Tumor necrosis factor α (TNFα) is a multifunctional cytokine with a potent pro-inflammatory effect. It is a validated therapeutic target molecule for several disorders related to autoimmunity and inflammation. TNFα–TNF receptor-1 (TNFR1) signaling contributes to the pathological processes of these disorders. The current study is focused on finding novel small molecules that can directly bind to TNFα and/or TNFR1, preventing the interaction between TNFα or TNFR1, and regulating downstream signaling pathways.Cheminformatics pipeline  was used to screen for novel TNFα and TNFR1 inhibitors in the Zinc database. The pharmacophore-based models were generated to screen for the best drug like compounds in the Zinc database.The 39, 37 and 45 best hit molecules were mapped with the core pharmacophore features of TNFα, TNFR1, and the TNFα–TNFR1 complex respectively. They were further evaluated by molecular docking, protein–ligand interactions and in silico ADMET studies. The molecular docking analysis revealed the binding energies of TNFα, TNFR1 and the TNFα–TNFR1 complex, the basis of which was used to select the top five best binding energy compounds. Furthermore, in silico ADMET studies clearly revealed that all 15 compounds  satisfied the Lipinski rule of five and had no toxicity.The new selective TNFα, TNFR1 and TNFα–TNFR1 complex inhibitors can serve as anti-inflammatory agents and are promising candidates for further research.The online version of this article (10.1186/s12967-019-1965-5) contains supplementary material, which is available to authorized users. Tumor necrosis factor α (TNFα) is a cytokine secreted by macrophages in response to septic shock, inflammatory agents and cachexia. TNFα plays a key role in the immune system and cell death  . TNFα isTNFα contributes to the pathogenesis of inflammatory, edematous, neovascular, and neurodegenerative diseases of the eye . InjectiA variety of TNFα antagonists, including infliximab, etanercept, adalimumab, certolizumab and glolimumab, were developed for therapeutic applications . HoweverTherefore, in the present study we used cheminformatics as part of the pipeline  to screen for novel, safe TNFα and TNFR1 inhibitors from the publicly available Zinc database.https://www.rcsb.org/). The TNFα–TNFR1 complex protein was downloaded from a public web site (http://www.cbligand.org/downloads/TNF_TNFR1.pdb). We removed all the hetero atoms and crystal water molecules from the target proteins and minimized the energy.We took the crystal structures of the target proteins: TNFα (2AZ5) with resolution 2.1 Å  and TNFRhttp://sts.bioe.uic.edu/castp/index.html?2pk9). CASTp measures and identifies pockets and pocket mouth openings, in addition to the cavities. We uploaded the target proteins as input to predict the ligand binding sites. The CASTp server predicted the key amino acids for binding interactions to the inhibitors [The active sites of TNFα, TNFR1 and the TNFα–TNFR1 complex were predicted using the CASTp (Computed Atlas of Surface Topography of proteins) server (hibitors .http://zincpharmer.csb.pitt.edu) is an online interface for searching the purchasable compounds of the Zinc database using the Pharmer pharmacophore search technology. ZINCPharmer can automatically extract a set of pharmacophore features from the molecular structure. Each feature comprises the feature type , a position, and a search radius [The structure-based pharmacophore technique can be used to advance the drug development process. For pharmacophore modeling, we selected three PDB (protein data bank) structures, i.e. TNFα (2AZ5), TNFR1 (1EXT), and the TNFα-TNFR1 complex (modelled protein) and their inhibitors (default inhibitor: 307), physcion-8-glucoside (ZINC33832439), Erythrosine B (ZINC08214556). The structure of the TNFα-307 complex was used as the fundamental of the pharmacophore modelling while TNFR1 with the physcion-8-glucoside and TNFα-TNFR1 complex with Erythrosine B complexes were used for pharmacophore design. ZINCPharmer  with the following settings: a maximum of 2,500,000 energy evaluations, a preliminary population of 150 randomly placed individuals, a maximum of 27,000 generations, a transmutation rate of 0.02, and a crossover velocity of 0.8, along with an exclusiveness rate  of one were designed for docking. The supposed Solis and Wets law was useful to a maximum of 300 iterations for each look for confined search. Default principles were thought to be designed for all the parameters not previously mentioned.http://www.molsoft.com/mprop/) and the FAF-Drugs4 server (http://fafdrugs4.mti.univ-paris-diderot.fr/). Drug-likeness was indicated by the Lipinski “Rule of 5’’ [LogP is a less than five, the number of groups within the compound that may give hydrogen atoms to hydrogen bonds is a less than five, and the number of group that may settle for hydrogen atoms to make hydrogen bonds is a less than 10 [The drug-likeness properties were analyzed using MolSoft Drug-Likeness explorer (e of 5’’ . Drug li than 10 .p-glycoprotein (p-gp) efflux, inhibitors of cytochrome P450 enzymes CYP2C9, CYP2C19, CYP2D6 and CYP3A4 and substrates for metabolism by CYP2D6 and CYP3A4. It has predicted drug likeness by Lipinski, Ghose, Veber, Egan, Muegge methods and medicinal chemistry parameters by the Pan-Assay Interference Compounds (PAINS), Brenk methods and other parameters.The physicochemical profiles of lead compounds can increase the quality of clinical candidates . The indTNFα is a cell signaling protein (cytokine) involved in systemic inflammation and is one of the cytokines that comprise the acute phase reaction. TNFα is produced chiefly by activated macrophages, although it can be produced by many other cell types. It is associated with a variety of important physiological processes and pathological conditions . To conthttps://www.rcsb.org/) [http://www.cbligand.org/downloads/TNF_TNFR1.pdb). All hetero atoms and crystal water molecules were removed from target proteins. We performed energy minimization by using the AutoDock Vina tool [The crystal structures of TNFα with a small molecule inhibitor (307) (2AZ5)  with ressb.org/) . The TNFsb.org/)  public wina tool  with theThe active amino acid sites of TNFα, TNFR1 and the TNFα–TNFR1 complex were predicted using the CASTp server. CASTp identified pockets, pocket mouth openings and the cavities of TNFα, TNFR1 and the TNFα–TNFR1 complex. TNFα had a pocket ID of 2, an area of 104.507 and a volume of 35.048. TNFR1 had a pocket ID-2, an area of 24.807 and a volume of 26.187. The TNFα–TNFR1 complex had a pocket ID of 3, an area of 521.964 and a volume of 26.187. The CASTp server predicted the binding site amino acids of TNFα  [The molecular binding process relies on several properties and features of the amino acids presenting in the active site . Accordihttps://zinc.docking.org/). The pharmacophore features acted as query parameters along with hit reduction, hit screening and subset selection, which we set. For examples, Max Hits per Conformation was set to 10, Max Hits per Molecular was set to 1, Max Total Hits was set to maximum, Max RMSD (root mean square deviation) was set to 2, molecular weight (MW) was set to 300–500, rotatable bonds was set to 0–10. All the hit reduction and hit screening parameters were applied against the purchasable Zinc subset. Accordingly, the success of a virtual screening performance can be quantified by the enrichment factor (EF) and hit rate (HR) when the percentage of active compounds in the screening database is known. The TNFα pharmacophore model obtained 39 hits, TNFR1 had 37 and TNF-TNFR1 complex had 45 hits. The pharmacophore hits were mapped with the pharmacophore models. All the hit molecules were submitted to molecular docking studies.The pharmacophore design models were used for screening molecules against the Zinc database  molecule may interact with electrostatic or Van der Waal bonds with − 6.8 kcal/mol−1 binding energy to the Ile58, Leu120, Gly121, and Tyr151 active site amino acids of TNFα  molecule OC, OH, OH, OH, OH, OH, OH and OH groups interacted with eight hydrogen bonds to the Arg77, Cys96, Cys96, Arg104, Arg104, Arg104, Tyr106 and Asn110 amino acid CN, CN, CO, CN, CN, CN, CO and CO functional groups with − 7.6 kcal/mol−1 binding energy respectively  molecule OC, and OC groups interacted with six hydrogen bonds to the Thr94 and Asn110 amino acid CO, and CN functional groups with − 7.2 kcal/mol−1 binding energy respectively  and FAF-Drugs4 (http://fafdrugs4.mti.univ-paris-diderot.fr/) tools. The compounds showed Log P ≤ 5, relative molecular mass ≤ 500, range of HBA (hydrogen bond acceptors) ≤ 10, and range of HBD (hydrogen bond donors) ≤ 5 considering the best ligand molecules were used as drug leads for biological activity , or the number of stereocenter properties and ADMET properties, which were all computed by FAF-Drugs4 , activated NK  and T cells, and by a diverse array of non-immune cells such as endothelial cells and fibroblasts . TNFα isThe trimer form of TNF binds to TNFR1, activates the downstream signaling, and predominantly promotes inflammation and tissue degeneration . The fivIn the present work, we established a pharmacophore model to recognize vitally assorted lead hits for TNFα, TNFR1 and the TNFα-TNFR1 complex. The recognized hit compounds were utilized to create novel, strong inhibitors for the targets, and further assessed by docking and in silico ADMET studies. Fifteen lead compounds satisfied all the criteria and serve as novel, structurally diverse inhibitors for TNFα, TNFR1 and the TNFα–TNFR1 complex.Additional file 1: Fig. S1. FAF-Drugs4 ADME results for the TNF-α best ligand molecules and their respective properties such as: 2D structure of each ligand atoms, physicochemical filter positioning, compound complexity, oral property space, oral absorption estimation and Pfizer 3/75 rule positioning. Fig. S2. FAF-Drugs4 ADME results for the TNFR1 best ligand molecules and their respective properties such as: 2D structure of each ligand atoms, physicochemical filter positioning, compound complexity, oral property space, oral absorption estimation and Pfizer 3/75 rule positioning. Fig. S3. FAF-Drugs4 ADME results for the TNF-α–TNFR1 complex best ligand molecules and their respective properties such as: 2D structure of each ligand atoms, physicochemical filter positioning, compound complexity, oral property space, oral absorption estimation and Pfizer 3/75 rule positioning. Table S1. TNF-α and its inhibitors to compute physicochemical descriptors as well as to predict ADME parameters, pharmacokinetic properties, druglike nature and medicinal chemistry friendliness properties predicted by SwissADME tool. Table S2. TNFR1 and its inhibitors to compute physicochemical descriptors as well as to predict ADME parameters, pharmacokinetic properties, druglike nature and medicinal chemistry friendliness properties predicted by SwissADME tool. Table S3. TNF-α -TNFR1 complex and its inhibitors to compute physicochemical descriptors as well as to predict ADME parameters, pharmacokinetic properties, druglike nature and medicinal chemistry friendliness properties predicted by SwissADME tool.


