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Asparaginase Erwinia Chrysanthemi (Erwinaze and Rylaze)
Asparaginase Erwinia chrysanthemi (Erwinaze)
Criteria for Initial Approval
Aetna considers Erwinaze (asparaginase Erwinia chrysanthemi) medically necessary as a component of a multiagent chemotherapeutic regimen for persons with the following malignancies:
Acute lymphoblastic leukemia (ALL) and Lymphoblastic lymphoma
For persons who are hypersensitive to Oncaspar (pegaspargase); or
As induction therapy for members age 65 years and older;
Extranodal Natural Killer/T-cell lymphoma
When both of the following criteria are met:
The member has previously received and developed hypersensitivity to an E. coli-derived asparaginase (e.g., pegaspargase); and
The requested medication is used in conjunction with multi-agent chemotherapy.
Aetna considers all other indications as experimental and investigational (for additional information, see Experimental and Investigational and Background sections).
Continuation of Therapy
Aetna considers of Erwinaze (asparaginase Erwinia chrysanthemi) therapy medically necessary in members requesting reauthorization for an indication listed in section I when there is no evidence of disease progression or an unacceptable toxicity while on the current regimen.
Note: Elspar (L-Asparaginase Escherichia coli) has been discontinued.
Asparaginase erwinia chrysanthemi (recombinant)-rywn) (Rylaze)
Acute Lymphoblastic Leukemia (ALL) and Lymphoblastic Lymphoma (LBL)
Aetna considers asparaginase erwinia chrysanthemi (recombinant)-rywn) (Rylaze) medically necessary for the treatment of acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL) in members 1 month or older who have developed hypersensitivity to E. coli-derived asparaginase (e.g., pegaspargase) and the requested medication will be used in conjunction with multi-agent chemotherapy.
Extranodal Naturual Killer (NK)/T-cell Lymphoma
Aetna considers asparaginase erwinia chrysanthemi (recombinant)-rywn) (Rylaze) medically necessary for the treatment of extranodal NK/T-cell lymphoma when both of the following criteria are met:
The requested medication will be used in conjunction with multi-agent chemotherapy.
Aetna considers continuation of asparaginase erwinia chrysanthemi (recombinant)-rywn) (Rylaze) therapy medically necessary in members requesting reauthorization for an indication listed in Section I when there is no evidence of unacceptable toxicity or disease progression while on the current regimen.
Asparaginase Erwinia chrysanthemi (Erwinaze) is available as 10,000 International Units as a lyophilized powder in single-dose vial for reconstitution for injection for intramuscular or intravenous use.
Acute lymphoblastic leukemia (ALL)
The recommended dosage is as follows:
To substitute for a dose of pegaspargase: The recommended dose is 25,000 International Units/m2 administered intramuscularly three times a week (Monday/Wednesday/Friday) for six doses for each planned dose of pegaspargase.
To substitute for a dose of native E. coli asparaginase: The recommended dose is 25,000 International Units/m2 administered intramuscularly for each scheduled dose of native E. coli asparaginase.
Source: Jazz Pharmaceuticals, Inc., 2019
Asparaginase erwinia chrysanthemi (recombinant)-rywn) (Rylaze) is available as 10 mg/0.5 mL solution in a single-dose vial for intramuscular injection.
The recommended dosage of Rylaze is 25 mg/m2 administered intramuscularly every 48 hours.
Experimental and Investigational
Aetna considers therapeutic drug monitoring of asparaginase activity experimental and investigational.
96401 Chemotherapy administration, subcutaneous or intramuscular; non-hormonal anti-neoplastic
96409 Chemotherapy administration; intravenous, push technique, single or initial substance/drug
+96411 intravenous, push technique, each additional substance/drug (List separately in addition to code for primary procedure)
96413 - 96417 Chemotherapy administration; intravenous infusion technique
J9019 Injection, asparaginase, (Erwinaze), 1,000 iu
J9021 Injection, asparaginase, recombinant, (rylaze), 0.1 mg
J9020 Injection, asparaginase, not otherwise specified, 10,000 units
J9266 Injection, pegaspargase, per single dose vial [Oncaspar]
C83.50 - C83.59 Lymphoblastic (diffuse) lymphoma
C86.0 - C86.6 Other specified types of T/NK-cell lymphoma
C91.00 - C91.02 Acute lymphoblastic leukemia [ALL]
C70.0 Malignant neoplasm of cerebral meninges
C71.0 - C71.9 Malignant neoplasm of brain
C81.00 - C83.39, C83.70 - C85.99, C88.0 - C90.32, C91.10 - C91.92 Lymphosarcoma and reticulosarcoma and other specified malignant tumors of lymphatic tissue [excluding ALL, lymphoblastic lymphoma, extranodal NK/T-Cell Lymphoma, nasal type]
D33.0 - D33.2 Benign neoplasm of brain
ICD-10 codes contraindicated for this CPB:
I66.01 - I66.9 Occlusion and stenosis of cerebral artery
I74.2 - I74.4 Embolism and thrombosis of arteries of the extremities
I81 Portal vein thrombosis
I82.0 - I82.91 Other venous embolism and thrombosis
K85.00 - K85.92 Pancreatitis
T50.995+ Adverse effect of other drugs, medicaments and biological substances [hypersensitivity to Erwinase]
U.S. Food and Drug Administration (FDA)-Approved Indications for asparaginase Erwinia chrysanthemi (Erwinaze)
Erwinaze is indicated as a component of a multi-agent chemotherapeutic regimen for the treatment of patients with acute lymphoblastic leukemia (ALL) who have developed hypersensitivity to E. coli-derived asparaginase.
Compendial Uses for asparaginase Erwinia chrysanthemi (Erwinaze)
Extranodal natural killer/T-cell lymphoma: as a component of multi-agent chemotherapeutic regimen
Lymphoblastic lymphoma (managed in the same manner as ALL)
Acute lymphoblastic leukemia (ALL) as induction therapy for adults aged 65 years and older as a component of multi-agent chemotherapeutic regimen, or as a substitute for pegaspargase in cases of systemic allergic reaction or anaphylaxis due to pegaspargase hypersensitivity
Pediatric acute lymphoblastic leukemia (ALL) as a substitute for pegaspargase in cases of systemic allergic reaction or anaphylaxis due to pegaspargase hypersensitivity
Asparaginase Erwinia chrysanthemi is available as Erwinaze (Jazz Pharmaceuticals, Inc.) and contains an asparaginase specific enzyme derived from Erwinia chrysanthemi and which catalyzes the deamidation of asparaginase to aspartic acid and ammonia thereby causing a decrease in circulating levels of asparagine. The mechanism of action is postulated on the inability of leukemic cells to synthesize asparagine due to lack of asparagine synthetase activity, resulting in cytotoxicity specific for leukemic cells that rely on an exogenous source of amino acid asparagine for their protein metabolism and survival (Jazz Pharmaceuticals, 2019).
Per the prescribing information, asparaginase Erwinia chrysanthemi (Erwinaze) is contraindicated in patients with a history of the following:
Serious hypersensitivity reactions to Erwinaze, including anaphylaxis
Serious pancreatitis with prior L-asparaginase therapy
Serious thrombosis with prior L-asparaginase therapy
Serious hemorrhagic events with prior L-asparaginase therapy
Per the prescribing information, asparaginase Erwinia chrysanthemi (Erwinaze) carries the following warnings and precautions:
Hypersensitivity reactions: Grade 3 and 4 hypersensitivity reactions following Erwinaze use has been reported in 5% of patients in clinical trials.
Pancreatitis: Pancreatitis has been noted in 4% of patients in clinical trials.
Glucose intolerance: Glucose intolerance has been noted in 5% of patients receiving Erwinaze in clinical trials.
Thrombosis and hemorrhage
The most common adverse reactions (occurrence ≥ 1%) per the prescribing information include: systemic hypersensitivity, hyperglycemia, abnormal transaminases, fever, pancreatitis, local reactions, vomiting, nausea, thrombosis, hyperbilirubinemia, abdominal pain/discomfort, and diarrhea.
In November 2011, the FDA approved Erwinaze (asparaginase erwinia chrysanthemi), an asparagine specific enzyme, as a component of a multi-agent chemotherapeutic regimen for the treatment of patients with acute lymphoblastic leukemia (ALL) who have developed hypersensitivity to E. coli-derived asparaginase. Erwinaze (asparaginase Erwinia chrysanthemi) catalyzes the deamidation of asparagine to aspartic acid and ammonia, resulting in a reduction in circulating levels of asparagine. The mechanism of action of Erwinaze (asparaginase Erwinia chrysanthemi)is thought to be based on the inability of leukemic cells to synthesize asparagine due to lack of asparagine synthetase activity, resulting in cytotoxicity specific for leukemic cells that depend on an exogenous source of the amino acid asparagine for their protein metabolism and survival.
The safety and efficacy of Erwinaze was established in Study 1, a single-arm, multi-center, open-label, safety and clinical pharmacology trial. Additional safety data were obtained in the Erwinaze Master Treatment Protocol (EMTP), an expanded access program. Study 1 enrolled patients treated on National Cancer Institute (NCI)-sponsored cooperative group ALL protocols who were unable to continue to receive pegaspargase due to hypersensitivity reactions. The main outcome measure was determination of the proportion of patients who achieved a serum trough asparaginase level greater than or equal to 0.1 International Units/ mL. Serum trough asparaginase activity ≥ 0.1 International Units/ mL has been demonstrated to correlate with asparagine depletion (asparagine < 0.4 mcg/mL or 3 µM) and to serum levels that predict clinical efficacy. Patients received Erwinaze 25,000 International Units/m2 intramuscularly for two weeks (total 6 doses) as a replacement for each scheduled dose of pegaspargase remaining on their original treatment protocol. Fifty-eight patients were enrolled in Study 1, of these 48 were evaluable for the main outcome measure based on availability of pharmacokinetic samples in Course 1. The median age was 11 years (2 to 18 years); 59% were male, 78% were White, 10% were Black/African American, 5% were Asian, and 7% were other or unknown. A total of 35% were Hispanic or Latino. Study 1 met its main outcome measure of demonstrating that greater than 50% of the patients achieved the prespecified trough asparaginase activity level of ≥ 0.1 International Units/ mL at 48 or 72 hours following the third dose. The safety and efficacy of intravenous administration were determined in Study 2 by characterizing the PK of a 25,000 International Units/m2 Erwinaze dose given 3 days per week on a Monday, Wednesday, and Friday schedule for up to 30 weeks. This open-label, single-arm, multicenter PK study enrolled 30 patients. The main outcome measure was determination of the proportion of patients with 2-day NSAA levels (48-hour levels taken after the fifth dose) ≥ 0.1 International Units/mL in the first 2 weeks of Erwinaze treatment. Of the thirty patients enrolled, 24 were evaluable for the main outcome measure based on the pharmacokinetic samples in Course 1. The median age was 7 years (1-17 years), 63% were male, 27% were Hispanic or Latino, 83% were White, 3% were Black/African American, 7% were Asian, and 7% were other (American Indian, Alaska Native, or Indian). In Study 2, serum asparaginase activity of asparaginase Erwinia chrysanthemi was determined in 24 evaluable patients (aged ≥ 1 year to ≤ 17 years) following intravenous administration of Erwinaze 25,000 International Units/m2 . Five minutes after the 60-minute infusion in Course 1, the mean asparaginase activity level was 12.65 ± 3.16 International Units/mL post-dose 1 and 12.11 ± 3.11 International Units/mL post dose 4. The main study objective was met with an asparaginase activity level of ≥ 0.1 International Units/mL 48 hours after the fifth dose observed in 83% of patients. The 72-hour post dose 6 asparaginase activity level of ≥ 0.1 International Units/mL was the secondary endpoint, with 43% of patients achieving this endpoint.
U.S. Food and Drug Administration (FDA)-Approved Indications for asparaginase erwinia chrysanthemi (recombinant)-rywn) (Rylaze)
Rylaze is indicated as a component of a multi-agent chemotherapeutic regimen for the treatment of acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL) in adult and pediatric patients 1 month or older who have developed hypersensitivity to E. coli-derived asparaginase.
Compendial Uses for asparaginase erwinia chrysanthemi (recombinant)-rywn) (Rylaze)
Extranodal NK/T-cell lymphoma
Asparaginase erwinia chrysanthemi (recombinant)-rywn) is available as Rylaze (Jazz Pharmaceuticals, Inc.) and contains an asparagine specific bacterial enzyme (L-asparaginase). Asparaginase erwinia chrysanthemi (recombinant)-rywn) initiates the conversion of the amino acid L-asparagine into aspartic acid and ammonia. The resultant pharmacologic effect is leukemic cell death due to a depletion of plasma asparagine. The low expression of asparagine synthetase in the leukemic cells and their diminished ability to synthesize asparagine cause their dependence on exogenous asparagine for survival (Jazz Pharmaceuticals, 2021b).
Per the prescribing information, asparaginase erwinia chrysanthemi (recombinant)-rywn) (Rylaze) is contraindicated in patients with a history of the following:
Serious hypersensitivity reactions to Rylaze, including anaphylaxis
Serious pancreatitis during previous L-asparaginase therapy
Serious thrombosis during previous L-asparaginase therapy
Serious hemorrhagic events during previous L-asparaginase therapy
Per the prescribing information, asparaginase erwinia chrysanthemi (recombinant)-rywn) (Rylaze) carries the following warnings and precautions:
Hypersensitivity: The incidence of hypersensitivity reactions reported in patients following Rylaze treatment was 25% in clinical trials with 2% being severe.
Pancreatitis: The incidence of pancreatitis in patients was 14% with 6% being severe in clinical trials of Rylaze.
Hemorrhage: Bleeding was noted in 17% of patients treated with Rylaze with 1% being severe.
Hepatotoxicity: The incidence of elevated bilirubin and/or transaminases was 62% in patients treated with Rylaze in clinical trials, and 12% had grade ≥ 3 elevations.
The most frequent adverse reactions (incidence > 20%) are abnormal liver test, nausea, musculoskeletal pain, fatigue, infection, headache, pyrexia, drug hypersensitivity, febrile neutropenia, decreased appetite, stomatitis, bleeding, and hyperglycemia (Jazz Pharmaceuticals, 2021b).
