G protein-coupled receptors (GPCRs) represent the largest family of therapeutic targets for FDA approved drugs. Therefore, understanding the molecular regulation of their signaling pathways is of paramount importance. Similarly, the mitogen activated protein kinase (MAPK) p38 is a critical mediator of proinflammatory disease. Yet despite decades of intense investigation, therapeutically viable inhibitors have struggled to make it into the clinic. New studies describing the regulation and activation of a GPCR dependent atypical p38 signaling pathway represents a novel therapeutic avenue to the treatment of many proinflammatory disorders. These recent studies have defined how thrombin and ADP can induce Src dependent activation of the E3 ubiquitin ligase NEDD4-2. Src dependent phosphorylation of a 2,3-linker peptide releases NEDD4-2 auto-inhibition and triggers the induction of proinflammatory atypical p38 signaling from the endosome. Activation of the atypical p38 pathway requires the direct interaction between an adaptor protein TAB1 and p38, that bypasses the requirement for the classical MKK3/6 dependent activation of p38. Therefore, providing a mechanism to specifically block proinflammatory GPCR atypical p38 activation while leaving basic p38 activity intact. Critically, new studies demonstrated that disruption of the TAB1-p38 interface is a druggable target, that would enable the selective inhibition of proinflammatory p38 signaling and ischemic injury. Atypical p38 signaling is linked to multiple clinically relevant pathologies including inflammation, cardiotoxicity, myocardial ischemia and ischemia reperfusion injury. Therefore, GPCR induced endosomal p38 signaling represents a novel understudied branch of proinflammatory p38 signaling and an ideal potential therapeutic target that warrants further investigation. There are over 800 G protein–coupled receptors (GPCRs) expressed throughout the body, playing critical roles in many physiological functions ranging from the control of taste and sight to the regulation of neuronal function and inflammatory responses. GPCRs are sensors for extracellular stimuli, and as such are activated by a wide array of ligands to induce intracellular signaling cascades, principally through the activation of the heterotrimeric G proteins. It is perhaps not surprising then that dysregulation in their expression or activities are lead causes in the onset and progression of many human diseases including hypertension, cancer and chronic inflammatory diseases such as rheumatoid arthritis and chronic obstructive pulmonary disease  domain containing E3 ubiquitin ligases. Additionally, there are the two smaller families, the E6-AP carboxy terminus (HECT) domain containing E3 ligases and the ring-between-ring ligases. The RING E3s act as adaptor molecules to bridge the substrate and the E2 ligase to facilitate substrate ubiquitination. Whereas, the HECT domain ligases contain a conserved cysteine residue required for the formation of a thioester bond with the C-terminus of ubiquitin and catalyzing substrate ubiquitination  p38 signaling pathway by GPCRs represents a new use of GPCR ubiquitination, while providing fundamental insight into the regulation of p38 signaling  ubiquitin binding domain, shown to specifically bind mono-ubiquitinated proteins , which in turn activate p38 via phosphorylation of the active loop  mediated activation of c-Src (An emerging concept in understanding how the catalytic activity of the NEDD4 family E3 ligases are regulated is that intramolecular interactions can hold the E3 ligases in inactive/inhibited confirmations that can only be released by phosphorylation or accessory protein binding. An example of this is where serine phosphorylation of NEDD4-2 (Debonneville et al., of c-Src . Activatof c-Src . Importaof c-Src , but notof c-Src . Releaseof c-Src . In a seof c-Src , howeverof c-Src . It is tof c-Src . While iof c-Src .1. Atypical p38 signaling has been shown to be critical for the induction of endothelial barrier permeability in vitro and vascular leakage in vivo, and is likely a major contributor to GPCR dependent tissue edema through regulation of endothelial cell adherens junctions and actin cytoskeleton remodeling (Grimsey et al., Thrombin and ADP rapidly induce the activation of ubiquitin-dependent proinflammatory p38 signaling by activation of PAR1 and P2YThe discovery of the ubiquitin driven endosomal signaling pathway was facilitated by the identification of the ubiquitin-independent lysosomal sorting of PAR1 (Dores et al., in vitro kinase assay (De Nicola et al., Targeting of the atypical p38 signaling pathway represents a novel, presently understudied therapeutic approach for the regulation of proinflammatory p38 signaling. The interaction between p38 and TAB1 is specifically required for the amplification of atypical p38 signaling (De Nicola et al., In summary, it is clear that GPCRs can induce a proinflammatory p38 signaling pathway as they undergo endocytic trafficking, with induction of this pathway being mediated by GPCR induced c-Src dependent tyrosine phosphorylation of the E3 ligase NEDD4-2. While significant steps have been made into defining the molecular regulation of this pathway, further investigations are critical and are likely to reveal novel therapeutic targets. Furthermore, peptide inhibitors or small molecules targeting the TAB1-p38 interaction offer a potentially paradigm shifting approach to the treatment of chronic proinflammatory diseases.JB and NG wrote and edited the manuscript. NG created the figure.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.


Several studies suggest that HTLV-1 infection may be associated with a wider spectrum of neurologic manifestations that do not meet diagnostic criteria for HAM/TSP. These conditions may later progress to HAM/TSP or constitute an intermediate clinical form, between asymptomatic HTLV-1 carriers and those with full myelopathy. Our aim was to determine the prevalence of HTLV-1-associated disease in subjects without HAM/TSP, and the relationship between these findings with HTLV-1 proviral load (PVL). Methods: 175 HTLV-1-infected subjects were submitted to a careful neurological evaluation, during their regular follow up at the HTLV outpatient clinic of the Institute of Infectious Diseases “Emilio Ribas”, São Paulo city, Brazil. Clinical evaluation and blinded standardized neurological screening were performed for all the subjects by the same neurologist (MH). Results: After the neurological evaluation, 133 patients were classified as asymptomatic and 42 fulfilled the criteria for intermediate syndrome (IS). The mean age of the enrolled subjects was 46.3 years and 130 (74.3%) were females. Clinical classification shows that neurological symptoms (p<0.001), visual disorders (p = 0.001), oral conditions (p = 0.001), skin lesions (p<0.001), bladder disorders (p<0.001), and rheumatological symptoms (p = 0.001), were strongly associated to IS, except for disautonomy (p = 0.21). A multivariate analysis revealed that HTLV-1 proviral load, oral conditions, bladder disorders and rheumatological symptoms were independently associated with the IS. Conclusions: We found some early alterations in 42 patients (24%), particularly the presence of previously not acknowledged clinical and neurological symptoms, among subjects previously classified as "asymptomatic", who we reclassified as having an intermediate syndrome. At least 5–10 million people live with the Human T-Cell Lymphotropic Virus type 1 (HTLV-1) worldwide, and around 0.25–5% of them may develop HTLV-1-associated myelopathy/Tropical spastic paraparesis (HAM/TSP), which is associated with chronic inflammation. In this study, involving 175 HTLV-1-infected subjects originally classified as asymptomatic, we found that 42 of them in reality presented some early clinical conditions, including alterations related not only to the neurological system, but also to the eyes and the skin. We called such conditions intermediate syndrome. Thus, it seems reasonable to suggest that all HTLV-1-infected subjects should be monitored for symptoms that may arise earlier in the course of their infection. HTLV-1, a human retrovirus, is the causative agent of Adult T Leukemia/Lymphoma (ATLL) and HTLV-1-associated myelopathy (HAM/TSP). HoweverSeveral neurological manifestations that are not explained by myelopathy have been described in so-called symptomatic persons, such as peripheral polyneuropathy, myositis, dysautonomia and cognitive alterations, as well as cranial neuropathies, movement disorders and an amyotrophic lateral sclerosis (ALS)-like syndrome . DespiteIn addition, peripheral neuropathy is significantly more frequent in the seropositive group. In a study with 153 HTLV-1-infected carriers, the presence of higher frequency of motor and bladder dysfunctions in HTLV-1 patients as compared with uninfected control subjects was found . Those dThis study aims to demonstrate that some clinical conditions, neurological finds and HTLV-1 proviral load may be associated with further development of full-blown HAM/TSP, in individuals considered free of the disease according to currently used criteria for its diagnosis. To do so we studied patients from a large cohort of asymptomatic HTLV-1 carriers who have been followed for more than twenty years.Our cohort has 659 HTLV-1-only infected subjects, with its inception dating back to 1997 . It is aClinical evaluation and a standardized screening neurological examination were performed by MH  for all subjects. Only symptoms/signals already associated with HTLV-I infection in previous reports were considered, and they should have no other clinical explanation. Each patient had at least one neurological/clinical evaluation, and a standardized questionnaire was used, with separate questions for clinical and neurological aspects .HAM/TSP diagnostic criteria was based on recommendations from an international consortium . BrieflyProbable HAM/TSP was defined by a monosymptomatic presentation: spasticity or hyperreflexia in the lower limbs or isolated Babinski sign with or without subtle sensory signs or symptoms, or neurogenic bladder only confirmed by urodynamic tests. For both definite and probable definitions, clinicians must exclude an array of disorders that can mimic HAM/TSP.We described a possible intermediate state of HAM not fulfilling the classical definition (16). To be considered as an intermediate syndrome case the patient must present more three signs, found during a neurological evaluation, with the investigator blinded to the patient’s HTLV status.For this present study, only clinical findings previously associated with HTLV-1 were considered, such as dermatological, ophthalmological, rheumatological, urinary, disautonomic, and oral changes . NeuroloA large clinical and laboratory database has been organized on an internet based platform using REDCap, software developed at the Vanderbilt University by an informatics core. All clinical data, which have been