Animals were provided rodent

diet and tap water ad libitum throughout the study. Research was conducted at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) and was in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals. USAMRIID is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. this website Mice were vaccinated SC or IM with fV3526 alone or formulated with adjuvant on Day 0 and 28. Due to restrictions in the volume of inoculum that can be delivered to a mouse via the IM route, the SC vaccinated mice received five times more viral protein (0.2 μg) per dose than IM vaccinated mice

(0.04 μg) (Table 1). For SC vaccination, 0.5 mL of inoculum was administered to the interscapular area. For IM vaccinations, 0.025 mL was administered into the muscle of each hind limb. C84 was administered according to the dosage (4 μg), route (SC), and schedule (0, 7, and 28) used in previously published animals studies [13] and [28] and as administered to human vaccinees [8] and [29] to allow comparisons between the data collected in this study to historical studies. Further, the dosage, route, schedule and use of adjuvants with C84 was not evaluated as the intent of the comparisons to be made with C84 were to show fV3526 formulations are as good as or better than C84 in its current formulation as the US government does not intend to fund further development of C84 as a VEEV vaccine. Sham-vaccinated mice received PCM either SC or IM and adjuvant control mice received Viprovex®, Alhydrogel™, SRT1720 cell line CpG or CpG + Alhydrogel™ at the same concentrations and on the same schedule as administered in experimental groups with fV3526. On Day 21 and 49 post-primary vaccination, blood was collected

from all mice for measurement of antibody responses. Mice were challenged on Day 56 with 1 × 104 pfu VEEV TrD by the aerosol or SC route. Aerosol exposures were conducted by putting mice in wire cages into a chamber where they were exposed to aerosolized virus for 10 min. Virus collected aminophylline in an all-glass impinger was titrated to determine the concentration of virus (pfu/L) in air using a previously described plaque assay method [30] and the volume inhaled was estimated using Guyton’s formula [31]. Mice were monitored daily for signs of illness for 28 days post-challenge at which time surviving mice were euthanized. One iteration of each vaccination-challenge study was conducted, unless otherwise noted. Virus-neutralizing antibodies in the immunized and control mice were determined as previously described [32] using live VEEV TrD virus as target antigen. Sera were serially diluted two-fold with virus and incubated overnight at 4 °C. The serum-virus mixtures were added to Vero cell monolayers for 1 h at 37 °C, after which the cells were overlaid with 0.

Other criteria identified include disability or quality adjusted life years lost, hospitalizations, morbidity, and epidemic potential for the disease in question plus issues of equity and the possibility of disease eradication. Many countries report that they rely more and more frequently on local data and where reported universally indicate a preference Ivacaftor cost for local data. Local data may be particularly relevant for diseases with highly variable epidemiology or for vaccines that behave differently in different populations. Committees not only use, or in some cases require local data but in most cases also make recommendations on additional local

research and data that are needed before a decision can be made. Economic evaluation data are considered by Selleckchem GSK1210151A all committees with the exceptions of Australia and Canada (where a separate advisory

committee evaluates economic issues). However, only the United Kingdom’s committee uses specific cost-effectiveness cut-offs for making recommendations on including vaccines in the public vaccination schedule. Five countries report that their committee considers financial sustainability when reviewing evidence (Iran, Korea, Oman, Sri Lanka, and Switzerland). The Sri Lankan committee reports that it does not recommend a vaccine unless it is certain that the country can sustain financing regardless of the availability of donor support such as through the GAVI mechanism. The other four committees do not report how financial sustainability issues affect committee

recommendations. In contrast to these five countries, the remaining countries included in the supplement indicate that why financing aspects are taken into consideration by the government after issuance of committee recommendations. In general countries use all sources of data available to them. This may include peer-reviewed articles, findings of other NITAGs, WHO documents, regional data (for example, Oman shares data with other gulf countries), and local data (published or unpublished). Beyond the use of data and publications from WHO, six countries report on the influence of WHO recommendations for final committee decisions. In three instances (Honduras, Oman, and Switzerland) the committee to date has supported all WHO recommendations. Three committees (South Africa, Thailand, and the United States) state that they modified WHO global recommendations to the local national circumstances. Twelve NITAGs indicate the process by which final recommendations are made and in seven cases this is by consensus and in five by voting. Among groups that vote, this usually occurs by majority vote. NITAG recommendations may have considerable implications for vaccine sales and thus most of the included manuscripts emphasize that committee members must be independent of pharmaceutical industry influence.

