conclusions: Low concentrations of UA (< 292 μmol/L or 4.91 mg/dL) is a potential risk factor for AD and PDD but not for VaD. The mechanism of different concentrations of the UA in dementia needs to be confirmed through further investigation. Dementia is a group of acquired clinical syndromes characterized by the progressive decline in cognition along with psychiatric and behavioral alterations of differing extents , regulated by diet and physical activities, is the final breakdown of purine nucleotides that plays a crucial role in human metabolism  determinant dementia in cases  had to be based on certain standardized diagnostic criteria, e.g., the National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer's Disease and Related Disorders Association (NINCDS–ADRDA) , PubMed (September 1974–July 2020), and Web of Science (March 1992–July 2020), which followed the developed guidelines of the preferred reporting items for systematic reviews and meta-analyses (PRISMA)  and odds ratio (OR), respectively. The statistical heterogeneity between studies was assessed by the I2 test, with a value higher than 50% considering heterogeneity  with a corresponding 95% confidence interval (CI) was identified by a A total of 1,202 articles were screened in the initial search, of which 96 studies were retained by inspecting titles and abstracts. Then, 73 articles were excluded through a comprehensive review of the full texts. Finally, 23 articles, which met the inclusion criteria, were included in the meta-analysis , the random-effect models were used to render the conservation of estimates by the weight disproportionation in small studies. The combined results of 23 studies showed lower levels of UA in patients with dementia compared with patients without dementia [SMD = −0.32  p = 0.04]  p = 0.933], smoking [OR = 1.08  p = 0.698], hypertension [OR = 1.32  p = 0.490], and diabetes mellitus [OR = 1.05  p = 0.806] between dementia and non-dementia controls; while older age [SMD = 0.51  p = 0.001] and lower educational attainment [SMD = −1.69  p = 0.002] appeared in patients with dementia than at in controls with non-dementia. The subgroup analysis of the type of dementia  p = 0.001] but not with VaD [SMD = 0.27  p = 0.466]. The UA quartile 1 [SMD = −1.17  p < 0.01], 2 [SMD = −0.72  p = 0.02], and 4 [SMD = 0.84  p = 0.028] were significantly related to dementia, whereas no relationship was noted in UA quartile 3 [SMD = −0.19  p = 0.093]  p = 0.02] with neurodegenerative dementia  on the relationship of UA with dementia; but age , BMI , diabetes mellitus , hypertension , and smoking  exerted no influence on the relationship.According to the presence of heterogeneity  indicated no evidence of publication bias , neurofibrillary tangles, and neuritic plaques  were negatively correlated with dementia and the subtypes of neurodegerative dementia, whereas a positive association of higher levels of UA (>316 μmol/L) with dementia was found after a smooth transition over a safe interval. This was consistent with previous studies that fluctuations in the concentrations of UA was linked to altered brain function and cognitive decline  were a potential risk factor for AD and PDD rather than VaD. Additional The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.The study was conceived by MZ, CZ, and ZZ. The literature search and selection were conducted by SZ and XZ. The data were extracted and analyzed by YL, KK, YX, RZ, and HQ. The rough manuscript was drafted by MZ, CZ, and ZZ. All authors reviewed and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Great Recession in Europe in 2002–2014. In contrast to previous studies, which largely concentrated at the country level, we use data for 251 European regions in 28 European Union (EU) member states prior to the withdrawal of the United Kingdom in January 2020. We apply three-level growth-curve model which allows for a great deal of flexibility in modelling temporal change while controlling for variation in economic conditions across regions and countries. Our findings show that fertility decline was strongly related to unemployment increase; this relationship was significant at different reproductive ages. Deteriorating economic conditions were associated with a stronger decline in fertility during the economic recession as compared with the pre-recession period. This evidence suggests the salience of factors such as broader perception of uncertainty that we could not capture in our models and which rose to prominence during the Great Recession. Furthermore, strongest fertility declines were observed in Southern Europe, Ireland and parts of Central and Eastern Europe, i.e. countries and regions where labour market conditions deteriorated most during the recession period. In Western Europe, and especially in the Nordic countries, fertility rates were not closely associated with the recession indicators.This study investigates how the changes in labour market conditions and economic growth were associated with