Canine histiocytic malignancies (HM) are aggressive tumors that occur with particularly high frequency in certain breeds including Bernese mountain dogs and flat-coated retrievers. Robust diagnosis of HM commonly utilizes immunohistochemical stains that are broadly ineffective on formalin-fixed tissues; thus the diagnosis is often one of exclusion. Clinical outcomes are generally poor, with frequent metastasis and therapeutic failure lowering overall survival at time of diagnosis to an average of less than two months in the majority of published work. The limited understanding of the molecular mechanisms underlying HM has hindered the development of more effective diagnostic modalities and the identification of therapeutic targets. A potential avenue exists for advancing clinical management of canine cancers through extrapolation from a close counterpart in human medicine. Historically, HM have been compared to the rare and understudied subset of human cancers involving the dendritic lineage, such as dendritic cell sarcoma or Langerhans cell sarcoma. Recent data have now thrown into question the cellular origin of HM, suggesting that the disease may originate from the macrophage lineage. This review summarizes existing knowledge of HM from the clinical, histologic and molecular perspectives, and highlights avenues for future research that may aid the development of novel diagnostic and therapeutic approaches. In turn, a more advanced appreciation of the mechanisms underlying HM should clarify their cellular origin and identify appropriate opportunities for synergistic extrapolation between related canine and human cancers. Canine histiocytic malignancies (HM) represent a spectrum of aggressive neoplasms that while relatively infrequent within the global domestic dog population, are associated with a remarkably high rate of mortality in a subset of breeds ,2. CliniWhile infrequent in the general dog population there is a particularly strong breed predisposition to HM in Bernese mountain dogs (BMD) and flat-coated retrievers (FCR), with odds ratios of 45.0 and 62.0 respectively . Other bClinical outcomes for HM are extremely poor ,7, with in vitro or as retrospective clinical trials [in vitro [SRC), demonstrated a modest ability to inhibit growth of canine HM cell lines [The past three years have seen marked progress in assessing alternative therapeutic options for canine HM, however these have either been performed l trials ,19,20,21in vitro . SubsequOne of the greatest challenges to the development of more efficacious therapies for HM is the lack of certainty regarding the biological origin of the disease. Morphologically, tumors can appear as sheets of large pleomorphic cells, whorls of spindle cells, or a mixture of the two . CellulaThe most comprehensive diagnostic scheme for HM has been based on IHC staining with CD1 and CD11c (indicative of cells derived from the dendritic lineage), CD18 and CD45 (pan-leukocyte markers) and MHC II , and the RB1 tumor suppressor gene on CFA22q11. Interestingly the MTAP locus has since been identified as a germline susceptibility locus for HM within the BMD breed using genome-wide single nucleotide polymorphism analysis [Molecular characterization of canine HM provides a complementary avenue for advancing diagnostic and therapeutic modalities and for clarifying the biological origin of the disease. To date there has been one study describing genome-wide somatic DNA copy number aberrations in HM, and three focusing on tumor-associated gene expression signatures ,9,10,26.analysis . Subsequanalysis ,29,30,31analysis , a re-viLUM and Col3aI were found to be significantly up-regulated in visceral and external tumors [SPIc and VCAM-1 due to their natural involvement in angiogenesis [Two of the three gene expression studies of HM were restricted to the FCR breed ,10. The l tumors . The intogenesis . CCL5 and CLEC12A and up-regulation of C6 in visceral tumors [CCL5 was considered unexpected due to the association of this gene with rapid tumor metastasis and cellular motility [CLEC12A down-regulation in visceral tumors was considered more consistent with expected findings, as the gene functions as a negative regulator of granulocyte/monocyte function. It was suggested that that these findings may better account for the increased cellular motility in visceral tumors while the up-regulation of C6 (complement component 6) may represent the interactions of the innate immune response to the tumor cells [The second study expanded on findings from the first, performing transcriptional profiling of localized HM cases to determine whether significant differences exist between visceral and limb forms of the disease in the FCR . Key finl tumors . Given tmotility . CLEC12Aor cells . Both thHMGB1 and RAGE, which are required for the development of dendritic cells [HMGB1 in disseminated HM specimens from other anatomical sites could not be shown definitively, due to conflicting results from the use of two independent housekeeping genes [HMGB1 and RAGE mRNA was reflected in a corresponding decrease in protein levels in tumor specimens when compared to control tissues, in combination with a lack of nuclear localization, demonstrating the potential for cytokine production and release in neoplastic cells. The impact of these findings was hindered by a small sample size, and the investigation of only disseminated HM in the BMD population [The final study took a more mechanistic approach, focusing on two genes, ic cells . In thisic cells . Unfortung genes . IHC anapulation . While these studies provide a useful foundation for the definition of molecular mechanisms underlying the pathogenesis of canine HM, their inherent limitations reinforce the need for alternative strategies. Numerous recent studies have supported the concept of advancing progress in clinical management of cancers through extrapolation of data between counterparts of the same disease in different species. HM has most commonly been compared to human sarcomas of the histiocytic and dendritic lineages, which together represent less than 1% of all hematopoietic and lymphoid neoplasms in people ,33,34. TAs with canine HM, robust diagnosis of tumors of the HS/DS lineage in human patients remains challenging. Tumors are diagnosed primarily on the basis of their cellular morphology, ranging from spindle to ovoid shaped cells arranged into whorls or sheets ,34. WhilMolecular characterization of HS/DS is a key requirement for recognizing underlying disease mechanisms and for improving outcome through targeted clinical management. A degree of conflict exists among the small number of reports describing the genome-wide profiles of HS/DS, with the majority of studies indicating relatively stable profiles in both diseases ,38, whilAssessment of immunoglobulin heavy chain (IGH) receptor clonality in HS/DS has shown the possibility that some cases of HS/DS may have arisen from a previously existing lymphoid neoplasm, based on IGH clonality in both the prior lymphoid disease and the current HS/DS ,41,42,43BRAF gene in HS/DS. BRAF is a member of the mitogen-activated protein kinase (MAPK) pathway that regulates a series of fundamental mechanisms involved in cellular proliferation, differentiation and apoptosis [BRAF, most commonly via substitution of valine for glutamic acid at codon 600 (BRAF V600E), have been shown to result in constitutive activation of the MAPK pathway in several human cancers, including malignant melanomas and thyroid tumors [BRAF V600E mutation as an initiator of tumorigenesis confers an effective therapeutic target via selective inhibition of the MAPK pathway [BRAF V600E was recently described in a series of canine cancers, with the highest frequency in prostatic and urothelial carcinomas (80% and 67% of cases respectively) [BRAF would be successful in the clinical management of these tumors [BRAF sequence and function, and that of its downstream targets, is required to determine whether canine HM and human HS/DS share dysregulation of the MAPK pathway as an underlying mechanism in disease development and progression. Broader mechanistic studies in canine HM may define alternative therapeutic targets for human HS/DS cases, particularly for the 80% of patients with dendritic tumors and 40% with histiocytic tumors that do not harbor the BRAF V600E mutation [Several recent studies have evaluated the mutational status of the poptosis ,45,46. A pathway . Recent  pathway ,45,46. Actively) . Interese tumors . Furthermutation ,45,46. et al [Despite several key advances in recent years, canine HM continues to represent a substantial clinical challenge due to the heterogeneity in disease presentation and consequent potential for misclassification, coupled with the absence of broadly effective treatment strategies. Continued efforts are needed to develop more sensitive and specific diagnostic markers that may be applied readily in routine clinical settings. This will require considerable investment in molecular characterization of HM at the genomic, transcriptional and translational level. Since translational dysregulation may offer options for targeted therapy, future studies should include quantitative assessment of members of the pathways highlighted by Boerkamp et al , particu


