Fertility is a priority for many young women with breast cancer. Women need to be informed about interventions to retain fertility before chemotherapy so as to make good quality decisions. This study aimed to prospectively evaluate the efficacy of a fertility-related decision aid (DA).A total of 120 newly diagnosed early-stage breast cancer patients from 19 Australian oncology clinics, aged 18–40 years and desired future fertility, were assessed on decisional conflict, knowledge, decision regret, and satisfaction about fertility-related treatment decisions. These were measured at baseline, 1 and 12 months, and were examined using linear mixed effects models.β=−1.51; 95%CI: −2.54 to 0.48; P=0.004) and improved knowledge , after adjusting for education, desire for children and baseline uncertainty. The DA was associated with reduced decisional regret at 1 year , after adjusting for education. Women who received the DA were more satisfied with the information received on the impact of cancer treatment on fertility (P<0.001), fertility options (P=0.005), and rated it more helpful (P=0.002), than those who received standard care.Compared with usual care, women who received the DA had reduced decisional conflict (These findings support widespread use of this DA shortly after diagnosis (before chemotherapy) among younger breast cancer patients who have not completed their families. Approximately 3–7% of women with early-stage breast cancer are under 40 years of age at diagnosis . Most woFollowing diagnosis of early breast cancer, women need to make rapid decisions regarding fertility interventions before commencing chemotherapy. Decision making about fertility preservation is complex with many ill-defined risk factors, such as fertility-treatment-induced increases in ovarian sex steroid levels, which are contraindicated in women with breast cancer  are educational materials designed to assist with treatment decision-making, addressing individual values and preferences . They inThe DA is a C5-sized booklet that contains information about breast cancer and female fertility, and a discussion of the different fertility options available. It also includes a series of values clarification exercises that list the advantages and disadvantages of each treatment option to be rated by participants as an option that they are either ‘leaning’ towards or against. Development and pilot testing of the fertility DA has been previously reported  and knowledge, over time.Compare decision-related outcomes, including decisional regret about treatment decisions, and informed choice at 1 and 12 months post diagnosis.Examine potential changes in anxiety and depression as a result of the use of the DA compared to usual care.The current study prospectively evaluated the efficacy of this DA compared with usual care among young women diagnosed with early breast cancer. Specific aims were to:in situ and metastatic disease) referred to 1 of 19 oncology clinics around Australia between 2006 and 2009. Approval was obtained from all institutional review boards, and all women provided written informed consent. Inclusion criteria were: 18–40 years of age; diagnosed with invasive early breast cancer (as defined above); proficient in English; pre-menopausal at time of diagnosis; not yet having commenced adjuvant therapy; and interest in having a child. Eligible women were invited to participate by breast care nurses (BCNs) at each site within a week of their diagnosis and before any medical oncologist or fertility specialist appointments.Participants were consecutive early breast cancer patients  or DA, which included completing the values clarification exercises (intervention), supplied in the sealed envelope before their oncologist appointment, if possible. Follow-up questionnaires were posted 1 and 12 months later. Participants were asked to complete paper-based self-administered questionnaires.Age at diagnosis, relationship status, parity, desire for more children, and highest level of education attained were recorded at baseline.in vitro fertilisation’. Participants could opt to include intended decisions for ‘other’ techniques .Two items, measured at baseline and 1-month follow-up, asked ‘At this point in time, are you leaning towards…’ (a) ‘waiting to see if fertility returns after treatment’ and (b) ‘The following outcome measures were administered at all three time points:Decisional conflict scale (DCS): The 10-item low literacy version of this scale assessed decisional conflict regarding ‘different fertility treatments’ : This assessed whether women made treatment decisions with adequate knowledge  in accordance with their values, assessed with an adaptation of the five-item scale to assess attitudes towards fertility treatment in regard to feeling ‘beneficial’/‘harmful’, ‘important’/‘unimportant’, ‘a good thing’/‘a bad thing’, ‘pleasant’/‘unpleasant’, and ‘worthwhile’/‘not worthwhile’ .2). Confounders  were incorporated into a statistical model if they were theoretically relevant and resulted in a >20% change in the treatment effect estimates. Confounders were also included if they were associated with substantial reduction in Akaike's Information Criterion and Schwarz's Bayesian Information Criterion. Depression scores were log-transformed, given their skewed distributions. For variables collected at 1 and 12 months only, raw scores were used for analyses. Linear regression was used to assess differences between groups for continuous variables (DRS), and logistic regression was used to assess group differences for binary outcome variables (MMIC).An intention to treat analysis was undertaken. Linear mixed effects models (with random baseline measurements and slopes) were used to examine the effect of the DA on outcome variables assessed across three occasions , adjusting for potential confounders and for curvilinear change (timeThe target sample size was 64 participants in each group. This would provide sufficient power (80%) to detect a difference of 0.5 between groups  were included in the analysis. This represented 83.3% of eligible subjects and 90.2% of those agreeing to participate. Due to slower than anticipated recruitment rates, the study was closed before reaching the target sample size; however, the sample size was large enough to detect differences.A total of 145 eligible women were invited to participate , of thesN=79). Of this group, 82% (N=65) reported that they planned to have children or that they were unsure whether they wished to have any children . Of the 35 participants who already had children, 46% (N=16) definitely planned to have more children and the rest  were unsure. Overall, most participants reported that they planned to have (more) children .There were no significant differences in demographics between DA and usual care groups . Most paUnadjusted mean scores and percentages are presented for all outcomes separately for the DA and usual care groups in The majority of participants (96%) who received the DA reported reading the materials, compared with 83% of participants allocated to usual care. The extent to which this was read is shown in β=−1.51; 95%CI: −2.54 to 0.48; P=0.004), in a curvilinear manner . Adjusted results identified a negligible mean difference of 1.46 in DCS between groups at 1 month . Decisional conflict scale scores were 15.30 units lower in the DA group at 12 months .Those who received the DA had a greater reduction in decisional conflict than those who received usual care over 12 months . Using aβ=0.09; 95%CI: 0.01–0.16; P=0.02) in participants who received the DA, compared with the usual care group. Adjusted results identified a negligible mean difference of 0.01 in knowledge scores between groups at 1 month . Knowledge scores were 0.94 units lower in the DA group at 12-month follow-up .Use of the DA significantly improved fertility-related knowledge . Using avs an uninformed choice about fertility based on group allocation at 1 month , and at 12-month follow-up , after adjusting for relationship status, education, parity, and desire for more children.There were no significant differences in the proportions of participants who made an informed β=0.02; s.e., 0.06; 95%CI: −0.10 to 0.14; P=0.73) or depression .There was no significant difference in anxiety between treatment groups after adjusting for education, parity, and desire for more children , or after adjusting for education . After adjusting for education, participants who received the DA had significantly lower decisional regret at 12 months .At 1 month, DRS scores regarding fertility-related decisions were not significantly different between groups  and on the different fertility options available . Participants were also significantly more likely to score the DA more highly on a helpfulness scale in terms of making fertility-related decisions .Sixty-seven percent of participants receiving usual care and 70.5% of participants allocated the DA were satisfied with the information provided on the impact of breast cancer treatment on fertility. Similarly, 54.4% and 77.3% were satisfied with the information given on the impact of treatment and fertility options, respectively. Compared with participants who received usual care, participants allocated the DA were significantly more satisfied with the materials they received on the impact of breast cancer treatment on fertility .Perceived thoroughness of discussions with oncologists and the extent to which the discussions were prompted by the educational materials are reported in χ2=0.48; P=0.49). One participant in each group sought a fertility specialist, although these patients were not referred by their oncologists. Of those who received a referral to a fertility specialist, 91.4% of controls consulted or were planning to see a fertility specialist, compared with 97.7% participants who received the DA .Sixty-two percent of participants who received the DA and 55.6% allocated to usual care were referred to a fertility specialist (P=0.31). Additionally, no differences in change scores were observed for intention to undergo IVF between groups (P=0.42). For the total patient sample, 79.7% of controls reported having made a decision about fertility treatment at 1 month compared with 91.1% of participants who were allocated the DA . At 12 months, 71.2% of participants allocated to usual care and 76.5% of participants allocated to the DA had made a decision about fertility preserving options .Leaning towards the ‘wait and see’ option did not change significantly from baseline to 1 month between groups , availability and cost of interventions, previous fertility as well as religious and personal beliefs . In addiReported discussions with oncologists and referrals to specialists did not differ, with or without the DA. Our findings suggested that most medical oncologists discussed fertility with breast cancer patients of reproductive age. These statistics are higher than the rate of ∼70% reported in previous studies , which is above the 51% previously reported as having their concerns addressed adequately (In addition to the limitations discussed above, the non-randomised design of the study may lead to bias. However, studies in similar populations using this study design have not shown bias as a result of time (Our results raise some interesting questions that may need further exploration. Most of our participants reported that their partners were involved in the fertility treatment decision-making process, yet very little is known about how partners perceive their role despite fertility-related decisions being a decision for both parties. Understanding the mechanisms of fertility-related decision making should incorporate this factor.Within individual items of our knowledge scale there were improvements of varying extent. Further exploration of the specific informational domains would assist in improving the information delivered and improve the DA. Other considerations include that the DA will require regular updating as reproductive technologies advance, bringing ongoing costs. Online resources may facilitate updating and reduce costs. Also, the DA is currently available only in English and is not appropriate for culturally and linguistically diverse populations. Further research into these issues would further advance support this body of work.We have shown that this fertility DA reduced decisional conflict and regret about fertility-related treatment options in young breast cancer patients. Lower decisional conflict and regret indicate increased satisfaction with choices with respect to personal values. The DA also improved fertility-related knowledge, which is an important aspect of informed decision making. The DA also improved satisfaction with information received and was considered helpful by patients. These findings suggest that this DA should be widely distributed to young breast cancer patients considering future pregnancies before chemotherapy.


