Plasmodium falciparum circumsporozoite protein (CSP) is a major vaccine antigen because it can be targeted by parasite neutralizing antibodies; however, little is known about this interaction. We used isothermal titration calorimetry, X-ray crystallography and mutagenesis-validated modeling to analyze the binding of a murine neutralizing antibody to Plasmodium falciparum CSP. Strikingly, we found that the repeat region of CSP is bound by multiple antibodies. This repeating pattern allows multiple weak interactions of single FAB domains to accumulate and yield a complex with a dissociation constant in the low nM range. Because the CSP protein can potentially cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T cell independent. However, while there was a modest response in mice deficient in T cell help, the bulk of the response was T cell dependent. By sequencing the BCRs of CSP-repeat specific B cells in inbred mice we found that these cells underwent somatic hypermutation and affinity maturation indicative of a T-dependent response. Last, we found that the BCR repertoire of responding B cells was limited suggesting that the structural simplicity of the repeat may limit the breadth of the immune response.The repeat region of the  Vaccines aim to protect by inducing the immune system to make molecules called antibodies that can recognize molecules on the surface of invading pathogens. In the case of malaria, our most advanced vaccine candidates aim to promote the production of antibodies that recognize the circumsporozoite protein (CSP) molecule on the surface of the invasive parasite stage called the sporozoite. In this report we use X-ray crystallography to determine the structure of CSP-binding antibodies at the atomic level. We use other techniques such as isothermal titration calorimetry and structural modeling to examine how this antibody interacts with the CSP molecule. Strikingly, we found that each CSP molecule could bind 6 antibodies. This finding has implications for the immune response and may explain why high titers of antibody are needed for protection. Moreover, because the structure of the CSP repeat is quite simple we determined that the number of different kinds of antibodies that could bind this molecule are quite small. However a high avidity interaction between those antibodies and CSP can result from a process called affinity maturation that allows the body to learn how to make improved antibodies specific for pathogen molecules. These data show that while it is challenging for the immune system to recognize and neutralize CSP, it should be possible to generate viable vaccines targeting this molecule. Plasmodium falciparum causes the deaths of around 430,000 people each year [Malaria caused by ach year . The mosach year . Phase IPlasmodium species are separated by a repeat region, which was the target of the original mAbs [P. falciparum, the CSP repeat has 38 repeats: 34 asparagine-alanine-asparagine-proline (NANP)-repeats interspersed with 4 asparagine-valine-aspartate-proline (NVDP) repeats that are concentrated towards the N-terminus [P. falciparum sporozoite neutralizing antibodies identified in these early studies was 2A10 which can block sporozoite infectivity in vitro [in vivo mouse models utilizing rodent P. berghei parasites expressing the P. falciparum CSP repeat region [Antibodies to CSP were first identified as potential mediators of protection following seminal studies that showed that immunization with irradiated sporozoites could induce sterile protection against live parasite challenge ,6. In thnal mAbs –11. In tterminus  though dterminus . One of in vitro  and in it region ,15.P. falciparum CSP mouse monoclonal antibodies (mAbs) identified some shared sequences [While CSP binding antibodies have been shown to be able to neutralize sporozoites and block infection, it has also been proposed that CSP is an immunological “decoy” that induces a suboptimal, but broad, T-independent immune response due to the CSP repeat cross-linking multiple B cell receptors (BCRs) . Howeverequences . In humaequences .We therefore set out to test the hypothesis that the CSP repeat can bind multiple antibodies or BCRs and drive a T-independent immune response. To do this we undertook a comprehensive biophysical characterization of the 2A10 sporozoite-neutralizing antibody that binds to the CSP repeat. We found that this antibody binds with an avidity in the nano-molar range which was unexpected as previous studies using competition ELISAs with peptides predicted a micro-molar affinity ,23. StriAB) fragment to test the thermodynamic basis of the affinity of 2A10 FAB towards CSP. Experiments were also performed on the 2A10 FAB fragment with the synthetic peptide antigen (NANP)6, which is a short segment of the antigenic NANP-repeat region of CSP (KD) were found to be -49.0 kJ/mol and 2.7 nM for the full 2A10 antibody with CSP, -40 kJ/mol and 94 nM for the 2A10 FAB with CSP, and -36.4 kJ/mol and 420 nM for the 2A10 FAB with the (NANP)6 peptide.We began our analysis by performing isothermal titration calorimetry (ITC) to understand the interaction between 2A10 and CSP. For ease of expression we used a recombinant CSP (rCSP) construct described previously which was slightly truncated with 27 repeats compared to 38 in the 3D7 reference strain ,24. ITC n of CSP ; Fig 1. AB domain:antigen binding stoichiometry (6 peptide was bound to by ~2 FAB fragments (2.8 repeats per FAB domain). With the rCSP protein we observed that ~11 FAB fragments could bind to each rCSP molecule, , we observe binding of 5.8 antibodies per rCSP molecule (4.7 repeats per antibody). Therefore all complexes exhibit approximately the same binding stoichiometry of two FAB fragments/domains per ~5 repeat units. These results suggest that the antigenic region of CSP constitutes a multivalent antigen and that repeating, essentially identical, epitopes must be available for the binding of multiple FAB domains.Surprisingly, we did not observe a typical 1:1 antibody/Fhiometry . We founAB domains. The FAB:rCSP complex and the 2A10:rCSP complex had similar enthalpy and entropy of binding (vs. 94 nM for FAB:rCSP). The observation that this antibody-antigen (Ab-Ag) interaction is primarily enthalpically driven is consistent with the general mechanism of Ab-Ag interactions [Kd) of a single FAB domain to the (NANP)6 peptide is substantially higher (420 nM), and that the avidity, the accumulated strength of the multiple binding events between the FAB domains of the antibody and the CSP repeat, is the basis for the lower Kd value observed in the 2A10:rCSP complex. Thus, the characteristic repeating pattern of the epitope on the CSP antigen allows multiple weak interactions with 2A10 FAB domains to accumulate, which yields a complex with a high avidity dissociation constant in the low nM range.It is not possible to separate affinity from avidity in this system, although it is apparent that there is a substantial benefit to the overall strength of binding between the antibody and antigen through the binding of multiple F binding , but theractions . It is c6 peptide. These results were inconsistent with a disordered random coil structure lgorithm . The onllgorithm , and whilgorithm . Thus, tAB fragment in two conditions -repeat region, we solved the crystal structure of the 2A10 Fnditions , yieldinnditions , except  binding . Indeed, binding .AB and the (NANP)6 peptide were unsuccessful; unlike binary Ab-Ag interactions, in which the Ab will bind to a single epitope on an antigen and produce a population of structurally homogeneous complexes that can be crystallized, in this interaction we are dealing with an intrinsically-disordered peptide, the presence of multiple binding sites on the peptide, and the possibility that more than one 2A10 FAB domain can bind the peptide. Therefore it is difficult to obtain a homogeneous population of complexes, which is a prerequisite for crystallization. Attempts to soak the (NANP)6 peptide into the high-solvent form of 2A10 FAB, in which there were no crystal packing interactions with the binding-loops, caused the crystals to dissolve, again suggesting that the heterogeneity of the peptide and the presence of multiple epitopes produces disorder that is incompatible with crystal formation.Attempts to obtain a crystal structure of a complex between 2A10 F6 peptide complex, the accurate structures of the 2A10 FAB fragment, the (NANP)6 peptide, and the knowledge that antibodies seldom undergo significant conformational changes upon antigen binding [AB fragment (AB:(NANP)6 peptide interaction was plausible, we performed site directed mutagenesis of residues predicted to be important for binding. Our model predicted that the interaction with (NANP)6 would be mainly between CDR2 and CDR3 of the light chain and CDR2 and CDR3 of the heavy chain  binding , allowed binding . Using t binding , we obtafragment . We thenfragment , S3 Fig.vy chain .via its hydroxyl group, to a number of backbone and side-chain groups of the peptide. Loss of this side-chain abolished binding. Y56 also forms part of the same proline-binding pocket as Y38, and loss of this side-chain also resulted in an almost complete loss of binding. R109 forms a hydrogen bond to an asparagine residue on the side of the helix; mutation of this residue to alanine results in a partial loss of binding. Y116 is located at the center of the second proline-binding pocket; since loss of the entire side-chain through an alanine mutation would lead to general structural disruption of the FAB fragment, we mutated this to a phenylalanine (removing the hydroxyl group), which led to a significant reduction in binding. Finally, S36A was selected as a control: the model indicated that it was outside the binding site, and the ELISA data indicated that had no effect on (NANP)n binding.In the light chain , Y38 is Within the heavy chain , mutatioAB fragment to the (NANP)6 peptide is centered on two proline residues from two non-adjacent NANP-repeats 6 repeat, the binding epitope of the peptide is 2.5–3 alternate NANP repeats, with a symmetrical epitope available for binding on the opposite face (AB fragments per (NANP)6 peptide. To investigate whether this binding mode was also compatible with the indication from ITC that ~10.7 2A10 FAB fragments, or six antibodies (containing 12 FAB domains) could bind the CSP protein  [9 repeats with streptavidin conjugated phycoerythrin (PE) or allophycocyanin (APC). To validate our tetramer approach, mice were immunized with either P. berghei CSPf or another line of P. berghei with a mutant CSP (P. berghei CS5M) that contains the endogenous (P. berghei) repeat region, which has a distinct repeat sequence (PPPPNPND)n. (NANP)n-specific cells were identified with two tetramer probes bound to different conjugates to exclude B cells that are specific for the PE or APC components of the tetramers which are numerous in mice [P. berghei CSPf sporozoites developed large tetramer double positive populations, which had class switched (n-specific cells (+ CD38- indicating that they are GC B cells in agreement with results from a recent publication [We next set out to determine the implications of our structure for the B cell response to CSP. Because the CSP protein could conceivably cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T-independent. As a tool to test this hypothesis we used (NANP)ei CSPf) . The tet in mice . We founswitched . In contic cells . Furtherlication   and measured serum (NANP)n specific antibody by ELISA and the B cell response using our Tetramers. CD28-/- mice have CD4+ T cells but they are unable to provide help to B cell responses [n responses in the CD28-/- mice and control animals n specific B cell responses using our tetramers, in particular examining the number and phenotype (plasmablast vs GC B cell) of activated IgD- Tetramer+ cells esponses . Interes animals , indicatr+ cells . In agremablasts . However-Tetramer+ responses 4 days post immunization in control and CD28-/- mice n by sequencing the BCRs of the identified cells. While the repeat structure of CSP has been hypothesized to induce a broad polyclonal response based on data that the CSP repeat can absorb most of the sporozoite binding activity of human sera from immune individuals [n-specific cells 35 days post immunization of BALB/C mice with sporozoites. We performed this analysis in BALB/C mice as this is the background of mice from which the 2A10 antibody was derived. We then prepared cDNA from the cells and amplified the heavy and kappa chain sequences using degenerate primers as described previously [n specific cells and the bulk B cells from