fertility before and during the The online version of this article (10.1007/s10680-020-09556-y) contains supplementary material, which is available to authorized users. Great Recession that started in autumn 2007 in the USA has hit almost all European countries, with many experiencing falling gross domestic product (GDP) and rising unemployment for most of the period of 2008–2013. The recession has destroyed many jobs, put a downward pressure on wages and induced a huge strain on government budgets, often resulting in spending cuts on social policies and families  that started around the turn of the century has peaked in 2008–2010. Thereafter, fertility rates declined or levelled off in most European countries, especially among young women below age 25, accelerating the postponement of births to higher reproductive ages , especially in the Baltic countries, Bulgaria, Croatia or Hungary, where it was either very strong initially or lasted particularly long. Economic output also fell sharply in Ireland. In five Southern European countries , GDP per capita contracted by over 10% in the period 2008–2013; in Greece the decline was as large as 32%. Economic contraction of more than 10% was also recorded in Ireland, Latvia, Croatia and Slovenia Eurostat . The falYoung adults below age 25 were hardest-hit by deteriorating labour market conditions. Their unemployment increased abruptly and even doubled or tripled in some of the countries, mainly in Southern Europe and in CEE  and Ireland OECD  and 2016Only few European countries were relatively unscathed by the recession. Among them, Poland did not experience a single year of declining GDP in 2008–2013, three out of the five Nordic countries , Belgium, France, Luxembourg and Malta saw only minor increases in unemployment and the three German-speaking countries  recorded very mild recession. Germany even displayed a decline in unemployment.The dynamics of economic recession varied strongly not only between countries, but also at sub-national level. Several countries  had both regions which experienced overall a positive economic growth over 2008–2013 and regions where the economic output was shrinking heavily. For instance, in the UK the economy declined by more than 10% in two regions and at the same time it grew by around the same amount in North Eastern Scotland  better account for both the local conditions and the country context than most of the past studies which focused on a national level only; (ii) focus on the fertility consequences of changing economic and labour market dynamics rather than simply studying the links between economic conditions and fertility; (iii) investigate whether fertility responses to changing economic conditions strengthened during the recession period; and (iv) assess which changes in economic and employment conditions contributed most to the observed fertility decline during the recession. To this end, we implement a three-level growth curve model, which allows us to account for national and sub-national context and separate the effects of changes in economic conditions on fertility from the cross-regional associations between the two variables.Our selection of data and indicators aimed to provide a wide coverage of European countries and sub-national regions to capture the effects of the changes in local economic conditions on fertility. We used data for NUTS-2 regions, which are roughly equally populated (population 0.8–3 million), and they are the smallest units for which we could access comparable and reliable data on fertility and economic conditions for the analysed countries. As of January 2017, there were 276 NUTS-2 regions in the 28 EU countries including United Kingdom.To measure fertility, we used the TFR, complemented by its age-specific components—cumulative age-specific fertility rates (ASFR) computed for the age groups 15–19, 20–24, 25–29, 30–34 and 35+. These data, published by Eurostat at the NUTS-2 level since 1999 onward, are computed by combining national vital statistics on births by age of mother and official data and estimates on female population by age. While birth registration is complete or almost complete in Europe, the fertility rates may be underestimated and the fertility time series can be distorted by series breaks and jumps in some countries due to incomplete registration of migration  and inconsistent and changing rules on the registration of births to women who left the country. These problems are most pertinent in the countries of Central and Eastern Europe with sizeable outmigration , therefore, our findings for these countries should be interpreted with caution.To measure the changing economic and labour market conditions, we use economic and labour market indicators, which closely reflect the economic cycle and which indeed deteriorated during the Great Recession. The selection of our explanatory variables was limited by data availability and reliability. First, we considered a range of labour market measures which indicate