T cells orchestrate the adaptive immune response, making them targets for immunotherapy. Although immunosuppressive therapies prevent disease progression, they also leave patients susceptible to opportunistic infections. To identify novel drug targets, we established a logical model describing T-cell receptor (TCR) signaling. However, to have a model that is able to predict new therapeutic approaches, the current drug targets must be included. Therefore, as a next step we generated the interleukin-2 receptor (IL-2R) signaling network and developed a tool to merge logical models. For IL-2R signaling, we show that STAT activation is independent of both Src- and PI3-kinases, while ERK activation depends upon both kinases and additionally requires novel PKCs. In addition, our merged model correctly predicted TCR-induced STAT activation. The combined network also allows information transfer from one receptor to add detail to another, thereby predicting that LAT mediates JNK activation in IL-2R signaling. In summary, the merged model not only enables us to unravel potential cross-talk, but it also suggests new experimental designs and provides a critical step towards designing strategies to reprogram T cells. The cells of the mammalian immune system do not exist in isolation, but rather form an integrated network that is constantly scanning the body for signs of ‘foreign’ invasion. Working together, these cells possess the ability to repel invaders and thereby establish protective immunity. One central population in this network are T lymphocytes; whose role it is to coordinate the activity of the adaptive arm of defense. However, T cells constantly receive multiple inputs and therefore it is not clear how they are able to reach a decision. Traditionally, these inputs are studied in isolation using a top-down or stimulus-response approach. Confounding this issue is that our knowledge of these input pathways is not cell-type-specific, but rather represents the sum of all knowledge related to a given stimulus. Therefore, we have undertaken to validate signaling pathways in primary human T cells. We are particularly interested to study the cross-talk between pathways to see how common elements are utilized to make specific decisions that determine cell fate. In doing so, we have identified new components in what were considered to be well-characterized receptor pathways. In vivo, cells are rarely exposed to only one signal at a time and therefore require the capacity to integrate multiple signals coming from many receptors simultaneously. Thus, a one-to-one relationship between receptor triggering and a functional outcome is usually not possible, as the result of receptor stimulation depends upon the temporal sequence of inputs from multiple receptors. Hence, in order to investigate the cross-talk between receptors, without testing all possible combinations of stimuli, strategies are required to efficiently derive the global signaling network taking advantage of the known isolated top-down pathways of receptors.A number of receptor signaling networks have been elucidated beginning with the proximal events at the receptor, initiated by ligand binding, and extending down to the level of transcription factor activation. However, this top-down approach to describe pathways usually ignores the potential input coming from other receptor systems. A system for which a number of receptors have been characterized and that provides both easy access to material and a short path to medically relevant applications are T cells. T lymphocytes are a central component of the immune system and orchestrate many aspects of the adaptive immune response. This function makes T cells an attractive target for therapeutic intervention, e.g. for treating autoimmune diseases, suppressing immune responses directed against organ transplants, and even in stimulating immune responses against cancer We initially focused our attention on the T-cell receptor for antigen (TCR), which recognizes peptides bound to HLA-molecules. The TCR ultimately determines whether a T cell will become activated or not The investigation of cross-talk immediately presents the problem of how to merge signaling networks. Therefore, in order to compare intervention in the TCR signaling pathway with the present therapeutic strategies, we created a tool to generate a merged model that combines our previous logical TCR-signaling network with the IL-2R network.The IL-2R exists in three forms. The low affinity variant consists of the IL-2Rα-chain (CD25) alone. The intermediate affinity receptor is composed of the IL-2Rβ-chain and the common γ-chain, which is shared with other cytokine receptors. The high affinity form of the IL-2R contains all three chains together and mediates the autocrine feedback-loop a priori not clear how the common signaling elements of these two pathways interact: can they be cross-activated to enhance signaling, are they used competitively leading to an effective inhibition, or do these modules function independently of one another. Here, our method to merge logical models of signaling networks allows us to identify potential points of receptor cross-talk in a semi-automated manner. To approach a validated version of the signaling network, the merged logical model enables us to design experiments to determine whether potential cross-talks exist or not. Following validation of the IL-2R network in human T-cell blasts, the merged model predicted that STAT signaling should also be initiated upon TCR triggering, which we then verified experimentally. Moreover, our model predicted that LAT should be activated following IL-2 stimulation, which we could verify as well. The ability to reveal new signaling elements in both TCR and IL-2R signaling opens the possibility of gaining new insights into the mechanisms of signaling in T cells that may ultimately identify new targets for T cell-specific therapy.A number of the key molecules in TCR signaling are also utilized by the IL-2R Approval for these studies was obtained in writing from the Ethics Committee of the Medical Faculty at the Otto-von-Guericke University, Magdeburg, Germany. Informed consent was obtained in writing in accordance with the Declaration of Helsinki.MEK activates ERK” as MEK→ERK and “STAT3 or STAT5 can induce Blimp-1 expression” as STAT3 OR STAT5→Blimp-1. The precise meaning of the ON-state for the logical variable representing a signaling component depends on the biological context. It can refer to an increase in the amount of a substance , its phosphorylation (e.g. LAT), or its recruitment to the membrane or association with other proteins   and the adaptation/shutoff of the signaling cascade (late time points).1 OR … OR An → BA that is transformed to the if-and-only-if (IFF) clause 1 OR … OR An A↔ B. This implies that B can only be active if at least one of the 1, …, AnA is active. We can formalize the standard signaling network in terms of IFF-clauses: Let the IFF-clauses of a given time horizon be denoted as iS with iS with the same time horizon should be valid at the same time to model the global behavior of the unit. Checking these amounts to solving a satisfiability (SAT) problem for the formula S, and each feasible solution represents one possible state of the signaling network. The fact that seemingly simple formulas with AND, OR, and NOT operations are used to represent the information is not a sign of low complexity: In fact, IFFSAT networks are able to encode the NP-complete 3-SAT problem We assume that in signaling networks a component cannot change its state from active to inactive or vice versa without the influence of either a change of state for other components or external stimulation. For some proteins inactivation may occur via intrinsic mechanisms, e.g. the intrinsic GTPase activity of RAS may result in its inactivation. However, as this activity is far slower than the catalyzed inactivation by GTPase-activating proteins (GAPs), for the purpose of simplification, it is excluded from the model. As introduced previously K of components nA is known to be ON or OFF. If this fixation pattern K of components is a possible solution of the network, i.e. every clause iS can be satisfied, then the fixation pattern is feasible otherwise the fixation pattern nA is called infeasible. An infeasible pattern, i.e. a conflict within the experimental data, can be due to a modeling error or result from a negative feedback loop that prevents signaling and should only be active at a later time point. The logical network allows us to compute a set of minimal interventions that permits a feasible solution for the fixation pattern K. These minimal interventions may represent a solution to the modeling errors, point out inconsistent data in the literature PAG ↔ CSK, NOT CSK ↔ LCKP1, LCKP1 ↔ FYN, FYN AND NOT TCRB ↔ PAG  and a Pan T-cell isolation kit II . After resting overnight, T cells were stimulated with plate-immobilized anti-CD3  and anti-CD28  in culture medium  for 48 hr. After washing twice with RPMI 1640, cells were rested in fresh culture medium for 24 hr.+ cells 1×106 T lymphocytes were washed twice with PBS and incubated with FITC anti-CD25 (BD Biosciences) for 15 min. at 4°C, followed by two additional washing steps. The number of apoptotic cells was determined using an Annexin V/FITC Kit (Bender MedSystems) according to the manufacturer's instruction. Cells were immediately analyzed by flow cytometry .To determine the number of CD25To identify proteins that became activated upon IL-2R signaling, rested T-cell blasts were stimulated with 100 U/ml IL-2  for the indicated time. To clarify the role of different kinases, T-cell blasts were pre-incubated for 30 min. with either 10 µM PP2, 1 µM wortmannin, 1 µM Gö6976, 1 µM Gö6983 or 1 µM Jak Inhibitor I , followed by the addition of IL-2 for the indicated time. Reactions were stopped by addition of ice-cold PBS .Cells were lysed  for 20 min on ice and centrifuged at 13000 rpm, 10 min, 4°C. The post-nuclear extracts were separated on 10% SDS-PAGE under reducing conditions. Proteins were transferred onto nitrocellulose membranes  and immunoblotting was performed using anti-pS473-AKT (93H12), anti-pT202-pY204-ERK1/2, anti-pT180-pY182-p38 MAPK, anti-pT183-pY185-JNK, anti-pY171-LAT, anti-pY705-STAT3 (D3A7), anti-pY694-STAT5 (C11C5); , anti-phosphotyrosine  and β-actin , followed by incubation with the appropriate secondary HRP-conjugated goat anti-mouse or goat anti-rabbit. Visualization was performed using enhanced chemiluminescence  according to the manufacturer's instructions. Films were scanned with an Epson Perfection 4990 Photo scanner .A number of studies have analyzed the effect of IL-2 on T-cell proliferation, survival, differentiation, and population dynamics The IL-2R network was then validated experimentally using human T-cell blasts. The cells were viable and expressed the high affinity receptor for IL-2 . First, c-fos and c-jun has been reported to be dependent on Lck One report suggests that PI3K is downstream of a Src family kinase (SFK) in IL-2R signaling Taken together, the Jak Inhibitor I and PP2 experiments suggest that SFK activity is largely downstream of JAKs  since boWe next investigated whether PI3K had any influence on other parts of the IL-2R signaling network by applying the PI3K inhibitor wortmannin (WM) . Figure Numerous studies performed two decades ago had demonstrated PKC activation upon IL-2R stimulation In analogy to TCR signaling, ERK depends strongly on SFKs, PI3K, and novel PKCs In summary, we present a validated IL-2R signaling network see  containiCross-talk between two receptors is mediated by molecules that are common to both pathways, but for which the upstream regulation is differentially organized. Thus, in each pathway the regulation of these common molecules is incompletely described from the perspective of a merged network that aims at describing both systems simultaneously. In our example of TCR and IL-2R signaling, synchronous stimulation of both receptors is likely to happen during clonal expansion. Here antigenic stimulation of T cells is still ongoing 30 hours after initial stimulation The prerequisites for merging are that the components of the systems are standardized, i.e. components with identical names refer to identical molecules, and the interpretation of their activity states is consistent see . Then thGAB2→SHP2 versus NOT ERK AND GAB2→SHP2. These clauses differ with respect to ERK. The question is whether ERK always inhibits SHP2 or whether this is a network-specific event involving (unknown) additional regulators that ensure receptor-specificity  versus PKCè which is one member of the