In vitro, betamethasone decreased lymphocyte viability and induced maturation-resistant dendritic cells, which in turn impaired γδ T cell proliferation and decreased IL-17 production. Prenatal betamethasone exposure caused thymus hypotrophy in newborn mice as well as alterations in immune cells subsets. Furthermore, betamethasone decreased β-cell growth, reduced C-peptide secretion and altered the expression of genes related to autoimmunity, metabolism and islet mass in T1D target tissue. These results support the protection against T1D in the betamethasone-treated offspring and demonstrate that this drug alters the developing immune system and β-cells. Understanding how betamethasone generates self-tolerance could have potential clinical relevance in T1D.Non-genetic factors are crucial in the pathogenesis of type 1 diabetes (T1D), a disease caused by autoimmunity against insulin-producing β-cells. Exposure to medications in the prenatal period may influence the immune system maturation, thus altering self-tolerance. Prenatal administration of betamethasone –a synthetic glucocorticoid given to women at risk of preterm delivery– may affect the development of T1D. It has been previously demonstrated that prenatal betamethasone administration protects offspring from T1D development in nonobese diabetic (NOD) mice. The direct effect of betamethasone on the immature and mature immune system of NOD mice and on target β-cells is analysed in this paper.  T1D is usually diagnosed during childhood or adolescence, and its incidence is steadily increasing an average of 4% per year2. Many autoimmune diseases display a similar increase in incidence, but so far, there is no explanation for this trend. The challenge in finding a cure is based on the heterogeneity of mechanisms that ultimately lead to the development of autoimmune reactions. For this reason, a better understanding of the influence of non-genetic factors in T1D susceptibility is needed.Type 1 diabetes mellitus (T1D) is a metabolic disease caused by the destruction of pancreatic β-cells by autoreactive T lymphocytes3 and other autoimmune diseases. Recently, special attention has been focused on pre- and perinatal factors. An indirect evidence of their contribution in this matter is that dizygotic twins show greater incidence of T1D (15–25%)5 than non-twin siblings (5–10%)6. The age of the mother is also relevant to the increasing incidence of T1D7. Moreover, the greater number of alloreactive microchimeric maternal cells in autoimmune diseases8 and specifically in the pancreas from patients with T1D can be related to the pathogenesis of the disease9. More recently, a preprimed CD4+ T cell population with capacity to respond immunologically to β-cell antigens was found in newborns, and these cells seem to be generated by an inefficient antigen presentation in the first months of life10.Predisposition to this disease relies mainly on genetic and environmental factors. Changes in the environment in the last decades have critically contributed to the increase in T1D incidence11. Betamethasone –a synthetic glucocorticoid widely used in obstetrics– induced foetal lung maturation, and significantly reduced mortality and morbidity in preterm newborns12. This drug easily reaches the foetus because it is not affected by inactivation by the placental enzyme 11β-hydroxysteroid dehydrogenase 213. Moreover, betamethasone also targets the ubiquitous glucocorticoid receptor, which is present in almost all cell types, including leukocytes, and may therefore affect the development of the immune system13.Considering the pivotal relevance of the prenatal time window and how extrinsic factors can determine the development of autoimmunity/T1D in later life, the use of medical interventions during this period can alter the immune response in adult life. In a spontaneous T1D experimental model, the non-obese diabetic (NOD) mouse, we have recently shown that prenatal administration of betamethasone reduced T1D incidence in the offspring and changed the TCR repertoire+ T cells and Vβ6 in CD8+ T cells) was reflected in the reduced immune cell infiltration in the pancreas and contributed to protection from T1D in the NOD mice11. These results encouraged us to analyse in depth the effects of prenatal betamethasone.One of the effects of betamethasone on T lymphocytes is the alteration of the T cell-receptor (TCR) repertoire. It has been previously demonstrated that prenatal betamethasone treatment results in a biased TCR repertoire, affecting the TCR (Vβ) relevant for T1D antigen recognition. In this scenario, the lower frequency of pathogenic TCRs  of 0.9887  from healthy donors. The results show that betamethasone affects leukocyte viability in vitro starting at 105 nM  and B cells (CD19+).To assess if this drug affects human leukocytes viability, we tested the In summary, betamethasone has a toxic effect on murine lymphocytes, and in human leukocytes, but at higher concentrations.+ cells . Betamethasone improved DCs viability after LPS maturation in comparison to untreated mature DCs (mDCs)  when compared to mDCs  CD3+ T cells was lower in 100betDCs condition when compared to mDCs condition  stained splenocytes from NOD mice. Although no differences were found in the percentage of B lymphocytes, the percentage of CD3ion Fig. , even th nM Fig. , demonst nM Fig. . In summin vivo effect of betamethasone in primary and secondary lymphoid organs, the weight of the thymus and spleen after birth, at day 1 (d1) and day 4 (d4), were determined. As expected, the weight of the thymus was lower in the betamethasone treated group when compared to the sham group (Δincrease = −62.05%) at d1 , reflecting a recovery, as reported14. According to these data, the number of thymocytes obtained after mechanical disruption was significantly lower in the betamethasone group at d1 when compared to the sham group. At d4, the number of thymocytes was similar in both groups.To assess the  d1 Fig. . At d4, −CD8+, CD8ISP) were the ones overrepresented in the betamethasone-treated samples at d1, while DN3 and DN4 percentages were maintained. At d4 after birth, the DN subpopulations were similar in treated and non-treated mice, except for the CD8ISP subset, which was significantly reduced in the betamethasone group.The effect of betamethasone in the cellular composition of the thymus after birth, at d1 and d4, was then analysed. Regarding thymocyte maturation, the percentages of thymocyte subsets were altered by prenatal betamethasone administration Fig. . As expe+CD4+ T cell subset. As expected, the percentage of this subset increased at d4 when compared to d1, both in treated and control mice. In contrast, betamethasone hindered the increase in the percentage of CD3+CD8+ T cell subset observed at d4 in the control group. Finally, betamethasone increased the percentage of γδ+ T lymphocytes at d1, recovering normal values at d4. A t-SNE analysis, performed with the data obtained from flow cytometry, show the composition of the thymocyte compartment at d1 and d4 in treated and sham mice  were examined Fig. . No effeice Fig.  in the bincrease = −16.32% at d1 and Δincrease = 21.5% at d4)  Fig.  while th+) was detected in the betamethasone group at d1 when compared to the sham group, but it subsequently normalised at d4. The TCRαβ+ T cell subset was reduced in percentage in the treated group at both time points. Likewise, the percentage of putative lymphoid tissue inducer cells  was reduced in the treated group at d1 and d4 when compared to the sham group. Due to the very low amount of T cells in spleen at d1, the T cell compartment was no further differentiated. At d4, no differences were found when comparing the CD4+ subset between the two groups, CD8+ cells were significantly decreased, and a compensatory increase in CD4−CD8− in the betamethasone-treated group was observed. Similarly to the previous thymus analysis, a t-SNE analysis of the spleen was performed, including lymphoid , Cd44, Cd14, Ccl2, Il22ra1, Cxcl2 (related to autoimmunity), and Tcf7l2, Pcsk1, Pten, Igfr1, Ptprj (related to metabolism and islet cell mass).To determine putative changes in gene expression induced by betamethasone, 13 genes known to be related to T1D were selected: Gad1, Ccl2, Cxcl2, Cd14, Il22ra1, Tcf7l2, Pcsk1, and Ptprj and a significant downregulation of Cd44 were observed in NIT-1 cells  caused fewer gene expression changes  genes through the effect of IL-2248. Again, the absence of the ligand in culture conditions could prevent further expansion.The phenotypic and functional effects of betamethasone was accompanied by changes in the expression of genes related to autoimmunity, metabolism and islet mass, both in NIT-1 cells and islets such as a downregulation of Ccl2 gene in T1D target tissue. This chemokine is involved in the recruitment of monocytes, macrophages, DCs, and activated T-cells, thus leading to islet destruction49 or tolerance50. Ccl2 gene was downmodulated in betamethasone-treated islets and in the pancreas of prenatally-treated pups, correlating with the reduction of destructive insulitis. The increased Ccl2 gene expression observed in NIT-1 cells can be due to its tumoral origin. Another important chemokine in T1D is Cxcl2. Cxcl2 gene was overexpressed by betamethasone in the three different substrates, indicating an increase of neutrophil recruitment to T1D target tissue. Neutrophils have an important role in β-cell destruction when accompanied by T cells in the microenvironment51. However, the here reported data suggest that T cell recruitment is halted by Ccl2 downmodulation. In this scenario, and because apoptotic islet cells removal by neutrophils has anti-inflammatory effects, neutrophils would contribute to reduce inflammation52. This alteration fits well with the increased percentages of splenic DCs and neutrophils after betamethasone treatment.Betamethasone also altered the expression of Gad1 gene –codifying for GAD67 autoantigen53– was strongly upregulated by betamethasone. It has been reported that glucocorticoids upregulate GAD6754. This is the most expressed isoform of GAD. The other isoform, GAD65 is a target autoantigen of very early autoimmune attack in NOD mice55 that is not affected by betamethasone. The role of GAD67 is less clear but immunization of prediabetic NOD mice with GAD67 prevents the onset of diabetes56. The here reported hyperexpression of Gad1, toghether with thymocytic apoptosis, suggests immunomodulatory effects.Furthermore, It has been previously demonstrated that prenatal administration of betamethasone causes changes in the TCR repertoire influencing the outcome of autoimmunity. The results from this study confirm that other relevant cell types in T1D are affected. Since NOD strain is diabetes-prone, the alteration of the immune system cells disrupts this predisposition. Moreover, the additional effect of betamethasone in reducing β-cell immunogenicity may explain the prevention of the disease by this drug.57 according to the route of administration . The results showed a weak