Lethal means and suicide prevention among military veterans

Dear Editor,Suicide amongst the military veteran population is a significant publichealthproblemintheUnitedStates.TheNational VeteranSuicidePreventionAnnualReportrevealedthat6261 died by suicide in 2019[1]. The lingering effects of the coronavirus disease 2019 (COVID-19) pandemic may account for an increase in veteran suicide rates[1].

Mapping and Functional Analysis of LE Gene in a Lethal Etiolated Rice Mutant at Seedling Stage

An EMS(ethy methanesulfonate)-induced lethal etiolated(le)mutant obtained from the rice variety Zhongjian 100 was characterized by lethal etiolated phenotypes,with significantly reduced levels of chlorophyll a,chlorophyll b,total chlorophyll,and carotenoids.Additionally,the mutant displayed a significantly decreased number of chloroplast grana,along with irregular and less-stacked grana lamellae.The le mutant showed markedly diminished root length,root surface area,and root volume compared with the wild type.It also exhibited significantly lower catalase activity and total protein content,while peroxidase activity was significantly higher.Using the map-based cloning method,we successfully mapped the LE gene to a 48-kb interval between markers RM16107 and RM16110 on rice chromosome 3.A mutation(from T to C)was identified at nucleotide position 692 bp of LOC_Os03g59640(ChlD),resulting in a change from leucine to proline.By crossing HM133(a pale green mutant with a single-base substitution of A for G in exon 10 of ChlD subunit)with a heterozygous line of le(LEle),we obtained two plant lines heterozygous at both the LE and HM133 loci.Among 15 transgenic plants,3 complementation lines displayed normal leaf color with significantly higher total chlorophyll,chlorophyll a,and chlorophyll b contents.The mutation in le led to a lethal etiolated phenotype,which has not been observed in other ChlD mutants.The mutation in the AAA+domain of ChlD disrupted the interaction of ChlDle with ChlI as demonstrated by a yeast two-hybrid assay,leading to the loss of ChlD function and hindering chlorophyll synthesis and chloroplast development.Consequently,this disruption is responsible for the lethal etiolated phenotype in the mutant.

Development of RAPD Markers and SCAR Markers Linked to Bentazon Susceptible Lethality Gene in Rice

Rice cultivar Norin 8 and its mutant Norin 8m harbour bentazon resistance trait and bentazon susceptibility trait respectively. A total of 360 arbitrary 10-mer oligonucleotide primers were screened on the genomic DNA of Norin 8 and Norin 8m with RAPD technique. Among which, five primers produced seven polymorphic RAPD bands between Norin 8 and Norin 8m. Amplified RAPD polymorphic products were cloned and sequenced. The sequences were used to design primers for PCR. Five SCAR markers, SCAR/G18/883, SCAR/G18/890, SCAR/G18/919/948, SCAR/D10/1237 and SCAR/F03/1186, were developed from OPG18/943, OPG18/972, OPD10/1248 and OPF03/1198. F-2 progeny of 320 individuals was analyzed to map SCAR markers in relationship to ben or Ben genes. SCAR markers of SCAR/G18/883, SCAR/G18/890, SCAR/G18/919/948 were shown to cosegregate with ben or Ben genes, and SCAR/D10/1237 to be linked of Ben gene with a distance of (14.8 +/- 2.1) cM. The genetic linkage to ben gene and SCAR markers was identified by a pair of near isogenic lines H121 and Hben121. Southern blotting analysis and segregation ratio of F-2 progeny revealed that OPG18/943 and OPG18/972 were single-copy in genome, and locus of OPG18/943 and OPG18/972 were allelic and sequence tagged sites. It is the first report on molecular markers linked to ben or Ben genes. The markers are useful to marker-assisted selection for the breeding and tag ben gene with map-based cloning.

Square Logistic Model for the Appearance of Lethal Recessive Homozygous Genotype in Random Mating Population

From the random mating population of maize, the relationship between heterosis and albino seedling was analyzed by means of mathematical theory, further the square logistic model for the appearance of lethal recessive homozygous genotype was deduced. The model shows that heterosis can determine the genetic structure and proportion of population. It approves, that the proportion of aa in each generation of maize seedlings shows a similar Logistic curve in growth process till equilibrium, so long as Aa containins lethal gene a from mutation presents heterosis. The ratio of equilibrium state of aa is determined by the ratio of AA-Aa fitness. Heterosis is helpful for the keeping of genetic diversity of population.