naïve mice we calculated the Shannon entropy for these populations. This analysis formally demonstrated that the diversity of the antigen specific B cells was significantly lower than the diversity of the repertoire in naïve mice (Our ability to identify and sort (NANP)ividuals ,38, an aeviously ,40. Heavïve mice . We furtïve mice . Similarïve mice . The V rïve mice , for exaïve mice  Tables tn specific cells in immune mice. This analysis showed that while, as expected, sequences from naïve mice contained few mutations, the sequences from immune mice had much higher levels of SHM. Importantly mutations were found to be concentrated in the CDR loops, and were frequently shared by immunized mice providing strong circumstantial evidence for affinity maturation  that would be indicative of B cells specific for CSP entering the GC. Taking advantage of the fact that our kappa chain primers capture the entire V-J sequences of the antibodies we sequenced we asked: 1) if the kappa chains shared between immune animals differed from the germline (providing evidence of SHM) and 2) if the mutations were conserved between different mice indicative of directed selection. Analysis of the reads from the kappa chains of the three immune mice showed that these had a much higher degree of mutation than bulk B cells from naïve mice, demonstrating SHM in the CSP-specific antibodies . We furtTo directly test if CSP-binding antibodies undergo affinity maturation we expressed the predicted germline precursor to the 2A10 antibody (2A10 gAb) in HEK293T cells. We identified the predicted germline precursors of the 2A10 heavy and light chains using the program V-quest  S5 and  Figs. ThTo identify the specific mutations that were important we introduced the mutations individually into the gAb light chain construct. We prioritized mutations that were shared with the 27E antibody which has previously been found to be clonally related to 2A10 having been isolated from the same mouse and which shares the same germline heavy and light chains as the 2A10 mAb . We founPlasmodium falciparum sporozoite-neutralizing antibody (2A10). Having obtained this structure we further modeled the binding 2A10 with its antigen target, the repeat region of CSP, and provide a thermodynamic characterization of this interaction. Finally, we used novel tetramer probes to identify and sort antigen specific B cells responding to sporozoite immunization in order to measure the diversity and maturation of the antibody response. We found that the avidity of 2A10 for the rCSP molecule was in the nanomolar range, which was much higher than the affinity previously predicted from competition ELISAs with small peptides [Here we provide an analysis of the structure of a peptides ,23. ThisAB fragments we too obtained a dissociation constant in the micro-molar range (0.42 μM). The difference between the FAB binding to the peptide and the tight interaction of the antibody binding to full length CSP appears to be driven by a high avidity, multivalent interaction. There is also additional enthalpic stabilization (per FAB domain) in the 2A10:CSP complex, although this is partially offset by the increased entropic cost associated with combining a large number of separate molecules into a single complex. One caveat of these data is that we used a slightly truncated repeat in our recombinant CSP, however it is likely that longer repeats will have further stabilization of the interaction that could result in even higher affinity interaction between CSP and binding antibodies.Using ITC we determined the dissociation constant of 2A10 for rCSP to be 2.7 nM, which is not unusual for a mouse mAb. However it is a tighter interaction than that predicted from competition ELISAs, which predicted a micro-molar affinity ,23. HoweAB fragments alone are sufficient to block invasion [The mechanism of sporozoite neutralization remains unclear, however our structural data may provide some insights. Repeat specific antibodies can directly neutralize sporozoites (without complement or other cell mediators) in the circumsporozoite reaction ,42. Moreinvasion . Howeverinvasion ,45. It hinvasion . Cleavaginvasion . AntibodOur results uncovering how neutralizing antibodies bind to CSP has several implications for understanding the development of the immune response to CSP. Notably the finding that the CSP molecule can be bound by multiple antibodies/B cell receptors raises the possibility that this molecule can indeed crosslink multiple BCRs and potentially act as a type-II T independent antigen . We findn repeat contradicts previous suggestions that the response to CSP might be broad and polyclonal [n repeat shares structural similarity with a self-antigen as is speculated to happen with meningococcus type B antigens [The finding of a limited repertoire in the BCR sequences specific for the (NANP)lyclonal . One explyclonal . The replyclonal . Anotherantigens , howeverOne area for future investigation is to determine the binding modes and sporozoite neutralizing capacities of other antibodies in the response. It is clear that not all CSP-repeat binding antibodies have the same capacity for sporozoite neutralization . As suchWhile our work has been performed with mouse antibodies, there are major similarities between mouse and human antibody loop structure . The maiOverall our data provide important insights into how the antibody response to CSP develops. Our results also help explain why relatively large amounts of antibodies are required for sporozoite neutralization and suggest that the ability to generate an effective B cell response may be limited by the very simplicity of the repeat epitope. These data support previous suggestions that CSP may be a suboptimal target for vaccination. However, we also find that CSP binding antibodies can undergo somatic hypermutation and reach high affinities. This suggests if we can develop vaccination strategies to diversify the repertoire of responding B cells and favor the GC response it may be possible to generate long-term protective immunity targeting this major vaccine candidate antigen.All animal procedures were approved by the Animal Experimentation Ethics Committee of the Australian National University (Protocol numbers: A2013/12 and A2016/17). All research involving animals was conducted in accordance with the National Health and Medical Research Council's (NHMRC) Australian Code for the Care and Use of Animals for Scientific Purposes and the Australian Capital Territory Animal Welfare Act 1992.-/- [4P. berghei CS5M sporozoites expressing mCherry [4P. berghei CSPf sporozoites dissected by hand from the salivary glands of Anopheles stephensi mosquitoes. Mice were either infected with live sporozoites and then treated with 0.6mg choloroquine IP daily for 10 days or immunized with irradiated sporozoites (15kRad). For immunization with rCSP, 30ug rCSP was emulsified in Imject Alum according to the manufacturer’s instructions (ThermoFisher Scientific) and delivered intra-peritoneally. All mice received only a single immunization in these experiments. To deplete CD4+ T cells mice were treated with two doses of 100ug GK1.5 antibody on the 2 days prior to immunization (BioXCell); control mice received an irrelevant isotype control antibody .BALB/C, C57BL/6 or CD28-/-  mice (br mCherry  or 5 x 19 tetramers conjugated to PE or APC. Antibodies were purchased from Biolegend while tetramers were prepared in house by mixing biotinylated (NANP)9 peptide with streptavidin conjugated PE or APC (Invitrogen) in a 4:1 molar ratio. Flow-cytometric data was collected on a BD Fortessa flow cytometer (Becton Dickinson) and analyzed using FlowJo software (FlowJo). Where necessary cells were sorted on a BD FACs Aria I or II machine.Single cell preparations of lymphocytes were isolated from the spleen of immunized mice and were stained for flow cytometry or sorting by standard procedures. Cells were stained with lineage markers  antibodies to B220 (clone RA3-6B2), IgM (clone II/41), IgD (clone 11-26c2a), GL7 (clone GL7), CD38 (clone 90), CD138 (clone 281–2) and (NANP)n tetramers and antibodies to B cell markers as described in the supplementary experimental procedures. Antigen specific cells were sorted on a FACS ARIA I or II instrument prior to RNA extraction with the Arturus Picopure RNA isolation kit (Invitrogen) and cDNA preparation using the iScript cDNA synthesis kit (BioRad). BCR sequences were amplified using previously described heavy and kappa chain primers including adaptor sequences allowing subsequent indexing using the Nextera indexing kit (Illumina). Analysis was performed in house using R-scripts and the program MiXCR as described in supplementary experimental procedures.Single cell suspensions from the spleens of immunized mice were stained with (NANP)Variants of the 2A10 antibody were expressed in HEK293 T cells  as described in the supplemental experimental procedures. Binding to the CSP repeat was tested by ELISA and ITC using standard techniques as described in the supplementary experimental procedures.p-value was < 0.05 for a tested difference. .Statistical analysis was performed using Prism6 (GraphPad) for simple T tests and one-way ANOVAs from single experiments. Where data were pooled from multiple experiments, analysis was performed using linear mixed models in R version 3.3.3 . Linear mixed models are a regression analysis model containing both fixed and random effects: fixed effects being the variable/treatment under examination, whilst random effects are unintended factors that may influence the variable being measured. If significance was found from running a linear mixed model, pair-wise comparisons of the least significant differences of means (LSD) was undertaken to determine at which level interactions were occurring. Statistical significance was assumed if the Sequence data generated in this paper is deposited at the NCBI sequence read archive (SRA) with accession number SRP092808 as part of BioProject database accession number PRJNA352758. Atomic coordinates and related experimental data for structural analyses are deposited in the Protein Data Bank (PDB) with PDB codes 5SZF and 5T0Y.S1 FigThe computational prediction of the spectra (A) was performed using DichroCalc , the exp(TIF)Click here for additional data file.S2 FigConformations were clustered by concatenating the trajectory and performing a Jarvis-Patrick analysis. The clusters are sorted by their RMSD from the first cluster (starting geometry). As shown, Run 2 is stable in the starting geometry for several ns, while Run 3 diverged, then reconverged to the starting geometry, where it was stable for several ns. These data suggest the quasi-helical structure observed from the ab initio calculations is stable, and can be spontaneously sampled, on a timescale of several ns.(TIF)Click here for additional data file.S3 Fig6:FAB complex. Root mean square deviation (RMSD) of the (NANP)6:FAB complex as a function of time. Independent simulations are shown in green, black and red.Molecular dynamics simulation of the (NANP)(TIF)Click here for additional data file.S4 FigP. berghei CSPf RAS, live P. berghei CSPf under CQ cover or rCSP. (A) 4 days later the IgM and IgG response to the (NANP)n repeat was analyzed by ELISA (B) At the same time the number of IgD- Tetramer+ B cells was quantified in the spleen. Data are from a single experiment, analyzed using linear models with immunization/treatment as the experimental factor.Mice either treated with an anti-CD4 depleting antibody or an isotype control were immunizaed with either (TIF)Click here for additional data file.S5 FigResidues that are mutated away from the predicted germline sequence in more one or more other antibody heavy chain (2E7 or 3D6) are highlighted in red, mutations that are predicted to be involved in binding to CSP are highlighted in blue.(TIF)Click here for additional data file.S6 FigResidues that are mutated away from the predicted germline sequence in both 2A10 and the related 2E7 antibody are highlighted in red, mutations that are predicted to be involved in binding to CSP are highlighted in blue.(TIF)Click here for additional data file.S1 Movie6 run 3. The trajectory was fitted to minimize alpha-carbon RMSD and then passed through a low-pass filter with a filter length of 8 frames to reduce temporal aliasing.Excerpt from (NANP)(MP4)Click here for additional data file.S2 Movie6 run 3. The trajectory was fitted to minimize alpha-carbon RMSD and then passed through a low-pass filter with a filter length 8 frames to reduce temporal aliasing.Excerpt from 2A10:(NANP)(MP4)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.