increase of the risk of developing T1D in the offspring, but the effects of each corticosteroid was not individually analyzed in this study. We found no specific studies that addressed the effect of the administration prenatal betamethasone in the development of T1D. Further studies should be performed in children prenatally exposed to betamethasone between the week 24 and 34 of pregnancy58 to determine the prevalence of autoimmune diseases.Since autoimmune diabetes is different in NOD mice and in humans, it would be interesting to analyse the effects of prenatal administration of betamethasone in children: whether it has the same protective effect or it predisposes to autoimmunity. In this sense, a study performed in a Danish cohort of children studied the effects of the prescription of several corticosteroids to pregnant women59, the prenatal immunological remodelling makes the immune system very susceptible to drugs. In fact, it has been reported that prenatal treatment with betamethasone has immunomodulatory effects in newborn infants in terms of acceleration of NK activity, decrease of T cell proliferation, and inappropriate cellular immune responses60. These effects last for weeks, and may be harmful for the newborn. Concerns have been raised about the effects of prenatal betamethasone exposure on the developing immune system. In this sense, the amount of betamethasone administered to pregnant women (2 doses of 12 mg) corresponds approximately to 900 nM (for 70 Kg of body weight), and this concentration has no in vitro deleterious effect in human leukocyte viability, suggesting that betamethasone is not detrimental for human leukocytes in prenatal treatment.Taking into account that the immune system of the newborn is developingGiven the increase in the incidence of allergies and autoimmune disorders in the last years, elucidating the mechanisms of action of prenatal glucocorticoids will allow the design of prevention strategies. In this sense, the results of this study show that the effect of betamethasone on the developing immune system influences the susceptibility to experimental T1D, by direct effect to immune system cells and to pancreatic β-cells. Understanding the effect of betamethasone in self-tolerance could have potential clinical relevance in T1D.Wild-type NOD mice, genetically prone to spontaneously develop autoimmune T1D, were obtained from The Jackson Lab  and kept under specific pathogen-free conditions. T1D incidence in NOD females was 67%. Mice were mated and the presence of a vaginal plug was considered day 0.5 of pregnancy (E0.5). On day 20 (E20) mice were i.p. injected with 0.1 mg betamethasone  in Phosphate Buffered Saline  or vehicle control . Betamethasone (Sigma-Aldrich) was diluted in ethanol  at 5 mg/mL. For further dilution, PBS (Oxoid Limited) was used.Newborn pups prenatally treated with betamethasone and controls (4 mice/group) were euthanised at days 1 and 4. Pancreas was removed at day 1 (48 h after betamethasone administration) and snap frozen in an isopentane/cold acetone bath for RNA isolation.61. Pancreases were removed and incubated at 37 °C for 30 min. After digestion, islets were hand-picked and groups of 10–20 islets were cultured. Culture medium (CM) was RPMI-1640  with 10% Foetal Bovine Serum , 100 U/mL penicillin , 100 μg/mL streptomycin , 2 mmol/L glutamine (Sigma-Aldrich), 1 mmol/L sodium pyruvate (Gibco), and 25 μmol/L β-mercaptoethanol (Sigma-Aldrich).To isolate islets, pancreases from NOD mice were perfused with collagenase  through the common bile duct62, was chosen because of its similarity to β-cells in terms of autoantigens  and cultured in CM. Viability was assessed by staining with annexinV (AnnV) PE  and 7-amino-actinomycin D (7aad) labelling  and analysed with FACS Canto II .NIT-1 cell line, established from NOD/Lt miceTo assess the changes induced by prenatal betamethasone in leukocyte subsets of newborn mice, thymi and spleens of pups euthanised at d1 and d4 were harvested and weighed. Single-cell suspensions were obtained by mechanical disruption. Cells were stained for flow cytometry  analysis with anti-CD3e eFluor 450 , anti-CD4 APC (BD Biosciences), anti-CD4 APC-Cy7 (eBioscience), anti-CD8α PE-Cy7 (eBioscience), anti-CD11b APC (eBioscience), anti-CD11c PE-Cy7 (BD Biosciences), anti-CD19 V450 (BD Biosciences), anti-CD25 PE , anti-CD44 APC (eBioscience), anti-CD45 APC-Cy7 (eBioscience), anti-TCRγδ chain PerCP-Cy5.5 (BioLegend), anti-TCRαβ chain PerCP-Cy5.5 (BioLegend), anti-Ly6g PerCP-Cy5.5 (BD Biosciences) and Pacific Orange dye  for viability. Data were manually analysed using FlowJo software . The dimensionality reduction algorithm t-SNE (t-distributed stochastic neighbour embedding) in R, using the CRAN package “Rtsne”, was used for direct comparison of treated and untreated samples.6 cells/mL with 0.1 nM, 1 nM, 2.5 nM, 5 nM, 10 nM, 50 nM, 100 nM or 1000 nM betamethasone or the corresponding controls (PBS). Cell viability was determined as abovementioned at 24 h using flow cytometry. Human PBMCs were obtained from 10 mL blood sample by means of Ficoll Paque  density gradient centrifugation. PBMCs were cultered at 106 cells/mL with 0.1 nM to 106 nM betamethasone. After 48 h, cell viability was determined in PBMCs using flow cytometry as detailed above, and three leukocyte subsets were analyzed using anti-CD3 APC, anti-CD14 APC-Cy7 and anti-CD19 PE (Immunotools).To study the effect of betamethasone, spleens from prediabetic NOD mice (8–10 weeks of age) were harvested and single-cell suspensions were obtained by mechanical disruption. After haemolysis, cell number and viability were determined by AnnV PE (Immunotools), 7aad (BD Biosciences) and Perfect Count Microspheres  using flow cytometry . Splenocytes were cultured at 106 cells/mL in CM with 1000 IU/mL mGM-CSF, and 50% of culture media was replaced on days 3, 5, 7 and 8 with CM supplemented with 10% bovine foetal serum (Gibco), 2 mmol/L glutamine (Sigma-Aldrich), 1 mmol/L sodic pyruvate (Gibco) and 2000 UI/mL mGM-CSF (Prospecbio). Betamethasone was added at increasing concentrations  at days 3–7. On day 8, DCs were cultured with insulin (Sigma-Aldrich) at 20 µg/mL for 2 h and then matured with 100 ng/mL LPS (Sigma-Aldrich) for 24 h or 1 µg/mL CpG  for 48 h. Untreated DCs were considered iDCs.DCs were derived from bone marrow precursors after 7 days of culture with mGM-CSF , with or without betamethasone (Sigma-Aldrich) from day 3 until the end of the differentiation protocol. Bone marrow was obtained from femur and tibia bones. After haemolysis, cell number and viability were determined by AnnV PE (Immunotools), 7aad (BD Biosciences) and Perfect Count Microspheres (Cytognos SL) using flow cytometry. Wells were seeded with 10To determine the phenotype, iDCs, mDCs, and mDCs generated with betamethasone at increasing concentrations  were harvested with accutase (eBioscience), stained with anti-CD11c PE-Cy7 (BD Biosciences), anti-CD25 PE (eBioscience), anti-CD40 APC (BD Biosciences), anti-CD86 PE (eBioscience), anti-Class I MHC eFluor 450 (eBioscience), anti-Class II MHC APC (eBioscience), AnnV PE (Immuntools), and 7aad (BD Biosciences), and analysed by flow cytometry  using FlowJo software (Tree Star Inc.). Corresponding fluorescence minus one staining was used as control.5 splenocytes were seeded and cultured with 1 nM, 5 nM, 25 nM betamethasone. To assess the proliferation capacity of splenocytes, 25 ng/mL PMA (Sigma-Aldrich) and 250 ng/mL Ionomycin  were added. After 3 days, the cells were harvested and stained with anti-CD3 V450 (BD Bioscience), anti-CD19 PE (BD Bioscience) and 7aad (BD Bioscience) to assess viability and lymphocyte subsets by flow cytometry .To determine the effect of betamethasone on lymphocyte proliferation, splenocytes from NOD mice were stained with 0.31 µM CFSE (ThermoFisher Scientific) and 2 × 105 splenocytes:104 DCs) for 4 days; CM and 25 ng/mL PMA plus 250 ng/mL IO were used as negative and positive controls, respectively. Cells were stained with anti-CD3 V450 (BD Biosciences), anti-CD4 APC-Cy7 (BD Biosciences), anti-CD8 V500 (BD Biosciences), anti-CD19 PE (BD Biosciences), anti-TCRγδ PE-Cy7 (BioLegend), and 7aad (BD Biosciences) to assess the viability and the different lymphocyte subsets by flow cytometry . Culture supernatant was collected and frozen at −80 °C until use. The concentration of IL17A after the co-culture of splenocytes with DCs was assessed using a Ready-Set-Go IL17A ELISA kit (Invitrogen).To determine the tolerogenic capacities of DCs generated under the effect of betamethasone, CFSE-stained splenocytes were co-cultured with autologous iDCs, mDCs, and with mDCs differentiated in the presence of increasing concentrations of betamethasone at a 10:1 ratio  for 2–10 days. CM was replaced every 2-3 days.5 NIT-1 cells were cultured for 2 days with graded concentrations of betamethasone (up to 10 μM) or CM. Cells were harvested using 0.05% trypsin EDTA (ThermoFisher) and stained for anti-CD44 BV786 (BD Biosciences), anti-Class I MHC eFluor 450 (eBioscience), AnnV PE (Immunotools) and 7aad (BD Biosciences). Median Fluorescence Intensity (MFI) and viability were assessed using flow cytometry . Corresponding fluorescence minus one staining was used as control. Data were analysed using FlowJo software (Tree Star Inc.). Culture supernatant from NIT-1 cells was frozen at −80 °C until use. Insulin secretion was assessed by measuring C-peptide concentration in the supernatant by ELISA .At least 5 × 10Ins2 (Mm00731595_gH), Gad1 (Mm04207432_g1), Gad2 (Mm00484623_m1), Cd44 (Mm01277161_m1), Ccl2 (Mm00441242_m1), Cxcl2 (Mm00436450_m1), Ptprj (Mm00501277_m1), Pten (Mm00477208_m1), Igf1r (Mm00802831_m1), Cd14 (Mm00438094_g1), Pcsk1 (Mm00479023_m1), Tcf7l2 (Mm00501505_m1), Il22ra1 (Mm01192943_m1). Relative quantification was performed by normalising the expression for each gene to that of the housekeeping gene GAPDH (Mm99999915_g1), as described in the 2−ΔCt method63. Values from NIT-1 and islets were normalised using their respective CM controls. The genes were selected using a gene-interaction database (Harmonizone)64 of glucocorticoid receptor, the list of genes differentially expressed in T1D patients65 and other β-cell specific genes66.To determine the effects of betamethasone on transcriptome, quantitative RT-PCR was performed. Briefly, RNA was isolated from NIT-1 cells, purified islets and whole pancreases using RNeasy Micro Kit , and was reverse-transcribed with a High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). The cDNA synthesis reactions were carried out using random hexamers  and reverse transcriptase Moloney-murine-Leukaemia-virus . For purified islets, targeted cDNA was pre-amplified prior to quantitative real-time PCR with TaqMan PreAmp Master Mix (ThermoFisher Scientific). Quantitative RT-PCR assays were performed by TaqMan universal assay (ThermoFisher Scientific) on a LightCycler® 480  using the following TaqMan assays: Statistical analysis was performed using Prism 7.0 software . Analysis of variance (Friedman’s test) was used for comparisons with several factors using a post-hoc Dunn’s test for the comparison between groups. For unpaired data, a non-parametric Mann-Whitney test was used; for paired data, non-parametric Wilcoxon test was used. A sigmoidal non-linear regression was used for IC50 assessment.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Generalitat de Catalunya, Catalan Government. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Germans Trias i Pujol Research Institute (Permit DAAM 8948) and has followed the principles outlined in the Declaration of Helsinki for animal experimental investigation. For human samples, all subjects gave written informed consent in accordance with the Decaration of Helsinki and the study was carried out after the approval and in strict accordance with the guidelines of Germans Trias i Pujol Research Institute and Hospital Ethical Committee.Supplementary Figures


