Chemodiversity and Bioactivity of the Essential Oils of Juniperus and Implication for Taxonomy.

The essential oils of Juniperus are highly beneficial medicinally. The present study aimed to assess the chemodiversity and bioactivity of Juniperus formosana, Juniperus przewalskii, Juniperus convallium, Juniperus tibetica, Juniperus komarovii, and Juniperus sabina essential oils from the Qinghai-Tibet Plateau. The results revealed 92 components in six essential oils: α-pinene (2.71-17.31%), sabinene (4.91-19.83%), and sylvestrene (1.84-8.58%) were the main components. Twelve components were firstly reported in Juniperus oils, indicating that the geographical location and climatic conditions of the Qinghai-Tibet Plateau produced the unique characteristics of Juniperus essential oils. The chemodiversity of Juniperus essential oils varied greatly, with J. sabina having the most recognized components (64) and the highest chemodiversity (Shannon-Wiener index of 3.07, Simpson's diversity index of 0.91, and Pielou evenness of 0.74). According to the chemodiversity of essential oils, the six plants were decided into the α-pinene chemotype (J. formosana), hedycaryol chemotype (J. przewalskii, J. komarovii, J. convallium, J. tibetica), and sabinene chemotype (J. sabina). PCA, HCA and OPLS-DA showed that J. formosana and J. sabina were distantly related to other plants, which provides a chemical basis for the classification of Juniperus plants. Furthermore, bioactivity tests exhibited certain antioxidant and antibacterial effects in six Juniperus oils. And the bioactivities of J. convallium, J. tibetica, and J. komarovvii were measured for the first time, broadening the range of applications of Juniperus. Correlation analysis of components and bioactivities showed that δ-amorphene, ß-udesmol, α-muurolol, and 2-nonanone performed well in the determination of antioxidant activity, and α-pinene, camphene, ß-myrcene, as well as (E)-thujone, had strong inhibitory effects on pathogenic bacteria, providing a theoretical basis for further research on these components.

Effects of changing culture medium on preimplantation embryo development in rabbit.

Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.

Sex-Related Differences on Chemical Composition, Anatomy, Histochemistry, and Biological Activities of Juniperus rigida.

Sex-related differences on phenolic profiles, chemical composition of essential oils, anatomy, histochemistry and biological activities (antioxidant and antibacterial activities) of Juniperus rigida needles collected from Yijun and Fugu region were first studied. In two regions, female and male had similar contents of total phenolic and total flavonoid. 10 phenolic compounds were analyzed by RP-HPLC, amentoflavone content was significantly higher in female than male in Yijun, and chlorogenic acid content was significantly higher in female than male in Fugu. 30 compounds (over 0.5 %) were detected in the essential oils, and the total contents of female were lower than male in Yijun. This difference mainly comes from Germacrene D, which was about twice as high in male as in female. Male needles had significantly larger mechanical tissue and phloem in Yijun. Histochemical analysis indicated that the phenols were stored in epidermal cells, sponge tissue, endodermis cells, edge of resin duct, stomatal bands, and the flavonoids were stored in epidermal cells, endodermis cells, edge of resin duct, stomatal bands. No sex-related differences were found in histochemical analysis, antioxidant activities (ABTS, FRAP) and antibacterial activities (9 strains). This preliminary study provided a reference for production practice and theoretical research of J. rigida.

Sperm-borne small RNAs improve the developmental competence of pre-implantation cloned embryos in rabbit.

The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit.

Vanillic acid mitigates the ovalbumin (OVA)-induced asthma in rat model through prevention of airway inflammation.

Asthma is a chronic allergic ailment affecting a considerably large population of the world. The aim of this study is to evaluate the ameliorative effects of vanillic acid against ovalbumin (OVA)-induced asthma in rat model. Asthma was induced in Sprague Dawley rats and vanillic acid was orally administered at 25 and 50 mg/kg body weight for 28 days. Rats challenged with OVA showed heavy signs of airway inflammation and remodeling similar to chronic asthma, evidenced by the increased differential cell counts and presence of inflammatory cytokines in the bronchoalveolar lavage fluid (BALF), along with elevated serum immunoglobulin levels, and the histological results. However, vanillic acid dose-dependently attenuated the manifestation of OVA-induced asthma (p < 0.05) through suppression of inflammatory mediators and modulation of immunoglobulin levels in rats. The asthma mitigating properties of vanillic acid might be due to suppression of oxidative stress and prevention of lung airway inflammation.

