The Epstein–Barr virus (EBV), Kaposi sarcoma herpesvirus (KSHV) and human T-lymphotropic virus (HTLV-1) are lymphomagenic viruses with region-specific induced morbidity. The RIAL-CYTED aims to increase the knowledge of lymphoma in Latin America (LA), and, as such, we systematically analyzed the literature to better understand our risk for virus-induced lymphoma. We observed that high endemicity regions for certain lymphomas, e.g., Mexico and Peru, have a high incidence of EBV-positive lymphomas of T/NK cell origin. Peru also carries the highest frequency of EBV-positive classical Hodgkin lymphoma (HL) and EBV-positive diffuse large B cell lymphoma, not otherwise specified (NOS), than any other LA country. Adult T cell lymphoma is endemic to the North of Brazil and Chile. While only few cases of KSHV-positive lymphomas were found, in spite of the close correlation of Kaposi sarcoma and the prevalence of pathogenic types of KSHV. Both EBV-associated HL and Burkitt lymphoma mainly affect young children, unlike in developed countries, in which adolescents and young adults are the most affected, correlating with an early EBV seroconversion for LA population despite of lack of infectious mononucleosis symptoms. High endemicity of KSHV and HTLV infection was observed among Amerindian populations, with differences between Amazonian and Andean populations. Neoplasms of an infectious etiology account for about 16% of all cancers, which amounts to about two million cases per year, considering virus-, bacteria- and parasite-derived cancers. Interestingly, this number is significantly higher for developing countries, in which it can be as high as 30%, while in highly industrialized countries, such as the US, it can be as low as 5% [Virus-derived cancers almost always originate from established chronic viral infections, because the mechanisms of viral persistence in the infected host are compatible with oncogenesis. Indeed, all human oncogenic viruses express proteins, and/or non-coding RNAs with the capacity to transform cells in culture and induce cancer in transgenic animals . Viral oThe Epstein–Barr virus (EBV) was the first oncogenic virus discovered 56 years ago by Dr. Anthony Epstein and Dr. Ivonne Barr. EBV was initially observed in samples of Burkitt lymphoma (BL) coming from the equatorial Africa, which represents the perfect example of the unequal geographical distribution of neoplasms of infectious origin. While EBV is responsible for close to 100% of the BL originating in this region, EBV only accounts for about 20% of all BLs in developed countries (see below). We later understood that whilst EBV is evenly distributed around the world, the African BL is also associated with repetitive infections with Plasmodium falciparum, an important co-factor of this neoplasm and a parasite endemic to this area. Today, BL is classified within three distinct clinical types, endemic , sporadic  and immunodeficiency-associated .The world distribution of the oncogenic viruses varies significantly, while most adults are already infected with EBV, HPV and MCPyV indistinct of the geographic region, the others tend to be more prevalent in specific populations . Even toLatin America (LA) comprises the land from Mexico to Argentina and the Spanish speaking Caribbean, countries with a complex mix of geographies, climates, politics, cultures, ethnicities and different levels of socioeconomic development, and, in which, a high prevalence of oncogenic viruses, acute tropical diseases and malnourishment collide. Early epidemiological studies documented a high seroprevalence of KSHV and HTLV-1 in some regions of LA, and to this day, it is common to find in the scientific literature that these viruses are endemic to LA (see below). However, the loco-regional estimation of their prevalence and induced morbidity remains poorly known. The RIAL-CYTED harbors a multidisciplinary Ibero-American network of clinical and basic researchers created to form a platform of multi-center cooperation focusing on increasing our knowledge of lymphoma, particularly for more underdeveloped or developing regions. This network aims to improve the diagnosis and prognosis of these neoplasms throughout LA by way of homogenizing its identification and classification. With that purpose, in this review, we sought to systematically organize and analyze the literature related to the lymphotropic and lymphomagenic viruses EBV, KSHV and HTLV-1, in order to better understand their loco-regional distribution and the risk our population carries in terms of developing lymphoma.To this end, we explored three web search engines, PubMed, Google Scholar and SciELO, to access all related scientific publications in English, Portuguese and Spanish. Searches were done with the name of each LA country plus the name of each virus individually: Epstein–Barr virus or EBV; KSHV, KSV, HHV8 or Kaposi sarcoma virus; HTLV-1, ATLV or human T-cell lymphoma virus 1. All collected publications were read and classified according to the content; sero-epidemiological studies of KSHV and HTLV-1 viruses were considered, in order to discuss their association with lymphoma in specific regions, while EBV sero-epidemiological studies were discarded, since it is well known that there is a worldwide high prevalence in both developed and developing countries. In case reports and when series of lymphomas were included, we assessed the methods of viral diagnosis, prioritizing those studies in which the presence of the virus was addressed in the lymphoma sample by means of immune or molecular methods. The search was carried out from February 2019 to December 2019, and, although we aimed to include all papers found, regardless of the date of publication, many journals do not have online versions of the documents prior to the 1990s. Moreover, many old studies did not comply with more recent lymphoma classification. The results are disclosed in the next sections for each particular virus.EBV is a human gamma-1 herpesvirus usually persisting as a harmless passenger; its growth-transforming ability is linked to a range of lymphoproliferative lesions and malignant lymphomas . EBV-assEBV infection has been associated with the following lymphomas in addition to BL: Hodgkin lymphoma (HL), lymphomas in immunosuppressed individuals (post-transplant lymphoproliferative disorders (PTLD) and HIV-associated lymphoproliferative disorders and T-cell and NK-cell lymphomas) . FurtherThere are several series of lymphomas reported in this region, particularly at the end of last century, which, however, were only classified as Hodgkin and non-Hodgkin (NHL), and which obviously did not follow the latest WHO classification. HL has a bimodal age distribution, there is an early peak occurring in adolescents and young adults, and a late peak after 50 years of age in industrialized countries. Developing countries also exhibit a bimodal distribution of the disease, but the early peak starts before adolescence . In the We found 36 studies of HL in LA, including pediatric and adult series from 12 countries. Pediatric series from Argentina exhibited a profile compatible with early cHL  ,25,26,27There is only one large pediatric cohort of 42 cases published from Mexico, with a median age of 5 yrs at diagnosis, and in which a male predominance (2.5:1) was observed . EBV posIn line with the above-described data, a 94% of EBV association was observed in pediatric HL in Peru , while iBL is a highly aggressive B cell lymphoma, characterized by the translocation of the MYC oncogene to the immunoglobulin loci. It affects mostly children and is also more predominant in males . EBV-assDLBCL is a highly aggressive neoplasm that can arise in almost any location of the body. It is very rare in pediatric patients and young adults, but is one of the most common NHLs in older individuals. Due to the median age of presentation of 70 yrs, it is usually associated with underlying immunosuppression. In the former 2008 WHO classification , EBV-posIn the 2016 WHO classification of lymphoid neoplasms, the age factor was eliminated from the association of EBV with DLBCL, leading to the substitution of the modifier “elderly” with “not otherwise specified”  . TherefoAlthough EBV tropism is mainly of B cells and in healthy individuals exclusively localizes in B cells , the revCAEBV corresponds to a group of reactive LPDs associated with a heightened EBV infection lasting longer than IM, and with the potential to progress to a systemic lymphoma. The clinical picture is diverse and includes the indolent, localized cutaneous form hydroa vacciniform-like LPD (HVL-LPD), renamed by the revised 2016 WHO classification . Severaln = 25). Remarkably, no ethnic differences were found between the patients with T/NK or B cell presentation [Extranodal NK/T cell lymphoma (ENKTCL) is an EBV-positive lymphoma more commonly derived from NK cells. Similar to CAEBV lymphoproliferations, this type of lymphoma has been described mainly in East Asia and LA, in which the ethnic composition includes a high proportion of Amerindians. EBV infection is also confirmed in virtually all cases. Series of cases have been reported from México , Peru 1,106, Chientation . Figure KSHV is a gamma-2 herpesvirus closely related to EBV, also called human herpesvirus 8 or HHV8. KSHV is the etiological agent of Kaposi sarcoma (KS), multicentric Castleman disease (MCD) and primary effusion lymphoma (PEL). The revised WHO classification of hematopoietic and lymphoid tissues also included two entities associated with KSHV, the HHV8-positive diffuse large B-cell lymphoma, NOS and the HHV8-positive germinotropic lymphoproliferative disorder. KSHV-associated neoplasms usually develop in severely immunocompromised patients, and, as such, KS is, in many countries, an AIDS defining marker. The incidence of KS has sharply diminished after the introduction of anti-retroviral therapy, but has since stabilized in many countries . In regiMCD and PEL are extremely rare neoplasms. There are two main presentations of Castleman disease (CD), one in which reactive B cells only affect a single group of close lymph nodes, denoted as unicentric (UCD) and MCD, in which more than one group of lymph nodes are reactive. CD is also classified according to its microscopic morphology as hyaline-vascular , and plasma cell and plasmablastic . CD is not a true lymphoma, but rather a polyclonal B cell expansion and is