nPKCs. Hence, whenever one isoform (PKCè) is activated, every interaction involving the more general class of proteins (nPKCs) may also be affected and vice versa see .This list contains specific examples, however a general problem occurs when the “local interaction” information taken into account while constructing signaling networks is of a different resolution with respect to the proteins involved. The different forms of inherently incomplete information result in obstacles for the merging of two networks, which can be formalized by considering the inverse problem: How can one extract the signaling network or an arbitrary subnetwork of one receptor from a merged network . However, it has been suggested that the different DAG species trigger different sets of effectors In the IL-2R signaling network the activation of SHP2 by recruitment to the adaptor GAB2 can be prevented by ERK. Engaging this question experimentally in primary cells has proved challenging due to a lack of suitable reagents directed against GAB2. Thus, it remains an open question whether ERK can modulate SHP2-GAB2 interactions in TCR signaling.Both receptors activate the SFKs Lck and Fyn. However, it is not clear whether common pools are used or if cross-activation is possible. Although we have excluded a role for SFKs in the activation of STAT3 and STAT5 by the IL-2R, the possibility that SFKs mediate STAT activation under other conditions still exists The pathways leading to p38 and JNK activation are known in quite some detail for the TCR network, but not for IL-2R signaling. This suggests that we experimentally verify whether some, all, or none of these elements are utilized by the IL-2R.in vitro and although being activated after IL-2 stimulation, Lck is not required for IL-2R-mediated STAT activation  between the four proteins demand that they are in the same state. This is in conflict with the inhibition of LCKP1 by CSK (dotted red line) which requires that one of them is active and the other inactive. As a consequence one or more of the interactions needs to be classified as a late implication formula. However, the additional input by TCRB allows a feasible solution by creating flexibility in the state of PAG.(TIF)Click here for additional data file.Figure S2Determination of CD25 and Annexin V/PI by FACS staining. After resting for 24 hr and before restimulation with IL-2, T-cell blasts were stained with a FITC-coupled anti-CD25 antibody to determine the percentage of CD25-positive cells (A) or a FITC-coupled Annexin V antibody and propidium iodide to determine the number of viable cells (B).(TIF)Click here for additional data file.Figure S3Dose response of IL-2. Human T-cell blasts were stimulated by the indicated dilutions of IL-2 . Cell lysates were analyzed by Western blotting for phosphorylated ERK and STAT3. β-actin was analyzed as loading control.(TIF)Click here for additional data file.Figure S4The reversible SFK inhibitor PP2 does not fully block AKT. The blot is a longer exposure of the blots of (TIF)Click here for additional data file.Figure S5The TCR/CD4/CD28 signaling network. To a large extent the signaling network was published previously in a different graphical layout (TIF)Click here for additional data file.Figure S6The merged network of TCR/CD4/CD28 and IL-2R signaling. The top layer represents input nodes. The bottom layer represents the output, i.e. molecules including transcription factors that become activated. Solid black arrows indicate activating interactions with a black circle denoting AND-connections. For clarity, activating influences with arrows pointing from the bottom to the top are drawn with dashed black lines. Red lines mark inhibitory influences that are expressed as NOT-conditions in the logical network. Note that some connections are based on below-quality-standard data , but are not highlighted separately (parately . The nod(TIF)Click here for additional data file.Figure S7STAT activation after TCR stimulation of mouse T cells. Primary mouse T cells and mouse T-cell blasts were stimulated as indicated in (TIF)Click here for additional data file.Figure S8IL-2R signaling does not affect TCR expression. Human T-cell blasts were either stimulated with IL-2 for 30 min or left untreated. The level of TCR (CD3) and IL-2Rα (CD25) surface expression were measured by flow cytometry. Unstained cells are indicated with the broken line, untreated cells are represented with the grey line, and stimulated cells indicated by the black line.(TIF)Click here for additional data file.Protocol S1Stimulation of mouse T cells. Method describing the isolation, culture, and stimulation of primary mouse T cells and mouse T-cell blasts.(PDF)Click here for additional data file.Text S1Model merging as an IFFSAT problem. Restating the problem of merging two networks as a projection of a larger network, including a formal proof of the algorithmic complexity.(PDF)Click here for additional data file.Text S2Protocol for Boolean model merging. A step-by-step protocol for the semi-automated merging of Boolean networks.(PDF)Click here for additional data file.Table S1List of network components. The components of the merged TCR-IL-2R network including biological names and interpretation of the ON state.(PDF)Click here for additional data file.Table S2List of clauses for the merged network. The implication formulas for each component of the merged network are provided along with references supporting the interactions.(PDF)Click here for additional data file.Table S3Activation states of the merged network upon different stimuli. Lists the state vectors for the merged network with TCR and IL-2R stimulation alone or in combination.(PDF)Click here for additional data file.


Human T-cell leukemia virus type-1 (HTLV-1) carriers co-infected with and hepatitis C virus (HCV) have been known to be at higher risk of their related diseases than mono-infected individuals. The recent studies clarified that IL-28B polymorphism rs8099917 is associated with not only the HCV therapeutic response by IFN, but also innate immunity and antiviral activity. The aim of our research was to clarify study whether IL-28B gene polymorphism (rs8099917) is associated with HTLV-1/HCV co-infection.The genotyping and viral-serological analysis for 340 individuals showed that IL-28B genotype distribution of rs8099917 SNP did not differ significantly by respective viral infection status. However, the IL-28B mRNA expression level was 3.8 fold higher in HTLV-1 mono-infection than HTLV-1/HCV co-infection. The high expression level was associated with TT , whiles the low expression was associated with co-infection of the two viruses . However, there was no association between down-regulation and ATL development .HTLV-1 mono-infection up-regulates the expression of IL-28B transcripts in genotype-dependent manner, whiles HTLV-1/HCV co-infection down-regulates regardless of ATL development. A retrovirus, human T-cell leukemia virus type-1 (HTLV-1), and a positive-strand RNA virus, hepatitis C virus (HCV), are completely different in terms of virologic characteristics. Nevertheless, they play a similar role in the pathogenesis of viral-induced malignant neoplasms, such as adult T-cell leukemia (ATL) in HTLV-1- infected individuals, and hepatocellular carcinoma (HCC) and B-cell lymphoma in HCV- infected individuals, during long-term chronic infections.Furthermore, it is known that co-infection with HCV and HTLV-1 is frequently observed in an area endemic for HTLV-1. HCV/HTLV-1 co-infected individuals have been reported to be at higher risk for developing HCC than those infected with HCV alone . AlthougIL-28B single nucleotide polymorphisms (SNP) [Recently, genome-wide association studies of patients with HCV have made great advances in viral clearance associated with ms (SNP) ,7. IL-28ms (SNP) . Althougms (SNP) , the amims (SNP) , innate ms (SNP) , HCV-relms (SNP) ,7.IL-28B gene would be associated with HTLV-1 infection. The aim of the present study was to examine the mutual association between IL-28B polymorphism (rs8099917 SNP) and mono-infected-HTLV-1 and co-infected HTLV-1 with HCV subjects.From these observations, we hypothesized that IFN-λ3 encoded from the All subjects were of Japanese origin living in Nagasaki City, an endemic area for HTLV-1 in Japan. For genomic specimens, 340 blood samples were randomly collected from patients who visited a liver clinic and liver transplantation center from April 2009 to March 2011 from the departments of Hepatology and a Hematology Clinic. One hundred and twenty-four of the 340 samples were also available for total RNA tests. Accordingly, most patients had either chronic liver disease (CLD) or adult T-cell leukemia (ATL). This study was done under informed consent after the approval of the Nagasaki University hospital IRB . Since the samples used here were un-linked materials, patient information was restricted.Eight HTLV-1-infected T-cell lines, Hut 102, MT-1, MT-2, ST1, KK1, KOB, OMT, and LMY-1, were used for IL-28B mRNA quantification. The first three were purchased and latter five were established in our laboratory .HCV and HTLV-1 infections were mainly serologically detected using commercially available kits, CLEIA-anti-HTLV-1, Lumipulse-II Ortho HCV . The confirming examination was genetically performed by the Cobas TaqMan HCV test  for HCV and in-house HTLV-1 proviral real-time RT quantifiable PCR . GenomicSNP genotyping was performed using multiplex PCR amplification and Pyrosequencing technology. To amplify target regions, newly designed biotinylated-primers were employed: sense and anti-sense for rs8099917, 5'-TCCTCCTTTTGTTTTCCTTTCTG-3' and 5'-AAAAAGCCAGCTACCAAACTGT-3'. Then, the amplicon was sequenced according to the manufacturer's instructions based on Pyrosequence technology . Biotin-labeled amplicons from the 1st PCR were captured by binding to streptavidin-coated Sepharose beads, and DNA was denatured to produce an ssDNA template for the Pyrosequencing assay. The ssDNA was released and combined with the sequencing primer, which was extended during the Pyrosequencing reaction to provide the sequence of the template DNA. Pyrosequencing data were produced in the form of Pyrograms, and genotypes were assigned by the peak pattern presented in the Pyrogram.™ RT System  was quantified by a LightCycler System  using newly designed sense and anti-sense primers, 5'-AAGGACTGCAAGTGCCGCT-3' and 5'-GCTGGTCCAAGACATCCC-3' (AY129149). A standard curve was generated using a tenfold dilution method with a reference material derived from pTAC-1.2735 inserted with 166 base fragments including the target. The amplicon was assayed by the Cyber green method. The raw data were normalized by abl mRNA density and evaluated as the relative value for abl gene expression calculated by IL-28B data/abl data × 104, modified from our previous mRNA real time RT qPCR method [mRNA for IL-28B transcribed into cDNA by a GoScriptCCC-3' AY9149. A sThe minor-allele frequency (MiAF) was set as the less frequent allele in a population for SNPs analyzed. Viral infectious status was divided into 4 groups of HTLV-1 mono-infection, HCV mono-infection, HTLV-1/HCV-co-infection, and non-infection . Differences in the genotype distribution of IL-28B SNPs among groups were compared using the Chi-square or Fisher exact test. The level of mRNA expression was compared using the Mann Whitney U test. Correlation analysis was performed by the Nonparametric Spearman's rank correlation method. The relationship between a factor and an outcome was estimated the magnitude of the association by the odds ratio with 95% confidence intervals (95%CI). Statistical analysis was performed using SAS 9.1. The statistical significance level was set at 0.05.Three hundred and forty samples were genotyped on IL-28B rs8099917 SNP and were serologically examined for viral infection of HTLV-1 and HCV. As summarized in Table p = 0.21). The prevalence of TT was not different statistically either (p = .495).Since the HTLV-1 mono-infection group consisted of 47 ATL patients and 26 HTLV-1 carriers, we stratified the two groups of ATL patients and carriers and the minor allele frequencies of the two groups were compared; the difference between that of ATL and carriers was not statistically significant  were the same in Figure p = 0.013). Therefore, to clarify whether ATL cells directly affect the expression of IL-28B mRNA, we compared the mRNA expression levels in mainly HTLV-1 carriers, ATL patients with ATL cells, and ATL cell lines. As shown in Figure P < 0.05) and ATL patients with TT having ATL cells than those of non-infected individuals . No high expression level was observed in two ATL or 16 carriers