Human T-cell leukemia virus type 1 (HTLV-1) has worldwide distribution and is considered endemic in southwestern Japan. HTLV-1 infection has been associated with adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) besides other diseases. This cross-sectional study aimed to investigate the prevalence, risk factors and molecular characterization of HTLV-1, among the world’s largest population of Japanese immigrants and their descendants outside of Japan, in São Paulo, Southeast Brazil, as well as to analyze the phylogenetic relationship among isolates of HTLV-1. From July to December 2017, 2,139 individuals from five Japanese associations were interviewed and submitted to blood collection. All serum samples were first tested for the presence of anti-HTLV-1/2 antibodies by ELISA and then peripheral blood from individuals with positive serological results were analyzed for the presence of HTLV-1 5’LTR proviral DNA. Partial sequencing of the 5’LTR region of HTLV-1 proviral DNA was performed by Sanger. The prevalence of HTLV-1 infection was 5.1% (CI 95%: 4.2–6.0). In the multiple logistic regression model, HTLV-1 infection was associated with age ≥ 45 years, female sex, being first and second-generation Japanese immigrants, and having sexual partners with history of blood transfusion. The phylogenetic analysis revealed that all HTLV-1 were classified as Cosmopolitan (1a) subtype. Of them, 47.8% were classified as Transcontinental (A) subgroup and 52.2% as belonging to the Japanese (B) subgroup. Although most HTLV-1-infected patients were asymptomatic (97.3%), blurred vision was associated with HTLV-1 infection. The high prevalence of HTLV-1 infection found in this studied population and especially the intra- and interfamily HTLV-1 transmission presents an urgent call for preventive and control responses of this infection in Brazil. What is the prevalence of HTLV-1 among the largest world population of Japanese immigrants and their descendants outside Japan? There are approximately 1.5 million people of Japanese descent in Brazil and most of them living in São Paulo state, Southeast Brazil. The prevalence of HTLV-1 infection was 5.1% among 2,139 individuals from five Japanese associations in São Paulo. The prevalence of 5.1% (95% CI: 4.2–6.0) of HTLV-1 infection found in this study is higher than those observed in other studies conducted in HTLV-1 endemic areas and in specific groups at high risk from Brazil. Among HTLV-infected individuals, the majority (86.2%) were unaware of their HTLV diagnosis. The HTLV-1 infection was associated with the age ≥ 45 years, being the first and second generation of Japanese immigrants, and having sexual partners with a history of blood transfusion. Epidemiological data, along with molecular results also demonstrated high occurrence of similar sequences transmitted intra- and interfamily. The high prevalence of HTLV-1 infection found in this studied population presents an urgent call for preventive and control responses of this infection in Brazil. Human T-cell leukemia virus type 1 (HTLV-1) has been associated with an aggressive malignant disease known as adult T-cell leukemia/lymphoma (ATL) and a chronic inflammatory neurological disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) .Although most people infected by the HTLV-1 remain asymptomatic, they are viral reservoirs and maintain the HTLV-1 transmission chain, transmitting it both to their children and to their sexual partners, once HTLV-1 is horizontally transmitted through close contact with fresh blood and unprotected sexual intercourse, as well as vertically through prolonged breast feeding –5.It is estimated that at least 5 to 10 million individuals are infected with HTLV-1 worldwide, but this might be underestimated , some paAs far as its genotypic variability is concerned, the Cosmopolitan subtype Transcontinental subgroup (HTLV-1aA) is recognized as the most prevalent in Brazil, followed by the Japanese subgroup (HTLV-1aB) ,14. The With the beginning of Japanese immigration to Brazil in 1908, mostly from provinces of Kumamoto, Hokkaido, Kagoshima, and Okinawa, Brazil is the country with the largest number of Japanese and their descendants outside of Japan. São Paulo state (Southeast region), followed by Paraná (South region) and Mato Grosso do Sul  concentrate the largest number of Japanese and their descendants in Brazil . In factSince South America has been considered as a very important focus of HTLV-1 carriers and associated diseases, correct estimates of the epidemiological and molecular aspects of the HTLV-1 infection in the city of São Paulo (the largest community of Japanese descendants outside Japan) are important to develop healthcare strategies to reduce new transmissions of HTLV-1.The study was approved by the Ethics Committee on Research Involving Human Subjects of the Federal University of Mato Grosso do Sul (CEP/UFMS), under protocol number 2.154.785 CAAE: 70626017.4.0000.0021. Written informed consent was obtained from all study participants prior to enrollment.This cross-sectional study was conducted among Japanese immigrants and their descendants living in São Paulo. After the project was disclosed to the community by means of educational lectures and distribution of flyers and leaflets with information on symptoms, prevention and diagnosis of HTLV-1/2 infection, participants were recruited from five Japanese Associations in the state of São Paulo (named A to E) between July and December 2017.Individuals were eligible to participate if they were Japanese immigrants (born in Japan), Japanese descendants (had any genetic relationship to a Japanese immigrant) or nondescendants who had a relationship to a Japanese immigrant by marriage, living in São Paulo and able to provide written informed consent. Based on an estimated prevalence of HTLV-1 infection of 6.8% , a signiA total of 2,139 volunteer individuals agreed to participate. Informed written consent was obtained after a detailed explanation of the study at the time of blood collection from all participants or their legal guardians in case of individuals under age 18 or with a mental disability. After written informed consent, each volunteer participant was interviewed on sociodemographic characteristics, risk factors associated with HTLV infection and on their history of medical past. Right after the application of the questionnaire, blood samples were collected. In addition to an anti-HTLV test, a free medical examination was offered to all participants and non-participants by the health campaign (named Kenko Kodo). Free testing for diabetes and blood pressure were also proposed.The diagnostic criteria to define the HTLV-1 infection included an initial screening test , and the repeatedly reactive samples by screening assay were checked by confirmatory analysis, using Polymerase Chain Reaction (nested PCR) or Western Blot (WB). The individuals with anti-HTLV seropositive results were contacted for further blood collection for retesting by ELISA. DNA was extracted from whole blood of HTLV seropositive samples using the QIAamp DNA Blood Mini Kit , according to manufacturer’s instructions. Nested PCR designed to amplify a 218 bp fragment of the tax gene of the both HTLV-1 and -2 was performed as previously described  to confiThe 5’LTR amplicons were purified using PureLink Quick PCR Purification Kit , according to the manufacturer’s instructions. The fragments were sequenced using BigDye Terminator Cycle Sequencing Ready Reaction Kit and ABI 1373  using Sanger’s method. The nucleotide sequences were edited in DNASTAR Lasergene software and aligned with published HTLV-1 sequences from various geographic regions using Mega X software . These pRegarding family pedigree, out of 109 HTLV-1 infected individuals, 12  became the index cases and 53 of their relatives who agreed to participate of this study, including mother, father, wives, husbands, sisters, brothers-in-law, sisters-in-law, children, nephews and nieces, were investigated.Being aware of being HTLV-infected was defined by self-report; those who had never been HTLV tested or who did not know their HTLV status were defined as being unaware of being HTLV-infected.2) to assess differences between proportions. Prevalence rate of HTLV-1 infection and 95% confidence interval (CI) were calculated. Bivariate analysis was performed to verify potential predictors of exposure to HTLV-1/2 infection (anti-HTLV-1/2 positive). Variables that presented a p-value < 0.20 were included in multiple linear regression. The selection of variables for the final model was performed stepwise, according to the number of events per variable (EPV). Hosmer-Lemeshow test was used to assess goodness-of-fit, choosing the best regression equation. P-values <0.05 were considered statistically significant.Data was entered into the Research Electronic Data Capture (REDCap) online database. Data analysis was performed in the statistical software Stata SE, version 13 . This study used the chi-square test , piercing (4.1%), having been in prison (0.3%), injection drug use (0.1%) and multiple sexual partners (4.2%). Nearly 7% of the participants reported having received a blood transfusion, and of these, almost half was before 1993, when compulsory HTLV-1 screening was implemented for Brazilian blood donors.Tax gene region by nested PCR, thus confirming the HTLV infection. The same 107 samples also amplified the HTLV-1 5`LTR region, thus confirming the HTLV-1 infection. The two positive samples remaining in the screening tests were confirmed for the HTLV-1 infection by Western Blot, since there was no whole blood sample from the respective individuals to perform the DNA extraction for the nested PCR. Therefore, considering that a total of 109 individuals were confirmed for infection by the HTLV-1, a prevalence of 5.1% (CI 95%: 4.2–6.0) of HTLV-1 infection was thus found , however one individual presented the chronic form of ATL and two of them depicted the possible factor qualification for HAM/TSP (HAM-PS). In addition, participants who reported blurred vision had 2.1-fold greater chance of being HTLV-1 positive compared with those without this symptom .The HTLV-1 proviral DNA was detected by amplification of the 5’LTR region by nested PCR in 98.2% (107/109) of anti-HTLV-1 positive samples. The remaining two positive samples were confirmed only by Western Blot (MP Diagnostics HTLV BLOT 2.4–Singapore), due to difficulty of venipuncture during the collection of new samples. Out of 107 samples with amplified LTR, 90 (84.1%) were successfully sequenced. The phylogenetic analysis of the HTLV-1 5’LTR region revealed that all isolates were classified as Cosmopolitan (1a) subtype. Of these, 47.8% HTLV-1 isolates were classified as belonging to the Transcontinental (A) subgroup and 52.2% to the Japanese (B) subgroup . The HTLThis large cross-sectional survey was conducted to investigate HTLV-1 infection in Brazil among the world’s largest Japanese population outside of Japan. There are approximately 1.5 million people of Japanese descent in Brazil and most of them living in São Paulo state . The preth century [This high rate (5.1%) found in the present study is similar to that observed in previous study involving Japanese community of Campo Grande, Mato Grosso do Sul State (6.8%)  probably century . Also, tSimilarly to findings from previous studies, the prevalence of HTLV-1 infection in our cohort was higher among females than in males (p = 0.04). Although vertical transmission is not sex-biased, sexual transmission is female-biased, which explains why females overall have a higher HTLV-1 carrier rate than males . MoreoveIncreasing age was associated with HTLV-1 infection in our cohort, probably due to prolonged exposure to HTLV-1 infection, which is a well-known characteristic and observed in other populations in previous studies ,13,39–41To reinforce these findings, when male and female were analyzed separately, HTLV-1 infection was independently associated with being a first or second (p = 0.01) generation immigrant for both males  and females . Moreover, for female participants having partners with a history of receiving blood transfusions was also associated with HTLV-1 infection (p = 0.033).A higher prevalence of HTLV infection was found in the population coming from Association E (8.9%). This fact can be explained by the higher proportion of first Japanese generation participants in this association when compared to the others (p<0.001). HTLV-1 infection is endemic in Japan, particularly clustered on the islands of Kyushu and Okinawa, in southwestern Japan . AlthougAs previously mentioned, having a partner with a history of receiving blood transfusion history was independently associated with HTLV-1 infection in our cohort. However, we should highlight that only three of the nine HTLV-1-positive women who reported so, informed their partners received blood transfusions before 1993. Of these three women, two were widows and therefore, we did not have access to results of their partners’ HTLV-1 antibody screening. The other one was married to an HTLV-1-non-infected individual. Among the other six women, five reported that their husbands’ blood transfusions occurred after 1993 and one did not know when it happened. Of these six women, 4 were married to subjects who participated in this study and of