Anti-bacterial activity and brine shrimp lethality bioassay of methanolic extracts of fourteen different edible vegetables from Bangladesh

Objective:To investigate the antibacterial and cytotoxic activity of fourteen different edible vegetables methanolic extract from Bangladesh.Methods:The antibacterial activity was evaluated using disc diffusion assay method against 12 bacteria(both gram positive and gram negative).The plant extracts were also screened for cytotoxic activity using the brine shrimp lethality bioassay method and the lethal concentrations(LC_(50))were determined at confidence intervals by analyzing the data on a computer loaded with"Finney Programme??Results:All the vegetable extracts showed low to elevated levels of antibacterial activity against most of the tested strains(zone of inhibition=5-28 mm).The most active extract against all bacterial strains was from Xanthium indicum which showed remarkable antibacterial activity having the diameter of growth inhibition zone ranging from 12 to 28 mm followed by Alternanthera sessilis(zone of inhibition=6-21 mm).All extracts exhibited considerable general toxicity towards brine shrimps.The LC_(50)value of the tested extracts was within the range of 8.447 to 60.323μg/mL with respect to the positive control(vincristine sulphate)which was 0.91μg/mL.Among all studied extracts,Xanthium indicum displayed the highest cytotoxic effect with LC_(50)value of 8.447μg/mL.Conclusions:The results of the present investigation suggest that most of the studied plants are potentially good source of antibacterial and anticancer agents.

Ethyl Methane Sulphonate (EMS) Induced Mutagenesis in Malaysian Rice (cv. MR219) for Lethal Dose Determination

Chemical and physical mutagenesis has been used to increase genetic variability in crop plants. More than 430 new varieties have been derived as mutants of rice (Oryza sativa L.) via the application of different mutagenic agents. Chemical mutagens such as ethyl methane sulphonate (EMS), diepoxybutane-derived (DEB), sodium azide and irradiation (Gamma rays, X-rays and fast neutrons) have been widely used to induce a large number of functional variations in rice and others crops. Among chemical mutagens, the alkylating agent, ethyl methane sulfonate (EMS) is the most commonly used in plants as it causes a high frequency of nucleotide substitutions, as detected in different genomes. In this study, seeds of potential genotype of the popular variety, (Oryza sativa L. spp. Indica cv. MR219) were treated with EMS at concentrations of 0.25%, 0.50%, 0.75%, 1%, 1.25%, 1.5% and 2%. Sensitivity to EMS was determined by various measurements on the M1 generation. As concentration of applied EMS increased, will decrease in germination, seedling height, root length and emergence under field conditions was observed in M1 generation as compared to the non-treatment control. Plant height and root length also decreased with increases in EMS mutagenesis in an approximately linear fashion. The LD25 and LD50 values were observed based on growth reduction of seedlings after EMS treatment with 0.25% and 0.50% on the rice variety (Oryza sativa L. spp. Indica cv. MR219).

Sub-lethal effects of Beauveria bassiana(Balsamo) on field populations of the potato tuberworm Phthorimaea operculella Zeller in China

The potato tuberworm Phthorimaea operculella Zeller, is one of the most important potato pests worldwide including China. Several reports indicate that P. operculella could be controlled biologically by the use of beneficial fungus such as Beauveria bassiana(Bals.-Criv) Vuill. However, limited information is available under growing conditions in China. Thus, this study evaluated the sub-lethal effects of B. bassiana on the offspring of P. operculella by the age-stage, two-sex life table. First instar larva of P. operculella were treated with 1×107 conidia m L–1 of the fungus, and several biological parameters were evaluated. The fecundity, duration of the egg stage, all larval stages, pre-adult stage, and total pre-oviposition period, were significantly shorter than the control treatment. Offspring of treated parents, presented a net reproductive rate and mean generation time of 17.43 per day and 24.98 days, respectively, compared to 65.79 per day and 26.51 days for the untreated ones. This study provides basic information to help understanding the potential long-term effects of entomopathogenic fungi on P. operculella.