Emadi et al (2014) stated that asparaginases are among the most effective agents against acute lymphoblastic leukemia (ALL) and are Food and Drug Administration (FDA)-approved for the treatment of pediatric and adult ALL. However, the effectiveness of these drugs for the treatment of other hematologic malignancies particularly acute myeloid leukemia (AML) is not well-established. The mechanism of action of asparaginases has thought to be related to a swift and sustained reduction in serum L-asparagine, which is required for rapid proliferation of metabolically demanding leukemic cells. However, asparagine depletion alone appears not to be sufficient for effective cytotoxic activity of asparaginase against leukemia cells, because glutamine can rescue asparagine-deprived cells by regeneration of asparagine via a transamidation chemical reaction. For this reason, glutamine reduction is also necessary for full anti-leukemic activity of asparaginase. Indeed, both Escherichia coli and Erwinia chrysanthemi asparaginases possess glutaminase enzymatic activity, and their administrations have shown to reduce serum glutamine level by deamidating glutamine to glutamate and ammonia. Emerging data have provided evidence that several types of neoplastic cells require glutamine for the synthesis of proteins, nucleic acids, and lipids. This fundamental role of glutamine and its metabolic pathways for growth and proliferation of individual malignant cells may identify a special group of patients whose solid or hematologic neoplasms may benefit significantly from interruption of glutamine metabolism. The authors stated that asparaginase products deserve a second look particularly in non-ALL malignant blood disorders.
Emadi and associates (2018) stated that depletion of glutamine (Gln) has emerged as a potential therapeutic approach in the treatment of AML, as neoplastic cells require Gln for synthesis of cellular components essential for survival. Asparaginases deplete Gln, and asparaginase derived from Erwinia chrysanthemi (Erwinaze) appears to have the greatest glutaminase activity of the available asparaginases. In this phase-I study, these researchers determined the dose of Erwinaze that safely and effectively depletes plasma Gln levels to less than or equal to 120 μmol/L in patients with relapsed or refractory (R/R) AML. A total of 5 patients were enrolled before the study was halted due to issues with Erwinaze manufacturing supply. All patients received Erwinaze at a dose of 25,000 IU/m2 intravenously 3 times weekly for 2 weeks. Median trough plasma Gln level at 48 hours after initial Erwinaze administration was 27.6 μmol/L, and 80 % (lower limit of 1-sided 95 % confidence interval [CI]: 34 %) of patients achieved at least 1 undetectable plasma Gln value (less than 12.5 μmol/L), with the fold reduction (FR) in Gln level at 3 days, relative to baseline, being 0.16 (p < 0.001 for rejecting FR = 1). No dose-limiting toxicities (DLTs) were identified; 2 patients responded, 1 achieved partial remission (PR) and 1 achieved hematologic improvement after 6 doses of Erwinaze monotherapy. The authors concluded that these findings suggested that asparaginase-induced Gln depletion may have an important role in the management of patients with AML, and supported more pharmacologic and clinical studies on the mechanistically designed asparaginase combinations in AML.
Brain Tumors (e.g. , Medulloblastomas)
Sanghez and colleagues (2018) noted that anti-metabolites are less myelosuppressive than DNA-damaging anti-cancer drugs and may be useful against brain tumors. These researchers evaluated the asparagine/glutamine-deaminating agent Erwinaze with/without temozolomide against brain tumor cells and mouse medulloblastomas. Erwinaze treatment of cell lines and neuro-spheres led to dose-dependent reductions of cells (reversible by L-glutamine), with half maximal inhibitory concentrations (IC50s) of 0.12 to greater than 10 IU/ml. Erwinaze at less than 1 IU/ml reduced temozolomide IC50s by 3.6- to 13-fold (300 to 1,200 μM to 40 to 330 μM). In this study, 7-week-old SMO/SMO mice treated with Erwinaze (regardless of temozolomide treatment) had better survival 11 weeks post-therapy, compared to those not treated with Erwinaze (81.25 % versus 46.15 %, p = 0.08). Temozolomide-treated mice developed 10 % weight loss, impairing survival. All 16 mice treated with temozolomide (regardless of Erwinaze treatment) succumbed by 40 weeks of age, whereas 5/8 animals treated with Erwinaze alone and 2/6 controls survived (p = 0.035). The authors concluded that Erwinaze enhanced cytotoxicity of temozolomide in-vitro, and improved survival in SMO/SMO mice, likely by reducing cerebrospinal fluid (CSF) glutamine. Moreover, temozolomide-associated toxicity prevented demonstration of any potential combinatorial advantage with Erwinaze in-vivo.
Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma (ALL/LBL)
The incidence of acute lymphoblastic leukemia is estimated at 5,700 patients annually with half of whom are children (FDA, 2021). According to Horton and Steuber (2021), acute lymphoblastic leukemia/lymphoblastic lymphoma (ALL/LBL) is the most common malignancy in childhood. Additionally, leukemia and lymphoma overlap in clinical presentation for the same disease. ALL involves the proliferation of immature lymphoid cells in the bone marrow, peripheral blood, and other organs. In general, patients with LBL benefit from ALL-like treatment regimens and should seek treatment in a center experienced with LBL (NCCN, 2022a).
On June 30, 2021, the United States Food and Drug Administration (FDA) approved Rylaze (asparaginase erwinia chrysanthemi (recombinant)-rywn) for use as a component of a multi-agent chemotherapeutic regimen to treat acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL) in pediatric and adult patients one month and older who are allergic to the E. coli-derived asparaginase products used most commonly for treatment. Additionally, Rylaze received Fast Track and Orphan Drug designations for ALL and LBL. The FDA approval for Rylaze was based on supporting clinical data from Study JZP458-201 (NCT04145531) (FDA, 2021; Jazz Pharmaceuticals, 2021a).
This study is an ongoing pivotal Phase 2/3 single-arm, open-label, multi-cohort, multicenter dose confirmation trial evaluating pediatric and adult patients with ALL or LBL who experienced and allergic reaction to E. coli-derived asparaginase and have not previously received asparaginase erwinia chrysanthemi. The study consists of two parts of which 102 patients with a median age of 10 years (age range, 1 to 24 years) and 94% of patients had experienced a hypersensitivity reaction to pegaspargase. Patients were treated with Rylaze at various dosages administered intramuscularly every Monday, Wednesday, and Friday for a total of 6 doses to replace each dose of pegaspargase. Treatment efficacy was based on the achievement and maintenance of nadir serum asparaginase activity (NSAA) above the level of 0.1 U/mL. The results of modeling and simulations demonstrated that for a Rylaze dosage of 25 mg/m2 administered intramuscularly every 48 hours, the proportion of patients maintaining NSAA ≥ 0.1 U/mL at 48 hours after a dose of Rylaze was 93.6% (95% Confidence Interval: 92.6%, 94.6%). The second part of this study is still active to confirm the dose and schedule for the intravenous route of administration. (Jazz Pharmaceuticals, 2021a; 2021b).
Therapeutic Drug Monitoring of Asparaginase Activity
Wurthwein et al (2020) stated that therapeutic drug monitoring (TDM) could identify patients with sub-therapeutic asparaginase (ASNase) activity [silent inactivation (SI)] and prospectively guide therapeutic adaptation. However, limited intra-individual variability is a pre-condition for targeted dosing and the diagnosis of SI. In the AIEOP-BFM acute lymphoblastic leukemia (ALL) 2009 trial, a total of 2,771 children with ALL were included and underwent ASNase-TDM in a central laboratory in Munster. Two bi-weekly administrations of pegylated ASNase during induction and a 3rd dose during re-induction or the high-risk block, which was administered several weeks later, were monitored. These researchers calculated the incidence of SI; and the predictivity of SI for SI after the subsequent administration. ASNase activities monitored during induction were categorized into percentiles at the respective sampling time-points. These percentiles were used to calculate the intra-individual range of percentiles as a surrogate for intra-patient variability and to evaluate the predictivity of ASNase activity for the subsequent administration. The overall incidence of SI was low (4.9 %). The positive predictive value (PPV) of SI identified by 1 sample was 21 % or less. Confirmation of SI by a 2nd sample indicated a high PPV of 100 % for bi-weekly administrations, but not for administration more than 17 weeks later. Sampling and/or documentation errors were risks for misdiagnosis of SI. High intra-individual variability in ASNase activities, with ranges of percentiles over more than 2 quartiles and low predictivity, was observed in approximately 25 % of the patients. These patients were likely to fail dose individualization based on TDM data. The authors concluded that ASNase activity is used as basis for clinical decisions, especially to identify patients with inadequate ASNase activity or to individualize dosing regimens. Therefore, not only specific and sensitive analytical methods are needed, but also standardized procedures for the daily clinical routine to avoid potential misinterpretation. In addition, the high intra-individual variability has to be taken into account. Therapeutic targeting at the lowest ASNase trough activities may result in under-dosing in a considerable number of patients. Model-dependent approaches, such as population pharmacokinetics, accounting for the intra-individual and inter-individual variability of the drug may be helpful in the future development of TDM concepts.
Pike et al (2021) stated that pegaspargase can cause anti-asparaginase antibody formation, which can decrease its effectiveness without causing any clinically apparent reaction (silent inactivation). When a patient has silent inactivation, a switch to Erwinia anti-asparaginase is needed; however, there is currently a global shortage of Erwinia. The only way to identify silent inactivation is to measure an asparaginase level. However, routine asparaginase level monitoring is not currently standard of care (SOC) at all Canadian centers. These researchers identified variations in practice regarding asparaginase level monitoring and Erwinia use. A 21-item survey was developed using OPINIO software and distributed to all Pediatric Hematology-Oncologists in Canada from February to October 2020. Respondents represented 15 hospitals across each region of Canada (response rate = 52 %). Only 39.2 % of respondents reported routinely measuring asparaginase levels, yet 53 % of respondents had modified therapy from pegaspargase to Erwinia in up to 50 % of their patients. The most common reason for not measuring asparaginase levels was not knowing how to use levels clinically (25.5 %). There was variation in the timing of levels and their target. The authors identified substantial variation in asparaginase activity monitoring practices across Canada. These investigators stated that future research should aim in developing a national practice guideline on asparaginase activity monitoring.
In a review on "Current evidence and place in therapy of asparaginase in the treatment of ALL in adults", Juluri et al (2022) stated that "Therapeutic drug monitoring (TDM) may allow for individualized asparaginase dosing and maximization of treatment efficacy with reduction of toxicity, but this is not yet widely practiced". Furthermore, these investigators noted that because asparaginase hypersensitivity is often associated with activity-neutralizing antibodies, recognition and mitigation is critical to ensure that therapeutic efficacy is not impacted. Multiple studies have established inferior outcomes with early discontinuation or dose reduction. This could be challenging since transient infusion reactions may mimic low-grade hypersensitivity reactions, and the use of pre-medication may mask a reaction that would signify the development of neutralizing antibodies. "Silent inactivation" of asparaginase could occur in the absence of detectable antibodies or clinical hypersensitivity. While there is evidence that TDM could be beneficial in these settings, no standard algorithm for TDM currently exists, and its use has generally been limited to the context of clinical trials. The authors concluded that ongoing areas of investigation include the incorporation of asparaginase into current adult treatment paradigms, combination with immunotherapy or targeted agents, novel formulations, as well as TDM.
Sidhu et al (2022) noted that in successive United Kingdom clinical trials (UKALL 2003, UKALL 2011) for pediatric ALL, polyethylene glycol-conjugated E. coli L-asparaginase (PEG-EcASNase) 1,000 iu/m2 was administered intramuscularly with risk-stratified treatment. In induction, patients received 2 PEG-EcASNase doses, 14 days apart. Post-induction, non-high-risk patients (regimens A, B) received 1 to 2 doses in delayed intensification (DI) while high-risk regimen C patients received 6 to 10 PEG-EcASNase doses, including 2 in DI. Trial sub-studies monitored asparaginase (ASNase) activity, ASNase-related toxicity and ASNase-associated antibodies (a total of 1,112 patients). Median (inter-quartile range [IQR]) trough plasma ASNase activity (14 ± 2 days post-dose) following 1st and 2nd induction doses and 1st DI dose was respectively 217 iu/L (144 to 307 iu/L), 265 iu/L (165 to 401 iu/L) and 292 iu/L (194 to 386 iu/L); 15 % (138/910) samples showed sub-threshold ASNase activity (less than 100 iu/L) at any trough time-point. Older age was associated with lower (regression coefficient -9.5; p < 0.0001) and DI time point with higher ASNase activity (regression coefficient 29.9; p < 0.0001). Clinical hypersensitivity was observed in 3.8 % (UKALL 2003) and 6 % (UKALL 2011) of patients, and in 90 % or more in regimen C. A 7 % (10/149) silent inactivation rate was observed in UKALL 2003. The authors concluded that PEG-EcASNase schedule in UKALL pediatric trials was associated with low toxicity but wide inter-patient variability; TDM potentially permits optimization via individualized asparaginase dosing.
Furthermore, the Prescribing Information notes that there is insufficient information to characterize the incidence of antibodies to Erwinaze; and there is no mentioning of TDM. https://www.accessdata.fda.gov/drugsatfda_docs/label/2011/125359lbl.pdf.
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Salzer WL, Asselin B, Supko JG, et al. Erwinia asparaginase achieves therapeutic activity after pegaspargase allergy: A report from the Children's Oncology Group. Blood. 2013;122(4):507-514.
Sanghez V, Chen M, Li S, et al. Efficacy of asparaginase erwinia chrysanthemi with and without temozolomide against glioma cells and intracranial mouse medulloblastoma. Anticancer Res. 2018;38(5):2627-2634.
Sidhu J, Masurekar AN, Gogoi MP, et al. Activity and toxicity of intramuscular 1000 iu/m 2 polyethylene glycol-E. coli L-asparaginase in the UKALL 2003 and UKALL 2011 clinical trials. Br J Haematol. 2022;198(1):142-150.
U.S. Food and Drug Administration (FDA). FDA approves component of treatment regimen for most common childhood cancer. FDA News Release. Silver Spring, MD: FDA; June 30, 2021
Vrooman LM, Kirov II, Dreyer ZE, et al. Activity and toxicity of intravenous Erwinia asparaginase following allergy to E. coli-derived asparaginase in children and adolescents with acute lymphoblastic leukemia. Pediatr Blood Cancer. 2016;63(2):228-233.
Wurthwein G, Lanvers-Kaminsky C, Gerss J, et al; AIEOP-BFM ALL 2009 Asparaginase Working Party. Therapeutic drug monitoring of asparaginase: Intra-individual variability and predictivity in children with acute lymphoblastic leukemia treated with PEG-asparaginase in the AIEOP-BFM acute lymphoblastic leukemia 2009 Study. Ther Drug Monit. 2020;42(3):435-444.


