Despite an increasing number of studies that identify leaders’ role in promoting employees’ voice behavior, little is known about the role that supervisors’ goal-focused leadership plays in this. The current study aims to address this research gap by using the role theory to explain how supervisors’ goal-focused leadership influences employees’ voice behavior and the conditions under which supervisors’ have maximum impact on employee voice. A field study of 197 employees and their immediate supervisors offered support for our model. The results indicated a positive association between goal-focused leadership and employees’ voice behavior that was mediated by leaders’ omission of reward and punishments. We also found that perceived helping and support from coworkers positively moderated the relationship between leaders’ reward and punishment omission and employees’ voice behavior such that the relationship was weaker when coworker helping and support was higher. The findings provide more comprehensive picture of the process by which goal-focused leadership influences employee voice and highlight how coworkers can buffer the negative effect of ineffective managerial reward and punishment omission. The practical implications of this research, its limitations and directions for future research are also discussed. Voice behavior is defined as “informal and discretionary communication by an employee of ideas, suggestions, concerns, information about problems, or opinions about work-related issues to persons who might be able to take appropriate action, with the intent to bring about improvement or change” , p. 172.Although there are benefits that flow from voice behavior, recent research indicates that engaging in voice behavior can be draining, and employees are often very hesitant to use their voice . Voice bOne contextual factor thought to play a prominent role in organizations’ success is goal-focused leadership, which is defined as a leadership style characterized by an emphasis on goal achievement that is supported by the setting appropriate goals, suggesting means and providing task structure and feedback . Not surAlthough prior studies have led to great advances in understanding of the effects of goal-focused leadership on follower outcomes , the appAccording to role theory, roles are generated by normative expectations and are related to identifiable social positions in organizational contexts . Role thSupervisors who offer goal-focused leadership are skilled in aligning individual roles with organizational goals and defining role responsibilities . They arFigure 1.As well as recognizing the importance of leadership, role theory also proposes that coworker help and support could help employees clarify their roles and thus buffer the negative impacts of leader’s omissions. We suggest, therefore, that coworker helping and support moderate the relationship between the omission of reward and punishments and voice behavior. Coworker help and support refer to “coworkers’ assisting an employee with his or her tasks when needed by sharing knowledge and expertise or providing encouragement and support” , p. 685.Subordinates who perform poorly will probably expect to be punished by their leader for this and so when the expected punished is not delivered they may become confused about what is expected of them . Role thHypothesis 1a: goal-focused leadership is negatively related to leaders’ punishment omission.Hypothesis 1b: leaders’ punishment omission is negatively related to employee voice.Hypothesis 1c: leaders’ punishment omission mediates the relationship between goal-focused leadership and employee voice.Goal-focused leaders are good at defining the responsibilities of individual roles . They atThe giving of reward by a leader is a form of supportive behavior and recognition of employees’ contribution to organizational goals . Liang eHypothesis 2a: goal-focused leadership is negatively related to leaders’ reward omission.Hypothesis 2b: leaders’ reward omission is negatively related to employee voice.Hypothesis 2c: leaders’ reward omission mediates the relationship between goal-focused leadership and employee voice.Previous research suggests that coworkers help and support colleagues by sharing knowledge and expertise, offering suggestions and guidance, and supplying task-focused feedback . EmployeHypothesis 3a: perceived help and support from coworkers positively moderates the relationship between leaders’ punishment omission and employees’ voice behavior such that the relationship is weaker when coworker help and support is high.Hypothesis 3b: perceived help and support from coworkers positively moderates the relationship between leaders’ reward omission and employees’ voice behavior such that the relationship is weaker when coworker help and support is high.We surveyed 32 workgroups  from a logistics company located in northern China. With the assistance of human-resource managers, we distributed subordinate questionnaires and supervisor questionnaires to 224 subordinates and their 32 immediate supervisor participants. No specific reward was given for questionnaire completion. Subordinate participants were asked to report their demographic information, their experience of supervisors’ goal-focused leadership and omissions of punishments and reward, as well as perceived coworker help and support; supervisor participants were asked to report their subordinates’ voice behavior. Participants completed the survey questionnaire voluntarily. Subordinate questionnaires and supervisor questionnaires were given identification codes to enable matching of responses. Respondents were assured that their responses would remain confidential. Participants sealed their completed questionnaire in the envelope provided with it and returned it 2 weeks later via a secure box outside one of the company’s meeting venues.SD = 0.48) and the average age of participants was 29.13 years (SD = 5.28). 23.9% of them had a junior high school degree, 21.3% had a senior high school degree, 18.8% had a junior college degree, and 34.5% had a bachelor degree.We excluded questionnaires with missing data, so the final sample consisted of 197 matched responses. Of the 197 subordinates, 63.5% were men  results. As shown, the baseline five-factor model fitted the data well . We compared the baseline five-factor model with a null model, three four-factor model, one three-factor model and one two-factor model. As shown in Table 1, the baseline model (five-factor model) fitted better than the null model and models 3–7, providing evidence of the construct distinctiveness of goal-focused leadership, leaders’ punishment omission, leaders’ reward omission, coworker help and support and employee voice behavior.Table 2 presents descriptive statistics and correlations between the studied variables. As anticipated, goal-focused leadership was negatively correlated with leaders’ punishment omission ; and with leaders’ reward omission , providing preliminary support for hypotheses 1a and 2a. In addition, leaders’ punishment omission and reward omission were both negatively related to employee voice behavior , providing preliminary support for hypotheses 1b and 2b.Table 3, goal-focused leadership was negatively associated with leaders’ punishment omission  and reward omission  after controlling participants’ gender, age, education, and working experience with leaders. Thus our hypotheses 1a and 2a were fully supported.As shown in We carried out regression analysis with voice behavior as the dependent variable, entering the variables in three steps :(1) the control variables ; (2) goal-focused leadership, mediators  and the moderator ; (3) the two-way interactive terms .Table 3, leaders’ punishment omission  and reward omission  were significantly related to employees’ voice behavior while the relationship between goal-focused leadership was also significant , indicating the partial mediation role of leaders’ omission behavior. To further examine the mediation effects of leaders’ punishment and reward omission, we conducted supplementary analysis using the bootstrap method with SPSS PROCESS Macro  and leaders’ reward omission × coworker help and support , were significant, providing support for hypotheses 3a and 3b.Hypothesis 3a and 3b proposed the moderating role of coworker helping and support in the relationship between leaders’ omission behavior  and employees’ voice behavior. As shown on in Figures 2, 3. As predicted: (a) the linear relationship between leaders’ punishment omission and employees’ voice behavior was weaker for the high coworker helping and support and stronger for low coworker helping and support; (b) the linear relationship between leaders’ reward omission and employees’ voice behavior was weaker for high coworker helping and support and stronger for the low coworker helping and support.Following In the present study, we developed and tested a model linking goal-focused leadership with employees’ voice behavior by investigating the underlying mechanisms as well as the boundary condition. We found that: (a) goal-focused leadership was positively associated with employees’ voice behavior via decreasing leaders’ punishment and reward omission; (b) perceived coworker helping and support positively moderated the relationship between leaders’ punishment omission and employees’ voice behavior in a way that the relationship was weaker when coworker helping and support is higher rather than lower; (c) perceived coworker helping and support positively moderated the relationship between leaders’ reward omission and employees’ voice behavior in a way that the relationship was weaker when coworker helping and support is higher rather than lower.The present study has several theoretical implications that should be noted. First, we apply role theory  to explaSecond, we have drawn on role theory  to introFinally, by specifying the role of coworker helping and support as an important moderator on the reward and punishment omission-voice relationships, we have improved the understanding of the boundary conditions of the link between goal-focused leadership and voice behavior. Drawing on role theory , we arguThis research has some implications for managerial practices. To begin with, our model suggests that goal-focused leadership can enhance employees’ voice behavior. This suggests that organizations should promote goal-focused leadership through training programs, to enhance leaders’ ability to encourage employees to use their voice. For instance, supervisors should link the organization’s policies to its goals, discuss goals with followers and give followers clear directions to encourage followers to voice their opinions, concerns and suggestions .Second, the finding concerning the mediating role of leaders’ omission of punishments and reward suggests that goal-focused leadership can promote employees’ voice behavior via minimizing leaders’ omission of punishments and reward. Leaders should therefore keep this in mind and pay extra attention to subordinates’ performance and provide appropriate feedback . More spFinally, as we have demonstrated that the help and support of coworkers helps to mitigate the effects of poor leaders, strategies that encourage employees to help and support coworkers should be developed and implemented. For example, organizations could create an atmosphere of trust, as coworkers who trusts their colleagues will be more likely to provide them with help and support . FurtherOur study has several limitations, which can be viewed as opportunities for future research efforts to improve, replicate, and extend our findings. The first limitation of the study is the cross-sectional design, which limits our ability to determine the causality of the detected associations we uncovered. Longitudinal research would be needed to do this. A second limitation is the limited generalisability of the findings. Because we collected data from just one company in China, it could be argued that our findings are contextually and culturally specific. Future research could include attempting to replicate our findings in other types of organizations and other cultures.From the perspective of role theory, our results highlight the importance of goal-focused leadership as an antecedent of voice behavior that is mediated by reward and punishment omission and the need for further research into the contingencies of this relationship. We therefore suggest that understanding of the mechanisms by which antecedents produce voice behavior would be advanced by research into the role of the mediation and moderation processes.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards with written informed consent from all subjects. This research was approved by the Human Research Ethics Committee (HREC) at Business School, Beijing Normal University.JQ and XL substantially contributed to the conception, the design of the work as well as the preparation of the draft. BS, WZ, MC, and YQ reviewed it critically and gave important intellectual input. BW contributed to the analysis and interpretation of the data.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.


