Insensitive Mechanisms Research Articles

Fracture behavior of fibrous network materials: Crack insensitivity and toughening mechanism

A 3D numerical model is established to characterize the fibrous network materials and the fracture behavior is investigated through the finite element method (FEM) analysis. The numerical results show that fibrous network material is insensitive to the crack when the crack length is less than a critical value, which is three times as much as the average length of fiber segments in the fibrous network. In addition, the toughening mechanism of the fibrous network material is revealed based on the FEM simulations. Due to the damage accumulation of bonds near the crack tip, the fibers around the crack tip are reoriented and become perpendicular to the crack path, which can further inhibit the crack propagation, and the deformation mode changes from bending-dominated deformation to tensile-dominated deformation, with the loading capacity of the fibrous network materials enhanced. The results in this paper demonstrate that the fibrous network materials show damage-tolerance to the small crack under the critical crack length, and the fibrous network formed by brittle fibers can exhibit ductile behaviors by controlling the microstructure and relative density, which is useful for the design and optimization of fibrous network materials.

Roscovitine differentially facilitates cerebellar glutamatergic and GABAergic neurotransmission by enhancing Cav 2.1 channel-mediated multivesicular release.

Synaptic vesicle exocytosis is triggered by Ca2+ influx through several subtypes of voltage-gated calcium channels in the presynaptic terminal. We previously reported that paired-pulse stimulation at brief intervals increases Cav 2.1 (P/Q-type) channel-mediated multivesicular release (MVR) at glutamatergic synapses between granule cells (GCs) and molecular layer interneurons (MLIs) in rat cerebellar slices. However, it has yet to be determined how Cav 2 channel subtypes take part in MVR in single axon terminal. This study therefore aimed at examining the effects of roscovitine on different types of cerebellar synapses that make contacts with Purkinje cells (PCs), because this compound has been shown to enhance Cav 2.1 channel-mediated MVR at GC-MLI synapses. Bath application of roscovitine profoundly increased the amplitude of excitatory postsynaptic currents (EPSCs) at GC-PC synapses by a presynaptic mechanism as previously observed at GC-MLI synapses, whereas it caused a marginal effect on climbing fiber-mediated EPSCs in PCs. At MLI-PC synapses, roscovitine increased both the amplitude and decay time of inhibitory postsynaptic currents (IPSCs) by enhancing multivesicular GABA release. When extracellular Ca2+ concentration ([Ca2+ ]e ) decreased, roscovitine became less effective in increasing GC-PC EPSCs. By contrast, roscovitine was able to augment MLI-PC IPSCs in the low [Ca2+ ]e . The Cav 2.1 channel blocker ω-agatoxin IVA suppressed the roscovitine-induced facilitatory actions on both GC-PC EPSCs and MLI-PC IPSCs. These results demonstrate that roscovitine enhances MVR at the GC-PC excitatory synapses in a manner dependent on the driving force of Cav 2.1 channel-mediated Ca2+ influx into the nerve terminal, while it also facilitates MLI-PC inhibitory transmission via Ca2+ -insensitive mechanisms.

Genome-wide screens reveal Escherichia coli genes required for growth of T1-like phage LL5 and V5-like phage LL12

The host factor requirements of phages and mechanisms of mutational phage insensitivity must be characterized for rational design of phage cocktails. To characterize host dependencies of two novel Escherichia coli phages, the T1-like siphophage LL5 and the V5-like myophage LL12, forward genetic screens were conducted against the Keio collection, a library of single non-essential gene deletions in E. coli str. BW25113. These screens and subsequent experiments identified genes required by phages LL5 and LL12. E. coli mutants deficient in heptose II and the phosphoryl substituent of heptose I of the inner core lipopolysaccharide (LPS) were unable to propagate phage LL5, as were mutants deficient in the outer membrane protein TolC. Mutants lacking glucose I of the LPS outer core failed to propagate LL12. Two additional genes encoding cytoplasmic chaperones, PpiB and SecB, were found to be required for efficient propagation of phage LL5, but not LL12. This screening approach may be useful for identifying host factors dependencies of phages, which would provide valuable information for their potential use as therapeutics and for phage engineering.

Severe growth failure associated with a novel heterozygous nonsense mutation in the GHR transmembrane domain leading to elevated growth hormone binding protein.

