Natural extracts, including nobiletin, have been reported to enhance the efficacy and sensitivity of chemotherapeutic drugs. However, whether and how nobiletin affects tumor growth and progression in renal cell carcinoma (RCC) are still unclear.in vitro. The nude mouse model was used to test the efficacy of nobiletin alone or in combination with palbociclib.Cell proliferation, cell cycle and apoptosis analyses, colony-formation assays, immunoblotting analysis, and qRT-PCR analysis were performed to investigate how nobiletin affected RCC cell proliferation in vitro and in an in vivo model.Nobiletin inhibited cell proliferation by inducing G1 cell cycle arrest and cell apoptosis in RCC cells. Mechanistically, nobiletin decreased SKP2 protein expression by reducing its transcriptional level. The downregulated SKP2 caused accumulation of its substrates, p27 and p21, which further inhibited the activity of the G1 phase-related protein, CDK2, leading to inhibition of cell proliferation and tumor formation. A higher SKP2 protein level indicated less sensitivity to the CDK4/6 inhibitor, palbociclib. A combination of nobiletin and palbociclib showed a synergistic tumor inhibition Nobiletin downregulated the SKP2-p21/p27-CDK2 axis to inhibit tumor progression and showed synergistic tumor inhibition effects with the CDK4/6 inhibitor, palbociclib, on RCC, which indicates a potential new therapeutic strategy. Despite a higher proportion of indolent and localized tumors identified partly due to widespread use of abdominal imaging techniques, there are still up to 17% of patients with distant metastases at the time of diagnosis2. In addition, approximately 25% of patients with localized RCC develop distant metastasis after radical surgery. Once progressed to metastatic RCC (mRCC), the treatment is still an intractable problem, despite the introduction of novel agents targeting different pathways including angiogenesis, mTOR inhibitors, and immune checkpoint inhibitors. Compared with cytokine treatment , targeted therapies have significantly improved the clinical outcomes of mRCC3. Sunitinib, as a first-line therapy for RCC, achieved significantly longer progression-free survival (11 vs. 5 months), overall survival (OS) (26.4 vs. 21.8 months), and a better response percentage (31% vs. 6%) than the interferon alpha group5. However, due to the intratumor and intertumor heterogeneities of RCC, the objective response percentages of current first-line agents are approximately 30% or lower6. Moreover, almost all patients eventually suffer drug resistance, which suggests that new targets and treatment strategies need to be identified to effectively treat these patients.Renal cell carcinoma (RCC) is the 6th most common cancer in men and the 10th in women, accounting for 5% and 3% of all new cancer cases, respectively8. Some extracts have anti-tumor pharmacological effects and can be used as a regulator of multi-drug resistance, to increase the sensitivity of cancer cells to chemotherapy9. Nobiletin is a typical example, which is an O-methylated flavonoid found mainly in citrus peel. Previous studies have shown that nobiletin significantly inhibited tumor cell growth and metastasis both in vitro and in vivo through multiple pathways11. Nobiletin can also significantly inhibit cell proliferation by attenuating the expression of cyclin D1, CKD2, CKD4, and E2F12. Nobiletin inhibits RCC cell viability and hypoxia-induced migration, which might be induced by the SRC/AKT, NF-κB, and Wnt/β-catenin signaling pathways14. Although nobiletin has been reported to induce G0/G1 cell cycle arrest in RCC cells, the mechanism of action is still unclear14.Many natural extracts are potential antitumor drugs because of their low toxicity and few side effects15. G1/S cell cycle transition is mainly regulated by two cyclin/CDK complexes, cyclinD-CDK4/6 and cyclinE-CDK2, which have been reported to trigger G1/S transition by promoting RB (retinoblastoma) phosphorylation16. Abnormal CCND-CDK4/6-INK4-RB signaling has been involved in tumorigenesis and in tumors21, and represents a valid therapeutic target16. Palbociclib is an orally active, potent, and selective inhibitor of CDK4 and CDK6, which blocks RB phosphorylation at low drug concentrations22. Palbociclib can inhibit RCC cell proliferation at nanomolar concentrations23, and has been tried in phase II studies to treat various types of solid tumors26. However, the effects of CDK4/6 inhibitors have been limited by the other bypass signal27. Some studies have shown that palbociclib blocked cells in the G1 phase by inhibiting CDK4/6 activity, but it did not inhibit CDK2 activity, and the inhibitory effect of RB phosphorylation could be reversed by CDK2 to produce drug resistance28, which indicated that a combination CDK2 inhibitor with palbociclib may increase the therapeutic effect.Cell-cycle dysregulation is prevalent in multiple malignancies, including RCCIn this study, we found that nobiletin induced G1-phase cell cycle arrest, apoptosis, and proliferative inhibition in RCC cells. Nobiletin downregulated mRNA expression of SKP2 by upregulating the transcription factor, FOXO3A, leading to accumulation of p21 and p27 and the reduction of CDK2, to inhibit cell proliferation and tumor formation. We further found that the sensitivity of the CDK4/6 inhibitor, palbociclib, was negatively associated with SKP2 protein levels. Furthermore, a combination of nobiletin and palbociclib showed synergistic lethality, which suggests this combined regimen may be a therapeutic strategy for RCC.2. All cell lines were tested and were shown to be free of mycoplasma contamination.Nobiletin, palbociclib, MG-132, and cycloheximide were purchased from Selleck Chemicals , and dissolved in dimethyl sulfoxide (DMSO)  and stored at -20 °C. The RCC lines  and the immortalized epithelial renal cell line (HK-2) were purchased from the Zhong Qiao Xin Zhou Biotechnology . The 786-O, 769-P, and OSRC-2 cell lines were routinely maintained in RPMI-1640 medium. The Caki-1 cell line was cultured in McCoy’s 5A medium, and the HK-2 cell line was cultured in KFSM medium. All media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin except KFSM medium. All media were purchased from Gibco . The cells were cultured at 37 °C in a humidified atmosphere containing 5% COFor direct IB analysis, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with phosphatase inhibitors. The following primary antibodies were used against: cleaved caspase-3 (Asp175) (5A1E) (#9664), cleaved caspase-8 (Asp391) (18C8) (#9496), RB (#9309), p-RB (Ser870/811) (#8516), CDK2 (#2546), p-CDK2 (#2561), Bcl-2 (#15071), Bax (#5023), p21 (#2947), p27 (#3686), cyclin D1 (#55506), cyclin E (#4136), p44/42 MAPK (Erk1/2) (137F5) (#4695), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP (#4370), Akt (pan) (40D4) (#2920), phospho-Akt (Ser473) (D9E) XP (#4060), PI3 kinase p110α (C73F8) (#4249), cyclin A2 (#91500), and cyclin B1 ( #4135), all purchased from Cell Signaling Technology . Antibodies to CDK4 (#11026-1-AP), CDK6 (#14052-1-AP), SKP2 (#15010-1-AP), p53 (1C12) (#2524), P16-INK4A (#10883-1-AP), EGFR (#18986-1-AP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#60004-1-Ig) were purchased from Proteintech . The anti-FLAG was obtained from Sigma-Aldrich . All secondary antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA.Cells were seeded into 6-well plates  at a density of 500 cells/well. After 24 h, cells were treated with or without nobiletin for 48 h. The nobiletin-containing medium was then removed and replaced by complete medium, followed by incubation at 37 °C for 10–14 days. The colonies were fixed with 4% paraformaldehyde, stained with Crystal Violet, and counted. The colonies composed of ≥ 50 cells were counted using a microscope. Each experiment was conducted in triplicate.For transient transfection, the cells were seeded in an antibiotic-free medium at 37 °C for 24 h and transfected with constitutively active SKP2 plasmid or empty vector DNA using Lipofectamine-2000 transfection reagent  according to the manufacturer’s instructions, and treated 48 h after transfection. The lentivirus used for SKP2 silencing was purchased from Genepharma . The cells were transfected with lentivirus according to the manufacturer’s protocol.An annexin V assay was performed according to the manufacturer’s instructions . Nobiletin- and/or palbociclib-treated cells were collected, and used for annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The samples were then analyzed using flow cytometry within 1 h. Each experiment was conducted in triplicate.Cells were collected at the indicated time points after nobiletin and/or palbociclib treatment and fixed with ice-cold 70% ethanol, then stored at 4 °C overnight. After washing twice with phosphate-buffered saline, FxCycle PI/RNAse staining solution  was used for detection of the cell cycle according to the manufacturer’s protocol. The stained cells