Listeria monocytogenes is a pertinent target for microbiological safety and shelf-life; due to its capacity to multiply in a broad range of food environments, is extremely complicated to prevent in fermented-sausage-producing plants. The frequent detection of L. monocytogenes in final products emphasizes the necessity for the producers of fermented sausages to correctly overcome the hurdles of the technological process and to prevent the presence of L. monocytogenes by applying novel control techniques. This review discusses a collection of recent studies describing pressure-induced elimination of L. monocytogenes in fermented sausages produced in the Mediterranean area. Although conventional microbial control techniques are currently employed and largely successful, their major drawbacks are related to their effects on quality of processed food. In recent years, there has been a growing demand for high-quality foods that are microbially safe and retain most of their natural freshness. Therefore, several modern and innovative methods of microbial control in food processing have been developed. High-hydrostatic-pressure (HHP) processing technology has been mainly used to enhance the food safety of ready-to-eat (RTE) products as a new pre-/post-packaging, non-thermal purification method in the meat industry.  In recent years, food business operators (FBOs) have been repeatedly required to meet the request of consumers for more unadulterated, preservative-free, and less-processed food products, without losing their microbiological quality and safety . AlthougHHP essentially involves the application of isostatic pressures, equally and immediately transmitted to food products by air-driven pumps through a liquid . HHP proClostridium botulinum. Furthermore, in fermented meat products, nitrites and/or nitrates are well known for their antagonistic outcomes against the main foodborne pathogens. [3 for traditional products without addition of nitrites, or 150/150 ppm NaNO3/NaNO2 in different cases [HHP has primarily been used in the ready-to-eat (RTE) meat products industry as a new pre-/post-packaging, non-thermal decontamination method . The impthogens. ,12,13,14nt cases . TherefoL. monocytogenes in several meat products [L. monocytogenes. The effects are related to food composition, time of submission, amount of pressure, and existence of antimicrobial substances [Numerous studies have investigated the use of HHP to avoid the presence of products ,22,23,24bstances ,26.L. monocytogenes is a small, non-spore-producing, permissive, anaerobic, Gram-positive rod. Peritrichous flagella enable a characteristic whirling mobility at 20–25 °C. In relation to the presence of somatic (O) and flagellar (H) antigens, thirteen serogroups have been determined: 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7. L. monocytogenes is catalase-positive, oxidase-negative, and grows between 0 and 45 °C, with an optimum temperature at 30–37 °C.w) of 0.92. L. monocytogenes is ubiquitous in the ecosystem; the main contamination sources are soil, forage, and water [L. monocytogenes can multiply at low temperatures, survives under modified atmospheres, and adheres to several surfaces, adapting to disinfectants [L. monocytogenes can resist NaCl (up to 12%) and nitrates, which are commonly lethal to other bacteria [The pathogen survives at pH values between 4.5 and 9.0 (optimum pH 6–8), and can grow in foods with a water activity ; Lineage II consists of isolates associated with sporadic human cases ; and Lineage III includes isolates hardly ever connected with listeriosis outbreaks or sporadic episodes. Most of the listeriosis outbreaks or sporadic episodes are known to be food-borne, and have an impact on the central nervous and circulatory systems and the gravid female uterus. Two types of human listeriosis have been reported, with symptomatology which includes febrile gastroenteritis in healthy people [y people , and sepy people ,41. Listeriosis is the fifth most common zoonotic disease in the European Union, with an annual incidence of 0.48 cases per 100,000 population and 2480 confirmed cases mostly acquired domestically . The higThe highest infection degrees have been outlined in elderly people, mainly aged above 84 years . ListeriL. monocytogenes, combined with the harshness of human listeriosis, give the presence and the enumeration of the pathogen a certain importance for FBOs of cold-stored RTE food products. L. monocytogenes has been isolated from several RTE foods [The morphology, physiology, and epidemiology of TE foods ,44,45 anTE foods ,46,47. L. monocytogenes in RTE food products are published in the EC Reg. 2073/2005. In RTE foods designated for infants and for special medical purposes, the absence of L. monocytogenes in 25 g is mandatory. In contrast, in RTE foods able to support the growth of L. monocytogenes (pH ≤ 4.4 or aw ≤ 0.92 or pH ≤ 5.0 and aw ≤ 0.94), L. monocytogenes cannot exceed 100 CFU/g throughout the product’s shelf life. In the same food products, the absence of L. monocytogenes in 25 g at the production stage is mandatory. However, the FBOs must confirm that these foods will not exceed the 100 CFU/g during their shelf life. In 2017, the highest level of non-compliance in several RTE foods was reported at the processing stage [L. monocytogenes occurrence was uppermost in fish and fishery products (6%), followed by salads (4.2%), meat and meat products (1.8%), soft and semi-soft cheeses (0.9%), fruit and vegetables (0.6%), and hard cheeses (0.1%) [L. monocytogenes at >100 CFU/g and consumed without further heat treatment should be considered a great public health concern [In the European Union, the food safety criteria for ng stage . The upps (0.1%) . Althoug concern . L. monocytogenes in HHP-processed foods [L. monocytogenes in meat products, mainly fermented sausages produced in the Mediterranean area. In recent years, much efforts have been put into developing HHP processing technology in order to ascertain the most effective inactivation kinetics of ed foods ,48,49,50L. monocytogenes has been reported in all the stages of the meat supply chain [L. monocytogenes, pork meat is extremely vulnerable to contamination by L. monocytogenes [The presence of ly chain ,52,53, ily chain ,55,56. Eytogenes ,58,59. L. monocytogenes contamination can be animal-related or through the slaughterhouse environment [L. monocytogenes in swine slaughterhouses, swine carcasses can be contaminated at any stage [L. monocytogenes is propagated to the carcasses from the pigs; it has been periodically found in feces and on the surface of animals in good health with the pathogen present in the intestines [The main routes of ironment ,61. Due testines ,63. L. monocytogenes contamination prior to slaughter [The occurrence in feces can reach 50%. Pigs with positive cecal content could have been infected on the farm or throughout transport or the pre-slaughtering interval in highly contaminated lairage areas, which can be considered the primary sources for laughter ,64,65,66L. monocytogenes is usually found at the end of dressing, due to handling of the carcasses with contaminated utensils and equipment [L. monocytogenes in the tongue (14%) and tonsils (7–61%). At the time of evisceration [L. monocytogenes can be around 60%–65% [L. monocytogenes in meat non-contact surfaces such as floor drains is generally around 20%–30%, and is probably correlated with inadequate cleaning procedures [The role of pigs as a niche of environmental contamination has been well demonstrated; the area of stunning/hanging is generally the most contaminated ,68,69,70quipment ,71. Sevequipment ,66 have ceration ,73, the  60%–65% ,60,74. I 60%–65% . The preocedures . Even thocedures . L. monocytogenes can persist in the same ecological niches in the slaughterhouses for months or years, becoming endemic and plant-specific through the formation of biofilms [The monitoring over time of the contamination of slaughterhouses over consecutive sampling sessions can be a useful tool to identify the emergence of new subtypes, as well as the persistence of resident “house strains” ,76,77. Sbiofilms . The mosbiofilms ,65,74,78L. monocytogenes contamination increases all along the meat production chain [L. monocytogenes in chopped meat ranges between 16% and 50% [L. monocytogenes on surfaces in contact and without contact with meat varies between 17% and 50% and 11% and 25%, respectively [Previous authors have reported that the degree of on chain ,79. The on chain ,80,81,82on chain ,74,80,83 and 50% . The amo and 50% ,74. At tectively .L. monocytogenes can survive in harsh physical and chemical conditions, forming biofilms on surfaces within the food-processing environment [L. monocytogenes  if sanitation is inadequate and/or infrequent. In addition, the more the surfaces show probable harborage niches , the more challenging they are to clean and disinfect and the more L. monocytogenes can settle and colonize them. The capacity of L. monocytogenes to grow in biofilms is thus fundamental for its persistence in food-processing facilities and is well known that these biofilms are the major source of contamination [ironment ,53,86,87mination ,58,59. L. monocytogenes biofilms; organic residues can be a niche for its accumulation and biofilm formation, as they are a source of cross-contamination [L. monocytogenes can survive and even increase in numbers as time progresses [L monocytogenes can adhere speedily and tightly to inert surfaces such as metal, glass, rubber, and plastic [L. monocytogenes strains with weak or moderate capacity to form biofilms on polystyrene surfaces. The most frequent serogroups of L. monocytogenes found in meat-processing environments are 1/2c and 1/2a, while serogroups 1/2b and 4b are generally less frequent. [Meat-processing plants represent a suitable environment for the growth of mination . In partmination ,91,92,93ogresses . L monoc plastic ,58,59. P plastic ,87,91 harequent. ,79,85,91L. monocytogenes strains are more resistant to stress than the planktonic cells [L. monocytogenes entrapped in biofilms is afforded unusual protection against sanitizers and disinfectants [L. monocytogenes growth in vitro after 24, 48, and 72 h of exposition to chlorines and quaternary ammonium at 37% concentration have been previously reported [Biofilms are a perpetual niche of contamination for foods, and it has been frequently shown that adhered ic cells ,90. L. mfectants ,94,95; pfectants . Decreasreported . w (<0.90), salt, and nitrites, chemical preservatives. [The manufacturing of fermented sausages in the Mediterranean area is a dynamic process characterized by biochemical, microbiological, physical, and sensorial changes occurring in minced ground meat formulation during fermentation and ripening . Meditervatives. ,97,98. Iw prevent colonization and/or prevent the growth of foodborne pathogens [The safety of fermented sausages is guaranteed by the combination of several environmental parameters, known as “hurdle technology” . The “huathogens . The simathogens . As a reathogens . These hathogens . Pseudomonas bacteria and other Gram-negative microorganisms, which easily grow and damage non-preserved meat in aerobic conditions [The microbial ecosystem of meat fermentation includes a complex and multispecies population; in the early steps, nitrite-curing salts inhibit nditions . Therefonditions ,103. In At the end of maturation, CNS are the second microbial group after LAB . CNS pla7–108 CFU/g and exceed one billion cells at the end of ripening [In Mediterranean sausages, the LAB consist mainly of homofermentative bacilli, while heterofermentative bacilli are less frequent . LAB areripening ,106. Theripening ,111,112.ripening ,114,115.w hurdle is reinforced with time, becoming predominantly responsible for the microbial safety of long-ripened fermented sausages [Throughout the fermentation stage, starters and protective cultures control the ecology of foods through so-called “selfish behavior” . This drsausages . The comsausages .L. monocytogenes can persist until the end of ripening or through the storage and marketing of dry-fermented sausages [L. monocytogenes tend to decline greatly during the ripening stage [L. monocytogenes may occur at many points of the technological process. The raw meat may be contaminated at the slaughterhouse, during the manufacturing, or by contact with unclean surfaces or workers [L. monocytogenes in ripened sausages [L. monocytogenes is not assisted. Despite this, in the production of several traditional sausages, empirical management of the “hurdle technology” happens sometimes; for instance, many FBOs are inclined to abbreviate the ripening stage to raise profits for their business [w of fermented sausages at the end of ripening are frequently below the levels necessary for the survival of L. monocytogenes [w values close to 0.92–0.94 [L. monocytogenes may persist during the fermentation stage, overcoming the various hurdles included in the production process [w were estimated as management tools at the ripening stage and used together with predictive mathematical models to assess the persistence of L. monocytogenes in fermented sausages. The decrease of aw to safe levels (<0.92) was proved to be closely related to persistence and was confirmed as an easy and useful ripening management tool. L. monocytogenes can replicate in fermented sausages with 12 day ripening only when the values of aw are not less than 0.92 [w, which can reduce the potential of L. monocytogenes survival (<0.92) [L. monocytogenes cannot compete with the prevailing LAB; therefore, the contamination levels of the pathogen are always ≤100 CFU/g [L. monocytogenes can grow up to >1000 CFU/g, constituting a primary public health problem [Depending on the fermentation conditions, sausages ; even song stage . Howeverng stage ,100,120. workers ,79 in th workers . Therefo workers . Previousausages ,127,128.sausages . Howeversausages . Even thbusiness . The pH .92–0.94  and L. m process . In a re process , weight han 0.92 . A corre (<0.92) ,119,128.00 CFU/g . If anta problem ,132. The problem ,84,85,91 problem ,134.HHP processing is a relatively new, non-thermal method of preserving and sterilizing foods, in which a product already sealed in its final flexible and water-resistant package is processed under very high hydrostatic pressure transmitted by water, leading to the elimination of several microorganisms and enzymes from foods . HHP proHigh pressurization of liquid or solid food products at room temperature is commonly followed by a modest temperature rise (3–6 °C for every 100 MPa increase in pressure), which is called adiabatic heating and is dependent on the food composition . Food prL. monocytogenes can present greater resistance than Gram-negative microorganisms such as Salmonella. Spores of both bacteria and molds are highly resistant to HHP inactivation. The resistance of viruses is mainly dependent on their morphological structure [Using HHP processing to kill bacteria has many advantages: in usual HHP operations, the high hydrostatic pressure spread to food products at room temperature will decrease the levels of most vegetative bacteria by up to 4 log units or greater, and inactivate certain enzymes with just tiny changes in the composition of foods ,140. Thetructure ,140,141.L. monocytogenes in several cured meat products [Frequently used food protection methods are essentially assessed based on their capacity to eliminate foodborne pathogens, thereby enhancing safety and broadening the shelf life of foods through the eradication of spoilage bacteria. HHP has a well-defined benefit in this regard, yielding food products of first-rate quality and nutritional value compared to thermally processed foods . Previouproducts ,142,143.L. monocytogenes in turkey breast meat. Vacuum-packaged turkey samples were treated at several processing combinations . A treatment of 500 MPa/1 min at 40 °C and 20 °C had significant effects on the reduction of L. monocytogenes . Patterson, Mackle, and Linton [Weissella viridescens strain on the elimination of L. monocytogenes in spiked (~2.2 × 103 CFU/g) cooked chicken samples stored for up to 105 days at 8 °C. HHP alone was insufficient to inactivate L. monocytogenes (>108 CFU/g at day 21), and W. viridescens substantially lengthened the lag phase of the L. monocytogenes strains that tolerated the HHP processing. In contrast, the association of sodium lactate and HHP successfully inhibited the occurrence of L. monocytogenes. Myers et al. [L. monocytogenes in spiked RTE turkey meat containing sodium nitrite (0 or 200 ppm) and sodium chloride (1.8 or 2.4%), treated at 0 and 600 MPa and stored up to 182 days. HHP (600 MPa/3 min) yielded an inactivation of L. monocytogenes between 3.85–4.35 log CFU/g in treated samples. No synergism between HHP and sodium nitrite/sodium chloride on the elimination of L. monocytogenes was shown. Stratakos et al. [L. monocytogenes in chicken breasts stored for 60 days at 4 °C. The association of coriander oil and HHP showed good synergism in keeping the L. monocytogenes count lower than 1.69 log CFU/g. Recently, Balamurugan et al. [L. monocytogenes in pre-blended ground chicken samples. Formulations with NaCl, KCl, and CaCl2 at three concentrations  and contaminated with L. monocytogenes (108 CFU/g) were treated with HHP . HHP at 100 or 300 MPa and the salts did not significantly decrease L. monocytogenes population. Only at 600 MPa was a synergism between salt and duration of HHP processing on L. monocytogenes inactivation shown. Formulations with increasing concentrations of CaCl2 produced a significantly greater L. monocytogenes inactivation rate.Chen et al.  showed td Linton  showed ts et al.  studied s et al.  assessedn et al.  assessedL. monocytogenes in sliced cooked ham treated with HHP (400 MPa) was evaluated by Jofré et al. [L. monocytogenes (4 log CFU/g) in all samples including bacteriocins. In contrast, HHP decreased L. monocytogenes in the control and lactate samples, with counts slowly raising to 6.5 log CFU/g at the end of storage. Marcos et al. [2) on the inactivation of L. monocytogenes in spiked cooked ham (104 CFU/g) stored for 90 days at 6 °C with temperature abuse. HHP and enterocin successfully precluded the growth of L. monocytogenes and kept it at undetectable levels (5 CFU/g) in comparison with untreated samples (8.2–8.8 log CFU/g). In another study, the same authors [L. monocytogenes in cooked ham stored for 84 days at 1 or 6 °C, and in presence of temperature abuse. Low temperatures, natural antimicrobials, and HHP caused a decrease of 2.7 log CFU/g of L. monocytogenes. In another study, the joined outcomes of nisin (800AU/g), potassium lactate (1.8%), and HHP (600 MPa/5 min/10 °C) on the inactivation of L. monocytogenes in spiked, vacuum-packed sliced cooked ham (4 log10/g) stored for 3 months at 1 and 6 °C was evaluated by the same authors.The synergism between enterocins A and B, sakacin K, nisin, and potassium lactate against spiked é et al. . HHP decs et al.  evaluate authors  studied L. monocytogenes [L. monocytogenes and widening the shelf life of treated samples at 90 days in comparison with control samples. More recently, Bover-Cid et al. [L. monocytogenes (107 CFU/g) in HHP-treated (600 MPa/3 min) sliced standard cooked hams. Standard hams were produced with organic acids and control samples without organic acids, but including potassium lactate and sodium diacetate. The inactivation of L. monocytogenes was evaluated in vacuum-packaging storage at 8, 12, and 20 °C. In the samples with potassium lactate and sodium diacetate, the growth of L. monocytogenes was barely extended by HHP. In standard cooked hams produced with organic acids, HHP significantly stimulated the growth of L. monocytogenes; at 20 °C, the growth of L. monocytogenes in samples with lactate was up to 4-fold greater than in the untreated products.The results highlighted that HHP and low temperatures can be successful at challenging survival of ytogenes . The sheytogenes . The comd et al.  evaluateL. monocytogenes on dry-cured hams (347–852 MPa/2.3 to 15.75 min/7.6 at 24.4 °C). The authors showed that pressure and time were the most important variables influencing the elimination of L. monocytogenes. Stollewerk et al. [L. monocytogenes (spiked at <100 CFU/g) in standard NaCl (28 g/kg) and NaCl-free (replaced with 15.31/kg KCl and 33.83/kg potassium lactate) sliced smoked dry cured hams stored at refrigeration temperature for 112 days. HHP successfully inactivated L. monocytogenes in standard samples after 14 days. In contrast, in NaCl-free samples, L. monocytogenes was persistent until 28 and 56 days, respectively. In another study, the same authors [L. monocytogenes (<2 log CFU/g), treated with HHP (600 MPa), and stored for 112 days. L. monocytogenes was only inactivated in HHP-treated acidified smoked standard ham slices.Bover-Cid et al.  conceivek et al.  showed t authors  manufactL. monocytogenes. In another study, Bover-Cid et al. [L. monocytogenes in dry-cured ham, in relation to HHP (347–852MPa/5 min/15 °C), aw (0.86–0.96), and fat quantity (10–50 %). The high fat level protected L. monocytogenes against HHP at