, 2014). In conjunction with findings in animal models, these results are consistent with the hypothesis that stress-associated changes in connectivity in large-scale brain networks are BKM120 chemical structure an important feature of depression and other stress-related neuropsychiatric disorders, and that resilience and vulnerability may be determined

in part by individual differences in the capacity for plasticity within these circuits. Understanding the mechanisms by which stress alters connectivity in vulnerable circuits may reveal new avenues for treatment. Undoubtedly, many factors are involved, and some of them have been reviewed elsewhere (De Kloet et al., 1998a, McEwen, 2000, De Kloet et al., 2005b, Arnsten,

2009, Joëls and Baram, 2009 and Chen et al., 2010). Here we focus on a factor that has received relatively little attention, namely, endogenous glucocorticoid oscillations and their role in regulating synaptic plasticity. Glucocorticoids are hormones that are released from the adrenal gland in response to signals originating in the pituitary and hypothalamus, which receives projections from distinct circuits for detecting physiological and psychosocial stressors (Herman and Cullinan, 1997 and Ulrich-Lai and Herman, 2009) (Fig. 2a). In the short term, glucocorticoids serve to mobilize energy resources and facilitate sympathetic nervous system responses to maintain homeostasis and adapt BI 6727 nmr to stress. In the long term, however, prolonged exposure to glucocorticoids in chronic stress states can have maladaptive effects, mediated in part by disruptions in negative feedback mechanisms (McEwen, 1998 and McEwen, 2003). Glucocorticoid activity also oscillates with diurnal activity rhythms, independent of external stressors (Fig. 2b): glucocorticoid secretion tends to peak in the early morning in diurnal animals (early before evening in nocturnal animals), remains relatively elevated for most of the active period of the animal’s

day, and becomes relatively suppressed for most of the night. In addition, recent reports (Stavreva et al., 2009a and Lightman and Conway-Campbell, 2010) have shown that an ultradian oscillation with a period of 1–2 h is superimposed on this circadian rhythm and has equally important consequences for glucocorticoid signaling (reviewed below). In previous fixed tissue studies, stress and glucocorticoid effects on dendritic arborization and spine density took weeks to develop (Magariños et al., 1996, Wellman, 2001, Vyas et al., 2002, Radley et al., 2004 and Radley et al., 2006), which would imply that glucocorticoid oscillations occurring on a timescale of minutes to hours were unlikely to play a direct role in these changes. However, recent studies indicate that glucocorticoids and related signaling molecules can have much more rapid effects on dendritic spines than were previously suspected.

4) on a magnetic stirrer at 37 ± 0.5° at 100 rpm. 5 ml

quantity of sample was withdrawn at different time periods and same volume of dissolution medium was replaced in the flask to maintain Cell Cycle inhibitor sink condition. The withdrawn samples were filtered and then the filtrate was diluted with phosphate buffer (pH 7.4). The samples were analyzed for drug release by measuring the absorbance at 249 nm using UV–visible spectrophotometer. The in vitro drug release studies were carried out in triplicate for each formulation. The in vitro release data of all the formulation were fitted with various kinetics models such as zero order, first order, Higuchi model and Korsmeyer–Peppas, 9 in order to predict kinetics and mechanism of drug release. The release constant was calculated from the slope of plots and regression

coefficient (r2), diffusion exponent (n) was determined. The stability study of freeze dried nanoparticles was carried out for D1 (1:2) to assess the stability of drug in nanoparticles. For this purpose the samples were taken in borosilicate vials and sealed and the vials were stored in room temperature (25°–30 °C) and refrigerator (3°–5 °C) over a period of 3 months. After specified period 0, 1, 2 and 3 months, the samples were checked for their physical appearance and drug content by UV spectrophotometer, as well as chemical stability by Fourier transform infrared (FTIR) studies. The biodistribution studies8 of ddi loaded albumin INCB024360 ic50 nanoparticles were carried out on healthy adult Wistar rats weighing 200–250 g and after obtaining approval from the local animal ethics committee and CPCSEA (DSCP/PH.D PHARM/IAEC/49/2010-2011). All animals were provided with proper care, food, water ad libitum