were subjected to cell cycle analysis using a flow cytometer and analyzed for cell cycle phases with the C6 Accuri software system . Each experiment was conducted in triplicate.3 cells/well, and the cells were seeded into 96-well plates, followed by 24 h of culture at 37 °C in an atmosphere with 5% CO2, then were treated with various concentrations of palbociclib or nobiletin and maintained in culture for 48 h. After removing the culture medium, the CCK-8 reaction solution was added according to the manufacturer’s instructions, and the absorbance was measured at 450 nm. The half-maximal inhibitory concentration (IC50) value is a critical index of the dose-response curve. Prism statistical software  was used to calculate the IC50 values and to plot dose-response curves. According to IC50 values, the cells was treated with the given concentration of nobiletin, and cultivated for a further 0, 1, 2, 3, and 4 days. At different times after cell plating, the cells were subjected to the CCK-8 assay, according to the manufacturer’s instructions. Each experiment was conducted in triplicate.The Cell Counting Kit-8 (CCK-8) assay  was used to assess the cell viability. The cell concentration was adjusted to 2 × 1029. Briefly, 786-O and 769-P cell lines (5 × 105 cells) were exposed to 100 μM and 25 μM nobiletin for 24 h, respectively, and followed by 24 h treatment with cycloheximide (50 μg/mL)  to stop de novo protein synthesis. SKP2 levels at 0, 4, 8, 12, and 24 h following cycloheximide co-treatment were then determined using IB analysis. Each experiment was conducted in triplicate.Cycloheximide chase analysis was performed as described previously to define the effect of nobiletin on the stability of the SKP2 proteinGAPDH, was used as a loading control. The following forward and reverse primers were used: SKP2 forward-CAGGCCTAAGCTAAATCGAGAG, SKP2 reverse-CTGGCAATGGT GGTGAAATG; GAPDH forward-AG CCTTCTCCATGGTTGGTGAAGAC, GAPDH reverse-CGGAGTCAACGGATTTG GTCGTAT. Each experiment was conducted in triplicate.Total RNA was isolated from control and nobiletin-treated cells using RNAisoPlus , according to the manufacturer’s instructions. Reverse transcription of the extracted RNA to corresponding complementary DNA was performed using a PrimeScript RT reagent Kit with gDNA Eraser (Takara). The qRT-PCR was performed with a QuantiNova™ SYBR® Green PCR Kit  on an Applied Biosystems 7900HT Real-Time PCR System . The housekeeping gene, 30. Briefly, after deparaffinization, rehydration, antigen retrieval, and blocking, the tissue slides were incubated overnight at 4 °C with the indicated antibodies. The following primary antibodies were used: anti-Ki-67 , anti-p27, and anti-cleaved-capase-3.IHC staining of mice tumors was performed as described previously6 786-O cells were mixed 1:1 with Matrigel (BD Biosciences) in a total volume of 200 μL, and were injected subcutaneously into the right flank side of nude mice. Nude mice were treated with vehicle, nobiletin , palbociclib , and nobiletin + palbociclib when the tumor size reached about 100 mm3. Nude mice were euthanized after 21 days of treatment. The growth of tumor was measured twice a week and the average tumor volume (TV) was calculated according to the equation: TV = (L × W2)/2. The protocol was approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University of China .Four- to six-week-old BALB/c athymic nude mice  were used, with each experimental group consisting of 5 mice. All animal experiments were conducted according to a protocol approved by the university committee for use and care of animals. A total of 2 × 10t-test. P < 0.05 was considered to be statistically significant. Both CalcuSyn software31 and Jin’s formula32 were used to evaluate the synergistic effects of drug combinations. Jin’s formula is Q = Ea + b / (Ea + Eb - Ea × Eb), where Ea + b represented the cell proliferation inhibition rate of the combined drugs, while Ea and Eb represented the rates for each drug, respectively. A value of Q = 0.85–1.15 indicated a simple additive effect, while Q > 1.15 indicated synergism. Combination index (CI) plots were generated using CalcuSyn software .All data are shown as the average ± standard deviation (mean ± SD) and each experiment was independently repeated at least 3 times. All statistical analyses were performed using the GraphPad Prism, version 5.0. The statistical significance of differences between groups was examined by one-way analysis of variance followed by Tukey’s multiple comparison procedure or Student’s Figure 1A. We first performed a CCK-8 assay to identify the effects of nobiletin in RCC and found that nobiletin significantly inhibited cell growth of RCC cell lines in a dose-dependent manner . The results showed that the IC50 response of nobiletin was lower in the 769-P cell line (IC50 = 20.22 μM) and higher in the 786-O cell line (IC50 = 90.48 μM) . We also found that nobiletin inhibited cell proliferation of 786-O and 769-P cell lines in a time-dependent manner .The chemical structure of nobiletin is shown in ) and promoted cell apoptosis in a dose-dependent manner in 786-O and 769-P cell lines . In addition, nobiletin significantly inhibited the colony formation ability of 786-O and 769-P cell lines . Nobiletin may have inhibited cell proliferation by inducing cell cycle arrest and apoptosis.We next determined whether nobiletin affected cell cycle distribution or apoptosis in RCC cells. The flow cytometry assays showed that nobiletin induced accumulation of G1-phase cells . Nobiletin had no effect on CDK2, CDK4, and cyclin D1 levels, but reduced the expressions of p-CDK2, RB, and p-RB levels with increasing doses, in RCC cell lines , indicating that G1/S transition was blocked. Nobiletin also attenuated the expression of cyclin E and p-RB . Our findings suggested that nobiletin induced cell cycle arrest during the G1 phase by increasing P21/p27, but inhibited CDK2 and RB activities. In addition, the decrease in Bcl-2 levels and the increase in Bax levels  confirmed the apoptotic effect of nobiletin on RCC cell lines.To clarify the mechanism of nobiletin involved in cell cycle arrest and apoptosis, we characterized the expression of G1 phase and apoptosis-related proteins upon nobiletin treatment. Consistent with G1-phase arrest, G1-phase checkpoint proteins p21 and p27 were increased by nobiletin treatment  and time-dependent manner . Next, we determined whether nobiletin downregulated SKP2 in a transcriptional and/or post-translational manner. To assess the effects of post-translational regulation, 786-O and 769-P cell lines were treated with nobiletin without or with co-treatment with a protease inhibitor MG-132, followed by SKP2 IB. In both RCC cell lines, MG-132 co-treatment did not rescue SKP2 expression to levels comparable to drug-free controls . To further confirm the SKP2 degradation caused by nobiletin, we conducted cycloheximide chase analyses, showing that cycloheximide only slightly changed the rate of degradation of SKP2 protein in nobiletin-treated RCC cell lines . However, we found that SKP2 mRNA levels were significantly reduced by nobiletin treatment of 786-O and 769-P cell lines . These results confirmed that nobiletin-induced SKP2 protein downregulation was mainly achieved by decreasing its transcriptional level. We also found that the transcription factor, FOXO3A, which is a negative regulator of the SKP2 and SKP2 SCF complex36, was gradually upregulated by escalating doses of nobiletin in 786-O and 769-P cell lines . Based on these results, we speculated that the downregulation of SKP2 may be associated with increasing FOXO3A levels.SKP2, a well-characterized F-box protein, acts as a classic oncogene by promoting proliferation and survival of cancer cells, mainly through targeted degradation of numerous tumor suppressive proteins, including p2128. Our results showed that the dose of palbociclib required to suppress 50% (IC50) of cell proliferation at 48 h was 0.4662 μM, 0.5548 μM, 1.256 μM, and 7.718 μM for the Caki-1, OSRC-2, 769-P, and 786-O cell lines, respectively . To determine the reasons for the higher concentration of IC50 reactions in 786-O and 769-P cell lines, we detected the expressions of related proteins in 5 renal cell lines, namely the Caki-1, 786-O, 769-P, OSRC-2 and HK-2 cell lines. Higher levels of CDK4, CDK6, CDK2, p-CDK2, and SKP2, and lower levels of p27 were found in 786-O and 769-P cell lines . The p27, as a tumor suppressor protein, can affect CDK2 activity28 and is directly degraded by SKP235. The inhibition of RB phosphorylation caused by the CDK4/6 inhibitor, palbociclib, can be reversed by CDK2, which results in drug resistance. We therefore speculated that RCC cells with higher levels of SKP2 might require higher concentrations of palbociclib to achieve a IC50 response. We first overexpressed SKP2 in the Caki-1 and OSRC-2 cell lines, and then treated them with palbociclib for 48 h. The results showed that SKP2 