pressure levels of ~700 MPa. At lower pressures, greater elimination of L. monocytogenes was shown when the fat level was raised over 30%. In another study, two different RTE dry-cured hams  stored for 60 days at 8 °C were treated with HHP (600 MPa/6 min) with or without use of nisin (200 AU/cm2) and examined for the elimination of L. monocytogenes (spiked at 107 cells/g). The inactivation of L. monocytogenes (from 1.85 to 3.58 log) was high in samples supplemented with nisin. Moreover, aw showed an antagonistic r effect in the elimination of L. monocytogenes: with low aw values the inactivation rates were limited [NaCl-free hams displayed delayed inactivation of d et al.  modeled of nisin 0 AU/cm2 L. monocytogenes, which was always below the detection limit in comparison with control samples. HHP elimination (from 300 to 800 MPa) of L. monocytogenes (107 CFU/g) on mortadella was developed by Hereu et al. [L. monocytogenes population declined near to the tail level. The high fat level of mortadella (∼17%) had a preserving effect on L. monocytogenes from HHP. The same authors [L. monocytogenes strains in mortadella inoculated T at 107 and 104 CFU/g) before HHP processing (400 MPa/5 min), and thereupon stored at 4, 8, and 12 °C. Freeze-stressed strains were more resistant to HHP and displayed a greatly extended lag phase following HHP processing. The high fat level composition of mortadella affected the survival of L. monocytogenes after HHP treatment. Valdramidis, Patterson, and Linton [w values (0.95–0.98). The presence of L. monocytogenes was affected by HHP, and HHP showed synergism with aw towards preventing the recovery of L. monocytogenes. The concentrations of sodium chloride/nitrite obliquely influenced the occurrence of L. monocytogenes by managing the aw values and, therefore, the shelf life of the samples. The lower the aw value, the lower were the inactivation rates caused by HHP.Jofré et al.  showed tu et al. . A clear authors  evaluated Linton  developeL. monocytogenes. Raw sausages were treated at 300 MPa for 10 min at 17 °C, and subsequently ripened at 12 °C for 27 days.In recent years, the implementation of HHP has been proposed to improve the safety and protract the shelf life of Mediterranean-style dry-fermented sausages , since tL. monocytogenes population in treated “fuet” from 2.26 log CFU/g to 1.42 CFU/g at the end of ripening was reported. In contrast, no effects in L. monocytogenes count were noticed in treated “chorizo” samples after 27 days of ripening. The authors highlighted that higher pressure degrees were necessary to eradicate L. monocytogenes cells in ripened fermented sausages. Jofré, Aymerich, and Garriga [L. monocytogenes (3 log CFU/g).A decrease in  Garriga  evaluateL. monocytogenes. In contrast, the application of HHP at the end of ripening did not yield the same result. Despite this, an increasing reduction of L. monocytogenes count was noticed throughout storage at room temperature. In another study, Porto-Fett et al. [L. monocytogenes (about 7.0 log CFU/g) in “Genoa” salami. Elimination of L. monocytogenes after fermentation ranged from roughly 1.1 to 1.3 log CFU/g. After drying, HHP at 600 MPa or at 483 MPa for 1 to 12 min at 19 °C decreased L. monocytogenes enumeration by a further 1.6 to ≥5.0 log CFU/g. After storage for 28 days at 4 °C, L. monocytogenes enumeration dropped by up to a further 3.0 log CFU/g. Ananou et al. [L. monocytogenes inactivation in the Spanish sausage “fuet” throughout ripening for 10 days at 15 °C and storage at 7 °C or at room temperature. AS-48 produced a substantial (5.5 log CFU/g) decline in L. monocytogenes population. After pressurization and storage, L. monocytogenes numbers stayed below 5 CFU/g in all “fuet” samples, including AS-48 (treated or not).Enterocins A and B displayed a prompt decrease of t et al.  assessedu et al.  investigL. monocytogenes outcome. Stollewerk et al. [® and HHP (600 MPa/5 min/13 °C) on inactivation of L. monocytogenes in NaCl-free acid (pH 4.8) and low-acid (pH 5.2) “chorizo” stored at refrigeration temperature for 91 days. HHP resulted in the inactivation of L. monocytogenes during the whole storage time. The reduction of NaCl could influence the safety of reformulated sausages. However, HHP could be a useful method to develop healthy and safe short-ripened, NaCl-free sausages. Marcos et al. [L. monocytogenes (107 cells/g) onto the outer layer of sausages. The combined outcome of HHP (600 MPa/ 5 min/12 °C) and antimicrobial wrapping with polyvinyl alcohol layers including nisin was evaluated for elimination of L. monocytogenes throughout a storage time of 90 days at 4 °C.HHP only had no anti-k et al.  evaluates et al.  producedL. monocytogenes throughout the storage time. Only the antimicrobial wrapping of fermented sausages with films including nisin caused an evident decrease of L. monocytogenes counts (1.4 log CFU/g). Association of HHP and nisin did not yield any extra protection against L. monocytogenes compared to nisin wrapping alone. Recently, Rubio et al. [w (0.79–0.92), pressure (349–600 MPa at 18 °C), and duration (0–12.53 min) on HHP-treated L. monocytogenes eradication in Spanish “chorizo” sausages. All the three components greatly affected HHP elimination of L. monocytogenes, as the pressure and holding time of HHP treatments increased.The results of this study showed that HHP alone did not preclude the rise of o et al.  implemenw showed a preserving effect on L. monocytogenes, and pressures below 400 MPa did not drive towards considerable pathogen decreases. More recently [L. monocytogenes occurrence on sliced “chorizo” treated or not with HHP after post-process contamination was developed. The effects of dissimilar degrees of L. monocytogenes initial contamination in the minced meat (−1.43–3 log CFU/g), HHP (400–600 MPa/18 °C/0–12 min) and nitrite amounts (0–150 ppm) on L. monocytogenes inactivation were evaluated through predictive models, existing references, and experimental data. In all the simulated outlines, compositions, and storage parameters, the population of L. monocytogenes on sliced vacuum-packed “chorizo” at the marketing stage was assessed to be below 100 CFU/g, and HHP at 600 MPa for 10–12 min was acceptable in non-contaminated samples.Slight degrees of arecently , a stochL. monocytogenes in Mediterranean-style ripened sausage emphasizes the necessity for the FBOs to implement a multi-hurdle strategy with HHP to enhance the safety of dry-fermented sausages [L. monocytogenes in fermented sausages is greatly dependent on the physicochemical parameters of the sausages. To define specific pressure treatments accomplishing the essential performance criteria, assessment and validation of the efficacy of HHP on specific Mediterranean-style fermented sausages is imperative [L monocytogenes in RTE meat products, as well as optimize the processing conditions of dry-fermented sausages.The frequent detection of sausages . The HHPperative . The use


Our data show that administration of rac-CE-123 30 min before learning blocks retroactive interference that has been experimentally induced at 3 h, but not at 6 h, post-learning. In contrast, S-CE-123 treatment 30 min before learning blocked the induction of retroactive interference at 6 h, but not 3 h, post-learning. Administration of S-CE-123 failed to interfere with proactive interference at both 3 h and 6 h. Analysis of additional behavioral parameters collected during the memory task implies that the effects of the new DA re-uptake inhibitors on retroactive and proactive interference cannot easily be explained by non-specific effects on the animals’ general social behavior. Furthermore, we assessed the mechanisms of action of drugs using intracerebral in vivo-microdialysis technique. The results revealed that administration of rac-CE-123 and S-CE-123 dose-dependently increased DA release within the nucleus accumbens of freely behaving mice. Thus, the data from the present study suggests that the DA re-uptake inhibitors tested protect the consolidation of long-term social memory against interference for defined durations after learning. In addition, the data implies that DA signaling in distinct brain areas including the nucleus accumbens is involved in the consolidation of SRM in laboratory mice.In the laboratory, long-term social recognition memory (SRM) in mice is highly susceptible to proactive and retroactive interference. Here, we investigate the ability of novel designed dopamine (DA) re-uptake inhibitors ( Social recognition memory (SRM) is the ability to distinguish between familiar and unfamiliar conspecific individuals  blocks the dopamine transporter (DAT) with high specificity and no adverse side effects  were selected to evaluate possible effects on retroactive or proactive interference during SRM consolidation. Further, additional parameters were monitored during the behavioral tests to allow a first identification of possible behavioral side effects of the treatment that might have affected the behavioral readout interpreted as “memory.”The present study was designed to assess the impact of CE-123 on the phenomenon of memory interference in SRM. In addition to the racemate, we used rac-CE-123 and S-CE-123 on extracellular DA levels in the mouse nucleus accumbens. Previous studies have shown that this brain area might be involved in the correct processing of short-term SRM in rats  with an age group of 9–16 weeks were used as experimental subjects. If not stated otherwise, they were housed in groups of five per cage (size: 20 × 37 × 15 cm) for at least 1 week before starting the experiments under standard laboratory conditions . Stimulus animals were C57BL/6JOlaHsd mice of both sexes with an age of 25–35 days. For microdialysis experiments, adult male C57BL/6J mice were used. These animals were kept under similar conditions and experiments starting at 08:00–08:30 h. All experimental manipulations were approved by the Committee on Animal Health and Care of the local governmental body  and performed in strict compliance with the EEC recommendations for the care and use of laboratory animals (2010/63/EU).ad libitum. As illustrated in i), a second, previously not encountered stimulus animal was presented for 4 min to the experimental subject during the 2nd sampling session. To measure retroactive interference, during retrieval (choice), the stimulus animal encountered during the 1st sampling was presented to the experimental subject together with a novel stimulus animal 24 h after the 1st sampling. To measure the proactive interference, during the choice session, the 2nd sampled stimulus animal was presented to the adult together with a novel stimulus animal. Significant longer investigation of the novel stimulus animal compared to the already encountered stimulus animal during choice was taken as evidence for an intact recognition memory  and 6 h (after the gap) for our studies.The social discrimination test performed has been described in detail in Engelmann et al. . In brieIn addition to the investigation duration also the