To report a novel mutation in GHR and to characterize a novel mechanism of nonclassical growth hormone insensitivity. Laron syndrome (LS) is a well-described disorder of growth hormone insensitivity due to mutations in the growth hormone receptor (GHR) that leads to short stature. Biochemically, LS patients classically have elevated levels of growth hormone (GH), but low levels of insulin-like growth factor (IGF)-1, IGF binding protein (IGFBP)-3 and GH binding protein (GHBP). Case presentation with in vitro functional studies. A young male Caucasian child with short stature was found to have growth hormone insensitivity manifested by elevated levels of GH and GHBP. Growth hormone stimulation tests revealed baseline GH level of 20.9µg/L and maximum stimulated GH level of 52.7µg/L and GHBP level of 4868pmol/L. GHR gene sequencing revealed a novel heterozygous nonsense mutation (c.800G>A, p.Trp267*) in the transmembrane domain of the receptor. Immunoblot analysis of transfected GHR p.Trp267* in HEK293 revealed inhibition of GH-induced STAT5 signalling that was overcome with increasing doses of recombinant human GH. Using an in vitro model, we show that elevated levels of GHBP inhibit the action of GH. Furthermore, our studies demonstrate that this inhibition by GHBP can be overcome by increasing doses of recombinant human GH. To our knowledge, this is the first study to demonstrate in vitro that elevated levels of GHBP attenuate the effect of GH and inhibit GH-induced signalling, thereby leading to short stature. Though this inhibition was overcome in vitro with supraphysiologic doses of GH, significantly above endogenously available GH, it remains to be seen whether such an effect can be replicated in vivo.

A modality-specific dysfunction of pain processing in schizophrenia.

Clinical observations showed that schizophrenia (SCZ) patients reported little or no pain under various conditions that are commonly associated with intense painful sensations, leading to a higher risk of morbidity and mortality. However, this phenomenon has received little attention and its underlying neural mechanisms remain unclear. Here, we conducted two experiments combining psychophysics, electroencephalography (EEG), and functional magnetic resonance imaging (fMRI) techniques to investigate neural mechanisms of pain insensitivity in SCZ patients. Specifically, we adopted a stimulus–response paradigm with brief stimuli of different sensory modalities (i.e., nociceptive, non‐nociceptive somatosensory, and auditory) to test whether pain insensitivity in SCZ patients is supra‐modal or modality‐specific, and used EEG and fMRI techniques to clarify its neural mechanisms. We observed that perceived intensities to nociceptive stimuli were significantly smaller in SCZ patients than healthy controls, whereas perceived intensities to non‐nociceptive somatosensory and auditory stimuli were not significantly different. The behavioral results were confirmed by stimulus‐evoked brain responses sampled by EEG and fMRI techniques, thus verifying the modality‐specific nature of the modulation of nociceptive information processing in SCZ patients. Additionally, significant group differences were observed in the spectral power of alpha oscillations in prestimulus EEG and the seed‐based functional connectivity in resting‐state fMRI (seeds: the thalamus and periaqueductal gray that are key nodes in ascending and descending pain pathways respectively), suggesting a possible contribution of cortical–subcortical dysfunction to the phenomenon. Overall, our study provides insight into the neural mechanisms of pain insensitivity in SCZ and highlights a need for systematic assessments of their pain‐related diseases.

Combined transcriptome sequencing reveals the photoperiod insensitivity mechanism of oats

Avena sativa L. is the most important cultivated oat species worldwide. Although photoperiod-insensitive oat varieties exist, the molecular mechanisms underlying their photoperiod sensitivity are poorly understood. This study investigated the effects of day length on the fioral transition of oats and the mechanisms underlying oat photoperiod insensitivity. Photoperiod-sensitive and photoperiod-insensitive varieties, including gp012, were used in shading experiments, and the developing leaves and main shoot apices (MSAs) of the HONGQI2 and gp012 varieties were used for sequencing. Leaves and MSAs were collected in 2016, and their transcriptomes were sequenced. The photoperiod-insensitive varieties headed under both short-day and long-day conditions, while the photoperiod-sensitive varieties headed only under long-day conditions. A total of 60673 transcript sequences were obtained, 7932 of which were differentially expressed; 3194 and 4738 transcripts were differentially expressed in the leaves and MSAs, respectively. A total of 25793 transcripts were classified into 123 pathways based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The carbon metabolism pathways were dominant, followed by ribosome and protein processing in the endoplasmic reticulum. In addition, 203 transcripts were classified into the circadian rhythm pathway. Compared with the expression of pseudo-response regulator protein 37 (PRR37) in photoperiod-sensitive varieties, that in photoperiod-insensitive varieties was upregulated. Among the differentially expressed transcripts (DETs), 8 MADS-box genes were identified. PRR37 is a key regulator of oat photoperiod insensitivity. The obtained transcriptome dataset may provide a reference for analyzing oat transcript expression, and the results should be used as a reference for oat breeding and production.