the instability of employment and persistence of joblessness, such as unemployment rate  and the proportion in long-term unemployment (unemployment lasting 12 months or more). These indicators originate from the European Labour Force Survey (ELFS), which is a comparative large-sample survey designed for collecting labour market data of high quality. These data are available in Eurostat Database as annual aggregate indicators provided for the NUTS-2 units for the period since 1999. Unfortunately, the database did not provide data on temporary employment at the NUTS-2 level.Second, we accounted for the proportion of young adults aged 18–24) not in employment, education or training (NEET), also collected in ELFS. The NEET indicator was criticized in the literature for being heterogeneous and consisting not only of the unemployed and disengaged, but also young care providers, persons with health problems and disabilities as well as persons who are actively involved in artistic activities or self-directed training Cavalca . In Nort8–24 not We lagged the economic and labour market indicators by one year.Overall, our sample comprised 251 NUTS-2 regions, nested within 28 countries. In this dataset, fewer than 5% of observations per variable are missing and fewer than 8% are classified by Eurostat as low reliability values. However, variables based on small populations, i.e. the youth unemployment rate and long-term unemployment, contain more missing values  and low reliability values (22–24%) (for details see Appendix 2). In order to minimize the number of missing values, we imputed the data with a cubic spline interpolation. We performed this imputation only if there were no more than four missing values in a row and if they were part of the time series, preceded and followed by known values.All indicators we considered show a sharp deterioration in economic conditions after 2007, especially in 2009. According to the conventional indicator of GDP growth, the economic recession was over by 2013, with more than half of the regions registering positive GDP change. However, the labour market indicators such as unemployment rate and long-term unemployment still increased in most regions in that year. Finally, the TFR trends show a reversal of fertility across most regions after 2007, but at the same time fertility rates did not become more differentiated across regions.Our data have a hierarchical structure. Year-observation units are nested within regions and regions are nested within countries. Using these data, we estimated three-level growth-curve models (GCMs) with regional TFR and ASFRs as dependent variables. The GCM offers a flexible way of modelling change in the dependent variable after accounting for the interdependency of observations nested within higher-level units , and Xi in a country c. Furthermore, the model in Eq. . Fertility response to an increase in long-term unemployment was, however, much weaker than to an increase in overall unemployment rate.Changes in the remaining two economic indicators—the rate of GDP decline and the share of NEETs—were only weakly related to changes in fertility when considered jointly with unemployment rate and long-term unemployment. Stepwise analyses showed that the GDP decline and the change in the share of NEETs were losing their association with fertility after we accounted for unemployment and long-term unemployment. This suggests that worsening of economic conditions is related to fertility mainly through employment-related conditions.In order to evaluate whether the correlation between changing economic conditions and fertility was stronger during than before the recession, we interacted the within-region variation in economic conditions entclass1pt{minimai was increasing (as opposed to remaining stable or declining)Why are the fertility reactions to changing economic and labour market conditions stronger during the economic recession than before? Is it because the relationship between economic conditions and fertility is nonlinear? For instance, Garfinkel et al.  found thOur empirical strategy allows us also to investigate variation in post-2008 fertility effects by country. To this end, we computed the empirical Bayes predictions of the random slopes So far, our analysis has focused on discussing how fertility changes were associated, on average, with a hypothetical 10 pp. change in each of the indicators of economic conditions we considered. Such hypothetical changes often differ widely from the actual dynamics in economic and employment conditions in individual regions. To obtain a more realistic picture of fertility responses to changing economic conditions, we predict total fertility in 2009–2014 under five scenarios, out of which the first four are hypothetical. To this end, we use our model presented in Eq.  with an In the first step, we compare the actual TFR with the TFR predicted under S5 . It turns out that our model predicts the actual TFR very well. The absolute difference between