SDH) or in von Hippel-Lindau (VHL) genes manifest a strong pseudohypoxic phenotype. Mutations in SDH are oncogenic, forming tumours in a select subset of tissues, but the cause for this remains elusive. Although elevated succinate levels lead to increase in hypoxia-like signalling, there are other phenotypes that are being increasingly recognised in SDH-mutated PPGL, such as DNA hypermethylation. Further, recently unveiled changes in metabolic re-wiring of SDH-deficient cells might help to decipher cancer related roles of SDH in the future. In this review, we will discuss the various implications that the malfunctioning SDH can have and its impact on cancer development.Hypoxia is a critical driver of cancer pathogenesis, directly inducing malignant phenotypes such as epithelial-mesenchymal transition, stem cell-like characteristics and metabolic transformation. However, hypoxia-associated phenotypes are often observed in cancer in the absence of hypoxia, a phenotype known as pseudohypoxia, which is very well documented in specific tumour types, including in paraganglioma/pheochromocytoma (PPGL). Approximately 40% of the PPGL tumours carry a germ line mutation in one of a number of susceptibility genes of which those that are found in succinate dehydrogenase ( In 2000, Hanahan and Weinberg introduced the concept of six universal hallmarks of cancer , also known as mitochondrial complex II (CII)  , RET and von Hippel-Lindau (VHL)  Else . InteresSDHx deficiency have been created using the Cre-lox system and have provided important information regarding the re-wiring of the metabolic network in response to loss of these key enzymes  dehydrogenases  activity for aspartate synthesis  transcriptional factors are formed of two constitutively expressed subunits—alpha and beta )  mutation in PPGL in 2012  are damaging molecules containing oxygen with an unpaired (free) electron that are capable of oxidising cellular macromolecules . These Ndufs2−/−) mice. In these mice loss of responsiveness to hypoxia was observed, which was suggested to be due to loss of a required ROS signal from complex I. Interestingly, the authors noted high SDH activity in glomus cells in wild-type mice, which was accompanied by high levels of succinate, suggestive of RET and ROS production  are oncogenic, forming tumours in a select subset of tissues. The reason for this tissue selectivity remains unknown—something that is not helped by the fact that despite continued attempts, mouse models driven by loss of these enzymes do not appear to form tumours  and EPAS1 (HIF2α) of which all directly control hypoxic signalling and with SDH and FH mutations further providing a link to hypoxia, pseudohypoxia seems to be an undeniable feature of PPGL. Intriguingly, pseudohypoxic HIF signalling has also been suggested as an important driver in the tumourigenesis of tumours with mutations observed in cluster 2 PPGL, as their signalling via Ras/MAPK, PI3K/AKT and mTORC pathways could result in increased HIF signalling (Jochmanova et al. Given the range of mutations observed in PPGL represented by


Multiple Myeloma is a malignant plasma cell disorder characterized by the development of end organ damages resulting in hypercalcemia, renal insufficiency, anemia and osteolytic lesions. The therapeutic options for multiple myeloma have markedly increased over the last decade. In particular, the development of proteasome inhibitors like bortezomib and carfilzomib and the immunomodulatory agents (IMiDs) thalidomide, lenalidomide and pomalidomide have become the cornerstones of current treatment strategies.In the past years, a distinct mode of action for lenalidomide and its analogs thalidomide and pomalidomide has been revealed. These compounds induce degradation of the transcription factors Ikaros IKZF1) and Aiolos IKZF3), that are essential for myeloma cell proliferation, via the E3 ubiquitin ligase complex CRL4 (CRBN) [ and Aiol, that arCRBN, IKZF1, IKZF3 and BSG in pretreatment samples of patients treated with a lenalidomide-comprising regimen [In order to determine whether expression of genes involved in the mechanism of lenalidomide has an impact on outcome, we measured RNA expression levels of  regimen . PatientIKZF1 expression levels in patients with International Staging System (ISS) stage III compared with ISS stage I and II. Patients who achieved a complete response or very good partial response at the time point of best response showed a trend towards lower pretreatment IKZF1 expression levels compared with patients with a partial response or stable disease. Using mRNA expression levels as continuous variables in univariate Cox regression analysis, we found that high IKZF1 levels were associated with an adverse PFS. Multivariable Cox regression analysis including genetic aberrations of known prognostic impact like t and del(17p) revealed that pretreatment IKZF1 expression levels are an independent marker for PFS. When we divided the groups by quartiles of IKZF1 expression, patients within the lowest quartile had an excellent outcome with an estimated 3-year PFS of 86% and an estimated 3-year OS rate of 100% as compared to patients in the higher quartiles . In the subgroup of cytogenetically defined standard-risk patients IKZF1, IKZF3, and BSG expression levels had a significant impact on PFS.We observed a trend for higher CRBN expression levels were associated with outcome in other studies, there was no impact on PFS or OS in our cohort. The major differences between all studies that may explain for these different observations are the patient population (newly diagnosed vs. relapsed/refractory), treatment , and assays applied .Our results suggest that multiple myeloma cells with low Ikaros expression are more sensitive to therapy. This may be counterintuitive on the first sight when considering a model where high expression of a gene indicates that it is relevant for the tumor cell and driving proliferation. However, almost all treatment-naïve multiple myeloma patients are primarily sensitive to lenalidomide and dexamethasone, suggesting that myeloma cells are in general Ikaros-dependent. Consistent with in vitro data in multiple myeloma cell lines, high expression of Ikaros, Aiolos and BSG may prevent lenalidomide-induced degradation of these proteins to critical low levels in those cells with high expression , 2, 4 F. HoweverIn order to determine the predictive and prognostic value of Ikaros and other cooperating members of the CRBN complex, future studies need to analyze gene and protein expression levels by standardized assays in larger patient cohorts treated with different protocols in controlled clinical trials. The identification of predictive markers for specific drugs like lenalidomide has the potential to further personalize treatment in multiple myeloma.


The cerebral cortex undergoes rapid folding in an “inside-outside” manner during embryonic development resulting in the establishment of six discrete cortical layers. This unique cytoarchitecture occurs via the coordinated processes of neurogenesis and cell migration. In addition, these processes are fine-tuned by a number of extracellular cues, which exert their effects by regulating intracellular signaling pathways. Interestingly, multiple brain regions have been shown to develop in a sexually dimorphic manner. In many cases, estrogens have been demonstrated to play an integral role in mediating these sexual dimorphisms in both males and females. Indeed, 17β-estradiol, the main biologically active estrogen, plays a critical organizational role during early brain development and has been shown to be pivotal in the sexually dimorphic development and regulation of the neural circuitry underlying sex-typical and socio-aggressive behaviors in males and females. However, whether and how estrogens, and 17β-estradiol in particular, regulate the development of the cerebral cortex is less well understood. In this review, we outline the evidence that estrogens are not only present but are engaged and regulate molecular machinery required for the fine-tuning of processes central to the cortex. We discuss how estrogens are thought to regulate the function of key molecular players and signaling pathways involved in corticogenesis, and where possible, highlight if these processes are sexually dimorphic. Collectively, we hope this review highlights the need to consider how estrogens may influence the development of brain regions directly involved in the sex-typical and socio-aggressive behaviors as well as development of sexually dimorphic regions such as the cerebral cortex. The complex neuronal organization and architecture of the cerebral cortex is thought to be responsible for the higher cognitive function bestowed upon mammals. The unique anatomical compartmentalization and lamination of discrete neurons arranges into horizontal layer identifiable with specific molecular markers  is the main biologically active form , we found several of the key molecular players involved in estrogenic signaling are expressed in the developing human brain, namely CY19A1 (aromatase), ESR1 (ERα), ESR2 (ERβ), and GPER1 (GPER1). Specifically, these genes were found to be expressed during the developmental period spanning embryonic to late prenatal in the dorsal frontal cortex (DFC), ventral frontal cortex (VFC), and the hippocampus (HIP). CYP19A1 expression was found to increase throughout development, within