Assessment of carbon footprint of a crop is an important component of sustainable crop production, as it helps in framing effectual and viable crop management strategies to minimize ecosystem ...tampering. Thus, in present investigation carbon footprint of potato production system in different agro-climatic zones viz. undulating plain zone, central plain zone and western plain zone of North-west India were estimated, and compared with the recommended practices of these zones. The carbon footprint was higher in undulating plain zone followed by central plain zone and western plain zone with values being 343, 296 and 220 kg CO2 eq./t tuber yield (TY), sequentially, whereas same were 198 kg CO2 eq./t tuber yield (TY) in case of recommended practices. The social cost of carbon (SCC), that represents economic damage from the CO2 emissions, was also estimated. The integrated net economic balance (net return from yield – SCC) was also better in case of recommended practices. The major sources of emission from potato production system were fertilizer (NPK) application (42 %), irrigation (20 %), seed (14 %), fertilizer production (13 %) and energy use (excluding Irrigation) (5 %). Top most in the list of carbon footprint contributors was fertilizer application which was due to imbalanced application of these, and for getting the clear picture of this imbalance as well as its impact, a new and exclusive index- Relative Imbalance Fertilization Index (RIFIcf) was developed and tested. Carbon footprints were also related to tuber yield and an empirical model was developed that can be used to predict tuber yield on the basis of carbon footprint of potato production system. An increase in tuber yield with increasing carbon footprint was noticed, which became somewhat static at higher emissions. The findings of this investigation provide a clear picture of quantitative GHG emissions due to imbalanced inputs that can be plummeted to some extent if already existing recommendations are followed.
Abstract
Background
Outcomes for patients with relapsed acute lymphoblastic leukemia (ALL) are poor and there is a need for novel therapies to improve outcomes. Targeted inhibition of WEE1 with ...small-molecule inhibitor adavosertib (AZD1775) has emerged as a therapeutic strategy to sensitize cancer cells to DNA-damaging chemotherapeutics, particularly in the context of
TP53
-mutated tumors. However, WEE1 inhibition as a potential therapeutic strategy for patients with high-risk and relapsed ALL, including those with
TP53
mutations, has not been definitively evaluated.
Methods
Anti-leukemic effects of adavosertib were investigated using a relapsed
TP53
isogenic cell model system, primary patient, and patient-derived ALL samples (
n
= 27) in an ex vivo co-culture model system with bone marrow-derived mesenchymal stem cells. Combination effects with drugs currently used for relapsed ALL were quantified by Excess over Bliss analyses. Investigations for alterations of cell cycle and apoptosis as well as related proteins were examined by flow cytometry and Western blot, respectively.
Results
Our study demonstrates the potent anti-leukemic activity of the clinically advanced WEE1 inhibitor adavosertib in a large majority (
n
= 18/27) of high-risk and relapsed ALL specimens at lower than clinically attainable concentrations, independent of
TP53
mutation status. We show that treatment with adavosertib results in S-phase disruption even in the absence of DNA-damaging agents and that premature mitotic entry is not a prerequisite for its anti-leukemic effects. We further demonstrate that WEE1 inhibition additively and synergistically enhances the anti-leukemic effects of multiple conventional chemotherapeutics used in the relapsed ALL treatment setting. Particularly, we demonstrate the highly synergistic and cytotoxic combination of adavosertib with the nucleoside analog cytarabine and provide mechanistic insights into the combinational activity, showing preferential engagement of apoptotic cell death over cell cycle arrest. Our findings strongly support in vivo interrogation of adavosertib with cytarabine in xenograft models of relapsed and high-risk ALL.
Conclusions
Together, our data emphasize the functional importance of WEE1 in relapsed ALL cells and show WEE1 as a promising p53-independent therapeutic target for the improved treatment of high-risk and relapsed ALL.
