Modification of atrioventricular node is a usual and necessary operation to cure atrioventricular nodal reentrant tachycardia (AVNRT). In this operation, atrioventricular block is the most severe complication and its prevention is of our great concern. This complication always occurs under some special circumstances with potential risk. So, it is very important to realize such conditions, as in this paper. A patient with paroxysmal palpitation for 10 years, aggravating to shortness of breath with chest distress for 1 year; cardiac electrophysiological examination found slow conduction in both antegrade and retrograde paths of reentrant loop, and typical AVNRT could be induced. During effective ablation there was no junctional rhythm. In some special cases, modification of atrioventricular node should not only rely on the junctional rhythm to determine the ablation effect, but also on the time of cardiac electrophysiological examination, as such to avoid the severe complication of atrioventricular block caused by excessive ablation. Many paroxysmal supraventricular tachycardia are due to atrioventricular nodal reentry, and typically, irritation conduct slowly in antegrade path and fast in retrograde path. Patients suffered from this kind of tachycardia always complain with repeatedly paroxysmal palpitation, and with sudden ending or beginning symptoms. Modification of atrioventricular node by radiofrequency catheter ablation is the most effective method to cure this disease. During this course, operators conventionally judge the ablation effect by the occurrence of junctional rhythm, and then determine the ablation time, so as to avoid injure of atrioventricular node by excessive ablation. But in some special conditions with unconventional electrophysiological phenomena, operators will confront additional difficulties and risk during ablating, such as in the case reported below.A 22-year-old male patient, with paroxysmal palpitation for 10 years and aggravating shortness of breath, chest distress for 1 year, presented with tachycardia with sudden ending or beginning. There was no other disease history provided. His symptoms used to occur once a year, but more frequently in recent 1 year and were associated with chest distress and shortness of breath. He was admitted with no abnormal physical sign. Ultrasound showed normal heart structure and blood flow. Electrocardiogram (ECG) showed no abnormal appearance in sinus rhythm but displayed narrow QRS wave tachycardia when onset, 167 beats per minute (bpm) and RP = 80 ms, RP < PR . These s1S1) increased to 110 bpm; therefore, atrioventricular bypass excluded. Right atrial S1S1 did not induce tachycardia. Right atrial programmed basic stimulus coupled with cycle length decreasing extrastimulus (S1S2) induced multiple AV conduction jumps, all of their prolonged AV intervals were more than 70 ms; and induced 2-3 atrioventricular reentrant waves, which showed the phenomenon of slow conduction in both antegrade and retrograde paths [1S2 scanning could induce typical AVNRT, with the characteristic of slow in antegrade and fast in retrograde conduction, and with the same rate and morphology of it in spontaneous PSVT. Examination suggested multiple reentrant paths in atrioventricular node. Subsequently, ablation of the slow conduction area of the atrioventricular node performed at several ideal target points inferior and superior to the ostium of CS, guided by inner ECG and fluoroscopic images. During this course of modification of atrioventricular node, there was no junctional rhythm [1S2 induced no AV conduction jump phenomenon, no reentrant wave or any tachycardia; and S1S1 at right atrial and right ventricular induced no tachycardia; Wenckebach point of atrioventricular nodal conduction induced by atrial S1S1 greater than 170 bpm. Examination demonstrated the successes of the operation.Intracardiac electrophysiological examination and ablation: mechanical irritation by catheter could induce paroxysmal supraventricular tachycardia (PSVT) during the operation, and its inner ECG shows that ventricular wave (V) and atrial wave (A) interfused at coronary sinus (CS) leads from proximal to distal , and it de paths . Fartherl rhythm , which aThree-month follow-up met no recurrence of tachycardia and no complication.PSVT due to AVNRT sometimes has unconventional manifestation. Electrop1S2 in the examination were featured by its slow conduction in both antegrade and retrograde paths. Moreover, during effective ablating there was no junctional rhythm, which always appears and been considered as the standard evaluation of the ablating effect. This special condition displayed by these two rare electrophysiological phenomena deserves our caution.From the intraoperative intracardiac electrophysiological findings and ablation results, the mechanism for the tachycardia is atrioventricular nodal reentry, slow in antegrade and fast in retrograde conduction. But the repeatable atrioventricular nodal reentries also induced by SImplications of the case: Much more judgment difficulties add to the radiofrequency ablation of AVNRT, if there is no characteristic junctional rhythm in some special complex condition which are not common, and the potential risk of severe complication also add to these cases. After a certain time of ablation by experience at chosen ideal target point guided by inner cardiac electrograms and fluoroscopic images, cardiac electrophysiological test should be performed in time to judge whether the ablation is accomplished or not, but can not only rely on the appearance of junctional rhythm to judge the effect of modification of atrioventricular node, in order to avoid the severe complication of atrioventricular block caused by excessive ablation.In addition, if there is any correlation is unknown between the absence of junctional rhythm during ablating and the peculiarity of slow conduction in both antegrade and retrograde path of its reentrant loop before ablation.


Innate and adaptive immunity plays important roles in the development and progression of cancer and it is becoming apparent that tumors can influence the induction of potentially protective responses in a number of ways. The prevalence of immunoregulatory T cell populations in the circulation and tumors of patients with cancer is increased and the presence of these cells appears to present a major barrier to the induction of tumor immunity. One aspect of tumor-mediated immunoregulation which has received comparatively little attention is that which is directed toward natural killer (NK) cells, although evidence that the phenotype and function of NK cell populations are modified in patients with cancer is accumulating. Although the precise mechanisms underlying these localized and systemic immunoregulatory effects remain unclear, tumor-derived factors appear, in part at least, to be involved. The effects could be manifested by an altered function and/or via an influence on the migratory properties of individual cell subsets. A better insight into endogenous immunoregulatory mechanisms and the capacity of tumors to modify the phenotype and function of innate and adaptive immune cells might assist the development of new immunotherapeutic approaches and improve the management of patients with cancer. This article reviews current knowledge relating to the influence of tumors on protective anti-tumor immunity and considers the potential influence that radiation-induced effects might have on the prevalence, phenotype, and function of innate and adaptive immune cells in patients with cancer. For many years, the paradigm on which the majority of immunotherapeutic approaches for the treatment of cancer has been based, is that adaptive immune responses to tumors are similar to those that are induced in the generation of immunity to infectious pathogens. Although this is, in part at least, the case, a fundamental difference is that responses induced by infectious pathogens are driven by exogenous (“foreign”) proteins/molecules, whereas those to tumors must be induced by endogenous (“self”) proteins. Although not all tumor antigens are “self” antigens , the majority are. For effective immunity, it is therefore necessary to overcome the well-developed capacity of the immune system to regulate responses to self-antigens and tissues.Tumors can produce immunosuppressive factors such as IL-10, TGF-β, and vascular endothelial growth factor (VEGF). Tumor-derived cytokines such as IL-10 and TGF-β might protect against the development of anti-tumor immunity by influencing the functional capacity of antigen presenting cells (APCs) such as dendritic cells (DCs), and by promoting the generation/differentiation/expansion of immunoregulatory T cell populations which have the capacity to control and prevent immune responses    cells and (2), naïve CD4+ T cells recognize a tumor-associated or tumor-specific antigen at extrathymic sites and, after being activated, develop into an inducible Foxp3+ Treg cell (iTreg or Tr1) under a variety of conditions that facilitate/enable tumor immune evasion. These conditions include not only antigen presentation under sub-immunogenic or non-inflammatory conditions, but also chronic inflammation and infections. The observed selective accumulation of Treg cells in the tumor microenvironment suggests that this process can also be driven by tumors.According to Savage et al. , the biode novo generation of iTreg cells.As hypothesized by Adema and colleagues  results in distinct properties of these cells which include differential stabilities  are phenotypically and functionally different to the population discussed above and resistant to apoptosis or oncological therapies   in patients with metastatic breast carcinoma  and basiliximab (Simulect®) are anti-human CD25 monoclonal antibodies (mAbs) which have been approved for use in autoimmune disease, transplantation, and cancer, including HTLV-1-induced adult T cell lymphoma/leukemia  antagonists  , a co-stimulatory molecule of the TNF receptor family  abolishes Treg cell function by down-regulating the Cdk inhibitor p27Kip1 and restoring Akt phosphorylation  agonists can also be considered as promising tools for blocking Treg cell-mediated immunosuppression. TLRs are involved in the recognition of pathogen-associated molecular patterns (PAMPs) and the activation of processes that lead to innate immune recognition. TLRs are expressed by a range of immune and non-immune cells, including Treg cells, and they play an important role in tumor immunotherapy  is a key immune-checkpoint receptor expressed by several T cell subsets including Treg cells which plays an important role in the balance and regulation of adaptive immune responses. Programmed death ligand 1 (PD-L1) is constitutively expressed by B cells, DCs, macrophages, and T cells and can also be found on different tumor cells of human cancer  has been used as single-agent in patients with advanced solid refractory tumors  cells via their production of TGF-β , IL-2 or IL-15, CD56highCD16− NK cells are the primary producers of IFN-γ and promote the activation of immune effector cells. An additional population of CD56highCD16+ cells which accounts for 5% of peripheral blood NK cells exhibit unknown functions  like receptor family (KIRs) possess two or three extracellular Ig-like domains and integrate incoming activating or inhibitory signals, but recognize MHC class I molecules with higher affinity than ligands of activating KIRs  by NK cells and apoptotic tumor activity are suppressed in patients with head and neck cancer. This suppression is tumor-dependent and possibly mediated by soluble TNF superfamily receptors (solTNFSFRs)   and melanoma  which is overexpressed in some cancers. PGE2 also has a negative influence on NK cell function via the receptors CD16, NCRs, and NKG2D  -κB and this has a number of downstream effects with regards to the expression of molecules that promote a pro-inflammatory environment. Radiotherapy stimulates the migration and function of leukocytes via the release of cytokines such as TNF-α . These receptors interact with exogenous PAMPs such as endotoxin and endogenous “alarmins” such as high-mobility-group-box 1 (HMGB1), hyaluronan, and heat shock (stress) proteins which together comprise the group of danger-associated molecular patterns (DAMPs) Bianchi, . The expin vitro and LPS responses in vivo, RP105 can be considered as being a negative regulator of TLR4 responses.The impact of PRR signaling on radiation responses has been documented by Shan and colleagues who demonstrated that radiation-induced release of IL-12 and IL-18 from macrophages is accompanied by NF-κB activation and an upregulation of CD14 and TLR4/MD2 expression, thereby implying the involvement of the Toll signaling pathway  expression and metastatic capacity in MGC-803 gastric carcinoma cells  ligand 22 (CCL22) levels in the tumor cell supernatants  anti-tumor effect than would be expected from simple additive cell killing  can be released from a variety of cell types and have been identified in the peripheral circulation in a number of healthy and diseased states  co-activation, it enhances the capacity of NK cells these cells to kill membrane Hsp70 positive tumor cells  boosting trafficking of APCs to the tumor site, (2) augmenting antigen uptake of irradiated tumor cells; (3) increasing the maturation of APCs, (4) inducing maturation of immune effector cells in order to generate a robust immune response, and (5) limiting the immunomodulatory capacity of Treg cell populations.Accumulation of Treg cells in cancer patients is a major factor in tumor immune escape. Targeting of these cells might thus provide a mechanism by which anti-tumor immune responses could be restored. Immunotherapeutic strategies which target Treg cells and deplete functional Treg cells from the tumor microenvironment cells appear to shift the immune balance in support of effective anti-cancer immune responses. It is likely that effective anti-cancer strategies will depend on multimodal approaches which combine immunotherapy with targeted therapies that stimulate protective immune effector activity and downregulate the suppressive effects of Treg cells.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.