Asthma is the first cause of children hospitalization and need for emergency and impose high economic burden on the families and governments. We aimed to investigate the economic burden of pediatric asthma and its contribution to family health budget in Iran.Overall, 283 pediatric asthmatic patients, who referred to two tertiary pediatric referral centers in Tehran capital of Iran, included from 2010–2012. Direct and indirect asthma-related costs were recorded during one-year period. Data were statistically analyzed for finding association between the costs and factors that affect this cost .P=0.011), and 7–11 yr old age group (P=0.018). In addition, we found significant association between total asthma costs and asthma control status (P=0.011).Ninety-two (32.5%) females and 191(67.5%) males with the age range of 1–16 yr old were included. We found the annual total pediatrics asthma related costs were 367.97±23.06 USD. The highest cost belonged to the medications (69%) and the lowest one to the emergency (2%). We noticed a significant increasing in boys’ total costs (The presence of an asthmatic child can consume nearly half of the health budget of a family. Our results emphasis on improving asthma management programs, which leads to successful control status of the disease and reduction in economic burden of pediatric asthma. Asthma is a chronic obstructive respiratory disease in all age groups and for both sexes. It happens after an allergen stimulation that leads to severe symptomatic attacks of bronchial constriction and recurrent episodes of coughing, chest tightness, wheezing and breathlessness .There is sharp rise in the prevalence of asthma in many parts of the world with adopted western lifestyle . OverallThe world health organization in a study on global burden of diseases estimated 13.8 million disability-adjusted life years (DALYs) per year because of asthma that encompasses 1.8% of total burden of all diseases . Asthma Investigation of burden of diseases published by WHO revealed a strong effect of economic aspect of asthma on quality of life of patients and their families . SeveralAsthma is responsible for death of many children in developed countries . A sharpThe global prevalence of asthma is different by reason of the lack of internationally accepted definition for asthma . AccordiEconomic aspect of asthma is important for both asthmatic patients and health administrators of countries for precise allocating the health budget. In studies on asthma economic burden, attention should be paid for both direct  and indirect costs (i.e. loss of productivity and premature death) , 17.In our previous study  on the eOverall, 283 asthmatic children entered this prospective study. The patients referred to Children Medical Center and Mofid Hospital, Tehran, Iran as the most important tertiary referral centers of pediatrics in Iran. The inclusion criteria for patients was the age ≤16 yr, diagnosis of asthma by pediatric allergist and immunologist based on GINA 2009 guideline , insurance type), the disease history ) and asthma control status . The costs of asthma recorded regularly during one-year follow-up period in second part of questionnaire. The recorded direct costs consisted of doctor visit, asthma medication, radiology, laboratory, skin prick test, spirometry, emergency, hospital admission. The only indirect assessed cost of asthma was transportation. The entire recorded price in questionnaire was as Rial for convenience. All the expenditure were converted to US Dollar based on the exchange data of the Iran Central Bank .t-test and one way ANOVA and post hoc test, Tukey were used to compare means of expenditures. The P-values less than 0.05 considered statistically significant.The data of this study were analyzed statistically by SPSS software version 19 . We used standard statistical descriptions such as mean, minimum, maximum, and standard error for each cost. Association of asthma economic costs according to age, sex, asthma control status, comorbidities and smoking status were investigated. Independent samples Among included asthmatic pediatric patients, 283 patients had successful one-year period follow-up; consisting of 92 (32.5%) females and 191 (67.5%) males, with the age range of 1–16 yr old (mean±SE=6.91±0.18). Only 33(11.7%) patients did not have any health insurance whereas 250 (88.3%) had health insurance. Assessment of parental education level showed that 118 (41.7%) of fathers had a university degree and 124 (43.8%) had high school diploma and others did not complete the high school. Whereas 132 (46.6%) mothers were graduated from university, 109 (38.5) mothers had high school diploma and the rest of mothers were educated less than high school. Patients had 1 to 34 d absent from school due to asthma problems during one year (mean±SE=1.7±0.21). Patients during one-year period had 1–18 times doctor visit (mean±SE= 4.44±0.18), they needed to emergency bed 0–3 times (mean±SE= 0.07±0.02), hospital bed day 0–11 times (mean±SE= 0.17±0.06). Patients performed X-ray radiography, skin prick test, laboratory test and spirometry, 0.31±0.03, 0.25±0.49, 0.32±0.03, 0.41±0.04 times respectively during this period.We estimated total pediatrics asthma related costs for one-year period as 367.97±23.06 USD. The details of expenditures demonstrated in .P=0.011). In addition, we found significant association between total asthma cost in different age groups (P=0.008), Tukey HSD revealed that 7–11 yr old asthmatic children have higher asthma related costs than <7 yr old group (P=0.018).P=0.14). Almost half of patients, 150(53.0%) had not any comorbid diseases of asthma but 55(19.4%) had sinusitis, 25(8.8%) nasal allergy, 24(8.5%) GERD and the rest of patients had other concomitant disease or combination of comorbidities, statistical analysis did not show any significant difference between total cost of these categories (P=0.16). Asthma control assessment showed that 22(11.0%) of patients had un-controlled asthma, 73(33.3%) had partly controlled and 122(55.7%) had well-controlled asthma. Comparing the means of different control groups showed significant association between control status of asthma and total asthma related costs (P=0.011), and Post Hoc test showed that this association is related to significant differences between partly-controlled and un-controlled groups (P=0.047).Sixty (20.2%) of asthmatic children had smoker parents and 223(78.8%) had non-smoker parents , which was the highest asthma related cost. The drug cost was the highest one for adult asthmatic patients  . MoreoveP=0.011).However, medication is the greatest part of asthma treatment regimens, but our results show that medication costs are a bigger proportion (69%) of asthma total costs, which reveals increasing usage of drugs in the therapeutic regimens in Iran. Increased usage of anti-asthmatic drugs maybe relate to poor control situation of asthma in our patients, therefore we investigate the association between total cost of asthma and control situation. We found a significant relationship between total cost of asthma and control situation of asthma , P=0.011Multiple comparisons indicate significant difference between total costs of partly controlled patients (388.69±38.39 USD) and well-controlled patients (329.53±38.79 USD). Our finding is in accordance with other studies that show higher economic burden for patients with severe asthma . In addiP=0.16). According to the relation of the literacy and economic status of families as well as access to medical facilities, we investigated the parents’ education level and economic burden of pediatric asthma but our data did not support any significant relation.Several concomitant diseases are frequently presented with asthma disease especially in cases with poor responses to treatment or severe asthma . ConcomiP=0.14).Although prenatally or after birth exposure to cigarette smoke is connected to increased risk of asthma-like symptoms , the resP=0.011). Before age of 14, male sex is a risk factor for asthma (P=0.08); Tukey test showed that this difference is related to significant differences of total costs between patients less than 7 yr old and those with 7–11 yr old. Non-specific and variable clinical symptoms of asthma in young children encounter the asthma management with some problems including difficulties in diagnosis and drug efficacy  have been completely observed by the authors.