these regions. Similarly, ESR1 trends toward an increase during over development, whereas ESR2 seems to be highly expressed during early corticogenesis before decreasing slightly then plateauing until late prenatal stage . Interestingly, the recently identified estrogen sensitive G-protein coupled receptor, GPER1  . BPA is a XenobioticMaternal consumption and/or exposure to xenobiotic compounds can enter into the amniotic environment of the fetus and effect neurodevelopment. Exposure to xenobiotic compounds at physiological levels and from the environment can be detected in the amniotic fluid, which introduces it to the fetus  in the ventricular zone (VZ) Figure  and sub-Asymmetric inheritance is also seen in Pax6 and Ngn2 positive RGCs  Figure . Beyond Glial-guided neurons use cellular locomotion to migrate to the CP. Once in position, neurons attach their leading strand to the marginal zone (MZ) and switch to somal translocation. Chemotaxis is mediated through membrane-bound receptors and extracellular matrix proteins, including Rac, brain-derived neurotropic factor (BDNF) and TrkB signaling Figure . CalciumThe lamination process relies on signaling pathways that halt migration and trigger neuronal maturation through terminal and somal translocation. Reelin, Dab1, Wnt/Fizzled, and SPARC-like1 (SC1) are essential for this process. Wnt/Frizzled and reelin signaling regulates boundary formation during lamination  is well documented Klinge, . EstradiIn a clinical setting, estradiol may act on miR to account for the gender disparity seen in the diagnosis of schizophrenia   worked through PI3K/Akt signaling pathway to provide short-term neuroprotection of neurons in the midbrain by upregulating BDNF (Yi et al., A comprehensive review of the literature in this area can be found in the references list (Srivastava et al., ERα regulates neuroglobin expression through genomic transcript region regulation (Guglielmotto et al., reeler mice show an abnormal steroid hormone profile with testosterone and estradiol increased but dihydrotesterone decreased (Biamonte et al., In hippocampal slice cultures, exogenous application of estradiol causes reelin expression to increase from Cajal-Retzius cells, which is facilitated by ERs. Furthermore, aromatase activity contributes to reelin expression. Impairment of aromatase activity causes reelin expression to reduce (Bender et al., Estradiol down-regulates ApoER2 in differentiating osteoblasts, which is reflected in other LDLR family members. Leading to the promotion of differentiation in osteoclasts (Gui et al., The cortical transcriptome can be manipulated epigenetically by estradiol, including functions ranging from memory (Frick et al., ERα has also shown to mediate subtle epigenetic mechanism that result in sex-specific differences in the prefrontal cortex in adulthood (Westberry and Wilson, In summary, many lines of evidence exist that suggest estradiol has many critical roles in corticogenesis Figure . HithertMCSD and DPS wrote the manuscript. NJFG and KJS edited and contributed to the writing of the manuscript. KJS and DPS oversaw the project.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.


Tumoral cells not only depend on oncogenic abnormalities to maintain its malignant phenotype but on non-oncogenic vulnerabilities. Targeting epigenomics can modify specific cellular functions required for malignant transformation. The Bromodomain (BRD) family mediates their effect by recruiting proteins of the transcription machinery, recognizing acetylated-lysine residues in nucleosomal histones. Bromodomain and extra-terminal (BET) inhibitors have shown to produce growth inhibition in several tumors through the inhibition of the expression of several transcription factors. In this review we will discuss the current knowledge regarding BET inhibitors in breast cancer. Recent data demonstrates their antiproliferative effect in several cancer subtypes, including the triple negative subtype, or when combined with cell signaling inhibitors. We will also describe options for therapeutic combinations or potential mechanisms of resistance, with special emphasis on their future clinical development. Breast cancer is one of the most prevalent tumors and a leading cause of death in women worldwide . AlthougBreast cancer has been classified in several different subtypes based on molecular and genomic findings . Around Epigenetic regulators are promising therapeutic targets in hematologic malignancies and solid tumors . For insThe efficacy of BET inhibitors was mainly explored in hematologic malignancies and pediatric cancers where well known transcription factors play a key role in the oncogenesis of the disease, as is the case of C-MYC in neuroblastomas or leukemia , 21. LatBET inhibitors have shown activity in different breast cancer subtypes including HER2 positive tumors, HR tumors and triple negative breast cancer (TNBC). For a comprehensive review of BET inhibitors in clinical development we suggest to read the article by Andrieu G et al. .BRD4 activity is necessary for the proliferation of breast and endometrial tumors expressing estrogen receptors as it regulates ERα-induced gene expression by affecting elongation-associated phosphorylation of RNA polymerase II (RNAPII) and histone H2B monoubiquitination . Indeed In HER2 and HR positive tumors, BET inhibitors have contributed to overcome resistance to currently approved therapies like lapatinib in HER2 positive tumors, or tamoxifen and everolimus in HR positive cancers, among other therapies –30. ThesGiven the fact that BET inhibitors can repress the expression of several transcription factors, the mechanism of action of these compounds is heterogeneous affecting several biological functions including: control of cell cycle, proliferation, stemness, metastatic spreading or angiogenesis, among others –34. Of nSeveral studies have described the function of Bromodomains (BRD) and particularly BRD2 and BRD4, in cell cycle regulation . Indeed,Inhibitors of BET proteins reduce cell migration and invasion, and recent data suggests that this mechanism is mediated by inhibition of the Jagged1/Notch1 signaling pathway through modulation of BRD4 . In addiFinally, several mechanisms have been reported in relation to resistance to BET inhibitors. The activation of casein kinase II (CK2) has been associated with resistance to BET inhibitors and agents targeting this kinase have shown to overcome such resistance . SimilarIf resistance to a given therapy is mediated by upregulation of specific transcription factors, BET inhibitors can overcome resistance to this specific therapy by reducing their expression. For instance, resistance to everolimus in estrogen receptor positive tumors was mediated by the expression of MYC, and reverted by treatment with BET inhibitors that downregulated MYC expression . Other sSimilarly BET inhibitors can overcome the resistance to PI3K inhibitors . In addiIn HER2 positive breast cancer, BET inhibitors have been shown to overcome resistance to lapatinib . ResistaPreclinical data from our group showed that BET inhibitors synergized with chemotherapies used for the treatment of breast cancer including antimitotic agents like taxanes or vinorelbine and DNA damaging compounds like cisplatin and carboplatin . In addiSimilarly BET inhibitors have shown a synergistic interaction with inducers of apoptosis, like BCL-X and BCL-2 inhibitors or with CK2 inhibitors, through the control or relevant phosphatases , 38.Combination of BET inhibitors with immunotherapies like checkpoint inhibitors is an attractive approach. Indeed some articles have suggested that PD-L1 expression is suppressed by treatment with JQ1, and this could be the bases to explore this regimen in several tumors .Also a potent inhibitory effect has been observed with the combination of BET inhibitors and HDAC inhibitors in triple negative and estrogen receptor positive breast cancer cells, mainly through an induction of the ubiquitin-specific protease (USP17) . And finAlthough not yet explored in breast cancer, in other tumor types like in leukemia the presence of stem cells is associated with resistance to BET inhibitors through an increase of the Wnt/β-catenin pathway . In addiTable When observing the clinical development of these compounds, it is interesting to see that there are multiple indications and not clear biological rationale for the development of BET inhibitors in some of them, what suggests that this family of compounds are developed as a tolerated but nonselective molecule.Strategies to augment the efficacy of these compounds include the designing of more potent agents or the identification of potential biomarkers of response. To increase the efficacy an approach is the development of bivalent BET inhibitors that are 100-fold more potent than monovalent antagonist . To idenIn summary, BET inhibitors have demonstrated preclinical activity in TNBC alone or in combination, and in some specific breast cancer scenarios mainly associated with resistance to existing therapies. Their effect goes beyond the regulation of oncogenes as they can act on a variety of cellular programs. The identification of patients whose tumors may benefit from these therapies together with studies to optimize the antitumoral properties of these compounds, represent important future challenges.