The objective of the present study was to prepare the fast disintegrating tablet of Salbutamol Sulphate, Cetirizine Hydrochloride in combined tablet dosage form for respiratory disorders such as ...bronchitis, asthma, and coughing for pediatrics and geriatrics. The tablets were prepared by direct compression technique. Superdisintegrant such as Sodium Starch Glycolate was optimized as 4% on the basis of least disintegration time. Different binders such as MCC and PVP K-30 were optimized along with optimized superdisintegrant concentration. 1% MCC was selected as optimum binder concentration on the basis of least disintegration time. The tablets were evaluated for hardness, friability, weight variation, wetting time, disintegration time, and drug content uniformity. Optimized formulation was further evaluated by in vitro dissolution test, drug-excipient compatibility, and accelerated stability study. Percent weight variation and content uniformity were within the acceptable limit. The friability was less than 1%. The wetting time and disintegration time were practically good for all formulations. FTIR studies and accelerated stability study showed that there was no interaction between the drug and excipients. It was concluded that, by employing commonly available pharmaceutical excipients such as superdisintegrants, hydrophilic and swellable excipients and proper filler, a fast disintegrating tablet of Salbutamol Sulphate, Cetirizine Hydrochloride in combined tablet dosage form, were formulated successfully with desired characteristics.
Abstract BACKGROUND Medulloblastoma (MB) is the most common malignant paediatric brain tumour, with 5-year survival rates >70%. Survivors frequently suffer a wide-range of late-effects due to their ...tumour and its treatment. Cranial radiotherapy (CRT) to the whole-brain, with posterior fossa boost (PFB), underpins treatment for non-infants, however, radiotherapeutic insult to normal brain tissue has deleterious consequences to neurocognitive and physical functioning, and causes accelerated ageing/frailty. Unfortunately, approaches to ameliorate radiotherapy-induced late-effects are lacking and a paucity of appropriate model systems hinders their development. METHODS We have developed a clinically-relevant in vivo model system that recapitulates the radiotherapy dose, delivery and late-effect profile of childhood medulloblastoma, at an equivalent developmental stage. Consistent with human regimens, age-equivalent (postnatal days 28-37) C57Bl/6J mice received targeted, CT image-guided, CRT (human-equivalent 38 Gy, n=12) or CRT with PFB (human-equivalent 49 Gy, n=12), via the small animal radiation research platform (SARRP) and were longitudinally assessed for over a year. Late-effects were compared to a sham-irradiated group (n=12). RESULTS CRT was well tolerated, independent of PFB receipt, and no mice suffered severe acute toxicity. Mice were significantly more frail following irradiation (frailty index; p=0.0002) and had reduced physical functioning; time to fall from a rotating rod and grip strength were significantly lower (rotarod; p=0.026 and grip strength; p=0.006, respectively). Neurocognitive deficits were consistent with those observed in childhood MB survivors; irradiated mice displayed significantly worse working memory (Y-maze; p=0.009) and exhibited spatial memory deficits (Barnes maze; p=0.029). Receipt of PFB did not induce a more severe late-effect profile. CONCLUSION We conclude that our in vivo model of childhood MB radiotherapy recapitulates the late-effect profile of MB survivors. Our clinically-relevant model will facilitate both the elucidation of novel/target mechanisms underpinning MB late-effects and the development of novel interventions for their amelioration.
Outcomes for adult patients with acute lymphoblastic leukemia (ALL) are dismal and have not kept pace with their pediatric counterparts, with five-year survival rates of less than 45%. TP53 mutations ...are infrequent in ALL, but activity of the oncoprotein MDM2 may otherwise phenotypically disrupt and circumvent normal p53 function, positing the p53-MDM2 signaling axis as a potential therapeutic target for the engagement of intrinsic cell death programs. Given the clinical safety and responses to p53-MDM2 antagonist idasanutlin (RG7388) in other hematological cancers and solid tumors, we aimed to evaluate the therapeutic potential of idasanutlin in ALL.