Convergent Evolution of C239S Mutation in Pythium spp. β-Tubulin Coincides with Inherent Insensitivity to Ethaboxam and Implications for Other Peronosporalean Oomycetes.

Ethaboxam is a benzamide antioomycete chemical (oomicide) used in corn and soybean seed treatments. Benzamides are hypothesized to bind to β-tubulin, thus disrupting microtubule assembly. Recently, there have been reports of corn- and soybean-associated oomycetes that are insensitive to ethaboxam despite never having been exposed. Here, we investigate the evolutionary history and molecular mechanism of ethaboxam insensitivity. We tested the sensitivity of 194 isolates representing 83 species across four oomycete genera in the Peronosporalean lineage that were never exposed to ethaboxam. In all, 84% of isolates were sensitive to ethaboxam (effective concentration to reduce optical density at 600 nm by 50% when compared with the nonamended control [EC50] < 5 μg ml-1), whereas 16% were insensitive (EC50 > 11 μg ml-1). Of the insensitive isolates, two different transversion mutations were present in the 239th codon in β-tubulin within three monophyletic groups of Pythium spp. The transversion mutations lead to the same amino acid change from an ancestral cysteine to serine (C239S), which coincides with ethaboxam insensitivity. In a treated soybean seed virulence assay, disease severity was not reduced on ethaboxam-treated seed for an isolate of Pythium aphanidermatum containing a S239 but was reduced for an isolate of P. irregulare containing a C239. We queried publicly available β-tubulin sequences from other oomycetes in the Peronosporalean lineage to search for C239S mutations from other species not represented in our collection. This search resulted in other taxa that were either homozygous or heterozygous for C239S, including all available species within the genus Peronospora. Evidence presented herein supports the hypothesis that the convergent evolution of C239S within Peronosporalean oomycetes occurred without selection from ethaboxam yet confers insensitivity. We propose several evolutionary hypotheses for the repeated evolution of the C239S mutation.

Status of insecticide resistance and its biochemical and molecular mechanisms in Anopheles stephensi (Diptera: Culicidae) from Afghanistan

BackgroundInsecticide resistance of Anopheles stephensi, the main malaria vector in eastern Afghanistan, has been reported previously. This study describes the biochemical and molecular mechanisms of resistance to facilitate effective vector control and insecticide resistance management.MethodsMosquito larvae were collected from the provinces of Kunar, Laghman and Nangarhar from 2014 to 2017. The susceptibility of the reared 3–4 days old adults was tested with deltamethrin 0.05%, bendiocarb 0.1%, malathion 5%, permethrin 0.75% and DDT 4%. Cytochrome P450 content and general esterase, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities were measured in the three field populations and the results were compared with those of the laboratory susceptible An. stephensi Beech strain. Two separate allele-specific PCR assays were used to identify L1014, L1014F and L1014S mutations in the voltage gated sodium channel gene of An. stephensi. Probit analysis, ANOVA and Hardy–Weinberg equilibrium were used to analyse bioassay, biochemical assay and gene frequency data respectively.ResultsThe population of An. stephensi from Kunar was susceptible to bendiocarb, apart from this, all populations were resistant to all the other insecticides tested. The differences between all values for cytochrome P450s, general esterases, GSTs and AChE inhibition rates in the Kunar, Laghman and Nangarhar populations were statistically significant when compared to the Beech strain, excluding GST activities between Kunar and Beech due to the high standard deviation in Kunar. The three different sodium channel alleles [L1014 (wild type), L1014F (kdr west) and L1014S (kdr east)] were all segregated in the Afghan populations. The frequencies of kdr east mutation were 22.9%, 32.7% and 35% in Kunar, Laghman and Nangarhar populations respectively. Kdr west was at the lowest frequency of 4.44%.ConclusionsResistance to different groups of insecticides in the field populations of An. stephensi from Kunar, Laghman and Nangarhar Provinces of Afghanistan is caused by a range of metabolic and site insensitivity mechanisms, including esterases, cytochrome P450s and GSTs combined with AChE and sodium channel target site insensitivity. The intensity and frequency of these mechanisms are increasing in these populations, calling for urgent reorientation of vector control programmes and implementation of insecticide resistance management strategies.