with HTLV-1/HCV co-infection. Surprisingly, these data were lower rather those from TG/GG. On the other hand, IL-28B mRNA expression in 8 HTLV-1-infected T-cell lines was undetectable in all but one (Hut 102). The genotype was TT in all cell lines.The expression levels of IL-28B mRNA were generally low in most cases with a median value of 5.2 in no-infection, 10.6 in HTLV-1 mono-infection, 3.9 in HCV mono-infection, and 2.8 in HTLV-1/HCV co-infection Figure . Notably2 = 0.0543, Figure In addition, there was no correlation between the IL-28B mRNA levels and HCV-RNA levels , but low expression was not associated with ATL development .As shown in Figure Although co-infection with HTLV-1 and HCV has been shown to result in higher rates of cirrhosis and increased death from liver diseases , the cauThe present study is the first to reveal that the IL-28B genotype is not associated with stratification based on the combination of HTLV-1 and HCV infection; no infection for both , HTLV-1 mono-infection, HCV mono-infection and HTLV-1/HCV co-infection. Similarly, the frequency of the major TT homozygotes was not associated among ATL patients and HTLV-1 carriers (Table Next, we found a strange phenomenon that the IL-28B mRNA expression levels in peripheral blood were lower in samples with HTLV-1/HCV co-infection than in samples with either HTLV-1 or HCV alone, especially significantly for HTLV-1 mono-infection. In particular, samples carrying TT homozygotes were strongly down-regulated, more than the minor TG hetero- and GG-homozygotes. Why are the mRNA expression levels different in mono- and dual-infection? Although it is not known how rs8099917 affects the action of IL-28B, presumably it alters the immune function to viruses. In addition to a common anti-viral IFN-stimulating signal pathway, HTLV-1 may use an alternative anti-viral pathway like HBV , becauseFrom our results, the IL-28B expression level was higher in HTLV-1 mono-infected individuals including ATL patients. IFN-λis usually up-regulated through activation of the NF-kappaB pathway after viral infection. Actually, the Sendai virus, an influenza A virus, and others have been demonstrated to activate the NF-kappa B pathway, resulting in up-regulated IL-28B expression ,20. AccoOf IL-28B producing cells in the literature, most cells in the blood are described as having a weak or absent expression under the steady state conditions. Li et al.  reportedIn conclusion, we found an unusual phenomenon in that the expression of IL-28B mRNA was affected by not only the IL-28B rs8099917 genotype, but also co-infected HTLV-1 with HCV. This will contribute to a better understanding the enigmatic impairment of immunity in the HTLV-1 carrier state, including co-infection with HTLV-1 and HCV.HTLV-1: Human T-cell leukemia virus type -1; HCV: Hepatitis C virus; SNP: Single nucleotide polymorphism; IFN: Interferon; PBMC: Peripheral blood mononuclear cell; PCR: Polymerase chain reaction; MAF: Minor-allele frequency.The authors declare that they have no competing interests.SK designed the study and wrote the manuscript, and SM, TU, KN, DS HH, KY, NU, YM analyzed the genotype, TK collected samples, and TK, KN, MI and SK organized and assessed the data. All authors interpreted the data and were financially supported. All authors read and approved the final manuscript.


Human T-cell lymphotropic virus type 1 (HTLV-1) has been identified as the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The virus infects between 15 and 20 million people worldwide of which approximately 2–5% develop ATL. The past 35 years of research have yielded significant insight into the pathogenesis of HTLV-1, including the molecular characterization of Tax, the viral transactivator, and oncoprotein. In spite of these efforts, the mechanisms of oncogenesis of this pleiotropic protein remain to be fully elucidated. In this review, we illustrate the multiple oncogenic roles of Tax by summarizing a recent body of literature that refines our understanding of cellular transformation. A focused range of topics are discussed in this review including Tax-mediated regulation of the viral promoter and other cellular pathways, particularly the connection of the NF-κB pathway to both post-translational modifications (PTMs) of Tax and subcellular localization. Specifically, recent research on polyubiquitination of Tax as it relates to the activation of the IkappaB kinase (IKK) complex is highlighted. Regulation of the cell cycle and DNA damage responses due to Tax are also discussed, including Tax interaction with minichromosome maintenance proteins and the role of Tax in chromatin remodeling. The recent identification of HTLV-3 has amplified the importance of the characterization of emerging viral pathogens. The challenge of the molecular determination of pathogenicity and malignant disease of this virus lies in the comparison of the viral transactivators of HTLV-1, -2, and -3 in terms of transformation and immortalization. Consequently, differences between the three proteins are currently being studied to determine what factors are required for the differences in tumorogenesis. Deltaretrovirus genus. Although there are currently four known types of HTLV, HTLV-1 is by far the most pathogenic of the group and has the distinction of being the first oncogenic retrovirus discovered in humans  was discovered in the early 1980s by two independent groups working in the United States . Each LTR contains three regions, the unique 3′ (U3), the repeated (R), and the unique 5′ (U5) regions Figure . The U3 cAMP response element binding protein interacts directly with the vCRE, thus making it a protein of paramount importance in Tax-mediated transactivation of the viral promoter  proteins are an interesting example of one of these associated host factors. TORCs enhance the interaction of CREB with the TFII130 component of Transcription factor II D (TFIID) thus enhancing the transcriptional initiation potential of CREB  coactivator proteins that share a high degree of homology  complex. Through a combination of chromatography and immunoprecipitation techniques, the work of Easley et al.  has demoFurther work examining the proteins associated with Tax-mediated transcription of the HTLV-1 LTR suggests that chaperone molecules such as Nucleosome assembly protein 1 (NAP1) may be involved in Tax-mediated chromatin remodeling. NAP1 is a histone chaperone molecule that has been shown to be involved in transcription-independent nucleosome eviction at the HTLV-1 promoter  has demonstrated their ability to counteract the action of HAT-activity containing factors. When overexpressed, HDACs interact directly with Tax and bind to the HTLV-1 promoter, thus repressing transcription by refolding chromatin fibers  is one of the major cellular proteins activated by Tax. Genes such as 6GG (termed the CArG box) when situated next to a transcription factor family (TCF) Ets element comprise the SRE. The SRF and TCF (members of which are part of the Ets family of proteins) proteins bind to the SRE creating a ternary complex at the promoter. Tax has been shown to interact with members of the TCF family, Elk-1 and SAP-1, and thus by interacting with the TCF protein of this ternary structure, Tax may dysregulate the SRF pathway It is well established that Tax interacts with the host transcription factor NF-κB, resulting in the activation of the NF-κB pathway, which is critical for transformation, proliferation, and survival of HTLV-1 infected cells. Due to the significance of Tax/NF-κB interaction on HTLV-1 disease states, NF-κB is given special focus in this review.The NF-κB family of transcription factors variably regulates gene expression as a response to dynamic post-translational modifications (PTMs), subcellular localization, and formation of homo and heterodimer complexes of the family members , where NF-κB transcriptional activation is at a maximum level of activity  or other cellular signaling proteins and kinases. As seen in Figure As with most cellular proteins, virally encoded proteins utilize PTMs to modify their enzymatic efficiency and expand their range of functionality. Modifiers allow viruses to carry out a diverse set of effects in the host cell with only a limited number of expressed proteins. In HTLV-1 infected cells, the virally encoded Tax protein is modified by numerous PTMs. Additionally, Tax acts upon host cell proteins to alter their PTM moieties. The modifiers taken on and generated by Tax allow it to affect numerous reactions within the host cell that are vital to the viral life cycle and also lead to many of the pathogenic outcomes indicative of HTLV-1 infections. Here we examine in detail the Tax-induced PTMs of host cell proteins, as well as review known PTMs of Tax and the mechanisms by which they are generated.Several key PTMs of host cell proteins induced via interactions with the HTLV-1 Tax protein have been elucidated. One vital regulatory complex that takes on numerous PTMs due to its association with Tax is the IKK complex. It has been shown that Tax can associate with all three subunits of the IKK, but that its interaction with the IKKγ subunit leads to IKKβ and IKKγ phosphorylation  at amino acids 18–52 of its N-terminus, Tax is predominantly a nuclear protein  4 and Cdk6 via its amino terminal domain  increased centrosome duplication; (ii) failure to complete cytokinesis; and (iii) incomplete or improper splitting during mitosis  and the subunit IL-2 receptor α chain (IL-2Rα) of its receptor complex  has recently become a protein of interest in the HTLV-1 oncogenesis story. HBZ, an antisense viral protein, was originally identified as associated with CREB-2 (an antisense transcription factor similar to CREB) which inhibited viral transcription at the 5′ LTR  and nucleotide (NER) excision repair, human mismatch repair (MMR), non-homologous end joining (NHEJ), and damage response signaling via ATR/CHK1 . Interestingly, Schavinsky-Khrapunsky et al.  report aCS) and the kinase domain of DNA-PK, Ku, a dimer composed of Ku70 and Ku80 subunits  and its target CHK2, both necessary for initiating the signaling involved in DNA repair of double stranded breaks , a derivative of epoxyquinomicin. Treatment with DHMEQ induces apoptosis in ATL and HTLV-1 transformed cells , a histone deacetylase inhibitor (HDACi), triggers Tax expression, thereby exposing the latent HTLV-1 reservoir to immune destruction , high levels of transcriptionally inhibited p53 are identified , Calattini et al. , interplay between Tax and miRNA during cellular transformation, and modulation of intricate cellular complexes (such as CBP/p300 and SWI/SNF) by Tax all provide promising avenues for further understanding of Tax-mediated cellular transformation requirements and, consequently, possible therapeutic targets. In fact, the promiscuity of Tax provides both great strength and weakness to the effectiveness of therapeutically targeting the oncoprotein in that direct targeting of Tax has a radiating effect on the major cellular pathways. It is important to note, however, that without a detailed understanding of the various branches of the Tax interactome, there exists the strong possibility of uncontrolled off-target effects. With improved understanding and more focused therapeutic approaches, modulating cellular Tax could have broad implications for the host cell and, consequently, be a first step in effective clinical treatments of HTLV-1 disease states.in vivo. The development of such models would also advance the study of possible therapeutic targets for both viral and cellular genes unique to infected cells. Finally, more fundamental concepts will require further study, including the development of a crystal structure for Tax and further study into histone degradation versus mRNA downregulation as seen in Tax expressing cells. Cumulatively, much has been learned about Tax over the past 35 years, but there remains much more to discover before this fascinating oncoprotein can be fully understood.Thanks to the excellent work of numerous talented researchers in the field, understanding the comprehensive role of Tax in HTLV-1 infections continues to be an evolving process. Many of the discoveries mentioned earlier in this review will provide promising avenues of future investigation. For example, a growing understanding of miRNA machinery has created another level of complexity in better defining cellular and viral protein regulation. Further research into the interactions between Tax and cellular miRNA (and possibly its machinery) and the resulting effect on oncogenesis will be required (Bellon et al., 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.