Survival of breast cancer patients with comorbidity, compared to those without comorbidity, has been well characterized. The interaction between comorbid diseases and breast cancer, however, has not been well-studied.From Danish nationwide medical registries, we identified all breast cancer patients between 45 and 85 years of age diagnosed from 1994 to 2008. Women without breast cancer were matched to the breast cancer patients on specific comorbid diseases included in the Charlson comorbidity Index (CCI). Interaction contrasts were calculated as a measure of synergistic effect on mortality between comorbidity and breast cancer.The study included 47,904 breast cancer patients and 237,938 matched comparison women. In the first year, the strongest interaction between comorbidity and breast cancer was observed in breast cancer patients with a CCI score of ≥4, which accounted for 29 deaths per 1000 person-years. Among individual comorbidities, dementia interacted strongly with breast cancer and accounted for 148 deaths per 1000 person-years within one year of follow-up. There was little interaction between comorbidity and breast cancer during one to five years of follow-up.There was substantial interaction between comorbid diseases and breast cancer, affecting mortality. Successful treatment of the comorbid diseases or the breast cancer can delay mortality caused by this interaction in breast cancer patients. Breast cancer patients with comorbidities have poorer survival than breast cancer patients without comorbidity -5. Few sa priori that the interaction may depend on the specific comorbid disease(s), and that the interaction may be different in the first year after breast cancer diagnosis than in subsequent years, since mortality in the first year is more likely affected by delayed breast cancer diagnosis and by treatment and toxicities.To resolve these limitations, we estimated the difference between the overall mortality rate and the expected mortality rate, given the baseline mortality rate, the effect of breast cancer on the mortality rate, and the effect of comorbidity on the mortality rate. We hypothesized This nationwide study included a cohort of Danish breast cancer patients aged 45 to 85 years who were diagnosed between 1994 and 2008, and a comparison cohort of women without breast cancer matched to the breast cancer patients on specific diseases included in the CCI . The popInternational Classification of Diseases, revision 7 (ICD-7) until 2003, when recorded diagnoses were converted to ICD-10. From the DCR, we identified female breast cancer patients diagnosed between 1994 and 2008 (ICD-10 code: DC50). We used the CRS to select up to five comparison women from the general population, matched to each breast cancer patient on age and history of the specific comorbidities defined below. The women in the comparison cohort had to be free of breast cancer on the date of breast cancer diagnosis for the corresponding case. The index date was defined as the breast cancer diagnosis date for cases in the breast cancer cohort and also for the women matched to them in the comparison cohort.The Danish Cancer Registry (DCR) contains nearly complete data on cancers diagnosed in Denmark ,11. DiagThe Danish National Registry of Patients (NRP) has recorded all non-psychiatric discharge diagnoses from inpatient admissions since 1977 and from outpatient clinic visits since 1995 . DiagnosWith linkage to the CRS, we followed the breast cancer and matched cohorts until death, emigration or 31 December 2011. Because members of the comparison cohort had no history of breast cancer, we did not ascertain breast-cancer specific or other cause-specific mortality.We calculated the frequency of women in the breast cancer cohort and the matched comparison cohort within categories of age , year of index date, CCI score , individual diseases included in the CCI index (presence/absence), and, for the breast cancer cohort, cancer stage .Crude mortality rates with 95% confidence intervals (CIs) were calculated within categories of baseline variables for 0–1 and >1–5 years of follow-up. The matching was dissolved when stratifying the follow-up period, so age-standardized mortality rates were calculated using age weights from the breast cancer cohort on the index date as the standard.We calculated the interaction contrast (IC), which measures the departure of the mortality rates from an additive model . It is ca posteriori repeated all interaction analyses excluding these diseases from the CCI.Although chronic pulmonary disease and “any tumor” were prevalent comorbidities in the breast cancer cohort, these diseases did not interact with breast cancer to affect mortality rates. We therefore The initial cohorts consisted of 48,292 breast cancer patients and 237,938 matched women from the general population. In the breast cancer cohort, 390 (0.81%) women were not matched with any woman in the comparison population. Of these unmatched breast cancer patients, 20% were between 81 and 85 years of age, compared to 9.1% of the matched breast cancer patients. A larger proportion of the unmatched breast cancer patients had a CCI score of ≥4 compared to the matched breast cancer patients (15% vs. 0.9%). Therefore, the combination of old age and multiple comorbidities precluded matching on both age and specific comorbid conditions, resulting in exclusion of these breast cancer patients from the analyses.Analyses were conducted with SAS version 9.2 . This study was approved by the Danish Data Protection Agency (2011-41-6174). No further permissions are needed to conduct studies with no intervention or participant contact in Denmark. Characteristics of the breast cancer and matched cohorts are presented in In the first year of follow-up, the interaction between breast cancer and comorbidity accounted for 17 deaths per 1000 person-years (PY)  for a CCI score of 1, 12 deaths per 1000 PY  for CCI scores of 2–3, and 29 deaths per 1000 PY  for a CCI score ≥4. These represented 17%, 9%, and 10% of total mortality rates, respectively, among the breast cancer patients with comorbid diseases. When the ICs were stratified on breast cancer stage, the interaction observed for the CCI score was primarily driven by distant and unknown stage cancer, as shown in Although history at index date of chronic pulmonary disease and “any tumor” were relatively common in the breast cancer cohort, the 0–1 year ICs were only 8.6/1000 PY  for chronic pulmonary disease and -13/1000 PY  for “any tumor.” When we repeated all analyses for the CCI scores without assigning weights to these two disease types, the 0–1 year overall estimates of the ICs rose from 17 to 21/1000 PY  for a CCI score of 1, from 12 to 31/1000 PY  for a CCI score of 2–3, and from 29 to 67/1000 PY  for a CCI score of ≥4. The ICs for the >1–5 year survivor cohort increased only slightly.The interaction contrasts between breast cancer and the specific Charlson comorbid diseases were larger during the first year of follow-up than during years one to five of follow-up. The disease with the largest IC in the first year of follow-up was dementia ). When we stratified analyses by breast cancer stage, the interaction between breast cancer and dementia was driven by interaction in the stratum of distant-stage cancers ). The ICs for dementia in the strata of local-stage  and regional-stage  cancers were much smaller. The stage distribution among breast cancer patients with dementia was skewed toward later stage at diagnosis compared with breast cancer patients without dementia. In the first year after breast cancer diagnosis, the mortality rate of breast cancer patients with dementia exceeded that of breast cancer patients without dementia in local-, regional-, and distant-stage strata, yielding a stage-adjusted MRR of 5.0 .In the first year after diagnosis, there was also interaction between breast cancer and other comorbid diseases, including metastatic solid tumors , mild liver disease , moderate to severe renal disease , and diabetes with end-organ damage .In the period one to five years after the index date, there was some interaction between breast cancer and leukemia , moderate to severe liver disease , mild liver disease , and diabetes with end-organ damage . Data for the individual Charlson diseases are presented in In this large, population-based cohort study from Denmark, including more than 47,000 breast cancer patients and 200,000 matched women from the general population, we found that overall mortality in the first year after breast cancer diagnosis was influenced by interaction between breast cancer and comorbid diseases present at diagnosis. The interaction was most pronounced in the strata of distant and unknown breast cancer stage. Among individual diseases, dementia interacted most strongly with breast cancer, but metastatic solid tumors, mild liver disease, moderate to severe renal disease, and diabetes with end-organ damage also showed strong interactions. In the >1–5 year survivor cohort, there was no strong interaction with the CCI summary comorbidity score, although some interaction was observed with leukemia, moderate to severe liver disease, mild liver disease, and diabetes with end-organ damage. A particular strength of this study is the inclusion of a comparison cohort free of breast cancer matched to the breast cancer cohort on specific comorbidities, which allows for the study of disease-specific clinical interactions between breast cancer and comorbidity. A concomitant limitation was our inability to study disease-specific causes of death, since members of the comparison cohort were unlikely to die of breast cancer. We included all women