Alexandrium catenella was detected in southern Chiloé, leading to the banning of shellfish harvesting in an extended geographical area (~500 km). On April 24, 2016, this bloom produced a massive beaching (an accumulation on the beach surface of dead or impaired organisms which were drifted ashore) of surf clams Mesodesma donacium in Cucao Bay, Chiloé. To determine the effect of paralytic shellfish poisoning (PSP) toxins in M. donacium, samples were taken from Cucao during the third massive beaching detected on May 3, 2016. Whole tissue toxicity evidence a high interindividual variability with values which ranged from 1008 to 8763 μg STX eq 100 g−1 and with a toxin profile dominated by GTX3, GTX1, GTX2, GTX4, and neoSTX. Individuals were dissected into digestive gland (DG), foot (FT), adductor muscle (MU), and other body fractions (OBF), and histopathological and toxin analyses were carried out on the obtained fractions. Some pathological conditions were observed in gill and digestive gland of 40–50% of the individuals that correspond to hemocyte aggregation and haemocytic infiltration, respectively. The most toxic tissue was DG (2221 μg STX eq 100 g−1), followed by OBF (710 μg STX eq 100 g−1), FT (297 μg STX eq 100 g−1), and MU (314 μg STX eq 100 g−1). The observed surf clam mortality seems to have been mainly due to the desiccation caused by the incapability of the clams to burrow. Considering the available information of the monitoring program and taking into account that this episode was the first detected along the open coast of the Pacific Ocean in southern Chiloé, it is very likely that the M. donacium population from Cucao Bay has not had a recurrent exposition to A. catenella and, consequently, that it has not been subjected to high selective pressure for PSP resistance. However, more research is needed to determine the effects of PSP toxins on behavioral and physiological responses, nerve sensitivity, and genetic/molecular basis for the resistance or sensitivity of M. donacium.In late February 2016, a harmful algal bloom (HAB) of  Alexandrium, Gymnodinium and Pyrodinium [Paralytic shellfish poisoning (PSP) is a neurotoxic syndrome caused by the ingestion of shellfish contaminated by saxitoxin and/or its analogues, causing a range of symptoms from slight tingling sensation or numbness around the lips to fatal respiratory paralysis (reviewed by Reference ). The torodinium ,3,4. PSTrodinium , which crodinium ,7, (d) drodinium , (e) hydrodinium ,9,10, anrodinium .Alexandrium species have been described, including changes in valve closure, filtration rate, feeding rate, byssus production, oxygen consumption, cardiac activity, neurophysiological effects, and pathological alterations [Mytilus meridionalis, oyster Crassostrea virginica, scallop Chlamys opercularis, cockle Cerastoderma edule, and clams Donax serra, D. variabilis, and Spisula solidissima [Some HAB species could affect shellfish, with the responses depending on a series of species-specific or individual characteristics of both phytoplankton (including toxin production) and shellfish . Severalerations ,23,24,25idissima ,26,27,28Aulacomya atra, whose toxicity was associated with the presence of the dinoflagellate Gonyaulax catenella (currently Alexandrium catenella) [A. catenella blooms and PSP toxicity outbreaks have been reported annually covering an extensive area between Magallanes Region to Los Lagos Region, specifically in Quellón, Chiloé Island  [PSP was detected in Chiloé for the first time in 1972, in Bell Bay, Magallanes Region . During that event, three fishermen died due the consumption of the ribbed mussel tenella) . Since t3°36′ W) ,31,32, w3°36′ W) ,36,37,38A. catenella was detected in Southern Chile. This outbreak was the worst event of all those recorded in Chile in terms of geographical extension and affected species, spreading, for the first time, from southernmost of the Chiloé Archipelago (43°50′ S) to Mehuín, Los Ríos Region (39°25′ S) [Mesodesma donacium and Gari solida and the crustaceans Austromegabalanus psittacus and Romaleon polyodon (Otaria flavescens), seagulls (Larus dominicanus), and dogs [In 2016, a late summer bloom of s Region °25′ S [3polyodon . Furthers Region °25′ S [Mand dogs  because Mesodesma donacium is an endemic species of the Pacific coast of South America, where it is distributed from Sechura Bay, Perú (5° S) to Chiloé Island, Chiloé (43° S) [ 43° S) ,42, inha° S ,42,  (43° S) . In Chil (43° S) ,46.M. donacium, and their possible link with the massive beaching of this species which took place during a large bloom of Alexandrium catenella. The aim of the current work was to describe the accumulation of PSP toxins, their distribution and profile in different tissues of −1. Two weeks later on April 6, an abrupt increase of toxicity was detected in all localities reaching values of 394, 351, 428 μg STX eq 100 g−1 for Chanquín, Palihue and Deñal, respectively, that exceeded the regulatory limit (80 μg STX eq 100 g−1). These toxicity levels remained stable for one week and then began to rise quickly, reaching toxicities higher than 1400 µg STX eq 100 g−1 . On April 24, the toxicity increased again to reach values between 3044 and 6614 μg STX eq 100 g−1. This increase was followed by the first massive beaching, which covered most Cucao beach (5 km long). At the end of April, the toxicity increased again to reach values near 5500 μg STX eq 100 g−1 in all localities and a second massive beaching was recorded. These toxicity levels remained approximately stable for 10 days from May 1 to May 11, a period during which at least three new surf clam beaching events were detected. The peak of shellfish toxicity was observed at May 20th, with values reaching 9059 μg STX eq 100 g−1  by October 29. On January 23, 2017 PSP was only detected in Chanquín (33 μg STX eq 100 g−1). Since June 3, the toxicity decreased quickly to levels between 3137 and 4014 μg STX eq 100 gM. donacium covering most of Cucao beach. During this episode, thousands of individuals were found dead, dying, or paralyzed, lying on the sand surface. Two days later, the inspectors reported a notable decrease in the beaching surf clams, estimating that only 20% of individuals were lying on the sand. On May 3, during a new beaching episode, the density of surf clams on the sand surface was 40–50 individuals m−2. Most of the specimens lying on the surface were alive with the foot or siphons extended or with the valve partially closed. A detailed examination revealed that the individuals were paralyzed because they had a weak reaction to mechanical stimulus on foot or siphons, with a slow and incomplete retraction. Three days later, the beaching surf clams decreased again, suggesting that some of the alive individuals recover the ability to re-burrow in the sand.On April 24, a seafood inspector of Laboratorio de Salud Pública Ambiental detected the first massive beaching of −1 (4699 ± 2971 μg STX eq 100 g−1), showing a bimodal distribution (−1. The toxin profile (% mole), was dominated by carbamoyl toxins that, in decreasing order of concentration, were GTX3 (24.64%), GTX1 (18.88%), GTX2 (14.79%), GTX4 (13.70%), neoSTX (7.46%), and STX (1.73%). Other toxins detected in less amount were N-sulfocarbamoyl, such as C1 (10.32%), C3 (5.89%), C2 (1%), and C4 (0.06%), and decarbamoyl, dcSTX being (1.17%) the most abundant toxins of this group, with dcGTX2 and dcGTX3 present in trace levels (<1%)  .−1. On a molar basis, the toxin profile was dominated by carbamoyl toxins, GTX3 being (32.1%) the most abundant, followed by GTX2 (16.2%), GTX1 (16.1%), GTX4 (13.3%), neoSTX (5.9%), and STX (2.74%). The second group in importance was that of N-sulfocarbamoyl toxins, with C1, C3, C2, C4, and GTX5, in decreasing order of abundance. The less abundant of the analyzed toxins were dcGTX3 and dcGTX2 (The most toxic organ was the digestive gland (DG), with 2221 ± 2193 μg STX eq 100 gd dcGTX2 .−1). The toxin profile was dominated mainly by carbamoyl toxins . In this case, the contribution of N-sulfocarbamoyl and decarbamoyl toxins were less relevant to the toxin profile  was the second in importance being approximately 1/3 as toxic as DG . Another relevant group was that of N-sulfocarbamoyl toxins, such as GTX5 (11.3%), C1 (6.9%), and C3 (6%). The toxins of the decarbamoyl group dcGTX2–3 were present in a very low molar percentage (Foot (FT) had about 1/8 the toxicity of DG, with values of 297 ± 157 μg STX eq 100 grcentage .−1. Its toxin composition (in molar proportion) was dominated by carbamoyl toxins , followed by other toxins present in low molar percentages as C1, C2, and dcGTX2  had a toxicity of 314 ± 8.5 μg STX eq 100 gd dcGTX2 .M. donacium tissues exposed to the HAB of A. catenella. Bacterial colonies were found in the foot  and Tagelus dombeii (262 μg STX eq 100 g−1), and similar to those reported in Gari solida (3286 μg STX eq 100 g−1) [Mytilus chilensis 22,000 μg STX eq 100 g−1 [Aulacomya atra in 1996  [−1 in M. chilensis [During the bloom, 100 g−1) . However 100 g−1 , ribbed 100 g−1) , and rechilensis  was atta−1 (average 4699 μg STX eq 100 g−1), revealing a high interindividual variability (CV = 67%) in PST bioaccumulation. This interindividual variation is similar to that reported in other shellfish, such as the clams Arctica islandica (56%) [Spisula solidissima (49%), and the scallop Placopecten magellanicus (44%) [Panopea abrupta (93%) [Mya arenaria, Ruditapes philippinarum, and Tagelus dombeii and the oysters Crassostrea virginica and Magallana gigas (Crassostrea gigas) decrease the clearance rate on the presence of the toxic dinoflagellate Alexandrium tamarense [M. gigas suggest that the feeding behavior is mainly responsible for interindividual variation in toxin bioaccumulation [Toxicity of the clams obtained during the third massive beaching ranged from 1008 to 8763 μg STX eq 100 gca 56%) , Spisulaus (44%) ,52, but ta (93%) . The intta (93%) . Diverse% , Spisuamarense ,58,59. Tmulation ,60,61.M. donacium, the toxin profile (in decreasing order) was dominated by GTX3, GTX1, GTX2, GTX4, C1, neoSTX, and C3, which contribute 95% of the total toxin content, while the remaining 5% corresponded to STX, dcSTX, C2, GTX5, C4, dcGTX2, and dcGTX3. The toxin profile is similar to those reported in the ribbed mussel Aulacomya atra from Darwin Channel, southern Chiloé (but differ in its higher proportion of GTX5) [M. chilensis from Errazuriz Channel, Magallanes region, dominated mainly by GTX2 [Venus antiqua and Tagelus dombeii , but differ by the absence of C toxins [Alexandrium catenella from Cucao Bay. The profile of M. donacium, however, showed characteristics which are very similar to that of strains of A. catenella isolated in other episodes, as the clones PFB36 from San Pedro Island (Los Lagos) and ACC02 from Coastal Channel (Aysén region). In both cases, the profile mainly differs by the presence of GTX6 in the dinoflagellates [In the whole tissues of of GTX5)  and M. c by GTX2 . SimilarC toxins . Unfortugellates ,63.M. donacium, all tissues had a toxicity higher than the regulatory limit (80 μg STX eq 100 g−1). In this shellfish, the relative contribution of the different tissues to toxicity (expressed as % μg STX eq) had the following pattern: digestive gland (DG) (68.4%), foot (FT) (23.7%), other body fractions (OBF) (7.4%), and MU (0.4%). In general, the anatomical distribution of toxicity in M. donacium