The global burden of ischemic heart disease attributed to high fasting plasma glucose: Data from 1990 to 2019.

Background: Ischemic heart disease (IHD) is the leading cause of death worldwide. High fasting plasma glucose (FPG) is an increasing risk factor for IHD. We aimed to explore the long-term trends of high FPG-attributed IHD mortality during 1990-2019. Methods: Data were obtained from the Global Burden of Disease Study 2019 database. Deaths, disability-adjusted life-years (DALYs), the age-standardized mortality rate (ASMR) and age-standardized DALY rate (ASDR) of IHD attributable to high FPG were estimated by sex, socio-demographic index (SDI), regions and age. Estimated annual percentage changes (EAPCs) were calculated to assess the trends of ASMR and ASDR of IHD attributable to high FPG. Results: IHD attributable to high FPG deaths increased from 1.04 million (0.62-1.63) in 1990 to 2.35 million (1.4-3.7) in 2019, and the corresponding DALYs rose from 19.82 million (12.68-29.4) to 43.3 million (27.8-64.2). In 2019, ASMR and ASDR of IHD burden attributable to high FPG were 30.45 (17.09-49.03) and 534.8 (340.7-792.2), respectively. The highest ASMR and ASDR of IHD attributable to high FPG occurred in low-middle SDI quintiles, with 39.28 (22.40-62.76) and 742.3 (461.5-1117.5), respectively, followed by low SDI quintiles and middle SDI quintiles. Males had higher ASMR and ASDR compared to females across the past 30 years. In addition, ASRs of DALYs and deaths were highest in those over 95 years old. Conclusion: High FPG-attributed IHD mortality and DALYs have increased dramatically and globally, particularly in low, low-middle SDI quintiles and among the elderly. High FPG remains a great concern on the global burden of IHD and effective prevention and interventions are urgently needed to curb the ranking IHD burden, especially in lower SDI regions.

Effect of Dietetic Obesity on Testicular Transcriptome in Cynomolgus Monkeys.

Obesity is a metabolic disorder resulting from behavioral, environmental and heritable causes, and can have a negative impact on male reproduction. There have been few experiments in mice, rats, and rabbits on the effects of obesity on reproduction, which has inhibited the development of better treatments for male subfertility caused by obesity. Nonhuman primates are most similar to human beings in anatomy, physiology, metabolism, and biochemistry and are appropriate subjects for obesity studies. In this investigation, we conducted a transcriptome analysis of the testes of cynomolgus monkeys on high-fat, high-fructose, and cholesterol-rich diets to determine the effect of obesity on gene expression in testes. The results showed that the testes of obese monkeys had abnormal morphology, and their testes transcriptome was significantly different from that of non-obese animals. We identified 507 differentially abundant genes (adjusted p value < 0.01, log2 [FC] > 2) including 163 up-regulated and 344 down-regulated genes. Among the differentially abundant genes were ten regulatory genes, including IRF1, IRF6, HERC5, HERC6, IFIH1, IFIT2, IFIT5, IFI35, RSAD2, and UBQLNL. Gene ontology (GO) and KEGG pathway analysis was conducted, and we found that processes and pathways associated with the blood testes barrier (BTB), immunity, inflammation, and DNA methylation in gametes were preferentially enriched. We also found abnormal expression of genes related to infertility (TDRD5, CLCN2, MORC1, RFX8, SOHLH1, IL2RB, MCIDAS, ZPBP, NFIA, PTPN11, TSC22D3, MAPK6, PLCB1, DCUN1D1, LPIN1, and GATM) and down-regulation of testosterone in monkeys with dietetic obesity. This work not only provides an important reference for research and treatment on male infertility caused by obesity, but also valuable insights into the effects of diet on gene expression in testes.

Unraveling the mechanism of raffinose utilization in Ligilactobacillus salivarius Ren by transcriptomic analysis.