Even with the push for 21st century skills, educators know that technology alone does not impact student achievement. Pearson's professional development leadership services focus on how to establish the vision for digital conversion and how to lead the change that is required to get there.
Explore the role that administrators play in developing a technology integration professional development plan.
Support and coach teachers through their change of instructional practice.


When you are injured due to the carelessness of another person, you deserve to be compensated for those injuries and your suffering. Our personal injury attorneys represent injured claimants throughout Massachusetts who have accumulated medical bills, lost time from work, or lost loved ones in accidental deaths. We help them recover maximum compensation for their injury claims.
Over the past 30 years, we have obtained numerous successful verdicts and settlements on our clients' behalf. We invite you to read a summary of some notable verdicts and settlements.
We represent people who suffer from serious injuries causing paralysis and other permanent disabilities. We also represent families who lost a loved one in a wrongful death. We focus on achieving favorable results on their behalf.
Do not hesitate to contact our firm if you suffer from one of these injuries or another injury not on this list. We offer free initial consultations to discuss your injury lawsuit. We accept no attorneys' fees until we recover compensation for your injuries. Evening and weekend appointments are available at our convenient office locations in Attleboro, Lexington, Medway, and Newton / Wellesley.


text:
Gays in the Israeli Military
By Yermi Brenner
The U.S. House of Representatives recently passed a defense bill that paves the way for gays to serve openly in the military for the first time.
The legislation -- which will face tougher opposition when raised in the Senate this summer -- has been strongly advocated by President Obama, who has pledged to change the "Don't Ask, Don't Tell" policy.
According to both the House and Senate, any repeal of "Don't Ask, Don't Tell" would take effect only after the completion of a Pentagon Working Group study (due Dec. 1, 2010) which will detail how the repeal would be implemented and how it would affect service members.
In Israel, restrictions on openly gay soldiers were lifted in 1993, on orders from then-Prime Minister Yitzhak Rabin.
The video report below shows what lead to the Israeli military adopting the liberal approach, and investigates whether the change of policy was implemented on the ground - within the military bases and the combat units.
Tomer Azaria -- the gay combat soldier interviewed in the story -- said he had never been discriminated while on uniform. He explained that during his obligatory three years service there was an homophobic atmosphere in his unit, but these days, when he is called to serve as a reserve soldier, it is much easier for him because his comrades accept and respect him.
Tomer Azaria (wearing blue shirt) during his reserve service
As mentioned in the story by one of the interviewees (Prof. Danny Kaplan) -- the military's liberal approach towards gays was a stepping stone for acceptance and recognition of homosexuals in other branches of the Israeli security forces and in the Israeli society in general.
Today, Tel Aviv is one of the most gay-friendly cities in the world, but outside of the liberal metropolis most Israelis are conservative concerning homosexuality.
There have been two violent hate crimes against gays in Israel in past few years; two young people were murdered and several others hurt when an unidentified gunman opened fire in the Tel Aviv Gay and Lesbian Association Center (August 2009), and three marchers were stabbed and injured during the gay parade in Jerusalem (June 2005) by an orthodox Jew who claimed he was acting in the name of god.
Within the orthodox community there is no acceptance of homosexuals at all. In case you missed -- check out my previous post/video report about an Israeli orthodox rabbi who came out of the closet last year and has been trying to change his community's approach towards gays from within.
•	House Repeals 'don't ask, don't tell', ABC News, 27.05.2010
•	Why Gays Serving in the Military is a Non-issue, WSJ, 08.02.2010
•	Gays in Military Succeeds Abroad, Palm Center, 19.02.2001
Follow Yermi Brenner on Twitter: www.twitter.com/yermibrenner
Yermi Brenner
Gays In The Military Jews Gay Rights Tel Aviv Gay Club Tel Aviv Israel

redpajama_set_name: RedPajamaCommonCrawl


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for finished goods, packing etc. We have our own QC for those processes.


Topix.net - 50 Cent News about 50 Cent, collected from various sources on the web.
.504 Plan News, profiles, and pictures for the Chicago punk-emo band.
VH1.com - 50 Cent Message Boards Active fan forums with an array of topics relating to the artist.
50CentFanForum Features news and events, imagess, media, biography and discography.
5ft Sonic Santa UK band. Biography, news, photos, MP3s, wallpaper, and links.
00:54 54 Seconds mailing list. Public archives and files. Posting for members. (Yahoo! Groups).
5000 Watts Energy riff pop. Includes free MP3 music and information.
BlinkBits.com - 50 Cent Features RSS feed with pictures, biography, news and views.
G-Unit Chat Features an unofficial 50 Cent and G-Unit chat.
50 Cent Online A fan site including news, biography, audio/video clips, pictures, tour information, and discography.
Blown Away Fan site offers news, photos, articles, and a discography.
Rachel Homepage The official site of 54 Seconds bassist Rachel Loy offers music downloads, photos, news, biography, and an online store.
50 Cent Official site from Sony including information directly from the source and media of many types. Discography.
5AM Underground rock band in Montreal; includes biographies, events, and pictures.
Rules My World Desktop themes, backgrounds, wallpapers, icons and browser skins.
MTV.com - 50 Cent Frequently updated news with artist release information, videos, ringtones, images, and album information.
50centz_Pocket Includes news, pictures, and a chat room.
54 Steps Four piece original, classic, and modern classic rock band from Hampton Roads, Virginia. Includes photos, schedule, and song list.
Bandnews.org - 50 Cent 50 Cent's news from official website and magazines.
MusicAustin: 54 Seconds Biography, album images, and two MP3 downloads.
Blown Away Club Yahoo! Group, includes, message board, photo files, calendar, and chat.
Here in My House Concert photographs.
5 Knuckle Shuffle Ska Punk from Sydney Australia. Includes biography, news, lyrics, and photos.
50¢ Headrush Official site of Detroit based band, playing reggae, punk, hip-hop and funk. Includes shows, biography, photos, MP3s, and lyrics.
Rock on the Net - 50 Cent Includes daily news updates, information on 50 Cent, and a weekly compilation of major music charts.
50 Shekel Parody artist; includes news, audio and video.
5 Knuckle Shuffle, The Profiles, sound file, and photos for the Meriden, Connecticut based skrap-core band.


text:
Rescuing Anchor Point
'Shocked and deeply saddened' by Hsu's felony charges, hedge fund of funds' future squarely in hands of Crowe
Christine Williamson
Anchor Point Capital co-founder Albert Hsu (left) is no longer employed by the firm, but Timothy J. Crowe is seen as being up to the challenge of saving the company.
It was Albert W. Hsu who pushed Anchor Point Capital Management LLC into the limelight when he was arrested and charged with allegedly trying to have his ex-mistress kidnapped and sexually assaulted. But it will be Timothy J. Crowe who saves the fledgling hedge fund-of-funds manager — if he can.
Mr. Hsu, the firm's chief investment officer, managing director and co-founder, was arrested on March 2 at his home in New Caanan, Conn. He did not enter a plea at his arraignment on March 5 in Norwalk (Conn.) Superior Court, said Cindy Dillon, deputy court clerk. His case was continued until March 23 and moved to Stamford Superior Court from Norwalk.
At press time, Mr. Hsu remained incarcerated at Bridgeport Correctional Center. He could not be reached for comment.
Robert S. Bello, an attorney with Bello Lapine & Cassone LLP, Stamford, Conn., who is representing Mr. Hsu, declined to comment. The phone was not being answered at Mr. Hsu's New Canaan home.
Industry sources agree that Mr. Crowe, Anchor Point's managing director, chief executive officer and co-founder, seems up to the task of saving the young, Coral Gables, Fla.-based firm.
Mr. Crowe did not return repeated calls seeking comment about the Hsu arrest or the future of Anchor Point. Mr. Crowe's son, Matthew Crowe, the firm's manager of investor relations, also did not return repeated phone calls.
No longer with the firm
In a letter dated March 7 and obtained by Pensions & Investments, Mr. Crowe told clients that Mr. Hsu was no longer employed by the firm. He said Anchor Point employees were "shocked and deeply saddened by the recent legal problems that have befallen Albert Hsu" and that he has not had contact with Mr. Hsu or his attorney since the arrest. Mr. Crowe also said in the letter that Anchor Point clients as well as consultants and friends have all been contacted and "to a person, each has been very supportive and encouraged us to persevere through this challenging period. We intend to do so."
Sources said Mr. Crowe's long investment experience as a foundation manager might end up being Anchor Point's saving grace.
Messrs. Crowe and Hsu founded Anchor Point in mid-2005. Mr. Crowe spent 15 years at the Miami-based John S. and James L. Knight Foundation. He retired from the then-$1.9 billion foundation as vice president and CIO early in 2005, along with many members of his nine-member in-house investment staff.
Mr. Hsu left his position as U.S. investment officer of the then-$3 billion Atlantic Philanthropies, New York. He had also worked at the Ludwig Institute for Cancer Research, New York; at Xerox Corp., Stamford, Conn.; and Common Fund, Westport, Conn.
It's difficult to learn much about Anchor Point Capital. The firm does not have a website. Anchor Point does not report data for P&I's manager surveys; it is not registered with the Securities and Exchange Commission as an investment adviser; it does not report information about its funds to major industry hedge fund databases.
Press reports and industry sources estimate Anchor Point's assets under management to be several hundred million dollars.
According to 13-F SEC filings, the firm manages an onshore and an offshore version of the Anchor Point Capital Flagship Fund and the Anchor Point Capital Global Macro Fund. A source with knowledge of the firm said the offshore version of the Flagship Fund has returned 12% since inception in January 2006.
One source, who asked not to be named, said Mr. Crowe is considered a thoughtful and credible investor, but "we thought it was odd that they (Messrs. Crowe and Hsu) had ever gotten together. We did not know Albert well, but Mr. Crowe was well thought of and that was what drew us to take a closer look."
Jay A. Yoder heads a similarly young specialist asset management firm, Tuckerbrook Alternative Investments LLC in Marblehead, Mass., which has $95 million in assets under management. Questions about the firm's stability are among those most often asked by prospective clients, Mr. Yoder said.
"It's a question we are asked all the time, and it's one that's very important to institutional investors. When I was managing (endowment) assets, I preferred to invest with people I had known for years or based on referrals from people I had known for years," Mr. Yoder said.
Mr. Yoder said Mr. Crowe's extensive investment experience, his long stint as a CIO and his successful track record at Knight "will help them survive. Anchor Point was not a one-man shop. It was a two-man shop. And one thing in their favor is that they are managing a portfolio of hedge funds, not securities, so there are not really day-to-day investment responsibilities."
But Mr. Yoder warned that bearing responsibility for both the investment management and administration of any firm is difficult. "Tim and Anchor Point can survive doing this short term. But these both are full-time jobs, requiring different skill sets. They probably will need to recruit either a CEO or a CIO."
'Key-man risk'
Mr. Yoder and hedge fund-of-funds managers agreed that while operational stability and so-called "key-man risk" — the risk of losing a critical employee or team — are most often thought of in relation to individual hedge funds, they also are extremely important issues for institutional investors in funds of funds.
Key-person risk is something that alternatives fund-of-funds manager Strategic Investment Group is constantly being assessed on, said Deborah Boedicker, principal. The Arlington, Va.-based firm is 20 years old, but questions from institutions about operational stability and succession planning, should something happen to President and CEO Hilda Ochoa-Brillembourg, remain standard.
"We all came out from a plan sponsor culture, and that fiduciary responsibility is something we're accustomed to," Ms. Boedicker said.
Read the print edition of P&I
How low is low? Projections say it's not low enough
FINRA honors Wharton's Olivia Mitchell with Ketchum Prize