is similar to those reported in other shellfish, in which DG is the initial repository of PST toxin during the intoxication phase [Mya arenaria (89.3%) [Spisula solidissima (85.6%) [Mercenaria mercenaria (80.9%) [Saxidomus giganteus (66.2%) [M. donacium had a high contribution to toxicity (23.71%) as compared with the clam species mentioned above, in which this tissue only contributes 1.4 to 2.2% [Mytilus edulis [Paratapes undulatus [M. donacium that corresponds to epimerization. In this case, the β-epimers  will gradually convert into their thermodynamically more stable forms that correspond to α-isomers  [Pecten novaezelandiae [M. edulis [M. galloprovincialis [Panopea globosa [M. donacium tissues to transform carbamoyl toxins GTX2, GTX3, and STX to their corresponding decarbamoyl derivatives and was demonstrated in clams Protothaca staminea [Spisula solida [Mactra chinensis, and Perodina venulosa [In the beached on phase . Some ex (89.3%) , Spisula 85.6%) , Mercena (80.9%) , and Sax 66.2%) . The FT  to 2.2% . All tis.2% . Thendulatus . The hig% , Merceand (DG) .4%, foot. edulis  M. galloincialis ,71, and  globosa . Finallystaminea , Spisulaa solida , Mactra A. catenella seems to have induced immunological and inflammatory responses in M. donacium. Histopathological analyses in different tissues of the individuals exposed to the A. catenella bloom revealed the presence of hemocyte aggregation in gill tissues of 50% of analyzed individuals, that could be associated with an immunological response to A. catenella cells during filtration processes. In bivalves, the gill is one of the most important organs because of it is involved in respiration and feeding, being the first organ that has contact with toxic phytoplankton cells [M. donacium, Estrada et al. [Nodipecten subnodosus exposed to the dinoflagellate PSP-producing Gymnodinium catenatum.Exposure to on cells . As in Ma et al.  observedMytilus edulis, M. gigas, and Nodipecten subnodosus exposed to A. fudyense, A. minutum, and G. catenatum [Inflammatory responses, as the observed haemocytic infiltration in connective tissues of digestive gland or other similar ones as diapedesis of hemocytes, were described in atenatum ,76,77  and the bimodal distribution of toxin concentration, suggest the presence of two groups of individuals, one of them sensitive to the toxins (incapable of burrowing and with low toxin accumulation capability), and another one (more) resistant to the toxins (capable of burrowing and with high toxin accumulation capability), as described in M. arenaria [++ influx in excitable cells and thus inhibiting the nerve action potential, leading to paralysis and death [Mya arenaria, in which differences were found between resistant individuals, from populations frequently exposed to toxic Alexandrium blooms , and sensitive individuals from areas with no record of PSP toxic episodes  [M. donacium population from Cucao Bay has not had a recurrent exposition to A. catenella and consequently that it has not been subjected to high selective pressure for PSP resistance. MacQuarrie and Bricelj [M. arenaria, reporting a maximum toxicity in resistant individuals  10-fold higher than the toxicity in sensitive ones (8200 μg STX eq 100 g−1).During the bloom of arenaria ,83. PSP nd death . Indeed, Canada) ,85. Cons Bricelj  found hiA. catenella toxic bloom in Cucao Bay was mainly due to incapability of burrowing—an indirect effect of PSP toxin-induced paralysis—which caused desiccation. However, more research is needed to determine the effects of PSP toxins on behavioral and physiological responses, nerve sensitivity, and genetic/molecular basis for the resistance or sensitivity of M. donacium.Our observations in this study suggest that the surf clam mortality during M. donacium) along a 20 km sandy beach. In general, this system is characterized by strong oceanic water influence, with salinity >31 despite the freshwater inputs from the Cucao River and other smaller tributaries (www.shoa.cl).Cucao Bay (42°38′ S–74°07′ W) is located in the oceanic coast of the Chiloé Island . This sibutaries . Water tWithin the framework of the regional monitoring program developed by the Laboratorio de Salud Pública Ambiental , surf clam samples were obtained periodically from Cucao Bay at least twice a month—from March 15, 2016, to January 23, 2017—in four sampling stations located at Cucao Bay: Deñal, Palihue, Chanquín, and Rahue .−1 sample using Sommer’s Table.Each sample consisted of at least 20 individuals of commercial size (>6 cm) collected by fishermen by means of hang-gathering. Samples were placed inside a plastic bag, then placed in a cool box at 10 °C and transported to the laboratory. Upon arrival to the laboratory, paralytic shellfish toxins (PST) were extracted from surf clam tissues following the official AOAC method 959.08 . For toxw/v) using an Ultra-Turrax T25 dispersing system  at 11,000 rpm for 3 min. After each extraction, the pH of the resulting emulsion was adjusted to 2–4, boiled at 100 °C for 5 min, centrifuged at 5000 g for 20 min , and its pH adjusted again to 2–4. To deproteinate the samples, a 1 mL aliquot was mixed with 50 μL of 30% trichloroacetic acid (TCA), vortex mixed, and then centrifuged at 9000 g for 5 min. To neutralize the solution, 70 μL of NaOH (1 M) was added, and then vortexed and centrifuged at 9000 g for 5 min. One 500-μL aliquot was filtered through a 0.2 μm Clarinert nylon syringe filter (13 mm diameter)  and stored into an autosampler vial. A second 500-μL aliquot was hydrolyzed in order to transform the sulfocarbamate toxins (if present) to the corresponding carbamate toxins , filtered through 0.2 μm nylon filter, and stored into an autosampler vial. For some analyses, extracts were diluted 5-fold or 10-fold with HCl 0.1 N to ensure the correct quantification of the most abundant toxins in the samples.Data: Surf clam samples were collected from Bahía Cucao during a massive beaching detected on May 3, 2016. The sample consisted of 20 individuals of commercial size (>6 cm) collected from the surface of the sand. To ensure that the shellfish were still alive, a mechanical stimulus was induced by means of a puncture in the foot using a steel needle to determine foot contraction, incapability, or reduction of valve closure and siphon retraction. Upon arrival to the laboratory, the shellfish were washed and distributed into two subsamples completely at random. The first subsample consisted of 10 clams and was used for individual whole sample analyses. The second subsample contained 10 individuals and was used to determine the anatomical distribution. These individuals were carefully dissected into foot  and the nonedible tissues corresponding to digestive gland (DG), adductor muscle (MU), and all other body fractions (OBF) . Each tissue was extracted following the official AOAC method 2011.02 , with sl−1 and the injection volume 10 μL. After separation, the toxins were derivatized in the post-column reaction system (85 °C) with a solution of 100 mM phosphoric acid, 5 mM periodic acid solution adjusted to pH 7.8 with 5 M sodium hydroxide, and, at the end of the reaction coil, mixed with a solution of 0.75 M nitric acid. The flow rate of both solutions was 0.4 mL min−1. Finally, the detection of the fluorescent derivatives of the toxins was carried out with an FL detector set to 330/390 nm excitation/emission wavelengths. PST concentration in the samples was quantified by comparing the obtained response with that of corresponding reference materials . The limits of detection of the technique were estimated for a signal/noise ratio of 3  using a Hitachi LaChrom Elite HPLC system equipped with a Hitachi FL detector L2485  and a post-column reaction system composed by two Waters pumps (Water Reagent Manager) and a 20 m Teflon coil (2 mL) in a heating reactor . The chromatographic separation was carried out using a Zorbax Bonus RP column   with a Zorbax Bonus RP guard column  at 40 °C. For the separation, two mobile phases were used consisting of 11 mM heptanesulfonate and 5.5 mM phosphoric acid solution adjusted to pH 7.1 with ammonium hydroxide (A) and 11 mM heptane sulfonate, 16.5 mM phosphoric acid, 11.5% acetonitrile solution adjusted to pH 7.1 with ammonium hydroxide (B). A gradient elution started with a proportion of 100% A, which was maintained for 8 minutes, followed by a linear increase to 100% B from minute 8.01 to minute 15, and then held for 4 min. The column was equilibrated in the initial conditions for 5 min previously to next run. The mobile phase flow was 0.8 mL mintio of 3 . FinallyUpon arrival to the laboratory, 10 individuals were carefully dissected for histopathological studies in siphons (SI), gill (GI), gonad (GO), digestive gland (DG), foot (FT), and mantle (MA). Each tissue sample was placed in a cassette, immediately fixed in Davidson´s solution  and kept


Dry eye disease (DED) is a multifactorial disease associated with ocular surface inflammation, pain, and nerve abnormalities. We studied the peripheral and central neuroinflammatory responses that occur during persistent DED using molecular, cellular, behavioral, and electrophysiological approaches.A mouse model of DED was obtained by unilateral excision of the extraorbital lachrymal gland (ELG) and Harderian gland (HG) of adult female C57BL/6 mice. In vivo tests were conducted at 7, 14, and 21 days (d) after surgery. Tear production was measured by a phenol red test and corneal alterations and inflammation were assessed by fluorescein staining and in vivo confocal microscopy. Corneal nerve morphology was evaluated by nerve staining. Mechanical corneal sensitivity was monitored using von Frey filaments. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous corneal nerve activity. RT-qPCR and immunostaining were used to determine RNA and protein levels at d21.We observed a marked reduction of tear production and the development of corneal inflammation at d7, d14, and d21 post-surgery in DED animals. Chronic DE induced a reduction of intraepithelial corneal nerve terminals. Behavioral and electrophysiological studies showed that the DED animals developed time-dependent mechanical corneal hypersensitivity accompanied by increased spontaneous ciliary nerve fiber electrical activity. Consistent with these findings, DED mice exhibited central presynaptic plasticity, demonstrated by a higher Piccolo immunoreactivity in the ipsilateral trigeminal brainstem sensory complex (TBSC). At d21 post-surgery, mRNA levels of pro-inflammatory (IL-6 and IL-1β), astrocyte (GFAP), and oxidative (iNOS2 and NOX4) markers increased significantly in the ipsilateral trigeminal ganglion (TG). This correlated with an increase in Iba1, GFAP, and ATF3 immunostaining in the ipsilateral TG of DED animals. Furthermore, pro-inflammatory cytokines , iNOS2, neuronal (ATF3 and FOS), and microglial (CD68 and Itgam) markers were also upregulated in the TBSC of DED animals at d21, along with increased immunoreactivity against GFAP and Iba1.Overall, these data highlight peripheral sensitization and neuroinflammatory responses that participate in the development and maintenance of dry eye-related pain. This model may be useful to identify new analgesic molecules to alleviate ocular pain. Dry eye disease (DED) is a multifactorial disease of the ocular surface and tears that result in symptoms of discomfort, visual disturbance, burning, pain, and