updated on a regular basis over the last 20 years, were entered into a specific REDCap database .4 peripheral blood mononuclear cells (PBMCs), as described elsewhere [HTLV-1 proviral load was quantified by real-time PCR, using primers and probes targeting the pol gene: SK110 and SK111, the internal HTLV-1 Taq Man probe was selected using Oligo . All samples were run in duplicate, and results expressed as HTLV-1 DNA copies/10lsewhere .The Ethical Board of the IIER approved the protocol (Number 86379218.6.1001.0061). We obtained signed informed consent from all participants prior to study inclusion, and all participants were adults.Statistical analysis was conducted using Student’s t-test for parametric data, and the chi-square test for proportions. Bivariate logistic analysis was performed to identify independent variables associated with the intermediate syndrome (IS). Variables associated with the outcome at a significance level of p<0.20 (IS) in the bivariate analysis were included in a multivariate logistic model, in a stepwise forward fashion. Such variables were: visual symptoms, skin lesions, oral conditions, bladder disfunction, and rheumatological conditions. The best fitting model was selected. The logistic analysis was performed with the aid of Stata 12 software .We enrolled 175 HTLV-1 patients on this study and classified them as having or not criteria for the diagnosis of the intermediate syndrome. Based on a thorough neurological examination, 42 patients met the criteria for making the diagnosis of the intermediate syndrome, whereas 133 did not and were called “asymptomatic” (not having the intermediate syndrome). All of them had intermediate symptoms that were classified as probable HAM/TSP at entry, primarily neurogenic bladder confirmed by urodynamic study. On a multivariate model analysis, including gender, age, and PVL and several clinical conditions, such as oral conditions, bladder disorders and rheumatological symptoms were independently associated with SI outcome. In this same model, all these variables and age were also significantly associated with the outcome, when included as continuous variables .This study aimed to define the early neurological disorders that can be present in HTLV-1-infected subjects. We found 24% of HTLV-1-infected patients from our outpatient service who were initially considered asymptomatic to have enough signs and symptoms putting them on a novel category, called intermediate syndrome. The correlation between some of their symptoms and the proviral load also reinforces the importance of such mild forms, which may constitute either an independent clinical intermediate syndrome or markers for an early diagnosis of HAM/TSP. A significantly PVL was also present in patients presenting three or more symptoms or signs.HAM/TSP is a chronic progressive myelopathy characterized by bilateral pyramidal tract involvement with sphincter disturbances. Why only a small proportion of HTLV-1-infected individuals develops classical HAM/TSP is not known . The maiWe and others have shown a correlation between proviral load and Tax gene expression with the presence of HAM/TSP ,19,20. WPerhaps the explanation for the observed neurological findings lies in the white matter. The white matter present in the CNS has the important function of transporting neural signals from subcortical regions to the cortex and from the cortex to the subcortical regions. In the CNS, ischemic and traumatic injuries often result in significant functional deficit, most of which can be attributed to white matter dysfunction –23. In tWe found that HTLV-1 infection is associated with a variety of clinical manifestations occurring in patients who either do not have or who did not have developed full HAM/TSP yet. The correlation between some of their symptoms and the proviral load also reinforces the importance of such milder forms, which may constitute either an independent clinical SI or markers for an early diagnosis of HAM/TSP. A significantly higher proviral load was present in patients presenting more than three symptoms/signs, a cut-off point that can constitute a surrogate marker for clinical progression.In conclusion, this preliminary report identified the presence of some clinical and neurological symptoms, in subjects classified originally as "asymptomatic," which may be promising markers for early HAM/TSP progression. This knowledge may contribute for a stricter clinical vigilance of the so-called asymptomatic HTLV-1 positive patients, prompting the introduction of a treatment for the infection, if and when it becomes available in the future.S1 Checklist(DOC)Click here for additional data file.


Using both data mining and network pharmacology methods, this paper aims to construct a molecule-target-disease network for medicines used for treating mastitis, mine out targets, and signaling pathways related to mastitis and explore the mechanism of Chinese materia medica (CMM) prescriptions in treating mastitis. A total of 131 CMM prescriptions for treating mastitis were collected from clinical practice and related literatures. A database of prescriptions for treating mastitis (DPTM) was then constructed. Based on data mining method, Traditional Chinese Medicine Inheritance Support System (TCMISS) was employed to mine out high-frequency CMM and key CMM combinations in DPTM. Subsequently, TCM Systems Pharmacology Database and Analysis Platform (TCMSP) and Traditional Chinese Medicine Information Database (TCM-ID) were searched for the targets of ingredients of high-frequency CMM. Then, Bioinformatics Analysis Tool for Molecular Mechanism of TCM (BATMAN-TCM) was searched for diseases and signaling pathways corresponding to the targets of key CMM combinations. The obtained results were denoted as results 1. In addition, human disease database MalaCards was searched for targets and signaling pathways related to mastitis. The obtained results were denoted as results 2. Results 1 and 2 were compared to obtain targets and signaling pathways included in both results, namely, mastitis-related targets of TCMs and mastitis-related signaling pathways that CMM involves in. Then, the biological functions of these targets and signaling pathways were investigated, on which basis the mechanism of CMM prescriptions in treating mastitis was explored. etc. were CMM with the highest frequency of use for treating mastitis. The potential targets of these high-frequency CMM in treating mastitis were intercellular adhesion molecule 1 (ICAM-1), interleukin-6 (IL-6), lipopolysaccharide binding protein (LBP), and lactotransferrin. The potential signaling pathways that key CMM combinations may involve in during mastitis treatment were NF-κB signaling pathway, immune system, PI3K/Akt signaling pathway, and TNF signaling pathway. A total of 12 key TCM combinations were identified. Taraxaci Herba, Glycyrrhizae Radix et Rhizoma, Paeoniae Radix Alba, semen citri reticulatae, From a perspective of network pharmacology, molecule-target-disease analysis may serve as an entry point for the research of mechanism of CMM. On this basis, we studied the mechanism of CMM prescriptions in treating mastitis by data mining and comparison of results. Our work thus provides a new idea and method for studying the working mechanism of CMM prescriptions. Zhenjiu Jiayi Jing  written by Huangfu Mi of the Jin Dynasty [ Mastitis is a disease commonly occurring in lactating women. The most frequent type of mastitis is acute suppurative mastitis with clinical symptoms of lumps in breast, swelling, pain, fever, and pus oozing out. In traditional Chinese medicine (TCM), acute mastitis is called “breast carbuncle”. This name was first seen in the book Dynasty . Since tFor the compatibility of medicines in CMM prescriptions, a “monarch-minister-assistant-messenger” rule should be followed. Various CMMs are used in combination to treating imbalance and disorders in the body. This is because the use of single CMM can hardly achieve high therapeutic efficacy, which indeed illustrates the idea of “multicomponents, multitargets, and systematic regulation” in TCM theory. Previous researches mainly attempt to explain the pharmacology of CMMs on the basis of the drug activity of single molecule and the effect of single target, which often fail to completely explain the working mechanism of CMMs.With the introduction of systems biology and the application of bioinformatics, network pharmacology is also proposed. Based on the interaction among diseases, genes, targets, and medicines, network pharmacology enables to comprehensively investigate the effects of medicines on diseases. If key CMM combinations  for treating mastitis are mined out, then a “key CMM combination-target-disease” network can be constructed. Subsequently, signaling pathway enrichment analysis of targets can be performed. Then, the mechanism of multiple compounds in the cooperative treatment of mastitis can be explained from the perspective of network pharmacology. This method agrees with the idea of holistic medicine and intuitively illustrates the mechanism of multisystem regulation in TCM. It also constructs a bridge between traditional Chinese medicine and western medicine since it enables investigating CMM prescriptions from a perspective of target-disease relationship, which is highlighted in western medicine.TCM prescriptions for treating mastitis were collected from clinical practice and related literatures and then a database of prescriptions for treating mastitis (DPTM) was constructed. On the basis of data mining method, Traditional Chinese Medicine Inheritance Support System (TCMISS) was employed to mine high-frequency CMMs and key CMM combinations in DPTM. Then, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Traditional Chinese Medicine Information Database (TCM-ID) were searched for the targets of high-frequency CMM. Later, Bioinformatics Analysis Tool for Molecular Mechanism of TCM (BATMAN-TCM) was searched for diseases and signaling pathways corresponding to the targets of key CMM combinations. The obtained results were denoted as results 1. Furthermore, human disease database MalaCards was searched for the targets and signaling pathways related to mastitis. The obtained results were denoted as results 2. Results 1 and 2 were compared to obtain the targets and signaling pathways included in both results, namely, mastitis-related targets of CMM and mastitis-related signaling pathways that CMM involve in. Then, the biological functions of these targets and signaling pathways were identified, on which basis the mechanism of CMM prescriptions in treating mastitis was explored. A flow chart is shown in http://www.ncbi.nlm.nih.gov) and CNKI (http://cnki.net/) with “mastitis” and “Chinese materia medica” as two keywords. Then, a total of 45 prescriptions for treating mastitis were collected from the papers. The above prescriptions were combined and after the same prescriptions were excluded, a total of 131 prescriptions were collected, on which basis DPTM was