these, 3 were anti-HTLV-1 positive. Serological screening for HTLV-1/2 became mandatory in blood banks in Brazil in 1993, effectively reducing the risk of HTLV-1 transmission by transfusion .The lack of association of HTLV-1 infection with STI differs from other studies conducted in Brazil ,33. ThisAccording to Kamoi and Mochizuki (2012), all patients with initial diagnosis of HTLV-1 infection should be strictly screened for ocular symptoms . AlthougThe characteristic clinical picture of the chronic spastic paraparesis is not always easily found, sometimes the patient depicts symptoms or signals that are incipient indicators of HAM/TSP . In BrazThe phylogenetic analysis revealed that all HTLV-1 belong to the Cosmopolitan (1a) subtype and 47.8% are from the Transcontinental (A) subgroup and 52.2% to the Japanese (B) subgroup. These two subgroups occur in Japan, with the prevalence of the Japanese –16. In BSome limitations found in this study should be considered. Firstly, recall bias may have impaired the reliability of information provided by study participants about risk behaviors, such as about having been breastfed in childhood. But it is noteworthy that cross-breastfeeding was a frequent past habit, reported to occur on ships that brought the first Japanese immigrants, according to our interviewees. Moreover, the number of symptomatic HTLV-1 carriers may have been underestimated, since difficulty in walking, the main clinical manifestation of the HAM/TSP, may have led to the non-participation of some subjects in the study. Despite these limitations, our study, having a large sample size, is representative of this population group and provides important novel insights about HTLV-1 infection in Brazil, where the largest population of Japanese and their descendants outside Japan live.In conclusion, our study demonstrates that the prevalence of HTLV-1 infection is high among Japanese immigrants and their descendants in the state of São Paulo, where the largest number of Japanese and their descendants outside Japan live. Epidemiological data, along with molecular results also demonstrated high occurrence of similar sequences transmitted intra- and interfamily. Therefore, our results on this neglected disease highlight the urgent need of preventive strategies to decrease spread and transmission of this virus in this area of Brazil, including i) effective HTLV testing strategies and routine systematic HTLV-1/2 antenatal screening and counseling of Brazilian pregnant women by the national health system ; ii) health educational programs to avoid sexual and other intrafamily horizontal transmissions; iii) systematic counseling of HTLV-1-infected individuals to reduce risk of viral transmission.


Polarized hyperspectral images can reflect the rich physicochemical characteristics of targets. Meanwhile, the contained plentiful information also brings great challenges to signal processing. Although compressive sensing theory provides a good idea for image processing, the simplified compression imaging system has difficulty in reconstructing full polarization information. Focused on this problem, we propose a two-step reconstruction method to handle polarization characteristics of different scales progressively. This paper uses a quarter-wave plate and a liquid crystal tunable filter to achieve full polarization compression and hyperspectral imaging. According to their numerical features, the Stokes parameters and their modulation coefficients are simultaneously scaled. The first Stokes parameter is reconstructed in the first step based on compressive sensing. Then, the last three Stokes parameters with similar order of magnitude are reconstructed in the second step based on previous results. The simulation results show that the two-step reconstruction method improves the reconstruction accuracy by 7.6 dB for the parameters that failed to be reconstructed by the non-optimized method, and reduces the reconstruction time by 8.25 h without losing the high accuracy obtained by the current optimization method. This feature scaling method provides a reference for the fast and high-quality reconstruction of physical quantities with obvious numerical differences. Due to reflecting the rich spectral and spatial characteristics, hyperspectral imaging is widely used in agricultural detection ,2, food In order to overcome the defects caused by the FTSP, the compressive sensing (CS) theory  has beenTo avoid the drawbacks of CCISP, we proposed the full polarization-compressed hyperspectral imaging (FPCHI) system. As shown in In this paper, we therefore propose a two-step reconstruction method for the FPCHI system to achieve the fast and efficient reconstruction of the full-Stokes parameters simultaneously under compressive measurement. It is well known that the first Stokes parameter represents the total light intensity, and the last three Stokes parameters represent various light intensity differences. Therefore, the first Stokes parameter and the last three Stokes parameters are not in the same order of magnitude. According to the inherent numerical difference of the four Stokes parameters, the reconstruction process is divided into two steps. Both steps are reconstructed based on the CS theory. Specifically, the first Stokes parameter comes from the first reconstruction, and the last three Stokes parameters come from the second reconstruction. Due to relying on the inherent properties of the four Stokes parameters, the two-step reconstruction method can be used to reconstruct the full-Stokes parameters of any targets. In addition, we verify this proposed method based on the polarized hyperspectral images measured in our laboratory. In order to further illustrate the effectiveness of the two-step reconstruction method, it is also compared with the above optimization method and the non-optimized method.As shown in Let M makes up the modulation matrix of the proposed system. Moreover, the values of M are all non-zero values. Thus, after Full polarization compression can be achieved by the combination of QWP and LCTF. The Mueller matrices of QWP and LCTF are denoted as n is less than four. Then, the In order to realize the compression measurement of the four Stokes parameters, G is a known matrix obtained by CMOS, and H is determined by the current system parameters. In particular, F is an unknown matrix to be reconstructed.The compression measurements of each spatial pixel in each spectral band are combined to form the full polarization-compressed hyperspectral images Inspired by polarization compression, we consider the polarization reconstruction of one spatial pixel in one spectral band. Since the first Stokes parameter is much larger than the other three Stokes parameters, it is difficult to reconstruct the four Stokes parameters at the same time. In this paper, the four Stokes parameters are reconstructed in two steps using feature scaling.R times to obtain a new modulation matrix as follows:In the first step, suppose that the first Stokes parameter Therefore, Equation  can be rThen, the deformed polarization information is reconstructed based on the CS theory. The deformed polarization information is first sparsely represented byIn this equation, Equation  into EquEquation , we can lgorithm , the spa follows:θ^R=argmiEquation , we can In the second step, the last three Stokes parameters are reconstructed again. Substituting the deformed first Stokes parameter into Equation , we can In Equation , HL∈Rn×3ented byfL=ΨL×θL,Equation , the newUsing the TwIST algorithm  again, tThen according to Equation , the lasR times the deformed parameter. In other words, let Finally, the required four Stokes parameters can be obtained by combining the reconstruction results from the above two steps. The last three Stokes parameters are taken from the reconstruction results of the second step. The first Stokes parameter is obtained by deforming the reconstruction result of the first step. According to the scaling rule in the first step, the first Stokes parameter is In order to measure the polarized hyperspectral images as the simulation input of the FPCHI system, we first established the definition-based polarization hyperspectral imaging (DBPHI) system . As showIn the experiment, the SFW is equipped with 18 spectral filters  whose center wavelengths are from 520 nm to 690 nm with an interval of 10 nm. The LP  works effectively from 510 nm to 800 nm, and the QWP  operates from 325 nm to 1100 nm. The CMOS detector  has Then, full polarization compression and reconstruction are simulated on the measured images of each spatial pixel in each spectral band. Assume that polarization modulation is performed twice. The linear polarization angle of the incident surface of LCTF remains 0, and the fast axis angles of QWP are 105 and 150, respectively. That is, Therefore, full polarization-compressed hyperspectral images As described in The discrete W transform (DWT) matrix  severs aBased on Equations –8), we , we 8), In the second step, the modulation matrix corresponding to the last three Stokes parameters isThe DWT matrix  is also Then according to Equations –12), th, th12), By integrating the reconstruction results based on these two independent steps, we can obtain the ideal four Stokes parameters In order to compare with feature scaling, we also reconstruct the polarized hyperspectral images not only with the sparse basis optimized by particle swarm optimization (PSO) , but alsIt can be seen from Equation  that theFurthermore, The reconstruction effect can be seen not only from the image details, but also from the PSNR values shown in In particular, there are two reasons why the two-step reconstruction method can quickly and accurately reconstruct polarization information. On the one hand, the first Stokes parameter contributes much more to the compressed image than the last three Stokes parameters. As a result, when the four Stokes parameters are reconstructed at the same time, the weight flooding problem makes the reconstruction of the last three Stokes parameters seriously affected by the first Stokes parameter. The two-step reconstruction method solves this problem. In the first step where all four Stokes parameters are involved, only the reconstruction result of the first Stokes parameter is retained. Then, the last three Stokes parameters are reconstructed in the second step, avoiding the interference of the first Stokes parameter. On the other hand, the reconstruction error can be better controlled under the two-step reconstruction method. In the two steps, different reconstruction algorithms can be used or the regularization weight value can be adjusted to accurately improve the reconstruction accuracy of each Stokes parameter. Moreover, there is no need to learn to optimize the sparse basis, which not only greatly improves the reconstruction efficiency, but also is widely applicable to various targets with different polarization characteristics.The FPCHI system can achieve both compression measurement of full-Stokes parameters and hyperspectral imaging. The QWP with adjustable fast axis angle unites the LCTF with selectable linear polarization angle to modulate the four Stokes parameters. Meanwhile, the LCTF also enables flexible selection among a large number of fine spectral bands to meet hyperspectral imaging. Then for each spectral band, the two-step reconstruction method can recover four Stokes parameters from the compressed measurements based on feature scaling. In the two steps, the first Stokes parameter is extracted from the first reconstruction, and the last three Stokes parameters are from the second reconstruction.Based on the polarized hyperspectral images measured in our laboratory, both the polarization-compressed hyperspectral imaging of the FPCHI system and the two-step reconstruction method are simulated sequentially. The simulation results perform well in the evaluation of image display, grayscale distribution and PSNR values. Compared with the optimization method using machine learning, the reconstruction accuracy of the two-step reconstruction method does not decrease and still maintains a satisfactory level. Further in terms of reconstruction efficiency, the two-step reconstruction method shortens the 9.9 h required for the optimization method to 1.65 h, which means a time reduction of 83.33%.The grayscale value of the first Stokes parameter is quite different from the last three Stokes parameters, so the two-step reconstruction method separates their reconstruction processes. This method is conducive to more accurately reduce the reconstruction error of each step. Since it is based on the inherent properties of the Stokes parameters, this method is suitable for various targets with different polarization characteristics. Moreover, for the compression of the spatial domain or other domains, this provides a reference for the reconstruction of parameters that are originally at different orders of magnitude. However, this approach still faces some limitations. Next, we will focus on solving the intelligent and appropriate selection of feature scaling values. Through further optimization, such as optimizing the polarization encoding pattern, more efficient reconstruction will be achieved to apply the polarization hyperspectral imaging to actual target detection and other monitoring fields.