instability of the tear film . AlthougUnderstanding the neuropathophysiological mechanisms of DED is essential for the development of new therapeutic strategies. One of the challenges of preclinical research involves the development and the characterization of relevant models that best mimic the human disease. Previous preclinical models have been developed to mimic DED using desiccative environment exposure , 14, theIn this context, we developed and characterized a preclinical mouse model of chronic DED by excision of the ELG and Harderian gland (HG) in adult female mice to affect not only stability of the tear film but also tear production, as the ELG produces the aqueous constituent of the tear film , 29, wheSeven- to 8-week-old adult female C57BL/6 mice (average weight 18.95 ± 0.06 g)  were maintained under controlled conditions . All animal procedures were performed in strict accordance with institutional guidelines for the care and use of experimental animals approved by the European Communities Council Directive 2010/63/UE (APAFIS #1501 2015081815454885 v2). A schematic workflow of surgical, clinical, molecular, behavioral, and electrophysiological experiments is provided in Fig. Unilateral (right side) ELG and HG gland excision was performed under ketamine  and xylazine (8 mg/kg i.p.) anesthesia. Before the surgery, a drop of lacrimal gel  was applied to both eyes. Under an operative microscope , an 8-mm skin incision on the temporal side was made to expose and remove the ELG. After dissociating the conjunctival tissue above the orbital cavity near the internal canthus, the HG was carefully removed. Complete removal was verified by inspecting the surgical area for any remaining glandular tissue. The skin incision was then sutured using 6.0 braided silk sutures . A drop of iodine solution was applied onto the incision to avoid bacterial infection. For sham animals, an incision was made in the same zone without touching the glands. The mice were placed in warm (30 °C) cages to recover from the surgery. All steps of the surgical procedure are illustrated in Fig. Tears were measured using cotton phenol red threads . In a quiet room, conscious mice were taken from their cage and the top of the phenol red threads placed on the lateral canthus of the eye for 30 s. The thread is yellow in color (acidic), but changes to light red when exposed to tears. After 30 s, the length (in millimeters) of the color change was determined using a scale, as previously described. Tear production was calculated by measuring the length of thread wetting and was evaluated before in vivo confocal microscopy.Spontaneous eye closure is itself a good index for monitoring spontaneous eye pain. Spontaneous eye closure or the eye ratio is one of the quantitative measures of the grimace scale, which is used to monitor spontaneous pain behavior –33. The Corneal fluorescein staining was performed before and every week after the surgery to assess the degree of corneal damage caused by HG and ELG excision. The extent of corneal damage (width and intensity of fluorescein take-up area) was scored according to the following scale: 0, absence of staining; 0.5, slight punctate staining; 1, diffuse punctate staining; 2, diffuse staining covering less than one third of the cornea; 3, diffuse staining covering more than one third of the cornea; and 4, staining covering more than two thirds of the cornea –33.An in vivo laser confocal microscope  II/Rostock Cornea Module ) was used to examine the entire cornea of anesthetized mice . The imaBoth DED and sham animals were weighed at d0 and d7, d14, and d21 after surgery.−ΔΔCt method was used to analyze the relative differences in specific mRNA levels between groups.Anesthetized animals were perfused with sterile 0.9% NaCl. The ipsilateral TG and TBSC were quickly dissected on ice and immediately frozen using liquid nitrogen and stored at − 80 °C before use. RNA extraction from the ipsilateral TG and TBSC was performed with a NucleoSpin RNA Purification II kit . RNA quality and concentration were then measured by the NanoDrop method . Reverse transcription was performed by high Capacity cDNA Reverse Transcription  according to the manufacturer’s instructions. Finally, cDNA was diluted in DNAse/RNAse-free water to a final concentration of 5 ng/μL. Real-time quantitative PCR was performed with 25 ng cDNA added to 15 μL of a solution of Applied Biosystems Mastermix  and primers for a final volume of 20 μL. The primers used were for interleukin-6 (IL-6) (Mm00446190_m1), tumor necrosis factor-α (TNFα) (Mm99999068_m1), FOS (Mm00487425_m1), ATF3 (Mm00476032_m1), chemokine (C-C motif) ligand 2 (CCL2) (Mm99999056_m1), interleukin-1β (IL-1β) (Mm00434228_m1), nitric oxide synthase 2 (iNOS2) (Mm00440502_m1), NADPH oxidase 4 (NOX4) (Mm00479246_m1), cluster of differentiation 68 (CD68) (Mm03047343_m1), glial fibrillary acidic protein (GFAP) (Mm01253033_m1), integrin alpha M (Itgam) (Mm00434455_m1), and hypoxanthine-guanine phosphoribosyltransferase (HPRT) (Mm03024075_m1). The reaction was performed on a 7300 Real-Time PCR System (Applied Biosystems). The HPRT gene was used as the endogenous reference for each reaction; mRNA levels were calculated after normalization of the results for each sample with those for HPRT mRNA. The 2Mechanical corneal sensitivity was monitored using von Frey filaments . VariousTwenty-one days after surgery, anesthetized mice were transcardially perfused with 10 mL 0.9% NaCl solution followed by 40 mL 4% (w/v) paraformaldehyde in 0.1 M PBS. The eyes were carefully removed and stored at − 80 °C. The brain and TG were carefully removed and immersed in the same fixative for 24 h. The TGs were immersed in 10% (w/v) sucrose in 0.1 M PBS overnight at 4 °C and then in 30% (w/v) sucrose in 0.1 M PBS before freezing in 7.5% gelatin and 10% sucrose. Transverse frozen TGs (14 μm) and cornea (12 μm) were cut on a cryostat (Leica CM 3050 S) and mounted on Superfrost slides. Free-floating sections (40 μm) of the TBSC were prepared using a vibratome . Sections were serially collected in 0.1 M PBS and used for immunofluorescent staining.After three washes in 0.1 M PBS, TG and TBSC sections were incubated for 1 h in a blocking solution of 0.1 M PBS containing 3% normal donkey serum and 0.1% triton X-100, followed by incubation with primary antibody at 4 °C for 48 (floating brainstem sections) or 24 h (frozen TG sections). Corneal sections from naïve, sham, and DED mice were fixed in 4% PFA for 15 min, washed in 0.1 M PBS, and then blocked for 2 h at room temperature in a blocking solution of 0.1 M PBS containing 3% normal donkey serum and 0.1% triton X-100. Next, corneas were incubated overnight with primary antibody at 4 °C.Griffonia simplicifolia isolectin IB4  overnight. All steps following incubation with the primary antibody were performed at room temperature. After three washes, ATF3, cFOS, and Piccolo staining were amplified using biotin-conjugated horse anti-rabbit antibody  and then biotin-conjugated horse anti-goat antibody  for 1 h and finally revealed by incubation with streptavidin-Alexa Fluor 488 . Iba1 was revealed using Alexa Fluor 594-conjugated donkey anti-rabbit antibody  and GFAP using Alexa Fluor 594-conjugated donkey anti-mouse antibody  for 1 h. β III tubulin was revealed using Alexa 594-conjugated donkey anti-mouse antibody . Finally, the sections were mounted onto glass slides and cover slipped.The primary antibodies used in this study were mouse anti-GFAP , rabbit anti-ATF3 , goat anti-cFOS , rabbit anti-Iba1  , rabbit anti-Piccolo , and rabbit anti-β III tubulin . Certain TG sections were incubated with fluorescein isothiocyanate-Tissue sections were examined using a Zeiss M1 epifluorescence microscope . The epifluorescence microscope was equipped with a digital camera  and image acquisition software . TIFF images were obtained. The microscope was calibrated with samples from the sham mice before acquisitions of those from the DED mice. For the quantitative analysis of GFAP, Iba1, and Piccolo immunoreactivity, TG and TBSC sections were analyzed under epifluorescence microscope using a × 20 objective and the same camera parameters  as previously described . Five ip2, 1.2 mM MgCl2, 16.3 mM NaHCO3, 1.3 mM NaH2PO4, and 7.8 mM glucose) saturated with O2 and adjusted to pH 7.4 by bubbling with 95% O2 and 5% CO2. Multi-unit extracellular electrical activity of the ciliary nerve was recorded using a suction electrode (Ag/AgCl). The signal was filtered (300–5000 Hz), amplified  , and digitalized by Spike 2 data analysis  at a sampling frequency of 10,000 Hz. The cornea was superfused with the physiological saline solution for 30 min to stabilize the preparation before performing the electrophysiological recordings. The extracellular spontaneous ciliary nerve fiber activity was defined as impulses per second (imp/sec).Spontaneous ciliary nerve fiber activity was determined at d0, d7, d14, and d21 as previously reported . Brieflyt test with Welch’s correction or nonparametric t test using GraphPad Prism version 7.00 . The difference in nerve fiber impulse activity between control and injured cornea was determined using the Mann-Whitney test or unpaired t test as appropriate. All P values were considered statistically significant when the values were < 0.05. All results are presented as the mean ± standard error of the mean (SEM).The data obtained from sham and DED animals after surgery were compared using the appropriate paired parametric or nonparametric statistical test, as indicated. For statistical analysis, the Kolmogorov-Smirnov test was performed followed by an unpaired P > 0.05)    Fig. c. Howeve01) Fig. c, which P > 0.05, at d0; 1.01 ± 0.20 vs 1.71 ± 0.21, P < 0.05, at d7; 1.10 ± 0.16 vs 2.55 ± 0.27, P < 0.001, at d14; and 0.95 ± 0.24 vs 2.55 ± 0.33, P < 0.001, at d21; Fig. We observed no difference between the two groups of animals by slit-lamp examination before surgery (d0) Fig. a. Slit lP > 0.05, at d0). However, the value for the sham animals was 0.75 ± 0.01, whereas that of the DED animals significantly dropped to 0.62 ± 0.02 (P < 0.0001 by d7). The spontaneous eye closing ratio remained lower in the DED animals to d21  and the corneal sensitivity remained stable over time in the sham animals , and 100% at d21 .We calculated the percentage of surface staining for Iba1 and GFAP for both groups (sham and DED mice). As the TG is located outside the blood-brain barrier, the infiltrating inflammatory cells labeled by the Iba1 antibody can only be derived from circulating monocytes, i.e., macrophages. This semi-quantitative analysis clearly showed significantly higher Iba1 immunoreactivity in the ipsilateral TG in the DED than the sham group , ATF3 , and GFAP  mRNA, as well as those of iNOS2  and NOX4  mRNA, were higher for DED than sham animals   Fig. b. The nu01) Fig. c, arrowsP < 0.001) and neuronal injury  markers , Itgam , and CD68  were significantly higher in DED than sham animals , IL-6 , and TNFα , and the oxidative stress marker iNOS2  were also significantly higher in DED than sham animals   Fig. b.Fig. 1Dryness and pain are common debilitating symptoms of DED, affecting the quality of life of 10% of the population worldwide –41. WomeThe excision of these two functionally different glands decreased tear production by 97% by d7 post-surgery, which remained low until d21 post-surgery. This reduction of tear production