Category Archives: TLR

Cancer Res

Cancer Res. cells. These findings suggested that SAHA treatment created a more drug-resistant state in residual ALDHbr cells. The imaging technique may facilitate searching and characterization of CSCs. by non-adherent suspension culture in serum-free medium, and they have been widely used to study underlying key molecular pathways [3]. Mounting evidences suggest that the tumor microenvironment is responsible for conditioning the stem cell status itself. The system has been questioned because of the entire differences between and systems in microenvironment. Side population (SP) technique and flow cytometry using cell surface markers have been applied to isolate CSC, but the specificity of these two methods is usually under debate. Previous studies reported that non-SP cells and CD133 cells can also generate tumors in NOD/SCID mice [4, 5]. Regarding the limitations in the isolation procedures, especially those used stem cell surface markers, would result in CSC injury, we designed an method using intracellular markers of stem cells which were identified in various human cancers to isolate CSCs from xenograft tumors in animal model. Aldehyde dehydrogenases (ALDHs) are detoxifying enzymes within a superfamily. In fact, Adamts5 the expression level of ALDH in stem cells usually high enough to protect them against oxidative insult, suggesting their well-known longevity. ALDH converts retinol to retinoic acid, a modulator of cell and stem cell proliferation. Elevation of the level of ALDH activity has been seen in stem cell populations of breast cancer [6], lung cancer [7], liver cancer [8] and colon cancer [9]. An ALDEFLUOR kit (Stem Cell Technologies) designed for precise identification and isolation of ALDH-bright CSCs using specific inhibitor for ALDH activity diethylaminobenzaldefyde (DEAB) Sofalcone was thus applied in this study. Histone deacetylase inhibitors (HDACIs) can induce hyperacetylation of specific proteins, recently considered as a new solution to inhibit cell proliferation and promote differentiation of various hematologic and solid tumors [10, 11]. Suberoylanilide hydroxamic acid (SAHA, Vorinostat), an HDACI, was approved by FDA for treatment of cutaneous T-cell lymphoma in 2004 [12]. Recent investigations Sofalcone exhibited that SAHA treatment can suppress the expression of the stem cell marker CD133 in glioma [13]. In addition, SAHA can also inhibit the ability to proliferation, self-renewal, migration, and invasion in human pancreatic CSCs by up-regulation of miR-34a [14]. These results implied that SAHA could be a potential agent for the therapy against CSCs. However, some studies revealed that SAHA leads to the increase of the stem cell markers in epithelialCmesenchymal transitions (EMT) phenotypic prostate cancer cells [15, 16]. These findings are in consistent with the clinical results of HDACIs, which have shown promise efficacy in hematological malignancies while disappointed effects in epithelial cell-derived cancers. The detailed mechanism of this phenomenon remains to be elucidated. In the current study, we aim to determine the significance role of SAHA in the mediation of CSCs in lung cancer. The ALDEFLUOR assay and FACS analysis were used to isolate CSCs from human lung carcinoma grown as xenografts on nude mice. The results showed that SAHA retards the growth of H1299 xenografts and decreases CSC population, but induces EMT phenotype and activates pluripotency associated program in the residual CSCs. Our results provide a possible mechanism for the limited treatment response of HDACIs in the clinical trials around the epithelial cell-derived cancer. RESULTS SAHA enhances the expression of CSC characteristics was examined in the H1299 human non-small cell lung cancer xenograft, which was inoculated subcutaneously in nude mice. The Sofalcone tumor progression rate was assessed by luciferase bioluminescent imaging. In the treatment group, the signals in the tumor are significant lower than that in vehicle-treated control tumor (Fig. ?(Fig.2A).2A). Daily administration of SAHA with the dosage of 100 mg/kg/day caused significant suppression of the growth of established H1299 tumors; reduction of 63% tumor volume compared with that of the vehicle-treated control animals (Fig. ?(Fig.2B).2B). Each animal receiving 100 mg/kg/day SAHA survived for at least 10 days. These results indicate that SAHA effectively reduces the tumor Sofalcone growth of H1299 xenografts at the dose of 100 mg/kg/day. Open in a separate window Physique 2 SAHA effectively inhibits the growth of H1299 tumor cells(A) Bioluminescent images of mice bearing H1299-CMV-Luc tumors before and after SAHA treatment. (B) Tumor growth curve of the subcutaneous H1299-CMV-Luc lung cancer xenograft in mice Sofalcone daily treated with vehicle alone or SAHA (100 mg/kg, i.p.). Data are presented as the mean tumor volume S.E. of the surviving animals in each groups..