overexpressed Caki-1 and OSRC-2 required higher concentrations of palbociclib to achieve an IC50 response, when compared with the control group (6C). The 786-O cells, a palbociclib highly-responding RCC cell line, was transfected with either control siRNA, or siRNA targeting SKP2 , followed by treatment with palbociclib for 48 h. The dose of palbociclib required to suppress 50% (IC50) of cell proliferation was 7.718 μM for the control group, which was at least 9-fold more than the SKP2 silencing group  (6F). From the IB results, we found that SKP2 overexpression significantly increased p-CDK2 levels by decreasing p27 levels in the Caki-1 and OSRC2 cell lines , and vice versa . Thus, we confirmed that the insensitivity of CDK4/6 inhibitor, palbociclib, was associated with higher SKP2 levels in RCC cells.Palbociclib, a CDK4/6-specific inhibitor, has shown clinical efficacy, but primary or secondary resistance has emerged as a problem37. As we previously discussed, nobiletin inhibited tumor progression by regulating the SKP2-p21/p27-CDK2 axis, and the insensitivity of the CDK4/6 inhibitor, palbociclib, was associated with higher SKP2 levels in RCC cells. Thus, we speculated that a combination of nobiletin and palbociclib may have the effects of dual inhibition of RCC cells. We selected 786-O and 769-P cell lines for the following experiments. Both CalcuSyn software and Jin’s formula were used as previously described to determine the synergy of the 2 agents. The 786-O was cultured with combinations of the 2 drugs at different doses but at a constant ratio  for 48 h. The combination of 6.25 μM nobiletin with 0.625 μM palbociclib in the 786-O cell line inhibited cell proliferation by 32.0%, compared with monotherapy of nobiletin by 15.1% or palbociclib by 11.2%, indicating synergism . Escalating doses, i.e., co-treatment with 12.5 μM nobiletin and 1.25 μM palbociclib  or 25 μM nobiletin and 2.5 μM palbociclib  or 50 μM nobiletin and 5.0 μM palbociclib , showed synergetic effects in 786-O cells . Those findings showed that nobiletin and palbociclib had synergistic lethality, because they inhibited both CDK2 and CDK4/6 kinases. Furthermore, a combination of the 2 agents strongly induced apoptosis, when compared with a single agent in the 786-O and 769-P cell lines , which was further demonstrated by the increase of cleavage of caspase-8 and caspase-3 . Similarly, a significant increase of p27 was observed with the combination treatment when compared with single agent , suggesting that simultaneously inhibiting CDK2 and CDK4/6 kinases did more to induce apoptosis.Clinical trials are currently ongoing with the single agent palbociclib in several advanced solid tumorsin vitro findings by using an in vivo xenograft model. A 786-O xenograft model was established and treated with vehicle, nobiletin, and/or palbociclib. Consistent with the in vitro results, the combination of nobiletin and palbociclib suppressed tumor growth significantly more than single agent treatment . The average tumor size and tumor weight at the end of the experiment (treatment for 21 days) were significantly lower in the nobiletin-palbociclib combination group (8C). The body weight of the xenograft model was unchanged during drug treatment, suggesting that the effect on normal tissues was minimal . Finally, immunohistochemical staining of tumor tissues revealed that compared with nobiletin or palbociclib single agent treatment, a combination of the two agents inhibited cell growth (decrease of Ki-67 and increase of p27) and induced apoptosis (increase of cleaved caspase-3)  significantly more.We validated the above 38, making them potential antitumor drugs7. Nobiletin is a common PMF that has been reported to prevent various tumor inductions and progressions40. Several studies have reported the antitumor activities of nobiletin, including inhibition of proliferation, induction of the cell cycle arrest, and promotion of apoptosis42.Polymethoxylated flavonoids (PMFs) have a variety of biological activities such as anti-cancer, anti-cardiovascular disease, prevention and treatment of obesity, and anti-inflammatory activitiesFigure 1) by inducing G1-S phase arrest in RCC cells . The causal role of nobiletin in G1/S arrest was due to an increase of p27 and p21 and inhibition of CDK2 phosphatase, ultimately leading to a decrease of p-RB . The p21 and p27 are crucial for restraining the G1-S phase transition43, and are regulated by the E3 ubiquitin ligase, SKP235. Nobiletin caused accumulation of p27 and p21 levels by downregulating SKP2 protein expression in time- and dose-dependent manners by decreasing its transcriptional level , which might be due to nobiletin-induced upregulation of FOXO3A. FOXO3A is a transcriptional repressor of SKP2 gene expression, which directly binds to the SKP2 promoter36. Small molecules targeting SKP2 activity or SKP2 complex assembly have been examined in leukemia cells and in xenograft tumor models, respectively, and have been proven to be effective as anticancer agents45. These findings not only provide new insights into the molecular understanding of nobiletin’s anti-proliferative effects, but also highlight the potential of nobiletin in the development of SKP2-targeted anticancer therapeutics.In the present study, we found that nobiletin inhibited tumor proliferation and progression in RCC by regulating the SKP2-p21/p27-CDK2 axis. Nobiletin inhibited the proliferation of RCC lines in dose- and time-dependent manners  and OSRC-2 (IC50 = 0.5548 μM), but showed less effects in 769-P (IC50 = 1.256 μM) and 786-O (IC50 = 7.718 μM) cells, especially the 786-O cell line . The insensitivities to 786-O and 769-P may be explained by the higher levels of p-CDK2 and SKP2 but lower levels of p27 . CDK2/cyclinE could be deactivated by p2728, which could be targeted for degradation by SKP235. A previous study confirmed that while estrogen receptor- (ER) positive breast cancer cell lines were inhibited by palbociclib, they quickly adapted because of degradation of p27 and a subsequent activation of CDK2, allowing compensatory phosphorylation of RB and passage into the S phase28. Another study also confirmed that insensitivity to CDK4/6 blockade was mediated by p-RB phosphorylation recovery induced by the noncanonical CDK2/cyclinD1 complex27. The cyclinE rebound is likely a consequence of CDK2/cyclinD1 activity and eventually triggering S-phase entry27. Based on these observations, searching for agents that reverse resistance of palbociclib represents a promising strategy to discover novel cancer therapeutics46. Herein, we showed that nobiletin downregulated SKP2 levels to suppress activation of CDK2 phosphatase . A combination of nobiletin with palbociclib further inhibited cell proliferation and induced cell apoptosis by increasing p27 and proapoptotic protein levels, when compared to palbociclib treatment alone . Similarly, a combination of nobiletin and palbociclib significantly inhibited tumor growth in a xenograft model (8E).49. It is also noteworthy that some natural compounds have been widely used in clinics, such as paclitaxel50. Nobiletin, as a natural extract, can be considered as a promising candidate for cancer therapy due to its multiple-targeting capabilities51. But the underlying mechanisms and bioavailability of nobiletin are still complex problems to understand52, which limits its application as a therapeutic agent. Our study demonstrated that nobiletin inhibited tumor progression by regulating the SKP2-p21/p27-CDK2-RB axis : SKP2 targets p27 for degradation, CDK2 can be reactivated upon p27 degradation, and phosphorylation of RB is recovered by activated CDK2, which leads to G1/S transition, tumor growth, and progression. However, nobiletin decreased SKP2 protein levels by reducing its transcriptional levels, and showed synergistic chemopreventive effects with palbociclib in RCC cells. The fact that a combination of nobiletin and palbociclib at their half doses produced stronger anti-cancer effects than nobiletin or palbociclib alone, provided the strong basis to utilize the nobiletin/palbociclib combination for renal cell carcinoma chemoprevention.It is noteworthy that natural compounds improved the sensitivity and efficacy of chemotherapy drugsNobiletin downregulated the SKP2-p21/p27-CDK2 axis to inhibit tumor growth and progression in RCC cells. A combination of nobiletin with a CDK4/6 inhibitor may be a new therapeutic strategy to overcome the resistance of a single agent CDK4/6 inhibitor in cancer  treatments.Click here for additional data file.