"Steal This MP3" appears on the 2012 CD, Meanwhile In Afghanistan. The acoustic version also appears on the acoustic version of the CD, which can only be found accompanying the download of the album on Bandcamp.
The music industry has been broken for a long, long time as far as promoting independent music goes, at least in the USA. You can be sure that whatever the big corporations are up to with the various trade deals related to intellectual property, patents, copyrights, downloading music, and so on, they're not interested in the independent musicians or other artists, and they're not interested in the well-being of independent culture in general. We need solutions to various problems involving the internet, free downloads, etc., but those solutions will not be found through these trade deals and laws being passed or proposed by the industries involved. What's good for GM is not what's good for America. Same goes for EMI.


Western culture no longer has a broad Christian understanding. In fact, much of what American media dishes out is pantheistic or anti-religious. Sociological research indicates that a multitude of beliefs have crept into Christian understandings of faith. This situation is not new. As early as the fifteenth century, Erasmus asserted that the greatest concern of his time was that Christians were most often "rank heathens" when it came to their orthodoxy. It is also apparent that attendance at Sunday school, Vacation Bible School, and other gatherings has fallen at a great rate. The author believes providing Christian education in the context of worship is therefore the best opportunity for reaching believers with historical elements of faith. A twenty-four-part lectionary-based catechetical curriculum is provided for worship. With it, congregations can dwell in a sermon series that is surrounded by supportive full-bodied worship.
James Åkerson is the pastor of a rural congregation and a district minister for the Virginia Mennonite Conference (MCUSA). He holds a DMin degree from Gordon-Conwell Theological Seminary in Massachusetts, and an MDiv from Eastern Mennonite Seminary in Virginia. He is a convinced Anabaptist. Born in Oregon, he grew up in a Lutheran tradition, where he gained an appreciation for high church practices of worship. He and his wife, Emily, have two grown sons.