thus considered an LPD. MCD is associated with HIV and KSHV co-infection, and if HIV is uncontrolled, MCD can have an aggressive clinical course and progress to an entity with cellular and viral monoclonality, in which it resembles a DLBCL . On the To our knowledge, there is no routine screening for KSHV infection in blood banks in LA countries, nor in the rest of the world, and the World Health Organization and the World Federation of Hemophilia do not recommend it . Thus, on = 25), Chile (n = 300) and Peru (n = 128) found 4%, 2.6% and 56.2% positivity, respectively [n = 497), finding a seropositivity of 42.4% [n = 171) found 1.2% KSHV positives [Argentina reports a prevalence of 3.6–7.4% ,162,163,ectively ,168,180.of 42.4% . Howeverof 42.4% ,183, incof 42.4% . There aositives . Mexico ositives ,186. Forn = 41) to 100%  seropositive [n = 339–1270) [n = 110) among Amerindian tribes from transitional zones in central Brazil from Amazonian to the savannah. On the other hand, 0–14% positivity has been found in Amerindians from the South of Brazil (n = 85) [n = 55) from Bolivia were 22% positive and 45% were positive in Paraguay [LA harbors distinct ethnic groups differentially distributed among different geographic regions in each country. Most urban populations are formed by mestizos and a minority of Caucasians, while some rural regions concentrate pure Amerindians or Afro-descendants. KSHV hyper endemicity is reported among LA indigenous populations and a hyper-endemicity area  is reported among tribes inhabiting the South American lowlands of the Amazonas and the savannah  ,197,198 n = 49) did not find any KSHV positive supporting the rarity of these kind of neoplasms [Although our main aim was to document the incidence of lymphomas mediated by tumor viruses in LA, there are no such studies for KSHV in many LA countries, with most studies being about the prevalence of KS in HIV/AIDS patients. Additionally, because many of the reports are from the 1980s and 1990s, the studies only addressed the overall frequency of lymphomas, and lymphomas were only classified as Burkitt, Hodgkin and non-Hodgkin. Thus, both MCD and PEL seem more common after HAART, but they may have been under-diagnosed in early studies. Furthermore, KSHV-associated neoplasms most often are reported based on their histological and clinical characteristics, lacking confirmatory tests for viral infection. KSHV-associated lymphomas are reported from Brazil, Argentina and Peru. We found five cases of MCD in Brazil, mostly of the plasmablastic variety, and one mixed plasmablastic and hyaline ,157,158.eoplasms . We founeoplasms ,172,173,eoplasms ,187. FigThere are five major KSHV genotypes described , mainly based on the hypervariable regions of the oncogenic K1 gene. Interestingly, in the series of studies from LA, while genotypes A, B and C ,201,202 HTLV belongs to the family Retroviridae, genus Deltaretrovirus, and, as such, its genome consists of two identical copies of positive sense single stranded RNA that during its life cycle is converted into double stranded DNA and inserted into the DNA of the human host cell. This integrated form is referred to as the provirus, and HTLV predominantly exists in the host as a cell-associated provirus. Indeed, infected cells hardly produce any virus and plasma viral load is usually undetectable. There are two main types of HTLV infecting humans, HTLV-1 that preferentially infects CD4+, but also CD8+ T-lymphocytes, and HTLV-2 that preferentially infects CD8+ T-lymphocytes. Two other HTLV types have been described in Africa, but today, it is not clear whether HTLV-3 and HTLV-4 can be transmitted from human to human or are exclusively zoonotic, and no clinical disease has been associated with these two novel isolates thus far .HTLV-1 and -2 are very similar in terms of genome structure and replication patterns, but they are strikingly different in terms of clinical impact and geographical distribution. HTLV-1 is present throughout the world with regions of high endemicity together with low prevalence in neighboring zones. Major endemic regions include Southwest of Japan, LA and some areas of West Africa ,209. TenHTLV-1 infection has been associated with adult T cell leukemia/lymphoma (ATLL) and a subacute myelopathy known as tropical spastic paraparesis or HTLV-1-associated myelopathy (TSP/HAM). Although most HTLV-1 carriers are asymptomatic, about 5% of them will develop clinical complications, including TSP/HAM and ATLL. Both diseases are severe and produce progressive incapacitation or death. In addition to ATLL and TSP/HAM, infection by HTLV-1 also leads to a broad spectrum of inflammatory diseases with significant morbidity, such as uveitis, arthritis, myositis, infective dermatitis, Strongyloides stercoralis infection and crusted scabies ,211. TheWhen assessing the viral prevalence, we found that the majority of studies are based on immunological methods that do not discriminate between HTLV-1 and HTLV-2 (referred to as HTLV). The Caribbean basin is usually considered endemic to HTLV infection, perhaps because early 1980s studies documented several cases of ATLL from black immigrants from the Caribbean islands ,213. TheZero to 0.39% HTLV positivity and no ATLL were found from Mexico ,342,343.Screening for HTLV infection in blood banks is mandatory in several South American countries; Brazil has done it since 1993, Peru since 1998, Uruguay since 2000, Argentina since 2005, Chile since 2009 and Colombia since 2014. This has allowed for studies based on a large number of samples, and for a more factual estimation of HTLV prevalence. In Caracas, Venezuela a 0.2% HTLV positivity and a rate HTLV-1: HTLV-2 of 11:1 was observed . Other sMultiple studies have addressed HTLV prevalence in Brazil and Argentina revealing regions of high endemicity. In the former, the highest prevalence was demonstrated in the North-North East region, in particular in Salvador de Bahia, ranging from 0.046% to 1.8% ,365,366,While the most pathogenic HTLV-1 strain is more prevalent among urban mestizo, Afro-descendants and Caucasian populations, HTLV-2 seems to be more prevalent among some Amerindian tribes. Interestingly, different endemic zones for HTLV-1 and HTLV-2 infections have been described among different tribes of Colombian Amerindians ,428,429,There are multiple reports of ATLL in South America, mostly describing a disease with similar features to Japan ATLL, but in younger individuals. In Colombia, 19 HTLV positive ATLL cases have been reported, 12 confirmed as HTLV-1, while, in the others, the HTLV subtype was not investigated ,248,249.The number of ATLL and TSP/HAM cases reported from Brazil is in agreement with a country of high HTLV endemicity. For instance, ATLL cases have been reported in the urban areas of North East Brazil, by and large in the Bahía state that presents the largest prevalence of HTLV-1 infection ,470,471.HTLV-1A has been found throughout South America ,486,487.The association between EBV infection and lymphoma varies worldwide. The most compelling observation is the higher incidence of EBV-positive cHL in LA than in industrialized countries, and also the relationship between the age at which EBV primo-infection occurs and the onset of cHL. In underdeveloped and developing LA populations, earlier exposure to EBV in the face of a relatively underdeveloped immune response might be a predisposing factor for EBV-positive cHL. Socioeconomic improvement in Brazil turned into a decreased incidence from 87% to 46% in a 54 year period . A similDDX3X, KMT2D, BCOR, BCORL1, TET2 and KM6A have been identified [EBV-associated T and NK LPDs represent a broad spectrum of diseases that occurs with higher incidence in Asian and LA populations. The incidence of these lymphomas in LA should grant EBV1 and EBV2 prevalence studies, particularly for Mexico and Peru, in which this lymphoma is highly prevalent. NKT LPDs often emerge within CAEBV disease in which infection of T- or NK- cells apparently occurred during primary infection. Genetic alterations are rare in CAEBV, but mutations in entified . Likewisentified . Gene exentified . No geneJAK3 (5–35%), STAT3 (6–27%) and STAT5B (2–6%) genes are characteristic of this lymphoma [TP53, as well as genes encoding proteins involved in epigenetic pathways  [STAT3 the most frequently mutated gene (22%). Mutations in BCOR, DDX3X  and TP53 were also identified, but with different frequencies than in Asian cohorts [ENKTL represents around 23% of all T-cell lymphomas in LA , compared with 4% to 5% in Europe and the United States. The clinical presentation is similar in all geographic regions; however, patients in LA tend to present with more advanced clinical stages . In the lymphoma ,499. Othd EP300) . In a st cohorts . These dWith respect to KSHV, available reports based on healthy populations and risk groups make it possible to infer an LA prevalence higher than in the US and non-Mediterranean Europe but lower than endemic regions of Africa and the Mediterranean. With the exception of Chile, LA exhibits global frequencies above 3%, and particularly above 10% for Cuba, Colombia, Argentina , Ecuadorian Afro-descendants and Peru. Indeed, large KS series  from Cuba, Colombia and Peru have been published ,501,502,In Amerindian tribes in which the KSHV genotype E is endemic, viral transmission starts very early, with 35 to 65% seropositives by age 10 ,197,505,While ATLL among Amerindians is very rare, TSP/HAM is commonly reported. TSP/HAM was first described in the Southern Pacific of Colombia in 1981 as a chronic non-hereditary spastic paraparesis, initially termed Pacific spastic paraparesis , later aReports of ATLL series were found only for Brazil and Chile see , which mWe observed a high incidence of EBV-positive lymphomas of T/NK cell origin for Mexico and Peru and of BL for North Brazil. Peru also carries the highest frequency of EBV-positive HL and DLBCL than any other LA country. The presentation of HL and BL was also earlier than for industrialized countries, coinciding with an early EBV primo-infection, in spite of there being no evidence of infectious mononucleosis in the region. High prevalence of KSHV and HTLV was found among Amerindian populations, with different distributions between low-land  and high-land  tribes. In spite of this, there were only scarce reports of MCD, PEL and ATLL. However, Peru reports KS as its third most common neoplasm.