with breast cancer diagnoses from the entire country and achieved complete follow-up through the CRS. Registration of breast cancer in the DCR is nearly complete . The valThe interaction between breast cancer and comorbidity was mainly observed during the first year after breast cancer diagnosis, possibly due to lack of focus on care for comorbid diseases during cancer treatment. A recent study based on SEER data showed equal quality of care for comorbid conditions in breast cancer patients and non-cancer controls, but this was at three years after the cancer diagnosis . In the Interaction contrasts were negative in some analyses, although often imprecisely measured. Negative interaction contrasts were observed most often in the local and regional stage categories. This pattern suggests that prevalent and well-managed comorbidities brought breast cancer patients to medical attention and diagnosis sooner, resulting in a stage-shift to earlier and more treatable breast cancers within the early-stage categories. In later stage categories, breast cancer patients with severe comorbidity may be diagnosed with breast cancer late and at an unfavorable cancer stage, as some comorbid conditions could mask evidence of this cancer . We haveTo our knowledge, this study is the first to report specific interaction contrasts between breast cancer and the CCI score or individual diseases included in the CCI that affect the mortality rate. Studies that lacked a cohort free of breast cancer have shown that comorbidity and associated suboptimal breast cancer treatment increase the risk of death without recurrence in older women . Other sIn summary, our study shows a clinical interaction between prevalent comorbidities and overall mortality in breast cancer patients—particularly within one year after breast cancer diagnosis and mainly in patients with distant and unknown stage breast cancer. There was substantial interaction between dementia and breast cancer, suggesting that these patients tend to have breast cancer diagnosed at later stages. Successful treatment of the comorbid diseases or the breast cancer can delay mortality caused by this interaction. Table S1Crude mortality rates, adjusted HRs, and interaction contrasts (ICs) by individual diseases in the Charlson Comorbidity Index for the breast cancer cohort and the matched comparison cohort during 0-1 year of follow–up.(DOCX)Click here for additional data file.Table S2Standardized mortality rates, adjusted HRs, and interaction contrasts (ICs) by individual diseases in the Charlson Comorbidity Index for the breast cancer cohort and the matched comparison cohort during 1-5 years of follow–up.(DOCX)Click here for additional data file.


Melatonin inclusion into in vitro oocyte maturation (IVM) protocols has been suggested because it possesses a powerful free radical scavenger capability that improves the quality of the oocyte used in in vitro embryo production (IVP). The aim of our study was to investigate the presence of melatonin membrane receptors (MT1and MT2) and MT3, which is the melatonin binding site of NQO2 enzyme, in both oocytes and hatched blastocysts to consider an additional subcellular mechanism responsible for the effects of melatonin on IVP.125I]-iodomelatonin (17 pM) in embryos. The kind of melatonin site was investigated in oocytes and embryos by immunocytochemistry. In vitro fertilized bovine embryos produced from in vitro maturated oocytes supplemented with melatonin (0.0001 to 1000 nM) were analysed to determine their cleavage and blastocyst formation rates.The presence of the high affinity melatonin receptors was investigated through an autoradiographic binding assay, using the non-permeable ligand [125I]-iodomelatonin (17 pM) binding in blastocysts was blocked by pre-incubation with melatonin (30000 nM), showing the presence of the high affinity melatonin receptors. MT1, MT2 and NQO2 immunoreactivity was observed in oocytes. MT1 immunoreactivity was observed in hatched blastocysts, however MT2 and NQO2 were not observed in this embryonic stage. Melatonin (pM) triggered significant difference in both cleavage and blastocysts formation rates.The [The high affinity MT1 melatonin receptor must be taking part in IVM events; furthermore it is the first melatonin receptor to appear during bovine embryo development in vitro. Environmental lighting controls temporal changes for organisms that live on earth. In mammals, the major biological clock is located in the suprachiasmatic nucleus (SCN). This major clock drives other minor biological clocks that are located in the peripheral tissues. In the dark phase of the circadian rhythm, the SCN signals the pineal gland to release indoleamine melatonin (N-acetyl-5 methoxytriptamine). During pregnancy, maternal melatonin crosses the placenta, signalling the day length for the fetus, and modulates fetal development -4.2O2) and the super-oxide anion (O2-). This neurohormone also has an indirect antioxidant role thought to be triggered by membrane receptors and binding sites, mainly resulting in the up regulation of the antioxidant enzymes  and the down regulation of the oxidant enzymes (NO synthase and lipoxygenases) -iodomelatonin  (17 pM) for 1 h. The pool used for non-specific binding was incubated in the presence of melatonin (30000 nM) for 30 min. After incubation, the embryos were washed with phosphate buffer solution , placed in slides and dried. The slides were exposed to [125I] sensitive film  for two weeks, and then the film was developed, fixed , rinsed and dried. The image was captured by scanning (HP Scanjet 2400) and then analysed using Image J .Two pools of six day 7 bovine blastocysts in vitro-produced from oocytes maturated without melatonin were incubated in a serum free medium containing the melatonin membrane receptors agonist -iodomelatonin (17 pM)  on bovine oocyte IVM and embryo development were analysed at day 2 (cleavage rate) and at day 7 (blastocyst yields). Melatonin improved the quality of bovine oocytes maturate in vitro, resulting in an increase in the cleavage rate and in significant blastocyst formation rate with an optimum concentration of 1 pM. However, the cleavage rate of embryos from oocytes treated with melatonin presented a gradual decline in response to increased melatonin concentration beyond 1 pM. Similar results were obtained when melatonin-treated blastocysts development was examined. The improvements of melatonin to blastocyst development decreased in a similar manner as observed for embryo cleavage over the same concentration range (Table In this study we showed that a pM concentration range of melatonin supplementation in IVM medium functionally increases cleavage and blastocysts formation rates. However, while increasing the melatonin concentration up to a μM range, the positive effects of melatonin decreased in comparison with the values of the control group. This profile is in accordance with the presence of the melatonin targets that bind with different affinities in oocytes, and the most effective concentration range is related to the MT1 melatonin receptor. In agreement with this pharmacological profile, MT1, MT2 and NQO2 immunoreactivity were visualized in oocytes for the first time in this study. Interestingly, in blastocysts, the autoradiographic binding assay showed a high affinity site, and immunocytochemistry assays only showed the presence of the MT1 melatonin receptor. Since the embryonic genome is activated at the 8-cell stage in bovine embryos, there is a complete absence of maternal genetic inheritance in day 7 blastocysts . Thus, tHerein, we also showed for the first time the presence of the cytosolic NQO2 enzyme in oocyte. This detoxifying enzyme is thought to produce free radicals when activated by xenobiotics, such as menadione and is inhibited by high concentrations of melatonin , as foun2+ waves, in parallel with the inhibition of the cAMP pathways [Despite powerful scavenger actions being considered the pivotal mechanism underlying melatonin protection in different cells ,38, melapathways ,42.2+ and PKC to bovine oocyte maturation [The mechanism of the oocyte self-maintaining in arrest is poorly understood, however, a review about its control shows that bovine oocyte maturation is modulated by inhibition of both cAMP and cGMP levels as well as by the energy sensor AMPK, which is activated by AMP. In addition, there are studies showing the importance of the Caturation . MT1 andturation . InteresAlong with the clear immunoreactivity to MT1, MT2 and NQO2 in oocytes, the addition of a melatonin pM concentration increased cleavage and blastocysts formation rates. We suggest this is due to the effects of the MT1 receptor. We suggest that this hormone does not only have free radical scavenger action, but it also has effects on oocyte maturation by a myriad of mechanisms, including membrane receptor activation.AMPK: Adenosine monophosphate-activated protein kinase; IVF: In vitro fertilization; IVM: In vitro maturation; IVP: In vitro production; NQO2: NRH:quinone oxidoreductase 2; MT3: Melatonin binding site; PKC: Calcium-activated kinase; SCN: Suprachiasmatic nucleus.The authors declare that they have no competing interests.RVS carried out the autoradiographic binding and IVP assays, and helped to draft the manuscript. SDBC carried out IVP and immunoassays. MSM participated in coordination, immunoassays, performed the statistical analyses and helped to draft the manuscript. OMO participated in its design and coordination and critically review the manuscript. LFSS conceived of the study, participated in its design and coordination, and draft the manuscript. All authors read and approved the final manuscript.