constructed.DPTM consists of two kinds of prescriptions: prescriptions used in clinical practice for treating mastitis and prescriptions from related literatures for treating mastitis. First, from January 2018 to May 2018, all prescriptions for treating diseases in both outpatients and inpatients in a provincial-level breast specialist hospital were collected. Among them, prescriptions for treating mastitis were screened out. After the same prescriptions were excluded, a total of 98 prescriptions for treating mastitis were collected. Second, we searched for papers in PubMed , KEGG pathway, and OMIM/TTD disease enrichment analyses. CMM ingredient-target-pathway/disease association network and biological pathways in which CMM's targets are significantly enriched were also shown.The composition of CMM is very complicated. The number of targets of CMM ingredients and the number of signaling pathways that they involve in are very large. In order to screen out highly associated targets and signaling pathways, their association with mastitis should be considered. In this paper, MalaCards was searched for targets and signaling pathways related to mastitis. If CMM can also act on the same targets or signaling pathways, then these targets or signaling pathways are taken as highly associated targets and signaling pathways. In this way, the slightly relevant and irrelevant targets and signaling pathways can be excluded Figures  and 3. FTCMISS was employed to statistically analyze the frequencies of all CMM in DPTM. DPTM includes 131 CMMs and their frequencies are shown in Association rules mining of DPTM was performed when support was set to ≥ 26 and confidence was set to ≥ 0.9 in TCMISS. The aim of association rules mining was to find frequent item sets, namely, key CMM combinations frequently appearing in the datasets. After repeated items were removed, 12 key CMM combinations for treating mastitis were obtained .MalaCards was searched with “mastitis” as the keyword for targets and signaling pathways related to mastitis. The results are shown in Tables After the 11 CMMs with the highest frequencies were input one by one into TCMSP and HIT (it is used to supplement the information of CMM which is unrecorded in TCMSP), the ingredients and their targets of 11 CMMs were obtained. The obtained targets were compared with those related to mastitis found in MalaCards. The same targets were screened out and taken as the mastitis-associated targets of CMM for treating mastitis . NotablyICAM-1 is a kind of membrane glycoprotein that participates in the interaction between cells or between cells and extracellular matrices . During  etc. [Interleukin (IL) is a kind of cytokine that is secreted by certain cells and has an effect on other cells. IL plays an important role in information transfer, activation and regulation of immune cells, activation, proliferation and differentiation of T and B cells, as well as inflammatory response. IL-6, as a member of interleukin family, mainly plays a role in the proliferation of B cells and antibody secretion, proliferation of T cells and CTL activation, formation of acute phase proteins by liver cells, inflammatory response, etc. . The mecLBP is a kind of glycoprotein existing in human and animal serum. LBP has a high affinity with lipoid A in lipopolysaccharide (LPS). It can function as a LPS carrier protein, catalyze the binding of LPS to CD14, stimulate monocytes and endothelial cells, and promote the release of inflammatory mediators such as TNF. LBP can also function as an opsonin, promoting monocytes to engulf conditioned LPS and gram-negative bacteria; thus LBP can regulate inflammatory response induced by LPS . The pos etc. [Lactotransferrin is a natural glycoprotein with immune functions that exists in breast milk. Its physiological functions include iron absorption promotion, immunomodulation, antibacterial, and antiviral effects, etc. . The mecBATMAN-TCM was employed to perform molecule-target-disease analysis of the 12 key CMM combinations. Diseases related to mammary gland and mastitis were screened out. Results show that each key CMM combination is related to two to four of the diseases including inflammation, inflammatory disease, breast cancer, and hormone-dependent breast cancer . HormoneκB signaling pathway, immune system, PI3K/Akt signaling pathway, and TNF signaling pathway. These four signaling pathways were the signaling pathways related to mastitis that CMMs involve in.BATMAN-TCM was used to perform signaling pathway enrichment analysis of the targets of 12 CMM combinations. Then the obtained signaling pathways were compared with those KEGG signaling pathways related to mastitis found in MalaCards. Four common signaling pathways were identified: NF-(1) NF-κB Signaling Pathway. Nuclear factor-κB (NF-κB) is a transcription factor widely existing in eukaryotic cells. NF-κB normally exists in nonactivated state in cells. When cells are stimulated by stimulating factors such as inflammatory mediators, viral infection, oxidative stress, etc., NF-κB will be activated and transfer to cell nucleus. It will then bind to the enhancer sites of target genes such as cytokines, growth factors, intercellular adhesion molecule, acute phase protein, etc. and enhance their transcription. Therefore, NF-κB signaling pathway plays a key role in regulating immune response, inflammatory response, cell proliferation/differentiation/apoptosis, etc. If the activation of NF-κB signaling pathway cannot be timely inhibited, various pathological responses may occur [κB signaling pathway and human diseases has received more and more attention. Research has shown that many CMMs which have significant efficacy in treating NF-κB-related diseases can inhibit the activity of NF-κB. By analyzing the working mechanism of CMM at cellular and molecular levels, it is found that CMM contain some active ingredients, which can regulate the activity of NF-κB at cellular or molecular levels and thus exert therapeutic effects. Among the CMM extracts that can significantly inhibit the activity of NF-κB, many are glycosides, including flavonoids, nonflavonoid polyphenols, and other glycosides [κB signaling pathway, block the NF-κB-mediated expression of various cytokines, and thus exert therapeutic effects on mastitis.ay occur . In receycosides . Therefo(2) Immune System. The inappropriate activation of NF-κB signaling pathway can not only cause inflammatory response, but also decrease the body's immunity [κB signaling pathway is closely related to immunomodulation, the key CMM combinations studied here may exert immunomodulatory effects by regulating NF-κB signaling pathways and immune system signaling pathways.immunity . When th(3) PI3K/Akt Signaling Pathways. Phosphoinositide 3-kinase (PI3K) and its downstream target Akt are important signaling molecules and key survival factors that control cell proliferation, apoptosis, and tumorigenesis [κB (IκB-α). In this way, IκB-α is separated from NF-κB, which is thus activated and enter cell nucleus. Then, it can induce the expression of many inflammatory factors such as IL-6, tumor necrosis factor-α (TNF-α), etc. and cause inflammatory response [α and IL-6, and mitigate inflammatory response [ρ-Akt and PI3K in colonic mucosa and exert therapeutic effects on colitis [igenesis . Researcigenesis . In addiresponse . Many CMresponse . Astraga colitis . The key(4) TNF Signaling Pathway. The activation of NF-κB signaling pathway can promote the release of proinflammatory factors such as TNF-α and IL-6. This can enhance inflammatory response and cause pro-/anti-inflammation imbalance, finally leading to further enhancement of inflammatory response and immune disorders [κB family are activated in response to various stimuli. The most characteristic inducers are TNF cytokine family [κB and block the development of inflammation. Red sage, Chinese angelica, moutan bark, peony root, sanqi, etc. are frequently used CMMs that can promote blood circulation. These CMMs not only can promote blood circulation and resolve stasis, but also have anti-inflammatory effects [isorders . Inducibe family . TNF is e family . CMMs wh effects , 24. TheκB signaling pathways. In addition, it can bind and activate PPAR- γ so that TLR4 can downregulate expression and inhibit the activation of downstream NF-κB signaling pathway. Finally, the expression levels of genes and proteins of inflammatory factors such as TNF-α, IL-1β, and IL-6 were decreased. Zhao Yongwang [α and IFN-γ compounds to maintain a certain level. It can not only participate in antibacterial immunity and prevent excessive inflammation of tissues, but also regulate cellular immunity and improve breast immunity.The treatment of mastitis in western medicine mainly adopts beta-lactam antibiotics to sterilize and prevent infection. The mechanism of drug action is to kill bacteria by destroying the cell wall. In comparison, there are many kinds of Chinese materia medica for treating mastitis, and the efficacy of CMM prescriptions also includes clearing heat and detoxification, removing swelling from breast, soothing liver and regulating qi, etc. Therefore, the study on the mechanism of action of CMM prescriptions in the treatment of mastitis is more complicated. Thus there are few related reports, most of which are about the mechanism of action of single Chinese materia medica. Gao Ruifeng  reportedYongwang  reportedIn this paper, data mining method was used to statistically analyze the frequencies of CMMs in prescriptions for treating mastitis and find association rules. Key CMM combinations for treating mastitis were obtained and can provide useful information for clinical therapy of mastitis. Network pharmacology was employed to obtain the potential targets of high-frequency CMMs and the potential signaling pathways that key CMM combinations involve in. This provides a new method for the research of the mechanism of CMMs in treating mastitis. However, the results are only based on already-known chemical composition of CMMs, related targets, and signaling pathways. With the development of technology, new ingredients and targets will be found in CMMs and there will also be more disease-related information. This will help enrich the results of this paper. In addition, according to CMM combination-target-disease analysis results, many CMMs for treating mastitis are also related to the treatment of breast cancer. Therefore, our future research will focus on the difference between CMMs used for treating mastitis and breast cancer.Due to the large number of ingredients in CMMs and the complicated interaction between CMM and human body, it remains difficult to elaborate the working mechanism of CMM. In fact, figuring out the working mechanism of CMM has become a bottleneck in the modernization and internationalization of CMM. From the perspective of network pharmacology, medicine-target-disease analysis may provide an entry point and a new strategy for further investigation into the working mechanism of CMM prescriptions.