These results may indicate that the PGC-1α induced VEGF/VEGFR2 pathway may trigger the neuronal survival signaling, and the PI3K/AKT and MEK/ERK signaling pathways. Thus, the axis of PGC-1α/VEGF/VEGFR2 (Flk-1) and the triggering of downstream PI3K/AKT and MEK/ERK signaling could be considered an endogenous neuroprotective effect against apoptosis in the hippocampus following status epilepticus. Status epilepticus may cause molecular and cellular events, leading to hippocampal neuronal cell death. Peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) is an important regulator of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2), also known as fetal liver kinase receptor 1 (Flk-1). Resveratrol is an activator of PGC-1α. It has been suggested to provide neuroprotective effects in epilepsy, stroke, and neurodegenerative diseases. In the present study, we used microinjection of kainic acid into the left hippocampal CA3 region in Sprague Dawley rats to induce bilateral prolonged seizure activity. Upregulating the PGC-1α pathway will increase VEGF/VEGFR2 (Flk-1) signaling and further activate some survival signaling that includes the mitogen activated protein kinase kinase (MEK)/mitogen activated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways and offer neuroprotection as a consequence of apoptosis in the hippocampal neurons following status epilepticus. Otherwise, downregulation of PGC-1α by siRNA against  Epilepsy is one of the most common serious brain conditions characterized by the recurrence of unprovoked seizures, affecting more than 70 million people worldwide . Status Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) have important roles in the formation, function, and maintenance of blood vessels and are essential regulators of angiogenesis and vascular permeability . Recent Both in the animal studies following sustained seizure activities ,19,23, aThe trigger for increased neuronal VEGF after seizures and brain insults is unclear. Beyond well-studied VEGF regulator HIF-1α ,18, actiRecent research showed that the upregulation of VEGF/VEGFR2 (Flk-1) occurred after sustained seizures and VEGF signaling offered neuroprotective effects against neuronal cell death in the hippocampus following status epilepticus ,19,23. WIn the present study, we propose that PGC-1α may be activated during experimental status epilepticus and regulate the VEGF/VEGFR2 signaling pathway, and further protects against apoptotic neuronal cell death in the hippocampus following status epilepticus through survival signaling and PI3K/AKT and MEK/ERK-dependent pathways.pgc-1α mRNA expression exhibited a significantly incremental change in the right hippocampal CA3 area 1 h after the elicitation of prolonged seizure activities, followed by a progressive decrement that returned to base line at 24 h  into the left CA3 subfield,  at 24 h a. For de at 24 h b.vegf mRNA extracted from the right hippocampal CA3 region was significantly augmented at 3 h, 6 h, and 24 h after administration of KA  into the hippocampal CA3 region significantly increased the expression of vegf mRNA  into bilateral hippocampal CA3 region, significant decrease of the vegf mRNA level was shown in our real-time PCR data  into the hippocampal CA3 subfield underwent a significant increment of expression of vegf mRNA  into the bilateral CA3 region, a significant elevation of the expression of vegfr2 mRNA  and AKT (p-AKT), and total levels of PIK3 and AKT were measured by western blotting analysis. The ratio of p-PI3K/total PI3K and p-AKT/total AKT were raised 6 h after microinjection of 3% dimethyl sulfoxide (DMSO) and KA into the left hippocampal CA3 region compared with controls  followed with KA microinjection into the hippocampus. Akt can be activated by p-PI3K and active PI3K  followed by KA administration into the left hippocampal CA3 region a,b. On tive PI3K c,d.pgc-1α and these proteins were analyzed by western blotting 6 h after KA-induced experimental status epilepticus. The ratio of p-ERK/total ERK and p-MEK/total MEK were increased 6h after microinjection of DMSO and KA into the left hippocampal CA3 region compared with control animals (pgc-1α (2 µg) followed by KA microinjection into the hippocampus  followed by KA microinjection into the hippocampus (pgc-1α (2 μg), followed by KA microinjection into the hippocampus.To further confirm the role of PGC-1α/VEGF/VEGFR2 signaling in neuroprotection from hippocampal damage due to prolonged seizure, we investigated the regulatory role of resveratrol on KA-induced hippocampal neuronal cell death. Significantly, pretreatment with resveratrol (100 μmol) attenuated the extent of cleaved caspases-3 expression in the hippocampal CA3 subfield 7 days after KA-induced status epilepticus a. Otherwpocampus b. Mitigapocampus a and quapocampus b analysepocampus c and quapocampus d analysiThe concept of neuroprotection under brain insults, such as prolonged seizures (status epilepticus), should consist of not only the conservation of structural neuronal cell death, but also preservation of neuronal networks and functions ,39. In rpgc-1α reduced VEGF and VEGFR2 expression and inhibited PI3K/AKT and MEK/ERK pathways, also accompanied by heightened caspase-3 activity, and augmented neuronal damage in the hippocampal CA3 subfield. We also noted that the mRNA levels for vegf and vegfr2 were reduced after pretreatment of siRNA against pgc-1α compared with the levels of control siRNA groups. This suggests an additional mechanism for seizure-induced activation following status epilepticus. Therefore, our results indicated that seizure activities triggered the upregulation of the VEGF/VEGFR2 (Flk-1) pathway, activation of PI3K/AKT and MEK/ERK signaling, and worked against the hippocampal neuronal apoptotic cell death following status epilepticus. Therefore, our results showed VEGF/VEGFR2 and related downstream PI3K/AKT and MEK/ERK signaling can exert endogenous neuroprotection in status epilepticus.The present study demonstrated that activation of PGC-1α activity regulated VEGF/VEGFR2 (Flk-1) signaling and showed the neuroprotective effect against neuronal cell death in the hippocampus following experimental status epilepticus. Particularly, we noted that PGC-1α expression was enhanced in the rat hippocampus following status epilepticus and upregulated VEGF and VEGFR2 (Flk-1) expression. Administration of the activator of PGC-1α, resveratrol, was accompanied by increased VEGF and VEGFR2 expression, and promotion of PI3K/AKT and MEK/ERK pathways and decreased neuronal cell death in the hippocampus following status epilepticus. Downregulation of PGC-1α by pretreatment of siRNA against Recently, our research revealed that PGC-1α is an important transcriptional regulator that acts through regulating the expression of reactive oxygen species (ROS), mitochondrial UCP2, superoxide dismutase 2, and mitochondrial biogenesis, which plays a beneficial part in neuroprotection in the CNS following global ischemia and status epilepticus ,28,41,47VEGFs are strong endothelial cell mitogens and major regulators of neurogenesis and angiogenesis ,56. In aVEGF can directly stimulate the proliferation of neuronal progenitors; however, the possible mechanism of seizure-induced activation of endogenous PGC-1α that promotes the following VEGF signaling that contributes to neuroprotection in the hippocampus following status epilepticus is not clear. Thus, we investigated two downstream pathways of the VEGF/VEGFR2 (Flk-1) signaling pathways that included PI3K/AKT and MEK/ERK signaling ,63,64,65Mitogen-activated protein kinases (MAPK) are considered to be a family of Ser/Thr protein kinases and conserved in eukaryotes and involved in many cellular programs. The MEK/ERK pathway is one of the MAPK cascades involved in transducing cell survival signals via growth factor receptors ,67. InteP2 (PIP2) to the second messenger, phosphatidylinositol -trisphosphate P3 (PIP3), on the plasma membrane. PIP3 directly or indirectly binds to AKT to induce structural changes and facilitate the phosphorylation of AKT amino acid residues under neurodegenerative diseases , where ΔΔCt = .The products of PCR were subsequently subjected to agarose gel electrophoresis for further confirmation of amplification specificity ,26,44. FWestern blot analysis was carried out on proteins extracted from total lysate or cytosolic fractions from hippocampal samples. The primary antiserum used included rabbit monoclonal or polyclonal antibodies against PGC-1α , VEGF , VEGFR2 , PI3 Kinase , Phospho-PI3 Kinase , AKT , Phospho-AKT , ERK1/2 , Phospho-ERK , Cleaved caspase-3 , and β-actin . The primary antibodies were diluted in 5% skimmed milk in TBST. The conditions for the membrane wash, detection of immunoreactive signals, and quantification of signal intensities on the blots were performed as previously described ,44,46. TDouble immunofluorescence staining ,44,46,84Hippocampal CA3 tissues were subject to measurement of the apoptotic DNA fragmentation following status epilepticus ,26,44,85p < 0.05 was considered statistically significant.The continuous variables were expressed as mean ± standard error of the mean (SEM). The statistical method was used to compare all the continuous variables with one-way analysis of variance (one-way ANOVA). Once the ANOVA was significant, for post hoc assessment, we used Scheffé multiple range test to assess the difference between groups, especially the experimental group and the control group. A pgc-1α will inhibit VEGF/VEGFR2 (Flk-1) signaling and suppress pro-survival PI3K/AKT and MEK/ERK pathways that also are accompanied by hippocampal CA3 neuronal cell apoptosis. These results may indicate that the PGC-1α induced VEGF/VEGFR2 pathway may trigger neuronal survival signaling and PI3K/AKT and MEK/ERK signaling pathways. Thus, the axis of PGC-1α/VEGF/VEGFR2 (Flk-1) and downstream PI3K/AKT and MEK/ERK signaling could be considered as an endogenous neuroprotective effect against apoptosis in the hippocampus following status epilepticus. Taking together, our previous research and the results from this study, new strategies for treatment, disease modification, and neuroprotection in epilepsy, particularly in status epilepticus, should be developed.In the present study, our results suggested activation of VEGF/VEGFR2 (Flk-1) by PGC-1α subsequently activates PI3K/AKT and MEK/ERK signaling by alternating the activation state of numerous of downstream proteins relevant to cell proliferation and inhibiting apoptosis in the hippocampus following status epilepticus. Otherwise, downregulation of PGC-1α by siRNA against to