Histone deacetylase inhibitors (HDACi) are small molecules that have important and pleiotropic effects on cell homeostasis. Under distinct developmental conditions, they can promote either self-renewal or differentiation of embryonic stem cells. In addition, they can promote directed differentiation of embryonic and tissue-specific stem cells along the neuronal, cardiomyocytic, and hepatic lineages. They have been used to facilitate embryo development following somatic cell nuclear transfer and induced pluripotent stem cell derivation by ectopic expression of pluripotency factors. In the latter method, these molecules not only increase effectiveness, but can also render the induction independent of the oncogenes c-Myc and Klf4. Here we review the molecular pathways that are involved in the functions of HDAC inhibitors on stem cell differentiation and reprogramming of somatic cells into pluripotency. Deciphering the mechanisms of HDAC inhibitor actions is very important to enable their exploitation for efficient and simple tissue regeneration therapies. Stem cells are distinguished from other cell types by their unique properties to self-renew and differentiate along multiple lineages . These pDNA methylation and histone modifications constitute major mechanisms that are responsible for epigenetic regulation of gene expression during development and differentiation –4. Among There are up to date 18 genes coding for histone (or epsilon lysine) deacetylases in the mammalian genomes. They are grouped in four families. Group I . IIa , IIb 6,10), III (SIRT 1–7), and IV (HDAC 11) [,10, III HDAC inhibitors (HDACis) are natural or synthetic small molecules that can inhibit the activities of HDACs. In spite of similarities in their enzymatic activities, loss of function experiments have attributed highly specific roles to individual members of HDAC proteins in the course of development and differentiation. In addition, HDAC inhibitors that have broad specificity towards their HDAC targets have shown highly specific effects depending on the target cell type . The profound events that govern stem cell differentiation and somatic cell reprogramming to pluripotency are mainly epigenetic . HDACis Analysis of knockout mice lacking HDAC genes has revealed their functions during mammalian development and differentiation . HDAC1 gSimilar to the cardiac differentiation, HDAC1 and 2 have essential but redundant roles in the differentiation of neuronal precursors into neurons . DeletioFinally, HDAC1 and 2 have important functions in hemopoiesis . HDAC1 aHDAC3 deletion is embryonic lethal due to deficient gastrulation –23 that Although class I HDACs are widely expressed, members of the IIa group show tissue-restricted expression. HDAC4 regulates skeletogenesis and knockout mice die in the first week after birth due to excessive ossification of endochondral cartilage which interferes with breathing . This efHDAC expression and activity are intimately associated with the emergence of neoplasias. In Acute Promyelocytic Leukemia (APL), fusions between Promyelocytic Leukemia (PML) and Retinoic Acid Receptor (RAR) recruit HDACs resulting in the repression of differentiation-related genes , 34. In HDAC class I and II inhibitors (HDACi) fall into discrete structural categories such as hydroxamic acids, cyclic peptides, benzamides, benzofuranone, and sulfonamide containing molecules , 38. TheDue to their activity in epigenetic regulation, HDACis have been widely used in order to alter the differentiation state of stem and somatic cells as shown in Differentiation is a process of gradual loss of potency that ends up to the point where specific cell fate is acquired. Embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts , 41, 42 In accordance with their effects on the differentiation of cancer cells, HDACis are able to promote the differentiation of ES cells. Treatment with Trichostatin A (TSA) promotes morphological and gene expression changes reminiscent of differentiation even in the presence of LIF , 52. InhFgf4 decreased after treatment with Valproic acid (VPA) and TSA. In contrast, the same treatment of P19 cells, which are more differentiated, caused the elevation of Fgf4 expression [In contrast to these studies, other studies have shown that HDACis increase self-renewal and interfere with differentiation. Low doses of TSA (10 nM) reverted mouse embryoid bodies towards the undifferentiated state  and emplpression . In agrepression . Hence, Directed differentiation of ES cells is not easy to control. Differentiation protocols generally rely either on the generation of ES cell aggregates (embryoid bodies) or on culturing on stromal cells. Effectiveness and selectivity need to be significantly improved in order for ES cell to be used as tools for cell-based therapies.HDACi treatment was used for directed differentiation of mouse ES cells towards the cardiomyocytic lineage. TSA added on embryoid bodies between days 7 and 8 potentiated cardiac differentiation due to hyperacetylation of GATA4  a masterAs indicated previously ablation of HDAC1 and 2 is postnatal lethal due to disorganization of brain structures . HoweverSpecifically, VPA was reported to increase neuronal differentiation of adult neural progenitor cells and inhibit astrocyte and oligodendrocyte differentiation . MoreoveThe molecular mechanism of VPA function was induction of NeuroD, a neurogenic bHLH transcription factor . DerepreMouse and human hemopoietic stem cells (HSC) self-renewal was potentiated by chlamydocin . In anotMesenchymal stem cells (MSCs) from adipose tissue and umbilical cord blood were treated with two HDAC inhibitors, VPA, and NaBu . PosttreCardiac side population cells isolated from rat hearts were coaxed in cardiomyocytic differentiation by TSA treatment . TSA indReprogramming differentiated somatic cells to pluripotent stem cells has emerged as a way of producing patient-specific stem cells. These cells can be possible candidates for regenerative medicine after their differentiation to a specific cell fate.A strategy used to reverse the differentiated state of cells was somatic cell nucleus transfer (SCNT) to enucleated eggs or oocytes , 93. ThiEarly reports have applied TSA to donor cells  or to thIn a pioneer work, the group of Yamanaka  reprograIn order to improve the efficiency of reprogramming, several strategies were developed  using smIn a recent study , human fAnother HDAC inhibitor NaBu used at low doses improved the generation of iPS cells by 50-fold by using retroviral or “piggyback” vectors for reprogramming human fibroblasts even in the absence of Klf4 and c-myc . In anotC. elegans as model [In addition to the typical iPS cells, reversion of differentiation was assisted by the addition of HDACi in other cell types. Dedifferentiation of primordial germ cells (PGC) into pluripotent embryonic germ (EG) cells was achieved using TSA to replace FGF-2 . A high-as model . The resStem cell methodologies have revolutionized modern therapeutic strategies that aim to replace damaged cells or tissues. Controlling the pluripotent stem cell fate  is depenMost importantly, HDACis have shown considerable activity in directing the neuronal, cardiomyocytic, and hepatic lineages differentiations. In most cases where the molecular mechanism was examined, it involved the induction of differentiation-regulating transcription factors. Moreover, HDACis were used in somatic cell reprogramming processes. Treatment of donor cells before transfer or embryos following transfer resulted in facilitation of embryo cloning and improvement of embryo developmental potential. These effects were due to enhanced histone acetylation, chromatin decompaction, increase of RNA synthesis, and inhibition of apoptosis. Due to the ethical issues raised by embryo cloning, these techniques are not yet applicable to humans. Therefore, the recent achievement of iPS generation has offered great expectations in custom-specific stem cells for human health. In that field, there is increasing effort in omitting retroviral vectors, oncogenes, and—if possible—all kinds of exogenous genetic material. Substituting transcription or signaling factors with simple small molecule reagents can render the therapies both safer and simpler. For that purpose, HDAC inhibitors have shown activity to enhance reprogramming and substitute for the presence of transcription factors, importantly the oncogenes c-myc and Klf4 . However


Accumulative disturbances can erode a coral reef’s resilience, often leading to replacement of scleractinian corals by macroalgae or other non-coral organisms. These degraded reef systems have been mostly described based on changes in the composition of the reef benthos, and there is little understanding of how such changes are influenced by, and in turn influence, other components of the reef ecosystem. This study investigated the spatial variation in benthic communities on fringing reefs around the inner Seychelles islands. Specifically, relationships between benthic composition and the underlying substrata, as well as the associated fish assemblages were assessed. High variability in benthic composition was found among reefs, with a gradient from high coral cover (up to 58%) and high structural complexity to high macroalgae cover (up to 95%) and low structural complexity at the extremes. This gradient was associated with declining species richness of fishes, reduced diversity of fish functional groups, and lower abundance of corallivorous fishes. There were no reciprocal increases in herbivorous fish abundances, and relationships with other fish functional groups and total fish abundance were weak. Reefs grouping at the extremes of complex coral habitats or low-complexity macroalgal habitats displayed markedly different fish communities, with only two species of benthic invertebrate feeding fishes in greater abundance in the macroalgal habitat. These results have negative implications for the continuation of many coral reef ecosystem processes and services if more reefs shift to extreme degraded conditions dominated by macroalgae. An