the predicted and actual TFR does not exceed 0.05 in 55% of cases and is smaller than 0.1 for 84% of observations in our sample. The distribution of the average regional absolute differences (Appendix 5) reveals that large differences are not geographically clustered, but are evenly distributed across the continent.Next, we map the maximum difference between predictions from scenarios S1 (no recession) and S5  for all analysed regions in 2009–2014 . In addition, there were relatively large regional differences in recession-related fertility declines in several strongly affected countries, especially Greece, where the recession-induced TFR change ranged from − 0.11 in Ionian Islands to − 0.20 in Crete.Finally, we investigated which economic factors contributed most to fertility decline during the recession. To this end, we compared total fertility predicted under the no-recession scenario to the total fertility predicted under scenarios S2–S5 . We present our findings in Fig. Our findings suggest that fertility decline was associated with an increase in unemployment and in Southern Europe and Latvia, in addition, with economic decline as measured by falling GDP. A different pattern was observed only in Ireland, where fertility decline was largely associated with an increase in long-term unemployment. In Germany, where an increase in fertility was recorded, it was achieved mainly in association with falling unemployment rates during the recession.t-2) in economic indicators in addition to the one-year lag (t-1) incorporated in our model. Results are displayed in Appendix 3 in the online supplementary material. Even though changes in some of the economic indicators at t-2 turned out to be significantly related to fertility (this was mainly the case of unemployment rate), introducing them into the model did not change the significance or the direction of the estimated coefficients at t-1 in the vast majority of cases.t-1 only, displayed a better fit.Our findings have passed several robustness checks. First, we verified whether lagging our economic indicators by one year is sufficient to capture fertility reactions to changing economic conditions. To this end, we introduced a two-year lag .Great Recession had a long-lasting impact on income, employment, health, well-being and family behaviour of Europeans. Our main focus was on investigating how economic and labour market dynamics relate to fertility dynamics while controlling for the variation in economic conditions between regions in each country and between countries. We covered a range of economic and labour market indicators to capture the multifaceted configuration of the economic recession and explore huge sub-national variation in its progression. To that aim, we have incorporated long-term unemployment and the share of young adults who are not in education, employment or training. Our models, including the full set of indicators, provided a close fit to the actual fertility trends in most of the sub-national regions in Europe.The Three general findings are consistent across wider groups of countries and are also in line with the past findings reported at a national level. First, the periods of economic downturns are associated with negative fertility dynamics across the countries and regions analysed. Second, the role of unemployment and long-term unemployment clearly dominates over the other economic indicators studied, which suggests that worsening economic conditions relate to fertility mainly through deteriorating labour market. In particular, changes in unemployment rate showed the strongest link to fertility dynamics across all ages: an increase in unemployment rate by 10 pp was associated, on average, with a decline in total fertility by nearly 0.04 in absolute terms. In addition to unemployment, GDP decline was closely associated with fertility decline in Southern Europe and Latvia. Third, fertility rates were negatively linked with deteriorating economic conditions across the whole range of childbearing ages, including late reproductive ages when women who postpone childbearing may not be able to realize their fertility plans later in life. The latter finding suggests that economic recession might have affected not only the timing of fertility, but also its quantum.We were able to provide a finer picture of the factors associated with fertility dynamics in individual countries and sub-national regions in Europe. Our results suggest that country context played a dominant role in recession-related fertility declines, as fertility change was strongly differentiated by country and in most countries, the regional variation in recession-related fertility change was relatively low. This is in line with large between-country differences in the severity of the economic recession and policy responses to it. Yet, there was notable regional variation in several countries, especially in Greece, as well as broader clusters of similar recession-induced fertility changes (or the lack of