latency between the introduction of the stimulus animal in the experimental subject’s cage and the first approach of the experimental subject towards the stimulus animal was monitored. Also, the duration of aggressive and sexual behavior of the experimental subject towards the given stimulus animal during the 1st and 2nd sampling was monitored.rac-CE-123 = 5-((benzhydrylsulfinyl)methyl)-thiazole methyl)thiazole  30 min before the 1st sampling session . For provia the drinking water  for post-surgery care and were housed individually. The evening before the microdialysis experiment, mice were shortly anesthetized with isoflurane and a microdialysis probe  with a molecular cutoff of 6 kDa  was inserted into the guide cannula of mice reaching into the nucleus accumbens. The probe was connected to a CMA/Microdialysis Syringe pump (CMA-4004) and constantly superfused with sterile artificial cerebrospinal fluid  at a flow rate of 0.5 μl/min. On the day of experiment, superperfusion rate was set to 1.0 μl/min and after 2 h of equilibration sequential microdialysis fractions were collected every 20 min into ice-cooled microtubes containing 6 μL of an antioxidative mixture , vortexed and stored at −80°C until further analysis. After three baseline samples (collected from −60 to 0 min), vehicle or drugs  were administered, and six samples were collected. Subsequently, vehicle or drugs were administered in a higher concentration  and another six microdialysates were collected. For the last two dialysates aCSF containing 100 mM KCl was used as a positive control to elicit local depolarization in order to confirm the functionality of the system. At the end of the experiment, mice were euthanized by an overdose of thiopental and brains were removed for histological verification of the placement of microdialysis probes. Data were only used from subjects with correct probe displacement  and placed in a stereotaxic frame . A guide cannula  was implanted unilaterally 1 mm above the right nucleus accumbens  according to the mouse brain atlas by Franklin and Paxinos  and fixement see .2EDTA at a pH of 5.6. Samples were injected via a SIL-20ACXR autosampler  and separated on a C18 reversed-phase column . The HPLC system was coupled to the DECADE II electrochemical detector . The column and detector cell were maintained at 35°C by a column oven as part of the electrochemical detector. The applied potential was set to +460 mV vs. reference electrode and was adjusted to a detection range of 100 pA/V with a filter frequency setting of 0.01 Hz. Substance amounts which yielded a detector signal corresponding to three times noise level were considered at detection limit. This allowed for the measurement of DA with a sensitivity of 0.25 fmol/5 μl sample. Instrument control and data acquisition were carried out by Lab Solution chromatography software . Calibration curves were constructed in the range of 50 pM to 1 nM (0.25–5 fmol of DA injected) and were consistently linear with correlation coefficients higher than 0.999.Dopamine was analyzed in 5 μl microdialysate fractions by high-performance liquid chromatography (HPLC) with electrochemical detection. The HPLC system consisted of a Shimadzu  system controller (CBM-20A), degassing unit (DGU-20A3R) and micro HPLC pump (LC-20ADXR) operated at a flow rate of 55 μl/min. The mobile phase consisted of 8% (v/v) methanol, 50 mM phosphoric acid, 50 mM citric acid, 2.36 mM octane-sulfonic acid, 0.1 mM Nat-test. The additional behavioral parameters  were analyzed using one-way ANOVA. For the microdialysis experiments, the DA content in each 20 min-microdialysate was expressed as a relative value to the mean content rates of the three samples preceding the administration of the drug or vehicle. Statistical analysis was carried out with Statistica Software v9  using two-way ANOVA for repeated measures followed by Fisher’s test. P < 0.05 was considered to be statistically significant.Data are presented as mean + SEM. Statistical analysis of the behavioral data was performed by GraphPad Prism 6.05 . Data obtained from the social discrimination experiments were analyzed using the paired Student’s rac-CE-123 at a Si = 6 h) had to be excluded from the analysis of the treatment conditions because the investigation duration during the 1st or 2nd sampling was <1 s and, thus, it is unreliable to assume that sufficient information was acquired for a successful recognition and interference, respectively. The data of the remaining animals show that the average investigation duration during both sampling sessions was sufficient to acquire the important information essential to establish long-term SRM and to produce an interference, respectively . At a Si of 6 h, rac-CE-123-treatment failed to significantly affect the investigation durations during choice . However, vehicle administered experimental subjects investigated the novel stimulus animal significantly longer than the 1st sampled stimulus animal during choice .When retroactive interference was introduced at a SS-CE-123 nor that of vehicle caused a significant difference in the investigation of the 1st sampled and the novel stimulus animal during choice at a Si = 3 h . If the same drug was administered at a Si = 6 h, during the choice session experimental subjects investigated the novel stimulus animal longer than the 1st sampled stimulus animal . Vehicle treatment failed to affect significantly the investigation durations .Neither administration of i of 3 h, neither vehicle  nor S-CE-123  showed a significant difference in investigation duration between 1st sampled stimulus animal and novel stimulus animal during the choice session.When testing proactive interference and introduced at Si = 3 h, no significant effects on any of the additional behavioral parameters monitored were detected (via ANOVA), independently upon the administered substance (vehicle or drug). In contrast, ANOVA statistical test revealed a significant effect on the latency to start investigating the stimulus animal during the 2nd sampling at a Si = 6 h in case of vehicle treatment only, for rac-CE-123 and its respective vehicle treatment only. Subsequent analysis via Student’s t-test failed to detect significant differences between the 1st and 2nd sampling under a vehicle and the respective drug treatment (data not shown). Thus, the differences detected via ANOVA resulted from different values measured during the 1st sampling vs. the 2nd sampling and did not reflect a specific treatment effect.The data collected from the additional parameters monitored during the behavioral testing are shown in F = 1.949, p < 0.05) and high doses  = 11.418, p < 0.001). While vehicle treatment failed to alter the DA concentrations, sc injections of both rac-CE-123 and S-CE-123 increased the concentrations of DA in the microdialysates. Specifically, the administration of 10 mg/kg of rac-CE-123 and S-CE-123 caused a moderate increase in DA concentrations for at least 20 min and 40 min, respectively, and returned to baseline levels within the subsequent 40–60 min (rac-CE-123 and of S-CE-123 caused a maximum increase in the DA concentration within 40 min (p < 0.01) after injections and remained elevated throughout the whole sampling period  protect the memory trace against retroactive interference by manipulating DA signaling in the brain. The action of the drugs cannot easily be explained by an alteration of the general social behavior of the experimental subjects  as the behavioral parameters analyzed here failed to differ between vehicle and drug treatment (1A receptors improved the consolidation of SRM in rats (Cavalcante et al., Previous studies have shown that 30 min after a peripheral injection of reatment . Researcreatment . Systemireatment . Intra-iS-CE-123 was able to block retroactive interference at a Si of 6 h (i of 3 h (S-CE-123 and rac-CE-123 administered via the same route and dose may affect differently the dopaminergic signaling relevant for SRM. This could be due to a different profile of washin and washout of the drugs targeting the brain tissue. Unpublished data show that compared to rac-CE-123, S-CE-123 is detectable in a ~5–10 times higher concentration in both liquor and brain tissue after intraperitoneal administration in adult male rats. The impression of a different duration of action of S- vs. rac-CE-123 is—to some aspect—supported by the microdialysis data. The release profile of the racemate at a dosage of 10 mg/kg differs from that of the S-enatiomer by showing elevated DA levels at the sample collected 20–40 min after treatment when rac-CE-123 is already indistinguishable from baseline (S-CE-123 differ to that of rac-CE123 in blocking the retroactive interference induced at 3 h vs. 6 h after the 1st sampling. Upon first view, this could result from a counter regulatory mechanism of the S-enantiomer in the administered racemate. However, such conclusions would be too premature without further studies investigating possible differences in the effects between the two CE-123 treatments including the analysis of molecular mechanisms involved.The enantiomer i of 6 h , but noti of 3 h . This inbaseline . In addibaseline . Thus, DS-CE-123 in the context with the induction of proactive interference failed to provide a protective effect of this enantiomer for a memory of the 2nd sampled stimulus animals (The results of the experiment in which we administered  animals . This sp animals , the previa which the modafinil analogues tested here affect SRM. The molecular signatures linked to the blockade of interference are likely to provide further insight into the neurobiological basis of this type of learning and memory in mammals.Taken together, the results of the present study, show for the first time, that modafinil-derived drugs increasing extracellular DA in the nucleus accumbens and acting at both the DAT and DA D1 receptor are able to make SRM resistant against retroactive interference. The drug- and time-dependent action suggests distinct action profiles of the different drugs and provides insight into the mechanisms underlying the consolidation of SRM which requires further investigations. Further studies will focus on the cellular mechanisms rac-CE-125, VD in that of S-CE-123. ME designed and supervised the behavioral experiments and contributed to the writing of the manuscript. GL initiated the project and contributed to the study design and to the writing of the manuscript. NS, SS and KE designed and performed the microdialysis experiments including statistical analysis and wrote the draft of the respective parts in the manuscript.JC-P performed the animal experiments, treated the animals, performed a part of the statistical analysis of the behavioral data and contributed to the manuscript draft. PK was involved in the design and synthesis of the drugs; he also contributed to the design of the study. HV analyzed part of the behavioral data and provided the first draft of the manuscript. NA was involved in the synthesis of 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.