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Dentistry Section DOI : 10.7860/JCDR/2018/37459.12342
Year : 2018 | Month : Dec | Volume : 12 | Issue : 12 Page : ZC06 - ZC12
Cymbopogon Citratus (Lemongrass Oil) Oral Sprays as Inhibitors of Mutans Streptococci Biofilm Formation
Kemthong Mitrakul1, Ratchapin Srisatjaluk2, Vimol Srisukh3, Pattamapan Lomarat4, Kadkao Vongsawan5, Theerachai Kosanwat6
1 Faculty, Department of Pediatric Dentistry, Mahidol University, Bangkok, Thailand.
2 Faculty, Department of Oral Microbiology, Mahidol University, Bangkok, Thailand.
3 Faculty, Department of Food Chemistry, Mahidol University, Bangkok, Thailand.
5 Faculty, International College of Dentistry, Walailak University, Bangkok, Thailand.
6 Faculty, Department of Oral and Maxillofacial Pathology, Mahidol University, Bangkok, Thailand.
NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Kemthong Mitrakul, 6 Yothee Street, Bangkok, Thailand.
E-mail: mkemthong@yahoo.com
Dental caries that occurs in young children or Early Childhood Caries (ECC) remains highly prevalent worldwide. Streptococcus mutans is not only one of the pioneer groups in plaque formation but is also crucial for its continuous development. It is important to prevent the presence of Mutans Streptococci (MS) for more effective prevention of dental caries.
To evaluate the susceptibility of Streptococcus mutans (ATCC 25175), Streptococcus sobrinus (ATCC 6715) and three Streptococcus mutans clinical isolates from Thai children to three oral spray formulations (2%L38, 4%L40, 6%L42) of Cymbopogon citratus extracted oil, and evaluate the inhibitory effect of each formulation on growth and biofilm formation of each strain.
S. mutans clinical isolations were obtained from children (12 males, 5 females aged 3.5-10-years-old) who came to the Paediatric Dental Clinic, Faculty of Dentistry, Chulalongkorn University, Bangkok. The inhibitory effect of each formulation on growth and biofilm formation was performed using a 96-well polystyrene plate biofilm assay. The inhibition effect was further analysed using fluorescent dyes (SYTO9 and propidium iodide) and Confocal Laser Scanning Microscopy (CLSM). All experiments were performed in triplicate. Data was expressed as mean±SD. For the determination of live/dead cells at the excitation wavelength of 485 nm, the green emission at 530 nm (live cells) and the red emission at 630 nm (dead cells) were measured and their ratio was calculated. The Kruskal-Wallis Test (Bonferroni and Dunn Test) was used to compare the experimental and control groups (significance at 95%).
All oral spray formulations showed excellent antibacterial activity. Results showed that 3 and 6 hours. preformed biofilms of all strains were sensitive to all formulations (100%) when compared to the controls. Biofilm formation of all strains was inhibited (100%) when compared to the controls by all formulations at different concentrations. Six percent (L42) oil formulations showed the maximum inhibition effect, followed by 4% (L40) and 2% (L38), respectively. Results from CLSM analysis of S. mutans (ATCC 25175) showed that 6% (L42) oil formulations gave the maximum inhibition effect, followed by 4% (L40) and 2% (L38), respectively. For S. sobrinus (ATCC 6715), 2% (L38) oil formulations showed the maximum inhibition effect, followed by 4% (L40) and 6% (L42). For all S. mutans clinical isolates, all formulations inhibited biofilm effectively and were not significantly different.
In this study, all oral spray formulations from Cymbopogon citratus essential oil showed excellent antibacterial activity and they might be an alternative inexpensive natural medication to prevent dental caries in the near future.
Kemthong Mitrakul, Ratchapin Srisatjaluk, Vimol Srisukh, Pattamapan Lomarat, Kadkao Vongsawan, Theerachai Kosanwat, .CYMBOPOGON CITRATUS (LEMONGRASS OIL) ORAL SPRAYS AS INHIBITORS OF MUTANS STREPTOCOCCI BIOFILM FORMATION.Journal of Clinical and Diagnostic Research [serial online]2018 Dec[cited:2019 Jul 18] 12 ZC06 - ZC12
Dental caries is an infectious disease that results from the dissolution of tooth mineral by acids derived from bacterial fermentation of sucrose and other dietary carbohydrates [1]. It is a multi-factorial disease that includes cariogenic microorganisms, substrate and a susceptible tooth/host. Microorganisms are one of the major components in dental caries progression. High risk caries children are colonised early in life by cariogenic bacteria transmitted from caregivers or other family members through salivary contact [1,2].
Early childhood caries is a destructive, debilitating and the most common chronic childhood disease that affect the young children. Not life-threatening but it affects normal health and well-being of the child [2]. Though dental caries prevalence has reduced worldwide, ECC prevalence is still high and is currently a WHO concern [2,3, 4]. Severe ECC as the name indicates is a severe, aggressive and debilitating form of this disease with ramifications.
Even though in Thailand, caries prevalence has been reported to be decreasing, it is still in a rate of high prevalence [5,6]. The consequences of ECC includes a higher risk of new carious lesions in both primary and permanent dentitions, hospitalisations, high treatment costs and diminished oral health-related quality of life [2].
Biofilm or dental plaque is a complex microbial community composed of numerous aggregated microorganisms that attach to a surface and are surrounded by an extracellular polymeric matrix [7,8]. The acidogenic bacterial species Streptococcus mutans, in combination with various lactobacilli, is recognised to be involved in cariogenic processes [9]. Over time, its attributed role changes from that of the pathogen (specific plaque hypothesis) to enhancer (active role) and/or indicator (passive role) in a sugar-triggered cariogenic vicious circle (extended caries ecological hypothesis) [8,9].
Epidemiological studies have consistently demonstrated that MS, which include Streptococcusmutans and Streptococcus sobrinus, are the primary cariogenic microorganisms associated with dental caries [10–13]. It was not found only in the high risk caries group but also in low risk or cavities-free groups [14,15]. Children who were either colonised early by MS or presented with persistently higher levels of MS experienced significantly more ECC [16,17]. Although S. sobrinus is less prevalent than S. mutans, studies have suggested that the presence of both species in dental plaque is positively correlated with ECC development [16,17]. MS is not only one of the pioneer groups in plaque formation but is also crucial for its continuous development. It is important to prevent the presence of MS for more effective prevention of dental caries.
Essential Oil (EO) has been reported for its effectiveness against bacteria embedded within the biofilm [18-21]. Lemongrass oil (Cymbopogon citratus) is a volatile oil obtained from lemongrass leaves. It is widely used in the perfume and cosmetic industries. It is mainly composed of citral, a natural mixture of geranial and neral and some myrcene, geraniol and geranyl acetate, which is a potential natural biocide for use as a disinfectant [22]. In this study, we developed three oral spray formulations, 2% L38, 4% L40, and 6% L42, to test. The efficiency of these oral spray formulations on the inhibition of biofilm growth and formation, along with a susceptibility test of S. mutans clinical isolations, should be further investigated before starting future clinical trials.
Confocal Laser Scanning Microscopy (CLSM) has been used to evaluate bacterial viability in numerous experiments. Fluorescence dyes are applied on the biofilm to differentiate live bacteria from dead bacteria, allowing the bacteria to be distinguished according to cytoplasmic membrane permeability. Moreover, CLSM is able to penetrate in a horizontal plane (X-Y axes) and evaluate the depth of biofilm from the outer part to the inner part [23-25]. CLSM can capture a series of image-scans showing changes in the viability of the bacterial cells over time, making the real-time visualisation of death of the microorganisms possible.
The objectives of this study were to: 1) Evaluate the susceptibility of MS and three clinical isolations from Thai children to different oral spray formulations of Cymbopogon citratus essential oil; 2) Evaluate the inhibitory effect of each oil formulation on biofilm growth and formation of each strain using a 96 well-polystyrene plate biofilm assay; and 3) Analyse the inhibition effect on biofilm using fluorescent dyes and CLSM.
Essential Oil Preparation
The Cymbopogon citratus (Lemongrass Oil) were purchased from Thai-China Flavours and Fragrances Co., Ltd., (510/3-4 Soi Ngamwongwan 25, Ngamwongwan RD., Ngamwongwan RD, Muang, Nonthaburi 11000). The components of the essential oils were analysed using GC-MS (Auxs) as described previously [26].
Main components in Cymbopogon citratus oil are E-Citral or Geranial (53%) and Z-Citral or Neral (38%), respectively [Table/Fig-1]. Three oral spray formulations (6%, 4%, and 2%) were prepared.
Chemical constituents of Cymbopogon citratus (Lemongrass Oil) by GC-MS and GC chromatogram of Cymbopogon citratus (Lemongrass Oil).
Retention Time (min)
Base peak (m/z)
Area (%)
1 10.734 93 2.13 β-Myrcene
2 20.013 44 1.40 Limonene oxide
3 22.751 41 38.13 Z-Citral or Neral
4 23.333 69 3.66 Geraniol
5 24.148 69 52.88 E-Citral or Geranial
6 29.169 69 1.80 Geranyl acetate
Subject Selection and S. Mutans Clinical Isolation
S. mutans clinical isolations were obtained from child subjects. This cross-sectional study was approved by the Ethical Human Research Committee, Faculty of Dentistry Chulalongkorn University, Bangkok Thailand (ethical approval number: 42/2010) [26]. Consents were signed from all child parents/legal guardians. All subjects were free to withdraw from the study at any time. S. mutans clinical isolations were obtained from 17 children (12 males, 5 females aged 3.5-10-years-old) who came to the Paediatric Dental Clinic, Faculty of Dentistry, Chulalongkorn University, Bangkok., For convenience sampling during the duration of the study within five months, after having obtained written and oral consents, oral examination was performed based on World Health Organisation definition. DMFT/dmft scores were recorded. In brief, stimulated saliva was collected and pooled in an equal proportion, and then cultured on MSA agar (Difco Laboratories, Detroit, MI) containing bacitracin (0.2 U/mL, Sigma Chemical Co., St Louis, MO), 0.001% (v/v) tellurite solution (Becton, Dickinson and Co., Sparks, MD) and 15% (w/v) sucrose (SigmaChemical Co.,). Subjects who had any systemic disease, were taking any kind of antibiotics, had professional fluoride application or any dental treatment within three months prior to the sample collection period were excluded. This was a once only collection of plaque samples so that participating in this study would not affect the subject's regular dental treatment. Ten colonies resembling S. mutans from each subject were then transferred to Brain Heart Infusion (BHI) broth and incubated at 37°C for 48 hours in an anaerobic jar. DNA samples from 280 isolates were extracted by washing and boiling for 10 minutes with TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0)22. All isolates were then confirmed to be S. mutans by biochemical analyses. Two standard strains (Streptococcus mutans ATCC 25175 and Streptococcus sobrinus ATCC 6715) were cultured on BHI agar or broth. For standard strains (S. mutans ATCC 25175, S. sobrinus ATCC 6715), they were cultured on BHI agar or broth and incubated for 48 hours at 37°C, supplemented with 5% CO2 for 18 hours.