After introduction of vaccination, some prevalences of nonvaccine types changed, without clear evidence for type replacement. We analyzed human papillomavirus (HPV) prevalences during prevaccination and postvaccination periods to consider possible changes in nonvaccine HPV genotypes after introduction of vaccines that confer protection against 2 high-risk types, HPV16 and HPV18. Our meta-analysis included 9 studies with data for 13,886 girls and women ≤19 years of age and 23,340 women 20–24 years of age. We found evidence of cross-protection for HPV31 among the younger age group after vaccine introduction but little evidence for reductions of HPV33 and HPV45. For the group this same age group, we also found slight increases in 2 nonvaccine high-risk HPV types (HPV39 and HPV52) and in 2 possible high-risk types (HPV53 and HPV73). However, results between age groups and vaccines used were inconsistent, and the increases had possible alternative explanations; consequently, these data provided no clear evidence for type replacement. Continued monitoring of these HPV genotypes is important. We excluded studies comparing HPV infection in vaccinated persons with HPV infection in unvaccinated persons as part of an individually randomized trial because such studies would not measure population-level effects. Similarly, we excluded studies in which HPV infection was compared only between unvaccinated and vaccinated persons in the postvaccination period. We also excluded studies in which only a small proportion (<2%) of the postvaccination study population was vaccinated . One author (D.M.) initially reviewed titles and abstracts of studies for eligibility; we reviewed in full those studies that appeared to address changes in HPV prevalence after introduction of HPV vaccination programs. We also compared search results with those identified in a recent related review  of sample collection, study setting and population, sample size, specimen type, assay used for HPV DNA testing, HPV genotypes included in the assay, demographic and sexual behavior data collected, and the measure of effect (and method used to determine any effect). For the postvaccination period, we also extracted data on the method used to ascertain estimated vaccination coverage.In addition, we assessed the potential bias in each study by considering the comparability of the study populations in the prevaccination versus postvaccination periods ; the extent of adjustment for potential confounders; the suitability of the specimen type to assess HPV DNA infection; the suitability of the assay used for accurate HPV DNA testing (and whether the suitability of assays differed between the prevaccination and postvaccination periods); and the method used to estimate HPV vaccination coverage. To assess external validity, we considered whether the study samples were population based. Each of these factors was scored as either low risk or high risk.When published data on HPV prevalence and prevalence ratios (PRs) for individual high-risk HPV types were unavailable, we contacted authors to request the HPV type-specific prevalences during the prevaccination and postvaccination periods and the PRs for the 2 periods for each nonvaccine high-risk HPV type. We requested PRs adjusted for demographic and sexual behavior data or the unadjusted PRs if data on confounders were unavailable; we calculated unadjusted PRs if authors provided raw data. By using data from a previously conducted validation study, 1 study included adjusted odds ratios rather than PRs to adjust for the change in assay used during the prevaccination and postvaccination periods (<19 and 20–24 years of age) because of expected lower rates of vaccination coverage and lower vaccine effectiveness in those vaccinated at older ages. Consequently, for each study, we requested data from authors for the same 2 age groups. One study included data for girls <13 years of age, so we requested data restricted to those 16–19 years of age . PRs within each subgroup were combined to obtain a summary PR by using a fixed-effects model if data were not shown to be heterogeneous; lack of heterogeneity was determined by a p value ,,–,,,,,<19 years of age and 23,340 women 20–24 years of age.After we eliminated duplications, we identified 4,648 unique articles in searches from all 4 databases . An init,,,The studies varied in methodologic quality on the basis of potential bias . Most st<19 years of age  but found little evidence of changed prevalences for HPV33 or HPV45 among this age group . Results were heterogeneous for HPV31, HPV33, and HPV45 in women 20–24 years of age; consequently, we did not calculate summary PRs   ; Table 2<19 years of age for HPV39 , HPV53 , and HPV73 . For women 20–24 years of age, evidence indicated increased prevalence for HPV39 .No consistent patterns appeared across the studies for other HPV vaccine types not used in the nonavalent vaccine  Figure 1>3 factors indicating high risk of bias) (>50%). For studies in settings that used the bivalent vaccine, we found evidence of increased prevalence between the prevaccination period and postvaccination periods among those <19 years of age for HPV52, HPV53, HPV56, and HPV70 ; by potential bias of the original study  ; and by <19 years of age, studies with a relatively low potential bias showed no evidence of increased prevalence for HPV52 or HPV39, although evidence existed when the studies were unstratified. For studies with relatively high potential bias, among this younger age group, evidence showed increased prevalences of HPV51 and HPV70, although these increases were not present in the unstratified analysis. In women 20–24 years of age, evidence showed decreased prevalence for HPV33 in those studies with a relatively low potential bias. No summary estimate was provided in the unstratified analysis because of heterogeneity of data. Studies with a relatively high potential bias showed evidence of increased prevalences of HPV52 and HPV58 among women 20–24 years of age. Among this older age group, evidence existed for decreased prevalence of HPV82 in those studies with both relatively high potential bias and relatively low potential bias, although those studies with relatively high potential bias had a larger decrease. Again, no summary estimate was provided in the unstratified analysis because of heterogeneity.Many of our analyses that were stratified by potential bias of the included studies had results similar to those in the unstratified analyses  Table 3.Vaccination coverage was high for the younger age group in all studies  Table 4.<19 years of age. Our main analysis showed increases in other nonvaccine HPV types , but these increases were inconsistent for the 2 age groups examined and the vaccines used.Comprehensive postvaccination surveillance should not only consider reductions of vaccine type–specific infection and associated disease but should also assess any other potential effects of reductions of targeted infections. We assessed changes in nonvaccine HPV types to determine evidence of cross-protection for individual HPV types and to investigate the potential concern that reductions in certain HPV types after the introduction of HPV vaccination in a population could create a niche that enables other nonvaccine high-risk HPV types to become more common . We found evidence of a reduction in the prevalence of HPV31 among girls and women A previous systematic review evaluated changes in high-risk HPV types combined and found evidence of a reduction in the prevalence of HPV types closely related to vaccine types  when they were considered as a single group   can lack sensitivity for detecting individual HPV types when multiple types are present, particularly if another HPV type with a higher viral load is present. In the postvaccination period, in the absence of HPV16 and HPV18, this lack of sensitivity could lead to an apparent but artificial increase in nonvaccine types because these types were underestimated in the prevaccine period because of the predominance of HPV16 or HPV18. Studies have shown this potential unmasking effect  vaccines. Conversely, type 1 errors can occur with multiple testing, so modest evidence for increases should be interpreted with caution.Given the low prevalence of some nonvaccine HPV types, assessing changes in prevalence for individual types since the introduction of HPV vaccination has been challenging. By combining data from several studies, we enhanced our power to consider changes in individual HPV types. However, even with data from 13,886 girls and women We decided against performing random-effects meta-analyses in the presence of between-study heterogeneity because, in most instances, inconsistency occurred in the direction of effect, making a summary estimate  uninformative , and some studies included populations with relatively low coverage, compared with nationally reported vaccination coverage for routine cohorts. Future studies should continue to monitor population-level prevalences of these HPV types. In particular, studies should consider populations vaccinated at young ages and having high vaccination coverage and, perhaps more important, should examine the absolute prevalence of cervical intraepithelial neoplasia 3 lesions attributed to each high-risk HPV type.Search strategies and additional details of a meta-analysis of changes in prevalences of nonvaccine human papillomavirus (HPV) types after introduction of HPV vaccination.