Soil N2O emissions from simulated cattle urine patches were quantified with closed static chambers and gas chromatography. At the regional level, rainy season cumulative N2O emissions (3.31 versus 1.91 kg N2O-N ha−1) and emission factors (0.42 versus 0.18%) were higher for low vegetative cover compared to adequate vegetative cover pastures. Findings indicate that under rainy season conditions, adequate vegetative cover through proper pasture management could help reduce urine-induced N2O emissions from grazed pastures.A decline in pasture productivity is often associated with a reduction in vegetative cover. We hypothesize that nitrogen (N) in urine deposited by grazing cattle on degraded pastures, with low vegetative cover, is highly susceptible to losses. Here, we quantified the magnitude of urine-based nitrous oxide (N Expanding livestock production is driven by a rapid increase in demand for cattle meat2. This increased demand for animal products together with the development of improved forage options to sustain higher levels of cattle productivity increases pressure on grasslands, the dominant cattle production systems of LAC, resulting in overgrazing and degradation of pastures3. According to Kwon3, an estimated 157 million ha  of the grazing area in LAC is degraded. In Brazil half of the 80 million ha of introduced tropical pastures are estimated to be in some state of degradation as they have, among other symptoms, low soil cover4.The livestock sector accounts for 46% of the agricultural gross domestic product of the Latin America and the Caribbean (LAC) region and grows at 3.7% annually2O) emissions, a powerful greenhouse gas (GHG)5. About 75–95% of cattle ingested N is excreted in either urine or dung, which provides N-rich substrate for nitrification and denitrification7. Cattle urine patches can contain very high amounts of soluble N , more than 2–3 times of the N uptake capacity of pastures8. Annually, about 1.5 Tg of total global anthropogenic N2O emissions (6.7 Tg N2O-N yr−1) are emitted from excreta produced by grazing cattle10 through both direct and indirect (from leached and volatilized excreta nitrogen) emissions. About 2% (0.7–6% uncertainty)11 of the nitrogen (N) in deposited urine is lost as N2O. Lower emission factors (EFs) (<0.7%), reported in other studies have been attributed to differences in climatic conditions, texture, soil moisture, and the N concentration in animal excreta12.Cattle excreta deposited on grazed pastures is estimated to represent 16% of global anthropogenic nitrous oxide 17.Pasture degradation may stimulate or constrain N losses. For example low vegetative cover, may reduce N sinks for deposited excreta and thus increase the vulnerability of N to loss through soil microbial processes and leaching. However, the low vegetative cover may also be associated with fewer plant root exudates and thus suppress microbial activity and N2O emissions from urine deposition can influence emission through multiple, often interacting, mechanisms and thus has produced contradictory results in the literature. Previous studies suggest that variations in soil N2O emissions from deposited urine patches in grazed pastures are driven by differences in several factors including ambient temperature18, urine volume and urine-N content19, soil drainage21, and soil moisture23. No previous studies have systematically explored the variation in urine-based soil N2O emissions associated with low vegetative cover in pastures.Clearly, the effect of pasture degradation on N2O emissions from cattle urine deposited on grazed pastures with adequate vegetative cover are less intense than those from pastures with lower vegetative cover by measuring soil N2O fluxes from urine patches deposited on different pastures located at seven contrasting sites, spread across five countries in the LAC region during rainy season.Here we tested the hypothesis that N−3 and was generally similar between the low and adequate vegetative cover pastures at each study location. The largest differences in bulk density, soil organic carbon and soil organic nitrogen between low and adequate vegetative cover pastures at the Estelí location in Nicaragua , Estelí (Nicaragua) and Taluma (Colombia) Table . Soil pHua Table .Table 1S2O monitoring period, with the exception of the Trinidad and Tobago site which received 31 days of rainfall  were higher in the AVC (59 ± 11 mg N2O-N m−2 day−1) compared to the LVC (45 ± 18 mg N2O-N m−2 day−1) pasture. The level of N2O emissions observed at Balcarce and Manfredi (Argentina) and Taluma (Colombia) sites were lower than 50 mg N2O–N m−2 day−1.Ntes Fig. ; Table 2−1  and AVC (0.48% of applied urine-N) pasture were at Estelí (Nicaragua) and Rio Grande do Sul (Brazil), respectively. On the other hand, the lowest N2O emission factors for LVC (0.02% of applied urine-N) and AVC pasture (0.01% of applied urine-N) were both observed at the Taluma site in Colombia  were observed at St. Augustine (Trinidad and Tobago) and Estelí (Nicaragua) and the lowest were observed at Taluma (Colombia)  in LVC (0.42 ± 0.19% SEM) than AVC (0.18 ± 0.08% SEM) pastures. Also at the regional level mean cumulative N2O emissions in the LVC (3.31 ± 1.09 kg N2O-N ha−1 SEM) were higher than those observed in the AVC (1.91 ± 0.78 kg N2O-N ha−1 SEM) pasture at the 10% level of significance (P = 0.08), based on results presented in Table 2O emissions at each individual site tended to be higher in LVC pastures than in the AVC pastures, with t-test detecting significant differences at the Nicaragua (Estelí) and Colombia (Patia) sites (Table 2O emission factors in the LVC (66%) pasture compared to the AVC (88%) pasture  compared to the AVC (0.0041) pastures suggests that urine deposited on LVC pastures is more vulnerable to high N2O losses when exposed to high rainfall. This may explain the significant differences between LVC and AVC pastures that were observed at Estelí (Nicaragua) and Patía (Colombia), where rainfall was high and the observed separation between the LVC and AVC pastures at the Rio Grande do Sul (Brazil) site where rainfall was also high.The key finding of our study is that, at the regional level of LAC, N2O emissions observed in LVC pastures compared to AVC pastures were likely due to lower plant N uptake from soil. Moreover, this may in part explain why AVC pastures generally resulted in lower net cumulative N2O emissions compared to degraded pastures. Despite the significant difference (P < 0.10) in net cumulative N2O emissions between LVC and AVC pastures, at a regional level, site-level comparisons showed that cumulative N2O emissions from LVC pastures were only significantly higher than those of AVC pastures in two sites, at Estelí (Nicaragua) and Patía (Colombia). The fact that soils within the LVC pastures at the Estelí site were more clayey than those under AVC pastures may have also contributed to the high net cumulative N2O emission in the former. Previous studies have reported higher N2O emissions from urine deposited on fine textured soils26. However, at Patía, where both the LVC and AVC pastures were on a clay soil observed differences suggest that despite the obvious influence of soil texture, pasture condition, based on vegetative cover is a driver of N2O emissions.High peaks of N2O observed at the two study sites in Argentina could have been due to the lower mean air temperatures. At the Balcarce location, mean air temperatures and the amount of N in applied urine were higher than at the Manfredi location. Low temperatures are known to reduce microbial activity and thus the rate of N transformation processes such as nitrification and denitrification in soil and, consequently, N2O production27. We assume that low temperatures may also be the cause of low net emissions and emission factors at the site in Brazil. It is important to note that the high soil pH (>7) at the Balcarce site, which would be expected to further increase with urine application may have also resulted in the inhibition of nitrification and high ammonia volatilization30. In addition, at the Balcarce site, the high urine-N levels may have resulted in microbial stresses with possible impacts on soil N transformation31 and thus contributed to the low N2O emissions and emission factors.Low emissions of N2O emission peak following urine application may be because the frequency of measurements at this site was insufficient to capture the expected N2O emission spike. Several other studies using manual static chambers have reported having missed the N2O peak, due to the low temporal resolution34. This problem can be resolved by increasing the frequency of monitoring using manual static chambers or switching to automated chambers. Alternatively, the N content in applied urine was also the lowest at Taluma, which implies that N2O fluxes could have been limited by N substrate availability. In addition, the forage grass (Brachiaria humidicola) that was used at Taluma had high nitrification inhibition capacity37, which could have also contributed to the observed low N2O emissions.At the Taluma location in Colombia, the absence of a N2O emissions, between LVC and AVC pastures at the St. Augustine (Trinidad and Tobago), Balcarce (Argentina), and Manfredi (Argentina) sites was possibly due to the fact that the spatial variation of vegetative cover of grass (soil cover) in the LVC (50–70%) pasture was high. As a result, local farmers based their classification on animal productivity differences which are influenced by both quantity (biomass) and quality (e.g. digestibility and crude protein content) of the forage on offer to animals. This further suggests that N2O emission differences between LVC pastures and AVC pastures are driven by differences in soil cover. With high plant density and greater plant vigour, we expect greater uptake of the urine-N by plants which could reduce the amount of N available for microbial transformations in soil such as nitrification and denitrification. It is therefore not surprising that when soil cover was high in the LVC pastures, there was no significant difference in N2O emissions with AVC pastures. This was however not the case for LVC pasture at the Patía (Colombia) site, which, though having similar soil cover (50–70%) showed significant differences between LVC and AVC pastures. This difference may be due to dissimilar vegetation types at the studied sites which would also affect N uptake and thus N availability for N2O emissions38.Absence of significant differences, in N11. During this short-term study, several of the emission factors were below the uncertainty range of the IPCC Tier 1 emission factor. While this may be due to the short gas monitoring period (1 month), several other studies conducted under temperate conditions42, reported a similar range of emission factors (0.02–1.63%) as we observed under warm temperate or sub-tropical conditions in Argentina and Brazil (0.02–0.7%). Similarly, the range of emission factors reported from this study under the tropical conditions in Colombia, Nicaragua and Trinidad and Tobago (0.01–1.2%) are in agreement with the range of values that have been reported from studies conducted under tropical conditions44.The IPCC Tier 1 emission factor for urine deposited on grazed forages is 2% with an uncertainty range of 0.7–6%2O emissions from cattle urine patches. When pasture degradation is associated with a reduction in vegetative cover, N2O emissions are expected to increase. Therefore, better regional understanding of the state of pasture degradation is vital for a robust understanding of N2O emissions from cattle urine deposits. More importantly, these findings suggest that improving soil cover/pasture condition through adoption of appropriate grazing and nutrient management practices may contribute towards mitigating excreta-based soil N2O emissions from grazed pastures during the rainy season. We expect findings from this regional study to contribute towards reducing uncertainties in future assessments on the importance of improving grassland management to achieve global commitments, such as the Bonn Challenge, 20 × 20 initiative45 and the Paris Agreement46.We conclude that in addition to the known effects of rainfall, temperature and the amount of urine-N, the pasture condition based on vegetative cover also influences NThe experimental plots were located at seven different sites in five countries in LAC spanning diverse climatic conditions and soil types  and adequate vegetation cover (>70%) pastures where soil cover is simply the proportion of soil covered by vertical projection of a plant canopy and vegetative biomass  or adequate vegetative cover (AVC). Pairs of LVC and AVC pastures were not always available at the very same location, but were always less than 1 km apart. We used a qualitative approach including expert knowledge, farmer perceptions and an arbitrary ranking system based on soil cover to define pasture conditions based on vegetative cover using criteria that combined those used by Hollman52 with five replicates per treatment  were demarcated within each pasture condition for making measurements of soil properties and N2O emissions. To simulate grazing, grass in each plot was cut to approximately 5 cm sward height, seven days prior to the beginning of the gas and soil sampling.On each pasture (low vegetative cover or adequate vegetative cover), experimental plots were organized following a systematic experimental