them) spanning across country boundaries, especially in Central Europe, where the recession was mild. Our model, which controls simultaneously for sub-national and country effects (regions are nested within countries) and accounts at the same time for the levels and changes in economic/labour market conditions, arguably constitutes an improvement to the models used in the past. We show that across Europe, both between-country and within-country variations in economic conditions are relevant for understanding fertility dynamics. The between-country dimension proved to be especially relevant, in agreement with the results of other recent studies . Moreover, in Central Europe and in the Nordic countries fertility rates were not closely associated with the recession indicators. This suggests that our indicators have tapped into the factors we could not capture in our models and which rose to prominence during the recession. These include rising poverty and economic deprivation, lower income, and also wider structural changes in the labour market characterized by falling opportunities for the middle-income workers and the spread of lower-paid temporary contracts  unemployment. In many countries, an increasing number of people—the emerging class of “precariat”—are facing uncertain lives, moving in and out of low-paid “stopgap” jobs that may give little meaning to their lives  of fertility that will also depress completed family size of women who were in prime reproductive ages around 2010. Even though the declines in fertility of women aged 35+ suggest some lasting influence on fertility levels among women approaching the end of their reproductive lives, these effects are rather small. Second, the range of available economic and labour market indicators is more restricted at a sub-national level, preventing us from delving deeper into different possible links between economic downturn and fertility. In addition, some survey-based indicators turned out to be too uncertain and unstable at the level of NUTS-2 regions. Third, our study does not consider the role of education as a potent moderator of the association between adverse economic circumstances and fertility trends. Clearly, the idea that all social groups are equally vulnerable to economic uncertainty is both logically thin and empirically tenuous Supplementary material 2 (XLSX 46 kb)Below is the link to the electronic supplementary material.


Relationship status is an important factor associated with condomless anal intercourse (CAI) amongst men who have sex with men (MSM).n = 4901) exploring factors associated with CAI via bivariate and multivariate multilevel logistic regression analyses.A multi-centre bio-behavioural survey with MSM was conducted in 13 European cities ; knowing their HIV status ; using substances ; being older ; successful sero-communication ; and, not having a recent HIV test . CAI with steady partners was associated with successful sero-communication ; not having a recent HIV test , and; being older .Understandings of partner type and/or relationship status in relation to CAI amongst MSM can potentially play an important role in the development of culturally appropriate HIV/STI prevention and risk-reduction efforts targeting at-risk MSM. Our results speak to the need to consider segmented and tailored public health and health promotion initiatives for MSM with differing CAI behaviours and relationship profiles. Indeed, research demonstrates that relationship status and/or partnership type is an important factor associated with condomless anal intercourse (CAI) and subsequent risk for HIV and sexually transmitted infections (STIs).10 For instance the number of partners as well as knowledge of own and partner’s HIV serostatus, the use of ‘negotiated safety’ agreements,11 serosorting, and the effective use of anti-retroviral drugs to lower viral load as well as the use of pre-exposure prophylaxis (PreP)13 may be contingent on the perception of the type of relations.However, the risk for HIV and other STI acquisition is dependent on other factors than just CAI. Kramer and colleagues have drawn attention to this issue and note that although prevention initiatives commonly target individualistic-behaviours thus regarding CAI as an inherently ‘risky’ sexual behaviour, such approaches can be unhelpful as they may mask more complex and dynamic issues occurring within MSM in both steady and casual or non-steady relationships including the use of risk-reduction strategies.Nevertheless, examining explanatory factors related to differences in CAI between MSM in steady and casual relationships can be important in order to interrogate segmented public health and health promotion initiatives for MSM with differing sexual behaviour and relationship profiles. Consequently, in this present analysis we utilize data from the EU-funded Sialon II study which was a large multi-centre biological and behavioural cross-sectional survey of MSM in community settings carried out across 13 European