The Hedgehog/GLI signaling pathway plays an important role in normal embryonic tissue development and has been implicated in the pathogenesis of various human cancers. In this review article, we summarize pre-clinical evidence supporting the suitability of targeting this signaling pathway in cancers. We review agents blocking both the ligand-dependent and ligand-independent cascades, and discuss the clinical evidence, which has led to the FDA approval of Hedgehog receptor Smoothened inhibitors, vismodegib, and sonidegib, in different malignancies. Finally, we provide an overview of published and ongoing clinical trial data on single agent or combination therapeutic strategies, targeting Hedgehog/GLI signaling pathway, in both advanced solid tumors and hematologic malignancies. The Hedgehog (Hh) signaling pathway plays an important role during normal embryonic tissue development. It precisely controls epithelial and mesenchymal cell interactions regulating cell proliferation and differentiation ,2. The HThe Hh pathway can be aberrantly activated through various mechanisms, which can lead to the pathogenesis of numerous human cancers. One of the most common ones is the Hh ligand upregulation in autocrine or paracrine fashion; this was reported in pancreatic, gastrointestinal, breast, lung, medulloblastoma, and prostate cancers ,5. OtherAlthough reinforcing endogenous negative regulation of Hh signaling, via PTCH1 and SUFU could be effective, current efforts center on developing new and refining existing inhibitors against SMO. In addition, efforts have been made to inhibit GLI transcription factors to tackle, for example, acquired resistance SMO to small molecule inhibitors via activating mutations or noncanonical Hh pathway activation. These efforts have culminated two FDA approved Hh pathway inhibitors in the clinic. Examples of pre-clinical evidence on targeting Hh signaling pathways are discussed below.Canonical activation of the Hh signaling pathway is most often inhibited by targeting SMO. Hh pathway inhibition, with small molecules, has been shown to decrease tumor growth in different models of various cancer types. For example, SMO inhibitor, saridegib, has been successfully used in combination with chemotherapy followed by maintenance therapy to suppress serous ovarian cancer xenograft growth . VismodeSimilar to the FDA approved vismodegib and sonidegib, glasdegib is a potent, selective small molecule inhibitor of SMO . In addiSaridegib, a semi-synthetic derivative of cyclopamine, is structurally distinct, compared to the previously discussed small molecule inhibitors. Saridegib has improved selectivity, affinity, and stability compared to cyclopamine. It is a potent and selective SMO inhibitor with an IC50 of 1.4 nM . In a meTAK-441 and BMS-833923 are other small molecule SMO inhibitors under clinical investigation. In preclinical studies, TAK-441 has a high anti-tumor activity with an IC50 of 4.4 nM in multiple tumor types . TAK-441Itraconazole, an FDA-approved antifungal drug, was discovered from screening a library of existing drugs for its ability to inhibit angiogenesis and Hh signaling pathway by antagonizing SMO . Recent The terminal effectors in the Hh signaling pathway, GLI1-3, can be activated through alternate mechanisms, apart from SMO activation. This ligand-independent pathway activation has been difficult to target previously, however, recent advances have been effective in directly inhibiting GLI factors, ultimately dampening the effects of active Hh signaling.In contrast to anti-SMO agents, GLI inhibitors like arsenic trioxide inhibits the Hh signaling pathway by preventing the accumulation of GLI2 and reducing the transcriptional induction of target genes of GLI2. In a mouse model of medulloblastoma, with Ptch+/− p53−/−, arsenic trioxide inhibited tumor growth at a similar serum level achieved in the treatment of acute promyelocytic leukemia . In a xeVarious small molecule SMO inhibitors, such as vismodegib and sonidegib, have been chosen as initial targets for the clinical evaluation of Hh pathway inhibition, due to their improved pharmacological properties from pre-clinical development and predictable Hh signaling pathway activation in BCC. Previous clinical trial results, and ongoing clinical trials evaluating Hh signaling pathway inhibitors, are summarized in Similar to other targeted therapies, a number of Hh inhibitors have been evaluated initially in advanced solid tumors. In a phase 1, first-in-human trial conducted with patients from western countries, sonidegib at doses from, 100 to 3000 mg daily or 250 to 750 mg twice daily, were administered to 103 patients with advanced solid tumors . A similSonidegib was evaluated in a phase 1 trial in 15 patients with extensive stage small cell lung cancer (SCLC) . SonidegVismodegib in combination with cisplatin and etoposide was compared to standard cisplatin and etoposide for patients with extensive stage SCLC in a randomized phase 2 trial . Fifty-tVismodegib and sonidegib were FDA-approved for patients with locally advanced and metastatic BCC in 2012, and 2015, respectively. A phase 1 study on vismodegib, in patients with locally advanced and metastatic BCC, determined the recommended dose for phase 2 was 150 mg daily. Vismodegib was very well-tolerated with only grade 3/4 fatigue and hyponatremia. No DLT was observed. Fifty four percent of patients had disease response and another 33% had stable disease. Tissue correlation studies demonstrated upregulation of Hh signaling pathway in 96% of patients as determined by elevated GLI1 mRNA expression. Vismodegib exposure led to a significant decrease in GLI1 expression . The phaAs for other agents in the phase 2 registration trial, patients with advanced or metastatic BCC were randomized into sonidegib at two dose levels: 200 mg daily or 800 mg daily. Sonidegib at 200 mg daily had lower grade 3/4 toxicities and lower dose interruption, reduction or discontinuation. Both dose levels provided equivalent ORR, comparable to that of vismodegib from previous reports . A phaseThe activity of vismodegib, in refractory metastatic medulloblastoma, was first reported in a patient whose tumor harbored a somatic mutation of PTCH1, thus activating the Hh signaling pathway . Phase 1Hh pathway inhibitors, including vismodegib and saridegib, have not demonstrated clinical benefit in addition to standard chemotherapy for metastatic pancreatic adenocarcinoma. Vismodegib was initially assessed for its effect on Hh pathway inhibition, cancer stem cells, cell proliferation, fibrosis, and clinical benefit. Although vismodegib effectively inhibited Hh signaling pathway activity, its addition to gemcitabine did not show additional clinical benefit . This waVismodegib was evaluated in clinical trials for a few other types of solid tumors, but it has not demonstrated additional clinical benefit. For example, vismodegib, in a phase I trial for patients with metastatic castration-resistant prostate cancer, showed effective Hh pathway inhibition, but no clinical response . VismodeGlasdegib was evaluated in phase 1 trials conducted in both Japanese  and westCurrently, there are only two FDA-approved Hh pathway inhibitors, Vismodegib and Sonidegib, which are limited to locally advanced and metastatic BCC. Recently, Glasdegib was granted priority review by the FDA in patients with previously untreated AML, in order to increase the number of inhibitors available to patients to three . Hh pathCurrent and future research efforts in targeting Hh signaling in cancer should focus on strategies addressing and overcoming these resistance mechanisms. The design and development of next generation therapeutics, targeting Hh signaling, should take into consideration both, acquired SMO mutations and downstream genetic variants, such as SUFU loss-of-function mutations and GLI gain-of-function mutations. To expand the scope of Hh signaling pathway regulation, GLI inhibitors such as arsenic trioxide and GANT61 have been evaluated in both pre-clinical and clinical settings ,31. To a


Taiwan and a few Asian societies have had among the lowest fertility rates in the world for the past decade. Understanding the reasons behind the low fertility and designing policies accordingly to improve fertility has been a priority of governments in the region. It what follows we examine the low fertility rate in Taiwan by studying the trend of actual fertility rate and desired fertility rate in Taiwan using an age-period-cohort (APC) model. Using the Knowledge, Attitude, and Practice (KAP) of contraception survey data between 1973 and 2004, we applied APC analyses on the actual fertility rate and desired fertility rate of married women. We found that youngest cohorts (the mid-cohort year 1983) had 10% higher actual fertility and 15% higher desired fertility compared to those who were born in 1959–1965, respectively. Additionally, we attributed current lowest-low fertility (at or below 1.3) to late marriages. There is a lag between the actual and desired fertility rates in KAP survey due to tempo effect. Furthermore, the trends of the cohort effects of both fertility rates in KAP surveys are reversing in Taiwan. Consequently, increase total fertility rate (TFR) should encourage marriage among the marriageable population and reward married and childbearing households. Maintaining stable population development is an indispensable factor of socioeconomic growth. Lowest-low fertility may cause problems, such as a labor force shortages, the ageing of society, a lack of staff for the long-term care of elderly persons, and a demographic dividend reduction ,2,3. In Research on fertility and increasing fertility has focused on appropriated mathematical models and statistical analyses of fertility data . DemograIn a series of studies, the Taiwan Provincial Institute of Family Planning examined the knowledge, attitudes, and practices (KAP) of contraception use among married women ,9. In TaThis study aimed to provide a clear goal, measurements and an appropriate approach for the construct analyses of psychological factors related to childbearing in Taiwan. Based on the identification and analyses described below, surveys were used to collect data about contraception knowledge and use, pregnancy experiences, fertility, and family planning  to examine the relationship of psychological fertility with actual reproduction. We investigated the influence of birth cohort on the changes in psychological and actual fertilities after accounting of age and the survey period. Furthermore, this paper explored age-period-cohort analyses to provide useful clues about factors that specifically influence actual reproductive behaviors.We used the KAP of contraception survey data collected by the Taiwan Provincial Institute of Family. The KAP survey in Taiwan has nine waves in total from the year 1965 to 2004, and collected data on contraception knowledge and behaviors, pregnancy experiences, fertility, family planning, family relations, and residence history . Our stuWe utilized two questions from the survey series to evaluate the actual and desired fertility rates. These two questions were, “How many live births have you had?” and “If you are currently married, how many children do you want to have in your life?” The formulas of actual and desired fertility are defined as follows:These formulas were used for the total 26,224 individuals data that were cross-tabulated with four seven-year