We aimed to investigate the association between serum uric acid (SUA) levels and obstructive sleep apnea-hypopnea syndrome (OSAHS) in patients with type 2 diabetes.  A cross-sectional study of 212 type 2 diabetes mellitus (T2DM) patients was conducted in Xiamen, China. All patients underwent polysomnography (PSG) recordings for OSAHS diagnosis. Patients were grouped according to the apnea-hypopnea index (AHI) as mild (5-14.9), moderate (15-29.9), and severe (≧30) OSAHS. Patients with AHI ≤ 4.9 served as the control group. Weight, body mass index (BMI), SUA, liver function, renal function, blood pressure, lipid profiles, and glycemic parameters were measured. r = 0.194, p = 0.016). The level of SUA was significantly higher among OSAHS patients compared to the control group . Multivariable logistic regression analyses showed that SUA was the independent risk factor for OSAHS .  A total of 158 patients (101 men and 57 women) with complete data were analyzed in this study. 127 patients were identified as OSAHS. Among the 127 patients with OSAHS, 56 (44.1%), 37 (29.1%), and 34 (26.8%) had mild, moderate, and severe OSAHS, respectively. Correlation analyses showed that the SUA level was significantly related to the apnea-hypopnea index (AHI) ( The SUA level is significantly associated with the severity of OSAHS and should be controlled when managing OSAHS. In recent decades, serum uric acid (SUA), which is the end product of purine metabolism in humans, has been consistently found to predict the development of metabolic syndrome (MetS) , 2. GrowObstructive sleep apnea-hypopnea syndrome (OSAHS) is a syndrome characterized by recurrent episodes of shallow or paused breathing during sleep and normally leads to intermittent hypoxia (IH) and sleep disruption due to partial or complete obstructions of the upper airway during sleep. OSAHS affects 3-7% of men and 2-5% women in the general population . IncreasPrevious studies observed an association between SUA levels and OSAHS. In a population-based survey study, a strong association was found between SUA and OSAHS even after adjustment for confounding factors such as gender, age, and BMI . In a laThe study was approved by the Human Research Ethics Committee of the Xiamen First Hospital. All subjects provided written informed consent.μmol/L were further excluded from the study. The remaining 158 patients (101 men and 57 women) were left for analyses. The flow diagram is shown in A total of 212 adult subjects with T2DM were recruited from the First Affiliated Hospital, Xiamen University, China, from June 2015 to June 2017. These subjects underwent face-to-face interview with uniform questionnaires. Subjects were admitted for routine check-up evaluations and underwent a detailed physical examination and overnight polysomnography (PSG). Exclusion criteria included the presence of hyperthyroidism or hypothyroidism, acute illnesses, serious heart diseases, uncontrolled hypertension, presence of cancer, craniofacial abnormalities, any respiratory disorder other than OSAHS, and current use of hypnotics or any treatment for breathing disorders. Among the eligible patients, 36 subjects were excluded because of the deficiency of SUA data. 18 patients with the creatinine level over 104 Anthropometric measurements include body weight, height, waist circumference (WC), blood pressure (BP), body mass index (BMI), and neck circumference (NC). NC was measured at the middle of the neck between the midcervical spine and the midanterior neck 0.5 cm below the laryngeal prominence. BMI was calculated as the weight in kilograms divided by the square of the height in meters. WC was measured at the midpoint between the inferior costal margin and the superior border of the iliac crest on the midaxillary line. Weight and height were measured with light clothes and without shoes to the nearest 0.1 kg and 0.5 cm, respectively.All blood samples were obtained after 12-hour fasting. Blood samples were tested in the central laboratory of the First Affiliated Hospital, Xiamen University, as described in details previously . BrieflyPolysomnography (PSG) is the gold standard method for diagnosing and assessing the severity of OSAHS. All patients underwent an overnight PSG study performed from 11:00 pm to 7:00 am according to standard techniques with monitoring of the electroencephalogram (EEG), electrooculogram (EOG), electromyogram (EMG), flow, thoracic and abdominal respiratory effort, oximetry, and body position. All PSG data were collected and stored using an E-Series digital system . Polysomnographic recordings were interpreted in accordance with the current American Academy of Sleep Medicine (AASM) guidelines . The recU test for continuous variables and the chi-square test for categorical variables. The correlation of SUA with AHI was analyzed using Pearson's correlation analysis. Multivariable logistic regression analysis was used to calculate the odds ratio (OR) for OSAHS in different models with adjustment for potential confounders. In model 1, no variables were adjusted for. In model 2, age, sex, BMI, and the waist/hip were adjusted for. In model 3, hypertension and dyslipidemia plus model 2 were adjusted for. All p values presented are two-tailed, and values less than 0.05 are considered statistically significant.Data were analyzed with the use of IBM SPSS Statistics 21.0. Results are expressed as mean ± standard deviation (SD). Differences between groups were analyzed using ANOVA or the Mann-Whitney 2, 0.97 ± 0.09 m, and 9.88 ± 2.45%, respectively. The overall prevalence rate of OSAHS was 80.37% in type 2 diabetes mellitus patients in the current cohort. Among the 127 patients with OSAHS, 56 (44.1%), 37 (29.1%), and 34 patients (26.8%) presented mild, moderate, and severe OSAHS, respectively. Comparing between the four groups , there were significant increases in BMI , NC , and SUA . Pearson's correlation coefficients with adjustment for gender, age, and BMI are shown in r = 0.194, p = 0.016).The baseline characteristics of the studied subjects are shown in 2 vs. 27.36 ± 3.90 kg/m2, p = 0.002) and TG  levels compared with the control groups.p < 0.01).μmol/L vs. 333.14 ± 80.52 μmol/L, p = 0.029). Furthermore, the SUA level was significantly correlated to the severity of OSAHS, with the Pearson's correlation coefficients between SUA levels and AHI of 0.194 (p = 0.016). With adjustment for potential confounding factors such as gender, age, and BMI, logistic regression analysis showed that SUA was independently associated with risk of OSAHS in patients with T2DM.The study showed a significant association between SUA and OSAHS in the patients with T2DM. The results suggested that type 2 diabetic patients with OSAHS had significantly higher levels of SUA than those without OSAHS (428.37 ± 123.58 μmol/L in the SUA level was associated with 16% increased risk of OSAHS (95% CI = 1.01-1.33). The OR is higher than we found in the current study, likely due to the different populations studied. In our study, we had excluded the patients with the serum creatinine level over 104 μmol/L and all the subjects were type 2 diabetes mellitus patients.Similar to our data, a previous study found that after adjustment for confounding factors such as gender, age, BMI, social class, ethnicity, cholesterol, triglycerides, blood pressure, and glucose, the patients with OSAHS had higher levels of SUA than those without OSAHS . It showOver the past few decades, hyperuricemia has been found to be a risk factor for atherosclerosis and hypertension . One of In the clinic, obesity is associated with insulin resistance and increases the risk for type 2 diabetes . But theIt was well known that obese individuals present higher incidence of OSAHS, which contributes to increased morbidity and mortality , 28. TheOur study also had a few limitations. First, the causal relationship between SUA and OSAHS could not be determined because of the cross-sectional study design. Second, the number of patients in this study was relatively small; therefore, our results should be confirmed in future prospective cohort studies with larger sample sizes. Third, the present subjects were not randomly sampled and the selection bias was obvious, so our results should be interpreted with much caution.The SUA level is significantly associated with the severity of OSAHS and should be considered in OSAHS management in the clinic.


Ageing, socioeconomic level, obesity, fertility, relaxed natural selection and urbanization have been postulated as the risk factors of ovarian cancer (OC56). We sought to identify which factor plays the most significant role in predicting OC56 incidence rate worldwide.bs) and urbanization. Partial correlation was used to compare contribution of different variables. Fisher A-to-Z was used to compare the correlation coefficients. Multiple linear regression (Enter and Stepwise) was conducted to identify significant determinants of OC56 incidence. ANOVA with post hoc Bonferroni analysis was performed to compare differences between the means of OC56 incidence rate and residuals of OC56 standardised on fertility and GDP respectively between the six WHO regions.Bivariate correlation analysis was performed to assess the relationships between country-specific estimates of ageing (measured by life expectancy), GDP PPP (Purchasing power parity), obesity prevalence, fertility (indexed by the crude birth rate), opportunity for natural selection  contains supplementary material, which is available to authorized users. Ovarian Cancer   ranks am2) may have a greater risk of developing OC56 because of their elevated levels of circulating estrogen [Although OC56 has been known to medical scientists for over 150 years , the aetestrogen –11. An aestrogen . Urbanizestrogen , but it estrogen  due to testrogen , 14, 15.bs) has been constructed to measure the opportunity for natural selection through differential mortality at the population level. The Ibs calculation combines life table function dx (number of deaths at age x) with the age-specific completed relative fertility rate sx : Ibs = 1 – Σdxsx [bs can be used as a way of measuring the opportunity for an individual born into a given population to pass on its genes to the next generation [bs has been postulated to reflect changes in the mutation-selection balance as a result of the effect of improved healthcare on relaxing natural selection and thus measure the magnitude of accumulation of the deleterious genes [Natural selection, as one of the key mechanisms of evolution, differentiates phenotypes’ survival and/or fertility that reflect genetic differences. The Biological State Index , obesity and socioeconomic factors (GDP and urbanization).To the best of our knowledge, despite that low fertility is a well-established risk factor for OC56, no research has compared the contributing effects of fertility to OC56 with other OC56 risk factors, such as ageing, IThere is significant variation in the incidence of OC56 between different geographic regions globally , 33–35. In this study, empirical macro-level data have been used to test the hypothesis that fertility (measured by the crude birth rate) is the principal determinant of developing OC56, and that it is fertility, instead of GDP, that is most important factor in shaping the regional variation of OC56 incidence rate.The GLOBOCAN 2012 estimates of incidence rate of female OC56 .The following country specific data published by the agencies of the United Nations were analysed for this study.GLOBOCAN provides contemporary population level estimates by cancer site and sex . This pr2.The World Bank published data on crude birth rate, per capita GDP PPP and urbanization OC56 incidence rate is expressed as the number per 100,000 females who were diagnosed with OC56 in 2012. The age-standardized OC56 incidence rate was selected in the interest of the data comparability between countries.Crude birth rate (CBR) indicates the number of live births occurring during the year, per 1000 population estimated at midyear. CBR was used to index the fertility in this study over a 20 year period (1992) to reflect