In the gastrointestinal tract, some dietary carbohydrates, such as xylose, raffinose and arabinose, are able to stimulate the growth of Lactobacillus and Bifidobacterium. In this study, the growth rate of Ligilactobacillus salivarius Ren in raffinose was 0.91 ± 0.03 h-1, which was higher than that in glucose (0.83 ± 0.01 h-1). However, limited information is available on specific transporters and glycoside hydrolases responsible for raffinose uptake and catabolism in L. salivarius. Transcriptomic analysis revealed the differential expression of 236 genes (∣log2FoldChange∣ > 0.8) in response to raffinose, which were mainly associated with raffinose transport, raffinose hydrolysis, galactose metabolism and pyruvate metabolism. Notably, gene rafP encoding lactose/raffinose permease was 101.86-fold up-regulated. Two α-galactosidase gene galA1 and galA2 were 117.82-fold and 2.66-fold up-regulated, respectively. To further investigate the role of these genes in raffinose utilization, insertional inactivation was performed using the pORI28-pTRK669 system. The growth assay of these mutants in modified MRS containing 2% (w/v) raffinose indicated that RafP played an important role in raffinose transport and GalA1 was the primary enzyme involved in raffinose hydrolysis. To our knowledge, this is the first report on the molecular mechanism of raffinose utilization in L. salivarius. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03280-6.

Loss of Renewal of Extracellular Vesicles: Harmful Effects on Embryo Development in vitro.

Background: Extracellular vesicles (EVs), as a promising platform for drug delivery, have attracted much attention. Degradation and regeneration of EVs maintain their homeostasis in vivo, but this regeneration is missing in the in vitro culture (IVC) system, which is likely to lead to negative effects. It is particularly concerning that most studies involving the addition of EVs in IVC seem to overlook this point. Methods: We used rabbit embryos and oviduct fluid EVs as a model of embryo development to examine the effect of loss or gain of EV functionality in an IVC system. Embryonic development ratios were determined in each group. Malondialdehyde and ammonium ions in the culture medium were measured. RNA-seq, reactive oxygen species (ROS) staining, immunofluorescence of LC3 and H3K36me3, and qPCR of oxidative stress-related genes and autophagy-related genes of blastocysts in the in vivo group, non-EVs group, con-EVs group, and R-EVsM group was implemented. Results: Incubation of embryos with 9.1×1011 EV particles/mL had a positive effect at 48 h and 72 h, which disappeared by 96 h, however. EVs at a concentration of 9.1×1012 particles/mL even showed a negative effect at 96 h. As culture time in the IVC system was increased, the amount of malondialdehyde and ammonium ions in the culture medium was increased, and there was a decrease in embryonic development activity of EVs. Lack of EV renewal in the IVC system impaired embryonic development competence, while replacement of EVs and medium during IVC could sustain embryonic development. Loss or gain of renewal in the IVC system affected EVs' influence on embryo transcriptome, embryonic ROS, autophagy, epigenetic state and apoptosis. Conclusion: Loss of renewal in the IVC system affected EVs' role in embryonic development by causing an imbalance in ROS, autophagy, abnormal H3K36me3 levels and apoptosis, while gain of renewal in the IVC system reduced these adverse effects and ensured the beneficial function of EVs.

Targeting the Mevalonate Pathway to Overcome Acquired Anti-HER2 Treatment Resistance in Breast Cancer.

Despite effective strategies, resistance in HER2+ breast cancer remains a challenge. While the mevalonate pathway (MVA) is suggested to promote cell growth and survival, including in HER2+ models, its potential role in resistance to HER2-targeted therapy is unknown. Parental HER2+ breast cancer cells and their lapatinib-resistant and lapatinib + trastuzumab-resistant derivatives were used for this study. MVA activity was found to be increased in lapatinib-resistant and lapatinib + trastuzumab-resistant cells. Specific blockade of this pathway with lipophilic but not hydrophilic statins and with the N-bisphosphonate zoledronic acid led to apoptosis and substantial growth inhibition of R cells. Inhibition was rescued by mevalonate or the intermediate metabolites farnesyl pyrophosphate or geranylgeranyl pyrophosphate, but not cholesterol. Activated Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) and mTORC1 signaling, and their downstream target gene product Survivin, were inhibited by MVA blockade, especially in the lapatinib-resistant/lapatinib + trastuzumab-resistant models. Overexpression of constitutively active YAP rescued Survivin and phosphorylated-S6 levels, despite blockade of the MVA. These results suggest that the MVA provides alternative signaling leading to cell survival and resistance by activating YAP/TAZ-mTORC1-Survivin signaling when HER2 is blocked, suggesting novel therapeutic targets. MVA inhibitors including lipophilic statins and N-bisphosphonates may circumvent resistance to anti-HER2 therapy warranting further clinical investigation. IMPLICATIONS: The MVA was found to constitute an escape mechanism of survival and growth in HER2+ breast cancer models resistant to anti-HER2 therapies. MVA inhibitors such as simvastatin and zoledronic acid are potential therapeutic agents to resensitize the tumors that depend on the MVA to progress on anti-HER2 therapies.