cities. The objectives of our analysis were to: (i) investigate CAI and explanatory variables amongst MSM in a large community sample; (ii) explore the differences in CAI between those participants who had steady partners with those who had casual or non-steady partners, and finally; (iii) potentially inform the development (and assist implementation) of risk-reduction initiatives targeting MSM.16 In summary, the Sialon II study was a complex multi-centre integrated bio-behavioural cross-sectional survey with a concomitant collection of behavioural data and biological data .Detailed study methods are reported elsewhere.n = 3596). TLS is a quasi-probabilistic method used to recruit members of a target population at specific times in set venues.17 In this study, the venues or settings for data collection included social and/or commercial venues and cruising settings preliminarily identified through formative research and which were then selected randomly for data collection sampling calendars.18 RDS was used in Bratislava, Bucharest, Verona, and Vilnius (n = 1305). RDS is similar to snowball sampling in that it requires the target population to be socially networked so participants can invite their peers to participate. However, RDS is different in that it incorporates numerous theoretical assumptions to reduce the numerous biases found in standard snowball sampling methods (see19). Enrolment for RDS in Sialon II was based on the individuals’ social network and for the data collection, locally accredited healthcare facilities  were used. In TLS cities, participants were recruited during 2013, whilst in RDS cities recruitment started in 2013 and finished in 2014. Prior to the survey we estimated a 50% response rate as part of the sample size calculations. A data collection procedure to record refusals was therefore developed for TLS only. However, not all sites collected this data (with exception of the Brighton site with a 59% response rate). Thus an overall response and/or refusal rate for the TLS survey is not reported.The survey was implemented in 13 European cities. The decision to use Time-Location Sampling (TLS) or Respondent Driven Sampling (RDS) to recruit participants was based on preliminary formative research. TLS  was used to recruit participants in Brussels, Sofia, Hamburg, Warsaw, Lisbon, Ljubljana, Barcelona, Stockholm, and Brighton (Participants were men present in the cities at the moment of data collection (2013–14) who met the inclusion criteria . Exclusion criteria were being younger than the legal age of consent (18 years old) or having already participated in the study.20 and previous EC-funded European projects (e.g.22) and then piloted amongst MSM in each study site. The English version of the questionnaire was translated into local languages and back-translated into English.A self-administered pen-and-paper questionnaire was used to collect behavioural data. The preliminary version of the questionnaire was designed by the Sialon II network in line with the Global AIDS Monitoring indicators (GAM)Research protocols were submitted to, and approved by, an institutional ethical review board in each participating city, as well as by the WHO Research Project Review Panel (RP2) and the WHO Research Ethics Review Committee (ERC). All participants were given a study information sheet and the details were read out to ensure they understood what the study involved, that participation was voluntary, and that they had the right to withdraw at any time without giving a reason. Those willing to take part then signed a consent form. For TLS and RDS, a dedicated barcode system was used in order to link anonymously the different types of data collected . For the TLS survey, respondents who wanted to collect their tests results could do so using their unique bar code ID. For the RDS survey where respondents were tested directly in a hospital/clinical setting, test results were available according to the local standards (including pre and post-test counselling).The primary focus of this analysis was to explore engagement in CAI measured as insertive/receptive unprotected anal intercourse in the last 6 months. Since we initially expected the ‘risk’ behaviour for those engaging in CAI in the last 6 months to differ depending on the relationship status , two separate ‘primary’ outcomes were created for two separate analyses. The first outcome indicated whether an individual had engaged in CAI with one or more (yes = 1) or zero (no = 0) steady partners. This included ‘boyfriends’ and ‘husbands’ (i.e. not being ‘single’) and excluded partners who were ‘sex buddies’. The second outcome indicated whether individuals had engaged in CAI with one or more (yes = 1) or zero (no = 0) casual partners. Casual partners were defined as: those with whom one had had sex with only once (e.g. a ‘one-night stand’); and those with whom one had sex with more than once but were not considered a steady partner (such as sex buddies). Some participants categorized