Establishing a model of primary bovine hepatocytes with responsive growth hormone receptor expression

The liver becomes resistant to growth hormone before parturition in dairy cows (uncoupling of the somatotropic axis). However, the mechanism of growth hormone insensitivity has not been fully described. The aim of the present study was to improve a previous model of adult bovine hepatocytes in a sandwich culture system to ensure growth hormone receptor (GHR) expression. First, we modified the protocol for hepatocyte retrieval and tested the effect of short (18 min) and long (up to 30 min) warm ischemia on hepatocyte viability. Second, we used medium additives that affect GHR expression in vivo-insulin (INS), dexamethasone (DEX), both (INS+DEX), or no hormone additives (CTRL)-to ensure the functionality of hepatocytes, as measured by lactate dehydrogenase activity and urea concentration in the medium. We also used reverse transcriptase PCR of hepatocytes to evaluate expression of albumin (ALB), hepatocyte nuclear factor 4α (HNF4A), nuclear factor-κ-B-inhibitor α (NFKBIA), cytosolic phosphoenolpyruvate carboxykinase (PCK1), and vimentin (VIM) mRNA. Moreover, we analyzed the expression of GHRtot (GHR), GHR1A, insulin-like growth factor-1 (IGF1), and IGF binding protein-2 (IGFBP2) in response to exposure to media with the different compositions. Modification of the protocol (changes in rinsing and perfusion times, buffer composition, and the volume and standardization of collagenase) led to increased cell counts and cell viability. Short warm ischemia with the modified protocol significantly increased cell count (4.7 × 107 ± 1.9 × 107 vs. 3.5 × 106 ± 1.5 × 106 vital cells/g of liver) and viability (79.1 ± 8.4 vs. 37.1 ± 8.9%). Therefore, we gathered hepatocytes from the liver after short warm ischemia with the modified protocol. For these hepatocytes, lactate dehydrogenase activity was lower in media with INS and with DEX than in media with INS+DEX or CTRL; urea concentrations were highest at d 4 for INS+DEX. As well, HNF4A and ALB were more highly expressed in hepatocytes cultured with INS and INS+DEX than in those cultured with DEX or CTRL, and the substitution of DEX suppressed VIM and NFKBIA expression but increased PCK1 expression. The expression of GHR, GHR1A, and IGF1 was suppressed by dexamethasone (DEX and INS+DEX), whereas INS distinctly increased GHR, GHR1A, and IGF1 mRNA expression. Hepatocytes in a sandwich culture showed influenceable GHR expression; this study provides a model that can be used in studies examining factors that influence the expression and signal transduction of GHR in dairy cows.

AlGaN avalanche Schottky diodes with high Al-content

AlxGa1−xN-based avalanche photodiodes with a Schottky-contact grown on AlN bulk substrate with an Al-content of x = 0.68 have been examined with respect to their structural and electro-optical properties. The Schottky diodes suitable for the solar-blind ultraviolet spectral regime show avalanche gain for voltages in excess of 25 V reverse bias in linear gain mode under frontside illumination. For 60 V reverse bias, avalanche multiplication exceeding 104 was obtained. The devices were operated well below breakdown; the measured current for frontside illuminated conditions exceeds the dark-condition characteristics by more than two orders of magnitude at this voltage. The Schottky-barrier height and the ideality n were extracted to eV and at room temperature, respectively. Temperature dependent measurements indicate a temperature insensitive dark current mechanism with an enhanced multiplication process towards lower temperatures.