Single-agent activity of idasanutlin was investigated in 17 high-risk ALL patient and patient-derived xenograft samples (aged 4 to 51 years), both B-ALL (n=15) and T-ALL (n=2), including KMT2A-rearranged, TCF3-rearranged, and Philadelphia-positive ALL. An ex vivo coculture of ALL blasts and hTERT-immortalized mesenchymal stem cells (MSC) was employed to support growth of the ALL blasts during short-term culture, complemented by a fluorescent image-based microscopy platform which identifies and discriminates the two cellular compartments using random forest machine learning algorithms based on cellular nuclear staining. Idasanutlin demonstrated sub-micromolar, dose-dependent anti-leukemic activity against 15 of 17 samples tested, with half maximal effective concentrations (EC50) in the range of 10 to 220 nM (mean EC50 = 45.1nM); the two exceptions were later determined to harbor homozygous inactivating TP53 mutations; p.Y220C and p.S241P within the p53 DNA binding domain. The idasanutlin EC50 concentrations determined are clinically achievable, well below the peak plasma concentrations reported in patients for other disease indications. Furthermore, idasanutlin concentrations below 10µM had no impact upon MSC survival.
Conforming to p53-MDM2 auto-regulatory feedback mechanisms, we demonstrated that idasanutlin efficiently stabilized and activated p53 at the protein level within 6 hours when treated with their respective idasanutlin EC50, to a level greater than 4-fold increased relative to their respective vehicle-only controls (p=0.001, n=7). Further, p53-regulated transcriptional target gene products, MDM2 and p21, were increased by 5-fold and 2-fold respectively, validating engagement of the p53 pathway by idasanutlin (p=0.036 and 0.125, respectively). By contrast, idasanutlin did not elicit increased expression of either p53-regulated transcriptional target gene product in the identified TP53-mutant patient samples (n=2). On-target specificity of idasanutlin was further confirmed in a NALM6 isogenic cell line model, whereby the TP53 wildtype line was sensitive to idasanutlin (EC50 = 74nM) and effectively activated p53 signaling whereas the TP53 homozygous null line was highly resistant (EC50 = ~10µM).
To determine whether the decreased cell numbers and engagement of p53 signaling observed were accompanied by cell death, the capacity of idasanutlin to induce apoptosis in the ALL samples was next investigated. Patient-derived ALL samples (n=6) were treated with vehicle or idasanutlin at their respective EC50s for 24 and 48 hours, and then analyzed by flow cytometry. There was an increase in annexin-V positive cells within 24 hours compared to the vehicle-only treated cells (mean±SD 14.3±6.6% vs 27.0±21.2% respectively (p=0.125). By 48 hours significant apoptosis was attained, with a mean±SD of 59.0±23.8% annexin-V positive cells compared to the mean of vehicle-treated cells at 29.1±11.6% (p=0.004). Cleaved poly(ADP-ribose) polymerase (PARP) levels were also increased greater than 3-fold compared to vehicle-only control cells as assessed by immunoblotting (p=0.045, n=3), corroborating these findings.
These data emphasize the potential of pharmacologically targeting the p53-MDM2 axis in ALL, demonstrating potent, on-target, cytotoxic activity in a range of high-risk ALL cytogenetic subgroups. Taken together, these findings support further preclinical investigations into idasanutlin and other p53-MDM2 antagonists and potential combinations to improve the treatment of adult ALL.
Irving:F. Hoffmann-La Roche: Research Funding.