Data Availability StatementThe datasets used and/or analyzed during the current research are available through the corresponding writer upon reasonable demand

Data Availability StatementThe datasets used and/or analyzed during the current research are available through the corresponding writer upon reasonable demand. survival times. To conclude, reduced DC-SIGNR manifestation in HCC cells may be another predictive biomarker of medical prognosis, not only ARP 101 is it a viable restorative focus on for HCC treatment. reported that secreted DC-SIGNR amounts in patient’s serum examples with cancer of the colon were considerably higher weighed against healthy controls; however no detectable upregulation was observed in DC-SIGNR expression in tumor cells relative to normal tissue (9). By contrast, Liu reported that serum DC-SIGNR levels in patients with lung cancer were lower compared with healthy controls, whereas in a subset of patients with brain metastases, serum DC-SIGNR levels were higher compared with patients without metastases (10). Another study identified significant increases in serum DC-SIGNR in patients with gastric cancer relative to healthy controls (11). Considering the limited number of studies assessing DC-SIGNR expression in HCC tissues, the aim of the present study was to compare the levels of this protein in tumor tissue samples and in adjacent non-cancerous tissues from patients with HCC. A combination of immunohistochemical and bioinformatics analyses was used to evaluate DC-SIGNR expression in HCC and characterize its potential functions. Materials and methods Patients and samples A total of 267 HCC samples and 166 adjacent non-tumor liver tissue samples were collected from patients who underwent surgery at Zhejiang Provincial People’s Hospital (Hangzhou, China) between January 2010 and December 2017. The tissues were verified to become non-cancerous or cancerous by medical center pathologists, set with 4% formalin for 24 h at space temperature and inlayed in paraffin. Info on individual sex, age group, tumor size, quantity, location, Edmondson site and quality of tumor metastasis was collected during individual hospitalization and treatment. Because some medical data had been unavailable or lacking, the total amount of some medical signals was <267. General survival (Operating-system) was established using either the day of individuals' loss of life or the last follow-up period ARP 101 point. This scholarly research was authorized by the Review Panel of a healthcare facility Ethics Committee, and written informed consent was from each participant to data collection prior. Immunohistochemical staining Paraffin-embedded specimens had been used to create three microarrays by using the Shanghai BioChip Co., Ltd. (Shanghai, China). Immunohistochemistry (IHC) was performed using the next process: Three cells section microarrays (5 m) had been warmed at 70C for 2 h, cleaned three times inside a xylene remedy to eliminate paraffin, rehydrated in reducing concentrations of ethanol (100, 95, 85 and 75%; each for 5 min), and boiled in Tris-EDTA (TE) buffer (Tris, 1.21 g/l; EDTA, 0.37 g/l; Tween-20, 0.5 ml/l) under ruthless (103 kPa) for 3 min to facilitate antigen retrieval. The examples had been incubated with 3% hydrogen peroxide for 15 min to avoid endogenous peroxidase activity, clogged with 10% goat nonimmune serum (reagent A; Histostain?-in addition Bulk kit; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 20 min to lessen nonspecific binding and incubated with anti-DC-SIGNR antibody (1:400, kitty. no. ab169783; Human being Compact disc299 Antibody; Abcam, Cambridge, UK) at 4C overnight. Sections were cleaned and incubated having a biotinylated supplementary antibody (reagent B; Histostain?-in addition Bulk kit; Thermo Fisher Scientific, Inc.) for 15 min. Examples were subjected ARP 101 to streptavidin-peroxidase (reagent C; Histostain?-in addition Bulk kit; Thermo Fisher Scientific, Inc.) for yet another 15 min and a chromogenic response was performed using the 3,3-diaminobenzidine color substrate remedy (OriGene Systems, Inc.; Beijing, China) based on the Rabbit polyclonal to ACSM2A manufacturer’s process. Color advancement was terminated whenever a brown sign indicative of staining was apparent in the test. Hematoxylin (kitty. simply no. C0107; Beyotime Institute of Biotechnology, Haimen, China).