Connexins (Cx) are largely represented in the central nervous system (CNS) with 11 Cx isoforms forming intercellular channels. Moreover, in the CNS, Cx43 can form hemichannels (HCs) at non-junctional membrane as does the related channel-forming Pannexin1 (Panx1) and Panx2. Opening of Panx1 channels and Cx43 HCs appears to be involved in inflammation and has been documented in various CNS pathologies. Over recent years, evidence has accumulated supporting a link between inflammation and cerebral neuropathies , Parkinson’s disease (PD), major depressive disorder, autism spectrum disorder (ASD), epilepsy, schizophrenia, bipolar disorder). Involvement of Panx channels and Cx43 HCs has been also proposed in pathophysiology of neurological diseases and psychiatric disorders. Other studies showed that following inflammatory injury of the CNS, Panx1 activators are released and prolonged opening of Panx1 channels triggers neuronal death. In neuropsychiatric diseases, comorbidities are frequently present and can aggravate the symptoms and make therapeutic management more complex. The high comorbidity between some neuropathies can be partially understood by the fact that these diseases share a common etiology involving inflammatory pathways and Panx1 channels or Cx43 HCs. Thus, anti-inflammatory therapy opens perspectives of targets for new treatments and could have real potential in controlling a cerebral neuropathy and some of its comorbidities. The purpose of this mini review is to provide information of our knowledge on the link between Cx43- and Panx-based channels, inflammation and cerebral neuropathies. The innate immune system mediates inflammation for a physiological response to insult, infection, or biological stress. In most central nervous system (CNS) diseases, a common underlying factor seems to be the triggering of the inflammatory cascade with release of inflammatory cytokines  and suicide is also observable  appeared to be supported by gap-junctional intercellular communication , are very present in the CNS in which cells have to be efficiently connected for treating incoming information from the body and its environment and controlling consequently adapted physiological responses. And indeed, several CNS cell functions  is activated and translocated to the nucleus and nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) are induced. Then, cytokines, prostanoids and NO, released in the subarachnoid space, promote an activation of the perivascular nociceptive trigeminal ending in pia mater that may induce headache. Pharmacological inhibition of neuronal Panx1 channels by carbenoxolone abolishes the inflammatory signaling cascade, perivascular nociceptive trigeminal activation and reduces CSD-induced mast cell degranulation. Even if these results suggest that neuronal Panx1 channels are a link between stressed neurons and subsequent inflammatory pathways, further investigations taking recourse to Panx1 knock-out mice are necessary to validate these findings , a slow wave of neuronal and glial depolarization, triggers migraine aura, activates the trigeminovascular system and is possibly responsible for migraine headache , the number of mastocytes in cortical and hippocampal areas early increases. Moreover, patients with AD present mastocytes near their amyloid plaques. Acute treatment with amyloid beta peptide induces rapid degranulation of cultured mastocytes via a Panx1 channel-dependent Ca2+ influx and this response is prevented by Panx1 blockers. In brain mastocytes, acute treatment with amyloid beta peptide also induces activity of Panx1 channels and Cx43 HCs, an effect that is associated with enhanced histamine release. In the presence of amyloid plaques, brain mastocytes of APPswe/PS1dE9 mice show high Panx1 channel and Cx43 HC activity  the intense neuronal activity elevates extracellular K+; (2) Panx1 channels are activated; (3) ATP is released; and (4) P2X receptors are activated leading to neuronal hyperactivity and this positive feedback mechanism amplifies seizures (Santiago et al., Experimental approaches demonstrated a prominent role of glial cells, activated during infectious and non-infectious causes of inflammation, in the mechanisms of seizure precipitation and recurrence. Both causes of inflammation share common pathways with activation of microglia and astrocytes releasing pro-inflammatory mediators that perturb glioneuronal communication and have proictogenic properties (Vezzani et al., A recent analysis of all available data on neuroinflammation in postmortem brains of schizophrenia patients revealed variable results in astrocytic and microglial markers, glial cell density and pro-inflammatory cytokine concentration (Trépanier et al., In the case of postmortem bipolar disorder patients, a post-mortem study revealed in their frontal cortex an increase of excitotoxicity and neuroinflammatory markers. This upregulation might explain the neurodegenerative component of bipolar disorder, with cell death, brain atrophy and cognitive decline. Markers of astrocyte and microglial activation are also upregulated in frontal cortex from bipolar disorder patients (Rao et al., Researching links between inflammation, neurological diseases and psychiatric disorders with Cx and Panx channels is at a very nascent stage but has the potential to improve our understanding of these diseases to establish effective therapeutics. For some diseases like migraine with aura or AD, a signaling cascade is proposed while for others the data are too sparse. Common inflammatory pathways can explain the high comorbidity between some neuropathies. Thus, anti-inflammatory therapy could have real potential in controlling a cerebral neuropathy and some of its comorbidities (Mazarati et al., Panx1 channels drive inflammation by the regulation of inflammasome, the release of pro-inflammatory cytokines and the activation and migration of leukocytes (Crespo Yanguas et al., The development of new therapeutic tools to inhibit selectively Panx1 channels and Cx43 HCs in cell subpopulations of the CNS will undoubtedly make them a promising target for anti-inflammatory therapy that could have real potential in prevention or delay of neurological diseases, psychiatric disorders and some of their comorbidities. However, such a strategy should not only focused on Cx43 but also to other highly expressed Cxs in the CNS like Cx36 which appears to be involved in several neuronal injuries (autism, ischemia, retina, etc.; Welsh et al., DS wrote and edited the manuscript, conceived and designed the major ideas developed in the manuscript. MM and CD have made substantial, direct and intellectual contribution to the work. All authors read and approved the final manuscript.The authors declare that the work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis thaliana suggest that thylakoid lipid biosynthesis triggers the expression of photosynthesis-associated genes in both the nucleus and plastids and activates the formation of photosynthetic machineries and chloroplast development. Meanwhile, galactolipid biosynthesis is regulated in response to chloroplast functionality and lipid metabolism at transcriptional and post-translational levels. This review summarizes the roles of thylakoid lipids with their biosynthetic pathways in plants and discusses the coordinated regulation of thylakoid lipid biosynthesis with the development of photosynthetic machinery during chloroplast biogenesis.The lipid bilayer of the thylakoid membrane in plant chloroplasts and cyanobacterial cells is predominantly composed of four unique lipid classes; monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG). MGDG and DGDG are uncharged galactolipids that constitute the bulk of thylakoid membrane lipids and provide a lipid bilayer matrix for photosynthetic complexes as the main constituents. The glycolipid SQDG and phospholipid PG are anionic lipids with a negative charge on their head groups. SQDG and PG substitute for each other to maintain the amount of total anionic lipids in the thylakoid membrane, with PG having indispensable functions in photosynthesis. In addition to biochemical studies, extensive analyses of mutants deficient in thylakoid lipids have revealed important roles of these lipids in photosynthesis and thylakoid membrane biogenesis. Moreover, recent studies of  Chloroplasts are prototypical members of plastids, the double-membrane organelles specific to plants and algae, with the ability to perform oxygenic photosynthesis. In addition to outer and inner envelope membranes, chloroplasts have an extensively developed internal membrane system, the thylakoid membrane, where photochemical and electron transport reactions take place. In the thylakoid membrane, thousands of proteins, pigments and photosynthetic cofactors are embedded in the bilayer formed by glycerolipids. Reflecting their evolutionary relationship, chloroplasts and cyanobacteria have similar lipid compositions in thylakoid membranes, with non-phosphorous glycolipids as major constituents Table , althougThe lipid composition unique to chloroplasts and cyanobacteria implies a special requirement of these glycerolipids for oxygenic photosynthesis. Thylakoid lipids provide a lipid bilayer matrix for photosynthetic protein–cofactor complexes and support the electron transport chain in the thylakoid membrane. The lipid bilayer avoids the free diffusion of ions across the membrane and enables generation of a proton motive force via photosynthetic activities. Furthermore, glycerolipids function as structural components of several photosynthetic complexes in the thylakoid membrane and are directly and indirectly involved in photosynthetic reactions, as described later in brief and in detail in comprehensive reviews  , under P-deficient conditions presumably by catalyzing transfer of glucuronic acid to diacylglycerol. GlcADG accumulates in response to P deficiency and may play a role in adaptation to P limitation, although its function and subcellular localization remain unclear   of Escherichia coli, was reported to be involved in PG biosynthesis in Anabaena sp. PCC 7120  homologous to the yeast PGPP (Gep4p) could complement a yeast PGPP mutant (Δgep4), which suggests that CrPGPP1 is a functional PGPP in Chlamydomonas. Arabidopsis has a gene (At3g58830) that is homologous to CrPGPP, and future studies are anticipated to reveal genes responsible for this step in higher plants.In plant cells, PG is synthesized in plastids, mitochondria and endoplasmic reticulum (ER) membranes  of ArabidopsisMGD1 by a T-DNA insertion resulted in severe loss of the thylakoid membrane along with a substantial reduction in content of both galactolipids , in which the level of total cellular PG was decreased by ~80 % as compared with the wild type. Under P-deficient conditions, the proportion of galactolipids and SQDG was substantially increased in pgp1-2, which led to the development of the thylakoid membrane in the mutant. However, pgp1-2 chloroplasts showed many vesicular membrane structures under P-deficiency, which are barely detected in wild-type chloroplasts  , a ~40 % decrease in MGDG level had no effect on intrinsic PSII activity but increased conductivity of the thylakoid membrane, which impaired thylakoid membrane energization and photoprotection , a further decrease in MGDG level (~80 % from wild-type levels) with partial DGDG reduction strongly impaired the PSII photochemical reaction and energy coupling between reaction centers and antenna complexes  to the secondary plastoquinone (QB) at the PSII acceptor side  , which resulted in 30 % reduction in the total PG content with 80 % reduction in plastidic PGP activity, caused no remarkable defects in photochemical and electron transport activities -dependent manner  in cotyledons was rapidly and transiently induced in response to light  probably because of reduced PA levels, which suggests the importance of PA metabolism for MGDG synthase activity in vivo. As discussed in Botella et al.  from the green sulfur bacterium Chlorobaculum tepidum. Masuda et al. , a central regulator of plastid signaling  needs to be elucidated to understand how entire sets of photosynthetic complexes are assembled with thylakoid lipids during chloroplast biogenesis. In addition, information on transcriptional and post-translational regulators involved in thylakoid lipid biosynthesis is severely deficient as compared with the increasing knowledge about regulators for chlorophyll and photosynthetic protein synthesis. The molecular mechanism linking the functional state of lipid biosynthesis with photosynthesis-associated gene expression in plastids and nucleus awaits future studies.