Aucubin protects against lipopolysaccharide-induced acute pulmonary injury through regulating Nrf2 and AMPK pathways.

Aucubin (Ai), a natural compound isolated from plants, including Aucuba japonica and Eucommia ulmoides, shows significant anti-inflammatory and anti-oxidative bioactivities. Here, we attempted to explore the protect effects of Ai on LPS-induced acute lung injury (ALI). Our results indicated that Ai increased the survival rate and ameliorated pathogenic processes in lipopolysaccharide (LPS)-induced mice. However, nuclear factor erythroid 2-related factor 2 (Nrf2) deletion may impede protective effect of Ai. Additionally, Ai reduced oxidative stress by down-regulating malondialdehyde (MDA) and O2· activity, and enhancing Nrf2-targeted signals, including heme oxygenase-1 (HO-1) and quinone oxidoreductase-1 (NQO-1). Also, Ai inhibited pro-inflammatory cytokines and phosphorylated-nuclear factor-κB (NF-κB) expression in LPS-administrated mice. However, these protective effects of Ai were suppressed in Nrf2-knockout mice. Importantly, Nrf2-deficiency showed no effects on phosphorylated AMP-activated protein kinase (p-AMPK) expression in mice treated with LPS and Ai. Similarly, in LPS-induced macrophages, Ai reduced reactive oxygen species (ROS) generation, elevated NQO-1 and HO-1 expression. LPS-stimulated pro-inflammatory cytokines and p-NF-κB were reversed by Ai. Of note, we found that Ai-induced Nrf2 activation was dependent on AMPK activation. Suppression of AMPK levels may inhibit Nrf2 activation, finally leading to up regulation of inflammatory response and oxidative stress. Thus, our findings indicated the crosstalk between Nrf2 and AMPK signaling pathways, and the interaction was essential for the anti-oxidant and anti-inflammatory effects of Ai in LPS-induced macrophages, which might be beneficial for finding new treatments against ALI.

HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer.

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies.Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition.Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2-irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivoConclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123-34. ©2017 AACR.

Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells.

Approximately 25% of breast cancers overexpress and depend on the receptor tyrosine kinase ERBB2, one of 4 ERBB family members. Targeted therapies directed against ERBB2 have been developed and used clinically, but many patients continue to develop resistance to such therapies. Although much effort has been focused on elucidating the mechanisms of acquired resistance to ERBB2-targeted therapies, the involvement of ERBB4 remains elusive and controversial. We demonstrate that genetic ablation of ERBB4, but not ERBB1-3, led to apoptosis in lapatinib-resistant cells, suggesting that the efficacy of pan-ERBB inhibitors was, at least in part, mediated by the inhibition of ERBB4. Moreover, ERBB4 was upregulated at the protein level in ERBB2+ breast cancer cell lines selected for acquired lapatinib resistance in vitro and in MMTV-Neu mice following prolonged lapatinib treatment. Knockdown of ERBB4 caused a decrease in AKT phosphorylation in resistant cells but not in sensitive cells, suggesting that ERBB4 activated the PI3K/AKT pathway in lapatinib-resistant cells. Importantly, ERBB4 knockdown triggered apoptosis not only in lapatinib-resistant cells but also in trastuzumab-resistant cells. Our results suggest that although ERBB4 is dispensable for naïve ERBB2+ breast cancer cells, it may play a key role in the survival of ERBB2+ cancer cells after they develop resistance to ERBB2 inhibitors, lapatinib and trastuzumab.