The acute lethality and endocrine effect of 1,2,3,7,8-PeCDD in juvenile goldfish (Carassius auratus) in vivo

1,2,3,7,8-PeCDD was administrated to juvenile goldfish （Carassius auratus） by peritoneal injections to explore the acute lethality and endocrine effects of 1,2,3,7,8-PeCDD in vivo. The value of acute median lethal dosage （LD50） of 1,2,3,7,8-PeCDD was determined in acute lethality tests. The endocrine effect of 1,2,3,7,8-PeCDD, whose exposed concentrations were determined based on the LD50 （1.84 mg/kg）, was studied by measuring the plasma vitellogenin （Vtg） content in juvenile male goldfish with enzyme-linked immunosorbent assays （ELISA）. Due to its significant induction of the plasma Vtg after one week＇s exposure in vivo in the 1/2 LD50 and LD30 groups, 1,2,3,7,8-PeCDD might be one of the important contributors to the estrogenic effect of PCDDs in the environment. The values of 1/2 LD50 and LD30 were within the range of the effective dosages of 1,2,3,7,8-PeCDD, indicating that there was a certain relationship between the estrogenic effective dosages and the LD50.

Synthetic lethal short hairpin RNA screening reveals that ring finger protein 183 confers resistance to trametinib in colorectal cancer cells

Background: The mitogen-activated extracellular signal-regulated kinase 1/2(MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer(CRC) is limited. Synthetic lethality arises with a combination of two or more separate gene mutations that causes cell death, whereas individual mutations keep cells alive. This study aimed to identify the genes responsible for resistance to trametinib in CRC cells,using a synthetic lethal short hairpin RNA(shRNA) screening approach.Methods: We infected HT29 cells with a pooled lentiviral shRNA library and applied next-generation sequencing to identify shRNAs with reduced abundance after 8-day treatment of 20 nmol/L trametinib. HCT116 and HT29 cells were used in validation studies. Stable ring finger protein 183(RNF183)-overexpressing cell lines were generated by pcDNA4-myc/his-RNF183 transfection. Stable RNF 183-knockdown cell lines were generated by infection of lentiviruses that express RNF183 shRNA, and small interference RNA(siRNA) was used to knock down RNF183 transiently.Quantitative real-time PCR was used to determine the mRNA expression. Western blotting, immunohistochemical analysis, and enzyme-linked immunosorbent assay(ELISA) were used to evaluate the protein abundance. MTT assay,colony formation assay, and subcutaneous xenograft tumor growth model were used to evaluate cell proliferation.Results: In the primary screening, we found that the abundance of RNF183 shRNA was markedly reduced after treatment with trametinib. Trametinib induced the expression of RNF183, which conferred resistance to drug-induced cell growth repression and apoptotic and non-apoptotic cell deaths. Moreover, interleukin-8(IL-8) was a downstream gene of RNF183 and was required for the function of RNF183 in facilitating cell growth. Additionally, elevated RNF183 expression partly reduced the inhibitory effect of trametinib on IL-8 expression. Finally, xenograft tumor model showed the synergism of RNF183 knockdown and trametinib in repressing the growth of CRC cells in vivo.Conclusion: The RNF183-IL-8 axis is responsible for the resistance of CRC cells to the MEK1/2 inhibitor trametinib and may serve as a candidate target for combined therapy for CRC.

DNA repair and synthetic lethality

Tumors often have DNA repair defects, suggesting additional inhibition of other DNA repair pathways in tumors may lead to synthetic lethality. Accumulating data demonstrate that DNA repair-defective tumors, in particular homologous recombination （HR）, are highly sensitive to DNA-damaging agents. Thus, HR-defective tumors exhibit potential vulnerability to the synthetic lethality approach, which may lead to new therapeutic strategies. It is well known that poly （adenosine diphosphate （ADP）-ribose） polymerase （PARP） inhibitors show the synthetically lethal effect in tumors defective in BRCA1 or BRCA2 genes encoded proteins that are required for efficient HR. In this review, we summarize the strategies of targeting DNA repair pathways and other DNA metabolic functions to cause synthetic lethality in HR-defective tumor cells.