Plants as sessile organisms are not able to move and must cope with adverse environmental conditions and stresses such as extreme temperatures, drought, high soil salinity, oxidative stress, pathogen attack, and so on. To respond in an appropriate manner to a specific environmental stimulus, plants possess signal transduction pathways, which are complex networks of interactions involving signal elements transmitting through the plant cell. In fact, cell signaling affects virtually every aspect of plant cell structure and function. For building elegant, complicated, and interconnected regulating networks, a huge number of components are involved, including receptors, secondary messengers, protein kinases, transcription factors, reactive oxygen species (ROS), and plant hormones that regulate or stimulate other components. Therefore, to unveil a global picture of plant cell signaling networks and underlying master regulators and machinery remains a challenge for researchers. For enriching our understanding of plant cell signaling with the assistance of modern molecular tools, this Special Issue “Cell Signaling in Model Plants” collects recent innovative original research and reviews, with an emphasis on the studies using model plants and crops. A total of 14 publications were published, which can be divided into five subtopics, as described below.A. thaliana to unveil how stress responses as well as plant development are regulated by signaling pathways related to different groups of RLKs [Transmembrane receptor-like kinases (RLKs) are conserved protein kinases that play critical roles in transducing the signal from the outside to the inside of plant cells, particularly to the nucleus. Jose et al. refined the recent advances of transmembrane RLKs in  of RLKs .FLOWERING LOCUS C (FLC) homologs in apple [FLC might be involved in flowering and associated with juvenility.To reveal the underlying mechanisms controlling the annual flowering of fruit trees, Kagaya et al. analyzed the function of in apple . They degai-1 mutant line of A. thaliana [gai-1 mutant line is more tolerant of drought than the wild type. They also found a strong interaction between GAI proteins and ABA-responsive element (ABRE)-binding transcription factors. Jung et al. characterized SLR1, encoding the DELLA protein in rice, using CRISPR/Cas-9 genome editing [20OX2 (gibberellin oxidase) and GA3OX2, as GA-related genes, was upregulated in the edited mutant lines. Finally, they indicated that in the created mutant lines, slr1-d7 and slr1-d8, cell elongation is limited. Gibberellin (GA) signaling plays a vital role in regulating plant growth and development. Wang et al. investigated the function of GAI-1, one of the proteins with the DELLA amino acid motif , as a negative regulator involved in GA signaling, using the thaliana . They in editing . They stMNP1 in Medicago truncatula as a model legume plant [mnp1 mutant line, the cell number of internodes and cell length are reduced. They also found that MNP1 is committed in GA biosynthesis.Dwarf phenotypes are widely used in crop breeding to increase resistance and yield. Guo et al. defined the function of me plant . They coA. thaliana [MicroRNAs (miRNAs) play a significant role in the regulation of gene expression. Ormancey et al. investigated the role of a miRNA-encoded peptide, miPEP165a, in Medicago truncatula using an ethylene-insensitive mutant called sickle mutant [AKR (Acyrthosiphon kondoi resistance), APR (Acyrthosiphon pisum resistance), and TTR (Therioaphis trifolii resistance).Ethylene is a gaseous phytohormone that is involved in response to biotic and abiotic stresses. Zhang et al. studied the effect of ethylene signaling on resistance to some aphids in e mutant . Their rBotrytis cinerea is a necrotrophic fungus causing grey mold disease in many plant species. Maqsood et al. examined the effect of iprodione, as a fungicide, on the molecular mechanisms of B. cinerea resistant mutant [t mutant . By analBrassinosteroids (BRs) as steroid hormones play critical roles in regulating the plant growth and development stages. Mao and Li reviewed the regulatory mechanisms of brassinosteroid-insensitive 1 (BRI1), BRI1-associated receptor kinase (BAK1), and brassinosteroid-insensitive 2 (BIN2), as three key kinases, involved in the BR signaling cascade . They stBRs have many interactions with other phytohormones that regulate the downstream pathways related to plant growth or response to stresses. Bulgakov and Avramenko reviewed the links between BR and abscisic acid (ABA) signaling that affect the stress-acclimation processes . They pr2+ signals through cyclic nucleotides-gated channels.The cyclic nucleotide, cAMP  as a signaling molecule, is involved in molecular processes linked to response to environmental stresses. Blanco et al. reviewed the current knowledge of cAMP signaling . They in2S) is a toxic gaseous molecule. Recent studies revealed that H2S plays a positive role in regulating plant growth. Xuan et al. considered the H2S roles in cellular processes and also indicated all possible crosstalk between H2S and phytohormones [2S may affect the protein activities and subcellular localization by contributing to post-translational modification. Hydrogen Sulfide (Hhormones . Interes+ ion accumulation through the expression induction of the Na+/H+ antiport and increasing the antioxidants.Selenium (Se) is identified as a beneficial element that can be included in improving the plant’s resistance facing adverse environmental conditions. Kamran et al. reviewed the protectant roles of Se in response to soil salinity . They in2+ and activation of anion channels.Tran et al. investigated the early cellular responses of bright yellow2 tobacco cultured cells under the application of calcite processed particles (CaPPs) . Their rThis Special Issue provides new and in-depth insights into molecular aspects of plant cell signaling in response to biotic, such as aphid- and grey mold disease-resistance, and abiotic stresses, such as soil salinity and drought stress, and additionally, functional analysis on signaling components involved in flowering, juvenility, GA signaling, and biosynthesis, and miRNA-regulated gene expression. Furthermore, plant acclimation was reported, with emphasis on mechanistic insights into the roles of brassinosteroids, cyclic AMP, and hydrogen sulfide, and the recent advances of transmembrane receptor-like kinases were refined. Clearly, plant cell signaling is an intensive topic and whether it is now or in the future, the emerging technology in functional analysis such as genome editing technologies, high-throughput technologies, integrative multiple-omics as well as bioinformatics can assist researchers to reveal novel aspects of the regulatory mechanisms of plant growth and development, and acclimation to environmental and biotic stresses. The achievement of such research will be useful in improving crop stress tolerances to increase agricultural productivity and sustainability for the food supply of the world.


The chloride-proton exchanger CLC-7 plays critical roles in lysosomal homeostasis and bone regeneration and its mutation can lead to osteopetrosis, lysosomal storage disease and neurological disorders. In lysosomes and the ruffled border of osteoclasts, CLC-7 requires a β-subunit, OSTM1, for stability and activity. Here, we present electron cryomicroscopy structures of CLC-7 in occluded states by itself and in complex with OSTM1, determined at resolutions up to 2.8 Å. In the complex, the luminal surface of CLC-7 is entirely covered by a dimer of the heavily glycosylated and disulfide-bonded OSTM1, which serves to protect CLC-7 from the degradative environment of the lysosomal lumen. OSTM1 binding does not induce large-scale rearrangements of CLC-7, but does have minor effects on the conformation of the ion-conduction pathway, potentially contributing to its regulatory role. These studies provide insights into the role of OSTM1 and serve as a foundation for understanding the mechanisms of CLC-7 regulation. Inside the cells of mammals, acidic compartments called lysosomes are responsible for breaking down large molecules and worn-out cells parts so their components can be used again. Similar to lysosomes, specialized cells called osteoclasts require an acidic environment to degrade tissues in the bone. Both osteoclasts and lysosomes rely on a two-component protein complex to help them digest molecules. Mutations in the genes for both proteins are directly linked to human diseases including neurodegeneration and osteopetrosis – a disease characterized by dense and brittle bones.For the main protein in this complex, called CLC-7, to remain stable and perform its roles, it requires an accessory subunit known as OSTM1. CLC-7 is a transporter that funnels electrically charged particles into and out of the lysosome, which helps to maintain the environment inside the lysosome compartment. However, due to the tight partnership between CLC-7 and OTSM1, how they influence each other is poorly understood.To determine the roles of CLC-7 and OSTM1, Schrecker et al. looked at the structure of the complex using a technique called single particle electron microscopy, which allows proteins to be visualized almost down to the individual atom. The analysis revealed that OSTM1 covers almost the entire inside surface of CLC-7, protecting it from the acidic environment inside the lysosome and contributing to its stability. When the two subunits are bound together, OSTM1 also slightly changes the structure of the pore formed by CLC-7, suggesting that OSTM1 may regulate CLC-7 activity.Schrecker et al. have laid the foundation for understanding more about the activity and regulation of CLC-7 and OSTM1 in lysosomes and osteoclasts. The structures described also help explain previous findings, including why OSTM1 is important for the stability of CLC-7. This coobic gap . Despite-/H+ exchange , for transport activity . CLC-7 aexchange . OSTM1 iexchange . However- cells as mEGFP-fusions, purified to homogeneity in the detergent lauryl maltose neopentyl glycol (LMNG), cholesterol hemisuccinate (CHS), 150 mM KCl and 50 mM Tris-HCl pH 8.0, and analyzed by cryo-EM. Vitrified human CLC-7 transporters displayed a strongly preferred orientation that was confirmed by two-dimensional classification (2) ggCLC-7 dimer interface  and human CLC-7, which are 86.4% identical, were expressed in HEK293S GnTIfication . Becausefication . Three-dfication . The finfication . Each prfication . Both thnterface .--conduction pathway and its three conserved Cl--binding sites  is at the luminal entrance and is formed by the side chains of Lys242, Glu467 and the backbone of Gly241. Together the three constrictions yield an occluded state for the transporter, sealing off the external and central Cl--binding sites from the cytosol and the lysosomal lumen.The transmembrane domain of ggCLC-7 adopts the canonical CLC architecture with each protomer possessing discrete ion permeation pathways that extend from the cytosol to the lysosomal lumen . Structung sites , and thi pathway . On the of 0.6 Å . The lumrictions . The firgate, whose conformation changes during the transport cycle. In previous structures of CLC transporters, the side chain carboxylic moiety of Glugate has occupied four different positions: ‘middle’ where it occupies the central Cl--binding site, ‘up’ where it moves toward the extracellular vestibule, ‘down’ where it occupies the central Cl- binding site and most recently ‘out’ where it reaches away from the Cl--conduction pathway toward the H+-conduction pathway (gate (Glu243) adopts the ‘up’ conformation, where it participates in establishing one of the luminal constrictions  is the strongest of the three Cl--binding sites and is nearly equivalent to that of backbone atoms of nearby residues, suggesting a high Cl- occupancy. The density for the central Cl- site has a slightly lower intensity (~12 σ) and is formed by the side chains of the highly conserved Ser200 from helix αD and Tyr598 on helix αS and the backbone nitrogens of Val509 and Phe510 of helix αN. The internal Cl--binding site, which is located in a solvent-exposed vestibule on the cytoplasmic side of the transporter, has the lowest intermediate intensity (~8 σ) and is formed by the backbone nitrogens of Ser200 and Gly201 and the side chain of Gln204, all on helix αD. The relative intensities of the three Cl--binding sites are consistent with structural and biochemical studies performed with E. coli CLC1 (ecCLC) that showed that the central and external binding sites have much higher affinity for Cl- ions than the internal site  on helix αG is located more than 15 Å away from Glugate, where it extends into a solvent filled cavity between the transmembrane and cytosolic domains that is continuous with the cytosol  is conserved in human CLC-7 as well as the closely-related human CLC-6 transporter (45% identity). The conservation is less clear in the more distantly related CLC-3, CLC-4 and CLC-5 transporters and no conservation is apparent in the CLC-1, CLC-2, CLC-Ka and CLC-Kb channels . Further- cell membranes. The PI3P molecule is located at the interface between the transmembrane domain and the cytosolic domain and interacts with residues from both domains. An amphipathic β-hairpin between helices αF and αG containing multiple positively charged residues surrounds the inositol phosphate head group and glycerol backbone, separating them from the rest of the membrane   . Based oatCLC-a) . In addi-/H+ exchange, we next co-expressed human CLC-7 and human OSTM1 in HEK293S GnTI- cells, purified the complex to homogeneity and analyzed its structure by cryo-EM. Similar to vitrified human CLC-7 by itself, human CLC-7/OSTM1 particles also adopted a preferred orientation in the ice. However, the effect was less severe for the CLC-7/OSTM1 particles and by collecting a large data set, we were able to resolve additional