Based on the predictor, hundreds of potential prkC-specific phosphorylation sites were annotated based on the known bacterial phosphorylation sites. prkC-PSP was the first predictor for prkC-specific phosphorylation sites, and its prediction performance was promising. We anticipated that these analyses and the predictor could be helpful for further studies of prkC-mediated phosphorylation.As an abundant post-translational modification, reversible phosphorylation is critical for the dynamic regulation of various biological processes. prkC, a critical serine/threonine-protein kinase in bacteria, plays important roles in regulation of signaling transduction. Identification of prkC-specific phosphorylation sites is fundamental for understanding the molecular mechanism of phosphorylation-mediated signaling. However, experimental identification of substrates for prkC is time-consuming and labor-intensive, and computational methods for kinase-specific phosphorylation prediction in bacteria have yet to be developed. In this study, we manually curated the experimentally identified substrates and phosphorylation sites of prkC from the published literature. The analyses of the sequence preferences showed that the substrate recognition pattern for prkC might be miscellaneous, and a complex strategy should be employed to predict potential prkC-specific phosphorylation sites. To develop the predictor, the amino acid location feature extraction method and the support vector machine algorithm were employed, and the methods achieved promising performance. Through 10-fold cross validation, the predictor reached a sensitivity of 91.67% at the specificity of 95.12%. Then, we developed freely accessible software, which is provided at  Bacillus subtilis [S. pyogenes adherence, invasion and in E. faecalis persistence [In 1992, the Nobel Prize in Physiology or Medicine was award to Edmond H. Fischer and Edwin G. Krebs for their discovery that reversible protein phosphorylation is a critical biological regulatory mechanism in biology . A numbesubtilis . Serine/subtilis , and it sistence . Furthersistence , bacterisistence  and glidsistence . HoweverTo understand the detailed biological functions and molecular mechanisms of prkC, identification of its substrates and sites is fundamental. Although the development of state-of-art proteomics technologies such as high-throughput mass spectrometry enabled leading scientists to carry out large-scale profiling of serine/threonine phosphorylation events in bacteria ,16, the et al. [In this study, we developed a novel predictor for prkC-specific phosphorylation. According to the 5-step rule defined by Chou et al. ,24, we cBacillus subtilis. Only the identified prkC-specific phosphorylation sites clearly described in the full text were reserved. In total, 36 phosphorylation sites in 14 substrates were obtained  are defined to extract useful features from the relevant training sequences. Since the length of peptides in the benchmark dataset is 31, 16 = S/T, and Ri can be any of the twenty native amino acids. Thus, the 31 components in its amino acid location feature vector are defined as follows.For each position of the fragment, we have1) is the occurrence frequency of R1 at position 1 for the positive peptide sequence of 2) is the occurrence frequency of R2 at position 2. n(R1) is the occurrence frequency of R1 at position 1 for the negative peptide sequence of 2) is the occurrence frequency of R2 at position 2, and so forth.In After deriving these amino acid location feature values from the training data, we use the SVM classifier LibSVM  to buildSn), specificity (Sp), accuracy (Ac), and Mathew’s Correlation Coefficient (MCC) were employed to evaluate the prediction performance. The four measurements were defined as follows:As previously described, four measurements of sensitivity  curves and AROCs (area under ROCs) were analyzed.To evaluate the prediction performance and robustness of prkC-PSP, the training data set was used to perform the http://free.cancerbio.info/prkc/.The online service of the prkC-specific phosphorylation sites prediction (prkC-PSP) software was implemented in Python and is freely available at Although a number of studies were carried out for prkC and its substrates, the sequence features and motifs for prkC substrate recognition are still to be dissected. With the collected prkC-specific phosphorylation sites , the seqSince the sequence preferences seemed to be evident, we tried to identify the potential motif for prkC-specific phosphorylation. The motif analysis for the dataset was carried out with the Motif-All software . HoweverTo develop an accurate predictor for prkC-specific phosphorylation, several widely used computational models, including amino acid location feature extraction (location) , PseAAC n-fold cross validations could represent the prediction of new or unknown sites, the results show that our prediction achieved promising performance. For the 4-fold cross validation, the prediction achieved an accuracy of 94.89%, sensitivity of 91.67%, specificity of 95.12%, MCC of 0.6989 and AROC (area under ROC) of 0.9798. For the 6-fold cross validation, the performance was an accuracy of 95.07%, sensitivity of 94.44%, specificity of 95.12%, MCC of 0.7159 and AROC of 0.9778. For the 8-fold cross validation, the prediction achieved an accuracy of 94.71%, sensitivity of 88.89%, specificity of 95.12%, MCC of 0.6817 and AROC of 0.9823. For the 10-fold cross validation, the performance was an accuracy of 94.89%, sensitivity of 91.67%, specificity of 95.12%, MCC of 0.6989 and AROC of 0.9770. From the results, it was observed that the performance was promising.To evaluate the performance of our prediction, the 4-, 6-, 8-, 10-fold cross validations were carried out. The ROC curves for these validations are presented in With performance taken into consideration, we developed a novel predictor of prkC-PSP (prkC-specific Phosphorylation Sites Prediction) software for online prediction service. The prkC-PSP was implemented in PHP and Python, and the prediction page was as shown in Bacillus subtilis odhB, ypfD and tal proteins  as examples to demonstrate the simplicity and precision of the prkC-PSP. These sequences were pasted into the text box, and the high threshold was chosen; then, we clicked the ‘Submit’ button, and the results were shown on the result page  and glucose-6-P in the pyk mutant [Escherichia coli played a stimulatory role in trans-translation through binding to tmRNA [in vitro by several kinases such as HipA and doc [The serine/threonine-protein kinase pknL (P9WI62) could phosphorylate the DNA-binding protein MT2231 in division . We predk mutant . Previouk mutant . We predto tmRNA  and coul and doc ,36. HereAs a dynamic regulatory mechanism, protein phosphorylation played important roles in regulation of various cellular processes in prokaryotes –9. IdentButt et al used statistical moments to extract features and Multilayer Neural Network (MNN) to predict membrane proteins [Akmal et al extracted the protein feature with multiple methods and combined with MNN to identify glycosylation sites [Ehsan et al used neuro network for classification of signal peptides [In this study, we carried out computational prediction for prkC-specific phosphorylation sites. The experimentally identified prkC-specific phosphorylation sites were manually collected, and the sequence preferences were analyzed. There are many feature extraction methods and algorithms developed for predicting biological features. For example, proteins , Akmal eon sites , and Ehspeptides . With ouTaken together, this study provides a start for kinase-specific prediction of phosphorylation sites in prokaryotes. Computational prediction will help advancing studies of serine/threonine phosphorylation in bacteria.S1 FigThe ROC curves of the 10-fold cross validation for different algorithms including the location model used in this study and other models such as PseAAC and CKSAAP. The AROC values were calculated and are shown.(TIF)Click here for additional data file.S1 Table(a) The phosphorylation sites in bacteria species that have prkC kinase. (b) The sites that were potentially phosphorylated by prkC kinase were annotated by the predictor prkC-PSP.(XLSX)Click here for additional data file.