Lethal and Sub-Lethal Effects of Transgenic Rice Containing cry1Ac and CpTI Genes on the Pink Stem Borer, Sesamia inferens(Walker)

Lethal and sub-lethal effects of transgenic rice containing cry1Ac and CpTI genes on the pink stem borer, Sesamia inferens, were studied to collect information for ecological risk assessment on insect-resistance of transgenic rice. In vitro insect-feeding bioassays were conducted to evaluate the lethal and sub-lethal effects of transgenic rice lines （II YouKF6 and KF6） containing cry1Ac＋CpTI genes on S. inferens at four different growth stages, viz., seedling, tillering and elongation, booting, and milk and maturing. Transgenic rice at seedling stage showed significantly high lethal effect on S. inferens with the shortest lethal duration for 50 and 100% individuals and the highest corrected mortalities after feeding on transgenic lines at this stage for 3 and 6 d. Followed by tillering and elongation stage, 50 and 100% S. inferens were dead after feeding on transgenic lines at this stage for 4 and 10 d, respectively. Moreover, corrected mortalities for 6 d feeding on transgenic lines at this stage were significantly higher than that at booting, and milk and maturing stages. Lethal effect of KF6 on S. inferens decreased significantly at booting stage. Lethal duration for 50% S. inferens significantly extended and its corrected mortalities for 6 d feeding also declined remarkably. However, lethal effect of II YouKF6 on S. inferens did not decrease significantly at this stage. Transgenic rice at booting, and milk and maturing stages did not show significant lethal effect to S. inferens and it showed the longest lethal duration for 50% individuals and the lowest corrected mortalities for 3 and 6 d feeding. A few larvae of S. inferens could survive, pupate and emerge on these two transgenic lines at booting, and milk and maturing stages. Sub-lethal effect of two transgenic lines on S. inferens also differed significantly between different developmental stages. Continuously feeding on transgenic rice lines at seedling, and tillering and elongation stages delayed the development of larvae and pupae and decreased pupation rate, but no effect was observed on eclosion rate. Larval development was significantly inhibited after feeding on transgenic rice at booting stage, but no obvious effect was observed in pupal stage, pupation and eclosion rate. There were no significant differences for larval and pupal development, pupation, and eclosion rates between feeding on transgenic and control rice lines at milk and maturing stage. Larval and pupal weights significantly declined, but no influence was observed on fecundity when S. inferens infested on transgenic rice at any stage. These showed that there were significant differences in lethal and sub-lethal effects of transgenic rice on S. inferens among developmental stages, and the effects gradually decreased with the increase of growth stages of rice plant.

A novel method for determination of lethal radius for high-explosive artillery projectiles

A new model has been defined that enables the estimation of the lethal radius(radius of efficiency)of HE(High Explosive)artillery projectiles against human targets.The model is made of several modules:CAD(Computer Aided Design)modeling,fragment mass distribution estimation,fragment initial velocity prediction,fragment trajectory calculation,effective fragment density estimation,and high explosive projectile lethal radius estimation.The results were compared with the experimental results obtained based on tests in the arena used in our country,and the agreement of the results was good.This model can be used in any terminal-ballistics scenario for high explosive projectiles since it is general,parametric,fast and relatively easy to implement.

Lethal-7-related polymorphisms are associated with susceptibility to and prognosis of gastric cancer

BACKGROUND The lethal-7(let-7)family members and their targets are involved in the development and progression of tumors.Let-7-related polymorphisms have been reported to be associated with tumorigenesis and prognosis.In gastric cancer,however,the related studies are limited.AIM To investigate the role of let-7-related microRNA polymorphisms in the tumorigenesis and prognosis of gastric cancer in a Chinese population.METHODS A total of 898 gastric cancer patients and 992 tumor-free controls were recruited into this study from 2008 to 2013.Gastric cancer patients were followed periodically.Ten single nucleotide polymorphisms(SNPs)in the let-7 gene region or their target mRNAs were genotyped using the MassARRAY system and their associations with the risk for or overall survival of gastric cancer were analyzed.RESULTS All the ten SNPs were in Hardy-Weinberg equilibrium.The C allele of the rs3811463 polymorphism in the 3’-untranslated region(UTR)of LIN28A was associated with a lower risk of gastric cancer[odds ratio(OR)=0.74,95%confidence interval(CI):0.61-0.88,P=0.001]after adjustment for age and Helicobacter pylori status.Seven hundred and thirty-five gastric cancer patients who had undergone radical tumorectomy were included in the survival analysis and their 5-year survival rate was 53.9%(95%CI:50.1%-57.6%).The rs10889677 in the 3’-UTR of IL23R was corresponded to the prognosis of gastric cancer in a dose-response manner,in which the death risk increased by 25%[hazard ratio(HR)=1.25,95%CI:1.04-1.45,P=0.011]with each increase in the number of C alleles after controlling for other potential clinicopathological parameters.CONCLUSION The let-7-related polymorphism rs3811463 in LIN28A is associated with the susceptibility to gastric cancer and the let-7-related polymorphism rs10889677 in IL23R is associated with the prognosis of gastric cancer.