High-temperature-resistant fiber Bragg gratings (FBGs) are the main competitors to thermocouples as sensors in applications for high temperature environments defined as being in the 600–1200 °C temperature range. Due to their small size, capacity to be multiplexed into high density distributed sensor arrays and survivability in extreme ambient temperatures, they could provide the essential sensing support that is needed in high temperature processes. While capable of providing reliable sensing information in the short term, their long-term functionality is affected by the drift of the characteristic Bragg wavelength or resonance that is used to derive the temperature. A number of physical processes have been proposed as the cause of the high temperature wavelength drift but there is yet no credible description of this process. In this paper we review the literature related to the long-term wavelength drift of FBGs at high temperature and provide our recent results of more than 4000 h of high temperature testing in the 900–1000 °C range. We identify the major components of the high temperature wavelength drift and we propose mechanisms that could be causing them. The majority of present-day industrial processes require complex monitoring and control. Over the last decade, the demand for sensors having high accuracy, repeatability, reliability and the capability to be easily distributed has increased significantly. While there is a great variety of sensors which exploit different principles and generally fulfill this demand, many sensors will simply fail or will not perform according to the requirements when used in high temperature processes and extreme environments, such as in the presence of intensive ionizing radiation, strong electromagnetic field, corrosive ambient etc.Traditionally, the majority of temperature measurements for applications in the 600–1200 °C temperature range were done with various thermocouples ,4,5 and (1)Regenerated fiber Bragg gratings (RFBG) made by using ultraviolet (UV) lasers and the phase mask technique ,(2)FBGs made by using femtosecond lasers and the phase mask technique (Type II FBGs) ,13.(3)FBGs made by using femtosecond lasers and the point-by-point (PbP) technique (PbP FBGs) .A relatively large assortment of FOSs, ranging from simple fiber devices that exploit Rayleigh , BrillouAll of the above types of FBGs  have good thermal stability up to 1000 °C, whereas the classical FBGs  are almost completely erased at 700 °C. RFBGs were discovered in 1997  as a resThe inscription in silica fibers of high temperature resistant FBGs using infrared (IR) ultrafast radiation and the phase mask technique was demonstrated for the first time in 2003 . The appBλ. The fulfillment of the Bragg condition leads to a simple relation between Bλ and the parameters defining the FBG structure, namely the effective refractive index of the core effn and the period of the refractive index modulation ΛG:The idea underlying the use of FBGs as temperature sensors is straightforward. A FBG temperature sensor is a narrow-band interference filter whose reflection spectrum is centered at a characteristic wavelength which is called the Bragg wavelength effn and ΛG are functions of temperature, Bλ is also correlated with the ambient temperature. Type II FBGs, for example, demonstrated near-linear wavelength drifts with temperature of 16.8 pm/ °C in the 800–1000 °C temperature range [As both re range .Using this basic principle, high temperature resistant FBGs were deployed in a considerable number of applications. RFBGs were used to monitor the temperature of a melt during metal processing , the temType II FBG arrays have been used in several applications: for monitoring temperature gradients of the flame tube of a low emission burner , to moniA number of tests were also performed in nuclear installations. As an example, RFBGs were placed in liquid sodium at 500 °C for high temperature monitoring of a sodium-cooled fast nuclear reactor . Four fiThe above published results confirm that FBG sensors can potentially replace thermocouples in some applications and, moreover, improve the quality of temperature measurements by leveraging the unique advantages of FOSs. However, at temperatures approaching 1000 °C, the silica glass of which the fiber is made of, starts changing its properties, which could potentially lead to degradation in the sensor’s performance. The first experiments to establish the survivability of FBG structures at high temperature mainly concentrated on monitoring the reflectivity of the FBGs under test . The nexeffn and ΛG due to several effects such as dopant diffusion from the core, devitrification  of the glass, and release of stresses that are built into the fiber during the fiber manufacturing process. Hence, a more specific aim of this study is to look at the most probable mechanisms of the Bragg wavelength drift in silica fibers and suggest ways to mitigate their effect. This becomes especially important because the initial proof-of-concept successes with 1000 °C-resistant sapphire FBGs [Studying the Bragg wavelength drift of high temperature resistant FBGs  at a constant temperature  is the main subject of this paper. It summarizes previously reported experiments and presents our recent tests performed with Type II FBGs. If no stress is applied to the fiber, the Bragg wavelength drift at a constant temperature (see Equation (1)) will be caused by changes in ire FBGs ,34,35 anire FBGs  were notBλ) is a function of the FBG’s period (ΛG) and the modal effective refractive index (effn). The latter is determined both by the fiber and the refractive index modulation inside the FBG, which in turn depends on the internal structure of the FBG. Therefore, one of the first questions to ask is whether the Bragg wavelength drift at high temperature is caused by transformations occurring in the optical fiber itself or in the FBG internal structure, or both. In order to answer this question the FBG internal structure should be known. Below we list the available information on the FBG internal structure.According to Equation (1), the Bragg wavelength of an FBG  having a core of a much smaller diameter that confines the light, preventing it to leak to the external region called cladding. The manufacturing process of optical fiber consist of drawing the fiber from a preform heated in a furnace to the glass softening temperature. When the fiber exits the furnace it starts cooling from high (above 1700 °C) temperature to room temperature after which it is coated and wound on a drum. Besides the intended effect of creating the core/cladding refractive index structure, the drawing and cooling processes result in a pattern of stresses  frozen into the fiber after the drawing process is finished . The froIn this section, we present a summary of experiments performed to study high temperature wavelength drift of: (1) RFBGs, (2) Type II FBGs and (3) PbP FBGs. Experiments on high temperature annealing of other structures such as (i) ablation FBGs in photonic crystal fibers (PCF), (ii) FBGs in chiral fibers, (iii) Fabry-Pérot fiber cavities and (iv) nanogratings in bulk glasses are also included in the discussion as they provide information about possible mechanisms, such as dopant diffusion or stress relaxation, that may cause a wavelength drift of FBGs. The long-term wavelength stability of RFBGs at high temperature was presented for the first time in 2005 . During During the test, the Bragg wavelength experienced a red drift of ~480 pm during the first 125 h of annealing and then a 190 pm blue drift  during the remaining 270 h. The initial red drift of 480 pm was likely the result of the regeneration process. The majority of the red drift took place in the first 25 h at a rate of ~16 pm/h. Looking for an explanation for the post-regeneration drift at 1000 °C, the authors examined samples of the annealed fiber for signs of devitrification using an optical microscope but did not find any crystalline regions in the fiber.A much longer annealing experiment at 1000 °C was performed on RFBGs in 2011 . The aut2-loaded SMF-28-type fiber  were annealed for 9000 h at different temperatures between 700 °C and 900 °C. The annealing temperature of each RFBG was continuously monitored by an individual thermocouple giving 760 °C, 810 °C, 850 °C and 890 °C. As can be seen in The longest high temperature test performed with RFBGs was reported in  when fouSome correlation between the evolutions of the Bragg wavelengths and the reflectivities of the RFBGs at 760 °C and 810 °C is also observable. The behavior of the RFBGs at higher temperatures was more erratic. For example, the Bragg wavelength of the RFBG at 890 °C experienced a red drift of ~500 pm in the first 500 h, then a blue drift of ~1200 pm in the next 3000 h, remained stable for further ~3000 h and finally experienced a red drift of ~700 pm. It is also to be noted that variations in the behavior the RFBGs became stronger and faster as the annealing temperature increased.In 2018, a simultaneous annealing in a tube furnace at 800 °C of a PbP FBG made in SMF-28 and a RFBG also made in SMF-28 was performed  see Fig. The 800Results of a 2200-h annealing test performed at 920–950 °C with four PbP FBGs were published in 2018 . Four PbThe earliest report of the wavelength drift at 1000 °C of Type