Dopamine is a brain neurotransmitter involved in the pathology of schizophrenia. The dopamine hypothesis states that, in schizophrenia, dopaminergic signal transduction is hyperactive. The cAMP-response element binding protein (CREB) is an intracellular protein that regulates the expression of genes that are important in dopaminergic neurons. Dopamine affects the phosphorylation of CREB via G protein-coupled receptors. Neurotrophins, such as brain derived growth factor (BDNF), are critical regulators during neurodevelopment and synaptic plasticity. The CREB is one of the major regulators of neurotrophin responses since phosphorylated CREB binds to a specific sequence in the promoter of BDNF and regulates its transcription. Moreover, susceptibility genes associated with schizophrenia also target and stimulate the activity of CREB. Abnormalities of CREB expression is observed in the brain of individuals suffering from schizophrenia, and two variants (-933T to C and -413G to A) were found only in schizophrenic patients. The CREB was also involved in the therapy of animal models of schizophrenia. Collectively, these findings suggest a link between CREB and the pathophysiology of schizophrenia. This review provides an overview of CREB structure, expression, and biological functions in the brain and its interaction with dopamine signaling, neurotrophins, and susceptibility genes for schizophrenia. Animal models in which CREB function is modulated, by either overexpression of the protein or knocked down through gene deletion/mutation, implicating CREB in schizophrenia and antipsychotic drugs efficacy are also discussed. Targeting research and drug development on CREB could potentially accelerate the development of novel medications against schizophrenia. The cAMP-response element binding protein (CREB) is localized in the nucleus and acts as a transcription factor, which binds to the cAMP response element (CRE) of the promoters of its target genes, upon phosphorylation at Ser133 by different receptor-activated protein kinases, such as protein kinase A (PKA), calmodulin-dependent protein kinase (CaMK), mitogen-activated protein kinases (MAPK), and other kinases . Once CRSchizophrenia is a severe mental illness that changes a patient’s way of thinking, feeling, and social behavior . The onsin vitro and animal studies show that dopamine receptor signaling increases the phosphorylation of CREB   of CREB . Activat of CREB , while o of CREB . Direct l models . (2) Datl models . As a dol models . Interesl models , while Gl models ; and (3)l models , an inte2+ ions facilitate the binding activity of bZIP to CRE by more than 25-fold  binding of dopamine to D1R elevates intracellular cAMP levels and activates PKA followed by the phosphorylation of CREB ; (ii) bi of CREB . Dopamin of CREB ; (iv) St of CREB ; (v) CRE of CREB , which m of CREB . It has  of CREB .2+/CaMK, PI3K/Akt/GSK3β, and cAMP/PKA pathways .Several different protein kinases phosphorylate CREB, making it a convergent target for multiple intracellular signaling cascades. The most important posttranscriptional modification is the phosphorylation of the amino acid residue Ser133 in the KID domain . This dopathways , which, Both cAMP and cGMP show regulatory functions in mental disorders  and bothIn vitro, binding affinities of cAMP for PKA and Epac are similar  is another cAMP-binding effector protein.  similar . cAMP ac similar .PEPCK, cytochrome c, and aminolevulinate synthase ), transcription , cell survival/cell cycle , and growth factors (such as BDNF and FGF6)  . A schempression .There are three phosphorylation sites existing in the amino-terminal of CREB, and the phosphorylation of Ser133 is the most studied. The phosphorylation of Ser133 leads to a 10- to 20-fold increase in CREB’s transcriptional activity . Functio2+ entry triggers the activation of Ca2+/calmodulin signaling and subsequently causes phosphorylation of PP2B, which acts as an inhibitory subunit of PP1  . The actThe “neurodevelopmental hypothesis of schizophrenia” emphasizes that the abnormalities of early brain development increase the risk for the subsequent emergence of clinical symptoms . In this2+ that leads to the activation of CaMKIV, which phosphorylates CREB. Neuronal exposure to BDNF also activates the Ras/Erk/Rsk pathway that causes CREB phosphorylation  receptor, were enriched in these mutated genes (Figure 3. Based on these findings, CREB1, CREM, PPP3CB, and PRKAR1A, are involved in the etiology of schizophrenia-related psychiatric disorders and regulated by the cAMP/PKA/CREB signaling pathway (de novo and transmitted mutations contribute to the development of schizophrenia. However, it is worthy of our attention that so far there has been no significant association with a gene. The penetrance of de novo mutation to chromatin regulation is yet unknown and deserves further clarification.Schizophrenia is a complicated central nervous disease, and the causes of schizophrenia include genetic factors and gene–environment interactions. In the last decade, a number of chromosomal regions and genes have been studied with molecular biology and genetic analyses. However, there has been no consistent single gene variation confirmed with the development of this illness, and the contribution of genetic factor remains obscure at this time . Genome-ed genes . These red genes . Collect pathway . Genetic pathway . Consist pathway . In summWith this background, we would like to address a few studies focusing on NRG-1 and the DISC-1 and dysbindin-1 genes in relation to schizophrenia. These genes play a role in both neural signaling and development, and are associated with schizophrenia . It woulCollectively, as we previously reviewed , all theBesides the susceptibility genes listed earlier, there are many other risk-genes that have been identified in schizophrenia . It is hIn vitro studies also support this conclusion. In SY5Y neuroblastoma cells, amisulpride, an atypical antipsychotic drug, stimulated neurite outgrowth by regulating D2R-mediated β-arrestin 2 signaling, and increased levels of CREB phosphorylation, with BDNF potentially involved in this process (Many studies addressed the expression and activity of CREB, a transcription factor regulated by cAMP/PKA, in animal models upon treatment with antipsychotic drugs. The CREB mRNA levels are not regulated by haloperidol in striatum of rats, while haloperidol induces a significant phosphorylation of CREB in striatum, indicating that CREB is transcriptionally active in response to haloperidol . In amph process . Similar process . A 4-wee process .in vivo situations. It was reported that haloperidol and eticlopride, selective D2R antagonists, stimulated the phosphorylation of CREB in the dorsal striatum. In contrast, clozapine reduced CREB phosphorylation, indicating that haloperidol and clozapine induce distinct patterns of CREB phosphorylation in the dorsal striatum (in vitro, but not at 10 days (We would like to propose that various antipsychotic drugs may have different roles on CREB phosphorylation in neurons in both culture and striatum . On the  10 days . These d 10 days .Figure 3. Dopamine, antipsychotic drugs, growth factors, and susceptibility genes could activate CREB and its downstream target BDNF via different pathways. The deficit of CREB/BDNF signaling impairs neurodevelopment and is implicated in the development of schizophrenia.Current studies suggest that CREB is a key integrator of diverse physiological processes in the CNS, including neurotransmission, neurodevelopment, neuronal survival, synaptic plasticity, and memory. Importantly, dysregulation of the CREB signaling has been implicated in a number of disorders in the CNS. Among these disorders, the relevance of CREB to the pathogenesis of schizophrenia has been most intensively investigated. The roles of CREB in the pathology of schizophrenia are depicted schematically in Given the important role of CREB activation in the CNS, it is reasonable to propose that activation of CREB signaling would have beneficial effects in schizophrenia therapy. However, sustained CREB activation also causes deleterious consequences. Hence, keeping a balance on CREB activation is a reasonable strategy in schizophrenia therapy. Pharmacological interventions, using transcriptional repressors, may serve this purpose. On the other hand, besides Ser133, CREB is also phosphorylated at many other sites. Investigating the relationship between these phosphorylation sites and schizophrenia may lead to novel pharmaceutical approaches. Among these sites, Ser129 and Ser 142 are close to Ser133, and it is tempting to suggest that these two sites will affect the transcription activity or binding of CREB to CRE. Moreover, the activity of CREB is modulated by additional signaling pathways, which due to antagonistic action may be manipulated for schizophrenia therapy. The signaling pathways involved in schizophrenia are complex. Both CREB and BDNF are key molecules implicated in the regulation of mood. Additional basic and clinical research is needed to further identify the specific role of the CREB in the pathology of schizophrenia, as this could shed new light on the effective management of this psychiatric disorder. Furthermore, CREB regulates the transcription of multiple genes, and extensive efforts are required to identify their involvement in the pathogenesis of schizophrenia and their myriad of combinatory effects.αΔ mutant mice is homozygous mice with a targeted mutation in CREB; the α and Δ isoforms of CREB are knocked down in this animal model, whereas the CREB β variant is upregulated (αΔ mutant mice support that enhanced CREB expression and phosphorylation promote neurodevelopment (The CREB-mutant mice, especially conditional knockout mice, represent a useful tool for examining its role in mediating neuropsychiatric behaviors. The CREBegulated . The funegulated . Currentelopment , while delopment . CREB-muelopment . We propelopment . On the elopment .In summary, targeting CREB proteins might promote synaptic plasticity and neurodevelopment in CNS, which can be beneficial or delay the development of schizophrenia. However, we also would like to point out that the increased risk of suicide should be take into consideration when targeting CREB, as data from human postmortem suggest that the numbers of phosphorylated CREB-positive cells were increased in amygdala in subjects, who had died by suicide, while lithium could significantly decrease the phosphorylation of CREB levels in the same region . Hence, HW initiated the research topic and discussed the literature and wrote the draft manuscript. WZ performed the conceptional design and writing of the final manuscript. JX wrote some part of the review. PL and RQ contributed constructive suggestions and extensive language editing.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.