Coseismic ultramylonites: An investigation of nanoscale viscous flow and fault weakening during seismic slip

Faults weaken during the propagation of earthquakes due to the onset of thermally-activated mechanisms, which vary depending on the rock type. Recent experimental work suggests that carbonate-hosted faults are lubricated by viscous flow in nano-granular aggregates having ultramylonitic textures. However, their frail nature has often hindered unbiased characterisation of the textures and deformation mechanisms operating at such extreme conditions (strain rates as high as 104), which remain so far poorly investigated and understood.We explore the formation, evolution and deformation mechanisms of coseismic ultramylonites in carbonate-hosted faults generated during high velocity (1.4 ms−1), displacement-controlled shear experiments in a rotary apparatus. Microstructures were analysed using integrated SEM and TEM imaging while detailed crystallographic fabrics were investigated using the electron back-scattered diffraction (EBSD) technique.Mechanical data show that the strength of the experimental fault decays dynamically with slip, according to a characteristic four stage evolution; each stage is associated with characteristic textures. Microstructural observations show that brittle processes dominate when the fault is strong (friction coefficients >0.6). Cataclasis, aided by twinning and crystal plasticity, operates forming an extremely comminuted shear band (mean grain size ∼200 nm). As the fault starts weakening, shear localises within a well-defined principal slip zone. Here, thermally-activated grain size sensitive (GSS) and insensitive (GSI) creep mechanisms compete with brittle processes in controlling fault strength. GSI mechanisms produce strong monoclinic crystallographic preferred orientations in the slip zone, while textures and crystallographic orientations in adjacent locations do not evolve from the previous deformation stage. By the end of the transient weakening stage, the slip zone has reached a steady state thickness (30 μm) and shows a nanogranular ultramylonitic texture. The intensity of the crystallographic preferred orientation in the coseismic ultramylonite is reduced compared to the previous stage, due to grainsize sensitive creep mechanisms becoming gradually more dominant. As the experimental fault re-strengthens, upon deceleration to arrest, the ultramylonite may be partially reworked by brittle deformation.Our findings show that the crystallographic orientations of transient microstructures are preserved in the slip zone of coseismic ultramylonites and in narrow, adjacent deactivated layers, where mirror-like surfaces are located. This shows that EBSD techniques can usefully be employed to determine the deformation mechanisms of coseismic ultramylonites and their evolution during earthquake slip in both experimental and, potentially, natural faults.

Combination of eribulin plus AKT inhibitor evokes synergistic cytotoxicity in soft tissue sarcoma cells

An activated AKT pathway underlies the pathogenesis of soft tissue sarcoma (STS), with over-expressed phosphorylated AKT (p-AKT) correlating with a poor prognosis in a subset of STS cases. Recently, eribulin, a microtubule dynamics inhibitor, has demonstrated efficacy and is approved in patients with advanced/metastatic liposarcoma and breast cancer. However, mechanisms of eribulin resistance and/or insensitivity remain largely unknown. In this study, we demonstrated that an increased p-AKT level was associated with eribulin resistance in STS cells. We found a combination of eribulin with the AKT inhibitor, MK-2206, synergistically inhibited STS cell growth in vivo as well as in vitro. Mechanistically, eribulin plus MK-2206 induced G1 or G2/M arrest by down-regulating cyclin-dependent kinases, cyclins and cdc2, followed by caspase-dependent apoptosis in STS cells. Our findings demonstrate the significance of p-AKT signaling for eribulin-resistance in STS cells and provide a rationale for the development of an AKT inhibitor in combination with eribulin to treat patients with STS.

Quartz deformation across interlayered monomineralic and polymineralic rocks: A comparative analysis

Given the increased availability of EBSD and fabric analyzer facilities, quartz microstructural and CPO analysis have become ‘tools of-the-trade’ for studies investigating crustal tectonics. Therefore, improved understanding of the complexities of quartz deformation is needed to ensure that such analyses distinguish primarily between tectonic outcomes and artefacts of the quartz deformation behaviours in different deformed rock volumes. We contribute to this understanding by comparing quartz deformation between monomineralic rocks, which have simple compositions; and polymineralic rocks, which are more complex and representative of the crust. Mylonite quartzites and gneisses of the Main Central Thrust (NW Himalaya) are used for comparison.Under similar deformation conditions, quartz inside monomineralic rocks forms well-developed CPOs and dynamically recrystallized microstructures, typical of grain size insensitive (GSI) creep mechanisms. Quartz inside polymineralic rocks, by contrast, exhibits weak CPOs and fine-grained microstructures commonly interpreted as grain size sensitive creep; but also exhibits lattice distortions (e.g. undulose extinction, sub-grain boundaries), associated with GSI creep. We postulate that intrinsic factors, such as interphase boundaries, cause these ambiguous results. The lack of clear evidence for deformation mechanisms in polymineralic rocks casts doubt on the reliability of quartz microstructures and CPOs for rapid diagnoses of their rheology and broader tectonic significance.