Bangladesh has experienced a rapid decline in fertility in the past several decades, facilitated by proactive population policies, provision of contraceptives, and broader societal shifts, encouraging smaller families and use of contraceptive to achieve revised childbearing norms. This paper presents 18 years of data from the Sample Registration System, a demographic surveillance system operated by the Maternal and Child Health–Family Planning Extension Project in six study areas in Bangladesh. Prospective measurements of women's fertility preferences were used for classifying nearly 25,000 birth outcomes from 1983 to 2000 as intended, unintended, or ‘up to God/Allah’. Over the 18-year period, the level of unintended births varied from 22% to 38%, with the lowest levels in the mid-1990s. Fatalistic responses declined significantly from 25% in the mid-1980s to 1% by the late 1990s. Results of the comparison of two geographic areas of Bangladesh indicate differential declines in the levels of unintended pregnancies over the study period. Pros-pective measurements of unintended pregnancies were 2-3 times the magnitude indicated by retrospective estimates of unwanted births from the demographic and health surveys conducted during the study period. This unique dataset provides a rare opportunity to visualize the vast changes in fertility preferences and unintended births in Bangladesh from 1983 to 2000. Significant declines in fatalistic responses reflect broader social changes that occurred in Bangladesh to facilitate the fertility decline and contraceptive uptake. The drastic differences between prospective and retrospective measurements of fertility preferences highlight the importance of considering the strengths and limitations of each method when attempting to estimate the true level of unintended pregnancies and births in a population. Bangladesh is touted as a success story of demographic transition. In the 1970s, the total fertility rate (TFR) was just under seven births per woman, with 8% of women using contraception ,3. ThereBongaarts has described the changes in the proximate determinants of fertility as nations undergo a demographic transition and the subsequent impact on the levels of unintended pregnancies . IndividTo assess these broader societal changes, it is important to measure fertility preferences and their impact on actual fertility. Most work examining fertility preferences is derived from demographic and health survey data, which assess fertility preferences through retrospective assessments of fertili-ty preferences for births occurring within the past 3-5 years. Retrospective assessments are subject to rationalization bias as women are much less likely to characterize a pregnancy as ‘unwanted’ after the birth of the child ,10. ProsIn addition to concerns regarding the timing of measurements, existing studies also note difficulties in ascertaining valid measurements of fertility preferences due to differences in the way that fertili-ty is conceptualized by individuals and is believed to be under one's control. Studies across cultural settings document the existence of fatalistic or ‘up to God’ responses when asked about the desired number of children. Non-numeric responses may be more likely in populations in which fertility is not considered to be within ‘calculus of conscious choice’ of individuals , in popuTo assess fertility preferences and subsequent fertility, we used a unique, longitudinal dataset from the Maternal and Child Health–Family Planning (MCH-FP) Extension project sites in Bangladesh from 1982 to 2000. Using this powerful dataset, we were able to match prospective fertility preferences of women with nearly 20,000 subsequent birth outcomes and to assess the levels of and trends in unintended births over nearly two decades across six different areas of Bangladesh.In 1982, the International Centre for Diarrhoeal Disease Research, Bangladesh  established the MCH-FP Extension Project and the Sample Registration System (SRS) in Jessore (southwest) and Sirajganj  areas of Bangladesh . The MCHThe SRS was a quarterly surveillance system that registered demographic and health events in approximately 8,000 households across the study areas . In addiThe dataset used in this analysis was constructed by merging the cross-sectional data from the five KAP surveys with all the births registered during 1983-2000 in the SRS longitudinal surveillance database. A set of algorithms was established to link each birth to a prior measurement of fertility preference: (a) women with pregnancies or births without a prior measurement of fertility preference were excluded (Table); (b) births were linked to measurements of fertility preference from the closest, previous survey, up to two prior surveys; and (c) if multiple pregnancies occurred between KAP surveys, it was assumed that prior fertility preferences applied to all pregnancies.A member of the research team reviewed and hand-coded data relating to each study participant. The lead author cross-checked every tenth case. To decrease the number of excluded cases, the research team searched over 25,000 hard-copy files.The table presents the sociodemographic characteristics of the included versus excluded cases. The large majority (74%) of the excluded cases were women who had migrated into the study area upon marriage, for whom the SRS had registered a birth but without a prior measurement of fertili-ty preference gathered through the periodic KAP surveys. Aligning with this profile, the table shows that the excluded cases were younger, lower pari-ty, and more educated .any more children) compared to 13% of births that were retrospectively classified by the DHS as unwanted. In 1996 and 1999, the discrepancies between MCH-FP and DHS data are similarly large: 21-25% versus 11% of births in 1996 and 23-24% versus 14% of births in 1999 were classified as unwanted via prospective and retrospective measurements respectively.This unique, longitudinal dataset provides a rare opportunity to assess the prevalence of and trends in unintended births over an 18-year study period across six areas in Bangladesh, a setting characterized by progressive family-planning policies and programmes and rapid fertility decline. These analyses yield three key insights into the measurement of and trends in unintended pregnancies.First, these data clearly illustrate the significant decline in fatalistic or ‘up to God’ responses throughout the study period, documenting the broader transitions in childbearing norms that facilitated drastic changes in fertility rates and contraceptive prevalence during this time in Bangladesh. As first noted by Coale, the shift away from fatalistic responses over the study period is likely reflecting the notion of ‘calculus of conscious choice’ in which individuals and couples begin to identify fertili-ty as something that is within one's control and can be regulated . NumerouSecond, data from three of the six study areas mirror the trends in unintended pregnancies hypothesized by Bongaarts as individuals and couples first develop the idea of, then attempt to achieve, smaller family-sizes . Data 1. AlthougThird, these data provide a rare perspective by matching prospective assessments of fertility preferences with subsequent birth outcomes. Although these measurements have their respective limitations, results of the comparisons of prospective versus retrospective assessments indicate that retrospective assessments likely underestimate the true level of unintended pregnancies/births ,30. In tA few limitations of the study should be mentioned. The measures of fertility preference that were used across the MCH-FP surveys were usually trichotomous . Results of more recent studies have pointed to the utility of a scaled measure to better represent the nuances of fertility preferences ,30. In aThis unique dataset provides a rare opportunity to visualize the vast changes in fertility preferences and unintended births in Bangladesh from 1983 to 2000. The significant declines in fatalistic responses reflect broader social changes that were occurring in Bangladesh to facilitate the fertility decline and contraceptive uptake. The drastic differences between pros-pective and retrospective measurements of fertility preferences highlight the importance of considering the strengths and limitations of each method when attempting to estimate the true level of unintended pregnancies and births in a population.The authors acknowledge funding from the United States Agency for International Development (USAID), Dhaka office (GHS-A-00-03-00019-00) for this project. They thank several team members from ICDDR,B, who were integral to the collection, compilation, and analysis of the MCH-FP Project data, including Khorshed Mozumder, Ahsan Kabir, Mizanur Rahman, Rezaul Kabir, Siddiqur Rahman, Subash Das, and Carel van Mels.


Characterizing cortical computation has been a key challenge in neuroscience. It has been suggested that the cerebral cortex is involved in unsupervised learning  and the The hierarchical temporal memory (HTM) model of cortical computation  was usedFor the current work we are investigating tactile perception and implementing the HTM model using sensory inputs from artificial whiskers. The HTM model emphasizes that temporal patterns in inputs are extremely important features for learning and inference in the brain. This is especially the case with tactile perception since very little information is available at any single instant; how sensory inputs evolve over time conveys indispensable information about stimuli in the world. In modeling unsupervised learning, ‘time’ is commonly treated as indexing sequential inputs . HoweverWe are suggesting that cortical networks can dynamically maintain local oscillations to best match the relative frequencies with which inputs tend to occur. Assuming that all inputs to any cortical network are superimposed on its ongoing activity, these local oscillations can discretize the continuous stream of sensory inputs in signaling to subsequent levels in the hierarchy, as well as modulate the signal to suppress or enhance effects of specific components in the inputs based on their timing. We are demonstrating these effects in our implementation of HTM.