Single-crystal X-ray analysis revealed that the introduction of O atoms into the structure of Ti8Bi9 retains the space-group type P4/nmm. The oxygen site is located within a Ti4 tetrahedron (point group symmetry m2) that is vacant in the Ti8Bi9 crystal structure. The occupancy of this site is 0.25 (4), and the O—Ti distance is 1.8824 (11) Å.Single crystals of Ti In addition to the three Ti1 sites, each Ti1 site is surrounded by six Bi atoms at distances of 2.945 (4)–3.074 (5) Å, and by two Ti2 sites at a distance of 3.017 (2) Å. The O atom of Ti8Bi9O0.25 is located in the Ti14 tetrahedron at a site with symmetry m2 and with a site occupancy of 0.25 (4). The partial occupation by the O atoms changes the Ti1—Ti1 distances in the tetrahedron to 2.992 (2) and 3.1142 (19) Å, representing increases of 1.9% and 1.3%, respectively. The Ti1—Bi2 distance is also increased by 1.4%, although the changes in the Ti3—Bi and Ti4—Bi distances are both less than 0.4%.The crystal structure and atomic arrangement for TiÅ Table 1. The Ti33+ and O2− (1.91 Å) and Ti2+—O2− (1.845 Å), based on ionic radii of 0.67 and 0.605 Å for Ti3+ and Ti2+, respectively, in sixfold coordination, and an O2− radius of 1.24 Å in fourfold coordination  for Ti4+ (1.815 Å), Ti3+ (1.815 Å) and Ti2+ (1.734 Å) and B = 0.37  to 2.291 (3) Å, all of which exceed the value of 1.8824 (11) Å for O1—Ti1 in Ti8Bi9O0.25. The BVSs calculated for the O sites in Tiδ12–GaxBix3–O10 using the parameters for Ti3+ and Ti2+ were found to be in the ranges 2.18–2.21 and 1.87–1.89 v.u., respectively  Å is intermediate between the sums of ionic radii for Ti2 powder  and 1.5 mmol Bi powder  in an agate mortar and subsequent pressing into a pellet (Ø 6 mm) under atmospheric conditions. The pellet was placed in a Ta boat that was then transferred into a stainless-steel tube and sealed with a cap in an Ar-filled glove box . The sealed stainless-steel tube was heated to 1073 K at a rate of approximately 400 K h−1, maintained at this temperature for 10 h, and subsequently cooled to 723 K at a rate of 10 K h−1. Below 723 K, the sample was cooled to room temperature by shutting off the electric power to the heater of the furnace. The resulting sample was crushed and single-crystal fragments of Ti8Bi9O0.25 were extracted. A single crystal for XRD analysis was sealed in a glass capillary. The crushed sample was also analyzed by electron probe microanalysis . Only Bi, Ti and O were found in the bulk. The O concentration was greater than the expected values, indicating that some oxidation had occurred while transferring the specimens to the EPMA instrument. In addition to fragments with a Ti:Bi atomic ratio of approximately 8:9, some Bi-rich (>85%) portions and fragments with a Ti:Bi ratio of approximately 3:2 were also identified.A sample containing the title compound was prepared by combining 0.85 mmol Ti powder , 0.125 mol TiO8Bi9O0.25 were initially analyzed using the Ti8Bi9 model , which corresponds to the 2a site in the Ti14 tetrahedron. The O-atom occupancy of this site was refined to be 0.25 (4), resulting in a decrease in R[F2 > 2σ (F2)] from 0.045 to 0.020. For this site an isotropic atomic displacement parameter was considered.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901801188X/wm5456sup1.cifCrystal structure: contains datablock(s) I, Ti8Bi9O0.25. DOI: 10.1107/S205698901801188X/wm5456Isup4.hklStructure factors: contains datablock(s) I. DOI: 1863407CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Polynyas are sections of the polar ocean that remain relatively ice-free during winter, imparting significant physical and biological impact on the region. The North Water polynya (NOW) situated between Ellesmere Island and Greenland is the largest recurring Arctic polynya. Historically, the NOW forms every season when Arctic Ocean floes moving southward through Nares Strait become congested and form an ice arch that defines the northern border of the polynya. This blockage usually forms during winter and breaks down in spring. It is conjectured that the polynya is maintained by latent heat of fusion from the continuous formation of new ice as floes are swept southward from the ice arch by wind and ocean currents. Analysis of four decades of satellite imagery indicates a growing instability in the location of the ice arch, challenging previous models of polynya maintenance. A linear trend of the data indicates the number of days of Nares Strait blockage has decreased 2.1 days/year between 1979 and 2019 with wide interannual variations. Prior to 2007, ice arches blocked Nares Strait an average of 177 days/year compared to 128 days/year since that time. The overall trend of reduced ice arch duration is a contributing factor to the dramatic loss of multiyear ice in the Arctic basin. For a latent heat polynya, ice is removed from the area by winds and/or ocean currents as quickly as it forms. Heat loss to the atmosphere is balanced by latent heat of fusion released by the continual formation of ice. For a sensible heat polynya, oceanic heat provided by the vertical mixing of warmer water from depth due to wind-induced upwelling, or through advection from ocean currents, prevents the formation of ice. Polynyas impart significant oceanographic, climatic and biological impact on the environment. They dominate the regional heat budget in winter with an ocean-to-atmosphere heat flux approximately two orders of magnitude higher than the surrounding pack ice1. The continuous formation of ice in polynyas leads to salt rejection and the subsequent formation of water masses and local currents. Interannual changes in polynya characteristics may contribute to the variability of the polar climate, reflecting large-scale climatic changes2. Polynyas constitute key habitats for seabirds and marine mammals5, serving as areas of high productivity and biodiversity7. As a result, polynyas are considered oceanographic ‘windows’ that allow an evaluation of the state of the polar marine ecosystem9.Polynyas are areas of the polar ocean that are covered by open-water and thin ice under climatic conditions that would normally dictate thick ice cover. These regions are generally classified as 13. Historically, pack ice transported southward from the Arctic Ocean through Nares Strait becomes congested and forms a blockage, or ice arch, across the narrow head of Smith Sound. The formation of ice arches is common in the narrow water passages of the Canadian Arctic Archipelgo (CAA)14 and this process is considered a critical aspect of the NOW’s formation and maintenance20. The Smith Sound ice arch usually forms in winter or early spring at which point ice is continuously swept southward from the blockage by prevailing winds and ocean currents23. The subsequent formation of new ice releases latent heat, which is an important mechanism for maintaining the polynya24. Other factors contributing to keeping the area relatively free of ice include strong tidal fluctuations25 and potential sensible heat influx through the upwelling of warm water near the Greenland coast26. While the Smith Sound ice arch sharply defines the northern limit of the polynya  situated between Ellesmere Island and Greenland in northern Baffin Bay is the largest recurring polynya in the Canadian Arctic Fig.  and one nya Fig. , the sou−1 24. The strongest tides in the Canadian Arctic occur north of the NOW in Kane Basin with modeled amplitudes reaching 135 cm29, which can reverse the Nares Strait current and significantly change the ice dynamics on a diurnal basis25. On the eastern side, the relatively warm West Greenland Current (2.0 °C) flows northward along the Greenland coast with a surface current speed of 3 to 5 cm s−1 until crossing the southern portion of the NOW to join the Nares Strait outflow and form the southward moving Baffin Current24. Nares Strait, which averages 30 to 50 km in width and extends 500 km from the Arctic Ocean to the NOW, transports an annual average area of 40,000 km2 of ice from the Arctic Basin into Baffin Bay30 with multiyear ice contributing a significant proportion to this amount31. Multiyear ice with drafts exceeding 5 m constitute up to 16% of the observed sea ice traversing Nares Strait, draining the last reservoir of old multiyear ice in the Lincoln Sea north of Ellesmere Island32. The formation and duration of ice arches in the Nares Strait system impacts the rate of ice loss from this reservoir, which is part of a region that climate models predict will be the last to lose perennial ice cover in the Arctic Ocean known as the Last Ice Area33.The western part of the NOW is characterized by cold outflow (−0.6 °C) from Nares Strait, with a mean surface current speed ranging from 10 to 15 cm s34. The diverse ecological system of the NOW made it possible for the prehistoric Thule Inuit to settle and their descendants survive in the region since 1250 AD35. Historical references to the NOW extend back to the voyages of William Baffin from 1612 to 1618 in which he discovered a great “open sea” south of what is now known as Kane Basin36. In 1861 Isaac Hayes, an American Arctic explorer, wintered at the narrowest section of Smith Sound where his sailing progress was stopped by the ice arch37. Air ice reconnaissance in the 1950s and 1960s allowed the Smith Sound ice arch to be drawn on charts with some precision38. The first scientific journal to feature the NOW emphasized that the most striking aspect of the polynya was the “extreme” stability of the “ice bridge” that defines the northern border39. The consistency of the Smith Sound ice arch from 1968 to 1979 is illustrated by ice charts created during this era in which it averages a seasonal latitude of 79.2 ± 0.5°N.  and volume (254 km3) of Arctic Ocean ice to pass through Nares Strait in a one-year period31. In 2009 the Smith Sound ice arch failed to form again, but a different ice arch at the northern terminus of Nares Strait  wavelengths permitted accurate tracking of the annual evolution of the NOW ice arch. In recent years, satellite data has revealed atypical ice configurations in the NOW. In 2007 the Smith Sound ice arch failed to consolidate Fig. , which aait Fig.  preventeThe changing ice arch dynamics of the NOW impacts the state of multiyear ice in the Arctic Ocean as well as the delicate ecosystem of the region and the people who have come to rely on this Arctic oasis. This research analyzes the NOW ice arches using visible and thermal IR satellite imagery from 1979 to 2019. The location and duration of ice arches over the past forty years are investigated and trends in the data examined.41. NOAA satellites equipped with the AVHRR sensor have been monitoring the Earth continuously since 1978 with the latest version (NOAA-19) launched in 2009. Between 2006 and 2018 the European Space Agency launched three MetOp satellites  that also carry an AVHRR. Eighteen satellites in total have operated an AVHRR, six of which are in operation in 2019. The latest NOAA polar orbiting satellite (NOAA-20) launched in 2017 replaced the AVHRR with the Visible Infrared Imaging Radiometer Suite developed from NASA’s Moderate Resolution Imaging Spectroradiometer flown on the Aqua and Terra satellites. Table Advanced Very High Resolution Radiometry (AVHRR) imagery was chosen for this study owing to its long heritage, continuity, availability and ease of processing. The AVHRR sensor is capable of imaging the Earth in visible and thermal IR wavelengths with a spatial resolution of 1.1 km at nadir. The first AVHRR was launched in October 1978 on the Television Infrared Observation Satellite - Next Generation (TIROS-N), which was put into