In this study, the effects of static and multi-pulsed mild-intensity high hydrostatic pressure (HHP) treatments  on the extractability and accumulation of phenolics and carotenoids in whole carrots were evaluated. HHP treatments were applied for the time needed to reach the desired pressure  either as a single pulse or multi-pulse . Likewise, a single sustained treatment (5 min) applied at 60 or 100 MPa was evaluated. Individual carotenoids, free and bound phenolics were quantified after HHP treatment and subsequent storage . As an immediate HHP response, phenolic extractability increased by 66.65% and 80.77% in carrots treated with 3P 100 MPa and 4P 60 MPa, respectively. After storage, CUT 60 MPa treatment accumulated free (163.05%) and bound (36.95%) phenolics. Regarding carotenoids, total xanthophylls increased by 27.16% after CUT 60 MPa treatment, whereas no changes were observed after storage. Results indicate that HHP processing of whole carrots at mild conditions is a feasible innovative tool to enhance the nutraceutical properties of whole carrots by increasing their free and bound phenolic content while maintaining carotenoid levels. HHP treated carrots can be used as a new functional food or as raw material for the production of food and beverages with enhanced levels of nutraceuticals. Horticultural crops contain high levels of health-promoting compounds, also known as nutraceuticals . These nIn this context, based on previous reports mainly using plant cell cultures, we recently proposed that nonthermal processing technologies such as ultrasound, high hydrostatic pressure (HHP) processing and mild-intensity pulsed electric fields could emulate a wound-like stress response in horticultural crops ,7,8. ThiRecently, it was reported that HHP processing come-up time CUT, 60–100 MPa), which requires less processing time and energy cost, could be used as an innovative tool to increase the extractability and induce the biosynthesis of free and bound phenolic compounds in carrots during storage (3 d at 15 °C) 0 MPa, wh. Thus, iTherefore, the objective of this study was to evaluate the effects of different static and multi-pulsed mild intensity pressure treatments  on the extractability and biosynthesis of phenolic compounds and carotenoids in whole carrots immediately after processing and after storage (48 h at 15 °C).v/v] were obtained from Fisher Scientific . Ethyl acetate, hexane, sodium hydroxide (NaOH), and orthophosphoric acid [(H3PO4) 0.097% v/v] were acquired from Desarrollo de Especialidades Químicas . Mili-Q® ultrapure water was obtained from a purification system . Chlorine  was purchased from a local supermarket . High barrier pouches (27 × 35 cm) made of nylon and low-density polyethylene , used for vacuum packaging carrots before HHP treatment, were acquired from Filmpack® . All other chemicals were obtained from Sigma-Aldrich® .Ethanol (HPLC grade), methanol (HPLC grade), acetone (HPLC grade), methyl tert-butyl ether  and hydrochloric acid [(HCl) 98% Carrots were obtained from a local market . Prior to HHP treatments, carrots were washed with tap water, sanitized with a chlorine solution  for 5 min, and dried using a paper towel. Sanitized carrots were vacuum-packed (67.7 kPa) in pairs using high barrier pouches and a Multivac R230 series 542 . High hydrostatic pressure was applied using a pressurization system , equipped with a 2 L chamber. Tap water was used as pressure transmission fluid. Temperature inside the chamber varied from 21.5 °C to 24.27 °C depending on the pressure treatment applied.−1 over 15.33 ± 2 s to reach 60 MPa and 4.61 MPa s−1 for 20.67 ± 2 s to reach 100 MPa. Decompression of the chamber was instantaneous (<1 s). For storage studies, samples were removed from vacuum-sealed bags after processing and placed in hermetic plastic containers (3.8 L). To keep the temperature constant (15 °C) containers were maintained in a Symphony incubator  for 48 h. For each HHP treatment and the control group, 6 carrots of ~130 g each were used per replicates. Three independent experiments were conducted. Carrots were collected at 0 h and 48 h to quantify individual free and bound phenolics as well as individual carotenoids.The immediate and late response of different HHP conditions, on the content of bioactive compounds, was evaluated. HHP conditions evaluated were: come-up-time (CUT) as the time needed to reach the expected hydrostatic pressure, two pulses (2P), three pulses (3P), four pulses (4P) and sustained pressure for 5 min, each at 2 levels of hydrostatic pressures: 60 and 100 MPa. Pressurization rate was 4.06 MPa sg, 20 min), which remained in the supernatant while the bound phenolics were extracted from the pellet (carrot solids). To remove the solvents, samples were carefully dried using a vacuum evaporator . Samples were dissolved using a methanol solution  prior to filtration using a nylon membrane  and chromatographic analysis.Free and bound phenolic compounds in samples were extracted and identified using the method described by Viacava et al. ,13. Briev/v, 2 mL), filtered  and analyzed with a HPLC-DAD 1260 Infinity System . The determination of individual phenolics was performed as previously described by Viacava et al. [Bound phenolics were extracted from free phenolic precipitate using 10 mL of 2 M NaOH and high temperature (95 °C) as indicated by Viacava et al. . The hyda et al. .−1. The identification of individual phenolic acids was performed by comparing their absorption spectrum with authentic standards. The detector was a diode array programmed to record signals at 280, 320 and 360 nm. For quantification of individual free and bound phenolic compounds, standard curves were performed (0.25–500 ppm). Finally, phenolic concentration was calculated as mg kg−1 dry weight (DW). The moisture content (%) of the samples was determined by the air–oven method (AACC 44-15A) at time 0 day, as well as the weight loss of samples, which was obtained during storage, in order to calculate the percentage (%) of moisture content.Free and bound phenolics profiles from carrot samples were assessed by chromatographic separation using a C18 reverse-phase column with dimensions 4.6 mm x 250 mm, 5 µm particle size  with its corresponding C18 guard cartridge. Mobile phases were composed of water (phase A) and methanol:water  both adjusted to pH 2.4 with ortophosphoric acid. Injection volumes were 10 µL for each replicate and the solvent gradient to elute the compounds was 0/100, 3/70, 8/50, 35/30, 40/20, 45/0, 50/0, 60/100 (min/% phase A) with a flow rate of 0.8 mL min−1 BHT) for 30 s using a tissuemizer . Homogenates were filtrated through a Whatman No. 1 filter paper under vacuum. This procedure was repeated 5 times for each sample to ensure complete color extraction. Filtered extracts were collected on a 100-mL volumetric flask and volume was completed with the acetone solution. Extracts were filtered with PTFE membranes  and placed in vials whose headspaces were filled with nitrogen to prevent oxidation of carotenoids.Carotenoids were evaluated as indicated by Viacava et al. . Extract−1. Carotenoids were recorded at 450 nm and individual quantification was carried out with standard curves from β-carotene (0.1–30 ppm) for carotenoids and lutein (0.125–12 ppm) for xanthophylls. The identification of carotenoids was performed by comparison of elution order and maximum absorption wavelength (λmax) with those reported in previous reports [Carotenoids were separated on a C30 reverse-phase column  , coupled to a corresponding C30 guard cartridge. Vial chamber and column temperatures were 4 and 30 °C, respectively. The mobile phase consisted of 50% methanol, 45% MTBE, and 5% water. Injection sample volume was 25 μL. The system was isocratic, where total elution time was 25 min at a flow rate of 0.5 mL min reports ,14.p < 0.05). Likewise, to compare between treatments at different storage times Student’s t-test pair comparisons were performed using Minitab software .Results were expressed as mean values ± standard error of the mean. To evaluate significant statistical differences between means a one-way ANOVA was carried out, followed by a Tukey test  treated samples B. HoweveO-caffeoylquinic acid (3-O-CQA), 5-O-caffeoyquinic acid (5-O-CQA), 3,5-di-O-caffeoylquinic acid , 4,5-di-O-caffeoylquinic acid , ferulic acid, 3,4-O-diferuloyquinic acid  and isocoumarin. The free phenolic compounds identified agree with previous reports on the identification of free phenolic profiles in carrots [The free phenolics compounds identified included gallic acid hexoside (GAH), 3- carrots ,13.When comparing the control against HHP processed samples, 3,4-diFQA was the free phenolic that showed a marked increase after pressurization . This inAfter storage, samples treated with HHP showed a significant increase in GAH, ferulic acid, 3,4-diFQA and isocoumarin as compared with the control before storage (CBS). The increases in GAH were detected in samples treated with CUT 60 MPa (97.45%) and 4P 60 MPa (149.69%). On the other hand, carrots treated with CUT at 60 MPa and 100 MPa showed increases in ferulic acid by 120.16% and 175.14%, respectively, as compared with CBS. Regarding 3,4-diFQA, all HHP treated samples showed a significant increase in its concentration, where samples treated with CUT 100 MPa presented the highest increase (2080.45%). Finally, the samples treated with 2P 60 MPa showed 60.42% of increase in isocoumarin content as compared with CBS .O-coumaroylquinic acid (4-O-CoQA), 5-O-coumaroylquinic acid (5-O-CoQA), caffeic acid, and p-coumaric acid, rutin and quercetin. The bond phenolic profile identified in whole carrots is in agreement with previous reports [The effect of HHP and storage time on the content of individual bound phenolic compounds in whole carrots is shown in  reports ,13.p-coumaric acid increased by 124.7% in samples treated with 4P 60 MPa, whereas the levels of the other bound phenolics remained unaltered. After storage (48 h at 15 °C) the bound phenolics that showed an increase in HHP treated samples as compared with CBS were the caffeoyl glucose, 4-O-CoQA, and p-coumaric acid. For caffeoyl glucose, the treatment that showed a significant increase (84.98%) as compared with CBS was the whole carrots treated with 100 MPa for 5 min. On the other hand, the 4-O-CoQA showed increases by 150.74%, 127.6%, and 68.65%, for whole carrots treated with 3P 60 MPa, 5min 60 MPa, and CUT 100 MPa, respectively, as compared with CBS. Finally, the p-coumaric acid increased by 68.65%, and 99.19% for 3P 60 MPa and CUT 60 MPa, respectively, as compared with CBS; whereas whole carrots treated with 100 MPa increased by 79.06%, 45.97%, 76.9%, and 43.88% as compared with CBS, for the sample treated under that pressure for the CUT, 2P, 3P, and 4P, respectively  in 3P 60 MPa treatment as compared with the stored control, whereas no significant difference was detected between stored HHP treated carrots and samples before storage. Regarding zeaxanthin content, the CUT 60 MPa and 2P 100 MPa showed 42.19% and 42.9%, respectively, higher levels as compared with the stored control samples; whereas all HHP treated samples showed higher zeaxanthin content than the CBS. However, a non-significant difference was detected in the stored HHP treated samples as compared with samples before storage. A similar trend was observed for β-cryptoxanthin content quantified after storage in HHP treated samples, where the 60 MPa CUT and 1P/5min treatments showed 31.21% and 33.41%, respectively, higher β-cryptoxanthin content as compared with the stored control. Likewise, the 100 MPa 2P, 4P and 1P/5min showed 32.75%, 42.82%, and 38.04%, respectively, higher β-cryptoxanthin content as compared with the stored control. Regarding carotenes (α- and β- carotene) content, no significant differences were detected between HHP treated samples and the control after storage.HHP is commonly applied in foods to induce microbial and enzymatic inactivation and enhance the shelf-life of processed products, while preventing undesirable changes in the sensory, nutritional and physicochemical properties . HoweverThe stress-responses in plant tissues can be divided as immediate and late stress responses ,6,16. InAs earlier described, the higher quantification of bioactive compounds is one of the early stress responses of horticultural crops to mild intensity HHP treatments. Thus, higher levels of free and