Characterisation of Resistance to Fenitrothion Insecticide and Associated Mechanisms inCulex pipiens pipiens(Diptera: Culicidae) Collected in Three Districts Across Tunisia

Previous studies on Culex pipiens pipiens mosquitoes collected from Tunisia between 1990 and 1996 revealed an alarming resistance to organophosphate insecticides. However, since then very few studies have assessed the evolution of insecticides resistance in this species of mosquitoes. Three populations of Cx. pipiens pipiens mosquitoes were collected in three districts across the country between 2003 and 2005 and subjected to evaluation of their fenitrothion resistance/susceptibility status and associated mechanisms. Studied populations showed moderate resistance to the used insecticide which ranged from 7.59 to 11.8. The use of synergists revealed the lack of detoxification enzymes as metabolic resistance. However, starch gel electrophoresis indicated that resistance at the Ester locus was attributed to different Ester alleles. On the other hand, the positive cross-resistance to organophosphate fenitrothion and carbamate propoxur insecticides indicating a high prevalence of insensitive AChE1 mechanism in field populations. AChE activity showed that all populations exhibited a significant excess of heterozygotes ([RS]) compared to other detected genotypes ([SS] and [RR]) in the studied samples. Our analysis suggests that the heterozygotes often have a fitness advantage over the homozygotes.

Growth Hormone Did Not Activate Its Intracellular Signaling Molecules in Rats' Liver Hepatocytes During Early Life Period.

BackgroundAlthough growth hormone (GH) has essential roles in the growth of animals, it has no growth-promoting effect during infancy period. The molecular mechanism underlying lack of growth-promoting effect of GH during infancy period remains unclear. Important signaling pathways are mediated by GH, including Janus kinase 2 (JAK2), extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducers, and activators of transcription 5, 3, and 1 (STATs 5, 3 and 1).ObjectivesThis study explored the underlying molecular mechanisms driving to the lack of growth-promoting effect of GH in the early stage of life by in vivo assessment of intracellular signal response (STAT5/ 3/ 1, JAK2 and ERK1/ 2) to GH at different physiological stages.MethodsIn this study, five age groups of rats (1-, 4-day-old, and 1-, 2-, 3-week-old) were selected. The rats were anesthetized using pentobarbital (100 mg/kg) and then received the rat GH (2mg/kg) via inferior vena cava injection. The control rats were injected with normal saline during the same period. The intracellular signal response to GH was assessed by Western blot analysis.ResultsJAK2 and STAT5 were expressed in 1-day and 4-day-old newborn rats and their expression levels were comparable with the levels of the 1-, 2-, and 3-week-old rats; however, JAK2/STAT5 phosphorylation was not observed in 1-day-old and 4-day-old newborn rats after stimulation with GH in the liver. Similar to JAK2 and STAT5, we did not detect STAT3/1 activation during infancy stages although basic STAT3 and STAT1 were also expressed in hepatocytes from newborn rats. In addition we detected ERK1/2 activation in 4-day-old, 1-, 2-, and 3-week-old rats but not in 1-day-old rats.ConclusionsJAK2, STAT5, STAT3, STAT1, and ERK1/2 were not simultaneously activated by GH in newborn rats; this finding may be one of the underlying mechanism of GH insensitivity in newborn rats.

Tyrosine kinase inhibitor-induced IL-6/STAT3 activation decreases sensitivity of EGFR-mutant non-small cell lung cancer to icotinib.