Upregulation of ER Signaling as an Adaptive Mechanism of Cell Survival in HER2-Positive Breast Tumors Treated with Anti-HER2 Therapy.

PURPOSE: To investigate the direct effect and therapeutic consequences of epidermal growth factor receptor 2 (HER2)-targeting therapy on expression of estrogen receptor (ER) and Bcl2 in preclinical models and clinical tumor samples. EXPERIMENTAL DESIGN: Archived xenograft tumors from two preclinical models (UACC812 and MCF7/HER2-18) treated with ER and HER2-targeting therapies and also HER2+ clinical breast cancer specimens collected in a lapatinib neoadjuvant trial (baseline and week 2 posttreatment) were used. Expression levels of ER and Bcl2 were evaluated by immunohistochemistry and Western blot analysis. The effects of Bcl2 and ER inhibition, by ABT-737 and fulvestrant, respectively, were tested in parental versus lapatinib-resistant UACC812 cells in vitro. RESULTS: Expression of ER and Bcl2 was significantly increased in xenograft tumors with acquired resistance to anti-HER2 therapy compared with untreated tumors in both preclinical models (UACC812: ER P = 0.0014; Bcl2 P < 0.001 and MCF7/HER2-18: ER P = 0.0007; Bcl2 P = 0.0306). In the neoadjuvant clinical study, lapatinib treatment for 2 weeks was associated with parallel upregulation of ER and Bcl2 (Spearman coefficient: 0.70; P = 0.0002). Importantly, 18% of tumors originally ER-negative (ER(-)) converted to ER(+) upon anti-HER2 therapy. In ER(-)/HER2(+) MCF7/HER2-18 xenografts, ER reexpression was primarily observed in tumors responding to potent combination of anti-HER2 drugs. Estrogen deprivation added to this anti-HER2 regimen significantly delayed tumor progression (P = 0.018). In the UACC812 cells, fulvestrant, but not ABT-737, was able to completely inhibit anti-HER2-resistant growth (P < 0.0001). CONCLUSIONS: HER2 inhibition can enhance or restore ER expression with parallel Bcl2 upregulation, representing an ER-dependent survival mechanism potentially leading to anti-HER2 resistance.

Systems analysis of drug-induced receptor tyrosine kinase reprogramming following targeted mono- and combination anti-cancer therapy.

The receptor tyrosine kinases (RTKs) are key drivers of cancer progression and targets for drug therapy. A major challenge in anti-RTK treatment is the dependence of drug effectiveness on co-expression of multiple RTKs which defines resistance to single drug therapy. Reprogramming of the RTK network leading to alteration in RTK co-expression in response to drug intervention is a dynamic mechanism of acquired resistance to single drug therapy in many cancers. One route to overcome this resistance is combination therapy. We describe the results of a joint in silico, in vitro, and in vivo investigations on the efficacy of trastuzumab, pertuzumab and their combination to target the HER2 receptors. Computational modelling revealed that these two drugs alone and in combination differentially suppressed RTK network activation depending on RTK co-expression. Analyses of mRNA expression in SKOV3 ovarian tumour xenograft showed up-regulation of HER3 following treatment. Considering this in a computational model revealed that HER3 up-regulation reprograms RTK kinetics from HER2 homodimerisation to HER3/HER2 heterodimerisation. The results showed synergy of the trastuzumab and pertuzumab combination treatment of the HER2 overexpressing tumour can be due to an independence of the combination effect on HER3/HER2 composition when it changes due to drug-induced RTK reprogramming.

Feedforward and feedback regulation of the MAPK and PI3K oscillatory circuit in breast cancer.

Although the theoretical possibility of oscillations in MAPK signalling has long been described, experimental validation has proven more elusive. In this study we observed oscillations in MAPK and PI3K signalling in breast cancer cells in response to epidermal growth factor receptor-family stimulation. Using systems level analysis with a kinetic model, we demonstrate that receptor amplification, loss of transcriptional feedback, or pathway crosstalk, are responsible for oscillations in MAPK and PI3K signalling. Transcriptional profiling reveals architectural motifs likely to be responsible for feedback control of oscillations. Overexpression of the HER2 oncogene and inhibition of transcriptional feedback increase the amplitude of oscillations and provide experimental validation of the computational findings.