views. Two-dimensional and three-dimensional classification revealed the presence of intact CLC-7/OSTM1 complexes in the data set as well as a minor population of free CLC-7 dimers  on OSTM1 . As prevWe next compared the structure of ggCLC-7 with the structure of the human CLC-7/OSTM1 complex to determine how OSTM1 binding influences the conformation of CLC-7. Overall, the CLC-7 dimers show good alignment (RMSD 0.4 Å) in the presence and absence of OSTM1 . The cyt- and H+ transport, we compared the ion conduction pathways in the CLC-7/OSTM1 and ggCLC-7 structures  and Phe514 (Phe510 in ggCLC-7), both of which are highly conserved among CLC family members and whose mutation in human CLC-7 leads to defects in Cl-/H+ exchange coupling, voltage-dependence and activation  narrows the constriction of this pathway to a minimum radius of 0.8 Å, which is too narrow to allow water molecules to enter the hydrophobic gap  is potent modulator of ion transport proteins whose abundance is tightly regulated (2 concentrations are very low (<0.1% of cellular PI content), but in yeast can rise more than 20-fold upon hyperosmotic shock P2 on atCLC-a. Consistent with the PI lipids influencing transporter activity, a mutation of Tyr715, which is located near the PI3P-binding site, to cysteine was recently identified in the gene encoding human CLC-7 that causes a novel lipid storage disease without osteopetrosis  lipid species enriched in endolysosomal membranes that constitutes between 0.1% and 0.5% of the total PI content in cells . While Pegulated . Under bic shock . In endol TRPML1  and the nel TPC1 . In planf ~10 nM . Becauses effect . In the petrosis . Functiopetrosis .For decades CLC-7 was perhaps the most enigmatic CLC family member . FunctioCLCN7 from Gallus gallus was synthesized (SynBio) and subcloned into a BacMam expression vector with a C-terminal mEGFP-tag fused via a short linker containing a PreScission protease site (– cells (ATCC: CRL-3022). For a 1 L cell culture 1 mg plasmid and 3 mg PEI 25K were used. After 24 hr incubation at 37 °C, sodium butyrate (Sigma) was added to a final concentration of 10 mM, and cells were allowed to grow at 37 °C for an additional 72 hr before harvesting. Cell pellets were washed in phosphate-buffered saline solution and flash frozen in liquid nitrogen. Membrane proteins were solubilized in 2% lauryl maltose neopentyl glycol , 0.2% cholesteryl hemisuccinate tris salt , 20 mM HEPES pH 7.5, 150 mM KCl supplemented with protease-inhibitor cocktail  and spatula of DNaseI for 1 hr. Solubilized proteins were separated by centrifugation 75,000 g for 40 mins, followed by binding to 2.5 ml anti-GFP nanobody resin for 1 hr, which was equilibrated with washing buffer containing 0.1% LMNG, 50 mM Tris-HCl pH 8, 150 mM KCl, 2 mM DTT (BufferA). Anti-GFP nanobody affinity chromatography was performed by 20 column volumes of washing with BufferA, followed by overnight PreScission digestion, and elution with wash buffer. Protein sample was concentrated to a volume of 500 µl using CORNING SPIN-X concentrators (100 kDa cutoff), followed by centrifugation 10,000 g for 10 mins. Concentrated proteins were further purified by size exclusion chromatography on a Superose 6 Increase 10/300 GL  in BufferA. Peak fractions were pooled and concentrated to ~2 mg/mL using CORNING SPIN-X concentrators (100 kDa cutoff).The gene encoding ase site . The plaCLCN7 and OSTM1 were synthesized (SynBio) and subcloned into BacMam expression vectors with C-terminal mCerulean- and mVenus- tags, respectively, fused via a short linker containing a PreScission protease site  was added to induce expression at a final concentration of 2.2 mM. Equal amounts of plasmids encoding CLCN7 and OSTM1 were added to the reaction mix. Cell pellets were washed in phosphate-buffered saline solution and flash frozen in liquid nitrogen. Membrane proteins were solubilized in 2% lauryl maltose neopentyl glycol , 0.2% cholesteryl hemisuccinate tris salt , 20 mM HEPES pH 7.5, 150 mM KCl supplemented with protease-inhibitor cocktail  and spatula of DNaseI for 1 hr. Solubilized proteins were separated by centrifugation 75,000 g for 40 min, followed by binding to 2.5 ml anti-GFP nanobody resin for 1 hr, which was equilibrated with washing buffer containing 0.01% LMNG, 50 mM Tris-HCl pH 8, 150 mM KCl, 2 mM DTT (BufferB). Anti-GFP nanobody affinity chromatography was performed by 20 column volumes of washing with BufferB, followed by overnight PreScission digestion, and elution with wash buffer. Protein sample was concentrated to a volume of 500 µl using CORNING SPIN-X concentrators (100 kDa cutoff), followed by centrifugation 10,000 g for 10 min. Concentrated proteins were further purified by size exclusion chromatography on a Superose 6 Increase 10/300 GL  in BufferB. Peak fractions were pooled and concentrated to ~2.5 mg/mL using CORNING SPIN-X concentrators (100 kDa cutoff).Genes encoding human ase site . TransieGallus gallus (ggCLC-7), 3 μl of purified protein at a concentration of 2 mg/ml was applied to glow-discharged Au 400 mesh QUANTIFOIL R1.2/1.3 holey carbon grids (Quantifoil), and then plunged into liquid nitrogen-cooled liquid ethane with a FEI Vitrobot Mark IV (FEI Thermo Fisher). Grids were transferred to a 300 keV FEI Titan Krios microscopy equipped with a K2 summit direct electron detector (Gatan). Images were recorded with Leginon (2.For CLC-7 from  Leginon  in superHomo sapiens (hsCLC-7/OSTM1), 3 μl of purified protein at a concentration of 2 mg/ml was supplemented with 1 mM ATP and 0.1% LMNG and was applied to glow-discharged Au 400 mesh QUANTIFOIL R1.2/1.3 holey carbon grids (Quantifoil), and then plunged into liquid nitrogen-cooled liquid ethane with a FEI Vitrobot Mark IV (FEI Thermo Fisher). Grids were transferred to a 300 keV FEI Titan Krios microscopy equipped with a K3 summit direct electron detector (Gatan). Images were recorded with SerialEM  of ggCLC-7 were gain corrected, Fourier cropped by two and aligned using whole-frame and local motion correction algorithms by MotionCor2  1.0723 . Approxi40-frame super-resolution movies (0.532 Å/pixel) of hsCLC-7/OSTM1 complex were gain corrected, Fourier cropped by two and aligned using whole-frame and local motion correction algorithms by MotionCor2 (1.064 Å/pixel). Approximately 500 particles were manually selected to generate initial templates for autopicking that were improved by several rounds of two-dimensional classification in Relion and autopicking using Relion, resulting in 15,288,379 particles. False-positive selections and contaminants were excluded through iterative rounds of heterogeneous classification in cryoSPARC v2 using models generated from the ab initio algorithm in cryoSPARC v2, resulting in a stack of 932,232 particles. Heterogeneous classification in cryoSPARC v2 was then used to identify 327,619 particles displaying the luminal, transmembrane and cytosolic domains. After particle polishing in Relion and local CTF estimation and higher order aberration correction in cryoSPARC v2, a reconstruction was determined to 2.8 Å. 3D variability analysis in cryoSPARC v2 was then employed to characterize conformational heterogeneity . To inteCyanidioschyzon merolae (cmCLC) (The structure of CLC from  (cmCLC)  was manu (cmCLC) . The mod (cmCLC) . Atomic  (cmCLC) .The refined ggCLC-7 structure was manually docked into the CLC-7/OSTM1 density map using Chimera . The humFigures were prepared with UCSF Chimera , UCSF Ch In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The structures presented in this manuscript provide interesting new insights into this fascinating family of membrane transporters and channels. They show binding sites for ATP, which is known to regulate some CLCs, and for phosphatidyl inositol, which is not known to regulate CLCs but is of interest given the conserved binding site. In addition, the structures demonstrate how OSTM1 likely stabilizes CLC-7 in lysosomes and osteoclasts, where CLC-7 is expressed physiologically.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Raimund Dutzler (Reviewer #2).Thank you for submitting your article "Cryo-EM structure of the lysosomal chloride-proton exchanger CLC-7 in complex with OSTM1" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:Schrecker et al. present cryo-EM structures of the CLC-7 chloride/proton antiporter. Chicken CLC-7 is solved without the accessory subunit OSTM1, and human CLC-7 is solved in complex with OSTM1. These are the first structures of a mammalian CLC transporter homolog and the first of any CLC with an accessory subunit. The data are of high quality. The structures reveal a mechanism by which OSTM1 stabilizes CLC-7 in lysosomes and osteoclasts . The cytoplasmic domain of ClC-7 carries a nonprotein density that the authors suggest, with strong support, represents a bound Mg-ATP. A highly interesting aspect of the new structure is another bound ligand, modeled as the phophoinositide PI3P. Although identification of PI3P in the sample by chemical methods or mass-spectrometry would further confirm its presence in the sample, the proposed binding model is reasonable given the shape of the residual density and the abundance of basic amino acids in the pocket; thus, additional support of this model is not essential at this time. The identification of this putative site for phosphatidyl inositol, which is not known to regulate CLCs, is of high interest given the conserved nature of the binding site. Altogether, these structures offer important new insights into CLC-7 molecular properties and provide a template for future structure-based functional investigations.Essential revisions:1) The moderate effects of OSTM1 binding to the human CLC-7 structure, as judged from the comparison with the chicken CLC-7 structure, is remarkable. The proposal that the OSTM interaction might alter the conformational properties of two Phe residues in vicinity of the ion binding path is interesting but it could also be a consequence of local differences between the two different orthologs. Moreover, the claim that this conformational difference might be connected to increased transport activity of the complex is noteworthy but speculative.2) The authors point out the heterogeneity in orientation of the lumenal domain of OstM1, yet we see only one orientation in the final structure. It would be interesting for the reader to know something about the scale of this heterogeneity--is it tilting by a few angstroms or rocking strongly back and forth? Would it be possible to provide some kind of analysis to reveal something like the envelope of possible orientations? How might all this impact ion access to the transporter? Could there be electrostatic effects of OstM1?E. coli occupied in the structure of chicken CLC-7. It is also interesting to see that the density of the central Cl- gets weaker in the structure of the human CLC-OSTM1 complex but it is unclear whether this reflects a lower occupancy or higher mobility of the ion or whether it is a consequence of the slightly lower quality of the data.3) The assignment of residual density in the data to bound chloride ions is justified in light of previously characterized ion binding sites of other CLC proteins. It is very interesting to see all three binding sites identified in the CLC from 2+ is convincing and provides novel insight into the interaction, which was not revealed in the structure of the isolated domain of CLC-5. The assumption that ATP would bind with higher affinity to full-length CLC-7 than to the isolated cytoplasmic domain of CLC-5, is supported by the fact that the nucleotide is copurified with the sample. In light of the high intracellular ATP concentration, the proposal that ATP might form an integral part of the structure that remains bound instead of a regulatory ligand which dissociates from the protein is reasonable but would ultimately have to be characterized in functional experiments. In that respect it might be interesting to know whether a similar role of ATP as structural component has been found in other proteins.4) The interaction of ATP with the cytoplasmic domain of CLC-7 is exciting and it provides further evidence on the role of nucleotide binding to the cytoplasmic domains of certain CLC transporters. The interaction of the nucleotide