Tyrosine kinase Inhibitors (TKIs) of epidermal growth factor receptor (EGFR) has considerably benefited for non-small cell lung carcinomas (NSCLC) harbor mutations in EGFR. However, the factors attenuating EGFR-TKI efficiency are obstacles to inhibit the proliferation of EGFR-mutant NSCLC cells successfully. Clarifying the insensitivity mechanisms of EGFR-TKI would help to develop new treatment strategy. In this study, the sensitivity of EGFR-mutant NSCLC cell lines, PC9 and HCC827, to icotinib was detected. Similar with other EGFR-TKIs such as gefitinib and erlortinib in previous research, the proliferation of two cell lines was apparently inhibited. However, we surprisingly found that contrast with the suppression of EGFR-AKT/ERK pathway, STAT3 was significantly activated in PC9 cells with the treatment of icotinib, but not in HCC827 cells. Further study confirmed that icotinib concomitantly induced IL-6 secretion and src activation in PC9 cells. Moreover, with the treatment of IL-6 neutralizing antibody or src inhibitor, dasatinib, icotinib-induced phosphorylation of STAT3 was reduced, as well as the sensitivity of PC9 to icotinib was also partially increased. Our results suggest that Src/IL-6/STAT3 bypass pathway is activated to maintain cell survival when the EGFR pathway was inhibited by TKIs, even in some EGFR-mutant NSCLC cells sensitive to TKIs. This finding provides a groundwork for potential combinatorial treatment with TKIs and Src or STAT3 inhibitor to improve icotinib sensitivity.

Abstract P4-04-12: Therapeutic targeting of CDK4/6 inhibitor resistant breast cancer

Abstract Background: Selective inhibitors of CDK4/6 kinases (CDK4/6i) were recently FDA approved for use in combination with endocrine therapy (ET), and represent the new standard of care. There are however patients who do not respond or develop resistance to these treatments, and therapies are required in this setting. While there is emerging data on the mechanisms of intrinsic insensitivity to CDK4/6i as monotherapies, which include cyclin E1 amplification, CDK6 amplification and Rb deletion, there is little data on mechanisms of resistance to combined ET and CDK4/6i. Methods: We established MCF7 cell line and patient-derived xenograft (PDX) models that are resistant to combined ET and Palbociclib (CDK4/6i) through long-term culture, allowing us to better understand mechanisms underlying CDK4/6i resistance and to model therapeutic strategies in this setting. We also evaluated our therapeutic strategy in vitro and in vivo using MCF cell lines that are resistant to ET, and in an ER+ PDX model derived from a patient who progressed on ET. Results: Cells resistant to CDK4/6i alone and in combination with ET show disrupted senescent pathways, and insensitivity to the induction of senescence. MDM2 inhibitors induce cells to enter into senescence, and consequently we are investigating the use of a new generation MDM2 inhibitor (CGM097, Novartis) either in combination with CDK4/6i treatment, or following acquisition of CDK4/6i resistance to prevent exit from senescence. We evaluated a CGM097 either in combination with CDK4/6i treatment, or in combination with fulvestrant following acquisition of CDK4/6i resistance to prevent exit from senescence. CGM097 was effective alone or in combination with fulvestrant in CDK4/6i resistant cells in vitro and in vivo, and resulted in a loss of G1 cells, and a reduction in B galactosidase, a senescence marker. Another mechanisms of CDK4/6i resistance that has been identified is CDK2 activation, which can occur through Cyclin E amplification. As a second therapeutic strategy, we screened a panel of pan-CDK inhibitors with CDK2 activity in our resistant lines, and identified that CYC065 (Cyclacel), a highly selective CDK2/9 inhibitor, had the most durable response and highest synergy with ET in long-term culture. The combined resistant models were sensitive to CYC065 in vitro and in vivo. CYC065 was mechanistically distinct to CDK4/6i's as it caused arrest in a different phase of the cell cycle and affected expression of different cell cycle proteins. Conclusion: An underlying mechanism of combined ET and CDK4/6i resistance is senescent escape, which allows for normal proliferation upon removal of the drug. Using our in vitro and in vivo models of combined ET and CDK4/6i resistance, we have identified two novel therapeutic strategies for this disease, which represents the next clinical challenge in ER+ breast cancer as the natural history of disease is changed with the increasing use of CDK4/6i. Citation Format: Lim E, Portman N, Alexandrou S, Haupt S, Haupt Y, Caldon E. Therapeutic targeting of CDK4/6 inhibitor resistant breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-12.