is much greater than that of 30% reported after ELG and infraorbital LG excision in rats , and 12.Ocular surface alterations and inflammation are the core mechanisms of DED , 46. HerWe also evaluated corneal integrity by in vivo confocal microscopy to monitor corneal inflammation, as well as changes that can occur in the different corneal layers , 49. TeaIn 2017, TFOS DEWS II updated the definition of DED and added the term neurosensory abnormalities, which have been increasingly featured in the recent literature [Electrophysiological studies show that mechanoreceptors represent 20 to 30% of corneal sensory neurons, which are the fastest conducting nociceptors stimulated by mechanical forces . Such coA link between corneal hypersensitivity, inflammation, and spontaneous corneal nerve fiber activity has been previously reported in two different mouse models of corneal injury . Indeed,Belmonte et al. elegantly showed that on-going ciliary nerve fiber activity results mostly from cold nerve fiber activity, which represents half of corneal sensory neurons , 60. ThuAccumulating evidence suggests that chronic changes in the activity of primary afferent neurons induce synaptic adaptations in the central nervous system and lead to functional remodeling of presynaptic sites . We thusInnervation of the cornea is provided by corneal neurons located in the dorso-medial part of the ophthalmic branch of the TG –10. The Consistent with the upregulation of inflammatory markers, we observed significantly higher mRNA levels of the oxidative stress markers iNOS2 and NOX4 in the TG of DED mice than sham animals. Induced by inflammation , 72, iNOSeveral studies in preclinical models of chronic pain have shown major activation of microglia , 78, whiRT-qPCR analysis revealed higher levels of mRNA for GFAP, pro-inflammatory cytokines (IL-6 and IL-1β), and oxidative stress (iNOS2) and neuronal (ATF3 and FOS) markers in the TBSC. Overall, persistent DED induced major central neuroinflammatory responses known to participate in chronic pain. Such central neuroinflammatory responses may participate in the development of chronic ocular pain observed in DED patients.Here, we report the molecular, cellular, behavioral, and electrophysiological changes in the cornea, TG, and TBSC in a preclinical model of persistent DED in mice Fig. . Our maj


Immunotherapy is currently being examined as a treatment modality for glioblastoma. Maintaining an optimal total lymphocyte count (TLC) after radiotherapy (RT) and using temozolomide may be beneficial in optimizing immunotherapy. However, conventional temozolomide-based chemoradiation is known to induce immunosuppressive effects, including lymphopenia. Therefore, this study aimed to identify potential clinical predictors of acute severe lymphopenia (ASL) in patients receiving chemoradiation for glioblastoma.We identified patients with glioblastoma treated with RT plus temozolomide from 2006 to 2017. ASL was defined as a TLC of < 500/μL within 3 months after initiating RT. Independent predictors of ASL were determined using logistic regression.P = .028). Multivariable analysis revealed that increased planning target volume (PTV) was independently associated with increased ASL incidence , while IMRT was independently associated with decreased ASL incidence . A propensity-matched comparison showed that the incidence of ASL was lower with IMRT than with 3D-CRT .A total of 336 patients were evaluated. Three-dimensional conformal RT (3D-CRT) and intensity-modulated RT (IMRT) were used in 186 (55.4%) and 150 patients (44.6%), respectively. TLC decreased during RT and remained persistently low during the 1-year follow-up, whereas the levels of other blood cell types recovered. In total, 118 patients (35.1%) developed ASL. During a median follow-up of 19.3 months, patients with ASL showed significantly worse overall survival than did those without ASL  contains supplementary material, which is available to authorized users. Radiotherapy (RT)-induced lymphopenia  has been reported in various types of tumors, such as glioblastomas, pancreatic cancer, and lung cancer –7. AlthoDespite multimodal treatment involving surgery, RT, and temozolomide, glioblastoma has a poor prognosis and almost all patients with glioblastoma eventually experience disease relapse . AlthougIn this context, identifying and modifying the factors associated with RT-induced lymphopenia can help maintain an optimal TLC, which may facilitate a synergistic effect between RT and immunotherapy. Accordingly, maintaining an optimal TLC may effectively improve treatment outcomes in patients with glioblastoma. Therefore, this study aimed to examine the potential clinical predictors of treatment-related lymphopenia in patients with glioblastoma treated with RT plus temozolomide.Between February 2006 and January 2017, we identified consecutive patients with histologically confirmed glioblastoma treated with temozolomide-based chemoradiation. We excluded patients who received whole-brain RT for gliomatosis cerebri or extensive disease, those who received whole-ventricle RT with a suspicion of ventricular seeding, and those without peripheral blood cell count results before and within 3 months after initiating RT.2 of body surface area per day, 7 days per week from the first to the last day of RT), followed by six cycles of adjuvant temozolomide (150–200 mg/m2 for 5 days during each 28-day cycle).Patients underwent either tumor resection or stereotactic biopsy. Thereafter, all patients received concurrent chemoradiation with temozolomide  was defined as a TLC of < 500/μL (grade 3/4 toxicity) within 3 months after beginning RT, as previously described .IDH1-R132H mutation (IDH1 mutation) status were also examined.Subventricular zone involvement was assessed via preoperative MRI using a standardized spatial classification system . The extP-value of <.05 in the univariate analyses were included in the subsequent multivariate analysis. All tests were two-sided, and significance was set at P < .05. The changes in blood cell counts over time were plotted using the R package “hexbin” [IDH1 mutation, PTV1, baseline TLC, and the cumulative dose of temozolomide. All statistical analyses were performed using the Statistical Package for Social Sciences version 23.0  and R software version 3.4.1 .Overall survival (OS) rates were calculated from the start date of treatment to the date of death or the latest follow-up visit by using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate analyses using Cox and logistic regression were performed to identify the predictors of OS and the development of ASL, respectively. Factors with a “hexbin” . Propens“hexbin” . Matchinn = 20), received whole-ventricle RT with a suspicion of ventricular seeding (n = 15), and had no peripheral blood cell count results before and within 3 months after initiating RT (n = 3). Thus, 336 patients who received partial brain RT were selected for the final analysis. Tumor resection was performed in 314 patients (93.5%), while stereotactic biopsy was performed in 22 (6.5%). Most patients  received a conventionally fractionated regimen with a total dose of 60 Gy in 30 fractions. Regarding the target volume, a limited field and a standard field were used in 49 (14.6%) and 287 patients (85.4%), respectively. A total of 186 (55.4%) and 150 (44.6%) patients received 3D-CRT and IMRT, respectively. Significant inter-group differences were observed between the 3D-CRT and IMRT groups in the age, sex, extent of resection, IDH1 mutation status, PTV, and number of fractions . The median PTV1 for the 3D-CRT and IMRT groups was 422 cm3  and 375 cm3 , respectively (P < .001). The mean cumulative dose of temozolomide for 3 months since the start of chemoradiation was not significantly different between the groups . The baseline patient characteristics and treatment details are provided in Table Of 374 consecutive patients, we excluded 38 because they received whole-brain RT for gliomatosis cerebri or extensive disease . Changes in hemoglobin concentrations and blood cell counts, including those of white blood cells, platelets, neutrophils, and lymphocytes, are shown in Fig. In total, 118 patients (35.1%) developed ASL. The median TLC at baseline was 1370/μL , with 3.0% of patients having grade ≥ 3 lymphopenia. At 1, 3, and 12 months after the initiation of RT, the median TLC was 1020/μL , 1120/μL , and 980/μL , respectively, and 10.1, 5.5, and 10.3% of patients developed grade ≥ 3 lymphopenia.P = .028; Fig. IDH1 mutation, MGMT methylation, and absence of subventricular zone involvement significantly indicated favorable OS . ASL was significantly associated with poorer OS on univariate analysis (P = .028), but it lost its significance for OS after adjustment for other confounders , the median OS was 20.5 months. Patients with ASL had significantly poorer OS than did those without ASL  and subtotal or partial resection (P = .001) were independently associated with increased ASL incidence. Among the treatment-related factors, an increased PTV1  was independently associated with increased ASL incidence. In contrast, the use of IMRT  was independently associated with decreased ASL incidence.The results of univariate and multivariate logistic regression analyses for ASL are shown in Table 3, ASL developed in 14, 18, 27, and 33% of patients in the IMRT group, respectively, and in 25, 39, 52, and 53% of patients in the 3D-CRT group, respectively. In total, ASL developed in 21.3 and 46.2% of the patients in the IMRT and 3D-CRT groups, respectively . In each PTV1 subgroup among the propensity-matched cohort, patients treated with IMRT had a lower ASL incidence  for glioblastoma and showed a large difference in the percent of blood receiving at least 0.5 Gy according to the target volume size. Our study confirmed the finding of this theoretical model regarding the effect of conventionally fractionated RT and PTV size on treatment-related lymphopenia by using clinical data of patients with glioblastoma.A mathematical computation model has supported the association between local RT for body parts without the bone marrow or lymphatic tissue and systemic lymphopenia . This mo0.5 Gy and V3 Gy were not significantly different between the 3D-CRT and IMRT groups, V5 Gy, V10 Gy, and V25 Gy were significantly lower in the IMRT groups