design54, McLean55, and Vogel56, respectively. A total of 20 cylindrical PVC static chamber bases (10 per treatment) were inserted at the center of each subplot to a depth of 5 cm, five days prior to the start of gas and soil sampling. For each treatment, chamber bases with an internal diameter of 25 cm and a height of 10 cm were distributed in five replicate plots. At each site, cattle urine samples (about 7 L) that were collected from at least 10 local dairy cows were pooled and, immediately, following setting aside a subsample for N analysis, 500 ml of the collected urine was applied to soils to simulate a urination event on soil within each static chamber base at a rate of 1.27 L urine/m2. Nitrogen concentration in urine was characterized in each of the study countries using the direct distillation method described by Hoogendoorn et al.57. Unfortunately, we were unable to quantify urine-N in Trinidad and Tobago as there are currently no laboratories that quantify N in animal urine samples.Prior to starting the experiment, ten soil subsamples (0–10 cm) were separately collected from LVC and AVC pastures, using augers with 5 cm diameter, and combined to give one composite soil sample for each pasture condition. Collected soil was characterized for texture, pH, total carbon (C) and organic and inorganic N as described by Gee and Bauder58. Yet, Davidson et al.59 reported the possibility of artefacts with both vented and non-vented chambers, making it difficult to know which chamber yielded the ‘true’ flux. Since similar non-vented chambers were used at all sites, the chambers did not affect observed differences between AVC and LVC pastures. However, the calculated N2O emissions factors may differ from those measured in other published studies that used vented chambers.Gas measurements were conducted from a total of 20 non – vented PVC static chambers (10 cm height and 25 cm diameter) fitted with two rubber septa (one for gas sampling and another for inserting a thermometer). On each sampling day, PVC chambers were fitted to the chamber bases and sealed with an air-tight rubber belt. Syringes (15 ml) fitted with hypodermic needles were used to collect four gas samples from each of static chambers following chamber closure and at 15, 30 and 45 minutes after chamber closure. Collected gas samples were transferred to pre–evacuated 10 ml headspace glass vials fitted with rubber butyl septa crimp caps. At each site, at least eight gas samples were collected between the months of November to December in 2015 for the localities of Patía (Colombia) and Rio Grande do Sul (Brazil), Estelí (Nicaragua) and St. Augustine (Trinidad and Tobago). For Taluma (Colombia) and Balcarce (Argentina) measurements were conducted between February to March (2016) and at Manfredi, Argentina in the month of May (2016). Sampling months were chosen to coincide with the wet seasons at each of the study sites. The use of non-vented chambers has been reported to cause bias in the flux estimates2O concentration was analyzed by gas chromatography (GC-2014 Shimadzu), within a month upon arrival. The daily N2O fluxes were calculated by regressing N2O emissions from each chamber on each sampling date against time in order to calculate the hourly flux which was then multiplied by 24 to determine the daily flux. Each calculated flux was corrected for temperature and barometric pressure according to Ideal Gas Law. Subsequently, cumulative fluxes were calculated from daily N2O fluxes by interpolation between measurement days60.Gas sampling frequency was as follows: once before the application of urine, 1 hour after urine application, daily for the first three days following urine application, twice a week during the second and third week and once during last week of the experiment. Due to logistical challenges, less frequent measuring campaigns were done at the Taluma site in Colombia. Immediately after the gas sampling campaign, vials were sent to the Greenhouse Gas Laboratory at the International Center for Tropical Agriculture (CIAT) in Colombia, where N2O–N emission factor for urine patches was calculated according to Sordi et al.61:2O–Nemitted and N2O–NControl are the cumulative N2O emissions from urine or control patches over the 18 to 24 days monitoring period. Napplied represents the amount of N in the applied urine.The N62. Cumulative N2O fluxes and emission factors were, correspondingly, log and square-root transformed to achieve normality and obtain homogenous variances. To determine effects at the region level, cumulative N2O fluxes were analyzed using a split-plot ANOVA where the main plot was the pasture condition  and split-plot was the nitrogen levels (with and without urine application) and the blocking factor was the location and the main plot error term was pasture condition nested within location. In addition, at the regional level the emission factor variable was analyzed using a one-way ANOVA where the treatment effect was the pasture condition and the blocking factor was the location (site). At the individual sites we used the t-test analysis for testing differences in emission factors as influenced by pasture condition.Statistical analyses were conducted using the PROC MIXED procedure of SASSupplementary information


Excess fluid balance in acute kidney injury (AKI) may be harmful, and conversely, some patients may respond to fluid challenges. This study aimed to develop a prediction model that can be used to differentiate between volume-responsive (VR) and volume-unresponsive (VU) AKI.AKI patients with urine output < 0.5 ml/kg/h for the first 6 h after ICU admission and fluid intake > 5 l in the following 6 h in the US-based critical care database ) were considered. Patients who received diuretics and renal replacement on day 1 were excluded. Two predictive models, using either machine learning extreme gradient boosting (XGBoost) or logistic regression, were developed to predict urine output > 0.65 ml/kg/h during 18 h succeeding the initial 6 h for assessing oliguria. Established models were assessed by using out-of-sample validation. The whole sample was split into training and testing samples by the ratio of 3:1.Of the 6682 patients included in the analysis, 2456 (36.8%) patients were volume responsive with an increase in urine output after receiving > 5 l fluid. Urinary creatinine, blood urea nitrogen (BUN), age, and albumin were the important predictors of VR. The machine learning XGBoost model outperformed the traditional logistic regression model in differentiating between the VR and VU groups .The XGBoost model was able to differentiate between patients who would and would not respond to fluid intake in urine output better than a traditional logistic regression model. This result suggests that machine learning techniques have the potential to improve the development and validation of predictive modeling in critical care research. Acute kidney injury (AKI) is common in the intensive care unit (ICU), and there is evidence that even a small increase in serum creatinine may be associated with increased risk of mortality –3. AKI cIntravenous fluid challenges are often used in critical care to restore blood pressure to improve urine output in patients with hypotension and oliguria, respectively , 6. ReceIn animal models, fractional excretion of electrolytes was found to perform well in early differentiation between VR- and VU-AKI . HoweverA large US-based critical care database named Medical Information Mart for Intensive Care (MIMIC-III) was analyzed . The MIMPatient eligibility was considered when urine output was less than 0.5 ml/kg/h for the first 6 h after ICU admission. This definition was consistent with the urine output component of the KDIGO criteria . To examThe urine output within an 18-h period following the initial 6 h for defining oliguria was used as the outcome. Patients were considered as VR-AKI if he/she had urine output greater than 0.65 ml/kg/h, corresponding to a 30% increase as compared with the baseline value. Otherwise, they were defined as VU-AKI.Routinely collected clinical and laboratory variables obtained within the first 6 h of ICU admission were assessed for their ability to predict volume responsiveness. For some variables with multiple measurements, both the maximum and minimum values were assessed. Age, gender, ethnicity, admission type, elective surgery, type of ICU and presence of infection, and vital signs including respiratory rate, blood pressure, heart rate, and temperature were analyzed. In addition, laboratory data including glucose, white blood cell count (WBC), hematocrit, chloride, potassium, sodium, lactate, creatinine, blood urea nitrogen (BUN), coagulation profile, PaO2, PaCO2, and pH were included.Because this was a hypothesis-generating epidemiological study, no attempt was made to estimate the sample size of the study; instead, all eligible patients in the database were included to maximize the statistical power of the predictive model. Because missing data may create bias, variables with > 70% missing values were excluded from further analysis. Other variables with a lesser degree of missing values were analyzed using multiple imputation method .t test or rank-sum test as appropriate. Chi-square test or Fisher’s exact test was employed to compare the differences of the categorical variables [Clinical characteristics between VR-AKI and VU-AKI groups were compared using either Student ariables . A stepwariables .Extreme gradient boosting (XGBoost) combined with decision trees was employed to predict VR versus VU. A classification tree was used as the weak learner, and the learning objective function was binary logistic. The boosting method works by iteratively refitting a weak classifier (decision tree) to residuals of previous models. Each successive classifier focused more on misclassified observations during the previous round of fitting . In thisOf the 10,795 patients with urine output < 0.5 ml/kg/h for the first 6 h after ICU admission, 7491 patients (69.4%) received fluid intake > 5 l within the following 6 h. A number of 809 patients were excluded because they received diuretics and/or RRT on the first day. A total of 6682 patients were included in our analysis; 2456 patients had VR-AKI, and 4226 patients had VU-AKI on day 1 in ICU . The maximum serum creatinine concentration  was higher, and the minimum bicarbonate concentration  and maximum hematocrit  were lower in the VU group. The VR group had higher urinary pH , lower urinary creatinine , higher systolic blood pressure , higher albumin , lower rate of mechanical ventilation , vasopressor use , and infection  than the VU group : learning rate = 0.04, minimum loss reduction = 10, maximum tree depth = 9, subsample = 0.6, and number of trees = 300. With these hyperparameters, bootstrap validation (BV) training log-loss decreases as the number of trees in an ensemble increases, and the BV testing log-loss was less than 0.693 and only slightly more than BV training log-loss as the tree grows . The XGBoost had a significantly greater AU-ROC than the logistic regression model , the latter is also associated with increased risk of VU-AKI. The utility of urinary biochemistry to predict AKI outcome has been controversial in the literature. Although urinary biomarkers such as creatinine and fractional excretion of electrolytes were significantly different between VR and VU groups in some animal and human studies , there aThird, we found that patients with AKI and oliguria after elective surgery were more likely to respond to fluid challenges in univariate analysis. Patients who underwent elective surgery are generally in better clinical condition than patients requiring urgent surgery or with a medical emergency. Postoperative oliguria can be explained by hypovolemia due to intraoperative and postoperative insensible fluid loss. As such, they will be more likely to benefit from a larger amount of fluid after major surgery. However, the association of elective surgery with volume responsiveness disappeared after adjusting for other physiological variables, indicating that hypovolemia can be represented by these variables such as systolic blood pressure, heart rate, and hematocrit. We found that an increased hematocrit within the first 24 h of ICU admission was also an independent predictor of VR (in both the logistic and XGBoost models). This result could be explained by the fact that hematocrit has a direct relationship with the intravascular plasma volume and a higher hematocrit may suggest a relative hypovolemic state . ConversThis study has some strengths and weaknesses. The XGBoost modeling is a novel technique that has not been widely adopted in critical care research. The XGBoost algorithm has been successfully used in some complex scenarios such as the prediction of the failure of the treatment for parapneumonic empyema , in whicIn conclusion, this hypothesis-generating study showed that some clinical factors were more likely to be associated with VR-AKI than VU-AKI. The XGBoost modeling technique could identify the predictors of VR-AKI that were not apparent using logistic regression, resulting in a better-performing predictive model to identify patients with VR-AKI. Further epidemiological studies using advanced machine learning techniques to validate our results will help us to identify the most suitable patients to be included in clinical trials assessing the benefits of fluid therapy in AKI.