Transport of PGF2α to the ovary results in release of OT by luteal cells in a positive feedback loop leading to luteal regression. This signaling cascade can be rapidly blocked by exposing endometrial cells to physiologic levels of P. This mini review will focus on the mechanisms by which P may act to block OXTR signaling and the luteolytic cascade in the ruminant endometrium, with special focus on both non-genomic signaling pathways and non-receptor actions of P at the level of the plasma membrane. While this review focuses on ruminant species, non-classical blockage of OXTR signaling may be important for fertility in women.Expression of the oxytocin receptor (OXTR) in the endometrium of ruminant species is regulated by the ovarian steroids progesterone (P) and estradiol (E). Near the end of the estrous cycle, long-term exposure of endometrial epithelial cells to P results in loss of genomic P receptors (PGRs), leading to an increase in E receptors (ERs). Genomic regulation of the OXTR is mediated via suppression of ER signaling by P. Upon OT binding at the plasma membrane of endometrial cells, a signaling cascade is generated stimulating release of prostaglandin F In mammals, signaling by the neuropeptide oxytocin (OT) via receptors (OXTRs) in the uterus results in initiation of parturition [as reviewed by Kamel ] and con2; OT is secreted primarily from the posterior pituitary, but also by the corpus luteum (CL) of several mammalian species such as swine, ruminants, and primates . HoweveIncreased expression of OXTR near parturition is noted in all mammalian species, including primates and ruminants; OXTR signaling is critical for initiation of myometrial contractions in ruminants  concentrations were all able to interfere with OT-stimulated PGF2α secretion and intracellular calcium release . The observed inhibition by P of Oxtr function is at the level of the plasma membrane: P conjugated to bovine serum albumin, which prevents P from crossing through the plasma membrane easily, also inhibited membrane binding of OT. Specific and high-affinity binding sites for P were reported only in transfected CHO cells expressing the Oxtr. Moreover, high-affinity binding of P to Oxtr-expressing membranes was regulated by the state of the G-protein coupling to the receptor, based on additional experiments performed with the G-protein uncoupling reagent GTPγ S. These observations indicate that the interaction of P with the rodent Oxtr is possibly a direct steroid-receptor interaction, either by altering the conformation of the receptor or directly blocking the binding site for OT on the OXTR . E, cortisol, testosterone, and arginine vasopressin failed to compete for the P binding site, however, P, R5020 (synthetic progestin), RU 486, and OT all bound to the same site on plasma membranes. A radioreceptor exchange assay demonstrated that pre-exposure of membranes to P significantly increased the number of P binding sites. Inhibition of OT binding to endometrial membranes by P was dose-dependent at a low (nM) steroid concentration , P might be acting to exclude cholesterol from the plasma membrane, thus altering membrane fluidity in these SLCs . The effect these mPRs might exert on OXTR signaling in uterine tissues remains to be investigated.Other novel, non-genomic membrane PRs (mPRs) have been identified in the ovine uterus which may act to block local OXTR signaling (Ashley et al., The genomic PGR also rapidly activates the MAPK pathway upon stimulation in breast cancer cells via a SRC kinase-like SH3 domain (Faivre et al., 2α from endometrial tissue by P, which could lead to delayed luteolytic events. The best-characterized tissue to date of regulation of OXTR expression and OT signaling is the ruminant uterus, however, rapid inhibition of OT action by P remains to be investigated in the ruminant uterus during pregnancy. Inhibition of OXTR signaling may be due to non-receptor mediated events related to steroid overloading of plasma membranes, or via specific binding of P to one of several membrane-associated P receptors (Figure situ, further studies are needed to elucidate any involvement of membrane P receptors in blockage of OXTR signaling. The role of an ovarian OXTR in regulation of primate luteal tissue remains unclear, but P may act to block local OXTR signaling. Given recent studies identifying functional OXTR in primate luteal cells and P inhibition of OT-signaling in bovine SLCs (Davis et al., There is evidence that P directly antagonizes OXTR signaling via a non-genomic mechanism, however, there are only a few studies, some with conflicting results (summarized in Table s Figure . NeverthThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human T-Cell Lymphotropic Virus Type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It has been estimated that 10–20 million people are infected worldwide, but no successful treatment is available. Recently, the epidemiology of this virus was addressed in blood donors from Maputo, showing rates from 0.9 to 1.2%. However, the origin and impact of HTLV endemic in this population is unknown.To assess the HTLV-1 molecular epidemiology in Mozambique and to investigate their relationship with HTLV-1 lineages circulating worldwide.Blood donors and HIV patients were screened for HTLV antibodies by using enzyme immunoassay, followed by Western Blot. PCR and sequencing of HTLV-1 LTR region were applied and genetic HTLV-1 subtypes were assigned by the neighbor-joining method. The mean genetic distance of Mozambican HTLV-1 lineages among the genetic clusters were determined. Human mitochondrial (mt) DNA analysis was performed and individuals classified in mtDNA haplogroups.LTR HTLV-1 analysis demonstrated that all isolates belong to the Transcontinental subgroup of the Cosmopolitan subtype. Mozambican HTLV-1 sequences had a high inter-strain genetic distance, reflecting in three major clusters. One cluster is associated with the South Africa sequences, one is related with Middle East and India strains and the third is a specific Mozambican cluster. Interestingly, 83.3% of HIV/HTLV-1 co-infection was observed in the Mozambican cluster. The human mtDNA haplotypes revealed that all belong to the African macrohaplogroup L with frequencies representatives of the country.The Mozambican HTLV-1 genetic diversity detected in this study reveals that although the strains belong to the most prevalent and worldwide distributed Transcontinental subgroup of the Cosmopolitan subtype, there is a high HTLV diversity that could be correlated with at least 3 different HTLV-1 introductions in the country. The significant rate of HTLV-1a/HIV-1C co-infection, particularly in the Mozambican cluster, has important implications for the controls programs of both viruses. Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of Adult T-Cell Leukemia/Lymphoma (ATL), the Tropical Spastic Paraparesis/HTLV-1-associated Myelopathy (TSP/HAM) and other inflammatory diseases, including dermatitis, uveitis, and myositis. It is estimated that 2–8% of the infected persons will develop a HTLV-1-associated disease during their lifetimes, frequently TSP/HAM. Thus far, there is not a specific treatment to this progressive and chronic disease. HTLV-1 has means of three transmission: (i) from mother to child during prolonged breastfeeding, (ii) between sexual partners and (iii) through blood transfusion. HTLV-1 has been characterized in 7 subtypes and the geographical distribution and the clinical impact of this infection is not well known, mainly in African population. HTLV-1 is endemic in sub-Saharan Africa. Mozambique is a country of southeastern Africa where TSP/HAM cases were reported. Recently, our group estimated the HTLV prevalence among Mozambican blood donors as 0.9%. In this work we performed a genetic analysis of HTLV-1 in blood donors and HIV/HTLV co-infected patients from Maputo, Mozambique. Our results showed the presence of three HTLV-1 clusters within the Cosmopolitan/Transcontinental subtype/subgroup. The differential rates of HIV-1/HTLV-1 co-infection in the three HTLV-1 clusters demonstrated the dynamic of the two viruses and the need for implementation of control measures focusing on both retroviruses. Human T-cell lymphotropic virus type 1 was the first oncogenic human retrovirus to be identified in 1980 HTLV-1 has a remarkable genetic stability when compared with other retroviruses such as HIV (Human Immunodeficiency Virus). However, based on the nucleotide diversity of its LTR region, HTLV-1 can be grouped in 6 major genetic subtypes (a–f), most of them linked to some geographic regions HTLV screenings performed in many African countries have been limited to sero-epidemiological surveys studies using ELISA and Western Blot criteria for HTLV typing The aim of this study was to investigate the molecular identities of HTLV in Mozambique and therefore to gain insights to HTLV infection in the country, focusing on a set of HTLV seropositive blood donors and HTLV/HIV co-infected individuals from Maputo city.Both Co-infection Protocol (148/CNBS/05) as well the Blood Bank Study (78/CNBS/06) were reviewed by National Center in Bioethics in Health (CNBS) located in MOH, Maputo, Mozambique. All subjects provided written informed consent and were included in the study.HTLV-1 positive samples were originated from a cross-sectional study conducted among 2019 repeat blood donors at the Maputo Central Hospital blood bank in Maputo city between August and December 2006, where the prevalence of the infection is 0.9% Pfu DNA polymerase (Stratagene). Human mitocondrial (mt) DNA hypervariable segment I (HVS-I) (302 bp) was accessed by PCR using conditions formerly described High molecular weight DNA was extracted from whole-blood samples of all 25 subjects using QIAamp DNA Blood Mini kit (Qiagen). The PCR for HTLV typing was carried out as described LTR sequences were editing and alignment using BioEdit v5.0.9.  and Clustal W programs, respectively. Neighbor-joining (NJ) tree was build by PAUP* software version 4.0b10 The mtDNA sequences were classified in haplogroups in agreement with the HSV-I nucleotide polymorphisms described gag and env genes were considered as infected by HTLV, according to the instructions provided by the manufacturer. Two samples were negative to p53 band and one to P19. All HTLV positive samples in our study population were reactive to rgp46-HTLV-I and typed as solely infected with HTLV-1 by Western blot with no reaction to recombinant rgp46-HTLV-II.The results of screening for anti-HTLV-1+2 antibodies and confirmed by a Western blot assay  are in The genetic analysis of 25 Mozambican LTR sequences, using 32 reference sequences representing all HTLV-1 subtypes and sub-groups, as well as African HTLV-1 sequences, clearly demonstrated that all isolates belong to the Transcontinental subgroup of the Cosmopolitan subtype . These nIn order to check the representativeness of this sampling in relation to Mozambican population, mtDNA haplogroup analysis was conducted. This analysis, based on HSV-I sequence motifs, revealed that all those Mozambican individuals belong to African macrohaplogroup L, including the sub-haplogroups L1a, L1c, L1d, L2a, L3d and L3e. The African haplotypes were detected at different frequencies of L1a (28%), L1c (8%), L1d (12%), L2a (36%), L3d (12%), L3e (4%) and indistinctly distributed within the 3 HTLV-1 LTR clusters described above.The two pandemic human retroviruses, HIV and HTLV, were originated as zoonoses in Africa at very distinct times. HTLV is an ancient virus while HIV emerged in the early 20th century The HSV-I mtDNA haplogroup analysis from HTLV-1 infected individuals revealed that all the Mozambican sequences belong to African sub-Saharan haplogroups. It was not possible to establish a correlation between HTLV-1 lineages and a specific ethnics group, since diverse mtDNA haplotypes were observed in the three HTLV-1LTR clusters. All African sub-haplogroups detected in this study  were reported as the major frequencies in Mozambique The sub-Saharan Africa is considered to be endemic for HTLV-1 infection, with overall seroprevalence rates in Western and Central African countries being similar to the ones observed in Eastern and Southern African countries Most of the genetic variability of HTLV-1 is represented in Africa, where the virus was originated th century as well as, consequence of the migratory movements between India and Austral Africa (1860) The genetic relationship of HTLV-1 Transcontinental subgroup strains from South Africa and Mozambique suggests a common origin of HTLV-1 in both countries, probably due to their common peopling and migration patterns and their intense commercial/migratory linkage and vicinity. It was interesting to find a cluster joining Mozambique, Middle East and India strains that can also be explained by a noticeable Indian population in Maputo city due to the Indians people migration started in the 19