current relationships as a mix of casual and steady partners since the two categorizations are not mutually exclusive.23Independent variables included: age (based on self-reported year of birth), education level , migrant status , ‘outness’ , overall perceived attitude towards gay or bisexual people at work/school and amongst parents/friends/acquaintances , HIV testing in the last 12 months and results known (no or yes), knowledge of own HIV status , sex role at last anal sex , number of substances (type specified in the questionnaire) used at last anal sex , frequency of visits to gay venues during last 3 months where sex-on-premises is possible , currently having sex with women (no or yes), serostatus communication at last anal intercourse , see.For continuous variables median and interquartile range (IQR) were used. For nominal variables count and percentages were used. The Chi-square test was used to examine the relation between CAI in casual partners and CAI in steady partners as well as to compare CAI rates between pairs of cities.For all bivariate and multivariate analyses, factors associated with CAI were identified using a two-level multilevel logistic regression model with a random intercept at the city level. The random component accounts for the hierarchical nature of the data. Analyses were carried out on all available cases.P < 0.05) variable first. The likelihood ratio test was used to compare the new model with the nested model. For all statistical tests, significance was indicated by P < 0.05. The final model estimated the adjusted odds ratios (AORs) and the corresponding 95% confidence interval (95% CI) for factors associated with CAI. We then used the resulting model to explore the relationship between age and risk of engagement in CAI for each city. Analyses were first carried out for modelling CAI with casual partners and then repeated for steady partners. Stata® Version 13 was used for all analyses .The first step to building a model was to identify those individual independent variables (from the full list above) that were statistically significantly associated with CAI using bivariate analysis. Variables from this pool of potential risk factors were then used for inclusion in the multivariate analysis. The variables were added to the null model one by one using a forward selection process choosing the most significant (P < 0.001) between participant reports of CAI with casual partners and CAI with steady partners. Those who had CAI with steady partners had 2.73 times higher odds of CAI with casual partners (odds = 0.862) compared to those who did not have CAI with steady partners (odds = 0.316).Of 4901 participants who completed the survey, 4340 (88.55%) had sex in the last 6 months and were included in the analysis. The median age was 32 years with an IQR of 15 years. 3624 (83.50%) had at least one casual partner, 2911 (67.07%) had at least one steady partner and 2195 (50.58%) had both. 1374 (31.66%) participants reported CAI with casual partners  and 1482 (34.15%) with steady partners  and 687 (15.83%) reported CAI with both types of partner . Median age for the 2171 (50.02%) who did not have CAI with casual or steady partners was 33 years (IQR 16 years). There was also a significant association (P = 0.001). Barcelona saw the lowest percentage rate of CAI with steady partners (23.81%) whilst Vilnius (40.34%) had the highest (P < 0.001). Table CAI varied between cities and by relationship status Table . Brusselpartners 3.81% whiP = 0.047); who knew their HIV status ; who used 1–2 substances ; and, who used two or more substances . Being older ; having successful sero-communication ; and, not having had a recent HIV test , were all associated with reductions in the likelihood of CAI.The results from the multivariate analyses are shown in Table P < 0.001) and for those who had not been tested for HIV in the last 12 months . It was also approaching significance for those who reported being out to a majority . Reduced likelihood of CAI with a steady partner was associated with increasing age for all cities .With reference to the multivariate analyses in Table P = 0.43 and this drops to P = 0.19 for older (78 yrs) MSM; Vilnius had the lowest probabilities and estimates ranged from P = 0.38 (18 years) to P = 0.16 (78 years). For steady partners, again Brighton has the largest estimated probabilities of P = 0.43 (18 years) and P = 0.26 (78 years); Bucharest had the lowest probabilities ranging from P = 0.35 (18 years) to P = 0.20 (78 years). Amongst MSM with steady partners, Barcelona, Brighton, Brussels, Hamburg, Lisbon, Ljubljana and Stockholm can all be grouped together as cities with consistently higher probabilities of CAI at each age; similarly Barcelona, Brighton, Brussels, Hamburg, Sofia and Stockholm all had higher probabilities at each age for CAI in casual partners compared to the other study sites.Figure A number of factors were associated with increased likelihood of CAI between MSM with casual partners