bound phenolics, and xanthophylls can be attributed to higher extractability and stress-induced biosynthesis of secondary metabolites ,19. HighAn additional explanation for increased quantification of hydroxycinnamic acids and xanthophylls as an immediate response to HHP, could be the pressure-induced biosynthesis of secondary metabolites. This phenomenon has been previously reported for papaya, where the application of HHP treatment increased the gene expression and accumulation of carotenoids as an immediate response to pressurization . SimilarAs a result of the production of primary and secondary stress signaling molecules in the immediate response to HHP, the secondary metabolism of plants is activated leading to the accumulation of secondary metabolites as a late stress response. In the present study, the accumulation of free and bound phenolics was detected after 48 h of storage at 15 °C. On the other hand, HHP treated whole carrots did not show significant accumulation of carotenoids, although stored HHP treated samples showed higher levels of carotenoids as compared with the stored control.The accumulation of free and bound phenolics in HHP treated whole carrots has been associated with a wounding-like response ,7,8,10. In this study, a multi-pulse HHP approach was evaluated as a potential strategy to induce the biosynthesis and accumulation of bioactive compounds in whole carrots. To the best of our knowledge, this is the first report in the literature that evaluated the effects of multi-pulse HHP treatments at physiological pressures (60 and 100 MPa) on the content of carotenoids and phenolics in whole carrots. The results showed higher total phenolics (free and bound) as the intensity of pressure and number of pulses incremented. As expected, whole carrots subjected to more stages of pressurization showed higher extractability of phytochemicals. On the other hand, lower pressure intensities elicited the biosynthesis of free and bound phenolics. The results presented herein, indicate that HHP could be used as an innovative tool to obtain the next generation of fresh produce that could be consumed as fresh food, as raw material for the production of processed foods or as a primary source of valuable natural antioxidants. Further studies should evaluate the shelf-life stability of HHP-treated whole carrots under the selected processing conditions, to validate the feasibility of incorporating the product in the fresh-produce market. Variables such as instrumental color change, microbiological analyses, sensory acceptability, texture analyses and enzymatic activities  should be included in the aforementioned shelf-life study.


Fertility problems may have a devastating impact on the people involved. Specifically, in highly pronatalist settings like Ghana, the personal and social consequences are high. This study focused on the relationship between stigmatisation because of fertility problems and quality of life among Ghanaian women and men, and the possible mediating role of coping strategies.Participants  were recruited with the help of a patient organisation and a hospital in Accra. Standardised instruments were used to measure the stigmatisation of having fertility problems, fertility quality of life and coping with fertility problems. Partial Pearson r correlations were conducted, followed by bootstrapped mediation analyses (PROCESS macro).Stigmatisation was negatively correlated with fertility quality of life, and fertility quality of life was negatively correlated with active-avoidance coping. Active avoidance coping partially mediated the relationship between being stigmatised because of fertility problems and fertility quality of life.Professionals working with people with fertility problems should pay more attention to how people are coping with experiences of stigmatisation. Prevalence rates estimate that 15% of Ghanaian women of childbearing age experience fertility problems in that they are not able to conceive a child after one year or more of regular unprotected sexual intercourse with their partner . What doThe values Ghanaians attach to having children are multifaceted . GhanaiaDue to the various roles children have and the high value placed upon having children and parenthood in Ghana, the consequences of having problems in becoming pregnant are very high . QualitaThe experiences of being stigmatised because of having fertility problems also influences the well- being of Ghanaian women with fertility problems. They report high levels of infertility related stress, and a high prevalence of depression . Less isAccording to the psychological mediation framework , coping Studies among Ghanaian women who have had to deal with fertility problem showed that these women used several coping strategies such as drawing on their religion, avoiding certain situations, getting the support of their partner, or becoming economically independent . HoweverIn total 49 Ghanaian men and women who were experiencing fertility problems in conceiving a child and were visiting a clinic participated in the study by filling in a questionnaire. Demographic characteristics and the fertility status and history of the participants are presented in Participants were recruited with the help of the Association of Childless Couples of Ghana (ACCOG), and a private hospital. Both were located in Accra . People who were visiting the ACCOG or had a doctor’s appointment in the hospital for their fertility problems were informed about the study, and were asked to participate in it by handing over an information letter, in which the aims and method of the study was described. In the information letter we emphasised that participation was voluntary and the decision to participate or not would not have any consequences on their relationship with ACCOG nor on their access to infertility treatments. It was also stated on the letter that participants may skip any questions that raised discomforts, or could stop filling out the questionnaire at any time. It was also mentioned that if a participant objected to any question, that she or he had the opportunity to contact the investigators about this. Contact information of a counsellor was also included in the information letter .After signing the informed consent letter, participants were asked to fill out the questionnaire on the spot. They could also make an appointment with one of the research assistants to fill out the questionnaire at another time and/or at place. When filling out the questionnaire. research assistants were present in case participants had questions. The research protocol was approved by the Institutional Review Boards of the Noguchi Memorial Institute for Medical Research, University of Ghana and of the Faculty of Social and Behavioral Sciences, University of Amsterdam.Data was collected with a paper-pencil questionnaire and included information on demographic characteristics and fertility status and history, experiences with stigmatisation of having fertility problems, coping with fertility problems, and quality of life in men and women experiencing fertility problems . The scales that were used were all standardised instruments and were selected based on the criteria that they:(a) showed good psychometric properties in previous studies, (b) were available in English and (c) have been used in multiple countries, including non-Western parts of the world.The demographic characteristics measured included questions regarding biological sex, age, relationship status and age of partner, place of residency, and religion. Regarding the fertility status and history, information was obtained on whether the participant had ever been pregnant, whether the participant had ever conceived a child from their current partner, how long she or he had been trying to conceive, and what the reasons were for their current fertility problems.Participants’ perceived experiences of stigmatisation as a result of having problems with conceiving or being childless was measured with a scale that has previously been developed and used by Donkor and Sandall . Donkor In this current study a coping questionnaire was used that has previously been developed by Schmidt et al. , 2005b. http://sites.cardiff.ac.uk/fertiqol/scoring) participants’ raw scores were multiplied by 25/k, where k is the number of items (24). The score for the total scale can be between 0 and 100 .The total score of the Core Fertility Quality of Life . Partial Pearson r correlations  were calculated to investigate the associations between experiences with stigmatisation of having fertility problems, the four coping strategies, and fertility quality of life. To examine the mediation effect of the four coping strategies on the association between stigmatisation and quality of life, a bootstrapped mediation through the PROCESS macro (model 4) as developed by Hayes  was usedActive-avoidance coping was the only proposed mediator that met our inclusion criteria for the bootstrapping mediation analysis. Therefore, it was only assessed whether this coping style mediates the association between stigmatisation for having fertility problems and fertility quality of life, and evidence was found for this mediating effect of active-avoidance coping . In this study we looked at the possible relationship between experiences of stigmatisation and fertility quality of life in a cross section of Ghanaian women and men with fertility problems. Moreover, we looked at the possible mediating role of coping styles to better understand the underlying differences in how infertile Ghanaian women and men are affected in terms of their quality of life. The moderate average level of stigma and the wide diversity in stigma experiences confirms the findings of a larger sample of infertile women from the South of Ghana . Mean scConforming to our hypothesis and confirming previous studies on the relationship between stigma and aspects of wellbeing , we founSome limitations need to be taken into account. First of all, we should be careful in drawing causal conclusions as we used a cross-sectional design in our study, whereas causality can be seen as a time-dependent concept. Although it is plausible, for example, that low levels of quality of life are a result of active-avoidance coping style, it might also be that problems related to quality of life might contribute to such a coping style. This can only be explored using a longitudinal study . SecondlThe recruitment through a hospital and a patient support group might also have influenced coping and quality of life. All participants actively sought support , which is a way of coping with infertility in itself. Additionally, seeking treatment might have given them a feeling of hope and agency, which may have had a positive influence on their quality of life . Based oTaking the aforementioned limitations into account, we would recommend further research to get a better picture of how the relationship between stigmatisation and fertility-related quality of life and the mediating role of coping strategies develop over time, using a longitudinal design, recruiting a bigger sample of Ghanaian men and women with fertility problems through multiple channels in multiple areas. A larger sample size might also enable us to distinguish possible differences in sex, education, work status, in the relationship between stigma and fertility-related quality of life and coping strategies, and the relationship between coping and quality of life. Insights into all these factors would give professionals working with people with fertility problems better guidelines to assist them.In the meantime, our cross-sectional finding of the mediating role of the active-avoidance coping strategy in the relationship between experiencing stigma and fertility quality of life, suggests that it would be helpful if professionals would teach infertile men and women to use coping strategies other than the active-avoidance strategy in reaction to stigmatisation. Reducing the use of this strategy may increase quality of life. An even more powerful way to improve the fertility quality of life of Ghanaian men and women would be to reduce stigmatisation. Increasing access to proper information about fertility problems, its causes, and to fertility treatments may might not only lead to reduced infertility and increased timely responses, but also to less stigmatisation of fertility problems and childlessness .