Supplementary MaterialsSupplementary data 1 mmc1

Supplementary MaterialsSupplementary data 1 mmc1. with no variations among three subgroups. Summary The IgM-IgG antibody check exhibited a good adjunct to RT-PCR recognition, and improved the precision in COVID-19 analysis of the severe nature of disease irrespective, which provides a highly effective go with towards the false-negative outcomes from a nucleic acidity check for SARS-CoV-2 disease analysis after onsets. valuevaluevalue /th th rowspan=”1″ colspan=”1″ /th th rowspan=”1″ colspan=”1″ n?=?44 /th th rowspan=”1″ colspan=”1″ n?=?52 /th th rowspan=”1″ colspan=”1″ n?=?37 /th th rowspan=”1″ colspan=”1″ /th /thead IgM29.1940.7623.250.446(17.04C61.02)(13.56C90.13)(8.67C104.5) br / br / IgG147.73148.63140.40.182(89.53C171.6)(130.95C167.7)(93.79C162.8) Open up in another window Notice: The focus device of antibodies in serum examples is AU/ml. The worthiness of AU/ml? ?10 is recognized as an optimistic reaction. 4.?Dialogue The outbreak of pneumonia quickly due to SARS-CoV-2 spreads, posing a significant threat towards the lives and wellness AZD5153 6-Hydroxy-2-naphthoic acid from the sociable people, which has turn into a serious global concern. SARS-CoV-2 is one of the coronavirus beta genus, having a linear single-stranded positive RNA, the seventh coronavirus recognized to infect human beings after SARS (2002) and MERS (Middle East respiratory symptoms coronavirus) (2012) [18]. There are many assays created to Mouse monoclonal to CD13.COB10 reacts with CD13, 150 kDa aminopeptidase N (APN). CD13 is expressed on the surface of early committed progenitors and mature granulocytes and monocytes (GM-CFU), but not on lymphocytes, platelets or erythrocytes. It is also expressed on endothelial cells, epithelial cells, bone marrow stroma cells, and osteoclasts, as well as a small proportion of LGL lymphocytes. CD13 acts as a receptor for specific strains of RNA viruses and plays an important function in the interaction between human cytomegalovirus (CMV) and its target cells detect different parts of SARS-CoV-2 genome using RT-PCR [10], [19]. In today’s study, we examined both antibody and nucleic acidity -centered diagnostic strategies on suspected individuals with moderate to important symptoms for COVID-19. Of the full total 133 patients had been examined, 68.4% (91/133) were positive regarding RT-PCR and 78.9% (105/133) regarding the antibody test. It had been also observed an elevated positive price in antibodies-based testing compared to that in nucleic acidity check in the analysis for COVID-19 individuals in various subgroups (moderate instances, severe instances, and critical instances). Our results suggested how the IgM-IgG antibody check has an effective go with towards the false-negative outcomes from nucleic acid check for COVID-19 analysis. Recently, upper body CT scans had been requested the rapid detection of SARS-CoV-2 induced COVID-19 [11], [20]. The chest x-ray or chest CT provides more information, but these are not conclusive as not all the patients with COVID-19 developed pneumonia and might produce false results as many other things can also cause pneumonia [21], [22]. Therefore, a more effective strategy such that testing antibodies or RNA is usually important. The conventional serologic assays, droplet digital (dd)PCR, CRISPR-based, and metagenomic next-generation sequencing (mNGS) techniques are also novel approaches for the detection of SARS-CoV-2. In fact, the optimal diagnosis ways for SARS-CoV-2 are usually selected based on the periods of illness onsets (eg. RT-PCR or serologic AZD5153 6-Hydroxy-2-naphthoic acid assays), the viral load of specimens (eg. RT-PCR or ddPCR assays), AZD5153 6-Hydroxy-2-naphthoic acid and the aim of pathogen identification of unexplained pneumonia (eg. CRISPR-based or mNGS techniques) [12], [23], [24], [25]. Hence, it is highlighted that this combined assessments on SARS-CoV-2 antibodies and RNA for the high accuracy of COVID-19 diagnosis according to the desired requirements. SARS-CoV-2 is an emerging kind of infectious pathogen and the immunological testing reagents have been recently developing [12]. It’s been established a higher awareness and specificity of SARS-CoV-2 IgM and IgG antibodies recognition in serum or plasma from COVID-19 sufferers, without cross-reactivity within examples from noninfected people [12], [26]. Even though the antibodies generated over time from the starting point of infections, their detections hand and hand with RT-PCR recognition were found even more promising as a precise detection technique in today’s situation. It’s advocated that serum antibodies-based exams could possibly be adjunctive to RT-PCR check successfully, especially for sufferers who got the significant length of disease, in whom RT-PCR may be unfavorable. The combining RT-PCR and IgM-IgG antibody detections significantly improved the sensitivity of pathogenic diagnosis for COVID-19 even in the early.