Mycobacterium tuberculosis infects over 10 million people annually and kills more people each year than any other human pathogen. The current tuberculosis (TB) vaccine is only partially effective in preventing infection, while current TB treatment is problematic in terms of length, complexity and patient compliance. There is an urgent need for new drugs to combat the burden of TB disease and the natural environment has re-emerged as a rich source of bioactive molecules for development of lead compounds. In this study, one species of marine sponge from the Tedania genus was found to yield samples with exceptionally potent activity against M. tuberculosis. Bioassay-guided fractionation identified bengamide B as the active component, which displayed activity in the nanomolar range against both drug-sensitive and drug-resistant M. tuberculosis. The active compound inhibited in vitro activity of M. tuberculosis MetAP1c protein, suggesting the potent inhibitory action may be due to interference with methionine aminopeptidase activity. Tedania-derived bengamide B was non-toxic against human cell lines, synergised with rifampicin for in vitro inhibition of bacterial growth and reduced intracellular replication of M. tuberculosis. Thus, bengamides isolated from Tedania sp. show significant potential as a new class of compounds for the treatment of drug-resistant M. tuberculosis. M. tuberculosis bacillus is extremely hardy and intrinsically resistant to acid, alkali, lysozymes, osmotic lysis and antibiotic killing3. This high tolerance to stress is due to the impervious quality of the mycobacterial cell wall, which is hydrophobic and contains few porins, thus reducing rates of transport of hydrophilic molecules and antibiotics into the bacillus5. For these reasons, effective antibiotics for tuberculosis (TB) are difficult to develop. The current treatment for TB consists of combinations of rifampicin (RIF), isoniazid (INH), ethambutol (EMB) and pyrazinamide (PZA), taken from twice-weekly to daily, over a period of six to nine months6. The persistence of infection causes many issues with patient adherence to the regimen and poses a significant obstacle to successful treatment, particularly in areas where the infrastructure required to ensure reliable drug supply, correct prescription and dedicated patient follow-up may be lacking. Intermittent and incomplete treatment increases the risk of relapse and incidence of multiple (MDR-TB) and extensively drug-resistant TB (XDR-TB)7, so there is an urgent and pressing need to develop new drugs which can shorten and simplify TB treatment in order to combat the burgeoning MDR-TB pandemic.The 8. Many current antibiotics are at their sixth or seventh generation of analogue development, and even these applications and approvals have declined steadily over time. One major factor behind the dearth of new antibiotics is the large but generally unsuccessful investment in genomic and target-based approaches by the pharmaceutical industry9. The failure of these approaches is likely due to difficulties in translating activity against a cell-free target to potency in more clinically relevant terms (such as the inhibition of whole cells), linked to adverse solubility or metabolic stability. Furthermore, the process of drug discovery and development is lengthy and expensive: it can take more than ten years and cost between US$800M and US$1Bn per drug10. The first-line treatment regimen for TB has not been updated to include new drugs for more than 50 years11.Since the golden era of antibiotic discovery during the 1940s and 1950s, when more than 20 new classes of antibiotics entered clinical use, only two novel classes have been discoveredPseudomonas aeruginosa12. Due to the extraordinary diversity and hit rates found when screening natural products13, there is currently increasing interest in returning to natural sources for drug discovery14. The marine environment has come under scrutiny as a relatively untapped cornucopia of novel chemical scaffolds displaying potent bioactivity15. An estimated 25% of all Earth’s biodiversity lies in marine species16 and an estimated 3.7 × 1030 microorganisms live in the marine environment17. For example, marine actinomycetes are unique to the ocean environment and produce numerous chemical metabolites distinct from those of terrestrial species18. Invertebrate species such as sea sponges and tunicates are other sources of novel drugs, including the FDA-approved cancer drugs trabectedin19, cytarabine20 and eribulin21 and the antiviral vidarabine22. In the search for new TB drugs, the marine environment offers a promising avenue of inquiry. In this study, by mining marine samples we have identified bengamide B as a TB drug development lead, being highly potent, synergistic with existing TB drugs and capable of inhibiting growth of intracellular and drug-resistant M. tuberculosis.Throughout history, the vast majority of antibiotics have been sourced from nature. The first antibiotic used in a clinical setting was pyocyanase, derived from M. tuberculosis H37Rv were identified. To determine this, 1434 diverse marine extracts23 were screened for their ability to inhibit M. tuberculosis growth in vitro. This identified 18 antimycobacterial hits, 11 of which derived from the Porifera phylum and five from the Chordata phylum  and 1.56 µg/mL (SN31927). Samples were screened for toxicity against differentiated THP-1 macrophages by determining CC50 values (the concentration at which cellular viability was reduced by 50%) , the two most effective antibacterial samples, SN31863 and SN31927, were non-toxic to THP-1 cells over a range of concentrations tested  revealed that insignificant amounts of cadmium (40 ppm) or zinc (0.037 ppm) were present in SN31863, the crude sponge extract, compared to values of 2000–15,000 ppm cadmium and 5000–5100 ppm zinc reported by Capon et al.24. This suggests that the antimycobacterial activity exhibited by samples SN31927 and SN31863 is due to a bioactive compound produced by the organism, and not general metal toxicity.Both SN31927 and SN31863 were derived from a M. tuberculosis H37Rv, while insufficient material was recovered from F7 and F9 for detailed bioassay analysis. MIC50 determination identified F10, F11 and F12 as the most potent, with F11 and F12 both active down to the low nanogram range (0.078 µg/ml)  is likely different to those of existing TB drugs, and as such, these fractions are promising drug leads.High performance liquid chromatography (HPLC) of SN31863 yielded 12 fractions, F1 to F12 Fig. , nine ofner Fig. . Testing32H58N2NaO8 [M + Na]+ (0.1 ppm) from the adduct ion, and five DBRE were calculated from this formula -CHCH = CHCH array . The presence of a single ring was deduced to be the fifth DBRE apparent in the molecular formula. Additional functional groups included three CH(OH) groups , a CH(O-CH3) group , a CH flanked by a carbonyl group and an amide NH , a second amide NH connected to a CH3 , two CH2 groups  and an aliphatic linear chain attached to a carbonyl . The COSY spectrum contained correlations for C-1 (δ 0.94(7), d) coupled to C-2 , which was in turn coupled to C-3 , C-4  and C-5   Fig. . The COSdd) Fig. . The COS-18 Fig. .Figure 325. Subsequent consideration of the NMR data confirmed this, through diagnostic peaks at δ 5.78, 5.44, 4.60 and 4.21, each of which corresponds to regions of the amide core structure  closely matched previously reported data, confirming F12 as bengamide B . Interestingly, bengamide B showed strong inhibition of EfMetAP1b, but this was not reflected in the results of bacterial inhibition screening, where bengamide B was not able to inhibit E. faecalis growth in vitro (data not shown). Similarly, although bengamide B was shown to inhibit HsMetAP1b, it was not cytotoxic against any tested human cell line in vitro (Table Bengamide B from various species inhibits activity of methionine aminopeptidase (MetAP), an enzyme that removes the N-terminal methionine from nascent proteinsM. tuberculosis H37Rv in combination with bengamide B at a range of concentrations . The results were analysed using the Chou-Talalay combination index (CI) equation29 and revealed strong synergy between bengamide B and rifampicin (CI = 0.1–0.3)  (1 nM) were incubated with .3) Fig. . Further.3) Fig. .Figure 430 and licensed for use by the FDA in 201231, and delamanid, licensed in 201432. Since then, the treatments which have progressed furthest through clinical trials are simply combinations of existing drugs33. Further work is urgently needed to bring novel and overlooked molecular scaffolds through development and into the TB drug pipeline. This study aimed to mine the marine biosphere in the search for potential TB drug leads using whole-cell screening techniques. The results reiterate that marine organisms are rich and underutilised sources of bioactive molecules. The most active antimycobacterial compounds identified in this study derived from sea sponges and sea squirts of the Porifera and Chordata phyla. As sessile filter feeders, these organisms rely heavily on secondary metabolites to gain competitive advantage in the struggle for space and resources in their benthic habitats34. Nine samples among the 18 antimycobacterial hits were broadly cytotoxic, and likely contain highly potent biological deterrents specifically produced by the organism, like other well-known examples such as cone shell poison, tetrodotoxin from puffer fish and stonefish neurotoxin36. Toxicity is the predominant cause of failure in marine natural product drug screening, with the cost of attrition increasing as samples progress through preclinical testing37. Consequently, any hits which resulted in cytotoxicity against any of the tested human cell lines were discounted from further investigation.Despite the heavy burden of TB disease and growing rates of drug resistance worldwide, efforts to develop new drugs for treatment have met with limited success. The two most recently approved drugs for TB are bedaquiline, discovered through investigation and optimisation of the diarylquinolone compound familyTedania sea sponges are known to cause blistering and peeling upon contact with bare skin38 and are therefore recognised as producers of potent bioactive molecules. T. ignis is the Tedania species that has been best documented in drug discovery studies, giving rise to several novel indoles39, an atisanediol40, a δ-lactam41, diketopiperazines39, the tedanolide family of potent antitumour macrolides42, and the tedanols, a group of anti-inflammatory diterpenes43. T. anhelans has given rise to some interesting pyrazole acids44, and T. digitis has been documented as the source of tedaniaxanthin45. All of these novel molecules and scaffolds isolated from Tedania spp. are larger (50–600 g/mol) aromatic compounds with some level of cytotoxicity. To date, the only antimycobacterial agents isolated from Tedania sponges were derived from a symbiotic strain of Penicillium chrysogenum with broad-spectrum inhibitory activity against S. aureus, P. aeruginosa, M. tuberculosis H37Ra, M. avium, M. smegmatis, M. fortuitum, M. vaccae, Aeromonas hydrophila, and Vibrio cholerae46. Purification of the active Tedania sp. sponge extract in the current study resulted in 11 active antimycobacterial fractions, one of which was resolved through mass spectrometry and NMR analysis as bengamide B. The other fractions either resulted in too little dry weight of material, or were not sufficiently pure for structure elucidation.Jaspis cf. coriacea sponges in 198625. Since then, the bengamide class of molecules has been found to have nanomolar antitumour activity47 due to the inhibition of methionine aminopeptidase (MetAP) type 1 and type 228, arresting cell growth at phases G1 and G2/M48. Bengamides have also been found to inhibit the NF-κB pathway, acting as potent anti-inflammatory compounds49. However the best-characterised bengamide, bengamide B, has gained most recognition as an antitumour agent. When used to treat MDA-MB435 human breast cancer xenografts on athymic rats in vivo, bengamide B significantly inhibits growth of MDA-MB435 human breast cancer xenografts49 and analogues of treatment have progressed to clinical trials for treatment of refractory solid phase tumours50. Interestingly, despite the levels of tumour cell line inhibition reported for bengamide B in the literature, the bengamide B natural product isolated in the current study