and were maintained under well ventilated in large spacious cages throughout the study. The rats were divided randomly into three groups with three animals per group and they were fasted at least 12 h before experimentation. Group 1 was injected with ddi (which was dispersed in water for injection) into the tail vein of rats, Group 2 was received ddi loaded albumin nanoparticles and Group 3 was administered polysorbate 80 coated albumin nanoparticles. All the formulations were given in a dose level equivalent to 20 mg/kg body weight. 7 One hour after injection, the rats were sacrificed by euthanized and organs such as liver, lung, kidney, Bumetanide lymph nodes, spleen, brain and blood were isolated. The organs were washed with clean buffer saline and absorbed dry with filter paper and then weighed. Prior to the analysis organs homogenates were prepared and was digested with 10% v/v trichloroacetic acid and was treated with 10 ml of acetonitrile to extract didanosine. Didanosine content in the various organs was estimated by reverse-phase HPLC method. BSA nanoparticles were prepared and loaded with didanosine by desolvation techniques with ethanol as it does not require an increase in temperature.

Catalepsy was measured at 30,

60, 90, 120, 150 and 180 min intervals after administering haloperidol, using the Bar test. The cut off time was five min (Murata et al, 1988). 14 The rats were divided into groups each containing five. Targeted compounds (SSP1-SSP10)/clozapine (5 mg per kg, i.p.) or vehicle were administered 30 min before the administration of lithium sulfate (200 mg/kg, i.p.) and the head Epigenetic activity twitches was observed and counted for 60 min after the administration of lithium sulfate (Wielosz et al, 1979).15 Present study was undertaken to synthesize some novel dibenzothiazepine derivatives and investigate their probable antipsychotic effects. Target compounds were obtained at four steps (Scheme 1). First of all, 2-[(2-nitrophenyl) sulfanyl] benzoic acid was prepared in presence of catalytic amount of copper powder. Secondly 2-[(2-aminophenyl)

sulfanyl] benzoic acid was prepared by reduction with sodium sulphide in methanol which was then cyclized to dibenzo [b, f] [1, 4] thiazepin-11(10H)-one (e). At final step, reaction of 4 with phosphorous oxychloride and subsequent condensation with substituted benzyl piperazines gave the title compounds (SSP1-SSP10). The structures of the synthesized compounds were elucidated by spectral data. Significant stretching bands in the IR spectra were observed at expected regions which are then confirmed by bending vibrations. The infrared spectral analysis of the compounds indicates the C N stretch between 1585 and 1610 cm−1 corresponding to the amidine CN, the aliphatic stretch of the methylene Lapatinib (–CH2) group has been observed between 2800 and 2900 cm−1 the IR spectra. The piperazinyl C–N stretching was observed Etomidate at 1170–1200 cm−1. All of the aromatic and aliphatic protons in the 300 MHz 1H NMR spectra were also recorded at estimated areas. The methylene protons of the benzyl group were observed at between 3.2 and 4.2 ppm while the tricyclic protons were observed downfield as compared to the benzyl aromatic protons. After haloperidol administration the induced catalepsy was measured up to 180 min. The maximum catalepsy was observed 150 min after haloperidol. In

SSP-9 treated group, maximum catalepsy was noted 30 min after haloperidol. It showed significant inhibition (p < 0.05) in haloperidol-induced catalepsy at 120 min and was extended significantly up to 180 min. The results are shown in Fig. 1. Lithium induced 40.2 ± 1.655 head twitches in 1 h. Clozapine (5 mg per kg) and SSP-9 (5 mg per kg) reduced the number of head twitches to 10 ± 0.7071 and 14.8 ± 0.8602, respectively. The derivative SSP-7 also showed moderate activity as compared to clozapine. The results of clozapine and SSP-9 were significant (p < 0.05) as compared to negative control. The results are shown in Fig. 2. The results of pharmacological investigation demonstrated that derivative SSP-9 has better antipsychotic potential amongst all.