Bacteria and Culture Conditions
A -20°C aliquot stocks of S. mutans (ATCC 25175) were grown on BHI and incubated for 48 hours at 37°C and supplemented with 5% CO2. After that, 3-5 colonies were picked and suspended in THB broth (Todd-Hewitt broth, Difco, USA) and incubated for 24 hours. Aliquot stocks (-20°C) out of a total of three S. mutans clinical isolations (N006, N029, N113) were grown as described above.
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
MIC and MBC were carried out using the Microbroth Dilution method, as described previously [27]. In brief, the oral spray solution was diluted in a series of two fold dilutions (ranging from 200 μg/mL to 1.5625 μg/mL) in Tryptic Soy Broth (TSB). Freshly grown bacterial suspensions in TSB were adjusted to approximately 106 CFU/mL. For MIC determination, the researcher added 100 μL of seeded broth to 8 wells and added 100 μL of test agent. The first test tube contained 100 μL of the seeded broth for eight wells. The solution of the test agent (100 μL) was added to the first tube to bring the volume to 200 μL. One hundred μL of this was transferred to the next tube and the process was repeated giving a two-fold serial dilution. The tubes were incubated at 37°C for 24 hours. The first assay well, with no apparent growth of the microorganism, contained the lowest dilution of the test essential oil and represented the MIC. The bactericidal effect was assessed by MBC determination. Samples were removed from the tubes that showed no turbidity and were dropped onto TSA plates. After incubation at 37°C for 24 hours the minimum concentration without any visible growth was reported as the MBC.
Biofilm Susceptibility Test
To examine the effects of various oral spray formulations on 3 and 6-hour-old biofilms and to determine whether different strains had different susceptibilities to the bactericidal activity of each formulation, a biofilm susceptibility test was performed. All strains were diluted with THB broth supplemented with 10% (w/v) sucrose. The starting cell density was 108 CFU/mL. The researcher transferred 2 mL of diluted cells into a new tube and incubated it for 3 and 6 hours. After incubation, planktonic cells were removed by washing with sterile water three times. Then, 2 mL of tested oral spray formulation were added into the same tube. After one minute this was washed out and ten 2×2 mm sterile beads and 2 mL of normal saline solution added, with one minute of vortex. 20 mL of the solution were pipetted and dropped on BHI agar. The numbers of CFU were obtained by plate counting. All experiments were done in triplicate. The colony was calculated as a percentage compared to the control. The effective bactericidal effect was significant, at more than 99.9% compared to the control.
Saliva-Coated 96 and 24-Well Plate for Biofilm Inhibition Assay
All plates in this study were coated with saliva to create an oral environment atmosphere. Stimulated pooled saliva was obtained from five volunteers by omitting from tooth brushing for more than 6 hours. and then chewing paraffin to collect the saliva. The pooled saliva was centrifuged and diluted to 1:10 with PBS buffer and filtered (filter paper, Millipore, USA). Each well containing 100 μL of diluted saliva was incubated at 37°C for 24 hours and then the saliva rinsed off.
Biofilm Inhibition Assay
This assay was to analyse the inhibition effect of oil formulations on biofilm formation. All strains grew in THB for 24 hours. and were then diluted with 5% (w/v) sucrose to the cell density of 106 CFU/mL. Each well contained 200 μL of solution. The researcher added oral spray formulations in each concentration with a pipet and mixed. All experiments were done in triplicate. After 18 hours of incubation, growth was measured using a micro-plate reader at the optimal density of 595 nm (Microplate Spectrophotometer, Biotek, USA). Then they were stained with 100 μL of crystal violet 0.05% (w/v) for 15 minutes, rinsed with water and placed in room temperature for one hour. Biofilm formation was read by adding 150 μL/well of 90% ethanol and put on a shaker at slow speed in order to remove all planktonic cells. A calculation of the mean percent inhibition of replicate tests and the IC50 was determined as a concentration of samples which inhibited 50% of biofilm formation in comparison to the control.
Fluorescence Staining and Confocal Laser Scanning Microscope (CLSM)
The researcher diluted all strains in BHI broth with 5% (w/v) sucrose to the cell density of 108 CFU/mL, then pipetted it into the 24-well polystyrene plate. Each well contained 600 μL and was incubated under the same conditions for 24 hours. After 24 hours, we pipetted and discarded all solution from each well and rinsed with PBS three times to remove non adherent bacteria on the cover glass. A 600 μL of each oral spray formulation was pipetted into each well and left it for five minutes. After five minutes, we pipetted and discarded all solution from each well and rinsed with PBS three times. We then added 600 μL PBS into each well to prevent biofilm dryness, And pipetted 3 μL of 1:10 dilutedSYTO-9 and propidium iodide (L7012 LIVE/DEAD® BacLight. A Bacterial Viability Kit *for microscopy and quantitative assays* (Invitrogen, USA) was put into each tested well and mixed with a pipette gently not to disturb the biofilm. This was stored in the dark for 15 minutes, then analysed under a confocal laser scanning microscope (CLSM, IX83ZDC, Olympus). The researcher adjusted the filter and lens of the fluorescent confocal electron microscope to the following parameters: live cell fluorescent imaging system IX83ZDC to standardise. Lens 60x green narrow 48%, PJ 46%, and PH 36.6% was used.
All experiments were performed in triplicate. Data was expressed as mean±SD. For the determination of live/dead cells at the excitation wavelength of 485 nm, the green emission at 530 nm (live cells) and the red emission at 630 nm (dead cells) were measured and their ratio calculated. Each well was measured three times. Mean and standard deviations were calculated, and the Kruskal-Wallis Test (Bonferroni and Dunn Test) was used to compare experimental and control groups (significance at 95%).
The MIC and MBC of Cymbopogon citratus oral sprays were in the range of 0.125-3 μg/mL and 0.25-6 mg/mL, respectively [Table/Fig-2]. Although the MIC and MBC varied among tested bacteria, the MIC in most cases was equivalent to the MBC, indicating a bactericidal action in the oil.
Minimum Inhibitory Concentration (MIC) and Minimum Bactricidal Concentration (MBC) of Cymbopogon citratus oral spray formulations on the inhibition of two standard strains and three clinical isolates.
Bacterial strains
2% (L38)(μg/mL)
S. mutans (ATCC 25175) 0.5 0.5 1 1 1.5 3
N006 0.5 0.5 1 1 3 6
N029 0.25 0.5 0.125 0.25 1.5 6
N113 0.5 0.5 0.125 0.25 1.5 3
S. sobrinus (ATCC 6715) 0.5 0.5 1 1 3 6
All strains of bacteria tested in this study were sensitive to all of the oral spray formulations (≥99.9%) when compared to the controls. All spray formulations had an inhibitory effect on 3 and 6 hour biofilm formation (100%) of all strains, compared to the control [Table/Fig-3,4].
Minimum amount of Cymbopogon citratus oral spray to inhibit biofilm formation (μL) of two standard strains and three clinical isolates.
Minimum amount of oral spray to inhibit biofilm formation (μL)
2% (L38)
S. mutans (ATCC 25175) 6.25 3.125 1.56
N006 3.125 3.125 1.56
N029 6.25 1.56 1.56
N113 6.25 3.125 1.56
S. sobrinus (ATCC 6715) 6.25 1.56 1.56
Example of 3 hour and 6 hour preformed biofilms by S. mutans (ATCC 25175), which was inhibited by 2% (L38) Cymbopogon citratus oral spray. All spray formulations had an inhibitory effect on 3 hour and 6 hour biofilm formations (100%) of all strains.
Most of the oil formulations had an inhibitory effect on biofilm formation (≥50.0%) compared to the control when stained with crystal violet solution 0.05% (w/v) At 108 CFU/mL the dose-dependent form showed an inhibitory effect. A 2% (L38) spray formulation inhibited growth and biofilm formation of S. mutans (ATCC 25175), S. mutans clinical isolates N029, N113 and S. sobrinus (ATCC 6715) at a concentration of 3.125 μL. For S. mutans clinical isolates N006 at a concentration of 1.56 μL. 4% (L40), spray formulation inhibited growth and biofilm formation of S. mutans (ATCC 25175), and of S. mutans clinical isolates N006 at a concentration of 3.125 μL. For S. mutans clinical isolates N029, N113 and S. sobrinus (ATCC 6715) at a concentration of 1.56 μL. A 6% (L42), spray formulation inhibited growth and biofilm formation of all strains at a concentration of 1.56 μL. Higher concentrations of low percentage oral spray was needed to inhibit growth and biofilm formation [Table/Fig-5].
S. sobrinus ATCC 6715 biofilm formation after staining with crystal violet solution 0.05% (w/v). Three Cymbopogon citratus oral spray formulations were added into wells containing cell inoculums of 108 CFU/mL and incubated for 24 hours. A 6% (L42) oil formulations showed maximum inhibition effect followed by 4%(L40) and 2%(L38).
Saliva-coated 24-well Plate and Biofilm Assay and Fluorescence Staining and CLSM
For S. mutans (ATCC 25175), 6% (L42) showed the highest inhibition rate, which gave the lowest percentage of live cells (11.28±2.90%), followed by 4% (L40)(11.56±0.82%), and 2% (L38)(12.18±2.08%), respectively [Table/Fig-6]. All formulae showed significantly (p=0.005) different inhibition rates compared to controls in the Kruskal-Wallis Test. When compared between each formula, there was no significant difference among the three formulations in inhibition of S. mutans (ATCC 25175).
A 24 hour. S. mutans (ATCC 25175) biofilm formation. Positive control: Live cells were stained and exhibited green fluorescent under CLSM, while dead cells exhibited red fluorescent under CLSM. Negative controls were 0.12% Cholorhexidine and 95% ethanol. Graph represents percent reduction of S. mutans biofilm by each spray formula.
Chemical n Mean Rank Test Statisticsab
Ratio neg 6 33.50 Peak Base peak
form2 6 18.08 Chi-square 15.449
form4 6 16.33 Df 5
form6 6 13.67 Asymp. 0.009
alc 6 13.33 Sig.
chx 6 16.08 a. Kruskal Wallis Testb.Grouping Variable:Chemical
For S. sobrinus (ATCC 6715), 2% (L38) showed the highest inhibition rate, which gave the lowest percent of live cells (8.19±0.69%) followed by 4% (L40)(10.64±1.05%) and 6% (L42)(12.23±2.00%), respectively [Table/Fig-7]. All formulae showed significantly (p=0.005) different inhibition rates compared to the controls. When compared between each formula, there was no significant difference among the three formulations in inhibition of S. sobrinus (ATCC 6715). When compared between each formula, 2% (L38) showed a significantly higher biofilm inhibition rate (p=0.013) when compared to 4% (L40) and 6% (L42) at the significance level of 0.05.
A 24 hour S. sobrinus (ATCC 6715) biofilm formation. Positive control: Live cells were stained and exhibited green fluorescent, while dead cells exhibited red fluorescent under CLSM. Negative controls were 0.12% Cholorhexidine and 95% ethanol. Graph represents percent reduction of S. sobrinus biofilm by each spray formula.
form2 6 6.17 Chi-square 28.442
alc 6 8.17 Sig.
For S. mutans clinical isolate N006, 4% (L40) showed the highest inhibition rate, which gave the lowest percent of live cells (11.16±1.14%), followed by 6% (L42)(11.50±2.57%) and 2% (L38) (12.31±1.82%), respectively [Table/Fig-8]. All formulae showed significantly (p=0.005) different inhibition rates compared to controls in the Kruskal-Wallis Test. When compared between each formula, there was no significant difference among the three formulations in inhibition of clinical isolate N006.