service by the National Oceanic and Atmospheric Administration (NOAA). The follow-on to TIROS-N was NOAA-6 in 1979, at which point the NOAA spacecraft took on numerical designations upon reaching operational statushttps://www.bou.class.noaa.gov/saa/products/welcome).All satellites equipped with an AVHRR are in low Earth polar orbits that pass over the Arctic approximately 14 times per day. The sensor has a swath of approximately 2,900 km, allowing the NOW region to be imaged five to seven passes in a 24-hour period. The 1.1 km spatial resolution at nadir degrades to about 8 km toward the edge of the swath but is still sufficient to define the ice arch structures. File formats offering the best spatial resolution are High Resolution Picture Transmission for the NOAA series and Full Resolution Area Coverage for the MetOp series. For earlier satellite images only Global Area Coverage format was available offering 4 km resolution at nadir. The reduced spatial resolution was found to be adequate for analyzing ice arch structures. All AVHRR data utilized in this study was retrieved on-line from NOAA’s Comprehensive Large Array-data Stewardship System for various AVHRR-equipped satellites from 1978 to 2019  and northern ice arch (head of Nares Strait) was determined for each season from 1979 to 2019 by visibly examining individual AVHRR images for each day. The term ‘season’ is used since the formation of an ice arch sometimes occurred in the previous calendar year of the ice arch break down. The darkness of Arctic winter requires the use of thermal IR imagery to determine ice arch formation, while visible wavelengths are superior for observing the break down of these structures. An ice arch was determined to be fully formed when it achieved the characteristic shape for the season, whereas the break down date was signified by the initial collapse of the arch structure. Figure et al. (2010) who used satellite synthetic aperture radar data to analyze ice flux though Nares Strait from 1997 to 2009. Discrepancies in the two datasets may be attributed to the sensor used, methods, temporal sampling and the different overall aim of the studies.Figures The following is a decadal and overall summary of the data in this study.During the 1980s the southern ice arch formed each year in a confined region just north of Smith Sound with an average latitude of 78.9 ± 0.2° Fig.  which is28.The 1990s experienced the lowest decadal continuous ice arch blockage of Nares Strait, averaging 126.5 days per season, with a range of 10 days (1993) to 229 days (1992). The location of the southern ice arch is more variable than observed in previous decades Fig.  and is h31. In 2008 the southern ice formed in the northern section of Kane Basin, approximately 100 km north of the normal position. This was followed by the formation of only the northern arch in 2009. This ice configuration, which lasted for 184 days, had never been observed before and led to a significant warm water anomaly in Baffin Bay in July primarily as a result of low ice coverage in the region40. The average continuous blockage of Nares Strait for the decade was 154.6 days per season, ranging from 0 days (2007) to 224 days (2005).Between 2000 and 2006 the southern ice arch showed great stability, forming at an average latitude of 78.8 ± 0.1°N Fig. . The norThe striking aspect of the 2010s is the wide spatial distribution of the ice arches Fig. . For thr2 value (0.104), which means that despite the downward trend of ice arch days it is statistically difficult to predict what will happen in any particular year in the future. The average continuous blockage of Nares Strait between 1979 and 2019 is 161 days per season. Prior to 2007 this value was 177 days compared to 128 days since 2007, which represents the beginning of an era of frequent ice arch anomalies.Figure 2 values for these trends are 0.039 and 0.112 respectively, indicating a low statistical significance for predicting the future as a result of the high variability of the data.The time of year for initial ice arch formation and the date of ice arch break down is shown in Fig. 31.Figure 28. The data suggests that other factors contributing to polynya maintenance play a more dominant role than the presence of the southern ice arch. For example, orographically channeled winds through Nares Strait accelerate through Smith Sound and lead to increased ice advection45. Additionally, the diurnal divergence and convergence of the pack ice in the NOW as a result of tides allow the escape of oceanic heat with the creation of leads in the ice25 while the formation of new ice during quiet tidal periods supply latent heat of fusion. Tides may also play a role in ice arch formation. For instance, during the consolidation of the 2009 northern ice arch, satellite data in this study showed a northward movement of floes in northern Nares Strait.Literature on the maintenance of the NOW indicates that the formation of the southern ice arch is a critical aspect of the polynya’s existence, but this does not appear to be the case. Since 2007 there have been five seasons when the southern ice arch did not fully consolidate, but the NOW has remained relatively ice free during all these years. Indeed, during the 2007 season when no ice arches consolidated in Nares Strait, the NOW reached record levels of winter polynya size48. Delays of up to 93 days for the southern ice arch formation were observed prior to 2009, but eventually Nares Strait would freeze completely once the southern ice arch formed. This leads to the conclusion that the freezing of Nares Strait is linked to the formation of the southern ice arch.The emerging prominence of the northern ice arch since 2009 is an indication of changing ice dynamics in the region. Historically, for years when both ice arches formed the southern ice arch would consolidate within approximately 30 days of the northern ice arch, which coincides with the amount of time it takes for a floe to traverse the length of Nares Straitin situ melting. These driving factors are affected by both large-scale and regional meteorological conditions. The trend toward younger, thinner ice in the Arctic basin makes it more vulnerable to melting as well as break up and export out of the region49. Arctic ice is generally transported southward through Fram Strait, the CAA and Nares Strait. The mean annual outflow through Fram Strait is 706,000 km2 50, which is significantly higher than yearly averages for Nares Strait 30 and the CAA 51. However, unlike Fram Strait the ice transported through Nares Strait is dominated by thick multiyear ice from north of Ellesmere Island and Greenland52. The Last Ice Area is losing ice mass at double the rate of the Arctic Ocean as a whole, which is augmented by reduced ice arch blockages in Nares Strait33. The ice arches have been observed to contain thick multiyear ice held together with thin younger ice31 and it is suggested that ice arches in the region are less stable in the absence of thick ice52. Consequently, the observed temporal and spatial variability of the Nares Strait ice arches may be a function of the thinning and depletion of the ice reservoir north of Ellesmere Island.The amount of Arctic Ocean ice loss during any particular year is complex, depending on the removal of ice by wind, ocean currents and 54 leading to a significant diminishment of ice in the Arctic Ocean over the past four decades. Physically and biologically, polynyas are considered a model for scientist to predict the Arctic system response to climate change9. After many years of stability, the iconic shape of the NOW as defined by the southern ice arch is no longer a certainty, reflecting the significant changes taking place in the Arctic icescape as a result of Arctic amplification. In 2009 the northern ice arch resulted in a virtually ice-free Baffin Bay in July, which led to unprecedented summertime sea surface temperatures largely as a result of decreased albedo and 24-hour solar insolation, affording insight to a seasonally ice-free Arctic Ocean40. Increased energy imparted to the ocean can lead to delayed winter freeze-up 55, while the additional sensible heat in the upper ocean results in enhanced heat transfer that may result in higher regional surface air temperatures56. The variability in the NOW ice arches since 2007 is a response to the warming climate. Continuing trends of anomalous ice arch location and diminishing duration will have an impact on the regional ecosystem and the storage of multiyear ice in the Last Ice Area.Arctic amplification is the observation that near surface air temperatures are increasing more rapidly in the Arctic than the global average


In developed countries, breastfeeding is not recommended for women living with human immunodeficiency virus (WLWH). However, lactation symptoms can be distressing for women who choose not to breastfeed. There is currently no universal guideline on the most appropriate options for prevention or reduction of lactation symptoms amongst WLWH. This review describes the evidence base for using cabergoline, a dopaminergic agonist, for the post‐partum inhibition of lactation for WLWH.A scoping review of post‐partum pharmaceutical lactation inhibition specific for WLWH was conducted using searches in PubMed, Medline Ovid, EBM Reviews Ovid, Embase, Web of Science and Scopus until 2019. A narrative review of cabergoline pharmacologic properties, therapeutic efficacy, tolerability data and drug interaction data relevant to lactation inhibition was then conducted.Among 1366 articles, the scoping review identified 13 relevant publications. Eight guidelines providing guidance regarding lactation inhibition for WLWH and two surveys of medical practice on this topic in UK have been published. Three studies have evaluated the use of pharmaceutical agents in WLWH. Two of these studies evaluated cabergoline and reported it to be an effective method of lactation inhibition in this population. The third study evaluated ethinyl estradiol and bromocriptine use and showed poor efficacy. Cabergoline is a long‐acting dopamine D2 agonist and ergot derivative that inhibits prolactin secretion and suppresses physiologic lactation when given as a single oral dose of 1 mg after delivery. Cabergoline is at least as effective as bromocriptine for lactation inhibition with success rates between 78% and 100%. Transient, mild to moderate adverse events to cabergoline are described in clinical trials. Few drug interactions exist as cabergoline is neither a substrate nor an inducer/inhibitor of hepatic cytochrome P450 isoenzymes. There are no reported clinically significant drug–drug interactions between cabergoline and any antiretroviral medications including protease inhibitors.Cabergoline is a safe and effective pharmacologic option for the prevention of physiological lactation and associated physical symptoms in non‐breastfeeding women. Future studies should focus on its safety, efficacy and acceptability among WLWH. No year restriction was imposed in the electronic search and the last updated search was performed on 12 February 2019. Studies were identified in the aforementioned databases using the following four groups of search terms: (1) “lactation disorder;” OR (2) “lactation” AND (3) “suppressing agent;” AND (4) “HIV.” These groups were combined and researched in the title, abstract and keywords of the studies published by the journals/documents indexed in the searched databases. Finally, a manual search was also conducted in: (a) the reference lists of the articles and (b) the reference list of the latest version of the US HIV perinatal