Ensiling is a simple and effective method for long-term preservation; however, less information exists about the ensilability characteristics of garlic stalk (GS). Therefore, the objectives were to examine the ensiling feasibility of GS.Lactobacillus plantarum KU5 in the presence or absence of 5% molasses and ensiled for 7, 14, and 28 d. As an alternative storage method, GS was co-ensiled with wet citrus pulp (CP) at different proportions . Analysis was made on physicochemical, fermentative, and nutritional parameters.The GS was ensiled alone or inoculated with The GS was found to be a biomass which is difficult to ensile. A combination of microbial inoculant and molasses was successful in the improvement of the silage fermentation quality of GS. Co-ensiling of GS with wet CP at the mixing ratio of 50:50 provided the most desirable silage fermentation parameters, including the substantial lactic acid formation, low final pH, minor effluent loss, and the more favorable organoleptic properties.Co-ensiling GS with CP appears to be a simple and viable method of conservation, enabling the more efficient utilization of these by-product resources over a prolonged period. The worldwide production of garlic amounted to 27 million tons in 2016, with China, India, and Korea being the major garlic producing countries . HoweverThe ensiling process is an effective and simple conservation technology that is known to acidify biomass, thus it inhibits the growth of spoilage-causing microorganisms, thereby providing safe and long-term conservation for moist forages . InoculaCitrus is one of the major fruits in Korea, which is mainly cultivated on Jeju Island and the southern coast line of the main land, with an estimated production of 0.68 million tons in 2011 . An estiLactobacillus plantarum (L. plantarum) inoculation without or with molasses would affect the silage fermentation patterns of GS. The second objective was to develop an alternative storage method through co-ensiling of GS with CP at different proportions, and determine the appropriate mixing ratio for efficient silage fermentation.To our knowledge, limited reports are available concern ing the silage fermentation characteristics of GS. Therefore, the first objective was to evaluate how L. plantarum KU5 (Accession No. HQ542227) inoculant was re-cultivated for 3 consecutive days on MRS (de Mann Rogosa Sharpe) broth  at 36°C for 24 h, before being applied onto the biomass.Different batches of sun-dried GS were provided from a garlic processing plant located in Uysung County, Kyung-Buk province and an agricultural marketing center located in Chungju city, Chung-Buk province in Korea. Before silage making, GS was cut into pieces of 20 to 30 mm and hand-mixed thoroughly, then divided into the respective treatments. L. plantarum KU5 (LAB) in the absence or presence of % molasses (LAB+ M). Ensiling duration was 7, 14, and 28 d at room temperature (20°C to 24°C). The detailed description of the silage making procedure is reported in our previous article [6 cfu/g of fresh mass. In a subsequent experiment, GS was mixed with CP at decremental proportions of 70:30, 60:40, 50:50, and 40:60 (wet weight), mixed manually, and then assigned to respective treatments. The packing density of approximately 200 kg dry matter (DM)/m3 was applied for all silages, before the sealing of the polyethylene silos. From the designated silo openings, the representative samples were collected randomly for the subsequent analyses.The GS was ensiled in nylon-polyethylene pouches (5 replicates per each treatment) without inoculant or with Contents of DM, crude protein (N×6.25), ether extract, crude ash, neutral-detergent fiber (NDF), and acid detergent fiber, both corrected for residual ash, were determined according to the standard methods of the Association of Official Analytical Chemists . True prData analyses were performed using the general linear model procedure . Treatment effects were evaluated with the analysis of variance in a completely randomized design. Each mini-silo was considered as the experimental unit. Microbial counts were transformed to log10-scale before statistical analysis. The difference among treatments was identified using Tukey’s multiple range test. Values less than 0.05 probability level were considered significant. Tendency was discussed at 0.05<p<0.1.3-N formation increased considerably (a 4.4-times increase after 28 d of ensiling), which indicates the microbial conversion of WSC into lactic acid and protein breakdown during ensiling, respectively [3-N [The silage fermentation metabolites and chemical composition of GS ensiled for 7, 14, or 28 d, are presented in ectively ,19. The ely [3-N .10 cfu of LAB per gram of fresh biomass is the required LAB number to ensure the fast decline of silage pH [10 cfu/g) to promote fast acidification and efficient ensiling, the silage fermentation was not successful, as evidenced by high NH3-N content and high silage pH after 28 d of fermentation. This suggests that GS is a difficult-to-ensile biomass. This can be justified by three explanations: i) The GS contains an inadequate content of WSC, which probably did not provide sufficient sugars for lactic acid production, and thus silage mass acidification [An approximate number of 8 log ilage pH . In the fication ,6. ii) Tfication . iii) GSfication  that incfication . These ffication . Based oThe silage fermentation dynamics of GS inoculated with LAB in the absence or presence of molasses in relation to the length of ensiling , are presented in 6 cfu/g of fresh GS). Surprisingly, LAB+M treatment promoted the more favorable fermentative patterns (with respect to a lower pH and less NH3-N production) than untreated or LAB-treated silage. After 28 d of ensiling, LAB+M-treated silage showed a 35% increase in lactic acid content and a 0.9-unit decline in pH, with respect to the LAB-inoculated silage. In support, Huisden et al [Inoculation with LAB prior to ensiling has been successful in the promotion of desirable fermentation patterns. However, when the epiphytic population of LAB exceeds the inoculant application rate, the domination of inoculant bacteria in the silage is difficult . In the en et al  reported3-N accumulation (631 ppm). However, when GS was ensiled with LAB+M, the silage pH dropped to 5.3 (after 28 d of ensiling) and as anticipated, less NH3-N was formed (3-N formation [Earlier findings confirmed that the combined use of the LAB inoculant and molasses improved the silage quality indices of various crops ,8,22. Fos formed . This obThe chemical composition of GS ensiled with LAB in the absence or presence of molasses in relation to the length of ensiling, is presented in This experiment evaluated the co-ensiling of GS with CP as an alternative storage technology, which has been successful in improving the silage fermentation quality of several feedstuffs . For exaThe pH of GS co-ensiled with incremental proportions of CP is illustrated in The silage fermentation, sensory, and physical parame ters of GS co-ensiled with incremental proportions of CP are shown in 3-N concentration lessened, which is indicative of diminished proteolysis and deamination during ensiling [3-N content accounted for 25.4% of the total N in GS 50% +CP 50% silage, which is approximately 70% less than the silages with 30% or 40% CP. The fast acidification of silage mass suppresses the growth of putrefying microorganisms, this probably occurred as the mixing ratio of GS:CP decreased, resulting in a lower formation of NH3-N, which is associated with the improved utilization of silage-N and thus microbial protein synthesis in the rumen [3-N formation.As the GS:CP mixing proportion decreased, NH ensiling . After 1he rumen . Decreashe rumen . It appeNo putrid odor or moldy spots were detected in the ex perimental silages , which sThe chemical composition of GS co-ensiled with incremen tal proportions of CP is shown in L. plantarum inoculant and molasses favorably promoted the silage fermentation quality of GS. As an alternate storage method, the effective silage fermentation was also achieved when GS was co-ensiled with CP at the mixing ratio of 50:50. Co-ensiling of GS with CP appears to be a simple and feasible method of preservation, which would provide the more efficient utilization of these waste resources over longer periods, especially in places where they are generated in large quantities.A huge amount of GS is generated during the harvesting season, an amount considerably exceeding the immediate use. This surplus is mainly composted into soil or incinerated, which is associated with serious environmental concerns. The simultaneous application of


GM-CSF-dependent macrophage polarization has been demonstrated in rheumatoid arthritis (RA). Our aim was to seek diagnostic/prognostic biomarkers for undifferentiated arthritis (UA) by analyzing GM-CSF expression and source, macrophage polarization and density in joints of patients with UA evolving to RA or PsA compared with established RA or PsA, respectively.