A 24 hour. S. mutans clinical isolate N006 biofilm formation. Positive control: Live cells were stained and exhibited green fluorescent while dead cells exhibited red fluorescent under CLSM. Negative controls were 0.12% Cholorhexidine and 95% ethanol. Graph represent percent reduction of S. mutans clinical isolate N006 biofilm by each spray formula.
For S. mutans clinical isolate N029, 4% (L40) showed the highest inhibition rate, which gave the lowest percent of live cells (11.10±1.47%), followed by 2% (L38)(11.45±2.81%) and 6% (L42)(12.98±2.42%), respectively [Table/Fig-9]. All formulae showed significantly (p=0.005) different inhibition rates compared to controls in the Kruskal-Wallis Test. When compared between each formula, there was no significant difference among the three formulations in inhibition of clinical isolate N029.
For S. mutans clinical isolate N113, 2% (L38) showed the highest inhibition rate, which gave the lowest percent of live cells (11.71±1.34%) followed by 6% (L42) (11.97±2.15%) and 4% (L40) (12.31±1.22%), respectively [Table/Fig-10]. All formulae showed significantly (p=0.005) different inhibition rates compared to controls in the Kruskal-Wallis Test. When compared between each formula, there was no significant difference among the three formulations in inhibition of clinical isolate N113.
[Table/Fig-10]:
It has been well established that S. mutans is the major etiological agent in dental caries [6]. Plant-derived essential oil are ideal for use in oral care products, including toothpastes, mouthwashes, spray and gels, because they are non-toxic and have antiseptic properties [28]. This investigation of Thai herbal oral spray formulations used an oil extraction from Cymbopogon citratus against dental caries, allowing for the possibility of lower costs to prevent dental caries in the Thai population. Chlorhexidine has been ranked as a gold standard among antibacterial agents effective in reducing oral Biofilm [26,29]. Nevertheless, it is an antiseptic agent which effectively kills bacteria by penetrating into a cell's cytoplasm and lyses cell components [27]. Previous reports have shown that bacteria might develop a resistant mechanism if used for a long time. Most researchers are trying to use herbal extractions as an alternative.
Cymbopogon citratus, or lemongrass, is a tropical plant that is easy to find in Thailand. The oil extraction of lemongrass has antioxidant anti-inflammatory effects which can be used in a variety of medicines. This work had demonstrated an antimicrobial efficacy in mono-specie of bacterium before stepping into a multi-species study. Single organism biofilms are advantageous in examining the mechanisms and actions of therapeutic agents on the interested specie. From a previous study by Manvitha K et al., they found that a Cymbopogon citratus oil extraction demonstrated anti-bacterial, fungal, protozoan, and other medical benefits such as being anti-inflammatory, anti-cancer, anti-oxidant, etc., [30]. Another study by Tofiño RA et al., tested the antibacterial effect of Cymbopogon citratus oil extraction against S. mutans (ATCC 35668) in vitro using MBEC-High-Through put screening (HTP) assay, their results showed that oil extraction at 0.1 mg/dL inhibited S. mutans at 95.4% and at 0.01 mg/dL inhibited S. mutans at 93.1% [31]. Also, study by Oliveira MAC et al., tested the antimicrobial activity Cymbopogon citratus oil against Actinomyces naeslundii, Lactobacillus acidophilus, S. gordonii, S.mitis, S. mutans, S. sanguinis and S. sobrinus. Results showed an inhibitory effect on all tested species. Cymbopogon citratus oil (10XMIC) reduced the number of viable cells of lactobacilli and streptococci biofilms (p<0.05). In addition, the essential oil inhibited adhesion of caries-related polymicrobial biofilm to dental enamel (p<0.01) [32]. Another study by Rego CB et al., investigated the effect of Cymbopogon citratus oil on S. mutans biofilm developed in hydroxyapatite discs surface. The biofilms of S. mutans were developed on the discs for five days and immersed daily in the three groups which were Cymbopogon citratus oil, BHI broth and control. Then biofilms were counted for CFU. The susceptibility test was indicating inhibition of microorganisms and the MIC value was 0.04 mg/mL. As for biofilm results, it decreased the bacterial growth in Cymbopogon citratus oil group when compared to control [33]. In this study, all oral spray formulations showed excellent antibacterial activity. Results showed that 3 and 6 hours preformed biofilm of all strains were sensitive to all formulations (100%) when compared to the controls. Biofilm formation of all strains was inhibited (100%) when compared to the controls by all formulations at different concentrations. Six percent (L42) oil formulations showed maximum inhibition effect, followed by 4% (L40) and 2% (L38), respectively. There were three different stages of biofilm formation, with accumulations at 0-5 hours active accumulation at 4-20 hours and a slower accumulation with a plateau-phase at 28 hours. The 3 hours biofilm was in the first stage, whereas the 6 hour. biofilm was in the second stage. The 6 hours biofilm was more complex and resistant because of a rapid increase in adherence [4]. This study demonstrated exceptional inhibition effects of all formulations, even in 6 hours preformed biofilm.
This study also tested the inhibitory effect on biofilm growth and formation of MS and three S. mutans clinical isolates. Results showed that all oral spray formulations exhibited an inhibitory effect on biofilm formation. The 6% (L42) formula reduced cell growth and inhibited biofilm formation at a cell density of 106 CFU/mL at all concentrations. The 4% (L40) was found to inhibit biofilm formation at 106 CFU/mL of S.mutans (ATCC 25175), and clinical isolate N006 at concentrations of 3.125 and 1.56 μL, for clinical isolates N029, N113 and S. sobrinus (ATCC 6715). The 2% (L38) inhibited biofilm formation at 106 CFU/mL at all concentrations of 3.125 μL, except for N006, which inhibited it at 1.56 μL.
In this study, biofilm was stained with fluorescence dye which penetrated through the cell cytoplasmic membrane. Live cells will exhibit green colour (SYTO9) and dead cells will exhibit red colour (propidium iodide). CLSM was used to evaluate bacterial viability in biofilms [31]. Fluorescence dyes were applied on the biofilm to differentiate live and dead bacteria, allowing the bacteria to be distinguished according to cytoplasmic membrane permeability [31]. Furthermore, CLSM can capture a series of image-scans showing changes in the viability of the bacterial cell over time, making the visualisation of real-time death of microorganisms possible [19]. Results indicated that all formulations showed significantly (p=0.005) different inhibition rates compared to the controls, and when compared between each formula, there was no significant difference among the three formulations in inhibition of all strains tested, except S.sobrinus (ATCC 6715). There are many possibilities in showing the various susceptibilities, such as the different genotypes of S. mutans strains, the components of each oil formulation, which might result in a different gene expression and lead to different aciduric and biofilm formation properties [15].
When evaluating 3D images with CLSM, all formulations were able to penetrate the biofilm layer to kill bacteria in the deepest layer and in all directions of biofilm structure. This result was better than our previous result using Citrus hystrix (makrut oil) oral sprays [23]. This result is similar to a study by Benjamin S et al., which studied the effectiveness of various dental products, including mouthwashes containing essential oils and dentifrice slurries of toothpastes using a CLSM camera which found that a mouthwash with a mixture of essential oils could penetrate the biofilm on all layers and in all directions and killed bacteria in biofilm in the first 30 seconds. The main antimicrobial agents in extracted Cymbopogoncitratus oil are α-citral (geranial), β-citral (neral), linalool, myrcene, Passenger α-citral (geranial), β-citral (neral), and linalool [34,35]. Myrcene is a substance in the essential oil which has an antibacterial and lipophilic property which can be absorbed through the cell via the lipid-bilayer and damage the cell membrane [36]. In addition, studies of the properties of citral (geranial) have been shown to reduce ATP within the bacterial cell, resulting in pH changes in cells and hyperpolarisation [37]. From previous study tested the cytotoxic effect of Cymbopogon citratus oil against a human keratinocyte (HaCaT) cell line was in the range of 0.25 to 25 mg mL-1. After exposure to 0.25 mg mL-1, the cell viability rate was 98% which did not affect the cell viability of 100% which showed low cytotoxicity to human keratinocytes.
S. mutans biofilm was developed as single-species plaque under the laboratory environment. This in vitro pre-established bacterial biofilm may not represent the actual oral environment of multi-species dental plaque with structural complexity and dynamic condition developed in oral cavity. Different responses of dental plaque to these oral spray essential oils in the toxicity aspect should be observed further in the future before starting the clinical study.
In conclusion, three oral spray formulations using an oil extraction of Cymbopogon citratus effectively inhibited biofilm formation and biofilm susceptibility. Oral sprays showed significant inhibition results even at a high cell density of bacteria. These formulations demonstrated satisfying results and need further clinical studies to verify its efficacy against ECC.
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When I decided that my next destination was going to be Gokarna, I applied for leaves, booked tickets, cut my hair, packed my bags and took off. Yeah, I had to mention my haircut because the bangs looked really bad for a long time. I went to Gokarna with a couple of my friends.
It was a small temple town that had two sides to it- a divine and spiritual side,and a breezy uber-cool side. Despite its proximity to Goa, Gokarna was fairly tranquil.
We took an overnight bus from Bangalore, reached Gokarna early in the morning and checked-in to our hotel. We made it to three of the five beaches in Gokarna. We took a stroll along the Gokarna beach, which was very close to the main town. The narrow old-world streets of Gokarna were very beautiful to look at.
We went to Kudle beach in the evening. The beach was dotted with a number of shacks and restaurants. We played for a while, clicked a few pictures, had yummy pancakes at a restaurant nearby, walked into a beach shack, booked a room, went back to our hotel, packed our bags, checked out and moved into the shack. Phew! We did not initially plan on moving out of the hotel. It was a nice hotel located in the main town with a great view, but Kudle was very tempting and we gave in.
We spent the night at the beach gazing at the stars. The other people on the beach were lost in their own sweet thoughts. It was a peaceful night. The next day, we walked to Om beach from Kudle. It involved climbing up a hill and walking down. It was a short walk but it was fun. Om beach was beautiful. We took a stroll on the beach, clicked a few selfies, treated our taste buds at Namste Cafe, returned back to Kudle, packed our bags and checked out. It was a lovely trip!