Zizyphus, Humulus lupulus, magnesium and vitamin B6), in otherwise healthy adults with mild insomnia. After a 7-day single-blind placebo run-in, eligible volunteers (n = 171) were randomized (1:1) to receive daily treatment for 2 weeks with LZComplex3 or placebo. Results revealed that sleep quality measured by change in Pittsburgh Sleep Quality Index (PSQI) score improved in both the LZComplex3 and placebo groups. There were no significant between group differences between baseline and endpoint on the primary outcome. The majority of secondary outcomes, which included daytime functioning and physical fatigue, mood and anxiety, cognitive performance, and stress reactivity, showed similar improvements in the LZComplex3 and placebo groups. A similar proportion of participants reported adverse events (AEs) in both groups, with two of four treatment-related AEs in the LZComplex3 group resulting in permanent discontinuation. It currently cannot be concluded that administration of LZComplex3 for 2 weeks improves sleep quality, however, a marked placebo response (despite placebo run-in) and/or short duration of treatment may have masked a potential beneficial effect on sleep quality.Acute, non-clinical insomnia is not uncommon. Sufferers commonly turn to short-term use of herbal supplements to alleviate the symptoms. This placebo-controlled, double-blind study investigated the efficacy of LZComplex3 (lactium™,  Insomnia is defined by disturbances in sleep quality together with impairment of daytime functioning, for example fatigue and low mood . DisturbMelissa officinalis), chamomile (Matricaria recutita), valerian , hops (Humulus lupulus), passionflower (Passiflora incanata), lactium™ (αS1-casein hydrolysate) and sour date (Zizyphus jujube var. spinosa) [S1-casein, the bioactive peptide α-casozepine [S1-casein hydrolysate containing the α-casozepine peptide. Clinical studies have demonstrated that lactium reduces some symptoms related to stress [Commonly used herbal supplements for insomnia often include single or combined formulations of lemon balm (spinosa) . A new cspinosa) . The milsozepine . Lactiumo stress ,12. Lacto stress ,13,14.Zizyphus jujube var. spinosa; alternative spelling Ziziphus) is a fruit used in traditional Chinese medicine for its mild sedative and calming properties, to relieve irritability and aid sleep [Humulus lupulus) have been used in traditional western medicine for the treatment of mood disturbances such as restlessness and anxiety and in sleep disturbances due to reported calming and sleep-promoting properties [γ-aminobutyric acid (GABA), and histamine [Sour date , the efficacy of the combined formulation as a treatment for sleep disturbance has not been investigated. We report here the results of a clinical trial the primary objective of which was to investigate the short-term effect of LZComplex3 on sleep quality, mood and cognitive function in individuals with sleeping difficulties not caused by a primary sleeping disorder or other diagnosed condition.This study was a placebo-controlled, double-blind, randomized, parallel group phase III trial with a single blind placebo run-in period . The triAssessments of sleep quality, daytime functioning and physical fatigue, mood and anxiety, stress-reactivity, and cognitive function were completed by participants during the baseline and end of treatment visits, as well as during a final follow-up visit one week after the end of treatment. All assessment visits followed a procedure identical to that used at the baseline visit. In addition, participants completed all subjective sleep, daytime functioning, physical fatigue, mood and anxiety assessments at home 1, 3 and 7 days after baseline. All participant data were collected either at the study site  or at the participants’ homes (interim assessments between baseline and end of treatment).Potentially eligible participants were identified in an initial telephone screen. Participant eligibility was confirmed at the initial screening visit; each eligible participant was allocated a unique participant number as soon as written informed consent was obtained and prior to any screening assessments. Mood questionnaires including the Hospital Anxiety and Depression Scale (HADS)  and StatEligible participants were healthy adults aged 18–65 years with no significant diagnosed diseases (as judged by the Investigator) who had self-reported sleeping difficulties over one month prior to the screening call. Following accepted practice , sleepinEligible participants entered a one-week single-blind placebo run-in period, during which a daily sleep questionnaire  was useAt the baseline visit following the placebo run-in, placebo responders were excluded and all other participants were randomly assigned to treatment for two weeks (placebo or LZComplex3 in a 1:1 ratio) based on a randomization list generated centrally by an external independent third party. Participants and investigators were blinded to study treatment in the treatment phase and did not have access to the randomization codes except under exceptional medical circumstances.Participants were invited at the baseline visit to wear an actiwatch to collect objective sleep data for exploratory cross-validation of the subjective sleep outcome measures. Participants who agreed were given an actiwatch to wear for the duration of the two-week treatment period.LZComplex3 tablets  and placThe primary outcome was the change in overall sleep quality after two weeks of daily supplementation with LZComplex3. The primary outcome was measured by the change in PSQI scores from baseline to end of treatment at day 14. Secondary outcomes were the safety of LZComplex3 and the change in sleep quality, daytime functioning and physical fatigue, mood and anxiety, cognitive performance, and stress reactivity at 1, 3, 7 and 14 days after treatment with LZComplex3 and after one week post-treatment. The secondary outcomes were measured using validated assessments as outlined in Screening assessments included the HADS, STAI-T, Leeds Sleep Evaluation Questionnaire (LSEQ), Bond-Lader Visual Analogue Scale (VAS), and the Stress and Fatigue Visual Analogue Mood Scales (VAMS). The HADS is a 14-item questionnaire designed to measure levels of anxiety and depression and was administered at screening to exclude participants with depression and/or anxiety . The STADetails of each assessment method are provided in With an anticipated drop-out/non-compliance rate of 33%, it was estimated that a total of 170 participants would be required for 80% power to detect a medium effect size of approximately 0.5 at the 5% level of significance, with respect to the primary outcome. All analyses were conducted on a modified intention-to-treat (mITT) population, representing a per protocol/completer analysis and defined as all participants who were randomized and who had valid PSQI measures at both baseline and end of treatment. The primary outcome was also measured in the per protocol (PP) population, which included all participants in the mITT population who were at least 80% and less than 120% compliant with randomized treatment medication and had no major protocol deviations. Safety analyses were conducted on the safety population, which included all participants who were randomized and received at least one dose of study drug.n = 16).All measures were analyzed using SAS software . A general repeated measures mixed model was fitted to explore the difference between placebo and