guidelines 2.2The review was restricted to human participants and to publications in English or French. Original studies, case studies, book chapters, conference proceedings, were selected if they reported the use of drug for lactation inhibition for WLWH. Non‐original studies, such as guidelines and reviews, were selected if they provided guidance regarding lactation inhibition for WLWH.2.3As recommended by the PRISMA Statement 2.4The same two authors independently extracted the information from the selected articles. The following information was extracted: (a) location (country) and year of publication; (b) design ; (c) inclusion criteria; (d) sample size; (e) type/dose of lactation inhibition agent; (f) antiretroviral use; (g) efficacy and tolerance. The information and the data extracted were reviewed and discrepancies were resolved by discussion.3The selection process is illustrated in Figure 3.1We have identified eight guidelines or reviews which provide some guidance regarding how to support breastfeeding avoidance in WLWH. While as early as 1998, De Ruiter suggested that WLWH “may benefit from the administration of bromocriptine or cabergoline” In the 2009 and 2012 Cochrane reviews about lactation inhibition, there is a specific discussion on lactation inhibition to prevent HIV vertical transmission 3.2Only five studies are specifically focused on this issue; the summary of their findings is provided in Table One study from Malawi reported that among 98 WLWH, all would like cabergoline to be available routinely, suggesting that they were all satisfied by the treatment No pharmacokinetic data in WLWH were identified.4As there were no published data on cabergoline pharmacology specific to WLWH, an additional narrative review was conducted to identify potentially relevant studies through an electronic search in Pubmed, Medline Ovid and Lexi‐drug Interact using the search terms “cabergoline,” “lactation inhibition” and “drug interaction.” A manual search of the reference list of identified articles was also conducted. The literature search was restricted to English language studies conducted in humans and updated until 20 February 2019. A review of the Canadian and US cabergoline product monographs was also conducted.4.1Prolactin is a hormone that is synthesized and secreted from lactotroph cells on the anterior pituitary gland, and it is responsible for the synthesis and maintenance of milk secretion. Hypothalamic control of prolactin production and release is mediated by the tonic inhibition of dopamine. Dopamine agonists act on dopamine D2‐type receptors on pituitary lactotroph membranes, leading to a decrease in the synthesis and release of prolactin Based on animal data, cabergoline is an ergoline dopamine D2 agonist with low affinity for dopamine D1‐, α‐1 and 2‐adrenergic, and 5‐HT1 and 5‐HT2 serotonergic receptors (it does have 5‐HT2B agonist activity) max) and area under the concentration curve (AUC) parameters have been observed in severe hepatic insufficiency (Child–Pugh score > 10) Cabergoline pharmacokinetics has been established based on data in non‐pregnant healthy volunteers and parkinsonian patients. Cabergoline is rapidly and well absorbed with peak concentrations achieved at approximately two to three hours and is unaffected by food 4.2Based on pharmacokinetic data, it was expected that a single cabergoline dose would be sufficient to inhibit lactation Dose‐ranging studies A recent drug use evaluation study on cabergoline use for post‐partum lactation inhibition was conducted at a women's hospital in Qatar 4.34.3.1Cabergoline has been well tolerated in clinical trials when used as a single dose for inhibition of lactation with most reported side effects being transient and mild to moderate in severity Table , 48, 49.In the largest randomized controlled study comparing single dose cabergoline to bromocriptine, there were numerically fewer adverse events reported in the cabergoline versus the bromocriptine group Table , howeverThe Canadian and US product monographs list uncontrolled hypertension, a history of pulmonary, pericardial and retroperitoneal fibrotic disorders, history of cardiac valvulopathy and known hypersensitivity to cabergoline or any ergot derivative as contraindications to prescription When bromocriptine was used for the inhibition of lactation there were concerns regarding the occurrence of post‐partum psychiatric reaction or relapse 4.3.2Cabergoline's metabolism is independent of the hepatic CYP P450 enzyme system, and it does not induce or inhibit CYP P450 isoenzymes According to the cabergoline product monograph Two papers, describing a potential drug interaction between clarithromycin/cabergoline and itraconazole/cabergoline have been published. The first paper Because protease inhibitors are among the first‐line antiretroviral medications recommended for pregnant WLWH 4.4There are a few limitations that have been identified in our scoping review. Only papers and abstracts that were published in English or in French were included. This especially limited our ability to extensively review existing guidelines focusing on WLWH perinatal care. We were, however, able to review several European and North American guidelines. Secondly, as there was no data specifically describing cabergoline pharmacokinetics in WLWH and/or women receiving cART, our conclusions have been extrapolated from findings in healthy volunteers and/or patients with Parkinson's Disease.5In most high‐income countries, where safe alternatives to breast milk are available, breastfeeding is not recommended for WLWH. Unfortunately, there are very few data and recommendations regarding lactation inhibition in this population despite the important need to provide optimal management for prevention of physical lactation symptoms in non‐breastfeeding WLWH. The dopaminergic ergot derivative, cabergoline offers an important pharmacologic option to achieve this. Compared to bromocriptine, it has similar efficacy at inhibiting lactation with associated advantages of easier dosing, fewer overall and serious side effects and fewer drug interactions. Based on our literature review, no interaction with cART is anticipated. However, while survey results indicate that HIV providers in the UK are routinely using cabergoline, there is a lack of data about cabergoline safety and efficacy in WLWH on ART. Moreover, the acceptability of drug inhibition and the psychosocial impact of breastfeeding avoidance in this context need to be considered. An ongoing prospective study in Canada is evaluating its safety, efficacy and acceptability in WLWH. Results from this, and future trials will help to address existing gaps in the literature and potentially support its incorporation into perinatal HIV guidelines.The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take responsibility for the content, including participation in the concept, design, analysis, writing or revision of the manuscript. Conception and design of study: KT, IB, DM, PD, FTR. Acquisition of data: KT, IB. Analysis and interpretation of data: KT, IB, DM, CE. Drafting manuscript: KT, IB. Critical revision of manuscript: KT, IB, DM, CE, PD, FTR. All authors have read and approved the final manuscript.Data S1. Review strategy.Click here for additional data file.


Temperature profiles of sea ice have been recorded more than a few decades. However, few high-precision temperature sensors can complete the observation of temperature profile of sea ice, especially in extreme environments. At present, the most widely used sea ice observation instruments can reach an accuracy of sea ice temperature measurement of 0.1 °C. In this study, a multilayer sea ice temperature sensor is developed with temperature measurement accuracy from −0.0047 °C to 0.0059 °C. The sensor system composition, structure of the thermistor string, and work mode are analyzed. The performance of the sensor system is evaluated from −50 °C to 30 °C. The temperature dependence of the constant current source, the amplification circuit, and the analog-to-digital converter (ADC) circuit are comprehensive tested and quantified. A temperature correction algorithm is designed to correct any deviation in the sensor system. A sea-ice thickness discrimination algorithm is proposed in charge of determining the thickness of sea ice automatically. The sensor system was field tested in Wuliangsuhai, Yellow River on 31 January 2018 and the second reservoir of Fen River, Yellow River on 30 January 2018. The integral practicality of this sensor system is identified and examined. The multilayer sea ice temperature sensor will provide good temperature results of sea ice and maintain stable performance in the low ambient temperature.  Arctic sea ice has been a sensitive indicator of climate change and provided the details of the complex atmosphere–ice–ocean interaction, which may affect the global climate and cause extreme weather ,2,3. To In these contexts, this paper focused on designing a multilayer sea ice temperature sensor with higher accuracy used in the polar regions. The system composition of the proposed multilayer sea ice temperature sensor is shown in As shown in 2C serial interface connected to the microcontroller by one cable. The low-power microcontroller  is in charge of data processing by inverting sea ice thickness using a sea-ice thickness algorithm and correcting measured temperature using a temperature correction algorithm. The temperature measurement accuracy of the sensor system is improved to better than 0.01 °C. The time response of the sensor system is 200 µs. The FPGA  is controlled by the microcontroller and can control the switching of the working and sleep modes of the constant current sources and the 32-channel switches. The power supply circuit provides voltage grades for the sensor system. The block diagram of the multilayer sea ice temperature sensor is illustrated in The inner structure and the cross-sectional view of the thermistor string are schematically shown in 1, D2, D3 and D4, respectively. And the width of four layers is D5, D6, D7 and D8, respectively. The size parameters of the four layers are indicated in As can be easily seen, the thermistor string is composed of four layers from outside to inside: a heat-shrink soft tube layer, a hot-melt adhesive layer, an expoxy resin layer, and a FPCB. As shown in In the polar region, low temperature is the primary challenge for operation of automated equipment. Our multilayer sea ice temperature sensor may affect by its instability and inadaptability at the range of temperatures (−50 to 30 °C). However, we do not fully be aware of the low temperature dependence of the sensor. The potential temperature dependence of the multilayer sea ice temperature sensor needs to be assessed. We focus on the current accuracy and stability of REF200. It is important for the ADC circuit to be evaluated and assessed of the accuracy of AD conversion. The performance of the amplification circuit at low temperature is in evaluation simultaneously.The schematic diagram of the experimental setup of constant current source is shown in The experiment and evaluation results are showed in The schematic diagram of the experimental setup of ADC circuit is shown in Similar to the experiment of the constant current source, we put the ADC circuit into the high-low temperature test chamber with same temperature range. And an auxiliary test circuit is built to complete experiment by providing the adjustable input voltages which simulate the actual detection process. The input voltage generated by a nominal 5 V voltage reference chip and a