with the N-terminus of the protein, which coordinates phosphate groups and Mg5) The PI3P in the chicken structure is very interesting and novel, yet the authors say nothing about whether they did or did not observe a similar lipid in the human structure. Especially given their argument about the conservation of the lipid binding site, it is worth noting explicitly what they see here. In the same vein, and considering their argument about PIP2, could there be differences between homologs in this site?6) The non-protein density modeled as CHS is less convincing than the modeling of the PI3P site, and its model displayed in Figure 5—figure supplement 2 appears distorted. Essential revisions:1) The moderate effects of OSTM1 binding to the human CLC-7 structure, as judged from the comparison with the chicken CLC-7 structure, is remarkable. The proposal that the OSTM interaction might alter the conformational properties of two Phe residues in vicinity of the ion binding path is interesting but it could also be a consequence of local differences between the two different orthologs. Moreover, the claim that this conformational difference might be connected to increased transport activity of the complex is noteworthy but speculative.We thank the reviewer for the acknowledgement of their positive assessment of our work. We agree with the reviewers that it is possible that the subtle differences in the resolved conformations of the phenylanine side chains may be due to local differences between the human and chicken orthologs. We also agree with the reviewers that the connection between the conformation of the phenylanine residues and increased transport activity is highly speculative, and we have modified the manuscript in the subsection “Effects of OSTM1 binding to CLC-7” to note these points.2) The authors point out the heterogeneity in orientation of the lumenal domain of OstM1, yet we see only one orientation in the final structure. It would be interesting for the reader to know something about the scale of this heterogeneity--is it tilting by a few angstroms or rocking strongly back and forth? Would it be possible to provide some kind of analysis to reveal something like the envelope of possible orientations? How might all this impact ion access to the transporter? Could there be electrostatic effects of OstM1?We agree with the reviewer that it would be beneficial for the reader to include more details on the nature of OSTM1 movement. For a deeper understanding of the heterogeneity of the CLC-7/OSTM1 complex, we performed 3D variability analysis using CryoSPARC without imposing symmetry. One of the primary components of heterogeneity identified was a rotation of the luminal domain of OSTM1 with respect to CLC-7. We have added a new figure, Figure 5—figure supplement 2, which compares the two extremes states with the midpoint. To quantify the displacement of OSTM1 in the three states, we docked in and rigid-body fit the luminal domain of OSTM1 separately from CLC-7. When aligned by the transmembrane domains, the periphery of luminal domain is displaced by up to 6 Å when the two extreme states are compared. The displacement is sufficient to blur the high-resolution features of the density map, but is unlikely to obstruct access of ions to the conduction pathway and thus it is unclear if the flexibility has any functional consequences.E. coli occupied in the structure of chicken CLC-7. It is also interesting to see that the density of the central Cl- gets weaker in the structure of the human CLC-OSTM1 complex but it is unclear whether this reflects a lower occupancy or higher mobility of the ion or whether it is a consequence of the slightly lower quality of the data.3) The assignment of residual density in the data to bound chloride ions is justified in light of previously characterized ion binding sites of other CLC proteins. It is very interesting to see all three binding sites identified in the CLC from - ions CLC-7/OSTM1 density map is hindered by its anisotropy and the resulting lower quality. Thus, we can only speculate that the lower density of the central Cl- ion is a result of lower occupancy or higher mobility. We have revised the text to “However, unlike in ggCLC-7, the central site in CLC-7/OSTM1 is the weakest and is only slightly above the background (~4 σ). While we must be cautious in interpreting the densities occupying the Cl- binding sites of CLC-7/OSTM1 because of the anisotropy present in the data set, the differences in relative intensities of the Cl- binding site peaks between ggCLC-7 and CLC-7/OSTM1 suggest that there may be a change in Cl- occupancy of the central site when CLC-7 is bound to OSTM1. A change in occupancy of the central Cl- site may be associated…”We agree with the reviewer that our ability to interpret the densities that we attribute to the Cl2+ is convincing and provides novel insight into the interaction, which was not revealed in the structure of the isolated domain of CLC-5. The assumption that ATP would bind with higher affinity to full-length CLC-7 than to the isolated cytoplasmic domain of CLC-5, is supported by the fact that the nucleotide is copurified with the sample. In light of the high intracellular ATP concentration, the proposal that ATP might form an integral part of the structure that remains bound instead of a regulatory ligand which dissociates from the protein is reasonable but would ultimately have to be characterized in functional experiments. In that respect it might be interesting to know whether a similar role of ATP as structural component has been found in other proteins.4) The interaction of ATP with the cytoplasmic domain of CLC-7 is exciting and it provides further evidence on the role of nucleotide binding to the cytoplasmic domains of certain CLC transporters. The interaction of the nucleotide with the N-terminus of the protein, which coordinates phosphate groups and Mg2+-coordination of CLC-7. We absolutely agree that further studies are required for the ultimate characterization of CLC-7 regulation by ATP/Mg2+. Whole-cell recordings by Leisle et al., 2011 showed that CLC-7/OSTM1 remains fully active in the absence of ATP, however due to the predicted slow off-rate, endogenous ATP likely remained bound in their recordings.We appreciate the acknowledgement of the interest regarding the ATP-Mg3 receptors and some of the prokaryotic RCK domain containing channels.We have included in the Discussion several examples of proteins that are proposed to bind nucleotides as structural components including AMPK, IP5) The PI3P in the chicken structure is very interesting and novel, yet the authors say nothing about whether they did or did not observe a similar lipid in the human structure. Especially given their argument about the conservation of the lipid binding site, it is worth noting explicitly what they see here. In the same vein, and considering their argument about PIP2, could there be differences between homologs in this site?We thank the reviewers for pointing out that we were unclear in our presentation of the ligands in the CLC-7/OSTM1 complex. To more clearly discuss the PI3P binding site within the CLC-7/OSTM1 complex. We have introduced a new figure  depicting an overlay of the ATP and lipid binding pockets of CLC-7 and CLC-7/OSTM1. We also show the density for the lipids in Figure 5—figure supplement 3. In the text we now state “Moreover, densities corresponding to ATP and PI3P molecules were resolved in their respective binding sites and the ligands interact with CLC-7 in a similar fashion regardless of the presence or absence of OSTM1 ” to emphasize that the ligand binding sites are conserved.6) The non-protein density modeled as CHS is less convincing than the modeling of the PI3P site, and its model displayed in Figure 5—figure supplement 2 appears distorted.We agree with the reviewer that modeling of CHS into the density is speculative. To avoid introducing potential errors into the model, we have removed the CHS molecule and have revised the text to “In addition to the direct protein-protein interactions, a non-protein density that may correspond to a cholesterol based on its size and shape was resolved at the interface between transmembrane domains of CLC-7 and OSTM1 . Notably, no density was present at this site in the ggCLC-7 map, suggesting that CLC-7 and OSTM1 together may form an additional lipid binding site.”. We have depicted the position of the density in the complex in a new figure .


Chlamydia species are causative agents of sexually transmitted infections, blinding trachoma, and animal infections with zoonotic potential. Being an obligate intracellular pathogen, Chlamydia relies on the host cell for its survival and development, subverting various host cell processes throughout the infection cycle. A key subset of host proteins utilized by Chlamydia include an assortment of host kinase signaling networks which are vital for many chlamydial processes including entry, nutrient acquisition, and suppression of host cell apoptosis. In this review, we summarize the recent advancements in our understanding of host kinase subversion by Chlamydia. Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae. C. trachomatis causes infections of ocular and genital tract epithelium .,86.Chlamd above) ,31,32,34ignaling . It was 1 (BAG1) ,53,58. Dnclusion . 14-3-3βed cells . Downstrpoptosis .C. trachomatis inclusion has been implicated to contribute to apoptosis resistance [C. pneumoniae Inc Cpn1027 has been shown to interact with members of the WNT-pathway, cytoplasmic activation/proliferation-associated protein-2 (CAPRIN2) and GSK3β. This interaction may contribute to apoptosis resistance by activating the transcription of pro-survival genes [C. trachomatis infection [C. trachomatis inclusion [Chlamydia’s requirements are met without triggering host apoptosis.PKCδ sequestration to the sistance . Recruital genes . Interesnfection . The T3Snclusion , howeverChlamydia has been epidemiologically linked with development of inflammatory diseases. C. trachomatis infection can lead to sequelae such as PID, infertility, and has been associated with development of cervical cancer [Chlamydia by the host cell results in an inflammatory response which is required for bacterial clearance but is also responsible for the immunopathology of chlamydial infections [C. trachomatis induces proinflammatory interleukin-8 (IL-8) during infection via activation of ERK and p38 kinase signaling [Chronic infection with l cancer ,5. Recogfections ,86,89. Cignaling ,50. Contignaling . It has ignaling . The eveC. trachomatis promotes host DNA damage by eliciting reactive oxygen species production while impairing the DNA damage responses (DDR). Even with impaired DDR, C. trachomatis keeps its host proliferating via ERK signaling  creating an environment that may predispose the host cell to malignant transformation [C. trachomatis has also been shown to reduce the levels of tumor suppressor protein p53 via PI3K/AKT signaling activation during infection to overcome DNA-damage-driven cytotoxic response [C. trachomatis induced epithelial-mesenchymal transition (EMT) has been implicated in molecular pathogenesis of sequelae associated C. trachomatis infections and its role as a risk factor in the development of cervical cancer [C. trachomatis infected cells demonstrated that C. trachomatis induced EMT was found to be mediated by ERK dependent phosphorylation of ETS2 repressor factor (ERF) and proto-oncogenic transcription factor ETS1 [C. trachomatis infected cells. Thus, ERK signaling is important for regulation of transcription factors that control EMT.ormation . C. tracresponse . C. tracl cancer . A combitor ETS1 . InhibitC. pneumoniae has been associated with atherosclerosis and found in atherosclerotic lesions suggesting a causal role in atherogenesis [C. pneumonaie-induced signal transduction pathways in multiple cell types relevant to atherogenesis. Endothelial cells infected with C. pneumonaie exhibited increased phosphorylation of p44/p42 MAPK and increased activation/translocation nuclear factor–κB (NF-κB) [C. pneumoniae in vascular smooth muscle cell (VSMC) proliferation. VSMC proliferation is responsible for intimal hyperplasia in early atherscelotic lesions. Specifically, C. pneumoniae and its HSP60 induces the proliferation of human VSMCs via Toll like receptor (TLR)4-mediated activation of p44/p42 (ERK1/2) kinase signaling [C. pneumoniae induced proliferation of coronary artery smooth muscle cells [C. pneumoniae HSP60 induces the inflammatory response via activation of MAPK kinase 3 (MKK3)/p38 signaling enhancing lung inflammation [ogenesis ,7. Early (NF-κB) . Heat sh (NF-κB) ,93. Suppignaling . The rolle cells . Additioammation . Furtherammation .Chlamydia subverts many host kinase signaling pathways to ensure its survival. MEK/ERK and PI3K signaling pathways are among the most prominent kinase signaling networks utilized by Chlamydia in regulating entry, host cell apoptosis resistance, immune response, and even pathology associated with chlamydial infections. Several chlamydial effectors likely interact with but may also phosphorylated by host kinases during infection suggesting their activity may dependent on host signaling pathways. Elucidating the role of chlamydial effector phosphorylation is an intriguing topic for future studies.Host kinases regulate host cell processes by phosphorylation of their target proteins. From entry and intracellular development to exit from the host cell,