ecosystem’s ability to recover from degradation is eroded by increases in frequency, intensity and array of disturbances All major coral reef regions of the world have undergone declines in coral cover Complex interconnections among organisms and with their physical environment, imply that changes to one aspect of the ecosystem may lead to a subsequent series of, often unanticipated, changes to the ecosystem’s community assemblage The interactions between the foundational structure upon which the live reef is built, the underlying substratum, and changes in the benthic community, may hinder essential ecosystem processes required for recovery, and perpetuate an alternative community. For example, coral recruit survivorship is considered an essential process for recovery Coral reefs of the Seychelles archipelago offer a unique opportunity to assess differing benthic communities. The inner Seychelles islands are geographically isolated, were severely impacted by the 1998 mass bleaching event, and there is a good record of post-disturbance degradation A research permit for this work was granted by the Seychelles government through the Seychelles Bureau of Standards; permit number A0347.2 area around the inner Seychelles islands  were surveyed in October 2010. Fishing practices in the inner Seychelles use non-destructive techniques  due to their high coverage. The underlying substratum, defined as the substratum below recorded benthic cover or the top 10 mm of sand/sediment, was categorised into loose dead coral rubble, consolidated rubble , solid carbonate pavement, or bommie . Fish species were assigned to 8 functional groups based on the literature and FishBase: obligate corallivores, browsing herbivores, other herbivores , planktivores, piscivores, non-coral invertivores (hereafter invertivores), omnivores  and generalist carnivores (fish and invertebrate feeders). Additionally, the level of exploitation sustained by different fish species was assigned at four levels: primary targets, important by-catch, occasional by-catch and non-fished species Twenty-one carbonate fringing reefs within a 3600 kmThe data were organized into four matrices; i) benthic habitat  that was natural log transformed to improve the spread of the data, and normalised to standardize the contribution of variables measured as percent cover and those measured on different scales, ii) underlying substrata cover (4 variables), iii) fish functional group abundances (8 variables) that were square-root transformed to downweigh abundant groups Variability in benthic composition among reefs was related to underlying substratum, and also the composition of fish assemblages, in two ways. First, data points ( = transects) on the underlying substratum and fish assemblage ordinations were colour-coded according to groups identified from the benthic cover hierarchical cluster analysis to visualize relationships. Second, the BEST BIO-ENV routine was carried out using a Spearman rank correlation between the different similarity resemblance matrices to identify the variable or group of variables that best explained similarities among the data matrices H’, which takes into account both abundance and the number of functional groups (maximum n = 8). The relationships between the benthic gradient  and fish functional group diversity (H’), fish species richness, total fish abundance, and individual functional group abundances were examined using General Additive Models (GAM). GAMs incorporate the possibility of non-linear relationships between the response and predictive variables A combination of cluster analysis and ordination showed the presence of contrasting benthic assemblages along a gradient from coral to macroalgae. To investigate whether there were any fish species that typified either assemblage, we ran a similarity of percentages (SIMPER) analysis using a subset of the fish species matrix that reflected the two extreme clusters of transects identified by the slice through the benthic cluster diagram. This represented transects dominated by macroalgae versus transects with high coral cover and structural complexity. An index of the fish functional group diversity was calculated using the Shannon-Weiner diversity index, Benthic cover of reef organisms was highly variable among the 21 reefs in the inner Seychelles. Live coral cover ranged from 0 to 47% (±5.1 SE) and macroalgae cover from 0 to 76% (±6.7 SE) per reef . The firs = 0.16, p<0.05) corroborated this pattern, finding a weak but significant correlation between the benthic and underlying substrata distance matrices, specifying the presence of pavement rather than rubble as the principal cause of similarity.The underlying substrata of the reefs varied from loose rubble to consolidated carbonate pavement. When highlighted on the underlying substrate PCA plot, transect groupings from the benthic cluster analysis were not apparent, however reefs found at both extreme ends of the benthic PC1 were associated with more stable substrata. It is to be noted that the stress level of the MDS was fairly high, so although general patterns are robust, details need to be interpreted with some caution s = 0.48, p<0.001). Fourteen fish species, including planktivores, invertivores, an obligate corallivore and non-browsing herbivores  contributed to 70% of the similarity within the cluster of transects at the high coral cover, high complexity end  of the benthic PC1 , Cheilio inermis and Lethrinus harak , contributed to 70% of the similarity within the cluster of transects at the high macroalgae cover, low-complexity end  of the benthic PC1  fish per 250 m2 at the reef with the highest overall coral cover and complexity. Moreover, two of the five primary fishing target species, and 19 important by-catch species The identified differences in the fish community with changing benthic condition are likely to have implications for ecosystem service provision Although we predicted that the stability of the underlying substratum would interact with the condition of the benthos, with stable substrata having higher coral cover than mobile rubble reefs, we found only weak relationships. Studies in rubble-dominant locations, such as former dynamite fishing areas, have found substantially lower coral cover on rubble versus stable rocky sites The multivariate gradient of benthic conditions found in this study indicates a continuum of reef states. However, in the absence of long-term time series data and/or experimental manipulations it is not possible to establish the stability of our extreme benthic state categories As coral reefs continue to degrade due to a range of anthropogenic drivers, and alterations in community compositions occur, it is imperative that we understand how changes in one aspect of an ecosystem affect the rest of the ecosystem. From a management perspective, the fact that many reefs do not exist in discrete states means that few generalisations are possible, and reef specific data may be required to implement necessary management plans Figure S1Proportional cover of benthic biota per site.(TIF)Click here for additional data file.


Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution.Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone.Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A.Chromosome number, The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm) from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic hybrid female still maintains its gynogenetic ability. Based on the present and previous findings, we discuss the association of rapid genetic changes and high genetic diversity with various ploidy levels and multiple reproduction modes in several unisexual and sexual complexes of vertebrates and even other invertebrates. Poecilia formosa, the first clonal reproduction vertebrate, was found in 1932[Since Amazon molly d in 1932, but it d in 1932,8, whichCarassius auratus gibelio) of goldfish, has been currently suggested as a separate species Carassius gibelio[Gibel carp, also commonly known as silver crucian carp or Prussian carp, which was previously nominated as a subspecies  to activate the eggs. As described previously, spawning was artificially induced by two intraperitoneal injections with a mixture of acetone-dried carp pituitary, hCG and LRH-A[Cyprinus carpio) sperm stimulation, because the offspring are all-females, and identical to the maternal. Moreover, they have been proliferated by 7 successive generations of gynogenesis with Xingguo red common carp (Cyprinus carpio) sperm stimulation, and more than several hundred millions of clonal individuals have been produced. From the fifth generation, the clonal strain as a new aquatic variety has been introduced into more than 10 hatcheries, and their gynogenetic offspring were cultured throughout China. As a stock hatchery, the successively gynogenetic offspring of 7 generations have been maintained in the Guanqiao Experimental Station of the Hydrobiology Institute in Wuhan, Hubei Province, and all the samples for genetic analysis were collected from the Station. Besides the maternal and paternal, generally, ten individuals were randomly sampled from each analyzed clone and generation, and their fin tissues and blood cells were sampled for DNA extraction and DNA content measurement respectively. The fin tissues were stored in 95% ethanol at -20°C and blood cells were fixed by 70% ethanol and stored at 4°C.Clone D female and clone A male were used as the maternal and paternal for the propagation experiments of gonochoristic sexual reproduction mating. Control gynogenetic individuals of clone D were inseminated by sperm from red common carp  of Cot-1 DNA was performed according to the method described previously[Chromosome metaphases were prepared from kidney cells of at least 5 individuals for each clone by the method of kidney cell-phytohemagglutinin (PHA) culture reviously, and imaEcoR I and Mse I and then selective amplification was performed using ten primer combinations[Ten microsatellite primers were applied to amplify microsatellites in genomic DNA and PCR amplification