Cedrela montana and Handroanthus chrysanthus showed the biggest stem shrinkage of up to 2 mm after 10 consecutive dry days. A comparison of daily circumference changes over 600 consecutive days revealed different drought responses between the families concerning the percentage of days with stem shrinkage/increment, ranging from 27.5 to 72.5% for Graffenrieda emarginata to 45–55% for Podocarpus oleifolius under same climate conditions. Moreover, we found great difference of recovery times after longer-lasting  VdN drought events between the two evergreen broadleaved species Vismia cavanillesiana and Tapirira guianensis. While Vismia replenished to pre-VdN stem circumference after only 5 days, Tapirira needed 52 days on average to restore its circumference. Hence, a higher frequency of droughts might increase inter-species competition and species-specific mortality and might finally alter the species composition of the ecosystem.Under drought conditions, even tropical rainforests might turn from carbon sinks to sources, and tree species composition might be altered by increased mortality. We monitored stem diameter variations of 40 tree individuals with stem diameters above 10 cm belonging to eleven different tree genera and three tree life forms with high-resolution dendrometers from July 2007 to November 2010 and additionally March 2015 to December 2017 in a tropical mountain rainforest in South Ecuador, a biodiversity hotspot with more than 300 different tree species belonging to different functional types. Although our study area receives around 2200 mm of annual rainfall, dry spells occur regularly during so-called “Veranillo del Niño” (VdN) periods in October-November. In climate change scenarios, droughts are expected with higher frequency and intensity as today. We selected dry intervals with a minimum of four consecutive days to examine how different tree species respond to drought stress, raising the question if some species are better adapted to a possible higher frequency and increasing duration of dry periods. We analyzed the averaged species-specific stem shrinkage rates and recovery times during and after dry periods. The two deciduous broadleaved species  Global change, especially climate change, affects forests worldwide, with adverse effects on biodiversity and ecological services like carbon sequestration. Hence, understanding forest responses to climate variability is key to conservation and protection of forest ecosystems . EspeciaDisentangling the various factors leading to stem circumference changes, such as swelling and shrinkage of phloem, xylem and bark due to water potential and elastic properties of tissues e.g.,  and radiAnalyzing the impact of climatic extremes on tree circumference provides important insights into tree functioning and carbon budget. Hence, analyzing responses of stem girth to extreme meteorological events is an important variable in climate-growth-modeling and helps to improve existing models . Stem ciTapirira and Vismia and draw inferences about possible consequences for forest composition in view of expected climate change. All add up to the main research question, how different humid mountain rainforest tree species respond to drought stress and if some species are better adapted to a possible higher frequency and increasing duration of dry periods.In this paper we analyze one of the largest dendrometer datasets existing in the tropics. Our study is separated into three parts. After examining the general response of stem size variations to climate parameters, we first analyzed how the different tree species in a moist tropical mountain forest in South Ecuador respond to different durations of rare dry spells. Second, we analyzed species-specific differences in growth dynamics and drought response over a 600-day period. Third, we compared recovery from drought in two contrasting tree species Melastomataceae, Lauraceae, Rubiaceae, and Euphorbiaceae  almost exclusively occur during VdN events, except one drought event during 30.06.2010–06.07.2010 . In oral-1 for the type DR and 0.0054 μm °C-1 for the type DRL26, respectively . Since the maximum temperature differences between consecutive nights in our study area do not exceed 3°C, no temperature correction was needed for the values derived from band dendrometers. Due to the higher thermal sensor coefficient of 3.29 μm°C-1 of the point dendrometers, their maximum error is the range of up to 62 μm in circumference. An error in this range does not question our main results. Data recorded by dendrometers of DR-type have only been used in the first part of the study. In all cases, dendrometers were installed at breast height (approx. 1.30 m). In case of thick-barked species, parts of the outer bark were removed without injuring the cambial zone to minimize the influence of bark swelling and shrinking due to water uptake or loss. Since active growth by wood formation and stem swelling due to water uptake cannot be differentiated by dendrometer data we will use the term “increment” in the following for any kind of stem diameter increase. To investigate the impact of drought on different species we selected dry intervals with a minimum of four consecutive days without rainfall during the periods July 2007 to November 2010 and March 2015 to December 2017. We analyzed the averaged stem shrinkage rates during periods from 4 to 9 days. To calculate stem shrinkage, only the daily maximum stem diameter values . From March 2015 to December 2017, logging band dendrometers (LBDs) with a built-in thermometer  were used. For comparability of the two dendrometer types, we converted the data of the point dendrometers into circumference values using the formula: circumference = 2 × π × radius, assuming symmetrical stem geometries of the studied individuals. Due to latest calibration tests, the used dendrometer types show different thermal sensor coefficients of 3.29092 μm°Cr values  were comPersea, Nectandra, and Ocotea. Hereafter, we use the genus names for the groups of trees comprising more than one species and the species names, if the genus is represented by only one species. For Prumnopitys, our data regarding dry spells is limited to 6 days due to data logger problems.In total, 40 tree individuals with stem diameters above 10 cm belonging to eleven different tree genera and three tree life forms were studied . Stem diThe dendrometer data were scanned for outliers and measurement artifacts (exceeding 0.5 mm stem circumference change within 30 min). For stem shrinkage and increment calculation we took the maximum daily circumference/diameter values and subtracted the values from the subsequent day. The distinguished groups for the second part of the study  differedH. chrysanthus and C. montana, with average circumference losses of 1.876 and 2.190 mm after nine dry days, respectively  in which no missing dendrometer measurement values, which might compromise the results, occurred. The species In G. emarginata showed the highest percentage of days with stem increment (68.1%) and also the lowest percentage of days with shrinkage (28.5%). These values are followed by V.cavanillesiana (65/29.8%), T. guianensis (62.3/35.8%), Ocotea (58.3/38.7%) and P. oleifolius (55.5/40.3%). The percentages missing to 100% is stagnation of circumference  until the pre-drought stem diameter was regained, but already at the beginning of December the species showed a flattening of the stem increment curve (★).Both species showed a very consistent response pattern among different individuals . Their rG.emarginata appeared very well adapted, showing the smallest absolute stem diameter shrinkage but standardized, it turned more to average. P. oleifolius showed the second smallest stem diameter change in response to ongoing drought, very similar to Ocotea and Inga. V.cavanillesiana responded very alike, but the daily changes of the last three dry days indicated differences in response, probably due to change in drought-coping adaptations like different degrees of leaf-water potential regulation , root and sapwood area (increase), and cavitation resistance (increase), which should protect trees against future droughts . ConsideThe datasets generated for this study are available on request to the corresponding author.AB and VR developed the concept of the manuscript. VR and KP collected the data in the field. VR and SS contributed statistics and graphics. All authors contributed to the text writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The morbidity of knee arthritis is increasing among aged people and total knee arthroplasty has been its mainstream treatment to date. Postoperative rehabilitation is an important part of the procedure. However, the intense pain during the functional exercise involved has always been a challenge for both patients and health care professionals. The aim of this study is to test the analgesic effect of a mixture of nitrous oxide/oxygeb (1:1) inhalation for patients who are doing functional exercise 1 month after total knee arthroplasty.This double-blind, randomized, placebo-controlled study will be implemented in the Rehabilitation Department in the General Hospital of Ningxia Medical University. Patients aged between 50 and 75 years who underwent a primary unilateral total knee arthroplasty are eligible for inclusion. The key exclusion criteria include: epilepsy, pulmonary embolism, intestinal obstruction, aerothorax. The treatment group (A) will receive a pre-prepared nitrous oxide/oxygen mixture plus conventional treatment , and the control group (B) will receive oxygen plus conventional treatment . Patients, physicians, therapists, and data collectors are all blind to the experiment. Assessments will be taken immediately after functional exercise begins (T0), 5 min (T1) after functional exercise begins, and 5 min after functional exercise has finished (T2). Patients will be randomly allocated between a treatment group (A) and a control group (B) in a ratio of 1:1. Primary outcome, including pain severity in the procedure, will be taken for each group. Secondary outcomes include blood pressure, heart rate, oxygen saturation, side effects, knee joint range of motion, Knee Society Score (KSS), rescue analgesia need, and satisfaction from both therapists and patients.This study will focus on exploring a fast and efficient analgesic for patients who are doing functional exercise after total knee arthroplasty. Our previous studies suggested that the prefixed nitrous oxide/oxygen mixture was an efficacious analgesic for the management of burn-dressing pain and breakthrough cancer pain. The results of this study should provide a more in-depth insight into the effects of this analgesic