Strong Additive Effect Research Articles

Enhancing Drug Efficacy against Mastitis Pathogens-An In Vitro Pilot Study in Staphylococcus aureus and Staphylococcus epidermidis.

Simple SummaryThe success rate of antibiotic treatment of mastitis is highly variable. Concurrently, the efficacy of available antibiotics is compromised by the rapid emergence of drug-resistant bacteria. Recently, it was reported that there has been a reduction in the presence of antibiotic-resistant bacteria in food-producing animals where interventions provide for restrictions in antibiotic use. In addition, societal concerns regarding the use of antimicrobials in food animal production are putting increasing pressure on all aspects of livestock production. Here, we have conducted a systematic procedure for the identification of conserved and unique drug targets. We propose that combination therapy with drugs that work synergistically against conserved and unique targets can help increase efficacy and lower the usage of antibiotics for treating bacterial infections. An in vitro pilot validation of our findings in vitro for the two most common mastitis-causing bacteria in North America—Staphylococcus aureus and the coagulase-negative Staphylococcus epidermidis—is presented. We identified that the dosage of ceftiofur, the mostly used veterinary antibiotic, can be significantly reduced when used in combination with phytochemical phosphorylcholine.Background: Bovine mastitis is one of the major infectious diseases in dairy cattle, resulting in large economic loss due to decreased milk production and increased production cost to the dairy industry. Antibiotics are commonly used to prevent/treat bovine mastitis infections. However, increased antibiotic resistance and consumers’ concern regarding antibiotic overuse make it prudent and urgent to develop novel therapeutic protocols for this disease. Materials and methods: Potential druggable targets were found in 20 mastitis-causing pathogens and conserved and unique targets were identified. Bacterial strains Staphylococcus aureus (ATCC 29213, and two clinical isolates CI 1 and CI 2) and Staphylococcus epidermidis (ATCC 12228, and two clinical isolates CI 1 and CI 2) were used in the present study for validation of an effective drug combination. Results: In the current study, we identified the common and the unique druggable targets for twenty mastitis-causing pathogens using an integrative approach. Furthermore, we showed that phosphorylcholine, a drug for a unique target gamma-hemolysin component B in Staphylococcus aureus, and ceftiofur, the mostly used veterinary antibiotic that is FDA approved for treating mastitis infections, exhibit a synergistic effect against S. aureus and a strong additive effect against Staphylococcus epidermidis in vitro. Conclusion: Based on the data generated in this study, we propose that combination therapy with drugs that work synergistically against conserved and unique targets can help increase efficacy and lower the usage of antibiotics for treating bacterial infections. However, these data need further validations in animal models of infection.

Anti-inflammatory effect of glycyrrhizin with Equisetum arvense extract.

Periodontal disease is the most prevalent infectious disease, and inflammatory mediators play critical roles in its progression. Therefore, controlling pro-inflammatory cytokine production, especially at initial disease stages, is essential to maintaining gingival and periodontal health. Glycyrrhizin (GL) has an anti-inflammatory effect and has been added to toothpaste and mouth rinse to prevent periodontal disease. However, there is a maximum dose for the use of GL. The aim of the present study is to screen plant extracts which can effectively enhance the effects of GL. The effects of extracts from six different plants on GL-suppressed TNF-α expression in Aggregatibacter actinomycetemcomitans (A.a.)-LPS-stimulated human oral keratinocytes (RT7) were examined. Results demonstrated that Equisetum arvense (EA) extract had the strongest additive effect on the suppression of TNF-α by GL at both mRNA and protein levels. In addition, GL downregulated the production of TNF-α by suppressing NF-κB p65 phosphorylation, but not JNK or p38 phosphorylation. In contrast, EA decreased JNK phosphorylation but not NF-κB p65 or p38 phosphorylation. The combination of GL and EA effectively attenuated A.a.-LPS-induced phosphorylation of NF-κB p65 and JNK. Furthermore, an LPS-induced periodontitis rat model showed that GL with EA supplementation significantly downregulated TNF-α mRNA in the gingival tissue. These results indicate that EA can suppress A.a.-LPS-induced pro-inflammatory cytokine production by inhibiting JNK activation and can promote the anti-inflammatory effects of GL. Our findings suggest that a combination of GL and EA may improve the development of new oral hygiene products aimed at enhancing periodontal health.