A combination therapy for KRAS-mutant lung cancer by targeting synthetic lethal partners of mutant KRAS

The KRAS gene is frequently mutated in multiple cancer types,but it fell off the drug discovery radar for many years because of its inherent "undruggable" structure and undefined biological properties.As reported in the paper entitled "Suppression of KRas-mutant cancer through the combined inhibition of KRAS with PLK1 and ROCK" in Nature Communications,we performed a synthetic lethal screening with a combinatorial strategy on a panel of clinical drugs;we found that combined inhibition of polo-like kinase 1 and RhoA/Rho kinase markedly suppressed tumor growth in mice.An increase in the expression of the tumor suppressor P21^(WAF1/CIP1) contributed to the synergistic mechanism of the combination therapy.These findings open a novel avenue for the treatment of KRAS-mutant lung cancer.

SCAR Markers Assisted Selection for a Bentazon Susceptible Lethality Gene (ben) in Rice

In progenies resulting from crosses involving rice cultivar Norin 8m susceptible to bentazon as the donor of ben gene, SCARs tightly linked to ben were utilized for selection of ben. The homozygous and heterozygous genotypes with ben could be identified with the SCARs. The molecular markers offer a powerful tool for indirect selection of ben and can accelerate the introgression of ben into current rice cultivars.

Specific Lethal Effects of Trace Neutral Red on Amoeba discoides and Trichodina

[ Objective] The aim of this study was to provide the reference for the artificial breeding of aquatic animals. [ Method] Amoeba discoides and Trichodina were treated by neutral red solution with different concentrations to observe the physiological activities of organelles in their death processes. Effects of neutral red solution on the growth of common aquatic animals such as Paramecium caudatum, Euglena viridis and Brachionus plicatilis were analyzed, and the specific lethal mechanism of trace neutral red to A. discoides and Trichodina was also preliminarily studied. [ Re- suit] The neutral red solution at a concentration of 0.5 mg/L damaged the physiological function of contractile vacuole in Trichodina and also had the specific lethal effect on Tdchodina, but it had no effect on the growth and reproduction of non-parasitic protozoa and B. p/icatilis. Neutral red so- lution with certain concentration led to disorder of the physiological functions of A. discoides, such as assimilation and rejection, which was a main factor that caused the death of A. discoides. [ Conclusion] With the advantages such as targeting, safety and easiness to be oxidized and decom- posed, neutral red is an ideal drug for treating the diseases caused by A. discoides and Trichodina, and its suitable concentration is 0. 5 mg/L.

Effects of Cold Acclimation on Several Enzyme Activities in Euonymus radicans ‘Emorald & Gold’and Its Relation to Semi-lethal Temperature

The changes in activities of superoxide dismutase (SOD), peroxidase (POD) and ATPase in the leaves of Euonymus radicans were studied when seedlings were cold-acclimated (at 4 ℃) for 1 week, 2 weeks, 3 weeks and then treated for 1 d under low temperature stress (at 5 ℃). The semi-lethal temperatures of acclimated and unacclimated seedlings were also investigated. The results indicated that the activities of the three enzymes in the leaves of the seedlings treated at 4 ℃ for 1, 2 and 3 weeks were all higher than those of unacclimated seedings (treated at 22 ℃ as controls). The activities of SOD and POD increased continuously with the prolongation of the time of cold acclimation, but stepped up to summits then down to the levels of the controls. The activities of SOD culminated at the first week, and the activities of POD at the second week. When acclimated and unacclimated seedlings were both treated at 5 ℃ for 1 d, the activities of the three enzymes in the leaves of acclimated seedlings were a little lower than those before stress, but higher than those of the controls. Moreover, the decrease rate of enzyme activities was greatly lower than that of the controls. The results showed that cold acclimation could enhance the stability of the three enzymes in the leaves of seedlings under low temperature stress; the semi-lethal temperature was 19.1 ℃ when the seedlings were treated at 4 ℃ for 3 weeks, but it was 5.4 ℃ when the seedlings were treated at 22 ℃. The decline of the semi-lethal temperature caused by the adaptive changes of enzyme activities was one of the foundations of enhancing the cold tolerance.