II FBGs made with infrared ultrafast radiation and a phase mask was presented in . SMF-28 2-loaded SMF-28 using the phase mask technique and 800 nm, 120 fs pulses. In this time frame, gratings fabricated in pristine SMF-28 experienced a ~300 pm red drift while the gratings made in H2-loaded SMF-28 exhibited a much more modest red drift of ~100 pm  test  at 1000 0 pm see .A very simple wavelength drift experiment  was desiλ = 800 nm was used to inscribe π-shifted Type II FBGs in SMF-28 using a π-shifted phase mask with a period of 3.21 μm. To test how the FBGs responded to high temperature annealing, a number of devices were temperature-cycled many times inside a tube furnace from 20 °C to 1000 °C in ambient air. During this testing  cavity made of a 60 µm section of SMF-28 with its end faces coated with a low reflectivity Cr-layer . The optIn order to directly analyze the magnitude of dopant diffusion, the radial distribution of the Ge-dopant after the annealing process was measured using Energy Dispersive X-ray analysis (SEM/EDX) on polished end surfaces of the fiber samples subjected to different temperatures. Another parameter that can affect the wavelength drift of FBGs at high temperature is the chemical composition of the host fiber. Commercially available silicate fibers include a pure silica core fiber that usually has a fluoride-doped cladding, silica fiber with low and high germanium concentration in the fiber core, fiber whose core is co-doped with boron and active fiber. There are no published comprehensive studies that would correlate the stability of the Type II FBGs with the chemical composition of the fibers, but individual studies have shown that pure silica core fibers and fibers with low germanium concentration are more stable at high temperature than fibers with high germanium concentration. A thorough study is very difficult and expensive to conduct as it would require customized fabrication of numerous samples of special fiber.2-SiO2 binary glasses and the final study focused on the effect of other dopants like TiO2- and B2O3. According to the results presented in An alternative approach was used in  where th2. There are only small differences between the four glasses, which the authors attributed to the fact that GeO2 and SiO2 form a relatively continuous mixture whose viscosity and, therefore, thermal stability are not changed much until a certain concentration of GeO2 is exceeded. Doping silicate glasses with other oxides (TiO2 or (B2O3) significantly affects the high temperature thermal behavior  using a cylindrical lens (CL) with a focal distance of 15 mm (see 1 = 1.07 µm (first-order mask) and Λ3 = 3.21 µm (third-order mask) were used to fabricate the FBG samples. Depending on the distance between the optical fiber (F) and the phase mask (M), the interference pattern at the fiber could be produced by the beams from mainly +1/−1 diffraction orders or the beams originating from all of the available diffraction orders, i.e., 0 and +1/−1 diffraction orders in the case of a first-order mask and 0, +1/−1, +2/−2 and +3/−3 diffraction orders in the case of a third order mask [The output Gaussian beam ~7 mm in diameter at the 1/5 mm see . A 7 mm-der mask . An examDuring inscription the π-shifted Type II FBGs were continuously monitored with a tunable laser system and the laser exposure was stopped when their strength reached approximately −15 dB in transmission. The prepared FBG samples were then spliced to fiber pigtails terminated with APC connectors and placed into a Lindberg Blue M Tube Furnace  for annealing. Three identical Lindberg furnaces were used, each accommodating 4–5 samples. An external Type K thermocouple  was placed in each furnace, in close proximity of the furnace’s built-in thermocouple, and monitored using data acquisition software. FBGs were centered within the furnaces, in-between the two thermocouples , loosely resting in a hollow fused silica semi-cylinder so that they were free from strain and not touching the furnace side walls. Using FBG optical interrogators, each FBG was monitored independently using two separate interrogator channels, with one channel monitoring reflection and the other monitoring the light transmitted through the FBG and an optical isolator.Full spectrum optical data was generally collected every two hours, with the Bragg peak wavelengths being logged every 5 s in order to facilitate data processing and provide additional resolution in time. Two interrogators were used: a Hyperion SI255 FBG Interrogator  with 16 Channels and 8 pm wavelength resolution and a Micron Optics SM125 FBG Interrogator with four Channels and 5 pm wavelength resolution.In a separate experiment, three π-phase shifted gratings, prepared under identical conditions to the ones above, were then packaged in a protective quartz tubing. Their long-term wavelength stability was studied in 0–1000 °C temperature range with 0.1 pm wavelength resolution using an 8164 Lightwave Measurement System  with an integrated tunable laser and detector modules. For the packaged fibers, only the wavelength drifts measured at 1000 °C in the horizontal tube furnace are reported in this paper, while the bulk of the results will be reported elsewhere. The temperature of the annealing furnace was controlled with an external S-type thermocouple. The tips of the packaged fibers with inscribed FBGs were placed in a massive Inconel block (to decrease temperature fluctuations below 0.1 °C) and placed in the middle of the furnace where the temperature profile of the furnace has a maximum. The drift rate at 1000 °C in pm/h was calculated by dividing the Bragg wavelength changes at 600 °C, which were measured in the caesium pressure-controlled heat pipe  before a(1)Type-a gratings made with 80 fs pulses (Fourier-transform-limited pulses) and a first-order phase mask;(2)Type-b gratings made with 400 fs pulses (chirped pulses) and a third-order phase mask;(3)Type-c gratings made with 80 fs pulses (Fourier-transform-limited pulses) and a third-order phase mask.Five independent annealing tests were run at 900 °C and 1000 °C. In each test, a set of either 4 or 5 Type II FBGs fabricated under the same experimental conditions and with similar spectral characteristics was annealed for a time duration of up to 1000 h. The purpose of the tests was to gather information regarding the influence of the laser writing conditions, annealing temperature and annealing history on the drift of the wavelength of the π-phase shift passband feature. The wavelength of the passband feature in the FBG spectrum will be called the ‘Bragg wavelength’ to avoid confusion. Three different types of gratings were tested, each of them being a Type II FBG made with different exposure condition and resulting in a different grating structure as presented above in Type-a, Type-b and Type-c FBGs were inscribed using a 0.7 mJ, 0.96 mJ and 1.03 mJ pulse energy, respectively. This choice of pulse energy was made in order to equalise the growth rate of the different types of FBGs. Type-a, Type-b and Type-c FBGs did not show any statistical difference between their spectra. A typical spectral shape of the pi-phase shifted FBGs fabricated for the present experiment is presented in Figure 7b of , with thThe wavelength of the π-phase shifted passband feature in the FBG spectrum was recorded every two seconds to measure the spectral drift, while the FBG spectra were recorded every two hours to evaluate the transmission loss as a function of time.Four FBGs of Type-a were placed in the furnace at 1000 °C and left at this temperature for 660 h. The FBGs were continouslly monitored both in transmission and reflection. Variations in grating reflectivity are shown in As can be seen in In the second part of this test, the temperature was decreased from 1000 °C to 900 °C in ~1 h and left at this temperature for ~500 h. For the entire second part of this test, the reflectivity of the FBGs remained constant, a proof that the refractive index modulation remained unchanged. There was however wavelength drift in the first 150 h and, as before, especially in the first 50 h (drift rate from 0.4 pm/h to 1 pm/h). For the remainder of the test, the FBGs were remarkably stable (less than 0.1pm/h drift rate), with only one FBG (green trace) showing a slight blue drift .The behavior of the five Type-b FBGs in this set is quite different from that of the Type-a FBGs. First, the grating structure of Type-b FBGs is much more stable than that of Type-a FBGs. Indeed, the reflectivity of Type b FBGs decreased only by a couple percent during annealing at 1000 °C for ~700 h a. SecondFive Type-c FBGs were tested at 1000 °C for ~500 h see a. There Following the annealing protocols for Type-a and Type-b FBGs, the temperature was decreased to 900 °C b. After As can be seen in For this experiment, a set of 5 Type-b FBGs was tested directly at 900 °C without pre-annealing at 1000 °C in order to compare the