LZComplex3 in unadjusted change of total PSQI across all PSQI assessments for the mITT population. Day numbers and treatment group were included as fixed effects, participant as a random effect, the change in PSQI from baseline as the dependent variable and the baseline value of PSQI as a covariate. Secondary endpoints except ISI scores were analyzed in the same form as the primary endpoint. For the STAI-S, Bond-Lader VAS and VAMS scores, the mixed model also included time point (before and after administration of the MTF) as a fixed effect. A multinomial distribution and cumulative logit link function using PROC GLIMMIX was used to explore the difference between placebo and LZComplex3 in unadjusted change in ISI. The intended analysis of actigraphy data was not performed due to insufficient participant numbers  were eligible for analysis in the mITT population  but no effect of treatment  and no interaction between treatment and day  in the mITT population. The results were similar in the PP population, with a significant effect of day , no effect of treatment  and no interaction between treatment and visit . The unadjusted PSQI scores between baseline and end of treatment are presented in The mean change in total PSQI across time in the mITT population is presented in Similar to the primary outcome measure, the majority of secondary outcome measures showed similar changes in the placebo and LZComplex3 groups across the study period . A signin = 9 ; p = 0.8125; n = 6; LZComplex3, n = 7) and gastrointestinal disorders . There were no deaths or other serious adverse events. Two of the 4 AEs related to treatment in the LZComplex3 group led to permanent discontinuation. There were two hospitalizations reported, both in the LZComplex3 group, neither of which fulfilled the criteria of a serious AE.There were 25 AEs  reported during the run-in period, none of which were serious . Overall, 25 AEs were reported during the treatment period . The proThis study evaluated the efficacy of LZComplex3 in improving sleep quality in otherwise-well individuals with sleeping difficulties. There were no group differences in the primary outcome. Improvements in sleep quality were seen over a two-week treatment period with LZComplex3, however a persistent placebo response was observed and there was no significant treatment effect compared with placebo. Although the study included a one-week placebo run-in period designed to identify and exclude placebo-responders, it appears that that the run-in period may not have been of sufficient length. It is also possible that the persistent placebo response occurring after randomization may have been due to participants’ increased focus on overall sleep hygiene as a result of study visits and assessments, filling in a daily sleep diary and observing the protocol-mandated study parameters regarding stimulant use and sleep times. This degree of attention was not required in the placebo run-in phase. It has previously been suggested that having a patient keep a sleep diary for 2 weeks will aid with identification of behaviors that may worsen insomnia, thus providing a useful behavioral intervention . ParticiAn improvement in sleep quality with LZComplex3 was expected, as previous studies have supported the use of individual components of the formulation as an aid for sleeping difficulties or insomnia (see Introduction). However, rigorous clinical studies are lacking. Effects of lactium on sleep quality have been investigated in a double-blind, controlled, parallel study of 32 Japanese patients experiencing poor sleep as determined by a global PSQI score greater than 4 . As in tZizyphus jujube var. spinosa) in treating insomnia has been investigated in one clinical trial in the form of suanzaorentang [Zizyphus jujube var. spinosa), Fu Ling (mushroom) (Poria cocos), szechuan lovage , Zhi Mu (Anemarrhenae rhizoma), and liquorice root (Glycyrrhizae radix) in a ratio of 7:5:2:1:1 [The efficacy of sour date  is considered to be a sedative agent, a view that originated from the observation of sleepiness in European hops-pickers [Valeriana spp.) and passionflower (Passiflora incanata) [Humulus lupulus) is listed as an approved herb for mood disturbances including sleep disturbances in The Complete German Commission E Monographs [Humulus lupulus) alone in the treatment of insomnia. Thus it remains unclear whether hops (Humulus lupulus) has independent sedative effects, works as a synergist, or lacks sedative activity.Hops (-pickers . It is cncanata) ,51. Althnographs , there aThe rationale for inclusion of magnesium and vitamin B6 in LZComplex3 is based on in vitro and animal studies suggesting they may promote sleep quality by supporting metabolic pathways involved in sleep regulation, rather than due to any direct sedative activity ,31,32,33It is possible that the two-week treatment duration in our study was not long enough to observe a treatment effect, and/or the one-week placebo run-in period was not long enough to eliminate all placebo responders. Another possibility is that the study population may have included individuals with chronic insomnia, which unlike brief or acute insomnia generally requires a cognitive behavioral approach to treatment . The stuOverall, the secondary outcome measures did not support a benefit of LZComplex3 over placebo during the two week treatment period. Participants completed a battery of questionnaires assessing various aspects of sleep quality, daytime functioning and physical fatigue, mood and anxiety, cognitive performance, and stress. Poor sleep can contribute to impairment of daytime functioning and physical fatigue thus these outcomes were anticipated to improve with better quality sleep. However, given that no significant difference between LZComplex3 and placebo was detected with respect to improvement in sleep quality as measured by the PSQI, it is not surprising that daytime functioning and physical fatigue were also not significantly different between the treatment groups. An improvement in ISI at day 3 favoring LZComplex3 and a between-group difference in stress reactivity as measured by change in STAI-S total score from pre- to post-administration of the MTF which appeared to be due to a slight increase in stress reactivity in the placebo group at baseline and at day 14 were the only significant treatment effects detected. Potential improvements in mood, anxiety and stress with LZComplex3 were anticipated as there is evidence that they may be positively impacted by some of the individual components of the formulation ,22,23,24The safety data collected in this study indicate that LZComplex3 is well-tolerated at the dose of two tablets prior to sleep. Overall the safety profile of LZComplex3 was similar to placebo, with similar proportions of patients reporting AEs in both groups, the majority of which were mild or moderate and none of which were serious. There were more severe AEs reported in the LZComplex3 group and only two were considered possibly, probably or definitely related to treatment .Despite finding a negative primary efficacy outcome, this study demonstrated an improvement in the PSQI and other measures of sleep quality for patients taking LZComplex3 with no deficits in cognitive or psychomotor function and a benign safety profile. These findings, taken into context with the marked placebo effect, short treatment duration and methodological limitations, suggest that further investigation of LZComplex3 is warranted.