was performed as the described method. As for binations. Productbinations. Transfebinations,21. To obinations were useClone A sperms were sampled from 8 individuals in reproduction season and 20 μl of them were respectively dropped into 1 ml of 70% ethanol. The fixed sperms at 4°C overnight were washed with PBS buffer for 3 times at 1000 rpm for 5 min. The pellets were resuspended in 0.5 ml of 0.5% pepsin in 0.1 M HCl. After the solution were incubated at room temperature for 30 min with gentle shaking, 100 μl of them was stained by adding 2 ml of propidium iodide solution (40 μgPI/ml) containing 4 kU/ml RNase (DNase-free ) at room temperature for 3 h in the dark. After the mixed cells were filtered through a special nylon mesh, the DNA content measurement was performed as described previously by PhoenAY771781). All sequences of transferrin and mitochondrial DNA were aligned and exported with CLUSTAL X [Microsatellite and AFLP amplification bands in the gibel carp clones were evaluated based on the electrophoretic patterns and the genetic diversities were calculated by Arlequin 3.11 software. The genLUSTAL X  and MegaLUSTAL X .Cyprinus carpio) sperm stimulation. Xingguo red common carp is a regional variety of common carp in Xingguo County, Jiangxi Province and has more than 1300 years of culture history. Owing to the significant growth superiority, the clone has been approved by National Certification Committee for Aquatic Varieties as a new aquatic variety that is suitable for aquaculture in China. From the fifth generation, the novel clonal variety has been introduced into more than 10 hatcheries, and their gynogenetic offspring were cultured throughout China. At present, more than several hundred millions of clonal individuals have been produced in the hatcheries. As a novel clone strain, it has been designated as clone A+, but its genetic organization and the mechanism of clone formation have been still unknown.Figure Cot-1 DNA fluorescent banding karyotype analysis of the clone A+ metaphases sampled from the fifth generation, and compared with that of clone D and clone A. Figure +  and Tf1162 (CAAGGGCATCTGCTTCCT) was designed according to transferrin cDNA sequences  of gibel carp because high polymorphism sites had been revealed within this fragment among various transferrin alleles. Using this primer pair, we amplified and identified five different transferrin alleles  from the three gibel carp clones (GenBank accession numbers: JF496199-JF496203). Tf2, Tf4 and Tf5 are 1170 bp in length, while the length of Tf1 and Tf3 are 1184 bp and 1177 bp respectively. From the aligned sequences, various mutations including deletion, transition and transversion are found among the five alleles , and contain the identical gene complement  and gene order as found in most vertebrate mitochondrial genomes[+ are all identical to that of clone D, and all different from that of clone A , the fifth generation (F5) and the seventh generation (F7) from the stock hatchery and comparatively analyzed the genetic profiles with the original maternal clone D and paternal clone A by nuclear markers of microsatellite and AFLP and cytoplasmic marker of mtDNA sequence. As shown in Figure + along with the seven generations.To further confirm the above data and to judge the validity, we randomly sampled 10 individuals in different generations including the third generation  from the maternal clone D. And, the selected fast-growing individual as clonal maternal might be a nucleo-cytoplasmic hybrid between clone A and clone D, which might be formed via androgenesis of the clone A sperm in the clone D ooplasm.In this study, we reported a detailed formation process of the novel clone strain, and analyzed and identified the genetic organization and background by nuclear and cytoplasmic genome markers. The current data clearly indicate that the nucleus of clone ACorbicula)[As far as we know, androgenesis has been observed to occur naturally in interspecific hybrids of Sicilian stick insects,48 and torbicula),50. In forbicula), common orbicula), loach[5orbicula),54, and orbicula), and in orbicula). Generalorbicula). In the orbicula), even thorbicula) in the pPoeciliopsis monacha-lucida[Poecilia formosa)[Corbicula that reproduce by androgenesis, ploidy changes including diploid, triploid, and tetraploid exist widely among different species and populations, and the higher ploidy levels have been suggested to be related to the origin of androgenetic reproduction[Ambystoma), Bogart et al. revealed a new reproductive mode of kleptogenesis by which unisexual salamanders steal sperm genome from donors of normally bisexual species[Carassius auratus, polyploid gibel carp have wide geographic distribution and occupy more multiple niches from northern Europe to Asia[Significantly, the selected nucleo-cytoplasmic hybrid female still maintains its gynogenetic ability, and thereby forms a nucleo-cytoplasmic clone with over several hundred millions of clonal individuals. In the past 20 years, we also obtained one allotetraploid clone with additional genome incorporation,59 and oha-lucida, and the formosa),61. Howe formosa). In inveroduction,62. In iroduction,63. Simil species,34. Perhe to Asia.Bufo viridis)[Pelophylax esculentus)[Indeed, the coexisting dual modes of unisexual gynogenesis and sexual reproduction in gibel carp might be indicative case of a change in reproductive mode from complete unisexual reproduction towards sexual reproduction. In gree viridis),65 and wculentus) involvinculentus). Therefoculentus) and nuclculentus),68, the culentus). Of courIn conclusion, we here identify a novel clone in polyploid gibel carp, and suggest that the novel clone might be a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm) from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic hybrid female still maintains its gynogenetic ability, and thereby forms a novel clone with over several hundred millions of clonal individuals proliferated by 7 successive generations of gynogenesis. The androgenetic nucleo-cytoplasmic hybrid clone should be the first case in vertebrates, and might be related to the high polyploidy level and the diverse reproduction modes in gibel carp. Based on the present and previous findings, we discuss the association of rapid genetic changes and high genetic diversity with various ploidy levels and multiple reproduction modes in several unisexual and sexual complexes of vertebrates and even other invertebrates. Therefore, the novel finding will be of great significance not only for exploiting the genetic breeding approaches in gibel carp but also for the studies of evolutionary genetics and ecology in other unisexual animals.The authors declare that they have no competing interests.JFG, ZWW and LZ conceived and designed the experiments. HPZ contributed to the chromosome data, DW and ZWW contributed to the AFLP data, FFJ and ZWW contributed to the transferrin allele data, and WG and ZWW contributed to the microsatellite data. ZWW analyzed the data. JFG and ZWW wrote the paper. All authors have read and approved the final manuscript.


Most patients with colon cancer are surgically treated, with postoperative association of chemotherapy and possibly immunotherapy in advanced cases. Surgical treatment is chosen depending on the evolution stage, tumor topography and the existence of complications, colonic surgery being dictated by colonic vascularization. The radical character of the surgical intervention can be assured only in the early stages of the tumor. Colostomy is rarely necessary in patients with colon cancer. In the period of the last 5 years (2007-2011), 307 patients with colon cancer were operated in “Colentina” Surgical Clinic, radical intervention being possible only in 219 cases. 48 cases were emergency interventions for occlusion or perforation with peritonitis. Every time the mechanical preparation of the bowel was not possible, the intraoperative washout technique was used. Postoperative complications were met in 27 cases, being represented by bleeding (3 cases), peritoneal abscess (5 cases), anastomotic fistula (7 cases), abdominal wound infection (12 cases). In 5 cases the operation was done laparoscopically. Preoperative mortality was of 13 cases. Postoperative chemotherapy was done in the great majority of cases. The topography is represented in 60% of cases by left colonic localization (among them 75% is only sigmoid topography), the rest of 40% being represented by right colon tumors; in 5% of the cases multiple (synchronous) tumors are met. The etiopathogeny of colon cancer recognizes both an hereditary predisposal  and a favorable influence of medium factors  [8]. The lately increase of colon cancer incidence in our country is due to the alteration of alimentary habits and life style of the people, more and more occidental, but also to the improvement of diagnostic means, with an increase of the diagnostic accessibility and better sanitary education of the population.Situated in the fourth place in the world regarding neoplazic frequency, colon cancer is responsible for 10% of the malignant tumors in the European Union, having the 3rd position among the digestive cancers, with a relative equality regarding the sex ratio, the highest incidence being met in the 6-7 decades of life :The clinical picture of colonic cancer associates manifestations that can be seen below ). Imagistic investigations are represented by: colonoscopy with biopsy and histopathological exam , irigography, plain abdominal radiography , abdominal echography possibly followed by CT-scan or MRI , urography, pulmonary radiography (to discover possible lung metastases), exploratory laparoscopy (for staging of disease). In case of colon cancer, the metastatic involvement is represented in decreasing order by: liver (35%), lung (19%), retroperitoneum (13%), bone (4%), ovary (1-2%), suprarenal (1-2%).Biologic exam can show: anemia, leukocytosis, thrombocytosis, inflammatory syndrome (increased levels for fibrinogen and VSH), hypoproteinemia, positive tumor markers  and colonoscopy. Each subject around the age