annealing behavior at this temperature with that of the Type-b FBGs that were first pre-annealed at 1000 °C for hundreds of hours and then monitored at 900 °C. The drift toward longer wavelengths  at the bThree Type-c FBGs were packaged in quartz tubing and the drift rates were measured in the 0–1000 °C temperature region to assess the influence of packaging on FBG performance. Only the drift rates obtained at 1000 °C in the horizontal tube furnace are shown in this paper in As explained in detail in The wavelength drift pattern presented as a general feature in the Literature Review section also holds true for the above results at 900 °C, 1000 °C, or at 900 °C after annealing the FBGs for hundreds of hours at 1000 °C. This pattern is made of a fast wavelength drift at the beginning of the annealing process and a slow blue secondary drift. The red drift rate for annealing at 900 °C and 1000 °C is 1–2 pm/h and 5–20 pm/h, respectively. The blue drift rate is 0.05–0.1 pm/h and 0.2–0.5pm/h for the annealing at 900 °C and 1000 °C, respectively. It is also characteristic to the wavelength drift pattern that in between the periods of red drift and blue drift there is a period of almost no drift for 50–300 h.While this is a general conclusion reached by analyzing the drift patterns, there is also a certain degree of variability among the results. In each set of Type-a and Type-c FBGs we tested, there was an outlier in terms of drift rate. This indicates that in these cases, and to a certain extent in the case of Type-b FBGs, there is variability in the internal structure of individual FBGs. This variability is most likely caused by the different exposure conditions of the fiber core to the femtosecond radiation. Type-a FBGs were written at a small fiber-to-mask distance (~50 μm) where the beams from 0 and +1/−1 diffraction orders strongly contribute to the resultant Talbot-like interference pattern . The samBeside the variability of drift rates within individual FBG sets, there is also a group variability among the three types of FBGs tested. While Type-b FBGs are quite different in their behavior compared to Type-a and Type-c FBGs, there are only small differences in the behavior of Type-a and Type-c FBGs, which is unexpected as the periods of FBGs made with first- and third order phase masks are obviously different.The most important results of the annealing tests are (i) the difference in the red drift rate for Type-b FBGs that were annealed directly at 900 °C 2 pm/h;  and thosBased on these observations, there is no straightforward answer to the question if the wavelength drift pattern is caused by temperature-induced changes in the refractive index of the optical fiber or re-organization of the grating structure. Nonetheless, the commonality of the red and blue drift patterns for widely different grating structures seems to indicate that the wavelength drift is primarily determined by the high temperature behavior of the optical fiber itself.The experimental results above, from previous reports and our new experimental studies presented in this paper, show that the accuracy of long-term temperature monitoring using FBGs will be strongly affected by isothermal Bragg wavelength drift if measurements are performed at high temperatures, i.e., at 800–1000 °C. This can be seen by converting the accumulated drift of the Bragg wavelength  into a fictitious short-term temperature variation  that would cause a Bragg wavelength drift of the same magnitude. More specifically, to compete with standard high temperature thermocouples in terms of accuracy, the wavelength drift of a FBG should not exceed a few tens of picometers if one assumes that a 16.8 pm change in the Bragg wavelength corresponds to a ~1 °C temperature variation in the 800 °C to 1000 °C temperature range . So far,As mentioned above, the experimental data presented in the literature, fall into two distinct categories: the wavelength drift patterns of FBGs during 12 to 9000 h of annealing at 900 °C to 1000 °C and the results of high temperature tests specially designed to study processes that may affect the wavelength drift, such as dopant diffusion, stress and glass chemical composition.The diverse results on the Bragg wavelength behavior at 1000 °C have nonetheless a certain degree of commonality. The most evident common feature of all the tests presented here is a very fast red drift (2–50 pm/h rate) in the beginning of 900 °C or 1000 °C annealing tests followed by a slower blue drift that can last for hundreds of hours. The red wavelength drift can also last for 100–200 h but the majority of the wavelength drift takes place in the first 10–30 h after the 1000 °C temperature has been reached. This trend is observed in ,48 at 10What is the cause of the rapid red drift? As it was mentioned earlier in the Introduction, there are several processes that can cause isothermal wavelength drift at high temperature. First, it is dopant diffusion, a well-known effect used in industry to enlarge the core of the fiber in a process called Thermally Expanded Core (TEC) . While TSecond, it is the fiber manufacturing process. During the drawing of the optical fiber and the fast cooling of the fiber, the internal stresses are frozen into the fiber (frozen-in stress). The residual stress induced during fiber drawing can be very large and thus cause changes in the refractive index of the optical fiber through the photoelastic effect ,74. AccoIt is possible that frozen–in stresses are released during annealing at ~1000 °C, causing an increase in the fiber refractive index and, as a consequence, a red drift of the Bragg wavelength at the beginning of the 1000 °C annealing process. High temperature pre-annealing at temperatures in excess of 1000 °C, such as 1100 °C or 1200 °C could release frozen-in stresses and thus mitigate the red drift during the first stage of annealing at a lower temperature ,60. ThisThe second stage of the annealing process at 1000 °C consists of a slow blue drift ranging between 0.1 and 0.5 pm/h that lasts for hundreds of hours. Based on the definition of the Bragg wavelength, Equation (1), the blue wavelength drift should result from either a decrease in the value of the effective refractive index or a reduction of the grating length. As it is hard to believe that the Bragg grating contracts at a high temperature, the blue drift should be mainly correlated with a slow change of the effective refractive index of the fiber substrate and/or the grating. The dopant diffusion could lead to a blue drift but, according to published reports, by the time the blue drift starts, , the diffusion process will have already been completed as it reaches equilibrium in less than 100 h . MoreoveFor FBGs to have a future as temperature sensors in the 900–1000 °C range, the phenomenon of Bragg wavelength drift needs to be addressed. According to the review of published results and our recent experiments presented in this paper, the drift exhibits two types of behavior: a fast red drift in the first 100–200 h and a slow blue drift after a “no-drift” period. As it seems that the red drift is caused by the release of frozen-in stresses generated during the fiber manufacturing process, its negative effect on the temperature measurement can be somewhat mitigated by pre-annealing the FBG sensor at a higher temperature. In our tests, annealing FBGs at 1000 °C for a few hundred hours reduced the wavelength drift at 900 °C to ~0.5 pm/h (0.03 °C/h), which, however, is still too high for accurate long-term temperature measurements. The same trend was reported in the literature when pre-annealing FBGs at 1100 °C and 1200 °C helped reduce wavelength drift at a lower temperature. The slow blue drift (0.1–0.5 pm/h rate) is more difficult to eliminate because of its elusive nature. However, it is probably possible to correct for the slow blue drift numerically as this phenomenon seems to be more predictable compared with the red drift. We would also like to emphasize that the Bragg wavelength drift at high temperatures should not affect FBG-based measurements performed on short time scales.