long exposure with delayed presentation of OC56. Terms “birth rate” and “fertility” are interchangeable in this paper.Socio-economic level has been associated with OC56 risk , 39, 40.3.The United Nations statistics division estimates of the life expectancy Urbanization has been postulated as a major OC56 predictor , 42 beca65, 2005–2010) [4.bs)The magnitude of OC56 gene accumulation in a population indexed with the biological state index   to indexbs was downloaded from the previous publication [bs) may have allowed accumulation of deleterious genes of non-communicable diseases [5.The WHO Global Health Observatory (GHO) data on obesity prevalenceThe country specific Ilication . It has diseases –19, suchdiseases .5.The WH2 in 2010 was extracted from the GHO data repository [Obese females may be at greater risk of developing OC56 than those who are not obese . The coupository .bs, obesity and urbanization were collated for all countries where data were available. We extracted OC56 incidence rates for 182 countries and then the other variables were matched individually with OC56.Country specific OC56 incidence rates, ageing, fertility, GDP, IEach country was treated as an individual study subject in the data analysis. Not all the countries (subjects) had information for all the variables.The relevant United Nations agencies offer free online access to data required for the analyses in this study. No ethics approval was required as there were no individual patients involved in the study.In order to avoid the inter-correlation between predictor variables, the multicollinearity statistics were calculated to test the correlations among the variables. Each variable was alternated as the dependent variable, and all the others were considered as the predictor variables in our analysis with the regression model. It was found that the collinearities between variables were not significant since only the tolerance of less than 0.20 and a VIF of more than 5 indicates a multicollinearity problem . Values Scatter plots were produced with the original data in Microsoft Excel® to explore and visualize the strength, shape and direction of correlations of OC56 to fertility and GDP respectively.Data were logarithmed to improve their homoscedasticity for linear regression analyses. Bivariate (Pearson’s r and nonparametric Spearman’s rho) correlations were performed to evaluate the direction and strength of the correlations between all the variables of all the subjects and effects possible effects of non-normality of distributions on the strength of moment-product correlations.bs, obesity and urbanization) as the independent predictor when all other five variables were included as the potential confounding factors.Partial correlation analysis of Pearson’s moment-product approach was performed. We alternated each of the six variables  when fertility was incorporated; and (2) excluded as a predicting variableStandard multiple linear regression (enter) was performed to describe the correlations between the dependent variable (OC56) and the predicting variables. In order to explore if low fertility can partially explain why ageing, GDP, ISubsequently, standard multiple linear regression (Stepwise) was performed to select the predicting variable (s) which have the greatest influence on OC56 in two versions: (1) when fertility was incorporated and (2) excluded as a predicting variable.2–0.504× + 14.816, R2 = 0.485) and GDP PPP  were used to calculate and remove the contributing effects of GDP PPP on OC56 incidence rate respectively by using regressions of OC56 residuals around fertility and GDP PPP. This allowed us to create two new dependent variables, “Residual of OC56 standardised on fertility” and “Residual of OC56 standardised on GDP PPP”The equations of the best fitting non-linear trendlines displayed in the scatter plots analysis of relationships between OC56 incidence and fertility  was conducted to detect the significant differences among the means of OC56 incidence rate, “Residual of OC56 standardised on fertility” and “Residual of OC56 standardised on GDP PPP” between the six WHO regions . FurtherBivariate correlations, multiple linear regression analysis (Enter and Stepwise) and ANOVA were conducted with SPSS v. 24. The raw data were used for calculation of mean OC56 incidence rate and “Residual of OC56 standardised on fertility” and “Residual of OC56 standardised on GDP PPP”. The significance was kept at the 0.05 level, but 0.01 and 0.001 levels were also reported. Standard multiple linear regression analysis criteria were set at probability of F to enter ≤0.05 and probability of F to remove ≥0.10.2 = 0.485, p < 0.001, n = 179, Fig. The relationship identified in the scatterplots between fertility and OC56 was noted to be polynomial with a strong, but inverse (negative) correlation   was tested by keeping the other five variables statistically constant in partial correlation analysis. Fertility was the only predictor showing a substantial significant correlation  with OC56 independent of the other five variables . The Fisher r-to-z transformation revealed that OC56 was in significant stronger correlation with fertility than with ageing . GDP, Ibs, obesity and urbanization showed significant correlation to OC56 in the bivariate correlation analyses respectively. However, none of these variables showed a significant correlation with OC56 independent of the other five predictors. This indicates that fertility is the only significant predictor of OC56 independent of the secondary association between OC56 incidence and Ibs (magnitude of OC56 accumulation) and environmental factors .The relationship between OC56 and each independent variable  analysis was applied to predict OC56 incidence when ageing, fertility, GDP, obesity and urbanization were included as the independent predicting variables.p < 0.001) and Ibs  were the two significant variables related to OC56 incidence. However, when fertility was included as an independent predictor, only the correlation between fertility and OC56 incidence was strong and significant. None of the other five predictors showed strong and significant correlation to OC56 .Table A subsequent ANOVA with post hoc Bonferroni procedure performed on the means of “Residual of OC56 standardised on fertility” in different WHO regions showed there was no significant difference among and between regions Table . Whilst bs, obesity and urbanization respectively, this study has shown that only fertility and aging were correlated with the OC56 incidence significantly, although the latter was not as strongly. Statistically, this may suggest that low fertility was the most significant risk factor for OC56 when compared to ageing, GDP, Ibs, obesity and urbanization. This finding is in agreement with three studies conducted by Hankinson et al. [The worldwide secular trend of increased OC56 incidence may have multiple etiologies, which may act through multiple mechanisms at different magnitudes. By examining the correlations of OC56 with low fertility, ageing, GDP, In et al. , Vachon n et al.  and Cramn et al.  respectiThe relationship between female reproductive performance and gynecological cancers has been known for over 300 years . PreviouThey may produce more oxytocin –69 due tThey may have less stress due to more positive psychological well-being from greater family size, reduces stress levels. This may make their neuroendocrine and immune systems more efficient to reduce the risk of cancer (developing OC56) –75.They are more likely to seek health service and maintain a healthy lifestyle –79, whicRecent studies suggested that women with greater fertility may receive the protection against developing OC56 because:This study revealed that low fertility determines the variation of OC56 incidence rate among the WHO regions. This finding contradicts the WHO and IARC’s statement that socioeconomic level is the determinant of regional variation of OC56 incidence rate , 6, 34. The strength of this study is that it uses an ecological study approach, different from hitherto used approaches, to demonstrate that low fertility is a significant determinant of OC56 risk.Each country was considered as a whole subject for the ecological study. The country-specific data included in this study may be different from those collected from individual participants. Therefore, the correlations identified from the data analysis may not hold true for all the individuals to have the risk in OC56 development.There may be some random errors that occurred when the United Nations and its agencies collected and aggregated data at country level. Data from developed countries may be more complete than those from developing countries.There are different categories of OC56, but we could not differentiate them for the correlation analysis due to the unavailability of such data.We need to note several limitations of this study:bs, obesity and urbanization. These findings may be helpful for governments, policy-makers, funders, clinicians and researchers when determining future screening and primary presentation strategies for the disease [Low fertility appears to be a significant and strong determinant of OC56 risk independent of ageing, GDP, I disease , 83, 84.Additional file 1:AF 1 Collinearity among the variables. (DOCX 16 kb)


This study aimed to investigate the association between severity of obstructive sleep apnea–hypopnea syndrome (OSAHS) and the neuroimaging consequences of cerebral small vessel disease (SVD).Patients with OSAHS and age‐ and gender‐matched healthy control subjects completed the mini‐mental state examination and underwent an evoked‐related potential study and overnight polysomnographic monitoring. Magnetic resonance imaging (MRI) was performed to detect markers of silent cerebral SVD, including Virchow–Robin spaces (VRS) rated on a five‐point scale, white matter lesions, lacunar infarcts, and deep microbleeds. Multinomial logistic regression models were used to examine the associations of the apnea–hypopnea index (AHI) and arousal index (AI) values, mean oxyhemoglobin saturation, the duration of snoring history, and MRI markers of small vessel disease with the incidence of enlarged VRS.p < .05), which is associated with neurocognitive impairment. Enlarged VRS were more prevalent in the basal ganglia and centrum semiovale of patients with OSAHS than in the control group. No significant between‐group differences were observed in the number of white matter lesions, lacunar infarcts, and deep microbleeds. Enlarged VRS were positively correlated with AHI and AI values in the OSAHS group .The study included 72 patients with severe OSAHS and 53 volunteers without OSAHS. The duration of snoring history ranged from 5 to 22 years in the OSAHS group. Smaller P3 amplitudes at Cz were found in OSAHS patients than control subjects (Silent cerebral SVD was more prevalent in patients with OSAHS than in the controls. Enlarged VRS observed in the basal ganglia and centrum semiovale were positively correlated with severity of OSAHS, which may contribute to cognitive impairment. At baseline, all participants completed the mini‐mental state examination (MMSE) and underwent a comprehensive interview to obtain information regarding age, sex, duration of snoring history, history of current and previous illnesses, and medical treatments. The interview questionnaire and PSG data were collected and analyzed by two independent investigators. Patients with a history of DM, hypertension, and atherosclerosis, defined as previous diagnosis according to standard clinical guidelines, and those with sleep disorders other than OSAHS or who had received treatment for OSAHS were excluded from the study. Additional exclusion criteria were congestive heart failure, intrinsic pulmonary disease, drug dependence, alcoholism, severe psychiatric disturbance, and pregnancy.2.3All patients underwent an evoked‐related potential (ERP) study. Electroencephalogram (EEG) data were recorded using a 256 channel HydroCel Geodesic Sensor Net  with Cz as the reference channel. All electrode impedances were monitored and maintained