+ macrophages by multicolor immunofluorescence. GM-CSF and activin A levels were also quantified in paired synovial fluid samples. CD163+ macrophage density was determined in all groups by immunofluorescence.Synovial tissue (ST) from patients with UA evolving to RA , PsA , persistent UA , established RA (n=12) and PsA (n=10), and healthy controls (n=6), were analyzed. Cell source and quantitative expression of GM-CSF and proteins associated with pro-inflammatory (GM-CSF-driven) and anti-inflammatory (M-CSF-driven) macrophage polarization  were assessed in ST CD163+ CD90+ fibroblast, CD90+ endothelial cells) and CD163+ sublining macrophages were the sources of GM-CSF. ST CD163+ macrophages from all groups expressed pro-inflammatory polarization markers . Expression of the M-CSF-dependent anti-inflammatory marker CD209 identified two macrophage subsets (CD163+ CD209high and CD163+ CD209low/-). CD209+ macrophages were more abundant in ST from healthy controls and PsA patients, although both macrophage subtypes showed similar levels of pro-inflammatory markers in all groups. In paired synovial fluid samples, activin A was detected in all patients, with higher levels in UA>RA and RA, while GM-CSF was infrequently detected. ST CD163+ macrophage density was comparable between UA>RA and UA>PsA patients, but significantly higher than in persistent UA.Synovial stromal cells (FAP+ FAP+ synovial fibroblasts, CD90+ activated endothelium and CD163+ macrophages in different types of arthritis. The polarization state of ST macrophages was similar in all UA and established arthritis groups, with a predominance of pro-inflammatory GM-CSF-associated markers. CD163+ macrophage density was significantly higher in the UA phases of RA and PsA compared with persistent UA. Taken together, our findings support the idea that GM-CSF is a strong driver of macrophage polarization and a potential therapeutic target not only in RA but also in PsA and all types of UA.GM-CSF is highly expressed by sublining CD90 Undifferentiated arthritis (UA) is frequent in early arthritis clinics, with a prevalence of 30%–50% –3. AfterRA and peripheral PsA are chronic inflammatory diseases mediated by the immune system and, although joint damage may be early and frequent in both diseases if not treated, their pathogenesis may be different regarding the relative implication of the adaptive and innate immune systems in each of them. This is illustrated by therapies that are useful in treating RA  but not PsA. Inversely, therapies inhibiting the IL-17/IL-23 pathway are successful in treating PsA, but do not work in RA patients  or synovitis, the primary target of inflammatory joint diseases . Synovitd damage , 11. In d damage , 13. Subynovitis  although and PsA .+ lining macrophages constantly repopulate from proliferating tissue resident interstitial macrophages and form a protective epithelial-like barrier that disintegrates during experimental arthritis  but low levels of the M-CSF-associated marker CD209  and M-CSF-driven (anti-inflammatory) monocyte-derived macrophages –23 has aer CD209 –23. Giveer CD209 –26, our Patients with UA and synovial biopsies were retrospectively selected when they met the classification criteria for RA or PsA during follow up. Synovial biopsies obtained by rheumatologic arthroscopy  from inf+ macrophages. CD115 and/or CD163 macrophage pan-markers were used to segment these cells in tissues and estimate the mean intensity of the proteins of interest. Pan-markers were intentionally acquired in saturated conditions to better depict macrophages areas. Macrophage density was normalized based on the selected tissue area (mm2). After background subtraction, data were plotted using GraphPad software .Multicolor confocal microscopy was performed as previously described , 29, 30.® Imaging processing program (Olympus®) as described previously in detail  using the following monoclonal antibodies: anti-CD3 , anti-CD20 , anti-CD68 , and anti-CD138  and an aminoethylcarbazole color reaction  as described previously in detail . Finallyn detail .6 cells/ml for 7 days in RPMI 1640 supplemented with 10% fetal calf serum, at 37°C in a humidified atmosphere with 5% CO2, and containing GM-CSF (1000 U/ml) to generate GM-CSF-polarized macrophages (termed GM-MØ), or M-CSF (10 ng/ml) for M-CSF-polarized macrophages (termed M-MØ). Cytokines were added every 2 days.Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats from normal donors over a Lymphoprep (Nycomed Pharma) gradient. Monocytes were purified from PBMC by magnetic cell sorting using CD14 microbeads (Miltenyi Biotech). Monocytes were cultured at 0.5 x 101 FITC/γ2a PE (BD) was included as a negative control.Phenotypic analysis was carried out by immunofluorescence using standard procedures. Mouse monoclonal antibodies used for cell-surface staining included FITC-labeled anti-CD14 (Miltenyi), anti-CD115 (R&D Systems) and FITC-labeled anti-CD163 . Alexa Fluor-647-labeled isotype-matched secondary antibody (Jackson ImmunoResearch) was used to stain anti-CD115. Control γ® 480 (Roche Diagnostics). Assays were made in triplicate and results normalized according to the expression levels of TBP. The results were obtained using the ΔΔCT method for quantitation. The oligonucleotides used to quantify mRNA transcripts were (5´-3´): CD209s cagagtggggtgacatgagtgac; CD209as gtgaagttctgctacgcaggag; INHBAs ctcggagatcatcacgtttg; INHBAas ccttggaaatctcgaagtgc; MMP12s tgtcactaccgtgggaaataag; MMP12as aacactggtctttggtctctcag; TBPs cggctgtttaacttcgcttc; TBPas cacacgccaagaaacagtga; TNFαs cagcctcttctccttcctgat; TNFαas gccagagggctgattagaga. For Gene Set Enrichment Analysis (GSEA) (http://software.broadinstitute.org/gsea/index.jsp) . Quantitative real-time PCR (qRT-PCR) was performed on a LightCyclerdex.jsp) , the gendex.jsp) . The predex.jsp) .GM-CSF  and activin A  were quantified using commercially-available ELISA.t test were used and Spearman’s correlation was determined. A value of p<0.05 was considered statistically significant . The analysis was made using GraphPad Prism software.The Mann Whitney test and Student’s Clinical, demographic and serologic data of the patients included are detailed in + activated endothelium, CD90+ FAP+ fibroblasts from the sublining layer around the endothelium, and in sublining CD163+ macrophages (+ or CD4+ T cells (data not shown). These results indicate that GM-CSF is detected similarly in the earlier and established stages of RA and PsA, and in persistent UA, and that GM-CSF-producing cells are pathogenic CD90+ FAP+ synovial fibroblasts  samples from patients with persistent UA (n=12), UA>RA (n=9), UA>PsA (n=7), RA (n=9) and PsA (n=9) indicated that GM-CSF levels were below detection levels in most SF samples, except for 4 out of 9 (44%) persistent UA patients who were csDMARD naïve, and one UA>PsA patient out of 7 analyzed (14%), who had increased GM-CSF levels . By contINHBA), the metalloproteinase MMP12 and the chemokine CCL17 , which differed in their respective MFI for CD209 fluorescence  (+ CD209+ cells (80%) than that found in UA  expressed the proinflammatory markers activin A, TNFα and MMP12 in all patients from the UA and established disease groups  (2 in UA>RA and 622 macrophages/mm2 in UA>PsA (2) showed significantly lower macrophage density than UA>RA and UA>PsA , CD20 (B cells), CD138 (plasma cells), and CD68 (macrophages), synovial tissue from each UA and definite arthritis group was categorized into three distinct synovial pathotypes: lympho-myeloid, diffuse-myeloid, and pauci-immune . ResultsSynovial tissue macrophages are key producers of cytokines relevant to the physiopathology of chronic arthritis and their changes correlate with disease activity, radiographic progression, and response to therapy –13. Howe+ macrophages isolated from synovial fluid of patients with active RA indicate that they exhibit a transcriptomic and protein profile compatible with GM-CSF-skewed macrophage polarization  had higher histological CD68+ macrophage scores than patients persisting with undifferentiated arthritis (Our results show that the density of CD163rthritis .neg) and that sustained disease remission in RA is actively maintained by MerTKpos macrophages, which are also predominant in healthy synovium. If validated with additional cohorts of patients with RA in remission, the ratio of MerTKpos to MerTKneg STMs could be useful as a predictor of disease flare versus sustained remission to help with treatment decisions in clinical practice , which could modify our findings, this factor was comparable in all groups. The study has the strength of the follow-up of UA patients until they met RA or PsA classification criteria, the group of persistent UA patients (treated or not with csDMARDs) as positive controls and the ST from healthy controls. Likewise, the results were consistent for all types of arthritis.+ macrophages from synovial tissue of UA patients evolving or not to RA or PsA compared with healthy controls. CD163+ macrophages expressed a pro-inflammatory profile of biomarkers  in synovial tissue from all arthritis subtypes studied, while in paired samples of synovial fluid only activin A levels were widely detected. CD163+ macrophages were significantly higher in the UA phases of RA and PsA compared with persistent UA. Taken together, our findings support the concept that GM-CSF is a strong driver of macrophage polarization and a potential therapeutic target, not only in RA but also in PsA and in UA. We also found that the ST sublining of undifferentiated and established arthritis patients showed a high percentage of CD163+ CD209low/- whereas healthy controls exhibited a high percentage of CD163+ CD209+, suggesting they may be resident macrophages with a potential anti-inflammatory function. Further studies using single cell technologies will definitively characterize phenotypes and function of synovial macrophages in undifferentiated arthritis.Our results show a high, generalized expression of GM-CSF in stromal cells and sublining CD163The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study was approved by the Ethics Research Committee of the Hospital Clinic of Barcelona, Spain (HCB/2014/0579) and signed informed consent was obtained from each patient. The patients/participants provided their written informed consent to participate in this study.AP-K and JC had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of data analysis. AP-K and JC were responsible for the study design. AC, RC, and JR performed clinical data acquisition. SF-R, LE-C, RS, RG-C, and AP-K were responsible for the analysis. SF-R, LE-C, AC, RC, AP-K, and JC performed the data interpretation. Manuscript preparation was by SF-R, LE-C, AC, AP-K, and JC. All authors contributed to the article and approved the submitted version.This work was supported by grants PI17/00037 from Instituto de Salud Carlos III/FEDER to AP-K, grants PI14/00785 and PI17/00993 from Instituto de Salud Carlos III to JC, grant RIER RD16/0012/0010 and RD16/0012/0007 FEDER to JC and AP-K, respectively, and co-financed by the European Regional Development Fund “A way to achieve Europe” (ERDF). SF-R is supported by a contract from Instituto de Salud Carlos III (FI18/00109) and AP-K is supported by FIBHGM. AC received grants from the Hospital Clinic of Barcelona “Emili Letang 2014”, and the Catalan Society of Rheumatology.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.