During pregnancy, the maternal cardiovascular system undergoes significant changes, including increased heart rate, cardiac output, plasma volume, and uteroplacental blood flow (UPBF) that are required for a successful pregnancy outcome. The increased UPBF is secondary to profound circumferential growth that extends from the downstream small spiral arteries to the upstream conduit main uterine artery. Although some of the mechanisms underlying uterine vascular remodeling are, in part, known, the factors that drive the remodeling are less clear. That higher circulating levels of estrogens are positively correlated with gestational uterine vascular remodeling suggests their involvement in this process. Estrogens binding to the estrogen receptors expressed in cytotrophoblast cells and in the uterine artery wall stimulate an outward hypertrophic remodeling of uterine vasculature. In preeclampsia, generally lower concentrations of estrogens limit the proper uterine remodeling, thereby reducing UPBF increases and restricting the growth of the fetus. This review aims to report estrogenic regulation of the maternal uterine circulatory adaptation in physiological and pathological pregnancy that favors vasodilation, and to consider the underlying molecular mechanisms by which estrogens regulate uteroplacental hemodynamics. In women, UPBF increases from 20–50 mL/min in the nonpregnant state up to 450–800 mL/min by late gestation , w, wCa patdilation  113]. A. ACa patUASMC are a target for estrogens that exert direct, acute vasorelaxant effects mediated by different mechanisms. In non-pregnant rats, this is due to nitric oxide produced from muscle (NOS1) in an ER-independent manner  116]..116].2+-activated K+ (BKCa) channels [2+ release events through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum of SMC. This type of Ca2+ release, termed sparks, activates BKCa channels, causing spontaneous transient outward currents (STOCs), and hyperpolarizing currents  and NO production. (2) Estrogens upregulate the receptors for adrenomedullin, RAMP1, and receptors for calcitonin gene-related peptide, CRLR, the former contributing to NO production. Estrogen by its receptors, ERα and ERβ, activates the ERK1/2-PI3K -eNOS phosphorylation (eNOS-P) pathway and increases NO production. (3) Estrogens by ERβ activate angiotensin II receptors (AT2R), which contributes to NO production; (4) Estrogens by upregulation of vascular endothelial growth factor (VEGF) induce hydrogen sulfide (H2S) production; (5) Estrogens activate the phospholipase A2 (PLA2)–cyclooxygenase 1 pathway to produce prostacyclin (PGI2). Preeclampsia (PE) inhibits the estrogens, RAMP1-NO and also ERs-ERK1/2-PI3K and eNOS-P-NO.In EC (from left to right in Ca++); (c) CRLR, which by the cyclic nucleotides cAMP and cGMP activates BKCa++; (d) demethylation of the BKβ1 subunit and activation of BKCa++; (e) H2S production; (f) ERK1/2, which inhibits PKC and actin polymerization. Hypoxia inhibits BKβ1 demethylation and also estrogens activation of ERK1/2.In SMC (from left to right in baboon pregnancy significantly decreased maternal serum estrogens, although UPBF and fetal growth were maintained at normal levels [Several recent studies have reported lower plasma estrogen concentrations in preeclamptic women compared with those having a normal pregnancy ,121,122.l levels .The lower circulating estrogen in PE prompted researchers to validate estrogen levels as a biomarker but also as a potential treatment for PE. Results from studies that have considered estrogen concentrations in pregnancy with other high risk factors to predict the risk for development of PE showed only a modest sensitivity and specificity . A theraovine UA [Low plasma estrogen concentrations were associated with shallower spiral artery invasion ,138, insovine UA , althougovine UA ,141. In ovine UA . sFlt1 iovine UA ,144,145.ovine UA ,147. sFlovine UA ,149. Theovine UA . Furtherovine UA . Becauseovine UA .Ca channels and reduces relaxations of UA [Ca channel inhibition in PE also occurs by DNA methylation of the BKβ1 subunit, an epigenetic mechanisms that is promoted by hypoxia (In PE UA, vascular tone is higher compared to normal pregnancy due to decreased endothelium-dependent relaxation, partly as a consequence of lowered response to NO-mediated dilation and augmented response to numerous constrictors ,153. Sevns of UA . BKCa ch hypoxia  [156,157In normal pregnancy, estrogens may, at least in part, drive maternal uterine vascular remodeling by influencing mechanisms associated with UA circumferential growth during pregnancy. In particular, estrogens play a role in mediating cytotrophoblast invasion of spiral arteries and subsequent increases in blood flow and shear stress. Estrogens have also been shown to act directly on EC to activate synthesis and production of vasodilators and growth factors such as NO and VEGF. The lower plasma estrogen levels and downregulation of ERs that have often been noted in preeclampsia may further attenuate spiral artery invasion, uterine vasodilation, and remodeling and contribute to placental underperfusion. A better understanding of the mechanisms by which estrogen regulates uterine vascular remodeling and uteroplacental hemodynamics could help in the development of new therapeutic approaches for the prevention and/or management of preeclampsia.


We previously constructed a perspiration ratemeter for the measurement of palmar sweating in human subjects. Although galvanic skin response (GSR) has been used to evaluate emotional responses in human subjects, little is known about the relationships between the phasic and baseline components in GSR and active palmar sweating. From the aforementioned, we aimed to investigate the relationships in human subjects with handgrip exercise and eyes closing or opening. Fifteen healthy volunteers (mean age: 26.9 ± 8.7 years) participated in the present experiments. We investigated the effects of maximal handgrip exercise, eyes closing or opening, and self-awareness of drowsy on the GSR, active palmar sweating, R-R interval in electrocardiograph (ECG), and percentage of α wave in EEG. The faster phasic component in GSR completely agreed with the starting point of active palmar sweating. Handgrip exercise induced significantly faster spike in GSR, active palmar sweating, and decrease in R-R interval in ECG. Eyes closing produced significant decreases in baseline GSR and active palmar sweating in all human subjects. The percentage of α wave in electroencephalograph (EEG) also increased. In contrast, eyes opening increased significantly the baseline GSR and active palmar sweating. In the equivalent electrical model of human skin, the eyes closing–mediated time-dependent decrease in the baseline GSR completely agreed with the hypothesis that the palmar skin voltage only in the model decreased time dependently to 0.4 of the control during 6 min. The self-awareness of drowsy in mid-night working with computer produced similar decreases in baseline GSR and active palmar sweating to the responses with eyes closing in all human subjects. In conclusion, the faster spike in GSR completely agreed with the starting point of active palmar sweating. Eyes closing and opening or self-awareness of drowsy significantly produced changes in baseline GSR and active palmar sweating, which may become useful tools for evaluating clearness or drowsiness in human subjects. Previously, we designed and constructed a high-sensitivity perspiration ratemeter using capacitive humidity sensors which is applicable to the direct measurement of palmar sweating in human subjects . The safIn contrast, galvanic skin response (GSR) is well known to reflect changes in the electrical properties between palm and forearm skin in human subjects, which are produced by noxious and emotional stimulation, and aroused alertness . The stiFrom the aforementioned, we aimed to (1) first clarify the relationships between the components in GSR and active palmar sweating using maximal handgrip exercise, and fluctuations in the R-R interval in electrocardiograph (ECG) recordings, and (2) observe active palmar sweating with handgrip exercise using a microscope. (3) Next, we investigated the effects of eyes closing and opening in human subjects according to self-control condition on the changes in tonic baseline GSR and palmar sweating, alongside simultaneous measurement of the percentage of α wave electroencephalograph (EEG) recordings. (4) We also evaluated the effects of self-awareness of drowsy during the operation of computer at mid-night on the changes in GSR, percentage of α wave in EEG, and R-R interval in ECG. In addition, to confirm the drowsiness of volunteers, research assistants checked the number of eyes closing and head down in the volunteers during the computer working behind a transparent wall. Finally, (5) we simulated the changes in the baseline GSR using an equivalent electrical model of human skin corresponding to the physiological and morphological properties of palmar and forearm epidermis in human subjects , 1956. TIn total, 15 healthy volunteers  without any diseases participated in the present experiments. The minimum number of volunteers was recommended by the ethical committee for clinical observation study. Hence, the number of participating volunteers was decided to be suitable for obtaining the conclusion. We selected the younger subjects because the older persons more than 50 years old have known to become less responsive to palmar sweating . The eth2). The amount of active palmar sweating was demonstrated as water loss per constant area and time using a calibration curve. With the step response of the ratemeter, the time to obtain a maximal response is ∼1.5 s. The changes in environmental temperature ranging from 15 to 35°C, and environmental humidity ranging from 30 to 70%, do not affect the output of the ratemeter. The correlation between water loss in active palmar sweating and changes in output voltage of the ratemeter is approximately linear, r2 = 0.983. The sensitivity of the electrical performance is 0.1 V/1 mg water loss/5 min.We previously designed and constructed a new perspiration ratemeter to directly measure skin perspiration on human subjects. We have demonstrated the properties of perspiration ratemeter in detail . Active We designed and constructed an apparatus capable of directly observing active sweating on the palmar skin alongside quantitative measurement of active sweating . The appWe designed and constructed the GSR system with Ag/AgCl disposable electrodes and adhesive gel , especially suitable for recording voltage changes in the baseline GSR. To evaluate the differences of neuronal activities of autonomic nerve fibers innervated on heart and the sudomotor nerve fibers in sweat glands in human subjects, the changes in R-R interval in ECG were evaluated  with a heart rate monitor . The recording and ground electrodes of the ECG were fixed on the skin surface at thorax and upper extremity of human subjects, respectively.To