below 50 kΩ. We used oddball stimuli with speech sounds using E‐Prime software . The auditory oddball task involved the presentation of a 1,000 Hz pure tone, with an oddball target stimulus presented at 2,000 Hz. The 1,000 Hz stimulus was presented in 85% of the trials, and the 2,000 Hz stimulus was presented in 15% of the trials. In total, the task consisted of 1,000 auditory stimuli at random inter‐stimulus intervals (ISI) ranging from 850 to 1,450 ms. The sound stimuli (75 dB) were delivered through two loudspeakers located 100 cm from the subjects. During the P3 cortical wave test, the subjects were instructed to close their eyes and press a button held in the right hand each time they detected a target tone.2.4Participants underwent high‐resolution three‐dimensional MRI to detect silent markers of SVD, including rated VRS, white matter lesions, lacunar infarcts, and deep microbleeds according to the definition provided by STandards for ReportIng Vascular changes on nEuroimaging  ≥4% or arousal. The apnea–hypopnea index (AHI) indicated the number of apnea and hypopnea events per hour during sleep, based on the results of the overnight PSG. Data without pulse SpO2 values, illegible recordings, and recordings <2 hr duration were excluded from analysis. OSAHS was rated as no OSAHS, AHI <5; mild, AHI 5 to <15; moderate, AHI 15 to <30; and severe, AHI ≥30  recordings were scored manually according to standard criteria by the same experienced technician. Respiratory events were scored according to the American Academic Sleep Medicine criteria: Apnea was defined as complete cessation of airflow lasting for ≥10 s; hypopnea was defined as either a ≥50% reduction in airflow for ≥10 s, or a <50%, but discernible, reduction in airflow accompanied by a decrease in oxyhemoglobin saturation . The Statistical Package for the Social Sciences version 16 (SPSS Inc.) was used to conduct the statistical tests. We first assessed univariate associations of the total VRS scores followed by a multiple linear regression analysis to determine independent associations of the total VRS scores with vascular risk factors, lacunar stroke subtype, and MRI markers of cerebral SVD. Two‐sided Student's 3The study included 72 patients with OSAHS  aged 29–58 years,  and 53 controls without OSAHS  aged 32–58 years . The duration of snoring history ranged from 5 to 22 years. The mean duration of snoring was 8.5 years, and the mean AHI value was 51.6 ± 12.1. The demographic and clinical characteristics of the participants are shown in Table p < .05), whereas the latency was not significantly different between groups.The MMSE scores of the patients with severe OSAHS were not significantly different from those of the control subjects. The ERP waveforms for the standard and target speech stimuli showing P3 components are shown in Figure 3.1Enlarged VRS, white matter lesions, lacunar infarcts, and deep microbleeds were observed in the brains of patients with OSAHS. Deep microbleeds were identified and rated according to the Microbleed Anatomical Rating Scale Figure . White mr = .63, p < .001) and AI  in the OSAHS group. Furthermore, the VRS total score was negatively correlated with the mean SpO2  in the OSAHS group, while the VRS total score was negatively correlated with P3 amplitudes .Enlarged VRS were most common in the bilateral basal ganglia regions Figure  and sign44.1The present study demonstrated that silent cerebral SVD was more prevalent in patients with OSAHS than in the controls. Enlarged VRS were observed in the basal ganglia and centrum semiovale. The P3 amplitudes of patients with severe OSAHS were smaller than those of the control subjects, which is an indicator of cognitive impairment. VRS volume was positively correlated with AHI and AI values, and was negatively correlated with P3 amplitudes, suggesting that enlarged VRS may contribute to cognitive impairment.4.2Mounting evidence suggests that OSAHS is associated with neurocognitive impairment (Chen et al., MRI‐visible VRS are potential neuroimaging markers of cerebral SVD, and several imaging markers of SVD have been independently associated with cognitive decline in a range of clinical cohorts (Hurford et al., However, there is little evidence to support an association between OSAHS and cerebral SVD. VRS detected by MRI have been associated with age‐related disorders and vascular risk factors (Zhang et al., 4.3Our finding of a negative correlation between VRS and mean oxygen saturation during sleep suggests that intermittent hypoxia may be an important contributing factor for cognitive dysfunction in OSAHS. Enlarged VRS are increasingly recognized as a marker of disease rather than simply a marker of aging; however, few studies have investigated the association between OSAHS and VRS. Our study provides further evidence that the P3 amplitudes of patients with severe OSAHS were smaller than those of the control subjects, and enlarged VRS volume negatively correlated with P3 amplitudes may contribute to cognitive impairment.4.4The main limitation of our study was its retrospective design, which restricted our analysis to available, routinely collected data. Secondly, we did not control for BMI in patients and control group, while high BMI is significantly related to the occurrence of OSA; thus, we consider the significant difference of BMI as inherent characteristics between groups. Additionally, PSG measurements obtained in the hospital may not provide a true representation of the normal sleep patterns exhibited at home in a more natural setting. A technological limitation of our study was the lack of a noninvasive reliable method for discriminating between arteries and veins in vivo using MRI. The strengths of our study include the use of high‐resolution MRI and our definition of enlarged VRS based on strict anatomical and imaging criteria, and the use of a highly reliable assessment method.5In summary, silent cerebral SVD was more prevalent in OSAHS patients. Neuroimaging consequences of cerebral small vessel disease were particularly found in the basal ganglia and centrum semiovale. The enlarged VRS and decreased P3 amplitudes may be a useful imaging biomarker of cognitive impairment in patients with obstructive sleep apnea–hypopnea syndrome.The authors declare that there is no conflict of interest statement.


The annual meeting “Signal Transduction—Receptors, Mediators, and Genes” of the Signal Transduction Society (STS) is an interdisciplinary conference open to all scientists sharing the common interest in elucidating signaling pathways in physiological or pathological processes in humans, animals, plants, fungi, prokaryotes, and protists. On the occasion of the 20th anniversary of the STS, the 22nd joint meeting took place in Weimar from 5–7 November 2018. With the focus topic “Signaling: From Past to Future” the evolution of the multifaceted research concerning signal transduction since foundation of the society was highlighted. Invited keynote speakers introduced the respective workshop topics and were followed by numerous speakers selected from the submitted abstracts. All presentations were lively discussed during the workshops. Here, we provide a concise summary of the various workshops and further aspects of the scientific program. The 22nd Joint Meeting “Signal Transduction—Receptors, Mediators and Genes” was held in Weimar, Germany, from 5 to 7 November 2018 [According to the focus theme “Signaling: From Past to Future” each workshop was introduced by an expert in the respective field, who gave an overview of how his or her research field has evolved. The first workshop “Growth factors, Cytokines and their Receptors” was opened by Manuela Baccarini , who presented her research on the non-canonical functions of ERK pathway components. Beyond their well-known functions as kinases of the ERK signaling module, RAF and MEK have several kinase-independent effects. These effects are mostly based on the ability of RAF/MEK to physically interact with other signal transduction proteins. Furthermore, the Baccarini group provided evidence for distinct in vivo functions of RAF and MEK, thereby laying ground for new possibilities in targeted anti-cancer therapy ,3.Manolis Pasparakis  opened the workshop on “Differentiation, Stress and Death” by presenting his work on receptor-interacting protein kinase (RIPK) signaling in cell death and its critical role in immune responses. There is a complex interplay between IKK/NF-kB and RIPK1 signaling modules influencing the balance between tissue homeostasis and inflammation in epithelial barrier tissues. In this context, IKK subunits can inhibit RIPK1 activity both NF-kB-dependently and –independently. Moreover, since it could be shown that RIPK1 and RIPK3 have kinase-dependent and kinase-independent effects, the Pasparakis group set out to elucidate the processes regulating these complex interplays and their in vivo significances. As an example, the deubiquitinating protein A20 targets RIPK3, MyD88, and NLRP-3 signaling molecules of the NF-kB pathway and absence of functional A20 in mice leads to severe multifocal inflammation, whereas specific ablation of A20 in myeloid cells leads to a spontaneous onset of destructive polyarthritis ,5,6.The introductory keynote presentation for the workshop on “Tumor Cell Biology” was held by Martin Eilers , who gave an informative and concise overview of the function of the transcription factor N-Myc in human neuroblastoma. Myc proteins are bound to virtually any active promotor in the human genome and can be found dysregulated not only in neuroblastoma, but in most human cancers. In this context, the Eilers group found evidence for a regulatory function of the kinase Aurora-A, which is expressed cell cycle-dependently and prevents N-Myc degradation by the ubiquitin ligase Fbxw7. During S Phase Aurora-A competes with RAD21 and TFIIIC2 for N-Myc binding, thereby preventing promotor escape and pause release of RNA polymerase II, which is mediated by the N-Myc/TFIIIC2/RAD21 complex. Thus, pharmacological inhibition of Aurora-A has the potential to inhibit proliferation of neuroblastoma cells overexpressing N-Myc, as demonstrated by increased tumor regression and improved survival in a mouse model of neuroblastoma ,9. Björn Lillemeier  presented his work on the effects of membrane dynamics of signaling molecules on T cell responses in the workshop “Immune Cell Signaling II” . He showed a plethora of exciting data on the organization of signaling molecules in nanometer scale plasma membrane islands, such as the T cell receptor (TCR) nanocluster and the nanoclusters containing the Zap70 substrate LAT. After activation of the T cells, the protein islands concatenate into microclusters. TCR signals are then relayed to LAT islands via translocation of Zap70 between the nanodomains in a ‘catch-and-release’ mechanism. For high-resolution imaging of the membrane dynamics, the Lillemeier group has developed sophisticated approaches combining single-molecule super-resolution and dynamic fluorescence microscopy with traditional biochemical and immunological techniques ,11,12. All of these keynote lectures were followed by a number of short talks selected from the submitted abstracts. As in the past, the stimulating blend of presentations given by young scientists, early career researchers and established scientists was an outstanding feature of the meeting.cis mode driven by classical G Protein signaling and a trans mode conveyed by the N-terminus of latrophilin in a non-cell-autonomous manner [The DGP junior group “Forum Junge Wissenschaft” organized the workshop “G Proteins and G Protein coupled receptors” with 5 early career researchers as speakers. Christoph Klenk  gave insights into his research concerning directed evolution of the human parathyroid hormone 1 receptor (PTH1R), which made it possible to obtain more thermally stable variants and subsequently a valid crystal structure of PTH1R . The focs manner ,19. The s manner .v1.7, which is dysfunctional in patients with the chronic pain syndrome erythromelalgia. Using induced pluripotent stem cells, which were differentiated into sensory nociceptors, he could establish a model system as a tool for development of new analgesics [v1 with the audience. Applying pH titration and targeted mutation of the channel, he identified amino acid residues which are crucial for proton selectivity and zinc binding [The “Young Physiologists” group of the DPG organized the workshop “Neuronal Signaling and Ion Channels” with 5 speakers recruited from their junior group. The session was opened by Jan-Philipp Machtens , who introduced the complex field of neuronal signaling and ion channels to the audience. To that end, he presented his work on excitatory amino acid transporters (EAAT), which he had analyzed using e.g., patch clamp techniques and computational electrophysiologic modelling ,22. Maddalgesics ,24. The  binding ,26.