Bioorthogonal Correlative Light-Electron Microscopy of Mycobacterium tuberculosis in Macrophages Reveals the Effect of Antituberculosis Drugs on Subcellular Bacterial Distribution.

Bioorthogonal correlative light-electron microscopy (B-CLEM) can give a detailed overview of multicomponent biological systems. It can provide information on the ultrastructural context of bioorthogonal handles and other fluorescent signals, as well as information about subcellular organization. We have here applied B-CLEM to the study of the intracellular pathogen Mycobacterium tuberculosis (Mtb) by generating a triply labeled Mtb through combined metabolic labeling of the cell wall and the proteome of a DsRed-expressing Mtb strain. Study of this pathogen in a B-CLEM setting was used to provide information about the intracellular distribution of the pathogen, as well as its in situ response to various clinical antibiotics, supported by flow cytometric analysis of the bacteria, after recovery from the host cell (ex cellula). The RNA polymerase-targeting drug rifampicin displayed the most prominent effect on subcellular distribution, suggesting the most direct effect on pathogenicity and/or viability, while the cell wall synthesis-targeting drugs isoniazid and ethambutol effectively rescued bacterial division-induced loss of metabolic labels. The three drugs combined did not give a more pronounced effect but rather an intermediate response, whereas gentamicin displayed a surprisingly strong additive effect on subcellular distribution.

A 6.5-kb intergenic structural variation enhances P450-mediated resistance to pyrethroids in malaria vectors lowering bed net efficacy.

Elucidating the complex evolutionary armory that mosquitoes deploy against insecticides is crucial to maintain the effectiveness of insecticide-based interventions. Here, we deciphered the role of a 6.5-kb structural variation (SV) in driving cytochrome P450-mediated pyrethroid resistance in the malaria vector, Anopheles funestus. Whole-genome pooled sequencing detected an intergenic 6.5-kb SV between duplicated CYP6P9a/b P450s in pyrethroid-resistant mosquitoes through a translocation event. Promoter analysis revealed a 17.5-fold higher activity (p<.0001) for the SV- carrying fragment than the SV- free one. Quantitative real-time PCR expression profiling of CYP6P9a/b for each SV genotype supported its role as an enhancer because SV+/SV+ homozygote mosquitoes had a significantly greater expression for both genes than heterozygotes SV+/SV- (1.7- to 2-fold) and homozygotes SV-/SV- (4-to 5-fold). Designing a PCR assay revealed a strong association between this SV and pyrethroid resistance (SV+/SV+ vs. SV-/SV-; odds ratio [OR]=2,079.4, p<.001). The 6.5-kb SV is present at high frequency in southern Africa (80%-100%) but absent in East/Central/West Africa. Experimental hut trials revealed that homozygote SV mosquitoes had a significantly greater chance to survive exposure to pyrethroid-treated nets (OR 27.7; p<.0001) and to blood feed than susceptible mosquitoes. Furthermore, mosquitoes homozygote-resistant at the three loci (SV+/CYP6P9a_R/CYP6P9b_R) exhibited a higher resistance level, leading to a far superior ability to survive exposure to nets than those homozygotes susceptible at the three loci, revealing a strong additive effect. This study highlights the important role of structural variations in the development of insecticide resistance in malaria vectors and their detrimental impact on the effectiveness of pyrethroid-based nets.

Cellular responses to deproteinized bovine bone mineral biofunctionalized with bone-conditioned medium

ObjectivesThe aim of the study was to investigate whether the osteoinductive properties of bone-conditioned medium (BCM) harvested from cortical bone chips within a clinically relevant short-term period can enhance the biologic characteristics of deproteinized bovine bone mineral (DBBM) in vitro.Materials and methodsTo assess the biofunctionalization of DBBM, the adhesive, proliferative, and differentiation properties of mesenchymal stromal ST2, pre-osteoblastic MC3T3-E1, and primary bone-derived cells grown on BCM-coated DBBM were examined by crystal violet staining of adherent cells, BrdU ELISA, and qRT-PCR, respectively.ResultsBCM extracted within 20 min or 24 h in either Ringer’s solution (BCM-RS) or RS mixed with autologous serum (BCM-RS + S) increased the adhesive properties of all three cell types seeded on DBBM. The 20-min BCM-RS preparation appeared more potent than the 24-h preparation. BCM-RS made within 20 min or 24 h had strong pro-proliferative effects on all cell types grown on DBBM. RS + S alone exhibited a considerable pro-proliferative effect, suggesting an impact of the serum on cellular growth. DBBM coated with BCM-RS or BCM-RS + S, made within 20 min or 24 h each, caused a significant induction of osteogenic differentiation marker expression with a higher potency of the BCM-RS + S. Finally, a strong additive effect of fresh bone chips combined with BCM-coated DBBM on the osteogenic differentiation of the three cell types was observed.ConclusionsAltogether, the data strongly support the biofunctionalization of DBBM with BCM extracted within a clinically relevant time window of 20 min.Clinical relevancePre-activation of non-osteoinductive biomaterials with BCM, prepared from autologous bone chips during a guided bone regeneration (GBR) procedure, bears the potential of an optimal treatment modality for bone defects in daily practice.