age groups , 5 seven-year time periods , and eight birth cohorts . Note that we discarded the childhood and adolescent groups (0–19 years old) from this analysis. Hence, we revised the formulas of actual and desired fertility, which listed as follows:However, the actual and desired fertility rate data for those aged 41–47 in 1972–1978 period was not available, hence the mid-cohort year 1934 cohort was not available for the analysis. In additional, we also calculated the ratio of actual fertility to the desired fertility. If the ratio is larger than one, it means the women gave birth to more children than desired. Conversely, if the ratio is less than one, it means the women did not have the desired number of children. We used the mid-cohort year 1962 cohort as the reference cohort. We also select the youngest age-group (aged 20–26) as the reference age-group because it has the lowest actual and desired fertility rates compared to other age-groups. Given the mid-cohort year 1962 cohort and the 20–26 age-group being the reference groups, the reference group for period was the period 1979–1985 because the exact linear relation of the age, period, and cohort (cohort + age = period). Moreover, we analyzed the fertility rate data of the childbearing women in Taiwan (after discarding the childhood and adolescent groups) following a similar approach to compare the results of the birth cohort effects (Department of Statistics (DS) 1950–2009). In this component of the study, there were 5 five-year age groups ranging from 20 to 44 years old , 8 five-year periods  and 12 cohorts . The divergent widths of the effects of the KAP surveys and fertility statistics had no bearing on the purpose of this study.ith age group and the jth period group be denoted by ijλ. The APC model is as follows:We used APC analyses used to measure the actual and desired fertility rate by parameters estimates . In this study, we partitioned the marital fertility into the effects of age, period, and cohort to measure childbearing probability. Let the marital fertility of the In epidemiology, one popular interpretation of the relationship of age, period, and cohort variables is that age and period interact to create unique generational experiences. Greenberg et al. proposed the conceptualization of cohort effects as an interaction of age and period ; a cohorWe used the median polish analysis to estimate cohort effects under the above conceptualization ,14. The ijR) for the i-th age category and j-th year is modeled as:−e to the rate of each age group. If the ratio of log-additive rate without and with cohort effects is close to one, then it is determined as the referent birth cohort. In words, more probable cohort could be reference group with less residual between before and after removing cohort effects from rate. The 95% confidence intervals (CIs) of rate ratios were also calculated with the corresponding standard errors.In this study, we conceptualized age and period effects as additive increases to the actual and the desired fertility rates. That is, the related fertility rates  and these points obtained by fitting segmented regression model. The segmented analysis via free R software performed to estimate the change point and standard error for estimates of the cohort effects with the Davies’ test for segmented regression model . The proFor age groups 27–33, 34–40, and 41–47, the actual fertility rates decreased faster than that in the 20–26 age-group. This non-parallel decreasing trend in actual fertility observed among these age groups indicated the existence of a cohort effect. Similar non-parallel trend for desired fertility were observed after the 1993–1999 period, suggesting a cohort effect on desired fertility as well. In addition, Among the birth cohorts, the women born near 1927 exhibited the highest rate ratio . The 30–34 age group can retrospectively be placed in the 1978–1982 age group that overlaps with the most recent cohort of 1983 (birth cohort = 1980–1986) in actual KAP birth rates. In this study, the women born around 1983 exhibited the highest fertility rate of childbearing among the birth cohorts and provided the greatest contribution to increasing future fertility rates. The question then becomes, “Did the integration of these effects actually increase the higher reproduction between years?” In response, we attribute the most substantial effects to the premeditated fertility behavior that occurred in the Dragon year. Consistently, the TFR immediately dropped to 1.07 in 2013, representing a decrease of 0.2 compared with 2012.The empirical findings of the current study indicating a behavior intention lag might lead to low fertility. Besides, women getting married later in life may also cause lowest-low fertility  in Taiwan . In the If the age-specific fertility rates of the population varied over time, then TFR may not be a precisely measure of completed fertility . Then, there is a stable effect which may exist on age-specific fertility rates, defined as a tempo effect. Additionally, the below-replacement level of fertility in Taiwan since the mid-1980s has primarily been attributable to the tempo effects  of postponing childbearing, which also reduced the TFR of the period . In thisIndeed, there are many uncertainties regarding the tempo of childbearing. Much of the existing literature has demonstrated that increased human capital expenditures per child are associated with lowest-low fertility ,26,27. THowever, the trends of the cohort effects of desired and actual birth rates in KAP surveys are reversing in Taiwan. Notably, the attitudes toward childbearing have changed among the more recent cohorts of married women. To be more specific, regarding the KAPs of recent birth cohorts, the cohort effects of actual births have increased after those of desired births. That is, there is a period lag between the recent cohort effects of desired and live birth rates and we can interpret it as behavior intention lag. The behavior intention lag also observed between the change points of cohort effect of actual birth rate in generation born from 1973 to 1979  and that of desired birth rate in generation born from 1966 to 1972 . Additionally, the cohort effect of the youngest birth cohort is lower than previously and consists of a continuous decline in the fertility rates of women of childbearing age in Taiwan. Specifically, the cohort effect of the actual birth rate is highest among the most recent birth cohort of 1980–1986 in the KAP, suggesting that the younger birth cohort of married women would have more births than that of the older cohort living in the same period. Furthermore, the last 6 stages of the KAP survey series reveal that the majority of women’s live birth rates are greater than the desired birth rates of the earlier period.The limitations of our study included the need for the median polish phase to achieve identification problem of the effects of age, period, and cohort. Unlike other approaches for modeling cohort effects ,28, the The cohort effects of desired fertility rates may ahead a period lag than that of actual fertility rates in KAP survey due to tempo effect. Although the trends of the cohort effects of both fertility rates in KAP surveys are reversing in Taiwan, we expect the total fertility will further to decline in the near future as evidenced in the results of the present study. Consequently, encourage the marriageable population to get married and reward childbearing married households may promote as fertility policies. Besides, simultaneously boost fertility intension and improve child mortality rate may also have a certain influence on fertility rates. Although these policies alone cannot fully restore the fertility rates to previous levels and ensure appropriate replacement, such policies should be a priority to retard the trend of declining fertility in Taiwan by encouraging, rewarding, and supporting childbearing.


Human T-lymphotropic virus 1 (HTLV-1) infection is commonly associated with neurological conditions like chronic progressive myelopathy and tropical spastic paraparesis (TSP) but rarely also reported with polymyositis, multiple sclerosis, and parkinsonism. It is important to recognize that HTLV-1 infection increases the risk of these neurological conditions. We present a case of 71-year-old female with HTLV-1 associated chronic progressive myelopathy with parkinsonism which signifies that it is under-recognized and not frequently reported due to lack of expert neurological assessment in these chronically debilitated patients. The human T-lymphotropic virus type 1 (HTLV-1) was discovered in 1980 and is one of the first retroviruses to be discovered. It affects about 10-20 million people around the world. It is transmitted by sexual contact, vertical transmission before birth either transplacentally or during C-section, after birth through breastfeeding . ParkinsA 71-year-old female with the history of HTLV-1 infection for 20 years, congestive heart failure, coronary artery disease, hypertension, diabetes type-2, peripheral vascular disease, chronic neck and back pain, nonambulatory for a year admitted to the medical service as dehydration and acute kidney injury. Neurology was consulted for worsening weakness and pain in the legs with paresthesia as well as evaluation for HTLV-1 myelopathy with pain, stiffness and gait problems. Neurological examination showed flat, mask-like face with a positive glabellar reflex. She was noted to have decreased power bilaterally in upper and lower extremities with brisk reflexes and hypertonia. Motor examination of upper extremities showed a strength of 4 x 5 with brisk reflexes and also noted to have resting as well as intention tremor. Lower extremity muscle power was 2 x 5 with brisk reflexes and bilateral clonus and bilaterally upgoing toes. Sensory examination was normal with generalized diffuse rigidity. Her workup included computed tomography (CT) scan of the brain showing bilateral basal ganglia calcifications and mild cortical atrophy, magnetic resonance imaging (MRI) brain T2W image demonstrating low signal intensity from iron accumulation in the red nucleus, and substantia nigra and atrophy of the cerebral cortex and superior vermis of the cerebellum  but an extensive literature search using Google Scholar and PubMed highlighted only one case report of HTLV-1 virus infection with myelopathy and parkinsonism . PD is aHTLV-1 infection can be diagnosed with serology, by detection of antibodies in the serum or in the cerebrospinal fluid (CSF) and by polymerase chain reaction (PCR) detection of proviral DNA . In our The HTLV-1 virus commonly affects the white matter in the thoracic and lumbar segments of the spinal cord but it also affects the brain white matter . The mosHTLV-1 commonly affects spinal cord but it can also affect the brain. Clinicians providing care should be vigilant in diagnosing these syndromes associated with HTLV-1 infection. Our patient presented with stiffness, mask-like face, bilateral hand tremors at rest and gait problems, and after a thorough neurological examination was diagnosed with parkinsonism and appropriate treatment was started. We are reporting the case so that these syndromes can be identified and treated appropriately. Future research should be targeted on HTLV-1 pathological changes in the brain so that its pathology can be better understood, improving patient care and outcome in these debilitated patients.