The hedgehog pathway, for which sonic hedgehog (Shh) is the most prominent ligand, is highly conserved and is tightly associated with embryonic development in a number of species

The hedgehog pathway, for which sonic hedgehog (Shh) is the most prominent ligand, is highly conserved and is tightly associated with embryonic development in a number of species. attempts at targeting this pathway, there are only three FDA-approved drugs for cancers that affect the Shh pathway. Two of these compounds, vismodegib and sonidegib, target SMO to suppress signaling from either PTCH1 or SMO mutations that lead to upregulation of the pathway. The other approved compound is arsenic trioxide (ATO), which can suppress this pathway at the level of the GLI proteins, although current evidence suggests it also has other targets. This review focuses on the efficacy and safety of these clinically-approved drugs targeting the Shh pathway along with a discussion on other Shh pathway inhibitors being developed. 1.?Introduction The hedgehog pathway is a highly conserved signaling pathway that is linked to many biological processes. This signaling pathway has been linked to development in many species, including humans (1). It has been linked to growth and patterning in many of these multicellular species including the development of the neural system and bone development (2, 3). The hedgehog pathway and its components have also been linked to several diseases, prominently including human cancer (4). Because of the importance of this pathway to human cancer, there have been several attempts to target this pathway for cancer therapies with few successes and many failures. In this review, we aim to provide an update on the successful agents targeting the hedgehog pathway that have been FDA approved for treatment in human cancers. We will also briefly discuss agents that are currently being developed to target this pathway for the treatment of cancer. 2.?The Hedgehog Pathway in Cancer Mammalian hedgehog signaling can be initiated by three unique ligands in Sonic Hedgehog (Shh), Indian hedgehog, and Desert hedgehog. However, Shh is the most widely expressed and also the most potent of these ligands (1, 5). The ligand Shh is expressed as an inactive full-length protein that is proteolytically cleaved to two proteins and the N-terminal 19 kDa fragment is the active Shh ligand (6). The receptor for this active Shh ligand is Patched1 (PTCH1), a 12-transmembrane protein that binds Shh ligand. Binding of Shh to PTCH1 relieves repression of Smoothed (SMO) by PTCH1 thereby activating SMO signaling activity (Figure 1). The activation of SMO ultimately decreases the interaction between suppressor of fused homolog (SUFU) and GLI proteins that allows GLI proteins to enter the nucleus and bind transcriptional targets to regulate cellular gene expression. There are three GLI isoforms in mammals in GLI1-3 wherein gene expression can be induced by GLI1 and repressed by GLI3 whereas GLI2 can regulate expression in either direction. The GLI proteins are the terminal effectors of the Shh signaling pathway and regulate genes that control organismal patterning and development. Many of the genes regulated by GLI proteins are co-opted by cancer cells as they regulate several cancer-related processes including proliferation, migration and invasion, as well as neovascularization (4). Open in a separate window Figure 1. The Sonic Hedgehog Pathway. A) In the absence of Shh ligand, PTCH1 suppresses SMO allowing for SUFU suppression of GLI1. B) In the presence of Shh ligand, PTCH1 repression of SMO is removed allowing for SMO to repress SUFU leading to the release and nuclear translocation of GLI1. GLI1, and the other GLI proteins then promote a gene expression program that promotes multiple cancer phenotypes. The inhibitors to this pathway, the FDA-approved inhibitors highlighted in KN-93 green, primarily have targeted SMO with some KN-93 KN-93 attempts to target Shh itself and the GLI proteins, but with little success. There have been numerous reports of genetic alterations in key components of the Shh pathway in different tumor types NOX1 that leads to constitutive signaling of this pathway and that paracrine signaling of Shh may be an important factor in multiple tumor types (7, 8). While there are reports of the Shh pathway being modified in several tumor types such as breast, pancreatic, colorectal, and rhabdomyosarcoma among several, genetic alterations in this pathway are most consistently seen in basal cell carcinomas (BCCs).