Strong additive and synergistic effects of polyoxyethylene nonionic surfactant-assisted protein MALDI imaging mass spectrometry

Protein MALDI imaging mass spectrometry (MALDI-IMS) holds a great promise to acquire spatial distribution information of proteins on biological tissue, but it suffers from the small number of proteins detected by direct MALDI-IMS detection. Ionic surfactants have been extensively used for protein extraction to improve the number of proteins detected in tissue samples by LC-MS analysis, but seldom by direct MALDI-IMS detection. Nonionic surfactants are milder than ionic surfactants and protein native structures are remained after extraction, which favors the spatial resolution of direct MALDI-IMS. However, nonionic surfactants are less effective than ionic surfactants. In this report, we utilized polyoxyethylene nonionic surfactants (PNS) to pre-incubate the tissue section, followed by the on-tissue trypsin digestion and then direct MALDI detection of in-situ formed peptides. For the first time, we observed that the additive effect of PNS and the synergistic effect of the mixed PNS in improving the number of peptides detected. Specifically, the peptides detected were 73.0–90.7% distinct when the different PNS (Tween 80 or Triton X-100 alone or their mixture) was used. Taking advantage of this additive effect, the 96 proteins including 12 transmembrane proteins were detected, corresponding to a ~10-fold improvement compared to MALDI-IMS without surfactant. When the mixed surfactants were used to replace Tween 80 and Triton X-100 alone, the optimized surfactant concentration decreased 20–100-fold and the number of peptides detected with m/z > 2500 Da was improved 15-fold. The additive and synergistic effects of PNS suggested that the interaction mode between each PNS and proteins is highly variable. Benefiting from the strong additive effect and diversity of PNS, further improvement of the number of proteins detected by MALDI-IMS is clearly feasible.

Identification and characterization of Fbxl22, a novel skeletal muscle atrophy-promoting E3 ubiquitin ligase.

Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of F-box and leucine-rich protein 22 (Fbxl22), and a newly identified splice variant (Fbxl22-193), in skeletal muscle homeostasis and neurogenic muscle atrophy. In mouse C2C12 muscle cells, promoter fragments of the Fbxl22 gene were cloned and fused with the secreted alkaline phosphatase reporter gene to assess the transcriptional regulation of Fbxl22. The tibialis anterior muscles of male C57/BL6 mice (12-16 wk old) were electroporated with expression plasmids containing the cDNA of two Fbxl22 splice variants and tissues collected after 7, 14, and 28 days. Gastrocnemius muscles of wild-type and muscle-specific RING finger 1 knockout (MuRF1 KO) mice were electroporated with an Fbxl22 RNAi or empty plasmid and denervated 3 days posttransfection, and tissues were collected 7 days postdenervation. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy. In vivo overexpression of Fbxl22 isoforms in mouse skeletal muscle leads to evidence of myopathy/atrophy, suggesting that both are involved in the process of neurogenic muscle atrophy. Knockdown of Fbxl22 in the muscles of MuRF1 KO mice resulted in significant additive muscle sparing 7 days after denervation. Targeting two E3 ubiquitin ligases appears to have a strong additive effect on protecting muscle mass loss with denervation, and these findings have important implications in the development of therapeutic strategies to treat muscle atrophy.