We investigated the prevalence of human T-cell lymphotropic virus types 1 and 2 (HTLV-1/2) infection in patients with hematological diseases from the western Amazon region of Brazil.Samples from 306 patients were submitted for the molecular diagnosis of HTLV-1/2 infection by real time PCR (qPCR), with amplification, sequencing, and phylogenetic analysis of the long terminal repeat (LTR) region. .A 29-year-old male carrier of sickle cell anemia with a history of multiple blood transfusions was diagnosed with the HTLV-2c subtypeThis study describes the first known occurrence of HTLV-2c in the urban area of Brazil’s western Amazon region. All patients were receiving consultation for hematological diseases and only those with a confirmed diagnosis were eligible to participate . PeripheHTLV-1/2 infections were diagnosed from proviral DNA extracted from patients’ peripheral blood mononuclear cells (PBMC). Samples were submitted to the amplification of tax gene sequences conserved across HTLV-1/2 isolates, as has been previously describedHTLV-2 subtyping was performed through amplification of the 788pb DNA fragment corresponding to the 5’LTR region, as has been described previouslyThe DNA was sequenced after cloning the PCR product into a pGEM®-T vector system . The 5’LTR region was sequenced using M13 primers and a BigDye Terminator v3.1 cycle sequencing kit . Phylogeny inference was performed with MEGA-6 software  applying the neighbor-joining method under dataset encompassing sequences retrieved from GenBank. Robustness of tree topology was evaluated with 1,000 bootstrap pseudoreplicates. ,We diagnosed one HTLV-2 infected patient (0.32%). A similar prevalence has been found in the general population of Brazilian cities such as Belo Horizonte (0.32%) and Rio de Janeiro (0.33%)The infected patient (JLM151) is a 29-year-old married male patient from a low-income family, with a high school degree and is a carrier of sickle cell disease. He has lived in Manaus his entire life, declared that he has no tattoo or body piercing, and had never used illicit injectable drugs. The patient has been receiving blood transfusions since early childhood. His spouse and parents refused to be tested for HTLV-1/2 infection. The patient does not have a history of sexually transmitted diseases and did not know about his infection. He was among the patient group with the highest blood transfusion rates in the past 12 months .,The 611bp 5’LTR fragment sequenced in this study shares over 99% genetic similarity with the HTLV-2c 5’LTR region sequences available in the GenBank database. The JLM151 sample (GenBank accession number MN 266910) was clustered within the subtype HTLV-2c, the same clade of the samples isolated from the Kayapo tribe (AF306731 and AF306731) .The distConsidering that the virus was detected in a patient suffering from sickle cell disease and a frequent recipient of blood transfusions, the infection could plausibly have been transmitted through a transfusion. Brazil has performed serological screening for HTLV-1/2 infection in blood supplies since 1993. However, a study conducted at blood centers from 2007-2009 estimated a residual transfusion risk of 5.0 per 10In conclusion, this study demonstrated the prevalence of the HTLV-2c virus in a new geographic area of the western Amazon region. The emergence of HTLV-2c in new urban areas points out the need for expanded public health surveillance of HTLV-1/2 infections. Better and updated information regarding the epidemiology of HTLV-1/2, especially related to the risk of transmission through transfusion, must be regularly monitored by blood centers to guarantee the safety of vital blood transfusions.


To report the outcomes of two cases of infectious scleritis with severe scleral thinning managed using combined mechanical debridement, cryopreserved umbilical amnion (UA) and amniotic membrane (AM) grafts, and antimicrobial therapy.Pseudomonas aeruginosa subsequent to pterygium surgery and intravitreal injection, respectively. Surgical debridement was followed by UA grafting, AM transplantation, and antimicrobial therapy. Epithelialization and vascularization were seen within 1–4 weeks post-operatively. At last follow up of 5–6 months, the structural integrity of the sclera remained preserved with no infection recurrence, graft necrosis, or wound dehiscence.Two patients presented with severe infectious scleritis with progressive scleral melts secondary to This case series highlights the utilization of UA grafting along with AM transplantation and topical and systemic antibiotics to preserve the globe in cases of severe infectious scleritis. He had a history of pterygium surgery with mitomycin-C in this eye 5 years prior. The visual acuity was 20/200 and intraocular pressure (IOP) was 11 mmHg. Examination revealed 9 mm × 5 mm scleral necrosis nasally with purulent material and scleral plaques. There was significant scleral thinning with uveal show but no uveal prolapse . There wThe patient was taken to the operating room. Toothed forceps and a 57-blade were used to carefully dissect the nasal scleral plaque, for which pathologic evaluation demonstrated dense calcification. The necrotic scleral material was then debrided with a 57-blade and sent for additional cultures. Dilute ceftriaxone was used to wash out the area of necrotic sclera. A 0.75 cm × 1.0 cm UA patch graft  was placed over the area of scleral thinning and secured into place using 16 interrupted 7-0 Vicryl sutures. The graft was trimmed at the edges so that it was flush with the edge of the corneal limbus. Next, a 2.5 cm × 2.0 cm AM  was cut in half. The first half was placed over the UA patch graft, and was secured in place with Tisseel fibrin glue . The other half was placed over the first AM and secured with 4 interrupted 7-0 Vicryl sutures at the corners of the graft. Subconjunctival aliquots of ceftriaxone were administered surrounding the area of infection, and the eye was patched and shielded. Post-operative drop regimen included fortified tobramycin 14 mg/ml and topical moxifloxacin 0.5% every 2 h with a taper over six weeks, oral ciprofloxacin 500 mg twice daily for four weeks, and cyclopentolate 1% twice daily for six weeks. Topical prednisolone acetate 1% four times daily was started three weeks after surgery and was tapered off over the ensuing 10 weeks.On post-operative day 1, the patient's vision was 20/300. The UA with overlying AM remained in good position. Slit lamp examination revealed 3+ conjunctival injection, inferior KPs, a small hyphema, and 2+ NS. At 1 week, the visual acuity was counting fingers due to worsening of the cataract, and IOP was 8 mmHg. Examination revealed that the UA remained in good position with staining inferiorly and dissolution of the overlying AMs. There was 3+ conjunctival injection diffusely, a chemotic plica, an area of depression between the plica and border of the AM graft, inferior KPs, a sliver hyphema, and 2–3+ brunescent NS. By 1 month, the visual acuity remained at counting fingers, and IOP was 10 mmHg. Examination revealed similar findings as 1 week, however the UA showed early vascularization with no staining present. The conjunctival inflammation had decreased to 2+ injection and hyphema had resolved. At 3 months, visual acuity had slightly improved to 20/400, and IOP was 12 mmHg. Examination revealed that the UA had dissolved with residual vascularization and no staining, a quiet conjunctiva, cornea with no infiltrate or epithelial defect, and 3+ brunescent NS with pigment on the anterior capsule . Six mon2.2Pseudomonas aeruginosa that had failed topical antibiotics and had progressed to suspected endophthalmitis. The patient underwent an intravitreal tap and injection for presumed keratitis-related endophthalmitis, and subsequently developed scleritis two weeks later in the location of the intravitreal injection, at which point she was referred to our practice. The visual acuity was hand motion and IOP was 4 mmHg. Slit lamp examination was notable for a 4 mm × 6.5 mm inferotemporal scleral abscess that was non-contiguous with the cornea, a diffusely edematous corneal stroma with patchy tissue loss (ranging from 40 to 80%), a 9 mm × 10 mm corneal epithelial defect, and a 3.5 mm hypopyon . At 5 months, examination revealed a white and quiet conjunctiva, well vascularized area previously covered by UA, and similar corneal findings . The vis3Pseudomonas aeruginosa being the most common infectious agent.,Pseudomonas aeruginosa.Bacterial etiologies comprise the majority of IS cases, with ,,In less than 20% of cases of IS, medical therapy alone  results in complete resolution of infection. There are several possible explanations for this relatively low success rate, including the sclera's dense collagen fibers and avascularity. As a result, surgical debridement is often used adjunctively to decrease the microbial load and improved antibiotic delivery.Pseudomonas-associated keratoscleritis occurring after pterygium excision, early debridement, topical and systemic antibiotics, and low-dose oral prednisone resulted in successful outcomes.In this case series, mechanical debridement, UA and AM grafting, and antimicrobial therapy successfully preserved the globe in two cases of necrotizing IS and scleral melt. Case 1 developed IS after pterygium surgery using mitomycin-C and Case 2 developed IS secondary to an intravitreal injection for presumed keratitis-associated endophthalmitis. Scleral melting and IS are well reported complications following pterygium surgery.Pseudomonas aeruginosa using debridement, scleral graft, tenonplasty, and AM transplantation. Finger et al. described the use of super-thick amniotic membrane grafts for ocular surface reconstruction after resection of conjunctival melanoma, squamous cell carcinoma, sebaceous carcinoma, and atypical pterygium, and for scleral necrosis; retention and complete epithelialization were achieved in all cases.Various tissues and materials have been used to cover the sclera following debridement of large areas.,Anatomically, the UA contains the AM as its outer layer and a loose, proteoglycan-rich Wharton's jelly as its inner layer. Both the UA and AM contain collagen, fibronectin, glycosaminoglycans, and HC-HA/PTX3, however UA is about 10 times thicker than AM alone.4This case series highlights the utilization of UA to maintain the scleral integrity and, along with AM transplantation and antibiotics, preserve the globe in cases of severe IS.Written consent to publish deidentified medical information and clinical photographs was obtained from the patients. This report does not contain any personal information that could lead to the identification of the patients.No funding or grant support.All authors attest that they meet the current ICMJE criteria for Authorship.CJR: Consulting: Avedro, Bio-Tissue, Kala, Sun Ophthalmics, Shire.The following author has no financial disclosures: ZAS.