Supplementary Components1

Supplementary Components1. to find out how chronic GDC-0834 surprise tension (CSS) GDC-0834 impacts electrophysiological and neurochemical properties of Type I, Type II, and Type III neurons within the BNSTALG. That CSS can be reported by us led to adjustments in the insight level of resistance, time constant, actions potential waveform, and firing price of Type III however, not Type I or II neurons. Additionally, just the sort III neurons exhibited a rise in mRNA along with a reduction in striatal-enriched proteins tyrosine phosphatase (usage of water and food. Experiments had been performed in 63 rats (discover Shape 2A for experimental style). For information on strategies, see Supplementary Components. Open in another window Shape 2. Experimental style (A). ASR: acoustic startle response; NS: no tension; CSS: chronic surprise tension; OF: open up field; EZM: raised zero maze. CSS rats froze even more in the surprise framework than NS rats on day time 8 (NS: = 24; CSS: = 23) and day time 13 (NS: = 8; CSS: = 8) (B). CSS rats got an increase amount of fecal boli through the entire tension paradigm, indicative of improved anxiousness and emotionality (NS: = 24; CSS: = 23; C). On day time 13, CSS rats demonstrated a higher upsurge in startle amplitude compared to the NS rats (NS: = 32; CSS: = 31; D). You can find no significant variations between the NS and CSS groups in time spent in the open portion of the EZM (NS: = 12; CSS: = 12) on days 13 and 14 respectively (E). Panel B: t-tests. Panel D: Mann-Whitney and (GluRl)Rn00709588_m1(GluR2)Rn00568514_m1(GluR3)Rn00583547_m1(GluR4)Rn00568544_m1(CRF)Rn01462137_m1(STEP)Rn01480059_m1(PP1A)Rn00580546_m1(PP1B)Rn00565033_m1(PP1C)Rn04339209_m1(Calcineurin A)Rn00690508_m1(Calcineurin B)Rn00566864_m1(Calcineurin C)Rn01465907_m1(DARPP-32)Rn01452984_m1 Open in a separate window 2.5. Statistical Analysis Statistical analyses were carried out using Prism 6 (GraphPad Software Inc., San Diego, CA), R (R version 3.2.3, RStudio v. 0.98.1103), and Matlab (The MathWorks, Narick, MA). Power transformations, Students = 52) had a significantly lower input resistance (A) and time constant (B) than Type III cells from the NS group (= 42). Type III cells from the CSS group also had a longer action potential rise time (C) and longer action potential half-width (D) than Type III cells Mouse monoclonal to HDAC4 from the NS group. There was no significant effect of stress on any of the parameters measured in Type I (NS: = 33; CSS: = 31) or Type II cells (NS: = 52; CSS: = 49; A-D). Recordings from 24 NS and 23 CSS rats. Error bars show SEM. * p 0.05, ** p 0.01. Open in a separate window Figure 5. CSS led to a loss GDC-0834 of sensitivity to Naspm (100 M) in the BNST; however, Type III neurons did not show this GDC-0834 effect of CSS. Type III cells in the BNST exhibited a depression of EPSC amplitude with Naspm application; however, there was no difference in the response between NS (open circles; = 8) and CSS (black squares; = 6; A). Non-type III cells, including only Type I and Type II cells showed a depression in EPSC amplitude with Naspm application, and there was a significant difference in the response between NS (open circles; = 9) and CSS (black squares; = 19; B). Error bars show SEM. Data from 9 CSS and 14 NS rats. Insets, representative average of three consecutive EPSCs from Type III (A) and non-Type III (B) cells from NS and CSS rats before (grey) and after (black) application of Naspm. Scale bars: 20 pA and 20 ms. 3.?Results 3.1. Context fear and anxiety-like behavior After seven days of CSS, the percentage of time rats spent freezing in the shock context was measured as an indicator of contextual fear learning. CSS rats froze significantly more than NS rats (606 % of time compared to 91 %; = 37, = 28; CSS: = 28; A), Type II (NS: = 47; CSS: = 47; B), and Type III (NS: = 37; CSS: = 44; C) cells from CSS (black squares) and.