The content of yellow pigments in durum wheat (Titicum durum Desf.) grains: biosynthesis, genetic control, marker selection

Зерно с высоким содержанием каротиноидных пигментов ценится за ярко-желтый цвет пасты, производимой из него, и провитаминную (витамин А) и антиоксидантную активность пигментов. Цель настоя- щего обзора – обобщение современных знаний о биосинтезе и генетическом контроле накопления пигментов в зерне твердой пшеницы и оценка основных результатов исследований и селекции за последние двадцать лет за рубежом и в России. Признак «концентрация каротиноидных пигментов в зерне» (Ypc) относится к раз- ряду количественных. Тем не менее превалирование сильных аддитивных эффектов генов и высокая насле- дуемость способствовали значительному прогрессу в селекции по этому признаку. Методами молекулярного маркирования локусов количественных признаков (QTL), контролирующих синтез каротиноидных пигментов и значения индекса желтизны (IY), установлено их распределение по всем хромосомам генома твердой пшеницы. Основные генетические локусы, определяющие более 60 % варьирования признака, были картированы в хромосомах 7AL и 7BL. Вклад этих локусов связан с аллельными вариациями, влияющими на активность фермента фитоенсинтетазы (PSY). В других хромосомах были локализованы минорные генетические факторы, из которых наиболее значимы QTL, расположенные в хромосомах 3AS (ассоциирован с геном LCYE-ликопин- ε-циклаза) и 4ВS (аллель Lpx-B1.1c). При этом показано, что аллель Lpx-B1.1c вносит вклад в снижение актив- ности липоксигеназы, окисляющей каротиноиды в процессе изготовления конечных продуктов. Рассмотрены и обсуждены проблемы использования молекулярных маркеров в селекционных программах, нацеленных на увеличение концентрации пигментов в зерне и улучшение цветовых характеристик пасты.

Defoliation and neighbouring legume plants accelerate leaf and root litter decomposition of Leymus chinensis dominating grasslands

Dominant plant species coexist with other species from the same or other trophic levels (e.g. neighbouring plants and herbivores), which may give rise to neighbourhood and defoliation effects on litter decomposition. Yet, little information exists on if and how neighbouring plant species and herbivore defoliation interactively affect the decomposition of dominant species, and further whether decomposition of leaves and roots has similar responses. Here, we simultaneously examined both the leaf and fine root litter decomposition of the dominant grass species, Leymus chinensis, in the presence and absence of a neighbouring plant species, Lathyrus quinquenervius (a functionally dissimilar legume species), and simulated herbivore defoliation by mowing. We found that both neighbouring legume plants and defoliation significantly accelerated both leaf and root litter decomposition of L. chinensis, and that neighbouring legume plants exerted stronger effects on litter decomposition than defoliation. The positive effects of neighbouring legume plants occurred across all study periods, whereas defoliation only had a positive effect during the first 15 days of litter decomposition. Neighbouring legume plants and defoliation exerted strong additive effects on root litter decomposition, and interactive effects on leaf litter decomposition. These findings highlight the significance of functionally dissimilar species coexistence to facilitate nutrient cycling in grasses - dominated grasslands.

Erythrosine B – coated gold nanoparticles as an analytical sensing tool for the proper determination of both compounds based on surface-enhanced Raman spectroscopy

An analytical sensor for gold nanoparticles (AuNPs) and erythrosine B, based on surface-enhanced Raman spectroscopy (SERS) using a specially designed nanostructured substrate is reported. It is well documented that highly conjugated organic molecules, such as food dyes, display a strong additional charge-transfer effect when adsorbed on the AuNPs metallic surface, exhibiting good abilities to act as Raman reporters. In this work, five synthetic food dyes were selected and studied as AuNPs sensor molecules. Erythrosine B showed the highest AuNPs-binding affinity due to its greater enhancement of the Raman signal with respect to the other dyes tested. Thus, this dye was selected to be part of the sensor. This fact allowed the development of an analytical sensor for both AuNPs and erythrosine B. The optimal performance conditions of the sensor for both analytes were studied, showing to be especially sensitive for the determination of AuNPs. A linear relationship exists between the SERS signal intensity and AuNPs in the 1.0 to 12.0 ng L−1 range and between 5.0 and 150.0 µg L−1 for erythrosine. Detection limits (LODs) were 0.3 ng L−1 and 1.4 µg L−1 for AuNPs and erythrosine B, respectively. The potential of this analytical method was demonstrated by applying it to the quantification of AuNPs in enriched environmental, biological and cosmetic samples, and of erythrosine B in food samples. The proposed analytical approach satisfies the need for a simple, quick and sensitive methodology to determine AuNPs in samples of interest, thus contributing to analytical nanometrology.