+ Treg cells. Väth showed that ablation of STIM1/2 in mature Foxp3+ Treg cells significantly impairs the further differentiation into follicular Treg (Tfr) cells and tissue-resident Treg cells. In vivo, mice with SOCE deficient Treg cells exhibited a severe inflammatory phenotype with a variety of autoantibodies and fatal inflammation in multiple solid organs underlining the critical role of STIM1/2 during differentiation of effector Treg cells [The “Young Immunologists” group of the DGfI participated in the organization of the workshop “Immune Cell Signaling I”, where they had chosen Martin Väth  as keynote speaker. He spoke about his extensive analysis of the stromal interaction molecule (STIM) 1 and STIM2 in the context of the effector differentiation of regulatory T (Treg) cells. STIM1/2 mediates the first wave of cytosolic calcium increase during store-operated calcium entry (SOCE) signaling in Treg cells, which is known to control the thymic development of Foxp3eg cells ,28. The In 2018, the STS grant committee chose 9 master, MD, or PhD students to receive travel grants of 250 € each to allow their meeting attendance. One of the travel grants was sponsored by Biomol, the Silver Sponsor of the meeting. As part of the well-known “one minute—one transparency” session, each poster-presenting scientist had the opportunity to introduce their research. Subsequently, the audience had the possibility to meet the presenting scientists at their posters in a casual atmosphere, discuss their data, and establish contacts. Overall, 7 poster prizes to a total value of 750 € were awarded to the most excellent posters, which had been selected by the chair people of the various workshops at the meeting.The STS Science Award is meant to honor outstanding research by a post doc or junior principal investigator of the STS. It was first introduced at the annual STS Meeting in 2005 and ever since it has become a regular element of all following STS Meetings. This year, Dirk Brenner  received the STS Science Award for his comprehensive research on the glutathione-dependent regulation of metabolic responses in B and T cells. The award, with a total sum of 1500 €, was donated by the STS and OMNI Life Science GmbH. Annually on the occasion of the STS Joint Meeting, the GBM announces the GBM Innovation Award for Young Scientists to the value of 500 €. The Award is meant to honor young scientists working on an innovative and interesting method in the field of signal transduction. An award jury consisting of the speakers of the GBM-Study Groups “Biochemical Pharmacology and Toxicology” and “Receptors and Signal Transduction” selects appropriate candidates from the applications. In 2018, Theresia Gutmann  received the award for developing an in vitro membrane-mimetic system by embedding purified full-length insulin receptors in so-called lipid nanodiscs, and directly visualizing the conformational change induced by insulin. This minimal in vitro system is a valuable technique for addressing signaling mechanisms of the insulin receptor or other receptor tyrosine kinases, respectively.The STS Honorary Medal was introduced in 2010 in order to honor outstanding scientists in the field of signal transduction. After a laudation for the awardee and the festive presentation of the medal, the laureate concludes the award session with the “Honorary Medal Lecture”. Previous winners of the award are Anthony Pawson, Tony Hunter, Carl-Henrik Heldin, Klaus Rajewsky, Jules Hoffmann, Mina Bissell, Tak Wah Mak, and Michael Reth. Since 2017, the medal is awarded by the STS in collaboration with the International Journal of Molecular Sciences (IJMS).In 2018, Prof. Dr. Karen Vousden  received the STS Honorary Medal Award for her lifetime contributions on the functions and regulation of the tumor suppressor p53. Not only has Prof. Vousden discovered MDM2 as a decisive regulator of p53 expression, but she has also uncovered how p53 induces cell death and how it regulates cell metabolism ,30,31. Ihttps://www.sigtrans.de and on the STS Facebook account.The preparations of the 23rd STS Meeting have already started. Again, the meeting will take place at the Leonardo Hotel in Weimar from 4 November to 6 November 2019. Further details and regular updates on the schedule and contents of the meeting can be found at


Studies have shown that the human T-lymphotropic virus 2 (HTLV-2) is endemic in several indigenous populations of the Brazilian Amazon and molecular analyses have shown the exclusive presence of HTLV-2 subtype 2c among the indigenous groups of this geographical region.The present study characterizes the prevalence of HTLV-2 infection in three new villages of the Xikrin tribe, in the Kayapo group, according to their distribution by sex and age. The study included 263 samples from individuals from the Kateté, Djujeko and Oodjã villages. Plasma samples were tested for the presence of anti-HTLV-1/2 antibodies using enzyme-linked immunosorbent assays (ELISA). Seropositive samples were confirmed using real-time PCR, nested PCR and sequencing.The serological and molecular results confirmed the sole presence of HTLV-2 in 77 (29%) samples, with a prevalence of 38% among women and 18% among men. In these communities, it was found that the prevalence of HTLV-2 infection increased with age. Nucleotide sequences  from eight samples were subjected to phylogenetic analysis by the neighbor-joining method to determine the viral subtype, which confirmed the presence of HTLV-2c.The results of the present study establish the presence of HTLV-2 infection in three new villages of the Xikrin tribe and confirm the high endemicity of the infection in the Kayapo indigenous group of the Brazilian Amazon. Retroviridae, are retroviruses with tropism for T lymphocytes [The human T-lymphotropic viruses 1 and 2 (HTLV-1 and HTLV-2), members of the family phocytes –10.env gene and the 5′ long terminal repeat (LTR) region [HTLV-2 is endemic in several African populations , 9 and i) region , 28, 29.HTLV-2 occurs with high prevalence among indigenous tribes of different linguistic families inhabiting the Brazilian Amazon, including in Kayapo villages, where subtype 2c was described for the first time , 17. LatThe geographical distribution of HTLV-2c in the Amazon region of Brazil ranges from the north (Tiriyo village) to the south (Kararaô and Gorotire villages), which suggests that its presence was derived during their settlement about 11,000 to 13,000 years ago during their migratory spread from the North to the South and the contact among different indigenous populations , 15. In n = 263) were collected from individuals living in the Kateté (n = 121), Djujeko (n = 113) and Oodjã (n = 29) villages of the Xikrin tribe, Kayapo group, Jé linguistic family, who live in the Cateté Indigenous Territory, bordered by the Itacaiúnas and Cateté rivers, in the municipality of Parauapebas, state of Pará . Following the CONEP’s recommendations, the project received, through formal written permission, the approval and consent of the communities through their leaders on behalf of the participants (adults and Children’s parents) and the National Indian Foundation (FUNAI), exempting individual consent in writing in accordance with national regulations.Plasma samples were tested for the presence of anti-HTLV-1/2 antibodies using an enzyme-linked immunosorbent assay . The samples with positive or inconclusive results  with values 20% below or above the cut-off value) were subjected to Real-Time PCR , for confirmation and differentiation between HTLV-1 and HTLV-2 infection.Blood Genomic DNA Miniprep extraction kit  to amplify the pol and 5′ LTR regions. The qPCR reactions were prepared using the TaqMan® Universal master mix according to the following protocol: 10 μL of MasterMix, 4.0 μL of water, 1 μL of Assay-by-Design (primer and probe set) and 5 μL of DNA in a final volume of 20 μL. The following cycling protocol was used: one cycle of 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Two target sequences, an endogenous control  and the non-homologous regions of the pol gene (186 bp) of HTLV-1 and HTLV-2, were used. The primer sequences used were: 5′-gaacgctctaatggcattcttaaaaccc-3′(HTLV-1F), 5′-gtggttgattgattgtccatagggctat-3′ (HTLV-1R), 5′-caaccccaccagctacagg-3′ (HTLV-2F), 5′-gggcatgacaggttttgcaatatta-3′ (HTLV-2R), 5′-gctcaactccctattgctattcaca-3′ (Albumin F), 5′-gggcatgagaggttttgcaatatta-3′; and the probe sequences used were FAM-5′-acaaacccgacctaccc-3′-NFQ (HTLV-1), FAM-5′-tcgagagaaccaatggtataat-3′-NFB (HTLV-2) and FAM-5′-ttgtgggtgtaatcat-NFQ (Albumin).DNA from the seropositive and inconclusive samples was extracted according to the manufacturer’s protocol of the AxyPrep™Eight samples that were confirmed as positive for HTLV-2 were subjected to nested PCR reactions for amplification of the 5’LTR region for the later analysis of nucleotide sequencing and construction of phylogenetic trees. The 1st and 2nd round PCR reactions were run in a final volume of 50 μL containing 500 ng of extracted DNA, 10 μM of each dNTP, 20 pmol/μL of each primer, 50 μM MgCl2, 1 x buffer  and 5 U of Taq DNA polymerase. In each amplification reaction, after initial denaturation at 94 °C for 5 min, 35 cycles were performed, with 40 s at 94 °C, followed by 30 s at 57 °C and 1 min at 72 °C. These 35 cycles were followed by a final extension for 10 min at 72 °C. The primers used were 5′-tcgcgatgacaatggcgactagcctc-3′ (F-IILTR) and 5′-gggggctttgggtattggagttggg-3′ (Long Gag) for the first round and 5′-gcctcccaagccagccac-3′ (Mo15) and 5′-gggaaagcccgtggatttgccccat-3′ (MSW-Gag) for the second round. The nested PCR products were visualized after electrophoresis (100 V/45 min) on 2% agarose gel in 1x TAE buffer  that contained 5 μL of ethidium bromide (10 mg/mL), using a transilluminator with an ultraviolet light source, and were subsequently purified using the manufacturer’s protocol of the QIAquick PCR Purification Kit  for improving the nucleotide sequencing.The analyses of the degree of genetic divergence between the nucleotide sequences and the phylogenetic profile of the strains were performed using the MEGA-6 - Molecular Evolutionary Genetics Analysis 6.0 software .The nucleotide sequences of the 5′ LTR region (642 nt) amplified in this study  were used to establish phylogenetic relationships with the HTLV-2 sequences that were previously described in the literature and are available in the GenBank. Sequence alignment was performed using the BioEdit software . The degAntibodies to HTLV-1/2 were detected in 113 (43%) samples and 3 (1.1%) were undetermined (Table p = 0.0030), corresponding to an infection prevalence of 32% in women and 8.5% in men. In the Kateté village, the percentage was 45% in women and 28% in men (p = 0.0609). In Oodjã, the prevalence rates  were not statistically significant (p = 0.3391).The prevalence rate according to sex was 38% in women and 18% in men Table . This dip = 0.0005, Table p = 0.0411).A significant difference  and those over 70 years (p = 0.0019).A further association of infection with both age and sex was significant only in women  [NRA) [The sequences included samples from the three villages and were aligned and compared to each other, as well as with the prototype strains of subtypes 2a (HTLV-2Mot.)  and 2b (.) [NRA) . The samIn the present study