Recent progress in maize lethal necrosis disease:From pathogens to integrated pest management

Maize(Zea mays),as a staple food and an important industrial raw material,has been widely cultivated for centuries especially by smallholder farmers.Maize lethal necrosis disease(MLND)is a serious disease infecting maize,which caused devastating damage in the African region recently.MLND is induced by co-infection of maize chlorotic mottle virus and one of several cereal-infecting viruses in the Potyviridae family,with the symptoms ranging from chlorotic mottle to plant death at different infection stages.Integrated pest management for MLND needs strengthening detection,focusing on prevention and effective control.Early detection system of MLND has been successfully established by serological methods,nucleic acid-based methods,next-generation sequencing,etc.The practices,such as using certified seeds,sanitary measures,crop rotation,tolerant or resistant varieties etc.,have been considered as the effective,economical and eco-friendly way to prevent and control MLND.

Influence of low temperature on lethal time extension for different life stages of Cryptolestes ferrugineus (Stephens) with strong resistance to phosphine fumigation

Knowing the time extension degree of full mortality in phosphine fumigation at low temperature significantly contributes to successful insect pest control,especially for reducing fumigation failure and inhibiting resistance development.The comparison ofmortality and lethal time on eggs,larvae,pupae and adults of Cryptolestes ferrugineus(Stephens)was conducted,and the strain with 1043 times of resistance factor to phosphine was assayed during fumigation with 300 mL/m^3 of phosphine concentration at 18,23 and 28℃.The LT50 values to eggs,larvae,pupae and adults of C.ferrugineus at 18℃ were postponed by 2,2,1 and 5 d comparedwith that at 23℃,and 5,4,5 and 7 d compared with that at 28℃,respectively.The LT99 values to eggs,larvae,pupae and adults at 18℃ were 5,1,2 and 7 d longer than that at 23℃,and 6,5,5 and 10 d longer than that at 28℃,respectively.The lethal time of different life stages of the C.ferrugineus strain significantly increased with temperature decreasing.The exposure time of full mortality on pupae at 300 mL/m^3 of phosphine at 18℃ reached 38 d,which was 12 d longer than that of larvae.The order of tolerance of different life stages of C.ferrugineus exposed to 300 mL/m^3 phosphine from high to low was pupae,eggs,adults,larvae at tested temperatures.

COVID-19 Coronavirus: Is Infection along with <i>Mycoplasma</i>or Other Bacteria Linked to Progression to a Lethal Outcome?

Most patients with COVID-19 disease caused by the SARS-CoV-2 virus recover from this infection, but a significant fraction progress to a fatal outcome. As with some other RNA viruses, co-infection or activation of latent bacterial infections along with pre-existing health conditions in COVID-19 disease may be important in determining a fatal disease course. Mycoplasma spp. (M. pneumonaie, M. fermentans, etc.) have been routinely found as co-infections in a wide number of clinical conditions, and in some cases this has progressed to a fatal disease. Although preliminary, Mycoplasma pneumoniae has been identified in COVID-19 disease, and the severity of some signs and symptoms in progressive COVID-19 patients could be due, in part, to Mycoplasma or other bacterial infections. Moreover, the presence of pathogenic Mycoplasma species or other pathogenic bacteria in COVID-19 disease may confer a perfect storm of cytokine and hemodynamic dysfunction, autoimmune activation, mitochondrial dysfunction and other complications that together cannot be easily corrected in patients with pre-existing health conditions. The positive responses of only some COVID-19 patients to antibiotic and anti-malaria therapy could have been the result of suppression of Mycoplasma species and other bacterial co-infections in subsets of patients. Thus it may be useful to use molecular tests to determine the presence of pathogenic Mycoplasma species and other pathogenic bacteria that are commonly found in atypical pneumonia in all hospitalized COVID-19 patients, and when positive results are obtained, these patients should treated accordingly in order to improve clinical responses and patient outcomes.