School Absenteeism Associated With Child Protection System Involvement, Maltreatment Type, and Time in Out-of-Home Care.

Greater school absenteeism is associated with numerous negative educational outcomes. We used a retrospective cohort design with linked administrative data on 296,422 children to examine the relationship between school absenteeism and child protection system (CPS) involvement. Children with substantiated maltreatment had 4.1 times more unexplained and problem absences than children with no CPS involvement. In multivariate analyses, children with substantiated maltreatment had significantly greater "chronic" truancy (OR = 3.41) and less "acceptable" levels of absences (OR = 0.74) compared to children with no CPS involvement. Greater absenteeism was seen for children with substantiated neglect and who had their first CPS notification earlier in life. Being in out-of-home care for 3+ years was a protective factor for children who had a CPS notification before age 5. Additional adversities had a strong additive effect with CPS involvement on absenteeism and chronic truancy. This study demonstrates the potential scope for reducing problem absenteeism and helps inform the public debate regarding how the type and timing of CPS involvement might ameliorate or exacerbate harm for children.

Genetic Characterization of Leaf and Stripe Rust Resistance in the Brazilian Wheat Cultivar Toropi.

Leaf and stripe rust are major threats to wheat production worldwide. The effective, multiple rust resistances present in the Brazilian cultivar Toropi makes it an excellent choice for a genetic study of rust resistance. Testing of DNA from different seed lots of Toropi with 2,194 polymorphic 90K iSelect single nucleotide polymorphism markers identified significant genetic divergence, with as much as 35% dissimilarity between seed lots. As a result, further work was conducted with a single plant line derived from Toropi variant Toropi-6.4. A double haploid population with 168 lines derived from the cross Toropi-6.4 × Thatcher was phenotyped over multiple years and locations in Canada, New Zealand, and Kenya, with a total of seven field trials undertaken for leaf rust and nine for stripe rust. Genotyping with the 90K iSelect array, simple sequence repeat and Kompetitive allele-specific polymerase chain reaction markers resulted in a genetic map of 3,043 cM, containing 1,208 nonredundant markers. Significant quantitative trait loci (QTL) derived from Toropi-6.4 were identified in multiple environments on chromosomes 1B (QLr.crc-1BL/QYr.crc-1BL), 3B (QLr.crc-3BS), 4B (QYr.crc-4BL), 5A (QLr.crc-5AL and QYr.crc-5AL), and 5D (QLr.crc-5DS). The QTL QLr.crc-1BL/QYr.crc-1BL colocated with the multi-rust resistance locus Lr46/Yr29, while the QTL QLr.crc-5DS located to the Lr78 locus previously found in a wheat backcross population derived from Toropi. Comparisons of QTL combinations showed QLr.crc-1BL to contribute a significantly enhanced leaf rust resistance when combined with QLr.crc-5AL or QLr.crc-5DS, more so than when QLr.crc-5AL and QLr.crc-5DS were combined. A strong additive effect was also seen when the stripe rust resistance QTL QYr.crc-1BL and QYr.crc-5AL were combined.

Combining the oncolytic peptide LTX-315 with doxorubicin demonstrates therapeutic potential in a triple-negative breast cancer model

BackgroundImmunochemotherapy, the combined use of immunotherapy and chemotherapy, has demonstrated great promise in several cancers. LTX-315 is an oncolytic peptide with potent immunomodulatory properties designed for the local treatment of solid tumors. By inducing rapid immunogenic cell death through the release of danger-associated molecular pattern molecules (DAMPs), LTX-315 is capable of reshaping the tumor microenvironment, turning “cold” tumors “hot” through a significant increase in tumor-infiltrating lymphocytes.MethodsWe investigated the potential of LTX-315 to be used in combination with standard-of-care chemotherapy (doxorubicin, brand name CAELYX®) against triple-negative breast cancer in an orthotopic 4 T1 mammary fat pad model. Tumor growth curves were compared using one-way ANOVA analysis of variance and Tukey’s multiple comparisons test, and animal survival curves were compared using the log-rank (Mantel-Cox) test. We considered p values ≤0.05 to indicate statistical significance.ResultsWe found that LTX-315 displayed a strong additive antitumoral effect when used in combination with CAELYX®, and induced immune-mediated changes in the tumor microenvironment, followed by complete regression in the majority of animals treated. Furthermore, imaging techniques and histological examination showed that the combination induced strong local necrosis, followed by an increase in the infiltration of CD4+ and CD8+ immune cells into the tumor parenchymal tissue.ConclusionsOur data demonstrate that LTX-315 is a promising combination partner with CAELYX® for the treatment of triple-negative breast cancer.