the prevalence of 29% of HTLV-2 infection was found in three villages of the Xikrin tribe, Kayapo group: Kateté, Djujeko and Oodjã. The finding is consistent with previous results that demonstrated that the Kayapo group presents one of the highest endemic rates of HTLV-2 infection in the world , 14, 15.Considering the results found for other indigenous peoples of Brazil, the prevalence rate found in the present study was higher. In the state of Paraná, in the Guarani group, the prevalence was previously reported as 5.8% . More spThe prevalence found in the present study is similar to that observed in previous studies involving indigenous populations in the Americas. In Venezuela, the prevalence among the Guahibo  was estiBy contrast, the prevalence observed here was lower than those of several indigenous groups in the Americas. In Venezuela, the communities of Yaruro and Guahibo  had an eThese differences in prevalence are expected and likely due to numerous factors, such as the: (i) differences in the methodologies adopted, which may have different sensitivities and specificities; (ii) differences in the sample sizes analyzed in each study; and (iii) differences in socio-cultural aspects that favor or limit the spread of the virus in the local population , 16. IndIt is generally agreed that HTLV infection is more easily transmitted from men to women, which would likely explain the higher prevalence of infection among women , 25, 37.In the present study, there was a significant difference in infection prevalence between women and men, with the prevalence rate among women nearly twice the value in men. These results are in agreement with other findings in indigenous populations that found a predominance of infection among women. In the Peruvian Amazon, among the Shipibo-Konibo group, the reported seroprevalence was 3.1% in women and 2.5% in men . In VeneHowever, the results also contrast with those of several studies conducted among indigenous groups in the Americas. Ishak et al.  did not It is generally accepted that social and cultural characteristics are responsible to the differences of prevalence rates among native Indian communities, but these studies and the present results refer to three Kayapo Indians who share similar cultural behavior and common origin , 15. ForVertical transmission and breastfeeding are also common routes of spread among epidemiologically closed communities , 14, 15,When the infection prevalence was analyzed by age group, a significant difference was observed between sexes. The highest prevalence was found in individuals between 31 and 40 years, and it was seven times higher than that observed in individuals aged 11 to 20 years and three times higher than that found in those aged 51–60 years. These findings are consistent with other studies conducted in other indigenous populations , 38. AmoThe phylogenetic analysis is in agreement with previous studies that have demonstrated the endemic presence of HTLV-2, subtype c, in indigenous and urban populations of the Amazon , 15. TheA comparison of the nucleotide diversity of the 5’LTR region among the samples amplified in the present study identified a genetic similarity of 99.9% between them. When the analysis was performed considering the HTLV-2a Mot and HTLV-2b NRA prototype strains, the mean similarity was 96.7 and 94.6%, respectively. These results are similar to those observed among the Gorotire, Kararaô and Tiriyo indigenous communities and in the urban region of Belém . This suThe results of the present study reveal the incidence of HTLV-2 in three new villages of the Xikrin tribe, thus confirming the high endemic nature of the infection in the Kayapo indigenous group of the Brazilian Amazon.


N-methyl-d-aspartate (NMDA) receptor (NMDAR) antagonist ketamine, which affects a range of signaling pathways in ways that are distinct from the mechanisms of typical antidepressants. Because ketamine and similar drugs hold the promise of dramatically improving treatment options for depressed patients, there has been considerable interest in developing new ways to understand how these compounds affect the brain. Here, we review the current understanding of how rapid-acting antidepressants function, including their effects on neuronal signaling pathways and neural circuits, and the research techniques being used to address these questions.Depression is a chronic, debilitating, and common illness. Currently available pharmacotherapies can be helpful but have several major drawbacks, including substantial rates of low or no response and a long therapeutic time lag. In pursuit of better treatment options, recent research has focussed on rapid-acting antidepressants, including the Major depressive disorder (MDD) affects an estimated 5% of the global population at any given time, and is the leading cause of disability worldwide . In addiFirst, the drug ketamine, which had long been used in high doses as an anesthetic, was found to have a rapid antidepressant effect in low, subanesthetic doses . It reliThis article will review the current state of research on the mechanism of action of antidepressants, including key signaling pathways, the emerging understanding of the role of neural circuits, and the cutting-edge approaches and agents that are helping researchers understand these mechanisms.In order to understand how antidepressants relieve the symptoms of depression, it is helpful to discuss how the brains of depressed people differ from those who are not depressed. This question has been difficult to study due to the wide diversity of clinical presentations that meet criteria for MDD according to the Diagnostic and Statistical Manual of Mental Disorders (DSM) . DerangeHuman neuroimaging studies have consistently demonstrated reduced brain volume in key areas associated with mood regulation, including the frontal cortex, cingulate cortex, and hippocampus . Most ofN-methyl-d-aspartate (NMDA) receptors (NMDARs) that are its primary target, but also to presynaptic metabotropic glutamate receptors (mGluRs). Activation of these presynaptic metabotropic receptors inhibits synaptic glutamate release, which leads to reduced postsynaptic glutamatergic signaling and ultimately reduced synaptic connectivity . Se. Se30]. Further supporting the idea that ketamine derives at least part of its antidepressant efficacy by blocking the response to spontaneous glutamate release, numerous studies have investigated an important role of NMDARs containing the GluN2B subunit, which is selectively activated by spontaneous glutamate release . Pharmacological studies report that GluN2B-selective antagonists produce rapid antidepressant effects in depressed patients  and in rKetamine also interacts with yet another facet of the plasticity regulating machinery through the glycogen synthase kinase (GSK) pathway . GSK con-hydroxynorketamine (HNK), is sufficient to produce a robust antidepressant response, even though it was reported that this metabolite does not show binding affinity for the NMDAR [A recent line of research has called into question the conclusion that NMDA antagonism is the functional mechanism of ketamine, based on the finding that one particular metabolite of racemic  ketamine, he NMDAR . This enhe NMDAR . Howeverhe NMDAR . NeverthKetamine has numerous points of interaction with signaling pathways that lead to increased synaptic plasticity and dendritic spine growth via new translation of the proteins needed to form new synapses, including the AMPA receptor subunit GluA1 . Rodent The ways in which other antidepressants comport with or diverge from this understanding of the mechanism of ketamine can lead to a more robust understanding of the necessary and sufficient conditions to relieve depression. Several rapid-acting antidepressants have been discovered in the past 20 years, some with efficacy similar to that of ketamine. All seem to share the key downstream mechanism of synaptogenesis and enhanced plasticity, but they achieve this effect in a variety of ways .Most similar to ketamine are several agents that bind to NMDARs in different ways. As discussed above, the GluN2B selective modulators are reported to have antidepressant actions in depressed patients  and in rScopolamine is a muscarinic acetylcholine receptor (mAchR) antagonist that has been found to have a rapid antidepressant effect in humans at very low doses, 4 µg/kg . Like keIt is also worth noting that ketamine has some molecular effects in common even with monoaminergic antidepressants, suggesting that those agents may utilize similar plasticity dependent mechanisms but in an indirect, less efficient way that cause them to exert an antidepressant effect much more slowly. In mice that have been genetically modified to express a mutated form of BDNF that impairs its expression, the antidepressant effects of both ketamine  and fluoAs the intracellular signaling pathways activated by antidepressants come into sharper and more detailed focus, the circuit level effects of antidepressants are beginning to be understood, thanks to new tools like optogenetics that enable the manipulation of specific brain circuits. The mood-regulating parts of the human brain have long been studied as an interrelated cortical-limbic system, and research efforts have identified correlates of these areas in non-human primates and rodents . A key rIt has been shown recently that optogenetic stimulation of glutamatergic neurones in the mPFC of rats, with a time course and intensity similar to that of ketamine, produces a robust and long-lasting ketamine-like synaptic and antidepressant behavioral response. Further, infusion of ketamine directly into the rat infralimbic cortex, thought to be a correlate of the human mPFC, was sufficient to produce an antidepressant effect similar to what is achieved when the drug is given systemically, and neuronal silencing of infralimbic PFC blocks the effect of systemic ketamine . These sFurther studies have begun to examine the importance of glutamatergic projections from the mPFC to other brain regions, including the dorsal raphe nucleus (DRN) and hippocampus, given that pyramidal neurones in the mPFC send axons to many other regions of the brain. The mPFC to DRN projection has been investigated using optogenetic stimulation of axon terminals in the DRN along with simultaneous behavioral analysis. These studies showed that activation of this projection produces a real-time antidepressant effect in rats . In prelThe ventral hippocampus has also been found to be relevant to the antidepressant effect of ketamine. A recent study used DREADDs (designer receptors exclusively activated by designer drugs) to mimic ketamine’s antidepressant effect by stimulating the ventral hippocampus to mPFC pathway. Further, the pharmacological or optogenetic inactivation of this pathway was found to block ketamine’s effect . These rThe scientific understanding of the mechanisms of antidepressants has progressed tremendously in nearly two decades that have passed since the discovery of rapid-acting antidepressants and the key role of glutamate. The intracellular signals that underlie the action of rapid-acting antidepressants have been elucidated, but gaps remain. In the near future, more precise and nuanced genetic modifications in rodents may allow researchers to test hypotheses about the progression from NMDA antagonism, to BDNF release and mTORC1 activation, to synaptogenesis in the action of ketamine. There is even more room for discovery related to the specific cell types that mediate these effects and how they affect their local circuitry as cell-type-specific viral vector systems and genetic modifications become more widely available. Such approaches should yield more concrete answers about the relative importance of the pyramidal-cell disinhibition hypothesis compared with blockade of spontaneous glutamate transmission via GluN2B-containing NMDARs as ketamine’s primary mechanism of action. Finally, advances in optogenetic and chemogenetic techniques are providing new insights into the role of specific circuits in the function of antidepressants.