Abstract BACKGROUND Medulloblastoma is the most frequent malignant brain tumour of childhood. Whilst advances in risk-stratification and upfront multimodal therapy have led to five-year survival ...rates around 70%, a subset of tumours remain refractory to current therapies. Group 3 medulloblastomas (MBGRP3) are enriched for amplification or overexpression of the proto-oncogene MYC which conveys a dismal prognosis (<10% survival), highlighting the urgent need for novel therapeutic strategies. Recent advances have revealed a role for metabolic reprogramming in MYC-amplified MBGRP3 tumorigenesis, providing novel opportunities for selective therapeutic targeting. METHODS & RESULTS To investigate MYC-dependent metabolic alterations in MBGRP3, we generated three independent isogenic MBGRP3 cell-based models with regulable MYC expression (iMBGRP3-MYC) using a doxycycline-inducible shRNA system. Using 1H high-resolution magic angle spectroscopy and stable isotope-resolved metabolomics, we identified upregulation of the de novo serine/glycine biosynthesis pathway (SGP), attributable to elevated expression of the rate-limiting enzyme PHGDH, as a novel therapeutic target. Genetic and pharmacological (NCT-503) inhibition of PHGDH induced greater cell death in MYC-expressing cells compared to MYC knockdown cells and increased survival across two independent MYC-amplified MBGRP3 mouse models. The decrease in tumour growth observed was moderate; we therefore hypothesised that a compensatory reliance on exogenous serine/glycine may play a role in sustaining tumour progression under therapy. To explore this, we investigated the impact of exogenous serine/glycine starvation on MBGRP3-MYC tumour development. Starvation resulted in a marked MYC-dependent reduction in cell viability in vitro across independent iMBGRP3-MYC models. Notably, dietary serine/glycine starvation was well-tolerated, delayed tumour progression, and increased survival (by 50%) in an MBGRP3-MYC mouse model (p=0.005), highlighting the metabolic plasticity of MYC-amplified tumours to utilise both de novo and exogenous serine/glycine sources to survive and proliferate. CONCLUSIONS These findings identify serine/glycine metabolism as a targetable therapeutic vulnerability within MYC-amplified MBGroup3 and a novel therapeutic strategy to treat this poor-prognosis disease group.
Acute lymphoblastic leukemia (ALL) is the most common type of childhood leukaemia. Recently improved risk stratification resulted in therapy optimization and extended survival for the majority of ...cases. Unfortunately, there is still a significant number of patients either relapsing or not responding to treatment with response to glucocorticoids being one of the most important prognostic indicators of treatment outcome.
In order to investigate the mechanism of dexamethasone resistance, we performed genome-wide CRISPR screens in patient derived xenotransplant (PDX) material from t(17;19)-positive ALL. Primary material was obtained from the patient at the presentation and at relapse stage of disease and corresponding PDX samples were generated in immunocompromised NSG mice. PDX cells were lentivirally transduced with the CRISPR knockout pooled ‘Brunello’ library and then subjected to dexamethasone pressure both ex vivo and in vivo. For the in vivo screen, CRISPR-modified cells were intrafemorally injected into immunodeficient NSG mice followed by either 7.5mg/kg dexamethasone or vehicle treatment. In parallel, PDXs were co-cultured with mesenchymal and endothelial-like human stromal cells generated from human bone marrow-derived iPSCs.
Data analysis performed with the MAGeCKFlute software identified the glucocorticoid receptor gene NR3C1 as a main driver of chemoresistance-mediated relapse in this high-risk ALL. Notably, a homozygous deletion of NR3C1 was present in the relapse PDX sample. Furthermore, we identified that loss of the NR3C1 gene in those cells was associated with an inferior engraftment potential in the absence of dexamethasone.
Interestingly, the whole-genome CRISPR screen in the relapse sample identified BCL2 and several genes associated with the mTOR pathway as crucial for leukaemic propagation. Knockout of NR3C1 in the diagnostic PDX also established dexamethasone resistance and further enhanced the already significant sensitivity towards mTOR inhibitors. To explore a potential synergism between BCL2 and mTOR inhibition, we assessed the effect of the BCL2 inhibitor ABT-199 and several mTOR inhibitors in both presentation and relapse PDX samples. PDX samples were co-cultured with MSCs and treated with drug combinations in a matrix format for 96 hrs followed by high-throughput fluorescence microscopy-based analysis. These experiments revealed substantial synergism of ABT-199 and mTOR inhibitors associated with increased cell death and prolonged growth inhibition in both presentation and relapse samples.
In conclusion, our studies (i) demonstrate that genome-wide CRISPR screens are feasible in PDX material both ex vivo and in vivo, (ii) provide an explanation for the relative rarity of NR3C1 mutations in relapsed material and (iii) identify drug combinations effective in both diagnostic and relapse PDX for further preclinical evaluation.
Vormoor:Abbvie (uncompensated): Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding; AstraZeneca: Research Funding.
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