potentiometer. The range of input is selected from 1 V to 3 V. We measured the output of the ADC circuit by the oscilloscope. At each input, the experiment was cover −50 to 30 °C range. The results in The results in 2 of the amplification circuit output is bigger than 0.999 at each measured temperature.The adjustable input voltages are generated by the auxiliary test circuit which is used to do the experiment of the ADC circuit. The range of input is selected from 0.2 V to 0.6 V which the interval of input is 0.1 V. We measured the output of the amplification circuit by the oscilloscope. Under the input voltage of 0.2–0.6 V, the output of amplification circuit has strong upward trend proportionately with the increase of the input. For each input, the RiceT, and resistance of PT1000 could be approximated as follows.As mentioned above, the evaluation reveals that low temperature performance of the sensor system maintained a strong stability at temperature range from −50 to 30 °C. However, we still need to develop an approach to correct and minimize the deviation of measuring temperature. The relationship between sea ice temperature, R(T) could be calculated by the output of the ADC circuit and the corresponding current ref(T)I, thus, Equation 1 could be described as follows.Since iceT and resistance of PT1000 could be converted to the relationship between sea ice temperature and the output of ADC out(T)V byThe relationship between sea ice temperature The schematic diagram of the experimental setup of temperature correction is shown in We put the whole thermistor string into the GDJS-series high-low temperature test chamber over a range of −50 to 30 °C and measured the accuracy of single point of temperature sensor. To simulate the sea ice temperature measurement site, the temperature sensor of the thermistor string was immersed in antifreeze in a stainless steel container. A high-precision measuring instrument of temperature, which is a resistance temperature detector (RTD), with accuracy 0.001 °C fixed next to the temperature sensor was used to synchronously collect and record temperature data in the test chamber. The thermistor string and RTD were immersed in antifreeze. The antifreeze has a very low freezing point and can provide a stable temperature field for temperature measurement experiments. In order to make the temperature of the antifreeze more stable, we installed a motor-controlled impeller at constant speed at the bottom of the vessel. During the experiment, the interval of measured temperature is set as 5 °C from −50 °C to 30 °C. The baffle of the test chamber was closed for reducing the heat exchange between the test chamber and the outside.2)a, C(T) can be calculated by directly measured temperature and the output of ADC circuit. The temperature measurement resolution of the thermistor string is 0.0001 °C and the sensitivity of the thermistor string is 0.1733 mV/ °C.The results of measured temperature of the thermistor string are shown in 2)a decreases approximately in a parabolic manner with increase of temperature (2)a are 788.76 at −50 °C and 786.72 at −10 °C, respectively. The temperature dependence of coefficient C(T) can be ignored. The value of the coefficient 2)a at the nonmeasured temperatures could be predicted by linear interpolation.The coefficient perature . The maxn points on the sea ice temperature profile.snowT, iceT, and oceanT are the temperatures of snow, ice, and ocean, respectively; snowμ, iceμ, and oceanμ are the expected values of temperature of snow, ice, and ocean, respectively; ke is the random error for every value; 1C and 2C are the locations of snow–ice and ice–ocean interfaces, respectively. Thus, the sea ice thickness can be calculated as follows.iceTH is the thickness of sea ice; R represents the vertical spacing of adjacent temperature values.The sea ice temperature profile observed by the multilayer sea ice temperature sensor includes a series of data that indicate the temperatures of air, snow, sea ice and ocean. Sea ice thickness can be calculated using the snow–ice interface and the ice–ocean interface . Based oC is the location of the interface; snow-iceT and ice-oceanT are the temperature value in a certain medium of snow or ice, and ice or ocean; snow-iceμ and ice-oceanμ and oceanμ are corresponding expected values and described as follows.The principle of the cluster theory is to take such an inflection point to minimize the sum of squares of the segment errors between the two subgroups which the inflection point is regarded as demarcation point, which target formula can be described as follows.kT  to get an inflection point 0C. Then a data set kT  is processed using the same method to get an inflection point 11C. For kT , we can obtain another inflection point 12C. Similarly, we use the Equation 6 to process kT  to get an inflection point 22C. Then a data set kT  is processed using the same method to get an inflection point 21C. So far we have got two sets of inflection points  and . 11C and 21C are potential snow–ice interfaces, and 12C and 22C are potential ice–ocean interfaces. As shown in 1 < E2E, 11C and 12C are two interfaces. Otherwise, 21C and 22C are two interfaces. The resolution of the calculated ice thickness is set 0.001 m.Accuracy and stability of the multilayer sea ice temperature sensor was tested and evaluated by a field experiment in Wuliangsuhai, Yellow River b. We desAs shown in The air temperature measured by the thermistor string from 1 February to 25 February is shown in The ice temperature measured by the thermistor string from 1 February to 25 February is shown in In The water temperature was presented a gradual upward trend. The maximum (minimum) temperature was 9.9 °C (6.29 °C), respectively. The average temperature was 7.7 °C. An increase in water temperature may accelerate the melting of the ice, resulting in a gradual decrease in ice thickness from 1 February to 25 February.Other instruments were installed near the thermistor string. However, the distance of temperature probes of the instruments differed from the thermistor string. The results of the comparison are shown in The multilayer sea ice temperature sensor was deployed in the second reservoir area of Fen River, Yellow River on 30 January 2018, as shown in A 4.5 m long thermistor string was designed and assembled 150 PT1000. The sensor spacing of the thermistor string was 0.03 m. The sensor system integrated an air temperature sensor , an air pressure sensor  and an Iridium module. The ice thickness was measured by drilled-hole once a day and two acoustic rangefinder sounders. The thermistor string and two acoustic rangefinder sounders were installed on the frozen lake ice. The field experiment lasted from 30 January to 15 February. The initial ice thickness was 0.29 m and there was no snow cover on ice. Water depth of installation position was 30.5 m. There were 25 temperature sensors exposed to the air, and others were fixed in the ice and water. The thermistor string was in charge of realizing temperature data collection every two hours. The air temperature and air pressure from 30 January to 15 February are shown in As shown in Typical temperature profiles are shown in Calculated ice thickness by algorithm (green lines), ice thickness measured by acoustic sounders (red lines), and ice thickness measured by drilled-hole (blue triangles) from 30 January to 15 February are shown in A multilayer sea ice temperature sensor has been designed in order to realize high-precision temperature measurement at the operating range of −50 °C to 30 °C. The sensor system composition, structure of the thermistor string, and work mode of the sensor system are depicted in detail. The multilayer sea ice temperature sensor includes a thermistor string for measuring sea ice temperature profile and a buoy, which is easy to be deployed and installed. The thermistor string is composed of four layers of a heat-shrink soft tube, a hot-melt adhesive, an expoxy resin, and a FPCB. We comprehensively evaluated low-temperature performances of this sensor system by experiments simulating low temperature in the polar regions. The experimental setups of the sensor system are built, which involves temperature dependence of the constant current source, ADC circuit and amplifier. The results of performance evaluation of sensor system indicate that the sensor system has excellent stability over −50 to 30 °C. Compared to traditional sea ice observation instruments, the sensor system has higher temperature measurement accuracy. And a correction algorithm based on the experiment model is developed to correct the biases to achieve temperature measurement error from −0.0047 °C to 0.0059 °C. Additionally, an automatic sea ice thickness discrimination algorithm is proposed. Field experiments were completed and the sensor system can be proved high precision and high stability in observation of ice temperature profile in Wuliangsuhai, Yellow River. During the field experiment in the second reservoir of Fen River, Yellow River, anomalies in temperature profiles were observed by the sensor system, which may be caused by releasing and storing water of dam. And successful observation of this phenomenon indicates that the sensor system has high sensitivity of measuring the multilayer ice temperature. Therefore, the multilayer sea ice temperature sensor is considered suitable for application of the multilayer temperature measurement of sea ice in polar regions.More design optimization can be realized to create a sensor system with better performance. In the future work, an integrated structure of the buoy will be designed and implemented, which the thermistor string will be integrated on the buoy, and the external cable can be removed. The length of the thermistor string can be further enlarged. For example, a 50 m long thermistor string can be designed to be in charge of measuring the ice cover temperature field in the Arctic Ocean and Antarctica. More sensors with different parameters, such as conductivity-temperature-depth (CTD) sensor, pressure sensor chain, etc, can be assembled into the sensor system to obtain a lot of data of sea ice and ocean. The sensor systems can be widely deployed and installed in the Arctic Ocean and Antarctica to improve the accuracy of temperature correction algorithm and the applicability of sea-ice thickness discrimination algorithm through in situ observation. The ice volume is calculated from the ice thickness and the sea ice concentration. Comparison of Satellite measurements (CryoSat-2) with some in-situ data sets reveals differences of less than 0.1 m in thickness . However


Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1  and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells , and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30–120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways , (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.Myosin regulatory light chain (LC H1152  with smooth muscle growth medium containing 5% FBS. Prior to treatment, cells were grown to 70% confluence and starved in FBS-free medium overnight. Cells were then stimulated with 5% FBS for times indicated in the figures  to observe their various responses to FBS. The treated cells were lysed immediately in Laemmli sample buffer and heated at 90 °C for 5 min. To investigate the effects of kinase inhibitors, cells were starved in FBS-free medium for 8 h to induce cell quiescence and then incubated with various inhibitors for ≥ 45 min prior to stimulation with 5% FBS for 2 min and then lysed in Laemmli sample buffer.Human coronary artery smooth muscle cells (CASMC: CC-2583) and human umbilical artery smooth muscle cells (UASMC: CC-2579), purchased from Lonza , were cultured in smooth muscle growth medium (Lonza SmGM-2: CC-3182) containing 5% fetal bovine serum (FBS), growth factors , gentamicin and amphotericin-B at 37 °C in a humidified incubator with 5% COCASMC and UASMC were transfected with human ROCK1, ZIPK or MLCK siRNAs or negative control siRNA (see below) using HiPerFect Reagent  according to the manufacturer’s protocol, as previously described in detail . Human R5'-GAGGAGUACUUCAGCAACAtt-3'sense: 5'-UGUUGCUGAAGUACUCCUCgt-3'.antisense: Human MLCK siRNA, purchased from Ambion/Thermo Fisher Scientific , has the following sequence:5'-GCCUCAUGUAAAACCCUAUtt-3'sense: 5'-AUAGGGUUUUACAUGAGGCtt-3'.anti-sense: Negative control siRNA  contains sequences that do not target any gene product.20 , rabbit polyclonal anti-pT853-MYPT1 , rabbit polyclonal anti-pT696-MYPT1 , rabbit polyclonal anti-pT163-Par4 , rabbit monoclonal anti-pS473-Akt (193H12) ; rabbit polyclonal anti-pT180/pY182-p38 MAP kinase , rabbit monoclonal anti-pT202/Y204-ERK1/pT185/pY187-ERK2 , rabbit anti-pS82-HSP27 , and rabbit polyclonal anti-α-actin . Polyclonal antibodies to purified, full-length chicken gizzard MLCK were raised in rabbits [Cells were lysed in Laemmli sample buffer  following treatments indicated in the Results section, heated at 90 °C for 5 min and resolved by SDS-PAGE (11% acrylamide). Proteins were transferred to nitrocellulose membranes in 25 mM Tris, 192 mM glycine, 20% methanol overnight at 30 V and 4 °C. Membranes were blocked with 5% non-fat dry milk in 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20 (TBST) for 1 h at 20 °C. Membranes were incubated for 2 h at 20 °C with primary antibody in 1% milk in TBST, washed, incubated with horseradish peroxidase (HRP)-conjugated goat polyclonal anti-rabbit IgG ; cat. no. AP132P) or donkey anti-goat IgG-HRP  in 1% milk in TBST and washed 3 times with TBST. Blots were developed with Supersignal West Femto enhanced chemiluminescence substrate (Thermo Scientific). Emitted light was detected and quantified with a chemiluminescence imaging analyzer  and images were analyzed with MultiGauge version 3.0 software (Fujifilm). Protein levels were expressed relative to a standard  as indicated in the text. The following primary antibodies were used in this study: rabbit monoclonal anti-ZIPK , rabbit polyclonal anti-ROCK1 , rabbit polyclonal anti-ROCK2 , rabbit polyclonal anti-pT18/pS19-myosin light chain 2 , rabbit anti-GAPDH , goat polyclonal anti-SM22α , rabbit polyclonal anti-LC rabbits .20 and ZIPK was analyzed by phosphate-binding tag SDS-PAGE [20 phosphorylation analysis, samples were resolved in SDS gels containing 12% acrylamide, 40 μM Phos-tag reagent and 0.1 mM MnCl2 at 20 mA/gel. Separated proteins were transferred to PVDF membranes  overnight at 28 V and 4 °C in 25 mM Tris-HCl, pH 7.5, 192 mM glycine, 10% (v/v) methanol. Proteins were fixed on the membrane by treatment with 0.5% glutaraldehyde in phosphate-buffered saline  for 30 min. For ZIPK phosphorylation analysis, samples were separated in SDS gels containing 6.5% acrylamide, 55 μM Phos-tag reagent and 0.1 mM MnCl2 (or 1–2 mM EDTA) at 20 mA/gel. Separated proteins were transferred to nitrocellulose membranes in 25 mM Tris-HCl, pH 7.5, 192 mM glycine, 20% (v/v) methanol at 28 V for 18 h at 4 °C. Membranes were then blocked with 5% non-fat dry milk in TBST for 1 h at 20 °C, washed with water and TBST and incubated for 2 h with primary antibody (anti-ZIPK) in Can Get Signal  prior to further washing in water and TBST and incubation for 1 h with secondary antibody in 1% (w/v) non-fat dry milk in TBST. Immunoreactive bands were detected with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).The phosphorylation of LCSDS-PAGE . For LC2n indicating the number of independent experiments performed. Statistically significant differences were determined by Student’s unpaired t-test or Dunnett’s post hoc test for multiple comparisons as indicated in the text and figure legends. Statistically significant differences were indicated by p < 0.05.Values are presented as mean ± SEM, with 20 phosphorylation in serum-starved human coronary and umbilical arterial smooth muscle cells by Phos-tag SDS-PAGE, which separates unphosphorylated, mono- and diphosphorylated LC20 species [i/mol LC20 (n = 7) in CASMC and 0.85 ± 0.02 mol Pi/mol LC20 (n = 6) in UASMC  in CASMC (i/mol LC20 (n = 6) in UASMC , not significantly different from the value at 2 min  (i/mol LC20 (n = 6), significantly lower than the value at 2 min   to steady-state levels significantly above resting values .Analysis of LC species , revealein CASMC  and 1.33in UASMC  at 2 min t-test) , whereas t-test) . Analysith CASMC  and UASMth CASMC  in respog values ; this isAnalysis of MYPT1 phosphorylation at T696 and T853 by western blotting with phosphospecific antibodies revealed a low basal level of phosphorylation at both sites under serum-free conditions in CASMC  and UASMAnalysis of Par-4 phosphorylation at T163 by western blotting with phosphospecific antibodies revealed a low basal level of phosphorylation under serum-free conditions in CASMC  and UASM20 phosphorylation, specifically the kinases Akt/PKB, ERK1/2 and p38 MAPK as well as the kinase substrate, heat shock protein HSP27. PI3K/Akt signaling in VSMCs is involved in the etiology of atherosclerosis [We also investigated the time courses of serum-induced phosphorylation events distinct from the signaling pathways involved in LCclerosis ,37. PhosThe ERK MAPKs, ERK1/p44 and ERK2/p42, are activated by a wide variety of growth factors and mitogens via phosphorylation at the T and Y residues (T202 and Y204 in ERK1 and T185 and Y187 in ERK2) within the activation loop TEY sequence . ERK1 anp38 MAPK has also been implicated in cell migration through activation by several growth factors, cytokines and chemotactic substances  and is aThe small heat shock protein HSP27 acts as an ATP-independent chaperone in protein folding, has been implicated in cell migration and cytoskeletal architecture, and is regulated by phosphorylation at multiple sites , includiTransfection with siRNA was used to investigate the involvement of MLCK, ROCK and ZIPK in serum-induced phosphorylation events. Transfection of CASMC with siRNA targeting MLCK reduced MLCK protein to ~55% of control levels (cells transfected with negative control siRNA) but had no effect on ROCK1 or ZIPK levels .Transfection of CASMC and UASMC with siRNA targeting ROCK1 reduced ROCK1 protein levels to ~27 and ~22% of control (cells transfected with negative control siRNA), respectively , confirmTransfection of CASMC and UASMC with siRNA targeting ZIPK reduced ZIPK protein to ~55% and ~50% of control levels (cells transfected with negative control siRNA), respectively , confirm20 phosphorylation following serum stimulation, we first examined the levels of smooth muscle MLCK in CASMC and UASMC. As shown in 20 diphosphorylation at T18 and S19 in response to serum treatment for 2 min, as shown by SDS-PAGE and western blotting with anti-2P-LC20 -induced contraction of de-endothelialized rat caudal arterial smooth muscle, as described previously [The MLCK inhibitors, ML7 and wortmannin, also had no significant effect on LCd by ML7 . Phos-tae of ML7 . Likewisrtmannin , which wrtmannin . In conteviously . We also2+ with EGTA to remove extracellular Ca2+ and deplete intracellular stores had no significant effect on serum-induced LC20 phosphorylation at T18 and S19. From western blotting with anti-diP-LC20, there was no significant difference between the normalized signal intensities for diP-LC20 from cells treated with serum in the presence of Ca2+ or EGTA  had a pronounced inhibitory effect on serum-induced LCnd UASMC . Phos-tand UASMC . ROCK innd UASMC . Similarnd UASMC .20 diphosphorylation was reduced to ~60% of control in CASMC and UASMC  at T696 and T853  resulting in decreased phosphatase activity ,48,65–70traction . While ttraction .2+ [ZIPK (DAPK3) is a member of the death-associated protein kinase (DAPK) family, which includes DAPK1, DAPK2, DAP-related protein kinase 1 (DRAK1) and DRAK2 ,75. Unli2+ . Seven p2+ . Phospho2+ . ROCK al2+ ,77–79, ain situ that contribute to cardiovascular diseases such as atherosclerosis [20 diphosphorylation and the kinases responsible, directly and indirectly, for its phosphorylation. We used two experimental approaches to investigate ROCK and ZIPK as likely candidates in the diphosphorylation of LC20 as well as MYPT1 and Par-4 phosphorylation in response to serum treatment: (i) siRNA-mediated knockdown of ROCK and ZIPK, and (ii) inhibition of kinase activity by well-characterized and selective inhibitors (GSK429286A and H1152 for ROCK and HS38 for ZIPK).Arterial smooth muscle cells undergo dedifferentiation when cultured, which involves transition from a non-proliferative, contractile phenotype to a proliferative, motile phenotype . These cclerosis . The majclerosis , with an20 at T18 and S19  that directly phosphorylates LC20 with the concomitant inhibition of MLCP activity. Furthermore, LC20 phosphorylation at both T18 and S19 reduces the rate of LC20 dephosphorylation by MLCP compared to that of LC20 phosphorylated exclusively at S19 [20.Exposure of serum-starved cells to serum resulted in the rapid phosphorylation of LCS19 Figs  and of PS19 Figs  were obsy at S19 . This he20 was MLCK itself. While S19 is the site of phosphorylation responsible for smooth muscle contraction, MLCK is well known to be capable of phosphorylating T18 as well [in vitro with purified MLCK at supra-physiological concentrations relative to the LC20 substrate [in situ and under physiological conditions MLCK does not phosphorylate T18 [20 phosphorylation in CASMC and found that: (i) western blot analysis confirmed the expression of smooth muscle MLCK in CASMC  ML8 or S19 , althoug8 or S19 , (iii) t8 or S19 , and ; n = 9 (B)). Significant differences from the value at time zero are indicated with their respective p values (Dunnett’s post hoc test).Human coronary (A) and umbilical arterial smooth muscle cells (B) were treated with serum at time zero, as described in the legend to (PDF)Click here for additional data file.S2 Fign = 8). Significant differences from the value at time zero are indicated with the actual p value or *p < 0.0001 (Dunnett’s post hoc test).Human coronary arterial smooth muscle cells were treated with serum at time zero, as described in the legend to (PDF)Click here for additional data file.S3 Fign = 9). Significant differences from the value at time zero are indicated with their respective p values (Dunnett’s post hoc test).Human umbilical arterial smooth muscle cells were treated with serum at time zero, as described in the legend to (PDF)Click here for additional data file.S4 Fign = 7). Statistically significant differences from the value at time zero are indicated with their respective p values (Dunnett’s post hoc test). No statistically significant differences were detected in panel D.Human coronary  and umbilical arterial smooth muscle cells  were treated with serum at time zero, as described in the legend to (PDF)Click here for additional data file.S5 Figpost hoc test.CASMC were serum starved overnight in the presence of H1152 (1 μM), GSK429286A , wortmannin (1 μM) or vehicle (control). Cells were lysed in Laemmli sample buffer for SDS-PAGE and western blotting with anti-pS473-Akt. Representative western blots are shown in panel A with cumulative quantitative data in panel B. Statistical analysis was carried out with Dunnett’s (PDF)Click here for additional data file.