Gallic Acid Potentiates the Antimicrobial Activity of Tulathromycin Against Two Key Bovine Respiratory Disease (BRD) Causing-Pathogens.

Bovine respiratory disease (BRD) is the most common infectious disease in dairy and beef cattle. It is associated with significant morbidity and mortality and causes a huge economic loss each year. In western Canada, a one-time injection of tulathromycin is commonly used as a metaphylactic procedure to reduce BRD incidence and eliminate potential BRD outbreak. With increased global concern on antimicrobial usage in dairy and beef products and bacterial resistance to antimicrobials, it is important to develop a novel strategy to eliminate the usage or decrease the dosage of antimicrobials. In this study, we showed that gallic acid was active against both Mannheimia haemolytica and Pasteurella multocida, two key BRD associated-pathogens, with the minimum inhibitory concentration (MIC) measured at 250 and 500 μg/mL, respectively. Co-administration of tulathromycin and gallic acid exhibited a strong additive or weak synergistic effect toward both M. haemolytic and P. multocida. Tulathromycin, gallic acid and their combination were also effective against the mixed culture of M. haemolytic and P. multocida. Furthermore, we showed that pre-exposure to tulathromycin generated bacterial resistance to the antimicrobial in M. haemolytica but not in P. multocida.

Enhanced estimates of carcass and meat quality effects for polymorphisms in myostatin and µ-calpain genes.

The objective of this study was to enhance estimates of additive, dominance, and epistatic effects of marker polymorphisms on beef carcass and quality traits. Myostatin (MSTN) F94L SNP and the µ-calpain (CAPN1) 316 and 4751 SNP haplotype have previously been associated with fat and muscle traits in beef cattle. Multiyear selection in a composite population segregating these polymorphisms increased minor allele (F94L L) and chosen haplotype (CAPN1 CC and GT) frequencies to intermediate levels resulting in more precise estimates of additive and nonadditive genetic effects. During the 3 yr after selection, 176 steers were evaluated for growth, carcass, meat quality, tenderness (n = 103), and meat color traits. The statistical model included year, age of dam, age of the steer, and genotype in a random animal model. The 9 genotypes (3 CAPN1 diplotypes × 3 F94L genotypes) affected marbling score, ribeye area, adjusted fat thickness, vision yield grade (all P < 0.001), slice shear force (P = 0.03), and CIE L* reflectance (P = 0.01). Linear contrasts of the 9 genotypes estimated additive, recessive, and epistatic genetic effects. Significant additive effects of the F94L L allele decreased marbling score, adjusted fat thickness, vision yield grade, and slice shear force; and increased ribeye area and CIE L* reflectance. The homozygous F94L FF and LL genotypes differed by 1.3 to 1.9 phenotypic SD for most carcass traits and by 0.8 to 0.9 SD for slice shear force and CIE L* reflectance but carcass weight differed by only 3 kg (0.1 SD). The L allele was partially recessive to F for ribeye area (P = 0.02) and the heterozygous FL means tended to be closer to the FF genotype than the LL genotype for other carcass traits but differences from additive were not significant. The CAPN1 additive × F94L additive effect on slice shear force was the only significant epistatic estimate. The F94L L allele is prevalent in Limousin but nearly absent in other U.S. purebreds. This allele had about half of the effects on birth weight, muscle, and fat traits reported for severe MSTN mutations in Belgian Blue and Piedmontese breeds. The interaction between MSTN and CAPN1 genotypes may reflect the strong additive effects of MSTN F94L L allele on fat and muscle traits interfering with the phenotypic effect of CAPN1 genotype on meat tenderness.