Self-assembly of proteins into amyloid fibrils is a hallmark of various diseases, including Alzheimer’s disease (AD) and Type-2 diabetes Mellitus (T2DM). Aggregation of specific peptides, like Aβ42 in AD and hIAPP in T2DM, causes cellular dysfunction resulting in the respective pathology. While these amyloidogenic proteins lack sequence homology, they all contain aromatic amino acids in their hydrophobic core that play a major role in their self-assembly. Targeting these aromatic residues by small molecules may be an attractive approach for inhibiting amyloid aggregation. Here, various biochemical and biophysical techniques revealed that a panel of tryptophan-galactosylamine conjugates significantly inhibit fibril formation of Aβ42 and hIAPP, and disassemble their pre-formed fibrils in a dose-dependent manner. They are also not toxic to mammalian cells and can reduce the cytotoxicity induced by Aβ42 and hIAPP aggregates. These tryptophan-galactosylamine conjugates can therefore serve as a scaffold for the development of therapeutics towards AD and T2DM. Paul et al. provide a rational design strategy to develop dual inhibitors, in the form of tryptophan-galactosylamine conjugates, against amyloid formation and aggregation of Aβ42 and hIAPP peptides. These conjugates are not toxic to mammalian cells and reduce cytotoxicity of Aβ42 and hIAPP aggregates, hence potentially could serve as scaffolds for development of Alzheimer’s disease therapeutics. Despite arising from different genetic, environmental and regulatory factors, most proteinopathies exhibit sub-cellular and molecular similarities, characterized by the accumulation of misfolded proteins that often, self-assemble into thermodynamically highly stable amyloid fibrils, which are resistant to proteolytic degradation and lack functionally stable amyloid fibrils7.Aberrant protein folding and consequent protein aggregation are hallmarks of a group of pathological conditions termed proteinopathies6. AD is the most common form of dementia in ageing populations9. A major triggering neuropathological cause of AD is the misfolding and accumulation of Aβ42, a hydrophobic peptide of 42-amino acids that is prone to self-assemble in the brain into toxic extra-cellular amyloid fibrils termed senile plaques10. Protein self-assembly is also a key feature in T2DM11. In this disease, pathological deposits of the peptide amylin, also called hIAPP, are found in the pancreatic islets of Langerhans of T2DM patients12. hIAPP is a 37-amino acid neuroendocrine peptide hormone produced by β-cells together with insulin14. When high insulin is required, such as in insulin deficiency, expression of hIAPP significantly increases, enhancing its propensity to aggregate into cytotoxic amyloid assemblies resulting in loss of β-cells and β-cell mass15.Alzheimer’s disease (AD) and type 2 diabetes mellitus (T2DM) are prime examples of proteinopathies17. Hyperglycemia increases the chance of developing AD by at least 2-fold by contributing to the accumulation of amyloid plaques on brain lesions18. AD brains are less capable of glucose uptake from the surroundings, resembling a condition of insulin resistance19. In both T2DM and AD, oxidative stress is exacerbated, mitochondrial functions are impaired and neuron integrity compromised23.Epidemiological studies indicate comorbidity of AD and T2DM25. In both Aβ42 and IAPP, the minimal aggregating sequence is a core of hydrophobic amino acids containing aromatic residues, KLVFFA and NFGAIL, respectively28. Aromatic amino acids have been identified as crucial in the formation of various amyloid structures. π-π stacking, rather than mere hydrophobicity, was shown to provide energy, order and directionality to promote amyloid assembly31. Based on these insights, peptide-based inhibitors were designed to contain aromatic residues whose side chains can intercalate the target amyloid aggregate and partially replace the original amino acids in π–π stacking36. Along the same lines, small aromatic molecules, e.g. polyphenols, have been amply studied as inhibitors of amyloid aggregation, where the aromatic ring appears to similarly interfere with amyloid self-assembly41.Intervening with the initial steps of protein misfolding and aggregation can thus be both a prophylactic measure and a means for modifying the course of these diseases46. However, a major limitation in the drugability of such molecules is their poor solubility in aqueous media49: Attaching a hydrophilic moiety for increasing solubility may enhance the overall therapeutic capacity of a drug48. Functionalization with glycans has been proven to enhance solubility, half-life and specificity of certain drugs in vivo50. The conjugation of glycans to peptides increases the overall interaction surface of the hybrid molecules, therefore increasing solubility by expanding the number of possible interactions with the solvent53. Along the same rationale, aromatic molecules, which display high potential as anti-amyloidogenic agents, but are by themselves hydrophobic, were conjugated to mannitol and glucosamine54.Combining the aromatic elements of amino acids and small molecules has also been attempted for amyloid inhibition. For example, a naphthoquinone–tryptophan hybrid was shown to be effective towards various amyloids  in vitro as well as in vivo56. Galactose , one of the most abundant monosaccharide in the human body, plays a vital role in numerous biological processes, modulating and mediating them58. To be able to speculate on the mechanistic role of galactose, we have included in the study GalNH2 and GalNAc modifications at C2 of galactose influencing its charge density.Here we examined this concept by synthesizing tryptophan-galactosylamine hybrid molecules and tested their ability to inhibit the aggregation of Aβ42 and hIAPP. Tryptophan was shown to be highly effective in intercalating the fibrils of various amyloidogenic proteins for inhibiting their aggregationsBy means of Thioflavin T (ThT)-binding assay, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM) and Congo red birefringence, we show that the presence of galactose is key for the improved efficiency of tryptophan as amyloid inhibitor. The tested compounds are not toxic to mammalian cell lines. These results suggest that tryptophan-monosaccharide may be developed as disease-modifying therapeutics to target simultaneously Aβ42 and hIAPP.2 and NAc groups. For simplicity, the hybrid molecules resulting from the conjugation of tryptophan and these sugars are hereafter referred to as WGal, WGalNH2 and WGalNAc. As a negative control, the dipeptide formed by two tryptophan, WW was included. The structure of these hybrids is shown in Fig. To examine the possibility of combining an amino acid and a glycan into a potentially improved amyloid inhibitor, we conjugated tryptophan to the N-glycoside 1-amino-1-deoxy-β-D-galactose  and to its variants derived from the substitution of the OH on C2 with NH60. The curve reached a plateau after ca. 8 h, indicating the completion of amyloid fibril formation. Next, Aβ42 was co-incubated with increasing doses of the tested hybrid molecules at peptide:hybrid molar ratios of 5:1, 1:1 and 1:5  was caused by the hybrid molecules towards either cell line, even at the highest concentration of 250 µM  with either human neuroblastoma (SH-SY5Y) or with human embryonic kidney (HEK-293) cell lines and assessed cell viability by XTT assay µM Fig. . However50 values for Aβ42 and hIAPP were found to be ~13 µM and ~10 µM, respectively, as compared to untreated control cells, in agreement with previous reports79. Various doses of the hybrid molecules  were added separately to pre-aggregated Aβ42 (13 µM) or hIAPP (10 µM). The mixtures were then independently applied to the respective cells for 24 h, and cell viability was further measured by XTT assay  to allow fibril formation. The peptide assemblies were then incubated for further 24 h with SH-SY5Y or HEK-293 cells and cell viability was monitored by XTT assay   Fig. . In agreely Fig. .2 and WGalNAc incremented the viability of 18 ± 5%, 61.7 ± 5%, 53 ± 5%, and 64 ± 4% respectively   Fig. . The calely Fig. .These results indicated that the hybrid molecules significantly reduced the cytotoxic effect induced by Aβ42 and hIAPP fibrils. WGalNAc was the most effective among them.In this work, we evaluated the potential of a new class of highly-soluble tryptophan-galactosylamine hybrid molecules to alter the progression of the aberrant aggregation of the disease-associated peptides Aβ42 and hIAPP, both in vitro and in mammalian cell lines.80. Indeed, tryptophan has been shown to be directly involved in the abrogation of protein aggregation by targeting the aromatic recognition interfaces of amyloidogenic proteins42. Tryptophan derivatives have been employed in the design of several inhibitors of amyloid formation. For example, tryptophan-containing peptides have been employed to reduce Aβ42-derived toxicity in animal models81. Tryptophan-coated gold and silver nanoparticles have been shown to inhibit both spontaneous and seed-induced aggregation of insulin82. Naphthoquinone–tryptophan hybrids displayed promising potential for mitigating the amyloidogenicity of several proteins and peptides83.The amino acid tryptophan was chosen in this study because it was reported to display the most amyloidogenic propensity among the proteinogenic amino acids, suggesting a potentially strong involvement of its aromatic side chain in the molecular associations leading to the formation of amyloid fibrils85.The present work aimed at improving the drugability of tryptophan by combining its ability to target amyloid species with a component that would confer the capacity to dissolve the target protein aggregates. Thus, we postulated