of 40 years, presenting intestinal transit alteration, dyspepsia or anemia, should be proposed for colonoscopy.Differential diagnostic of colonic cancer could be made with other intestinal diseases generating of occult bleeding  or diseases presenting like abdominal palpable tumor .In case of absence of correct treatment, evolution of colonic cancer goes toward developing of dangerous complications  and finally to exitus.11].Surgical treatment, determinant for the subsequent evolution, consists in radical interventions  or palliative (colostomy or colonic bypass in advanced non-resectable stages). Tumors with invasion in neighbor organs may lead to one block resections that associate distal splenopancreatectomy, gastrectomy, etc. Emergency operated cases usually demand a sequential solution, the initial time that remove the tumor and ends by colostomy being followed after a free interval of 3-6 months by a secondary time that reestablishes the digestive continuity [15]. These devices are represented by staplers of different types  and dimensions, allowing achievement of side-to-end, end-to-side or side-to-side anastomoses.A relatively recent option of using mechanic suture devices with a goal in digestive continuity reestablishment after removal of the intestine segment that carries the tumor is offering an increased degree of safety as well as a faster postoperative recovery . The fast-track surgery protocol presumes, after obtaining consent of the patient, the following attitude: normal diet in the day before operation, lack of mechanic preparation of the bowel, use of volatile anesthetic drugs with rapid induction, intraoperative restricted liquid intake, postoperative oxygen therapy, non-opioid postoperative analgesia, suppression of nasogastric tube at the end of the anesthesia and of the urinary probe in the first 24 postoperative hours, early postoperative feeding , active mobilization of the patient since operative day.Fast-track surgery concept, introduced in colonic surgery by Danish surgeon Henrik Kehlet, starts gathering more and more adepts, its goal being that of better postoperative recovery, with evident benefits both for the patient and for the economic standards of the health system [Oncologic treatment supposes postoperative submission of most patients in chemotherapy programs, the mainly used regimen consisting in association of 5-FU with folinic acid.Long-term results depend on tumor stage in diagnostic moment, the overall survival rate after 5 years being of around 45%. The follow up of the operated patients with colonic cancer presumes a clinical examination (every 3 months in the first 2 years and then every 6 months in the next 3 years), colonoscopy, and tumor markers determination and computer tomography (every 6 months in the first 2 years and then every year in the next 3 years).We realized an retrospective descriptive study lasting on the later 5 years (2007-2011), considering all cases of colonic cancer treated by surgical intervention in “Colentina” Clinical Surgery (307 patients), based on the analysis of medical papers and operatory registries.Sex ratio was almost equal, with little predominance of the males (162 cases); as regards the provenience environment, such clear majority of the patients from urban sites could be seen, this being explained by the alimentary habits centered on predominant intake of animal protein diet.Tumor topography: sigmoid colon - 112 cases, descending colon - 43 cases, transverse colon - 78 cases, cecum and ascending colon - 74 cases. The more frequent localization on left colon, mainly on sigmoid colon could be seen.In 49 cases, the presentation was made in an emergency status, 39 cases being with acute intestinal occlusion and 10 cases with peritonitis by tumor perforation or by cecum diastatic perforation.The clinical picture that brought the patient for medical help, except the cases presented in emergency, was carrying the following symptoms and signs (possible association of these):-alterations of the intestinal transit, with increased constipation or constipation-diarrhea alter-nation: 187 cases;-abdominal distension: 257 cases;-abdominal pain: 113 cases;-anemia of unknown reason: 97 cases;-weight loss: 215 cases;-digestive bleeding: 39 cases.The diagnosis was mainly based, in the great majority of cases (287 cases), on colonoscopy with biopsy and histopathology examination.The cases of our study group belonged to one of the following evolutionary stages:- stage I: 34 cases;- stage II: 75 cases;- stage III: 135 cases;- stage IV: 63 cases.In the great majority of cases, the histopathological result indicated the existence of an adeno-carcinoma with different grades of differentiation.Information needed for correct tumor staging was offered by investigations like abdominal echography, pulmonary radiography and abdominal computer tomography. The cases presented as emergencies benefited from diagnostic from plain abdominal radiography.We started lately with such timidity to apply the principles of fast-track surgery, even trying to realize a mechanic preparation of the bowel anytime possible. Regarding the cases operated as emergencies, we practiced intraoperative cleaning of the bowel by washout technique.The operations proceeded were represented by:- right hemicolectomy: 86 cases;- transverse segmental colectomy: 52 cases;- left hemicolectomy: 48 cases;- segmental sigmoidectomy: 112 cases;- subtotal colectomy: 4 cases;- total colectomy: 5 cases.Considering the emergency operated cases, they benefited from the following interventions:- those cases of occlusion by left colonic tumor and those with peritonitis by tumor perforation were solved in seriated interventions, the first time realizing the removal of the tumor with temporary colostomy, the second time for bowel continuity reestablishment being done 3 months later;- those cases with peritonitis by diastatic cecum perforation were subjected for total colectomy with ileostomy as first operative time, followed 3 months later by reintervention for bowel continuity reestablishment (ileorectostomy).We should mention here some cases with particular presentation and solution:- 2 cases of appendical carcinoid that 1 month after the initial intervention (appendectomy), when the histopathological report was ready, needed reintervention for right hemicolectomy with ileotransversostomy;- 13 cases with preexistent hepatic cirrhosis where the surgical intervention had to face with great hemorrhagic risks and the postoperative period was affected by decompensation of the hepatic function that had to be sustained;- 5 cases of colonic tumor developed on familial adenomatous polyposis, submitted for subtotal colectomy (3 cases) or total colectomy (2 cases) with ileal pouch;- 11 cases of splenic flexure colonic tumor with invasion in pancreatic tail and splenic hilum needed association of distal splenopancreatectomy;- 3 cases of hepatic flexure colonic tumor with duodenal invasion needed reconstructive techniques for duodenum after tumor removal;- 24 cases with 1-3 hepatic metastases associated wedge resections of the metastases.In the last period, we started to use the stapler devices in colon-rectal cancer more and more. Thus, from the total of 307 patients operated for colon cancer, we reestablished the bowel continuity by side to end, end to side or side to side anastomoses using the staplers of EEA, TA, GIA or Endo-GIA types. The advantages were represented by shortened duration of the surgical intervention, with accomplishment of surgical sutures that have not produced any postoperative complication until now.Surgical intervention was done in 5 cases by laparoscopic technique: right hemicolectomy - 2 cases, segmental sigmoidectomy - 2 cases, transversal colectomy - 1 case.Postoperative complications occurred in 27 cases, being represented by:- bleeding (3 cases),- peritoneal abscesses: 5 cases,- anastomotic fistula: 7 cases,- wound infections: 12 cases;11 cases need a surgical re-intervention.Preoperative mortality was of 13 cases, due to one of the following complications:- bronchopneumonia: 5 cases;- IMA: 2 cases;- hepatic failure: 3 cases;- sepsis: 3 cases.All the patients were subsequently submitted for oncologic treatment program with chemo¬therapy having the leading role.Follow up of operated patients was done by clinical controls every 3 months in the first 2 years and then every 6 months in the next 3 years, colonoscopy, tumor markers and imagistic investigations done every 6 months in the first 2 years and then every year in the next 3 years.1. Incidence of colonic cancer in our country has an increasing tendency, probably due to the alignment of our culinary habits to those of the western civilization.2. A majority proportion of cases discovered in advanced stages is still present, due to the persistence of poor sanitary education.3. Colonic cancer prognostic mainly depends on the stage of disease, but also on the performance of a correct therapeutic protocol. It seems that for equivalent stages of disease, the tumors on right colon have worse prognostic than the left ones.4. Surgical treatment represents the basic therapeutic link. In every case with invasion of the neighbor organs, it is important to realize a one-piece resection of the tumor mass.5. The use of mechanic stapler devices allowed the shortening of the duration of the surgical interventions, with positive effect on postoperative recovery.6. We are still reserved as regards the complete implementation of fast track surgery principles, but the even partial application of these encourages us to be more courageous in the future, considering the good results of centers with great experience in this domain.7. Laparoscopic colonic surgery has to be proceed more and more in the future, the old reluctance based on studies showing risks for tumor dissemination being denied by the latest data published in scientific reviews. 8. It is compulsory to submit all patients operated for colonic cancer to close periodic follow up with goal of early detection of local recurrences or distance metastases.