text:
Q: bootstrap datepicker, beforeShowDay and array of disabled dates I am trying to use bootstrap datepicker plugin (https://github.com/eternicode/bootstrap-datepicker/blob/release/docs/index.rst) with array of disabled days passed into it.
My code (partial):
 var disabled_dates = ["23.03.2014","21.03.2014"];
 $("#datepicker").datepicker({
      language: "pl",
      autoclose: true,
      startDate: '+1d',
      weekStart: 1,
      default: 'dd.mm.yyyy',
      beforeShowDay: function(date){
           var formattedDate = $.fn.datepicker.DPGlobal.formatDate(date, 'dd.mm.yyyy', 'pl');
           if ($.inArray(formattedDate.toString(), disabled_dates) != -1){
               return {
                  enabled : false
               };
           }
          return;
      }
  });

it works (almost) OK. It disables wrong date. Instead of disabling 23.03.2014 it disabled 24.04.2014.
I am not sure where is the issue, on timezone maybe? Any suggestions?

A: Looking inside the plugin, I found out $.fn.datepicker.DPGlobal.formatDate returns a UTC-formatted string instead of GMT. Try changing the setting of formattedDate variable to this:
var formattedDate = date.toLocaleDateString('pl',{day:'2-digit',year:'numeric',month:'2-digit'});

A: You just need to remove line "startDate: '+1d'," it does not make any sense using it when you have available dates array. Anyway, you can add new Date() function to get atual date.
It will be something like this:
 //here you store new Date() Object to get today's date formatted as you want dd.mm.yyy

 var disabled_dates = ["23.03.2014","21.03.2014"];
 $("#datepicker").datepicker({
      language: "pl",
      autoclose: true,
      //removed line: startDate: '+1d',
      weekStart: 1,
      default: 'dd.mm.yyyy',
      beforeShowDay: function(date){
           var formattedDate = $.fn.datepicker.DPGlobal.formatDate(date, 'dd.mm.yyyy', 'pl');
           if ($.inArray(formattedDate.toString(), disabled_dates) != -1){
               return {
                  enabled : false
               };
           }
          return;
      }
  });

redpajama_set_name: RedPajamaStackExchange


text:
How To Check Ehsaas Program Money Online. Know Here
how to check ehsaas program money online
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You can check here
Also, you can lounge a Complaint on this Portal if you are facing any issues regarding Ehsaas Program. by visiting this.
Hope this Article will help you to get your answer.
In conclusion,the Ehsaas Program is a great way to get money if you are in need. The application process is easy and can be done online. The program has helped many people in Pakistan and is continuing to help more every day. If you or someone you know is in need of financial assistance, be sure to check out the Ehsaas Program.
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