evaluate the neuronal activity of the central nervous system (CNS) in human subjects, changes in the percentage of α wave in EEG were measured with a portable EEG  equipped with a computer and software .n = 30). The R-R interval in ECG was simultaneously recorded with maximal handgrip exercise. Next, all subjects performed the eyes closing and opening with each self-control condition because of reducing emotional stress by the order of research assistants. The eyes closing and opening in the volunteers were also confirmed by the research assistants seated behind a transparent wall.All subjects were seated on comfortable chairs with back support and then relaxed in a silent air-conditioned room. All subjects were equipped with the ECG and EEG electrodes, placed on their skin surface of their thorax, forearm, and head. Their palm and forearm skins were also fixed with the Ag/AgCl electrodes for GSR measurement. The probe of the perspiration ratemeter was placed on the palmar skin at the bottom of the thumb at the same hand. First, the subject performed the 5-s handgrip exercise at 100% maximal voluntary contractions 4–5 times with 60-s interval to confirm the reproducible palmar sweating responses. The handgrip exercise with these parameters is known to produce no or little adaption in active palmar sweating . In addiNext, to evaluate the self-awareness of drowsy in human subjects, we recorded changes in the baseline GSR and active palmar sweating with simultaneous recordings of EEG and ECG when the 15 volunteers operated a computer at mid-night between 22:00 and 00:00. Simultaneously, the research assistants checked the numbers of the eyes closing and head down in the volunteers during the computer working behind transparent walls. After the experiments, the research assistants demonstrated the obtained data and the numbers of the eyes closing and head down measured simultaneously by the research assistants to all volunteers. The volunteers confirmed by themselves the self-awareness of drowsy with considering the changes in the all data.To clarify mechanisms of the eyes closing and opening-mediated changes in the baseline GSR, we simulated changes in baseline GSR level using the equivalent electrical model of human skin combined with morpho- and physiological properties of human palm and forearm skin; the thickness of epidermis, the length of duct of sweat gland, physiological function of eccrine sweat gland, i.e., reabsorption and secretion properties of water and electrolytes, especially sodium, chloride, and potassium ions .t-test for unpaired observations after checking that the data were suitable for a t-test distribution or a one-way ANOVA followed by a Duncan’s post hoc test, as appropriate. p < 0.05 was considered statistically significant. The associations between GSR output and water loss in palmar sweating, and between time delay in GSR and in active palmar sweating were compared by linear regression. Pearson’ ratio, r2 was obtained.All GSR data are represented as the mean ± SEM. Statistical significance was analyzed using Student’s n = 30). The absorbed time of the sweat into the palmar skin was 6.02 ± 2.19 s (n = 30).r2 = 0.94, n = 30; n = 30; water loss in palmar sweating 0.38 ± 0.04 mg/min, n = 30).n = 30). The corrected (1.5 s) time delay in active palmar sweating is shown as the time (s) measured between the starting points of handgrip exercise and palmar sweating , being minus 1.5 s. r2 = 0.75, n = 30). The 30 absolute values are shown in When the electrical time-delay properties (1.5 s) incorporated with the perspiration ratemeter were corrected by the equipped computer, the starting points for the handgrip exercise-mediated GSR and active palmar sweating were entirely consistent . The timn = 15, p < 0.01 each value vs. control). In contrast, n = 15, p < 0.01 vs. each value vs. control).On the equivalent electrical model of human skin, when closing the eyes with stopping active palmar sweating, the reabsorbed sodium and chloride ions in the extracellular tissues of palmar skin return gradually to the blood stream, resulting in the time-dependent decrease in palmar voltage E1 in the model. When the palmar electrical voltage, E1, decreased to 0.8, 0.4, and 0.2 of the control values over the time course of 6 min, we simulated the changes in baseline GSR. The simulated data are demonstrated in On the other hand, the changes in R-R interval of ECG were variable in the self-awareness of drowsy and the handgrip exercise to stay awake. Thus, when the volunteer confirmed the self-awareness of drowsy during 22:30 to 22:40, the R-R interval of ECG produced no or little change. When he tried handgrip exercise to stay awake at first trial, the R-R interval of ECG increased slightly. At the second trial, it decreased tentatively. Comparing with the changes in the baseline GSR and the percentage of α wave in EEG, the changes in R-R interval of ECG showed a marked variability.n = 15) with self-awareness of drowsy. Around 3–4 min after starting self-awareness of drowsy in human subjects, the baseline GSR significantly decreased which is quite similar to the response obtained with eyes closing in the subjects .n = 30). The absorbed time of the sweat into the palmar skin was 6.02 ± 2.19 s (n = 30). (3) The faster phasic component of GSR with maximal handgrip exercise entirely agreed with the starting point of active palmar sweating and with decreasing point of the R-R interval in ECG in all volunteers. (4) Eyes closing in human subjects induced a significant time-dependent decrease in the baseline GSR; in contrast, eyes opening produced a significant increase in the baseline GSR in all subjects. (5) Eyes closing diminished active palmar sweating and increased the percentage of α wave in EEG in all subjects. (6) On the simulation with equivalent electrical model of human skin, when the palmar skin voltage in the model, E1, decreased time dependently to 0.4 of the control during 6 min, the simulated changes in the baseline GSR was completely consistent with the actual changes in baseline GSR recorded in human subjects with eyes closing. (7) The self-awareness drowsy during mid-night working induced a similar decrease in the baseline GSR and increase in the percentage of α wave in EEG to those observed with eyes closing in all subjects.In the present experiments, we demonstrated that (1) the handgrip exercise–mediated secretion of sweat in palmar sweating was observed at the top region of fingerprints in the thumb and the secreted sweat was quickly absorbed into the epidermis of palmar skin. (2) The appeared time of the sweat after starting maximal handgrip exercise was 1.43 ± 0.15 s (+ and Cl– ions in the sweat produces a decrease in the skin surface electrical resistance between palm and forearm skin, resulting in decrease in the skin surface electrical voltage difference between palm and forearm. This evidence may clearly explain the phasic component of GSR which changes to a downward direction in the GSR output. In addition, the phasic component of GSR correlated to water loss in active palmar sweating in human subjects (In the present experiment, the faster spike in GSR completely agreed with the starting point of active palmar sweating in all subjects. The correlation relationship between the GSR output and water loss in active palmar sweating was approximately linear. The relationship between the time delay in GSR and the corrected time delay in active palmar sweating was also approximately linear. The active sweating with high concentrations of Nasubjects . Thus, tIn addition, the sweat secreted on the surface of palmar skin in human subjects with active palmar sweating was quickly absorbed into the epidermis of palm. The absorption of excreted sweat is a specific property of active palmar sweating, comparing with thermal sweating in the forearm, which may contribute to make the soft-cushion properties of palmar skin.On the changes in R-R interval of ECG, the maximum handgrip exercise decreased rapidly the R-R interval of ECG concomitant with changes in faster spike in GSR and active palmar sweating. In contrast, the self-awareness of drowsy produced no or little significant change in the R-R interval. The differences for the responses of R-R interval in ECG between handgrip exercise and aroused alertness may be related to the differences of contributed peripheral autonomic nervous activities between the exercise-mediated skin thermal perspiration and the drowsiness-mediated emotional palmar sweating. In addition, the changes in the R-R interval of ECG may be influenced by many other factors, i.e., the environmental stimulation included with light, the condition of vagal nerve activity, and a time-locked change in ECG. However, the detailed mechanisms of the variability will be needed in the future to investigate comprehensively.In summary, these findings strongly suggest that active palmar sweating and the first spike in GSR are available tools for the evaluation of emotional reactions in human subjects .+ and Cl– ions and water within the duct of sweat glands are quickly reabsorbed into the extracellular tissues of palmar skin during stopping active sweating, and then time dependently moved into the blood stream. Changes in the transport of electrolytes and water may be related to decrease in palmar skin electrical voltage in the electrical model of human skin.Another important finding in the present study is that eyes closing in human subjects caused a significant decrease in the baseline GSR. The equivalent electrical model of human skin suggests that the eyes closing–mediated change in the baseline GSR may completely agree with the hypothesis that the palmar skin electrical voltage, E1, decreases time dependently to 0.4 of the control during 6 min. The decrease in the palmar skin electrical voltage may be explained by the morphological and physiological findings that residual sweat in the duct of the sweat glands is produced with eyes closing–mediated stopping active palmar sweating. Thus, NaIn addition, eyes closing simultaneously increased the percentage of α waves in EEG recordings. The changes may be concomitant with the findings that the amygdala and hippocampus in the CNS are responsible for active palmar sweating. In contrast, eyes opening induced active palmar sweating with increases in the input stimulation to the CNS center.In support to the conclusion, The noteworthy finding in the present finding is that the self-awareness drowsy in human subjects at mid-night working with computer produced significantly a time-dependent decrease in the baseline GSR and increase in the percentage of α wave in EEG, the responses of which were quite similar to those obtained with eyes closing . The finIn conclusion, the faster phasic component in GSR completely agreed with the starting point of active palmar sweating. Changes in the baseline GSR in human subjects reflect closing or opening eyes. A similar decrease in the baseline GSR was observed with self-awareness of drowsy in human subjects. These changes will form the basis of useful tools for evaluating clearness and drowsiness in human subjects using GSR and active palmar sweating.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethical committee for human studies at Shinshu University School Medicine. The patients/participants provided their written informed consent to participate in this study.All human experiments were performed in the laboratory of HM, NM, EI, SK, MS, and TO. Photomicrographs and videos were produced in the laboratory of HM, NM, EI, SK, and MS. TO: conception and design of the experiment. HM and TO: acquisition, analysis, and interpretation of data. TO: drafting the manuscript and critically revising it for intellectual content. All authors approved of the final version of the article and agree to be accountable for all aspects of the work to ensure that questions related to the accuracy or integrity of any part of the work are appropriately addressed. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.HM was employed by company SKINOS Co., Ltd. NM and EI were employed by company Rubycon Co., Ltd. SK was employed by company NEC Solution Innovators Co., Ltd. The remaining 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.