did not display any notable cytotoxicity against THP-1 derived macrophages, HepG2, HEK293 or A549 cells 27. The two best-performing derivatives were potent MetAP inhibitors but resulted in modest MIC values of 122 and 53.9 µM respectively against mycobacteria, in contrast to the nM activity observed for Tedania-dervied bengamide B  was used for this study, along with the clinical M. tuberculosis 3410 (INH-resistant), 1863 (RIF/INH-resistant) and 2441 (RIF/INH/EMB-resistant) isolates. Middlebrook 7H9 media supplemented with albumin-dextrose-catalase , glycerol (0.2% v/v) and Tween-80 (0.05% v/v) was used to grow cultures which were incubated at 37 °C in a humidified 5% CO2 incubator.2 incubator at 37 °C until they reached ~80% confluency, at which point the cells were trypsinised if required and counted before resuspension at required cell concentrations.THP-1 (ATCC TIB-202), HepG2 (ATCC HB-8065), HEK293 (ATCC CRL-1573) and A549 (ATCC CCL-185) cells were used in this study. Frozen cell stocks were taken from liquid nitrogen, thawed and grown in cell culture flasks containing 25 mL RPMI for THP-1 cells, or DMEM for HepG2, HEK293 and A549 cells. All cell media was supplemented with 10% v/v foetal calf serum, 100 U/mL penicillin and 0.1% w/v streptomycin. The flasks were incubated in a 5% COTedania sp. was collected from the East Diamond Islet of the Tregrosse Reefs off the coast of Queensland, Australia. The sponge was deep-frozen immediately after collection and freeze-dried before extraction with methanol. This crude extract was then stored at −80 °C.Test samples consisted of 239 crude extracts obtained from marine organisms provided by the Australian Institute of Marine Science (AIMS). Crude extracts were sequentially fractionated in methanol by AIMS using a pre-equilibrated solid phase extraction Phenomenex Strata C18-E cartridge  to generate 1434 samples. All samples were supplied in 100% DMSO solution at 5 mg/mL and stored at -80 °C. The marine sponge 58. Bacterial strains were grown to log phase and diluted to a stock concentration of OD600 = 0.001. Test samples (0.5 mg/mL) or positive controls (5 μM rifampicin) were diluted in dH2O in 96 well plates and 10 μL of each sample dispensed into separate wells. Ninety μL of bacterial suspension was added to each well and incubated at 37 °C for 4 days, after which 10 μL of 0.05% resazurin was added to each well. After overnight incubation, fluorescence readings were taken at 590 nm after excitation at 544 nm using the FLUOstar Omega Microplate Reader . Percentage viability was calculated in comparison to the average of untreated control wells after normalising for background readings. Z’-factors were greater than 0.5 for all assays59. For the clinical isolates of drug-resistant M. tuberculosis, plates were incubated for 7 days before the addition of resazurin for 24 h and results of the resazurin reduction assay were read by eye. To determine MIC50 values, serial two-fold dilutions of test samples were made using dH2O to give final sample concentrations of 500 µg/mL to 0.01 µg/mL, which were tested as described above, and MIC50 was determined according to percentage survival in comparison to untreated controls. To assess drug synergy, M. tuberculosis H37Rv (OD600 = 0.001) was added to wells containing suboptimal concentrations of rifampicin (1 nM) or isoniazid (0.25 µM) together with with 1:3 serial dilutions of bengamide B starting at MIC = 1 μM. The plates were incubated and treated with resazurin before fluorescence readings, as described above.The resazurin reduction assay is a quantitative, colorimetric means of determining cell viability and was employed throughout this study to determine growth inhibition5 cells/well) were treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) before seeding in order to stimulate differentiation into macrophages-like cells. All cells were left to adhere in a 5% CO2 incubator at 37 °C for 48 h before test samples were added to a final concentration of 50 µg/mL. Plates were incubated for 4 days, then treated with resazurin for 16 h and read for fluorescence as per the previous section. To determine CC50 values, serial two-fold dilutions of test samples were made using dH2O to give final sample concentrations of 500 µg/mL to 3.9 µg/mL, which were tested as described above.Cells were seeded into 96-well plates at THP-1 cells  analyses were performed on the Perkin-Elmer OPTIMA 7000 DV Inductively Coupled Plasma-Optical Emission Spectrometer equipped with an axial torch, cyclonic spray chamber, and Meinhard® Type C concentric nebuliser. The operating conditions were RF Power 1300 W, plasma flow 15 L/min, auxiliary flow 0.2 L/min, nebuliser flow 0.8 L/min and a viewing distance of 15.0 mm. Each element was initially analysed by three spectral lines and then a single spectral line that exhibited low interference and high analytical signal and background ratios were selected for each element. These spectral lines were 228.804 nm (Cd) and 213.860 (Zn). Each element was quantified by the average of two readings.Prior to analysis, solid sponge sample was dissolved in concentrated nitric acid, then adjusted to 14% v/v concentration of nitric acid using Milli-Q HTedania sp. sample was carried out on a reversed-phase column , 100 µL sample injection and a gradient elution with 0 to 100% acetonitrile–H2O at 1 mL/min over 80 minutes, monitored at 215 nm. Fractions were collected, freeze dried and dissolved in DMSO at 5 mg/mL for testing against M. tuberculosis H37Rv and THP-1 derived macrophages. Electrospray Ionisation (ESI) High Resolution Mass Spectrometry (HRMS) was recorded in a positive ion mode on a Fourier transform ion cyclotron resonance mass spectrometer  with an Apollo II electrospray ionisation (ESI) ion source. The solvent for HRMS was CH3CN. NMR spectra were recorded on a Bruker AVANCE III 500 (1H at 500.13 MHz and 13C at 125.21 MHz). Spectra were referenced to residual solvent resonances (CHCl3 δ 7.26 (1H) and δ 77.16 (13C); DMSO δ 2.50 (1H) and δ 39.52 (13C)). All samples were dissolved in approximately 45 μL of DMSO-d6 or CDCl3 and transferred to 1.7 mm NMR tubes. All data were collected and processed using Bruker Topspin 3.5 software. Chemical shifts (δ) are reported in ppm, coupling constants in Hz, and for signal multiplicities: s = singlet, d = doublet, dd = doublet of doublet, ddd = doublet of doublet of doublet, dddd = doublet of doublet of doublet of doublet, qqd = quartet of quartet of doublet, t = triplet, m = multiplet.Semi-preparative HPLC of 5 cells/well and left to differentiate as described above. Cells were then infected with 106 M. tuberculosis H37Rv in complete RPMI for four hours. The media was removed and the cells gently washed 3 times with 2% FBS in PBS. One hundred μL complete RPMI was then added to each well along with test samples at a final concentration of 50 µg/mL. Rifampicin at 5 µM was used as a positive control for intracellular inhibition. The plates were incubated at 37 °C in 5% CO2 for 7 days, following which cells were lysed with cold dH2O and bacteria enumerated by plating on Middlebrook 7H11 agar supplemented with oleic-acid-albumin-dextrose catalase  and glycerol (0.5% v/v) for 3 weeks at 37 °C.THP-1 cells were grown to ~80% confluency in cell culture flasks and then counted, dispensed into 96-well flat-bottomed wells at 2 × 10Synergism between bengamide B and rifampicin was determined by calculating the CI . CI values of <1, =1, and >1 indicate synergism, additive effect, and antagonism of drugs, respectively. The DRI for both bengamide B and rifampicin were calculated using the multiple drug effect equation (DRI = Dx1/D1), where DRI values quantify how many folds of dose reduction result from drug combination in comparison to single drug treatment. Both CI and DRI were plotted against fraction affected (FA) to generate a Chou-Talalay and Chou-Martin plot providing visual illustration of synergism and dose-reduction.MtMetAP1c, EfMetAP1b and HSMetAP1b enzymes were expressed in E. coli and purified62. Samples were tested at a single concentration of 50 µM and enzymatic activity was monitored by fluorescence readings following hydrolysis of the fluorogenic substrate, methionyl aminomethylcoumarin, at room temperature. Percentage inhibition was calculated in comparison to negative controls.Statistical analysis was performed using GraphPad Prism 6 or 7 software . Differences between two groups were analysed by Student’s t-test, or between multiple groups by ANOVA with Tukey’s Multiple Comparisons test and were considered significant when the P values were ≤0.05.Supplementary Information


Work to date has largely focused on GR actions in forebrain excitatory neurons; however, recent studies suggest a potential role mediated by interneurons. Here, we targeted GR deletion in forebrain GABAergic neurons, including the cortical interneurons, using a Dlx5/6-Cre driver line to test the role of forebrain interneuronal GR in HPA axis regulation and behavior. Our data indicate that GR deletion in GABAergic neurons causes elevated corticosterone stress responsiveness and decreased cross-over latencies in a passive avoidance task in females, but not males. Dlx5/6-Cre driven gene deletion caused loss of GR in interneurons in the prefrontal cortex (PFC) and hippocampus, but also in select diencephalic GABAergic neurons . Our data suggest that GR signaling in interneurons is differentially important in females, which may have implications for GR-directed therapies for stress-related affective disease states.The glucocorticoid receptor (GR) is critically involved in regulation of stress responses [inhibition of the hypothalamic-pituitary-adrenal (HPA) axis], emotional behavior and cognition  The glucocorticoid receptor (GR) is critical for control of hypothalamic-pituitary-adrenal (HPA) axis reactivity, affective state and emotional memory in mice. Many mouse genetic knockdown/knockout (KD/KO) models have been created to elucidate the role of forebrain GR in these processes. In particular, numerous GR KO/KD models have been designed to assess the role of GR in potentiation of psychiatric illness and stress regulation. Pan-neuronal GR KOs  and forebrain glutamatergic neurons (FBGRKO) all increase anxiety-like and/or depressive-like behavior  is lost after chronic stress concomitant with deficits in behavioral control flox/flox mice with transgenic mice expressing Cre recombinase behind a Dlx5/6 promoter 1Kc/J; Stenman et al., ad libitum access to food and water in a humidity and temperature controlled vivarium with males on one side of PIV rack, females on the other. Due to the individual filtered ventilation it is unexpected that scent and auditory cues could be transferred. Experiments occurred 1–6 h after housing room lights came on. We split mice into two cohorts so all mice would be at least 8 weeks of age at the start of behavioral testing, as to mitigate confounds of adolescents. As cohorts were subsequently, the age of the mice differed between cohorts. Mice in cohort 1 started the experiment at 9–10 weeks while mice in cohort 2 started the experiment at 15–16 weeks. Cohort 1 consisted of n = 7 female Dlx5/6 GRKD, n = 9 female Control, n = 11 male Dlx5/6 GRKD; n = 15 Control. Cohort 2 consisted of n = 10 female Dlx5/6 GRKD, n = 10 female Control, n = 9 male Dlx5/6 GRKD, n = 10 male Control. Female cycle was not monitored. All experimental