Confocal imaging verified the significantly lower skin permeability of nanoencapsulated FITC compared to Rh B. The effect of % initial loading on skin permeation of nanoencapsulated Rh B and FITC is shown in Fig. 10. Transdermal delivery of Rh B increased significantly (P < 0.05) with the increase learn more in dye loading. For 5% Rh B loading (F8), Q48 and flux values were 1.78 ± 0.63 μg/cm2 and 2.53 ± 0.87 μg/cm2/h,

respectively. Increasing loading to 10% w/w (F7) and 20% w/w (F6) caused a significant increase (P < 0.05) in both Q48 (2.99 ± 0.26 and 5.40 ± 0.39 μg/cm2, respectively) and flux (4.29 ± 0.42 and 6.19 ± 0.77 μg/cm2/h, respectively). Differences between Q48 and flux values obtained at 10% w/w and 20% w/w initial load were also statistically

significant (P = 0.001 and 0.030, respectively). On the other hand, increasing initial% FITC loading (5%, 10% and 20% w/w, F9, F10, and F11, respectively, Table 1) led to reduced skin permeation ( Fig. 10 and Table 2). NP formulations F9, F10, and F11 showed average Q48 values of 0.13 ± 0.04, 0.09 ± 0.01, and 0.06 ± 0.02 μg/cm2, PFT�� manufacturer respectively ( Table 2). This corresponded to an average flux of 0.17 ± 0.05, 0.12 ± 0.02, and 0.09 ± 0.03 μg/cm2/h, respectively. Differences between Q48 and flux values obtained at 5% w/w (F9) and 20% w/w Carnitine palmitoyltransferase II (F11) initial load were statistically significant (P = 0.026 and 0.041, respectively). Notably, increasing the initial FITC loading of NP stabilized with 1% w/v DMAB from 5% to 20% w/w was associated with an increase in particle size with a higher PDI for F11 and a decrease in zeta potential. The literature information provided proof of concept of enhanced transdermal delivery

of drugs encapsulated in nanocarriers, particularly liposomes [9] and polymeric NPs [10] across MN-treated skin, promoting the transdermal delivery enhancing effect of either approach used separately. A better mechanistic insight is needed for optimization of this combined strategy for diverse drug delivery applications. At the outset, it could be postulated that the flux of a nanoencapsulated drug across MN-treated skin is a complex multifactorial process involving possible in-skin transport of the nanocarrier and the released drug through MN-created aqueous filled microchannels and deeper skin layers.

Present results are obviously similar to the results explained above which shows that bilirubin level increases due to malarial infection. Present study also shows that hypoglycemia is more common in sever malaria patients. 68% of patients were found with hypoglycemia. We detected hypoglycemia in nearly 11% of the patients with sever falciparum malaria. Shah et al 11 reported hypoglycemia in 2 out of 20 cases (10%) of severe falciparum malaria from Karachi (Pakistan). The occurrence of malaria in adults is due to mal-absorption of glucose from intestine. Thai adults with sever malaria had

greatly reduced absorption capacity for sugar transport both actively and passively. 12 Most of our patients have hypoglycemia before quinine administration. selleck inhibitor This suggests that other causes may also be responsible for hypoglycemia. 13 All authors have none to declare. We are very thankful to Professor Dr. Salman Akbar Malik Chairman

Department of Biochemistry, QAU Islamabad, Pakistan for his valuable suggestions. “
“Multi-particulate (MP) modified release drug delivery systems have several performance advantages over single unit dosage forms. MP formulations generally have a more reliable in vivo dissolution performance, resulting in more uniform bioavailability and clinical effect. 1 Pelletization is an agglomeration process that converts fine powders or granules of bulk drugs and excipients into small, free flowing, spherical or semi spherical Carnitine palmitoyltransferase II units, referred to as pellets. 2 Pellets offer a high degree of flexibility and can be divided into desired dose strengths without formulation or process changes. 3 Pellets are in selleck chemicals a size range between 0.5 and 1.5 mm and are produced primarily for the purpose of oral controlled release dosage forms having gastro resistant or sustained release properties or the capability of site-specific drug delivery. 4 The pelletized products can improve the safety and efficacy of the active agent, showing a number of advantages over the single unit dosage

system. 5 Extended release formulations are designed to allow at least twofold reduction in dosing frequency or significant increase in patient compliance or therapeutic performance when compared to a conventional immediate release dosage form. 6 Sustained release pharmaceutical pellet is one of the most popular approaches among the various types of extended release dosage forms as it offers several manufacturing and biopharmaceutical advantages. 7 Pellets are also less affected by gastric emptying. 8 After administration, the coated pellets spread uniformly throughout the gastrointestinal tract resulting in a consistent drug release with reduced risk of local irritation and dose dumping of the drug can be avoided. NSAIDs are a group of drugs of diverse chemical composition and different therapeutic potentials.9 Most NSAIDs are weak acids, with a pKa values in the range of 3.0–5.0 and contain hydrophilic groups and lipophilic ones.