Under physiological conditions, cAMP signaling plays a key role in the regulation of cardiac function. Activation of this intracellular signaling pathway mirrors cardiomyocyte adaptation to various extracellular stimuli. Extracellular ligand binding to seven-transmembrane receptors  with G proteins and adenylyl cyclases (ACs) modulate the intracellular cAMP content. Subsequently, this second messenger triggers activation of specific intracellular downstream effectors that ensure a proper cellular response. Therefore, it is essential for the cell to keep the cAMP signaling highly regulated in space and time. The temporal regulation depends on the activity of ACs and phosphodiesterases. By scaffolding key components of the cAMP signaling machinery, A-kinase anchoring proteins (AKAPs) coordinate both the spatial and temporal regulation. Myocardial infarction is one of the major causes of death in industrialized countries and is characterized by a prolonged cardiac ischemia. This leads to irreversible cardiomyocyte death and impairs cardiac function. Regardless of its causes, a chronic activation of cardiac cAMP signaling is established to compensate this loss. While this adaptation is primarily beneficial for contractile function, it turns out, in the long run, to be deleterious. This review compiles current knowledge about cardiac cAMP compartmentalization under physiological conditions and post-myocardial infarction when it appears to be profoundly impaired. The beating heart ensures an essential function by supplying an oxygen- and nutrient-rich blood flow to the organs. Efficient blood flow distribution in both the pulmonary and systemic circulations allows the exchange of oxygen and carbon dioxide. Pump function is maintained by the automatic cardiac excitation that spontaneously triggers rhythmic and periodic contractions. This dynamic process is modulated in response to various physiological and pathophysiological stimuli such as hormones, neurotransmitters, cardiac injuries and stresses that adapt/maladapt heart function ,2. CardiEvidence of cardiac cAMP signaling compartmentalization relies on experiments performed in the late 1970s ,7,8. A hIschemic heart diseases or coronary heart diseases (CHDs) are the leading causes of death in industrialized countries and kill 15 million people worldwide each year . ACS, whThis section details the current knowledge about major cardiac GPCRs that directly modulate cAMP signaling and for which the regulation of cardiac function under physiological conditions and post-MI has been reported . Other cα and Gβ,γ. In the inactive state, these three subunits form a heterotrimer with GDP-bound Gα. Upon GPCR stimulation, GTP replaces GDP, which leads to a conformational change and releases the Gα subunit. The nature of the Gα protein  and Gβ,γ ensure specificity in the activation of intracellular signaling. GTP hydrolyzation in GDP by the Gα subunit triggers GPCR inactivation ..AKAP9, PsAC produces cAMP in the mitochondrial matrix, which triggers PKA-dependent phosphorylation of cytochrome-c, regulating ATP production ,97. In mIn the heart, Protein Kinase-A (PKA) is the major cAMP downstream effector. The PKA holoenzyme is a heterotetramer composed of two regulatory (R) and two catalytic (C) subunits. R subunits contain an N-terminal docking and dimerization (D/D) domain, a linker region with a PKA C subunit inhibitor site and two cAMP binding sites . The D/D domain locates within the first 45 residues of each R subunit, which mediates R dimerization and A-kinase anchoring protein (AKAP) docking . The PKAPKA plays a central role in the regulation of cardiac function and adaptation to stress ). Upon β-AR stimulation and cAMP production, PKA phosphorylates CEC substrates and modulates positive chronotropic, inotropic and lusitropic responses . In SAN,Ca channel opening . E. E2+ rels (CICR)  [120]. Hs (CICR)  121]..2+ releaFinally, the role of Epac in cardiac pathophysiology remains ambiguous as it has been reported to have effects that are both pro- and anti-apoptotic, both pro- and anti-fibrotic and both pro- and anti-hypertrophic, with pro-arrhythmic effects ,123. ThePost-MI, the profibrogenic factor TGF-β1 decreases Epac1 expression in cardiac fibroblasts located at the border zone, which inhibits fibroblast migration and induces collagen synthesis . In cont+ and K+) and Ca2+ with distinct properties  proteins are plasmalemma proteins that are encoded by three different genes  133]. P. P133]. The spatial regulation of intracellular PKA signaling is orchestrated by its compartmentalization to a precise subcellular subset mediated by direct interaction with A-kinase anchoring proteins (AKAPs) . AKAPs aAKAPs, which are widely expressed in various cell types and tissues, form precise macromolecular complexes to ensure the specificity in PKA-dependent signaling cascades ,145,146.AKAP1  is a dual-specificity AKAP that distributes to the mitochondrial outer membrane (AKAP1 exhibits a PP1 (Protein Phosphatase 1) binding sequence and a KH-Tudor domain that restricts and favors translation of specific mRNAs . Furthermore, AKAP1 anchors CaN , PDE4 and Drp1 (GTPase Dynamin-related protein 1) to regulate mitochondrial fission and fusion  is a PKA type II-specific anchoring protein for which roles and functions have been extensively investigated over the past years in cardiac physiological and pathophysiological conditions  , myocyte apoptosis and an enhanced hypertrophy with pulmonary edema . T. TAKAP6 ack loop C. Activay PDE4D3 C 159]. . AKAP6  is a PKA type II-specific anchoring protein highly expressed in the heart and anchors PKA RIIα with higher affinity than RIIβ  is upregulated at the border of infarction zones, which exhibits cardioprotective functions against I/R-induced apoptosis ..AKAP10  is a PKA type II-specific anchoring protein that locates in the cytoplasm or associates with the cytoskeleton network and plasma membrane , PDE4D, CaN and β-Ars (β-Arrestins)   . . AKAP12 f AKAP12 D. Of notreviewed .AKAP7  is a PKA type II-specific anchoring protein with several isoforms which have been reported to be present in the heart including shorter  and longer variants ). All isoforms present a common C-terminus that includes the AKB domain. Differences in AKAP7 isoforms occur in the length of the N-terminus. The distribution at the plasma membrane of shorter AKAP7 isoforms is facilitated by palmitoylation and myristoylation of N-terminal residues. The leucine zipper motif present in the C-terminus mediates AKAP7α interaction with LTCC . . PDE2A g3-AR located in the T-tubule network induces NO synthesis by eNOS, which, in turn, triggers cGMP production . . PDE3A ad PDE3B) B 224]. . PDE3A ad PDE3B) B 225]. . PDE3A a fashion B 226]. . PDE3A aOf note, pan-PDE3 inhibitors  have been clinically used to treat heart failure patients . Despite2+ load and leak  that generate all alternatively spliced variants. These alternative splicings provide PDE4s with different sizes and molecular masses that are classified into long, short, super-short and dead-short isoforms. PDE4s exhibit a unique N-terminal domain (TD), a central region containing upstream conserved regions 1 and/or 2 (UCR1 and UCR2) and a highly conserved C-terminal catalytic domain. The N-terminal domain confers to PDE4s a specific subcellular location, binding to signalosome complexes and the modulation of enzymatic activity  A 233]. . 233]. Pand leak B [235,23and leak . PDE intPDE4D KO mice exhibit an increase in the cardiac cAMP level at the Z-line, which triggers PKA-dependent RyR2 hyperphosphorylation and calcium leak . InteresFinally, the PKA-dependent phosphorylation of Hsp20 (at Ser16) has been reported in rats to provide cardioprotection and to reduce myocardial apoptosis after I/R injury ,244,245.In addition to the major types, other PDEs hydrolyzing cAMP co-exist in the heart but exhibit lower expression . Their roles in the regulation of cardiac physiology or post-MI remain unclear and need to be further characterized. Beside its detailed role in CF, PDE1 also modulates the cAMP-PKA signaling pathway in the SAN and regulates pacemaker activity . PDE8A rCyclic AMP signaling is one of the best characterized signaling pathways and critically involved in many cellular and physiological processes. It is now widely accepted that in the heart, cAMP plays a central role in the regulation of cardiac function. This includes the binding of an extracellular ligand  to GPCR, which, via G proteins and AC, leads to an intracellular increase in cAMP content. Next, this triggers activation of intracellular downstream effectors , which, in a synergistic process, mediate the adaptation of cardiomyocytes to distinct stimuli. The temporal regulation of the cAMP signaling depends mainly on AC and cAMP-PDE activities. AKAPs provide the means to achieve both spatial and temporal regulation of PKA signaling. By scaffolding signaling effectors and modulators in the vicinity of substrates, AKAPs ensure spatial specificity in signal transduction. In addition, they also facilitate temporal regulation of the cAMP signaling pathway by placing the kinase optimally vs. the cAMP nanodomains. Furthermore, by assembling multiprotein signal complexes including phosphatases and PDEs, AKAPs facilitate discrete temporal control of the cAMP signalosome .Acute β-AR signaling is triggered to compensate MI-induced loss of cardiac pump efficacy. This process increases myocardial cAMP, which favors positive cardiac inotropic effects and left ventricular function. Historically, considering these beneficial effects of acute β-AR signaling, the use of β-adrenergic agonists or PDE inhibitors has been speculated to be beneficial for patients with MI and heart failure ,250. DesAlmost two decades ago, the overexpression of cardiac AC6 in mice revealed unexpected promising outcomes post-MI and for which a reduction in the mortality rate, a decrease in cardiac remodeling and protection of cardiac function were reported . PreciseHuman clinical trials demonstrated in various pathologies benefit from small interfering RNAs mediating a specific gene knockdown (reviewed in ). HoweveAn elegant alternative with specific PDE activators would increase myocardial cAMP hydrolysis in distinct cAMP nanodomains and thus inhibit deleterious PKA signaling post-MI. However, such molecules are still in development or under characterization and have not yet been tested under cardiac pathological conditions .IS) would lack specificity and should not be considered as potential therapeutics. Alternatively, PPI disruptors targeting the anchoring domain between AKAPs and PKA substrates demonstrate higher specificities and efficiencies. Among them, a PPI-disrupting peptide competing in the AKAP18δ/PLB interaction in cardiomyocytes alters PLB phosphorylation and calcium re-uptake [Finally, targeting protein–protein interactions (PPI) in macromolecular signaling complexes orchestrated by AKAPs with PPI disruptors  offers new exciting therapeutic options . Peptidee-uptake . In addie-uptake . Finallye-uptake . Therefos coupling. In this context, β2-AR-pepducin ICL1–9, a cell penetrating lipopeptides derived from the amino acid sequence of the first β2-AR intracellular loop, exhibits promising β-Ars-biased agonist properties. It selectively activates β2-AR-associated β-Ars signaling, internalizes β2-AR and promotes adult cardiomyocyte contractility [The beginning of this century has seen the emergence of innovative therapeutic approaches focusing on the engineering of allosteric modulators and “biased” ligands to selectively activate one of GPCR-associated intracellular signaling events . Therefactility ,266. In actility .It is noteworthy that cAMP signaling displays ubiquitous distribution in all cells and organs over the body, and caution should be noted as the design of medications regulating such signaling could introduce side effects. To avoid adverse effects, new therapeutic molecules developed to treat cardiac diseases could, with benefit, be delivered efficiently and rapidly directly to the myocardium in a tissue-specific manner. Such a strategy would improve the treatment of cardiovascular diseases in general  and would abolish or minimize undesirable side effects. In addition, heart-targeting systems would support the development of new therapeutic agent classes, such as PPI disruptors  or nucleic acids (siRNA) ,269,270.In conclusion, although the management of patients post-MI is well established, strategies to improve disease outcome are still under investigation. Even if β-blockers remain the cornerstone therapy in CHD, they also exhibit unwanted effects and are inappropriate for some patients . As desc