Rationale: Individuals with chronic illness are usually asymptomatic; therefore, their condition is definitely very easily overlooked

Rationale: Individuals with chronic illness are usually asymptomatic; therefore, their condition is definitely very easily overlooked. and a bacterial infection. The patient experienced progressive respiratory failure and was placed on a ventilator. He was immediately treated with albendazole when was found in samples of his sputum and feces. Outcomes: The patient died despite treatment with albendazole and antibiotic therapy. Lessons: It is essential to consider the possibility of illness in immunosuppressed individuals with nephrotic syndrome. Given the lack of classic manifestations and high mortality rate of advanced disease, continuous monitoring, early analysis, and proper treatment are imperative. is an intestinal parasite that spawns larvae in the dirt and primarily infects humans. Most instances of are distributed in tropical, subtropical, and temperate areas.[1] From 1973 to 2013, 330 Risedronate sodium cases were reported in China, mainly in the southern regions.[2] Those chronically infected with are usually asymptomatic and are easily overlooked by healthcare workers. The immunosuppressed population is more vulnerable to disseminated infection and is more likely to develop hyperinfection. Many studies on have focused on organ transplant recipients and patients with malignant tumors, since these individuals often receive multi-target immunosuppression treatment and therefore have severe immunodeficiency. We reviewed the literature and record a complete case of the fatal hyperinfection in an individual with nephrotic symptoms. 2.?Case record A 70-year-old man suffered progressive generalized edema after consuming stale crabs, with just mild abdominal distress no fever or additional symptoms. Before this, he was healthful and didn’t possess Risedronate sodium a history background of digestive illnesses, diabetes, or chronic obstructive pulmonary disease. The person was a indigenous of Chongqing, the subtropical region in southwest Tcfec China. He utilized to be always a soldier; he fought in the Vietnam Battle and joined the authorities force after time for his hometown. In a healthcare facility, his preliminary vitals had been BP 108/78 mmHg, HR 111, respiratory price 22, and air saturation 98%. Preliminary laboratories included white bloodstream cells 13.32 109/L (neutrophils% 77.5%; lymphocytes% 13.31%; eosinophils% 0.5%), normal platelets and hemoglobin, albumin (ALB) 14.5?g/L, globulin (GLB) 19.9?g/L, alanine aminotransferase (ALT) 78.7?IU/L, aspartate aminotransferase (AST) 90.9?IU/L, creatinine (Cr) 134?mol/L, 24-hour urine protein 9.61?g, and bad antinuclear antibody range (ANAs) and anti-neutrophil cytoplasmic antibodies (ANCA). Upper body X-ray showed gentle emphysema but no indication of disease. The individual was identified as having nephrotic symptoms but was struggling to go through pathological biopsy because of a renal cyst. He was given full-dose glucocorticoid therapy only, with no additional immunosuppression. Three weeks later on, while under this treatment still, the patient experienced lower limb cellulitis. His procalcitonin (PCT) was 0.3 ng/ml, and he was administrated mupirocin IV and ointment cefuroxime. After those remedies, his position improved and he continued to take oral glucocorticoids after discharge from the hospital. However, Risedronate sodium over the next ten days, the patient seemed to get worse and had to return to the hospital due to persistent fever, cough, and intermittent abdominal pain. Initial vitals on admission were temperature 37.8?C, BP 90/60 mmHg, HR 125, respiratory rate 26, and oxygen saturation 95%. Laboratory tests showed white blood cells 12.36 109/L (neutrophils %: 83.5%; eosinophils %: 0.7%). Sputum smear and culture were negative. Imaging examinations included CT scans of the chest, which reported interstitial pneumonia (Fig. ?(Fig.1),1), and the abdomen, which reported no specific findings. The patient was diagnosed with normal gastrointestinal discomfort and pulmonary bacterial infection. A proton pump inhibitor, cefoperazone sodium, and sulbactam sodium were administered. However, the patient then started display hemoptysis, passed occult blood-positive stool, and gradually fell into a state of hyperpyrexia and drowsiness. Soon, (which was sensitized to the previous antibiotic), and unexpectedly, a large number of larvae (Fig. ?(Fig.2)2) were found in repeated sputum specimens. Meanwhile, the parasite was.