procedures were conducted in accordance with the National Academy of Science’s Guide for the Care and Use of Laboratory Animals and were approved by the University of Cincinnati Institutional Animal Care and Use Committee.Two cohorts of mice were bred and ran through behavioral assays Figure . Mice weMice were restrained in well-ventilated 50 mL conical tubes for 30 min and then released into their home cage. Blood samples were taken from tail clip at the moment of restraint, 0 min, for basal levels, 15 min during restraint, 30 min during restraint (released from restrainers), 60 min, (freely moving in home cage), and 120 min (freely moving in home cage) from initiation of restraint. For tail clip at the 0 min time point, a sterile razor blade was used to nick less than 1 mm from the distal most part of the tail. Pressure was used to stop bleeding after each blood collection and a gentle rub with sterile gauze was used to remove the clot and reinitiate bleeding at subsequent time points. Plasma was kept on ice and immediately stored in −20°C after the experiment. Plasma samples were analyzed with I-125 radioimmunoassay (RIA) kit from MP Biomedicals. Data was pooled for each sex from both cohorts of mice.For open field, mice were placed in a white plastic open field arena with an open top measuring 50 cm W × 50 cm L × 22 cm H under ambient room lighting. Mice were placed in a corner of the open field  and allowed to freely explore the arena for 10 min while being video recorded. Time the mouse spent in the center area (18.34% of the inner area) and total distance moved throughout the total arena was later analyzed with CLEVER software .For forced swim testing, mice were placed gently into 2L beakers filled 3/4 full with tap water at 25°C (±2°C) for 10 min under ambient lighting. Mice were video recorded and later analyzed by hand by a blinded observer. The animal’s behavior, either swimming or immobile (defined as the minimum motion required for the mouse to keep afloat) were noted every 5 s and then calculated as % of total time bins.For passive avoidance testing, on day 1, mice were placed in the lighted compartment of a light/dark shuttle box  with the center door closed. After 20 s the center door was raised manually and mice were free to enter the dark compartment. Once a mouse crossed all four paws over to the dark compartment, the door was manually closed and time to cross recorded. After a 2 s delay a 0.5 mA shock was administered for 2 s. Mice were removed from the dark compartment 30 s after the shock was administered back to their home cage. On day 2 (approximately 24 h after shock), mice were returned to the light compartment with the center door raised and time to cross to the dark compartment was measured. After crossing with all four paws to the dark compartment, the center door was closed and mice were removed 1 s later with no shock administered.Mice were injected with sodium barbital and transcardially perfused with 0.1 M PBS followed by 4% paraformaldehyde for 3–5 min. The thymus, heart, and adrenals were extracted and weighed after perfusion. Although differential changes in weight can be seen with perfusion, mice were given equal volumes of both PBS and paraformaldehyde. Brains were extracted and incubated in 4% paraformaldehyde for 24 h and then placed in 30% sucrose in PBS.2O2 for 10 min at room temp. Slices were washed 5 × 5 in PBS and then incubated at 4°C overnight in blocking solution  with GR antibody  at 1:250 dilution and Gad67  at 1:1,000 dilution. The following day slices were washed 5 × 5 in PBS and then incubated in secondary antibodies Cy3 anti-mouse  and cy5 anti-rabbit , both at 1:500 in blocking solution for 1 h at room temperature. Slices were washed 5 × 5 in PBS and mounted onto slices in PBS +1% gelatin.Brains were sliced on a sliding microtome on a frozen stage  at 35 μm in series of 6. Slices were preserved in cryoprotectant , and 30% Ethylene glycol, in 0.1 M PBS) until assayed. For GR and glutamic acid decarboxylase 67 (Gad67) co-expression, slices were washed five times for 5 min (5 × 5) in PBS, and incubated in 1% sodium borohydride in PBS for 30 min, room temperature. Slices were washed 5 × 5 in PBS and then incubated in 1% HFor Cre Recombinase immunohistochemistry slices were washed 5 × 5 in PBS and blocked for 1 h room temp in 4% goat serum, 0.2% BSA, 0.4% TritonX. Primary incubations of Cre (EMD Millipore MAB 3120) occurred overnight at 4°C. On the following day, slices were washed 5 × 5 in PBS and incubated in Cy3 anti mouse  secondary for 1 h room temperature. Slices were then mounted in PBS +1% gelatin. Images were captured with a Nikon Confocal microscope  at 10× to 20× objective. Basal Lateral Amygdala images were taken at an additional 1.55× zoom and the hippocampus at 1.6 zoom for clarity.post hoc analysis for differences at individual time points. Data from both cohorts were pooled in respective groups for the corticosterone assay after a three-way repeated measures ANOVA (cohort × time × genotype) showed no significant effects between cohorts  = 0.665; p = 0.418). Similarly, open field data was pooled as no significant cohort differences were evident . Due to the fact that the male data for the passive avoidance test did not pass equal variance and/or normality, both male and female data were analyzed with a Mann Whitney U non-parametric test (Rank Sum Test). Outliers were excluded from the analysis if they lied greater than 2 standard deviations from the mean. When data passed equal variance and normal distribution, parametric statistics were used. Male and female data were analyzed separately because female rodents naturally secrete higher corticosterone.Sigma Plot/Sigma Stat 13.0 software  was used to analyze all data. Data for weight and corticosterone measurements were analyzed with a two-way repeated measures analysis of variance (ANOVA). Fisher’s LSD was used for n = 6) and control  mice with GR KD. Due to the limitations of immunohistochemistry, complete KO cannot be assumed, and therefore cells without clear GR staining in the cell nucleus were considered KD (thus Dlx5/6 GRKD nomenclature will be used). We found the number of Gad67 cells containing GR in the cortex are significantly decreased in Dlx5/6 GRKD mice compared to control mice (T(6) = 7.196; p < 0.001; Figure Due to the small, diffuse cell population of Dlx5/6-Cre neurons, we first assessed the level of GR KD in Dlx5/6 GRKD mice, using dual immunofluorescence. The Dlx genes (including Dlx5 and 6) are critical for migration of interneurons from the lateral ganglionic eminence to cortical regions  = 2.645, p = 0.045). Cohort 2 females only had a significant effect of time  = 13.635; p < 0.001; Figure F = 2.852, p = 0.02, Time F = 53.779, p < 0.001, genotype F = 6.625, p = 0.02)]. Cohort 2 mice only had a significant effect of time, no interaction  = 43.042; p < 0.001; Figure post hoc comparisons revealed a significant weight difference between male Dlx5/6 GRKD and controls at each week. Dlx5/6 may be expressed in other tissues of the body that are commonly affected by stress, such as the adrenals and thymus. Importantly, there were no differences in males nor females in weights of hearts, thymi, nor adrenals  = 2.868, p = 0.032) in response to acute restraint challenge . (B) In contrast, male mice showed no differences in corticosterone during or after restraint  = 0.372; p = 0.828; Figure F = 42.752; p ≤ 0.001). Female controls had higher corticosterone compared to male controls at the 15 min (p = 0.003), 30 min (p < 0.001), and 60 min (p = 0.022) time points. In Dlx5/6 GRKD mice females had higher corticosterone at the 15 min (p = 0.031), 30 min (p < 0.001), and 60 min (p < 0.001) time points. There was no sex × genotype interaction  = 0.290; p = 0.592).Limbic forebrain regions including the PFC, amygdala and hippocampus are critical for negative feedback inhibition of the HPA axis after a psychological stress. These studies tested whether KD of GR in inhibitory neurons of the forebrain would cause similar, or perhaps opposing, effects on corticosterone. In females, there was a significant genotype by time interaction  = 5.041; p = 0.041). On the first day, neither male nor female mice showed a difference between control and Dlx5/6 GRKD to enter the dark compartment before shock [Females: genotype within day 1 p = 0.968, Males: genotype within day 1 p = 0.204 , whereas male Dlx5/6 GRKD have no significant change from day 1 to day 2 (p = 0.761). Female mice with Dlx5/6 GRKD had a lack of ability to inhibit cross over behavior, or were unable to recall the experience of shock in the dark compartment, as shown by a decreased latency to enter the dark compartment compared to controls on day 2 . Although male Dlx5/6 GRKD mice were not different from controls on day 2, male Dlx5/6 GRKD mice do not differ in time to cross from day 1 to day 2  = 1.024; p = 0.318, Sex × Day F = 0.046; p = 0.83, Sex × Genotype F = 0.506; p = 0.481) while the genotype × day effects still were present  = 6.248; p = 0.017). However, the design of the experiment, with males ran on a different day than females, may not have been optimal for detection of statistical significant sex differences. Additionally, to see a statistical sex difference, females Dlx5/6 GRKD mice would have had to cross over to the dark compartment much faster than male mice with Dlx5/6 GRKD which was not observed.To test if a change in behavioral control or impaired memory in an aversive context can be seen with GR loss in the forebrain Dlx5/6-derived neurons alone (in the absence of stress), we tested Dlx5/6 GRKD and control mice in the passive avoidance assay. Rodents have an inherent preference for dark confined spaces as opposed to open, bright areas that may carry environmental threats. Control mice will inhibit the compulsory behavior to escape to the dark compartment if previously given a foot shock in that chamber. A two-way repeated measures ANOVA revealed a day × genotype interaction for the females (T(17) = 0.296; p = 0.771; Females T(17) = −0.995; p = 0.334; Figures T(17) = 0.139; p = 0.891; Females T(17) = −0.0133; p = 0.990; Figures T(16) = 1.044; p = 0.312; Females T(14) = −1.317; p = 0.104; Figures We used the open-field assay to test for anxiety-like behavior. Mice will generally spend more time along the perimeter of the arena, and entry into the center is generally thought to represent decreased anxiety. Our male and female Dlx5/6 GRKD mice showed no differences from controls in center time  GR is required for normal regulation of stress reactivity and behavioral inhibition in females. Males appear to require GR in excitatory neurons for stress and emotional regulation as KO in CaMKIIα neurons produce higher corticosterone and deficits in fear conditioning, elevated plus, and dark/light box, forced swim test, and sucrose preference (Boyle et al., JS conceptualized and designed the study, acquired the data, analyzed the data, interpreted data and drafted the manuscript. PM and RM bred mice, acquired data, contributed to the design. LF acquired data and assisted in data analysis. BP acquired data and contributed to execution of assays. KC critically revised the manuscript for intellectual content. JH conceptualized study, interpreted data, and critically revised the manuscript for intellectual content for the final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.