Several mechanisms underlying the molecular basis of ARVD/C type 1 have been proposed. Evaluating previously described mechanisms might elucidate how TGFβ3 contributes to disease progression in ARVD/C type 1. Here we review how TGFβ3 can induce fibrogenesis through Smad and/or β-catenin signaling. Moreover, the role of apoptosis is addressed. Finally the extent to which the immune system has been demonstrated to be a modulating and amplifying agent in the onset and progression of ARVD/C in general is discussed.Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited cardiomyopathy associated with cardiac arrhythmias originating in the right ventricle, heart failure, and sudden cardiac death. Development of ARVD/C type 1 has been attributed to differential expression of transforming growth factor beta 3 (TGF Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), also known as ARVD, ARVC, and ARVC/D, is an inherited cardiomyopathy. It is associated with cardiac arrhythmias originating in the right ventricle, heart failure, and sudden cardiac death . DemandiV1–V3), are major criteria. In addition, depolarization or conduction anomalies can be observed, for example, epsilon waves and late potentials, as well as arrhythmias, such as ventricular tachycardia [To help establish a diagnosis, Task Force Criteria have been proposed by McKenna et al.  and revihycardia . Fibrofahycardia .DSP gene), plakophilin 2 (PKP2), desmoglein-2 (DSG2), desmocollin-2 (DSC-2), and junctional plakoglobin (JUP). The nondesmosomal proteins related to ARVD/C are desmin (DES), transmembrane protein 43 (TMEM43), transforming growth factor β-3 (β3TGF), lamin-A/C (LMNA), titin (TTN), phospholamban (PLN), and α-T-catenin (CTNNA3). Even though these genes are associated with the different types of ARVD/C, often the underlying mechanisms resulting in the disease phenotype remain uncertain.As part of the Task Force Criteria, genetics may be a helpful tool in establishing or confirming a diagnosis. Several mutations associated with ARVD/C have been described to date. Five proteins, out of a total of twelve that are related to ARVD/C, are part of the desmosomal complex , and thuOver the years, it has become clear that ARVD/C is not a structural defect present at birth, explaining the original term dysplasia, and that the disease can also be biventricular or even left dominant . The afoβ3. The genetic origin of the disease was first mapped in 1994 [β3 gene [β3 [β3 expression in vivo has not been verified. Nevertheless, it seems reasonable to presume that TGFβ3 overexpression causes ARVD/C type 1.The present review will focus on potential mechanisms involved in the development of ARVD/C type 1 (OMIM: 107970). ARVD/C type 1 is a rare form of ARVD/C , found o in 1994 , but the in 1994 . For two[β3 gene . An earlgene [β3 . As the gene [β3  demonstrβ3.In the more prominent forms of ARVD/C, resulting from mutations in desmosomal proteins, occasionally multiple mutations were observed in a single patient , 29. Preβ3 (GeneID: 7043) is a secreted protein involved in tissue development and differentiation as well as fibrogenesis [β3 overexpression in fibrogenesis, cell-cell adhesion abnormalities, apoptosis, and the role of inflammatory injury in general in ARVD/C will be discussed.TGFogenesis . Howeverβ3 is directly involved in promoting fibrofatty infiltration in the myocardium, or that the replacement of myocardial cells is an indirect result of other processes, such as apoptosis, remains to be established.The extent of fibrosis in the heart is correlated with the severity of conduction anomalies, such as delayed activation . Howeverβ3 is known to be involved in tissue regeneration, accelerating healing and improving tissue strength, and it has been used to reduce scar tissue formation in wound healing [β3 in extracellular matrix organization. Active TGFβ is thought to affect fibroblasts through TGFβ receptor 1 and 2 (TGFBR1 and 2), activating the Smad signaling pathway, as reviewed by Leask [β receptor complex, and Smad 2 and 3 subsequently relay the signal to the nucleus, altering gene expression, as illustrated in β1) overexpression induces cardiac fibrosis, resulting in an increased collagen content in the heart [β3 exerts its function through the same signaling pathway, it seems possible that TGFβ3 overexpression will also induce fibrosis in the myocardium. This is further corroborated by the presence of both TGFβ 1 and 3 during cardiac fibrogenesis [β overexpression might excessively activate cardiac fibroblasts, resulting in fibrosis.TGF healing . This wa healing , implyinby Leask , and Shiby Leask . In thishe heart . As TGFβogenesis . Moreoveogenesis , 40. Thuper se might be insufficient to lead to ARVD/C. Persistent fibrosis resulting from TGFβ overexpression requires cofactors [However, Smad activation ofactors , which mAn alternative explanation for excessive fibrofatty tissue formation in the myocardium is episodes of apoptosis/necrosis. This would provide cofactors required for persistent fibrosis, as corroborated by several works, in which expression of these factors was observed after heart failure, dilated cardiomyopathy, and myocardial infarction , 43. Morβ3-activated Smad signaling, on one side and the biochemical environment created in the tissue to sustain fibrosis on the other side. Disturbing this equilibrium by excessive TGFβ3 expression may result in the augmented levels of fibrosis seen in ARVD/C.Thus, it is likely that the myocardial fibrosis is the result of a complex interplay of intracellular signaling pathways, among which TGFβ-catenin is an integral part of the adherens junctions, loss of cell-cell adhesion inhibits Wnt/β-catenin signaling. This perturbed cell signaling pathway can result in induced cardiac myocyte apoptosis and development of fibrofatty tissue [Clear signs of gap junction remodeling have been observed in ARVD/C patients –47, whicy tissue , 45. Yety tissue . Furthery tissue , 24.β3 and desmosomal proteins affect β-catenin signaling [β-catenin is stabilized through Wnt signaling [β-catenin translocates to the nucleus, regulating gene transcription  and subsequently Nemo-like kinase (NLK) in the heart. In turn, NLK inhibits Wnt/β-catenin signaling by counteracting β-catenin-induced gene transcription [β 1 and 3 have been shown to decrease E-cadherin expression via the Smad signaling pathway [β-catenin signaling, as was found in ARVD/C caused by mutations in DSP and JUP. Thus, TGFβ inhibited β-catenin signaling could stimulate adipogenesis in cardiac tissue.TGFcription . Moreove pathway , 60. Thiβ3 overexpression might be expected to have an activating role in the Wnt/β-catenin pathway. In human renal proximal tubular cells, TGFBR2 was shown to dissociate from adherens junctions upon TGFβ1 activation [β3 is known to bind to the same receptor, possibly eliciting a similar process in the heart. Incubation with TGFβ1 yielded increased levels of β-catenin, and dissociation of β-catenin from adherens junction complexes was observed [β-catenin was stabilized by TGFβ activity, thereby promoting Wnt/β-catenin signaling [β-induced fibrosis [Alternatively, TGFtivation . TGFβ3 iobserved . In addiignaling , 59. Thiignaling  or mightfibrosis .β3 overexpression. Several pathways have been associated with TGFβ signaling, sometimes with counteracting effects, as illustrated in β-catenin signaling, as the effects of Wnt signaling are strongly dependent on the cell type and signaling context [β overexpression on Wnt signaling remain obscure. Further research is necessary to elucidate the complex interplay of TGFβ signaling and Wnt signaling.At this point, the molecular mechanisms that might be involved in organ fibrogenesis are not properly defined . There i context . For thiDsp and Jup deficient mice, increased levels of apoptosis were observed [β-catenin signaling, which has been shown to be involved in regulation of apoptosis [β-catenin signaling might be, at least in part, responsible for the increased levels of apoptosis observed in ARVD/C [Finally, in both observed , 53. Thipoptosis . Hence, n ARVD/C –66.β3 might be exerted through its influence on cell-cell adhesion integrity, thereby altering β-catenin signaling in a variety of ways. Ultimately this can result in fibrofatty tissue development and apoptosis.Therefore, one of the effects of TGFMyocardial damage is one of the hallmarks of ARVD/C. Signs of increased levels of apoptosis in myocardial tissue are frequently observed in patients suffering from ARVD/C –67. Evenβ3 remains to be established. TGFβ signaling has been implicated in both cell growth and apoptosis, dependent upon the concentration of the cytokine and the signaling context. Myocardial cells of patients suffering from ARVD/C were shown to express and overexpress proapoptotic caspase 3 (CASP3) and Bax, respectively [β-induced apoptosis in other tissues, as reviewed by Schuster and Krieglstein [β to a heterodimer of TGFBR1 and 2, Smad proteins are activated. Ultimately, CASP3 and subsequently the caspase cascade are activated through this pathway. Thus, TGFβ3 overexpression might induce CASP3 expression, thereby lowering the threshold to apoptosis. Alternatively, altered Wnt/β-catenin signaling might also be involved, as discussed earlier.However, the origin of the increased level of apoptosis remains uncertain. Whether apoptosis in ARVD/C type 1 is a direct or secondary effect of the increased levels of TGFectively , 66. Expeglstein . Throughβ3 overexpression. Unraveling the source of myocardial degeneration is complicated by the subtle interplay of signaling pathways, and the multiple functions of TGFβ3 [β3 remains an enigma, as most other tissues seem to be unaffected [Nevertheless, at this point it remains uncertain whether the aforementioned pathways are the main processes involved in myocardial apoptosis resulting from TGFof TGFβ3 . Moreoveaffected . More reAnother contributor to the onset and progression of ARVD/C in general is inflammatory injury. Infiltration of inflammatory cells in necrotic or degenerative cardiac tissue has been reported in ARVD/C patients . For exaA frequent finding upon pathological examination of ARVD/C biopsies is myocarditis , 71. It α (TNF-α) and interleukin 1-β (IL1-β) converting enzyme result in production of nitric oxide synthase (iNOS) [Nevertheless, the role of viruses cannot entirely be excluded. Multiple studies report the presence of the enteroviral genome in the myocardium of ARVD/C patients with myocarditis, thereby supporting the possibility of its role in ARVD/C , 72. Seve (iNOS) . Ultimate (iNOS) .Genetic predispositions may amplify damage to myocytes caused by myocarditis, which in turn might quicken the progression of ARVD/C. However, it remains unclear whether inflammation and/or infection could be a primary cause of myocyte damage or a secondary response to degenerative loss of myocytes. Moreover, inflammation of cardiac tissue is not limited to the infiltration of inflammatory cells. Cardiac myocytes themselves produce inflammatory mediators and might thus contribute to immune-independent degeneration of cardiac tissue . As discβ3 overexpression, as in ARVD/C type 1, can be amplified. Such interference might contribute to severe myocardial injury, as observed in ARVD/C patients [Another possible mechanism by which inflammation plays a role in progression of ARVD/C might be the effect of inflammatory mediators on desmosomal proteins. As set out in the introduction, ARVD/C is predominantly caused by mutations in genes associated with cardiac desmosomes , 14. Intpatients .