Fracturing, fluid flow and shear zone development: Relationships between chemical and mechanical processes in Proterozoic mafic dykes from southwestern Montana, USA

AbstractFluids can play an important role in the localization of deformation in the deep crust, yet the specific mechanisms active during the complex interactions between metasomatism, metamorphism and deformation remain elusive. Precambrian metagabbronorite dykes in southwest Montana contain fractures filled with Hbl±Grt and discrete cm‐scale shear zones with well‐preserved strain gradients. This system offers an ideal opportunity to constrain the chemical and mechanical processes that facilitated strain localization. An early M1 assemblage of Grt1+Cpx1+Pl1+Qz developed at conditions of 0.51–0.85 GPa and 500–700°C and is preserved largely as a static replacement of relict igneous phases (Opx, Pgt, Pl) in coronitic textures. An M2 assemblage characterized by Grt2+Pl2±Cpx2+Hbl+Scp+Qz developed at 0.86–1.00 GPa and 660–730°C coincided with fluid flow and deformation associated with shear zone development. Microstructural observations in marginal protomylonite/mylonite and laminated ultramylonite suggest a shear zone evolution that involved (1) nucleation from pre‐existing fractures that were sites for major fluid infiltration, (2) initial widening coincident with grain‐size reduction by microfracturing, dislocation creep, and synkinematic metamorphic reaction by solution transfer, and (3) a switch in the dominant deformation mechanisms active in the ultramylonite from grain‐size insensitive mechanisms to grain‐size sensitive granular flow accommodated by fluid‐assisted diffusion. Throughout this evolution, the effective bulk compositions of the rock volumes responding to metamorphism changed through a combination of mechanical and metasomatic processes.

Microstructural and rheological evolution of calcite mylonites during shear zone thinning: Constraints from the Mount Irene shear zone, Fiordland, New Zealand

Layers of calc-mylonite in the Mount Irene shear zone, Fiordland, New Zealand, show substantial variations in thickness due to deflection of the shear zone boundaries around wall rock asperities. In relatively thick parts (c. 2.6 m) of the shear zone, calcite porphyroclasts are internally strained, contain abundant subgrain boundaries and have a strong shape preferred orientation (SPO) and crystallographic preferred orientation (CPO), suggesting that deformation occurred mainly by dislocation creep involving subgrain-rotation recrystallization. In relatively thin parts (c. 1.5 m) of the shear zone, aggregates of fine-grained recrystallized calcite surrounding flattened porphyroclasts have a weak SPO and CPO, and contain polygonal calcite grains with low degrees of internal misorientation. The recrystallized aggregates also contain microstructures (e.g. grain quadruple junctions, randomized misorientation axes) similar to those reported for neighbor-switching processes during grain-boundary sliding. Comparison of subgrain sizes in the porphyroclasts to published grain-size differential-stress relationships indicates that stresses and strain rates were substantially higher in relatively thin parts of the shear zone. The primary microstructural response to higher stresses and strain rates was an increase in the amount of recrystallization to produce aggregates that deformed by grain-boundary sliding. However, even after the development of interconnected networks of recrystallized grains, dislocation creep by subgrain-rotation recrystallization continued to occur within porphyroclasts. This behavior suggests that the bulk rheology of shear zones undergoing thinning and thickening can be controlled by concomitant grain-size insensitive and grain-size sensitive mechanisms. Overall, our observations show that shear zone thickness variations at constant P-T can result in highly variable stresses and strain rates, which in turn modifies microstructure, deformation mechanism and shear zone rheology.

High Plasmodium Infection Rate and Reduced Bed Net Efficacy in Multiple Insecticide-Resistant Malaria Vectors in Kinshasa, Democratic Republic of Congo.

Accounting for approximately 11% of all malaria cases, the Democratic Republic of the Congo (DRC) is central to malaria elimination efforts. To support vector control interventions in DRC, we characterized the dynamics and impact of insecticide resistance in major malaria vectors in 2015. High Plasmodium infection rates were recorded in Anopheles gambiae and Anopheles funestus, with Plasmodium falciparum predominant over Plasmodium malariae. Both mosquito species exhibited high and multiple resistance to major public health insecticide classes. The extremely high resistance to permethrin and DDT (dichlorodiphenyltrichloroethane) in An. gambiae (low mortalities after 6 hours exposure) is worrisome, and is supported by a reduced insecticidal effect of bed nets against both mosquito species in laboratory tests. Metabolic and target site insensitivity mechanisms are driving this resistance in An. gambiae, but only the former was observed in An. funestus. These findings highlight the urgent need for actions to prolong the effectiveness of insecticide-based interventions in DRC.