method. If this treatment proves successful, it could be implemented widely for patients doing functional exercise in the rehabilitation department.ChiCTR-INR-17012891. Registered on 6 October 2017.The online version of this article (10.1186/s13063-019-3472-7) contains supplementary material, which is available to authorized users. Knee arthritis is a chronic joint disease that leads to considerable morbidity in terms of pain, difficulties in performing daily life activities, sleep disorder, functional limitations, mental illness, and decreased quality of life , 2. KneeRehabilitation after TKA generally begins immediately after surgery and, therefore, sufficient analgesia is important. An ideal analgesia for post-TKA rehabilitation will permit adequate knee flexion with minimal pain and without motor impairment, leading to successful mobilization . In ChinOrally administered analgesics are often the main treatment in the immediate- to short-term postoperative period. Despite the effectiveness of opioids, they often lead to some undesirable side effects such as vomiting, constipation, and respiratory depression . It is rPopulation: patients who undergo total knee replacement and are do functional exerciseIntervention: nitrous oxide/oxygen inhalationComparison: oxygen inhalationOutcome: pain severity, adverse events, satisfaction from both patients and therapists, Knee Society Score (KSS), the time for ROM ≥ 90° and rescue analgesiaStudy design: a randomized controlled trial (RCT)  approach:Potential participants for the study will be sourced from the General Hospital of Ningxia Medical University, which is the biggest general hospital in Ningxia. Recruitment sources will be focused on patients who undergo TKA and are doing functional exercise. A member of the research team will contact the principals, directors, case managers and other relevant staff for information about the study. Should they wish their organization to cooperate with the study, staff members will be asked to provide written information to individual members whom they deem to be an appropriate candidate for the study. If that member or client is interested in participating, based on the information provided, they will then voluntarily contact the research team. Alternatively, should the potential participant wish to be contacted directly by a member of the research team, they may choose to complete a “consent to contact” form. At this point, it is made clear that patients are only consenting to the research team contacting them to discuss the possibility of participating in the study as opposed to actually consenting to participate in the study itself. Those who agree to contact the research team will be invited to attend a screening interview to see whether they meet the inclusion criteria . The study will be conducted in the rehabilitation department of the General Hospital of Ningxia Medical University. Patients in the intervention group will receive conventional analgesic regimens  with the premixed nitrous oxide/oxygen mixture inhalation while patients in the control group will receive conventional analgesic regimens  and oxygen inhalation when doing functional exercise  [The study will be implemented in the General Hospital of NingXia Medical University, which is a specialized tertiary hospital. A written informed consent form will be collected from all the participants after a thorough explanation of the procedure by the therapists. The researchers state that voluntary participation and all their demographic data and survey answers will be kept confidential. Patients are eligible if they are undergoing primary unilateral TKA (see Table actures) . The patAccording to the computer-generated random number table in a 1:1 ratio, all the participants will be randomized to the intervention group  or the control group . The allocation sequence of each patient will be decided by a computer-generated schedule, which will be numbered by a statistician. The randomization schedule will be kept and sealed in an independent research room. Apart from the project manager, who is responsible for gas distribution, no other nurses or data collectors will have access to the data allocation. Prior to, and during, the treatment period, the participants, therapists, and the investigators will all be blind to the allocation. Only the project manager will know the allocation. The two gases will be stored in identical-looking canisters labeled A and B  . During The primary endpoint measures will be pain severity at T0, T1, and T2. The anticipated pain score  during functional exercise will be a reduction from to 3 to 5 in the intervention group. Repeated-measures analysis of variance will be used for data analysis.Secondary outcomes will include physiological parameters: blood pressure, heart rate, oxygen saturation at T0, T1, and T2, adverse effects, and satisfaction investigated by a 5-point satisfaction scale  from both patients and therapists, KSS, and the time for ROM ≥ 90°(ROM will be measured by a goniometer). Rescue analgesia should also be an indicator and recorded.Respiratory adverse effects, such as desaturation (pulse oximetric saturation≦ 94%), arterial hypotension or bradycardia—as well as hallucination and gastrointestinal adverse effects  or any other discomfort—will be evaluated and recorded during and after the procedure. If any of these happen, patients will be given oxygen inhalation, and the discomfort should vanish within 5 min.P value of 0.05 , these calculations show that 14 patients are needed (seven per group). We finally decided on a sample size of 140 to meet the Chinese Food and Drug Administration standard for feasibility and the safety of staffs in implementing this analgesic [Pain relief was defined as a 30% decrease in the pain level in T1-T0  . Based onalgesic , 28.A DSMB will be established shortly after the project launch and will meet several times during the data collection period. Members will included two pain management specialists; four professional therapists and nursing officers; and a senior academic statistician who served as the Board’s Chair.2 test or Fisher’s exact test for categorical variables and the Mann-Whitney U test for non-parametric data. Statistical significance will be defined as P < 0.05.Before the study, all data collectors will be given unified training. The data will be analyzed using SPSS Statistics for Windows, Version 22.0. Chicago, IL, USA. Descriptive statistics will be analyzed by medians (inter-quartile ranges), means (standard deviations), and proportions . The baseline parameters will be compared between the two groups  using the chiKnee arthritis is associated with recurrent sharp pain, gait disturbance, joint deformity, and functional degeneration. TKA is the main surgery to solve the problem, but pain along with the need for rehabilitation after TKA has always been a significant clinical challenge for physicians, especially therapists in the rehabilitation department. Endurance has long been strongly represented in the Chinese consciousness. Patients are often told to just get over it, but it's reported that for women patients it is pretty hard –31. PoorThe use of adjunct, non-opioid agents is integral for pain control following TKA. The premixed nitrous oxide/oxygen displays its superiority. Its effective use in controlling pain and its rare side effects accelerates its wide use in various areas such as venipuncture, lumbar puncture, bone marrow aspiration, and laceration repairs , 32, 33.The limitations of this study must be acknowledged. The study will be conducted in a remote area of China. As a result, our findings may not reflect the national average. Due to the limited resources, the research will be carried out in a hospital only. The results may not represent an ideal research situation and further multi-center studies will be necessary in the future.Additional file 1: Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) 2013 Checklist: recommended items to address in a clinical trial protocol and related documents. (DOC 126 kb)Additional file 2: Equipment. (TIF 379 kb)


The ability of the quinoxaline derivatives to induce apoptosis in A549 cells was assessed using the Acridine Orange/Ethidium Bromide (AO/EB) and Annexin V-FITC/Dead Cell Assay. Of the four quinoxaline derivatives tested, 3- prop-2-ynyl methanosulphate (LA-39B) and 3- prop-2-yn-1-ol (LA-55) displayed a dose-dependent reducing power, free-radical scavenging activity, inhibition of cell viability, and stimulation of ROS production which was accompanied by induction of apoptosis in A549 lung cancer cells. None of the quinoxaline derivatives induced cell death or ROS production in non-cancerous Raw 267.4 macrophage cells. Cytotoxicity was observed in A549 lung cancer, HeLa cervical cancer, and MCF-7 breast cancer cells albeit inhibition was more pronounced in A549 cells. The results of the study suggest that 3- prop-2-ynyl methanosulphate and 3- prop-2-yn-1-ol induce apoptotic cell death in A549 lung cancer cells.Despite major advancements in the development of various chemotherapeutic agents, treatment for lung cancer remains costly, ineffective, toxic to normal non-cancerous cells, and still hampered by a high level of remissions. A novel cohort of quinoxaline derivatives designed to possess a wide spectrum of biological activities was synthesized with promising targeted and selective anticancer drug activity. Hence, this study was aimed at determining in vitro anticancer activity effects of a newly synthesized class of 3- prop-2-ynyl quinoxaline derivatives on A549 lung cancer cells. An assessment of the quinoxaline derivatives ferric reducing power, free radical scavenging activity, cytotoxic activity, and ability to induce reactive oxygen species (ROS) production was performed using the Ferric Reducing Antioxidant Power (FRAP), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 3--2,5-diphenyltetrazolium bromide (MTT) and 2’,7’-dichlorodihydrofluorescein diacetate (H Lung cancer remains among the leading causes of cancer-related death in the world affecting both males and females . Non-smaQuinoxalines are referred to as a benzopyrazines because their structure consists of a benzene ring and a pyrazine ring . They caIn this study, we document the antioxidant properties of 3- prop-2-ynyl methanosulphate (LA-39B), and 3- prop-2-yn-1-ol (LA-55) quinoxaline derivatives and their ability to induce cell death in A549 lung cancer cells. 1 , PdCl2(PPh3)2 , and CuI  were dissolved in 15 mL dry THF followed by the addition of Et3N  and propargyl alcohol . The reaction mixture was stirred at 50 °C for 18 h under nitrogen atmosphere, thereafter partitioned between EtOAc/water in (20 mL 3:1). The layers were separated and the aqueous layer was washed with EtOAc (3 × 20 mL). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified by recrystallization from acetone and gave 3-prop-2-yn-1-ol as a brown powder , mp = 139.2 to 140.8 °C (Lit 140 to 141 °C) 2; δH  4.610 , 7.789 , 8.077  and 8.887 ; δC  51.39, 83.03, 91.90, 128.44, 129.16, 130.72, 132.01, 138.72, 141.06, 141.96, and 146.90; Vmax (FTIR) 758, 946, 1014, 1127, 1229, 1429, 1694, 2228, 2922, 3275 cm−1; m/z MS (ESI): MH+ 185.1.Quinoxaline derivative LA-55  was synt1 , PdCl2(PPh3)2 , and CuI  were dissolved in 25 mL dry THF followed by the addition of Et3N  and 2-methylbut-3-yn-2-ol . The reaction mixture was stirred at 50 °C for 18 h under nitrogen atmosphere, thereafter partitioned between EtOAc/water in (20 mL 3:1). The layers were separated and the aqueous layer was washed with EtOAc (3 × 20 mL). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified by recrystallization from acetone and gave 2-methyl-4-but-3-yn-2-ol as a brown powder , mp = 155.3 to 158.4 °C; δH  1.695 , 7.778 , 8.076  and 8.871 ; δC  31.07, 65.51, 79.84, 98.21, 129.14, 129.18, 130.56, 130.75, 139.01, 140.93, 141.96, and 147.09; Vmax (FTIR) 762, 960, 1050, 1229, 1301, 1488, 1538, 2230, 2982, 3290 cm−1; m/z HRMS (ESI): MH+ 213.1020.Quinoxaline derivative LA-65C3  was synt, were mixed in 50 mL round bottom flask containing 10 mL dichloromethane and stirred for 30 min at room temperature. Following this, aqueous 10% Na2S2O3.5H2O (10 mL) and aqueous saturated solution NaHCO3 (6 mL) were added into the reaction mixture and stirred for further 5 min. The layers were separated and the aqueous layer was extracted with dichloromethane (10 mL × 3). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified on prep TLC 3:7 ethyl acetate/n-hexane and gave 3-propionaldehyde as brown powder , mp = 122.2 to 124.8 °C; δH  7.873 , 8.139 , 9.021  and 9.532 ; δC  88.18, 89.25, 129.46, 129.68, 131.37, 132.16, 136.41, 141.85, 124.31, 147.09, and 175.89; Vmax (FTIR) 752, 955, 1024, 1090, 1122, 1295, 1367, 1485, 1662, 2200 cm−1; m/z HRMS (ES): MH+ 183.0552.Quinoxaline derivative LA-16A  was synt3N  in 10 mL dry THF, cooled to 0 °C, followed by drop-wise addition of MeSO2Cl . The reaction was maintained at 0 °C for 2.5 h under nitrogen atmosphere. The reaction was quenched by the addition of aqueous saturated solution of NaHCO3 (10 mL), the layers were separated and the aqueous layer was extracted with Et2O (10 mL × 3). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified on prep TLC using 3:7 ethyl acetate/n-hexane and gave 3- prop-2-ynyl methanesulfonate as a brown solid , mp = 93.8 to 96.7 °C; δH  3.193 , 5.159 , 7.795 , 8.067  and 8.885 ; δC  38.95, 57.12, 84.54, 86.12, 129.25, 131.00, 131.20, 137.56, 141.33, 141,97, and 146.60; Vmax (FTIR) 523, 649, 765, 801, 938, 1008, 1172, 1355, 1490, 2953 cm−1; m/z HRMS (ES): MH+ 263.0481.Quinoxaline derivative LA-39B  was syntThe FRAP assay was carried out on the four quinoxaline derivatives to evaluate their reducing power potential. The DPPH assay was carried out to evaluate the free-radical scavenging abilities of the quinoxaline derivatives. The ability of quinoxaline derivatives to induce cancer cell death was assessed using the MTT assay after challenging various cancer cell types with the four selected quinoxaline derivatives. The two quinoxaline derivatives, LA-39B and LA-55, with high free-radical scavenging activities were tested on Lipopolysaccharide (LPS)-activated Raw 264.7 mammalian macrophages. Reactive oxygen species (ROS) profiles are used as an indicative measure of oxidative stress levels in mammalian cells ,15,16,17Free-radical levels in cancer cells play a crucial role in DNA stability and the ability to proliferate ,15,16,17Induction of apoptosis was analyzed through staining of A549 cells with Acridine Orange and Ethidium Bromide wherein double-staining with the two dyes display an orange fluorescence indicative of apoptotic cell death whereas green nuclear fluorescence designates viable cells. Apoptosis profiles are displayed in a quadratic format showing various stages of apoptosis after treatment of A549 lung cancer cells with LA-39B and LA-55. Despite major advancements in the management of cancer through chemotherapy, the progressive pledge in research of discovering new treatment strategies with improved efficacy is of crucial importance ,15. The Antioxidant properties have been identified for their preventive action against cancer through a number of cellular processes which include apoptosis . In thisApoptosis is a programmed and regulated process in induction of cell death which is characterized by numerous cellular and molecular changes . An apopThe FRAP assay is principled on the ability of antioxidant-containing compound to react with potassium ferricyanide to form ferrocyanide. This will then react with ferric chloride to form ferrous complex that has absorption maximum at 700 nm. Antioxidant activity of quinoxalines was assessed following the ferric reducing power assay protocol as previously described . BrieflyThe principle of the assay is based on the ability of antioxidant containing compounds to scavenge the DPPH stable free radical by reducing the purple colored DPPH to yellow color. The assay was carried out as described previously . Briefly2 environment in cell culture flasks. Raw 264.7 cells and A549 cells were maintained in RPMI-1640 culture medium supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine and 1x penicillin, streptomycin, and neomycin mixture (PSN). Cervical cancer HeLa and breast cancer MCF-7 cells were maintained under the same conditions but maintained in Dulbecco’s modification of Eagle medium (DMEM) medium supplemented with 10% FBS, 2 mM L-glutamine, and 1× PSN. Cell density was determined by diluting cells with 10× trypan blue dye and counted using a Life technologies countess II FL automated cell counter .Raw 264.7 macrophage cells, lung cancer A549 cells, breast cancer cells MCF-7, and cervical cancer HeLa cells were maintained at 37 °C, in a humidified 95% air and 5% CO4 pre-well in a 96 well-plate and incubated overnight. Cell were then treated with various concentrations of quinoxaline derivatives , 0.5% DMSO in culture medium and 20 μg/mL Actinomycin-D  for 24 hrs. Prior to the addition of the MTT reagent, cell imaging was conducted. MTT of 5 mg/mL  was added and after 4 h of incubation the aqueous medium was replaced with 100 µL DMSO. The blue formazan crystals were allowed to dissolve in DMSO by incubating in the dark for 30 min. Absorbance was then measured at 570 nm using GloMax-Multi microplate reader . Cell viability was determined using MTT  assay which is principled on the ability of mitochondrial succinyl dehydrogenase in viable cells to convert soluble yellow tetrazolium salt into an insoluble formazan product. Briefly, cells  were seeded at a density of 6 × 102DCFDA dye to permeate the plasma membrane and fluoresce to 2’,7’-dichlorofluorescein (DCF) upon encounter with ROS after removal of the diacetate moiety by cellular esterases. Briefly, cells were seeded in 12 well plates at a density of 6 × 105 cells/mL for 24 h. Oxidative stress was stimulated in Raw 264.7 cells using Lipopolysaccharides (10 µg/mL) for 24 h and in A549 cells using hydrogen peroxide (1 mM) for 1 h before staining. Cells were then treated with quinoxaline derivatives at 25 µM and Actinomycin D 20 µg/mL for 24 h. Cells were detached, centrifuged, and re-suspended in culture medium with H2DCFDA for 30 min at 37 °C in the dark. Analysis was then carried out using the Muse™ Cell Analyzer.This fluorescence-based assay is principled on the ability of the H5 cells/mL on coverslips in 6 well-plates overnight. Cells were then treated for 24 h with quinoxaline derivatives at 25 µM and Actinomycin D at 20 µg/mL Plates were centrifuged at 5000 rpm for 5 min then washed with PBS. Cells were then fixed with 2.7% paraformaldehyde for 30 min and centrifuged for 5 min at 5000 rpm. Cells were stained with Acridine Orange 100 µg/mL for 5 min, centrifuged and washed then stained with Ethidium Bromide 100 µg/mL for 5 min and washed again after centrifuging. Coverslips were then mounted onto slides and viewed under the microscope using the Nikon Ti-E inverted microscope.The extent of apoptosis in cells was assayed using the Acridine Orange/Ethidium Bromide assay. This assay is based on staining cells with Acridine Orange (AO) which stains the DNA and RNA of live cells and Ethidium Bromide (EB) which stains exposed DNA and RNA of dead/necrotic cells . The com® Annexin V and Dead Cell Assay kit was used according to manufacturer’s instruction. Briefly, cells were seeded in 12 well-plates at a density of 6 × 105 cells/mL for 24 h. They were then treated with quinoxaline derivatives at 25 µM and Actinomycin D 20 µg/mL for 24 h. Cell were detached, centrifuged, and resuspended in 1% FBS. Muse™ Annexin V and Dead Cell Reagent was then added to the suspension and incubated for 20 min in the dark. Analysis was then carried out using the Muse™ Cell Analyzer.This assay is principled on Annexin V and PI stain which is based on the staining of cells undergoing early apoptosis with Annexin V which binds to phosphatidylserine which is being expressed on an impermeable membrane, while with late apoptosis, PI will be able to bind to the DNA due to permeability of the membrane. MuseIn this study, the four quinoxaline derivatives were designed to possess the signature benzopyrazine core molecule made of a benzene ring and a pyrazine ring capable of triggering antimicrobial activities and induce anticancer properties due to their ability to bind DNA ,32. FourTM 3 software with variants test (ANOVA), Tukey–Kramer to compare samples. Each value represents the mean ± SD of three experiments performed in triplicate wherein p-value represents the level of significant changes and p < 0.05 is considered statistically significant. Levels of significance are denoted using an associated asterix as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.All experiments were carried out in duplicate and repeated three times to confirm consistency and determine level of variation. Data was reported as mean ± SD and statistical analysis was performed using Graph Pad InstatThe results obtained demonstrate that quinoxaline derivatives, 3- prop-2-ynyl methaonosulphate (LA-39B) and 3- prop-2-yn-1-ol (LA-55), can potently induce significant anticancer properties against A549 lung cancer cells while displaying no cytotoxicity against the non-cancerous Raw 264.7 macrophage cells. These newly developed series of quinoxaline derivatives may present new alternatives with potential abilities for use in lung cancer treatment.