including being ‘out’ to a majority, knowing one’s own HIV status, and using substances. Reductions in the likelihood of CAI were associated with being older, as well as successful sero-communication, and not having had a recent HIV test. Being older may be related to having experienced more intensive condom promotion and having witnessed the severe consequences of historically untreatable HIV infection. In terms of successful sero-communication: with casual partners sero-communication may be a surrogate for HIV-related concerns and higher intentions of self-protection, while sero-communication with steady partners may serve to confirm HIV sero-concordance and successful serosorting and to allow more ‘intimacy’ by practising CAI.For those with at least one casual partner, having sex with a female and being a migrant were not associated with the likelihood of CAI. Similarly for MSM with at least one steady partner, the likelihood of CAI was positively associated with successful sero-communication and not having had a recent HIV test within the last 12 months; it was also negatively associated with increasing age. Interestingly, regardless of partner type, our analysis indicated a downward trend in the probability of CAI with increasing age. The gradual declining trend, and smaller 95% confidence intervals at the margins, indicated that relationships amongst steady partners are more stable whilst casual partners are more variable. These data suggest that regardless of partner type, prevention strategies may benefit from disproportionately targeting younger MSM.26 Concurring with our own findings, prior studies have also found significant associations between CAI and age with younger MSM seemingly more likely to engage in CAI with steady partners.24 In our study this was also the case although irrespective of partner type.Previous studies have identified associations between CAI between MSM and relationship status.13 Future studies may therefore wish to consider the importance of understanding the complex dynamics of partner type/relationship status for the prevention of other STIs as well as considering how CAI behavioural stratification could be used to determine who might benefit from tailored health promotion interventions including HIV PrEP.Of potential relevance to our analysis, a recent study from Australia has shown that a rapid increase in pre-exposure prophylaxis (PrEP) use by gay and bisexual men in Melbourne and Sydney was accompanied by an equally rapid decrease in consistent condom use with casual partners.Understandings of how partner type or relationship status may shape sexual behaviour such as CAI amongst MSM in European cities may help to play an important role in the development of culturally appropriate HIV/STI prevention and risk-reduction efforts targeting at-risk MSM. Our findings indicate the need for further investigation on how partner type and other partnership characteristics and dynamics may influence CAI and HIV and/or STI transmission amongst MSM.27 However, TLS and RDS do nevertheless still represent one of the main and current approaches for recruiting most at-risk populations to bio-behavioural surveys.28 Survey data can of course be subject to specific biases related to the fact that some data were self-reported (excluding the data on HIV status when based on laboratory testing) limiting generalisability. This implies recall and social desirability bias given behaviours such as CAI were explored. The questionnaire has however been designed to overcome these potential biases, for instance through the active involvement of local gay NGOs in each site.29 It is also possible that although we provided descriptions of different partner types in the survey, variations regarding the interpretation of what constitutes a ‘steady’ versus a ‘non-steady/casual’ partner might not be uniform across study participants (e.g. see7).Due to the cross-sectional nature of the study design, no causality or temporality between the associations examined can be inferred. An important limitation relates to the sampling methodology. TLS and RDS methods are considered quasi-probabilistic approaches, targeting MSM through their attendance in gay venues (TLS) or via social networks (RDS). This means that such approaches are subject to specific shortcomings such as the possible over- or under-representation of potential MSM sub-samples.Finally, as an EC co-funded project, the Sialon II project was designed to include cities from countries with different social and cultural contexts. As in many such EC-funded projects, cities were selected on the basis of previous research and collaboration networks and on the basis of pragmatic financial/organisational issues; therefore, some key cities with sizable gay populations have not been covered by the survey.Despite the above limitations however, our analysis provides important information regarding the association between CAI and partnership characteristics amongst MSM in 13 European cities.