The Pan-PIM Kinase Inhibitor LGB321 Strongly Increases Ibrutinib Effects in CLL, By Blocking Anti-Apoptotic Pathways and Microenvironmental Interactions

Despite remarkable anti-tumor activity of ibrutinib in CLL, some patients develop resistance due to acquired mutations. By adding drugs which target survival pathways and the CLL protective microenvironmental interactions, we aim to increase ibrutinib efficacy and to prevent ibrutinib resistance. The three PIM kinases are involved in important disease mechanisms in CLL, with PIM1 regulating CXCR4 surface expression impacting the interaction with the protective microenvironment, and PIM2/3 affecting the apoptotic machinery by regulating BAD.Here, we investigated the effect of the Pan-PIM kinase inhibitor LGB321 in CLL in vitro and in vivo and its cooperative effect with ibrutinib. LGB321 was highly effective in inducing apoptosis in primary CLL cells, independent of prognostic factors or microenvironmental interactions, and reduced pBAD and total BAD protein levels in CLL. In vitro combination of ibrutinib and LGB321 demonstrated strong additive effects with doubled apoptosis rates compared to single treatment. Further, LGB321 treatment blocked the CXCR4/CXCL12 axis by dephosphorylating the CXCR4 receptor on Ser339, by reducing total CXCR4 protein levels, and by blocking the externalization of the CXCR4 receptor. Concordantly, LGB321 blocked CXCR4 functions regarding migration towards a CXCL12 gradient and homing of LGB321-pretreated primary CLL cells towards the bone marrow of NOG mice.In vivo experiments confirmed the efficacy of LGB321 in 5 different CLL xenograft studies. Transplantation of primary CLL cells into NOG mice and treatment with LGB321 for 2 weeks strongly reduced WBC counts, spleen size and spleen infiltration with human CLL cells in all 5 CLL cases, and blocked BAD as well as CXCR4 phosphorylation also in vivo.Xenograft studies combining ibrutinib with LGB321 demonstrated much faster reduction of WBC counts than ibrutinib single treatment and strongly reduced disease burden in the double treated mice, confirming the cooperative effects of those two inhibitors also in vivo.Our results demonstrate that LGB321 might be an effective treatment option for CLL patients by impairing PIM2/3 mediated CLL-cell survival, and by blocking the PIM1/CXCR4-mediated interaction of CLL cells with the protective microenvironment. Furthermore, LGB321 enhances ibrutinib treatment effects and might help to increase its efficacy and to avoid the development of ibrutinib resistance. DisclosuresNo relevant conflicts of interest to declare.

μ-Calpain (CAPN1), calpastatin (CAST), and growth hormone receptor (GHR) genetic effects on Angus beef heifer performance traits and reproduction

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. An Angus population was subjected to marker assisted selection for multiple years to equalize CAPN1 haplotypes, CAST, and GHR genetic marker frequencies. The objective was to estimate the pleiotropic effects of these carcass quality oriented markers for body weight, reproduction, and first calf performance traits in 174 replacement beef females which were managed under 2 post-weaning development protocols. Heifers were weighed at 11-, 12-, and 13-mo, at first breeding season pregnancy evaluation, and prior to first calving season. Pubertal status was determined at 11-, 12-, and 13-mo of age. Antral follicles were counted, reproductive tracts were scored, and tract dimensions were measured at 13-mo. Body condition and hip height were scored and measured at pregnancy evaluation and prior to calving season. Heifer pregnancy and weaning rates and ordinal birth date were recorded. Calf body weights at birth and weaning were analyzed. Single df linear contrasts for recessive effects of the GHR heterozygous genotype showed significant decreases of 2.5–3.6% in 11-, 12-, and 13-mo heifer body weights and heifer weight prior to calving. The additive differences between GHR homozygotes were small and not significant for all body weights measured but a 1 wk difference in calf birth date was significant. For all 13-mo uterine measurements, scores, and antral follicle counts, only the CAST dominance contrast for medium antral follicle count was significant. The CAPN1 haplotype with a strong additive effect for increased beef tenderness also had a significant additive effect on calving date. Heifers homozygous for the tender haplotype calved 7.9 days later than heifers homozygous for the tough haplotype. Most heifer reproductive traits were not significantly affected by CAST and CAPN1 markers that are widely used to improve beef tenderness by selection and breeders should not be concerned with how these markers affect reproduction and other heifer traits with the possible exception of CAPN1 effects on calving date.