that the conjugation of tryptophan to a glycan unit could increase solubility and target selectivity of the hybrid molecule88. For example, galactosylated nanocarriers have been shown to improve site-specific delivery of siRNA and anticancer drugs86. Indeed, galactose has been employed in studies for the treatment and diagnosis of several diseases, as well as vaccine development91. D-galactose, together with D-glucose, are actively transported across the blood-brain barrier (BBB), making these hexoses highly attractive for the specific delivery of drugs to the brain92. Galactose-conjugated dopamine was found to have better BBB permeability, bioavailability, and therapeutic effects against Parkinson’s disease95. Finally, galactose is a preferred glycan candidate for targeting hIAPP because, due to hepatic metabolism, insulin and glucose level responses to galactose are lower than for glucose97.The glycan galactose is involved in numerous biological processes, hence galactosylation is considered as a useful tool for delivery of therapeuticsTaken together, this evidence points to tryptophan and galactose as desirable building blocks for the design of potential hybrid molecules against amyloid formation.98. The formation of oligomers in the absence of other pre-existent larger peptide species is refer to as primary nucleation99. These oligomers can grow into fibers by the addition of other oligomers (elongation) or monomers (secondary nucleation)60. The kinetics of amyloid formation generally follows a sigmoidal trend, in which a stable lag phase, an exponential phase and a plateau, the stage by which no more monomers are available, can be distinguished101. According to the mechanism of action of a potential inhibitor, any of the phases of the aggregation process may be effected. All our in vitro analyses demonstrated an appreciable amyloid inhibitory effect on both Aβ42 and hIAPP by all the tryptophan-galactosylamine hybrids. The data collected for WGal, WGalNH2 and GalNAc show that there is no kinetic effect caused by the inhibitors on primary nucleation or elongation . Instead, the plateau levels of both Aβ42 and hIAPP are significantly reduced by the presence of the hybrid molecules. To speculate on the mechanistic role that these inhibitors are playing on the aggregation of the target peptides, it is important to bear in mind that there are many ways by which small molecules may model the aggregation pathway103. Among these, it has been reported that various inhibitors reduce the amount of amyloids by redirecting the aggregation towards alternative off-path products, rather than preventing the self-assembly of the monomers106. We believe that this is the case for the hybrid molecules studies here: the lack of an obvious kinetic effect on primary/secondary nucleation or elongation suggests the sequestration of the monomers and oligomers to a parallel alterative pathway that does not lead to amyloid formation but to smaller and often amorphous assemblies. This hypothesis is supported by the ca. 50% reduction of ThT-positive aggregates in the presence of the inhibitors; by the variation in Aβ42 and hIAPP’s secondary structures; by the lack of fiber-like assemblies as shown by TEM and Congo red birefringence; and by the significant lower cellular toxicity.At the core of the amyloidogenic process there are conformational changes of the native, soluble peptides into unstable oligomers that subsequently assemble into insoluble protofibrils and fibrils2, as indicated by the p-value calculations . These results could be explained by the amyloidogenic nature of tryptophan and by its ability to self-assemble into fibrils, resulting in an additive aggregation level80. The co-presence of aggregating peptides may have functioned as further nucleation agent for the π–π stacking of tryptophan, which, on its end, could also have consequently induced further Aβ42 and hIAPP aggregation into mature amyloid fibrils. This effect, however, did not translate into an increment of toxicity in the in-cell assays: albeit not as efficiently, WW, like the hybrid molecules, reduced the cytotoxicity induced by extra-cellular pre-aggregated Aβ42 and hIAPP. To explain the partial inhibitory effect of WW on Aβ42 and hIAPP-induced toxicity, we postulate that WW may have driven the aggregates of these peptides towards larger bundle of fibrils, which are reported to be less toxic than smaller oligomeric forms114. WW’s effect on cytotoxicity, however statistically significant, is very weak when compared to the hybrid molecules: EC50 of all hybrids are between 2.5 and 5-fold lower than the one calculated for WW.Alongside the designed hybrid molecules, all experiments performed in this work included the dipeptide WW, here referred to as “negative control” which allowed to assert the effect of the galactosylamine derivatives in comparison to the tryptophan alone. However, in some circumstances, WW displayed a stimulatory effect on the aggregation of Aβ42 and hIAPP. This behavior became more pronounced and statistically significant . hIAPP was purchased from Peptide 2.0 (USA). The inhibitors  were purchased from Aldlab (USA). WW was purchased from GL Biochem Shanghai (China). Lipids were purchased from Avanti Polar Lipids (USA). All chemicals and reagents were of analytical grade. Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich .Aβ42 and hIAPP were monomerized by a 10 min pre-treatment with HFIP and the solvent was evaporated using a Speed Vac. The resulting thin films were dissolved in PBS  and sonicated for 5 min to get a stock concentration of 50 µM, which immediately diluted to required concentration for an experiment. Stock solutions of ThT (4 mM in PBS) and hybrid molecules (10 mM in PBS) were prepared. All the stock solutions were diluted according to the requirement.44. For fibril disassembly assays, Aβ42 and hIAPP were first allowed to self-assemble until a ThT-associated plateau was reached; the hybrid molecules were then added separately to designated wells , and the fluorescence values were further followed until a new plateau was reached. Kinetics fluorescence data were collected at 37 °C in triplicate using Infinite M200 microplate reader , with measurements acquired at 15 min intervals. Excitation and emission wavelengths were set at 440 nm and 485 nm, respectively.For the aggregation kinetics, the stock solutions of Aβ42 or hIAPP were diluted in a 96-well black plate so that the final mixture (100 µl) contained 10 µM peptide and 20 µM ThT in 100 mM PBS. The experiment was performed according to the previous report42 and hIAPP) were placed in a quarz cuvette (path length 1 mm) and CD spectra were recorded on a Chirascan spectrometer between 190–260 nm. The blank was subtracted from the acquired CD spectra. For the CD study in the presence of the tryptophan-galacosylamine molecules, the spectra of hybrid molecules were also recorded separately and subtracted from the respective sample spectra. The normalized data were plotted using Origin pro 2015 software.To analyze the secondary structure of the aggregated or disassembled peptides, 300 μL samples (10 µM of AβSamples (10 µL) were placed for 2 min on 400-mesh copper grids covered with carbon-stabilized Formvar film (Electron Microscopy Sciences (EMS), Hatfield, PA). Excess fluid was removed, and grids were negatively stained with 2% uranyl acetate solution (10 µL) for 2 min. After excess fluid removal, the samples were visualized using a JEM-1400 TEM (JEOL), operated at 80 kV.5 cells/mL) were cultured in 96-well tissue microplates (100 µL/well) and allowed to adhere overnight at 37 °C. The conjugate molecules were dissolved in DMEM:nutrient mixture F12 (Ham’s) (1:1)  at different concentrations. The negative control was prepared as medium without hybrid molecules and treated in the same manner. 100 µL of medium with or without hybrid molecules were added to each well in triplicate. Following incubation for 24 h at 37 °C, cell viability was evaluated using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) cell proliferation assay kit  according to the manufacturer’s instructions. Briefly, 100 µL of activation reagent was added to 5 mL of XTT reagent, followed by the addition of 50 µL of activated-XTT solution to each well. After 2 h incubation at 37 °C, color intensity was measured using an ELISA microplate reader at 450 nm and 630 nm. Results are presented as mean and mean standard error. Each experiment was repeated minimum three times.The SH-SY5Y and HEK-293 cell lines  and type to 2 , according to reported methods121. From the XTT viability assays, EC50 and Emax values were determined from the dose-response curves of each tested molecule using non-linear least-square data fitting, according to literature122.For the quantitative data derived from ThT and XTT assays, statistical significance, expressed as Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary DataPeer Review FileReporting Summary