XRCC1) gene has been defined as essential in the base excision repair (BER) and single-strand break repair processes. This gene is highly polymorphic, and the most extensively studied genetic changes are in exon 6 (Arg194Trp) and in exon 10 (Arg399Gln). These changes, in conserved protein sites, may alter the base excision repair capacity, increasing the susceptibility to adverse health conditions, including cancer. In the present study, we estimated the frequencies of the XRCC1 gene polymorphisms Arg194Trp and Arg399Gln in healthy individuals and also in women at risk of breast cancer due to family history from Rio de Janeiro. The common genotypes in both positions (194 and 399) were the most frequent in this Brazilian sample. Although the 194Trp variant was overrepresented in women reporting familial cases of breast cancer, no statistically significant differences concerning genotype distribution or intragenic interactions were found between this group and the controls. Thus, in the population analyzed by us, variants Arg194Trp and Arg399Gln did not appear to have any impact on breast cancer susceptibility.The X-ray repair cross-complementing Group1 ( Many risk factors are involved in breast cancer, the genetic component being one of the most important affecting its frequency (XRCC1) gene has been defined as essential in the base excision repair (BER) and single-strand break repair processes  and in exon 10 (Arg399Gln). The frequency of variant 194Trp is very low in Caucasians and African Americans (5%-11%), while the frequency of variant 399Gln ranges from 32 to 48% in Caucasians , from Rio de Janeiro, in order to analyze possible differences between these groups regarding the distribution of XRCC1 variants.In the present study, we estimated the frequencies of Arg194Trp and Arg399Gln in the XRCC1 polymorphisms was performed as described by Peripheral blood samples were collected from 418 healthy individuals from the general population (population sample) undergoing routine medical tests at the Pedro Ernesto University Hospital, in the city of Rio de Janeiro, and from 104 women participating in the “Breast Cancer and Genetics Project – National DNA Bank” (BNDNA) of the Fernandes Figueira Institute-FIOCRUZ, located in the same city. This DNA bank contains genomic DNA from no consanguineous women who reported breast cancer in first- and/or second-degree relatives (women with a family history of breast cancer). The control group consisted of 240 individuals (72 men and 168 women) taken from the population sample who had no reported case of breast or ovary cancer in the family and were not affected by breast or any other type of cancer. All the study subjects were recruited between 1996 and 2001, and epidemiological data were obtained by standardized questionnaires. Based on phenotype characteristics, the individuals were classified into whites and non-whites (mulattoes and blacks). Written informed consent was obtained from each subject. This study was approved by the Ethics Committees of the Pedro Ernesto University Hospital and the Fernandes Figueira Institute. Genomic DNA was prepared from the blood samples by proteinase K digestion, according to standard methods , the control group was composed of both men and women from the population sample with no previous cases of breast or ovary cancer in the family. The distribution of XRCC1 polymorphisms was similar in both groups, although women with an FH of breast cancer showed a higher percentage of genotypes presenting the Trip allele (Arg/Trp + Trp/Trp) than the controls . This difference was more noticeable when we considered only women reporting at least one affected first-degree relative: 22% x 13.3% in controls. On the other hand, the Gln allele for codon 399 was more frequent in the control group. However, these differences were not statistically significant . The intragenic association study did not show any statistically significant difference concerning the frequency of a particular genotype combination for both polymorphic variants when women with an affected first-degree relative and controls were compared . All genotype distributions of the XRCC1 gene were in Hardy-Weinberg equilibrium.The characteristics of the study groups “Population Sample” (n = 418), “Women with family history (FH) of breast cancer” (n = 104), and “Controls” (n = 240) are presented in nificant . Women rXRCC1 polymorphisms: Arg194Trp, located in exon 6, and Arg399Gln, located in exon 10. Both substitutions produced structural changes in the protein molecule, probably altering its biological activity and consequently affecting the DNA repair efficiency. These observations led us to realize the importance of determining the distribution of these genetic variants in our population. Brazil has a large territory and the differences in colonization among different geographical regions resulted in different levels of miscegenation . So far, four papers have described the XRCC1 genotype distribution in Brazilian populations, all of them from the State of São Paulo . No statistical differences were observed in the genotype distributions or in the intragenic interactions of polymorphisms Arg194Trp and Arg399Gln. So, according to our data, these XRCC1 gene variants do not appear to have any impact on breast cancer susceptibility in the analyzed population. To elucidate the role of XRCC1 variants and cancer risk, several case-control studies have been conducted, but the results are inconsistent. Two meta-analysis studies described the 194Trp variant as being related to a decrease in cancer risk (XRCC1 Arg194Trp or the Arg399Gln polymorphisms, among other DNA repair genes, and risk of breast cancer. Repair genes are unequivocally important in the process of breast cancer susceptibility but, as several repair systems may superpose and act together, a clear picture in case-control studies is very difficult to achieve. In spite of our limited sample size, we found the same trend described in previous studies on breast cancer patients with an FH (XRCC1 gene variants in the genetic susceptibility to breast cancer.The XRCC1 protein is considered an essential part of both the single-strand break repair and the base excision repair systems (ão Paulo . These rand TP53 . In an a


Organo-fluorine compounds are the substances of considerable interest in various industrial fields due to their unique physical and chemical properties. Despite increased demand in wide fields of science, synthesis of fluoro-organic compounds is still often faced with problems such as the difficulties in handling of fluorinating reagents and in controlling of chemical reactions. Recently, flow microreactor synthesis has emerged as a new methodology for producing chemical substances with high efficiency. This review outlines the successful examples of synthesis and reactions of fluorine-containing molecules by the use of flow microreactor systems to overcome long-standing problems in fluorine chemistry. Fluorine is a key element in the development of materials and biologically active agents. The introduction of fluorine atoms into organic molecules often exerts influences upon physical, chemical, and biological properties –8. AlthoDespite increased demands in wide fields of science, synthesis of fluoro-organic compounds is still often faced with problems such as the difficulty in handling of fluorinating reagents and in controlling of chemical reactions. Furthermore, low stability of fluorine-containing intermediates and low selectivity  of the reactions have disturbed the progress of synthesis of fluorochemicals. New methodologies that can cure the weak points in synthesizing fluoro-organic compounds would open up a new perspective for fluorine chemistry.Meanwhile, flow microreactor synthesis, the use of microfluidic devices, has emerged as a new method for producing chemical substances with high efficiency –13. Now,This review deals with the successful examples on synthesis and reactions of fluorine-containing molecules by the use of flow microreactor systems. Useful applications using flow microreactor technology to overcome long-standing problems in synthetic organofluorine chemistry are showcased.2) is one of the most straightforward methods to make fluorine-containing molecules with high atom economy. However, handling F2 needs special care and there are a lot of practical difficulties associated with direct fluorination. Elemental fluorine is a poisonous pale yellow gas at room temperature (bp −188 °C). Reactions of organic substances with elemental fluorine are typically strongly exothermic and extremely fast, frequently explosive. So it is difficult to control direct fluorination reactions with F2 gas in conventional batch reactors. In 1999, the pioneering work using flow microreactor technology for direct fluorination was reported by Chambers and Spink (2 gas cause not only substitution (aromatic fluorination) but also side reactions such as fluorine-addition, polymerization and so on due to the high reactivity of F2 gas [Direct fluorination employing elemental fluorine (Fnd Spink  26]. Ut. Ut2) isf F2 gas –40. Diref F2 gas .2NSF3, abbreviated as DAST) was demonstrated. DAST is a commercially available liquid reagent that is utilized for the conversion of alcohols and carbonyl compounds into the corresponding fluoro derivatives. In spite of the effectiveness and convenience for nucleophilic fluorination, DAST is volatile and quite moisture sensitive, and readily undergoes disproportionation to SF4 and explosive (Et2N)2SF2 when heating at over 90 °C  (t1/2 = 110 min), protocols for rapid and selective synthesis of [18F]-labeled compounds have been required. In addition, on-site and on-demand chemical production of PET agents with automation and ease of in-line purification is suitable for hospital use. To date, continuous flow microreactor technology has shown potential for synthesis of [18F]-radiolabeled molecular imaging probes such as [18F]FDG, [18F]fallypride, [18F]annexin, and so on, which were made by nucleophilic substitution reactions using [18F]-fluoride ion  chemistry contributes to creating useful materials. In 2005, Comer and Organ reported SNAr reactions of 2-fluoronitrobenzene using a flow microreactor system with microwave irradiation (NAr reactions (N-dimethylamino)acrylate with 2,4,5-trifluorobenzoic acid chloride, followed by two kinds of library diversification involving 1) Michael addition of a set of primary amines and the subsequent nucleophilic ring closure to give the difluoroquinolone system, 2) intermolecular nucleophilic aromatic substitution with various amines and hydrolysis of ester moieties to afford a number of ciprofloxacin analogues in good overall yields with high purity.Nucleophilic aromatic substitution (Sadiation  69]. To. ToNAr) eactions  70]. St. StNAr) As aforementioned, the benefits of flow microreactor synthesis are 1) accurate control of reaction time, temperature, and pressure, 2) efficient mixing, 3) improved product selectivity (yield and purity), 4) rapid removal of heat of reaction for increased process safety, 5) ease of scale-up from lab to plant scale, and so on.The reaction times are governed by the residence times (concerning path lengths and flow rates) inside flow microreactor which allow the generation and reactions of reactive intermediates before decomposition. By virtue of these characteristic features, the residence time control in flow microreactors makes the generation of highly reactive species possible, including their reactions on a preparative scale within a second or less.Pentafluorophenylmagnesium bromide (PFPMgBr) is industrially produced by means of the halogen–metal exchange reaction of ethylmagnesium bromide and bromopentafluorobenzene . HoweverMolecular switches based on photochromic diarylethenes are one of the most promising electronic materials . In partF) groups are one of the most effective and general methods for the synthesis of perfluoroalkylated compounds [FLi avoiding the β-elimination [FLi and the reactions with electrophiles should be separated to give the desired RFSiMe3 and RFSnBu3 in high yields. To solve the issue, the use of the integrated flow microreactor system was effective; well-controlled generation of highly reactive RFLi intermediates in the absence of active electrophiles and transfer of RFLi into the next step were accomplished by taking short residence times in the reactors before they decomposed (The installation of perfluoroalkyl moieties into organic molecules can substantially changes the electronic properties of the parent organic compounds, and improves lipophilicity leading to enhanced solubility in fatty tissue and more efficient transport in the body. Nucleophilic introduction of perfluoroalkyl (Rompounds . Perfluoompounds , howeverompounds . Flow mimination . In the composed .The illustrious achievements have been made in developing organic reactions by the use of flow microreactor devices. Fluorinated organic compounds have been gaining a significant importance in wide field of science and technology, and there have been the associated growing need for new and more efficient synthetic methods. As highlighted here, each of these elegant flow microreactor syntheses surely supplies the innovative methods for the synthesis of organo-fluorine compounds. For the future, continuous flow microreactor chemistry will make a breakthrough in developing new chemical production with highly efficiency and unexploited fluorinated intermediates by the ingenious strategy, and will afford fluorine-containing useful materials.