Abstract B36: The response of colorectal cancer cells to 5-FU and oxaliplatin mimicking the clinical schedule involves the interplay between of apoptosis, senescence, and cytoprotective autophagy

Abstract Colorectal cancer (CRC) is among the most common and aggressive cancers worldwide. Primary therapy to CRC includes 5-fluoruracil (5-FU) and oxaliplatin (Oxa). Here, we aim to investigate the cellular mechanisms that mediate the response of CRC to the cotreatment with 5-FU and OXA, in a schedule that mimics the clinics, i.e., 48 h of exposure to the drugs followed by two weeks before the second treatment. We repeated this cycle twice. Our main objective was to understand the outcome of CRC cells after the period of exposure to the drugs, in order to understand the mechanisms of response and resistance to the treatment. To do this, we used the CRC human cell lines HCT116 and HT29. We found that acutely (48 h), drugs did not show additive toxicity. However, chronically the combination had a strong additive effect, reducing both the growth of the population of cells and the growth of single cells in a clonogenic assay. 5-FU induced apoptosis, peaking 3d after treatment, while Oxa induced senescence 7 days after treatment, both in higher extent in HCT116 than in HT29 cells. The cotreatment induced an intense, transitory autophagy in both cell lines, reaching a peak 5 to 7 days after the treatment. Pharmacologic suppression of autophagy during its peak of activation but not together with the chemotherapeutics strongly reduced cell growth. In summary, in the first cycle of treatment we found that the combination of 5-FU and OXA for 48 h had additive toxicity along two weeks by the combination of apoptosis (induced by 5-FU) and senescence (induced by Oxa). However, both cell lines displayed regrowth from day 7 after treatment onwards. In addition, suppression of autophagy strongly decreased cancer cells' growth and clonogenicity. Then, we performed a second cycle of 5-FU and Oxa in the cells. Interestingly, cells were more sensitive to the second cycle of treatment, suggesting that resistance was not established. However, along the second cycle of treatment we found that (i) senescent cells (induced in the first cycle of treatment) survived to the second cycle of treatment, while nonsenescent cells were sensitive, and (ii) that cells acquired a phenotype that resembles the epithelial to mesenchymal transition, which is a hallmark of cancer progression. The next step, which is ongoing, is the evaluation of autophagy in the second cycle of treatment, as well as the long-term analysis of those cells that were suppressed to autophagy in the first cycle of treatment. Translationally, our data suggest that the rational modulation of autophagy may increase the toxicity of 5-FU plus Oxa cotreatment. Furthermore, we found that senescent cells, which can have a protumor role due to their secretome, resisted to the second cycle of treatment, so that the elimination of these cells could improve the efficacy of the combined therapy with 5-FU and Oxa in CRC. Citation Format: Andréa Baldasso Zanon, Nayara Franco, Patrícia Luciana da Costa Lopez, Guido Lenz, Eduardo C. Filippi-Chiela. The response of colorectal cancer cells to 5-FU and oxaliplatin mimicking the clinical schedule involves the interplay between of apoptosis, senescence, and cytoprotective autophagy [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B36.

The use of Anthrax and orthopox therapeutic antibodies from human origin in biodefense

The use of Anthrax and orthopox therapeutic antibodies from human origin in biodefense

Changes in habitat associations during range expansion: disentangling the effects of climate and residence time

The distributions of many species are not at equilibrium with their environment. This includes spreading non-native species and species undergoing range shifts in response to climate change. The habitat associations of these species may change during range expansion as less favourable climatic conditions at expanding range margins constrain species to use only the most favourable habitats, violating the species distribution model assumption of stationarity. Alternatively, changes in habitat associations could result from density-dependent habitat selection; at range margins, population densities are initially low so species can exhibit density-independent selection of the most favourable habitats, while in the range core, where population densities are higher, species spread into less favourable habitat. We investigate if the habitat preferences of the non-native common waxbill Estrilda astrild changed as they spread in three directions (north, east and south-east) in the Iberian Peninsula. There are different degrees of climatic suitability and colonization speed across range expansion axes, allowing us to separate the effects of climate from residence time. In contrast to previous studies we find a stronger effect of residence time than climate in influencing the prevalence of common waxbills. As well as a strong additive effect of residence time, there were some changes in habitat associations, which were consistent with density-dependent habitat selection. The combination of broader habitat associations and higher prevalence in areas that have been colonised for longer means that species distribution models constructed early in the invasion process are likely to underestimate species’ potential distribution.