, 2010 and Hammes and

Schmid, 2009). Highly weathered soils, which account for approximately 10% of Taiwan, are some of the most common types of agricultural soils in Taiwan. Quisinostat This is particularly true in northern Taiwan (a subtropical climate), an area of rice and tea production, and in southern Taiwan (a tropical climate), an area of rice and pineapple production. Under humid subtropical and tropical climates, highly weathered soils with intensive cultivation are characterized by a very low pH (≤ 5.0), and low soil organic matter (≤ 1%), CEC, and base saturation percentage (BS). Huang (1986), Lin and Hung (2000), and Lin (2002) studied the soil erosion rates of highly weathered soils in Taiwan, and indicated that the soils have moderate to serious soil Galunisertib supplier losses ranging from 10 to 280 tons ha− 1 yr− 1. Similar climates and soil degradation problems appear in Trinidad and Tobago (Wuddivira and Camps-Roach, 2007 and Wuddivira et al., 2009), where the most critical factors influencing the degradation are SOM content and soil aggregation stability. Previous studies on amending soils with biochar typically focused on restoring soil fertility and crop production. Few studies have discussed the influences of biochar on the physical

properties of soil and erodibility in highly weathered soils. The objectives of this study were (1) to evaluate the effects of wood biochar on the physical properties and erosion potential of highly weathered soils, and (2) to assess the relationships between soil properties and soil erosion potential. Soil samples (0–25 cm) were collected from a terrace located at field erosion Casein kinase 1 experimental plots at the National Pingtung University of Science and Technology, southern Taiwan (about E 120°37′11″; N 22°38′54″). The soil was classified as a Typic Paleudults based on Soil Taxonomy (Soil Survey Staff, 2010). Pineapple (Ananas comosus (L.) Merr.) is the dominant crop on this terrace. The biochar used in this study was supplied by Taiwan Forestry Research Institute (TFRI) and was produced from the wood of white

lead trees (Leucaena leucocephala (Lam.) de Wit). The waste wood of the white lead trees, which are commonly invasive plants, was collected from a clearcutting program in Kenting National Park, southern Taiwan. The biochar was produced at a pyrolysis temperature of 700 °C based on the recommendation of Lehmann (2007). After pyrolysis, the biochar was ground to pass through a 2 mm sieve to ensure that all biochar had the similar particle size in subsequent experiments. Incubation experiments were conducted to evaluate the effects of biochar on the physiochemical properties of soil. Fifteen kg samples of the study soils were placed in plastic pots (measuring approximately 30 cm in width and 40 cm in depth) and then mixed with biochar at three application rates (0%, 2.5% and 5% (w/w)).

5 ml. Ninety-six well plates were coated with HPV16, HPV18, HPV31

or HPV45 L1 VLPs (0.5 to 1.5 μg/ml) overnight at 4 °C, and blocked with 1% bovine serum albumin, 0.1% Tween-20 in phosphate-buffered saline. For the determination of the chaotropic agent concentration, coated wells were incubated with 0–8 M NaSCN for 15 min at room temperature. After a washing step, wells were incubated with biotinylated V5 (1.56 ng/ml; anti-HPV16) or J4 (6.25 ng/ml; anti-HPV18) monoclonal antibodies for 90 min at 37 °C. For the avidity ELISA, coated wells were incubated with serum samples (12 serial 2-fold dilutions) for 1 h 30 min at 37 °C. After a washing step, wells were incubated with 0 or 1 M NaSCN for 15 min at room temperature. After another washing step, wells were then incubated with biotin-conjugated anti-human IgG (Jackson; Studies 1 and 2) or Ig (Amersham; check details Study 3). Biotinylated antibody detection used the colorimetric readout based on streptavidin-horseradish peroxidase (Amdex, GE Healthcare) and O-phenylenediamine substrate (Sigma). Optical densities were read at 492/620 nm