Genetic engineering, also called genetic modification, is facing with growing demands of aquaculture and aquatic products. Although various genetically modified (GM) aquatics have been generated, it is important to evaluate biosafety of GM organisms on the human health before entering into our food chain. For this purpose, we establish a zebrafish wild adult feeding Flk1-transgenic larvae model to examine the predatory fish's histology in multiple tissues, and the global gene expression profile in the liver. 180 days of feeding trial show that there are no significantly morphological changes in intestine, liver, kidney, and sex gonads between fish fed with Flk1 transgenic fish diet (TFD) and fish fed with regular food meal (RFM). However, a characteristic skin spot and autofluorescence increase in the theca of follicle are observed in F1 generation of TFD fish. Liver RNA-sequencing analyses demonstrate that 53 out of 56712 genes or isoforms are differentially transcribed, and mostly involved in proteolysis in extracellular region. According to GO enrichment terms these deregulated genes function in catalytic activity, steroid storing, lipid metabolic process and N-Glycan biosynthesis. These results suggest that a long term of Flk1-transgenic fish diet could alter certain metabolic pathways and possibly cause related tissue deformation. Compared to the previous reports, our feasible transgenic dietary assess system could evaluate subchronic and potential health impact of transgenic fish diet by combining multi-tissue histology and liver transcriptome analyses. Oreochromis niloticus  is expressed in hemangioblasts. Then Flk1 expression is stronger in developing angioblast/ endothelial precursors than in mature vessels  expression construct Flk1: GFP transgenic zebrafish (Flk1-transgenic fish) were home cultured under an ambient temperature of 28.5°C as previously described  and stained with hematoxylin and eosin (HE) according to the standard protocol. As autofluorescence can provide information about morphological and pathological states of tissues and cells , and low qualities were filtered out by Trim Galore . Tiltered reads were mapped to reference genome using TopHat; gene assembly and differential gene expressions were determined using Cufflinks . After homogenization, RNA was extracted following the manufacturer's protocol and stored at 4°C until cDNA libraries construction. For each group (TFD fish or RFM fish), two libraries were constructed for RNA-seq analysis . Data process and analysis were conducted as previously reported  of 1 based on the reads counts and the length of the gene mapped to the model gene  by Gene Ontology  and the Kyoto Encyclopedia of Genes and Genomes databases  , using the Cuffnorm to calculate the expression of each sample with the corrected We projected a 180 days of feeding trial to test whether eating the transgenic fish would induce any dietary harm. The feeding trial started at the juvenile period, 30–40 day post-fertilization (dpf), when the AB line zebrafish were developed big enough to eat the transgenic larvae. We randomly chose 40 fish and divided them into two groups. All fish were fed with equivalent amount of F0 TFD or F0 RFM twice per day according to the growth rate and the body weight. After 90 days, we calculated the length of the body, spawning, the growth rates and the death rates. The results showed no significant differences in the above parameters and swimming behaviors between two groups, although many F0 TFD fish had a little bit lower weight and smaller ovary than F0 RFM control Figure .F0 TFD fish intestine showed higher intense fluorescence signal than the F0 RFM control individuals. To rule out this possibility that the digested tissues of the Flk1: GFP transgenic fish in diet may invade into the predatory intestine fish, we starved the TFD fish for 24 h to keep their intestine empty, and then fed with RFM. As a result, the fluorescence signal in the intestine became much weaker than TFD fed fish Figure . This reThe histological analyses showed that after 180 days feeding trial, the Flk1-transgenic fish diet didn't change tissue structures and cell morphology of the intestine and kidney in comparison with RFM diet Figures . The mucHaving observed small ovaries in TFD fish, we then tested the fish's reproductive capacity. Seven adult female F0 TFD fish mated with wild type AB lines and produced dozens of F1 generations with normal phenotype. Neither the F1 embryos nor the F1 larvae of TFD fish expressed GFP, confirming that the Flk1-GFP transgene products were digested, and failed to be seen in the next generation of TFD fish.Intriguingly, most of the female F1 generation had an obvious white or deep tissue-deficient patch appeared on the left side of the abdomen skin adjacent to the ovary. Under inverted phase contrast microscope, the patch was seen in dark red color. As the fish grew, this patch became enlarged Figure . The darTo better distinguish different cell types and stages of ovarian follicles, we combined HE staining method with fluorescent microscopy. In comparison with F1 RFM ovary, the oocytes in F1 TFD ovary showed similar fluorescence, but the follicle stroma, particularly in the theca of follicles showed stronger autofluorescence and structure clarity by the corresponding excitation light source Figure . The difLiver is one of the most important digestive organ responsible for multiple dietary metabolisms, such as storing the polysaccharide glycogen, excreting excess nitrogen, detoxification and synthesis cholesterol fatty acids and triacylglycerols Blann, . To testAfter generated all of the raw reads by high-throughput sequencing, the lower quality sequences were trimmed and removed from the reads  were higher than that of RFM fish (80.7%), the multiple mapped reads and splice reads were lower in TFD fish than RFM fish  are involved in collagen and elastin metabolisms, and four genes  are related to pathways of cytokine or inflammatory reactions  and lipid metabolisms. Five deregulated genes  under the flk1/vegfr2 promoter , and exhibits a green fluorescent vasculature. After feeding Flk1:GFP transgenic larvae, the predatory fish show developed soft tissue defects in the ovary and adjacent skin hypoderm in the F1 generation. However, this defect was uninheritable. Compared to the previous reports reviewed by Snell et al. , our feaJY conceived the idea. JY, YW analyzed the data and wrote the manuscript. YW, LH, and JC performed the experiments, and analyzed the data. CW supervised the project and contributed to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Next-generation sequencing (NGS) is now more accessible to clinicians and researchers. As a result, our understanding of the genetics of neurodevelopmental disorders (NDDs) has rapidly advanced over the past few years. NGS has led to the discovery of new NDD genes with an excess of recurrent de novo mutations (DNMs) when compared to controls. Development of large-scale databases of normal and disease variation has given rise to metrics exploring the relative tolerance of individual genes to human mutation. Genetic etiology and diagnosis rates have improved, which have led to the discovery of new pathways and tissue types relevant to NDDs. In this review, we highlight several key findings based on the discovery of recurrent DNMs ranging from copy number variants to point mutations. We explore biases and patterns of DNM enrichment and the role of mosaicism and secondary mutations in variable expressivity. We discuss the benefit of whole-genome sequencing (WGS) over whole-exome sequencing (WES) to understand more complex, multifactorial cases of NDD and explain how this improved understanding aids diagnosis and management of these disorders. Comprehensive assessment of the DNM landscape across the genome using WGS and other technologies will lead to the development of novel functional and bioinformatics approaches to interpret DNMs and drive new insights into NDD biology. Every human inherits approximately half of their genetic information from their mother and half from their father. However, a small number of changes, referred to as de novo mutations (DNMs), are not observed in the genome of either parent. These mutations are either newly formed during gamete formation or occur very early in embryonic development and, thus, are unique to the child when compared to the parent. DNMs can range in size from a single nucleotide change to large >50 kbp) genomic deletions, duplications, or rearrangements Table . Errors 0 kbp genNeurodevelopmental disorders (NDDs) are a collection of heterogeneous phenotypes diagnosed during early childhood that persist throughout life and include but are not limited to autism spectrum disorder (ASD), intellectual disability (ID), developmental delay (DD), and epilepsy. Combined, NDDs are thought to affect 2–5% of children , 16. DifGenomic technologies, such as microarray and next-generation sequencing (NGS), have enabled a more comprehensive interrogation of the entire genome. Recent reductions in cost and more rapid implementation due to improvements in bioinformatics have led to routine use of these assays for diagnostics and genetic testing, particularly for families with children affected with NDDs . The traHere, we review the current state of NDDs in the context of DNMs with an emphasis on the implicated genes and genomic regions. Although NDDs may encompass a wide array of phenotypes that affect the developing brain, such as adult neuropsychiatric conditions, we focus here on disorders with pediatric onset. We consider a range of mutations from large CNVs to single-nucleotide variants (SNVs) and explain how the rapid growth of population genetic resources and technology improvements have increased specificity for disease-gene discovery. We summarize functional networks and pathways consistently identified as enriched for DNMs in NDDs, which includes evidence that implicates different regions and cell types of the developing brain. We conclude with a discussion of how this information could improve diagnostics and guide future therapeutics, with specific emphasis on the value of whole-genome sequencing (WGS) over whole-exome sequencing (WES) in both clinical and basic research.Table MAPT and KANSL1 . Re. Re48]. De novo CNVs often arise mechanistically as a result of elevated mutation rates in regions flanked by segmental duplications (long DNA sequences with > 90% sequence similarity that exist in multiple locations across the genome)  due to uSNVs (single base-pair changes) and indels  are the most common forms of genetic variation in the genome Table  76]. Pa. Pa76]. LGD or protein-truncating variants are the best-characterized class of DNMs because of their straightforward mechanism of action and abundance in children with NDD. For example, there was a twofold excess of LGD DNMs in ASD patients versus their unaffected siblings , 79. LGDSPAST, S100G, MLANA, LSM3, HMGN2, WDFY3, and SCN1A. SPAST is a common causal gene of autosomal dominant hereditary spastic paraplegia, a phenotype that is very distinct from the characteristic traits of individuals with ASD [Curation of a DNM database of NDD and other disease studies has facilitated the identification of genes . We findwith ASD . Severalwith ASD , 87, 88.Although there are overlapping genes between ASD and ID/DD phenotypes, gene sharing does not necessarily result in identical phenotypes across patients. For example, the DDD reported that 56% of their cohort carried an LGD or missense DNM in a known epilepsy gene even though only a quarter of these individuals had reported epilepsy or seizure phenotypes , 89. AltGATAD2B have been observed exclusively in ID/DD cases whereas LGD mutations in CHD8 have been biased toward ASD cases, which means that some cases reported as ID/DD also carry an ASD diagnosis  sequence reads. Not all regions or types of genetic variation can be readily assayed using this platform alone , 133 and