β3 overexpression has not been established. Finally, inflammatory mediators in ARVD/C patients may be useful in risk stratification and possibly the development of anti-inflammatory therapies to ameliorate quality of life.Taken all together, inflammation and/or infection may play a significant role in ARVD/C, possibly as a modulating and amplifying agent in disease progression. However, further research is required to elucidate the order in which certain pathogenic events occur; that is, whether inflammatory effects are a primary causal agent or a secondary response to already affected cardiac tissue. Moreover, a direct link to TGFβ3 overexpression, which is associated with ARVD/C type 1, might excessively activate cardiac fibroblasts through Smad signaling, resulting in fibrosis. Furthermore, TGFβ3 overexpression may affect cell-cell adhesion integrity by dissociating β-catenin, thereby interfering with cell signaling pathways, resulting in fibrogenesis and apoptosis. Concerning the latter, overexpression of TGFβ3 may stimulate CASP3 expression, resulting in a lowered threshold for apoptosis. Finally, inflammatory injury seems to play a significant role in progression of ARVD/C in general. The putative mechanisms seem to be interrelated, but at this point it is unknown whether these processes occur in concert, all induced by TGFβ3 individually, or that some of the observations result indirectly from TGFβ3 overexpression.The myocardial damage observed in ARVD/C is to a large extent due to the fibrofatty replacement and apoptosis of ventricular myocardium. TGFβ3TGF gene, one may question whether TGFβ3 overexpression is the actual molecular basis of ARVD/C type 1. One concern is that in vivo data on the (over) expression of TGFβ3 in ARVD/C type 1 patients are lacking. Another concern involves the unsuccessful efforts to identify mutations in β3TGF in the two families of the original linkage analysis by Rampazzo et al. [PKP2 gene was identified. A further concern is the apparent cardioselective phenotype of ARVD/C type 1, despite the widespread tissue expression of β3TGF. The recently generated Tgfb3 conditional knockout mouse [Although Beffagna et al.  demonstro et al. . Of noteo et al.  recentlyut mouse  may provβ3 and signaling pathways, their role is largely speculative. Therefore, further research is required to fully elucidate and verify the specific role TGFβ3 induced signaling and the immune system in ARVD/C type 1 disease progression. A better understanding of ARVD/C can ultimately lead to better means for diagnosis and possibly it can offer possibilities for treatment of the disease.Revealing the molecular basis for ARVD/C remains to be a challenging task. In this review, an overview of putative mechanisms associated with onset and progression of ARVD/C, in particular type 1, has been provided, defining different directions to better understand the aforementioned molecular basis of the disease. However, research on ARVD/C type 1 is hindered by the small number of patients. This explains, at least in part, that the role of the aforementioned mechanisms has not been established. Even though the putative mechanisms are based on observations in ARVD/C patients as well as known interactions between TGF


In the last decades, an outbreak of HIV/AIDS occurred in Mozambique (MZ), and since HIV and HTLV share the same routes of virus transmission/acquisition, we decided to estimate the actual prevalence of such retroviruses.Blood samples were collected from 208, 226 and 318 individuals from Northern, Central and Southern MZ, respectively, of all socioeconomic and demographic strata, attending Public Health Centers. Sera were assessed for HIV-1/2- and HTLV-1/2–specific antibodies by enzyme immunoassays and confirmed by Western blot. HTLV-1/2-seropositive blood samples were submitted for PCR  and real-time PCR (pol) assays, and sequenced.An overall HTLV-1/2 prevalence of 2.3% was observed, and increased with age. Regional variation in the prevalence of HIV and HTLV-1/2 was observed; 32.2%, 65.5% and 44% of individuals tested HIV-positive in Northern, Central and Southern MZ, respectively, and 2.4%, 3.9% and 0.9% tested HTLV-1-positive in the same regions. No association between HTLV-1 infection and socio-demographic variables or HIV status was detected. One woman aged 54 years, born in Northern MZ, HIV-negative, who complaints of rheumatic pain, presented high HTLV-1 proviral load, and phylogenetic analysis of env and LTR sequences clustered HTLV-1-isolate into Cosmopolitan subtype, transcontinental subgroup A (GenBank Accession numbers HM770441 and JF271853).These data suggest different risk factors and epidemiologic correlates of HIV and HTLV-1 transmission in MZ. HTLV-1 is endemic in this country, and arthritis was associated with HTLV-1 infection in one patient.


New ideas and approaches will be needed in the future to address the pressing questions of how ROS as signaling molecules can participate in the coordination of stress adaptation and development and how they are involved in the chatter of the organelles.The production of reactive oxygen species (ROS) in different plant subcellular compartments is the hallmark of the response to many stress stimuli and developmental cues. The past two decades have seen a transition from regarding ROS as exclusively cytotoxic agents to being considered as reactive compounds which participate in elaborate signaling networks connecting various aspects of plant life. We have now arrived at a stage where it has become increasingly difficult to disregard the communication between different types and pools of ROS. Production of ROS in the extracellular space, the apoplast, can influence their generation in the chloroplast and both can regulate nuclear gene expression. In spite of existing information on these signaling events, we can still barely grasp the mechanisms of ROS signaling and communication between the organelles. In this review, we summarize evidence that supports the mutual influence of extracellular and chloroplastic ROS production on nuclear gene regulation and how this interaction might occur. We also reflect on how, and  During their life plants face a vast set of environmental challenges: extreme changes in ambient illumination, temperature, and humidity, differences in soil salinity, attack by pathogens and herbivores, mechanical wounding, etc. To withstand all these challenges, plants have developed a repertoire of signaling pathways that is unparalleled in its complexity among living organisms. Signaling through plant hormones , cell su1O2), superoxide (2O2), and hydroxyl radical (•OH) are unavoidable by-products of aerobic metabolism . For example, ROS that are produced in the chloroplast have been implicated as intermediates in retrograde signaling from chloroplast to nucleus during acclimation of photosynthesis  can also be regarded as a part of a larger network where apoplastic signals induce the generation of ROS in the chloroplast, which in turn leads to regulation of nuclear gene expression by several still uncharacterized, but at least partially chloroplast-derived, ROS-dependent retrograde signals.A critical role in this signal integration and decision-making is played by a class of reactive forms of the molecular oxygen, collectively referred to as reactive oxygen species ROS; . ROS, intabolism  which hatabolism  as well tabolism . During tabolism , the roltabolism  and is ntabolism . Yet uncynthesis . Intriguynthesis . Thus, tFigure 2; 2O2, either spontaneously or in a reaction catalyzed by the superoxide dismutase  under physiological conditions and in many respects resembles a molecule of water. Its dipole moment, similar to that of H2O, limits passive diffusion of H2O2 through biological membranes. Possible candidates for the import of apoplastic H2O2 are aquaporins , a ubiquitous family of channel proteins that has undergone an extensive expansion in vascular plants . Those putative receptors or other sensory systems for extracellular ROS in plants have so far remained elusive, but for example, several classes of receptor-like protein kinases (RLKs) with cysteine-rich extracellular domains  have been suggested to be involved in ROS perception .What happens in the plant cell after an extracellular oxidative burst has been triggered? A connection of apoplastic and chloroplastic ROS into common signaling networks during the plant stress response is evident in various model systems and processes , althougFigure 2). The second increase in ROS production happens several hours after the pathogen attack  varies according to changing photosynthetic electron transfer and CO2 fixation rate. Extracellular stimuli, such as recognition of bacterial components by the plasma membrane receptors, can rapidly regulate chloroplastic functions. During plant–pathogen interactions the cues perceived in the apoplast trigger MAPK cascades  and result in fast down-regulation of photosynthetic genes and accumulation of H2O2 in chloroplasts that is necessary for initiation of HR-mediated cell death  at an early stage of pathogen recognition. This decrease in NPQ makes chloroplasts more predisposed to the production of ROS, which might be a priming mechanism for chloroplast ROS signaling at later stages of immune response  and on the other hand to a retrograde signal (soldat8 mutation in the chloroplastic RNA polymerase SIGMA SUBUNIT 6 (SIG6) gene increases the tolerance of seedlings to 1O2  biosynthesis, and hormonal control . For exall death . Anotherresponse . Severalresponse . For exae signal . Anothere signal . The sols to 1O2 , which ls to 1O2 . GUN1, as to 1O2 . The hems to 1O2 . The dis1O2) as a chloroplastic signal involved in the regulation of plant cell death. PS II and its light-harvesting antennae produce 1O2 when light-excited chlorophylls adopt the rare triplet state and then reduce triplet oxygen (1O2 is enhanced when the light-excited electrons cannot escape PS II chlorophylls because the downstream components of electron transfer chain (mainly the plastoquinone pool) are already over-reduced, a situation typical of excessive illumination. 1O2 readily reacts with lipids, proteins, and pigments and is rapidly quenched by water, which makes its diffusion distance from the site of production shortest among all ROS  conserved in higher plants are involved in this process, although their exact role is unknown . Regulation of CAS activity is linked to the state of photosynthetic electron transfer chain. CAS has earlier been shown to be involved in high light acclimation of the green alga Chlamydomonas reinhardtii , peroxiredoxin, and glutathione peroxidase. H2O2 produced in chloroplasts gives rise to retrograde signals. The signaling is not well understood and might be a combination of passive diffusion of H2O2with indirect pathways including hormonal (abscisic acid) signaling  seem to be involved in this H2O2 leakage  have similarities to the profiles induced by ozone, while 12 h after flg22 treatment the expression profile resembled that of chloroplastic ROS production induced by MV . It is unlikely that, for example, merely a change in the cytoplasmic redox state could carry the information about the subcellular source of H2O2. Therefore, as proposed  the apoplast, the chloroplast, and the nucleus. To achieve the elaborate and fine-tuned responses to biotic and abiotic stimuli that we observe on transcriptional, biochemical, and physiological level, intense and strictly controlled communication between the subcellular “crime scenes” must take place. While some components of this information exchange have been proposed, we still lack a thorough understanding on how the apoplast, the chloroplast, and the nucleus keep in touch. It will be perhaps one of the major challenges of ROS research in plants to understand ROS-induced signaling pathways between different organelles. Once we find out which components transmit information under specific conditions, we will be able to generate an integrated view of ROS signaling and its role in environmental adaptation.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.Research in the Plant Stress group of Professor Jaakko Kangasjärvi is supported by Biocentrum Helsinki, the Academy of Finland, the University of Helsinki, and the ERA-PG.