and antibody concentrations were calculated relative to a standard antiserum reference using SoftMaxPro software (4-parameter equation) and expressed in EU/ml. An avidity index (AI2) was calculated as a ratio of the antigen-specific antibody concentration determined after 1 M NaSCN treatment divided by the antigen-specific antibody concentration without NaSCN treatment. All statistical analyses were not part of the objectives of the BMN 673 molecular weight clinical trials from which the samples were taken

and therefore were considered as exploratory. Parametric analyses were performed using SAS software on log10 transformed data. The Shapiro–Wilk test, Skewness and Kurtosis calculations were used to confirm normality. Differences were identified by ANOVA followed by Tukey’s test. All comparisons were two-tailed. Pearson’s r statistic was used to identify correlations between (log10 transformed) AIs and antibody concentrations. Significance was ascribed to p-values <0.05 (and in the case of antibody concentrations, to ≥2-fold differences). second AIs are described to two-significant figures in the text. The HPV16 L1 and HPV18 L1 conformational epitopes that are important epitopes for neutralising antibodies [7] and [26], were evaluated in an ELISA using monoclonal antibodies V5 and J4, respectively. Both epitopes were not significantly denatured by 15 min pre-incubation with <4 M NaSCN (Fig. 1). However, 10% of HPV16 L1 conformational epitopes were denatured by 2 M NaSCN. Therefore, a 15 min incubation with 1 M NaSCN in the ELISA was selected to assess the antibody avidities with serum samples from HPV-16/18 vaccine recipients. In Study 1 and 2, the AIs of HPV16 L1- and HPV18 L1-specific antibodies were assessed in samples taken from vaccinated girls and women one month post-Dose 2 (Month 2) and one month post-Dose 3 (Month 7).

Our data suggest that most severe episodes of gastroenteritis are not seen at health facilities.

It is in such settings that the potential life-saving impact of rotavirus vaccination can be most fully realized. PATH’s Rotavirus Vaccine Program, funded, through a grant from the GAVI Alliance, and Merck & Co., Inc. This study, under protocol V260-015, was designed, managed, conducted, and analyzed by the co-sponsors in collaboration with the site investigators and under the supervision and advice TSA HDAC of the Data and Safety Monitoring Board (members listed below). This manuscript is published with the permission of the Director, KEMRI. We acknowledge the volunteers and their families because without their participation this seminal research would not have been LY2157299 possible. At Merck, we thank Michele L. Coia, Stephen J. Rivers, Donna Hyatt, and Florian Schödel. At PATH, we thank Kristen Lewis, J.C. Victor, and A. Duncan Steele. KEMRI/CDC is a member of the INDEPTH Network. Conflict of interest statement: MJD is an employee of Merck & Co., Inc. and owns shares in the company. MC was an employee

of Merck & Co., and owned shares in the company when the study was conducted. No other conflicts of interest are reported. “
“Rotavirus is a leading cause of hospitalization and death from diarrhea among infants and children younger ever than five years of age in Africa [1], [2] and [3]. More than 80% of the hospitalizations and deaths resulting from rotavirus happen in resource-poor countries in Sub-Saharan Africa and South Asia [2]. HIV infection rates are high among infants and children in many African countries where severe outcomes from rotavirus gastroenteritis are also common. Given that diarrheal disease is an important cause of morbidity and mortality among HIV-infected children [4], [5], [6] and [7], a safe and effective vaccine against rotavirus is a particularly important public health tool in areas in areas where

HIV/AIDS is common. Following removal from the market in 1998 of RotaShield®, a live, oral rotavirus vaccine, because of concerns about vaccine-associated intussusceptions [8] and [9], two live, oral, attenuated rotavirus vaccines were licensed in the mid-2000s: the pentavalent rotavirus vaccine (PRV), RotaTeq® (Merck and Co., Inc. Waterhouse Station, NJ) [10] and the monovalent human rotavirus vaccine Rotarix® (GlaxoSmithKline Biologicals, Rixensart, Belgium) [11]. Large phase III clinical trials in the United States and numerous European countries and countries in Latin America demonstrated that these two vaccines were safe and highly efficacious [11], [12] and [13], and they are in routine use in the US, Americas, Europe, and Australia.