Identification of Acyl-Protein Thioesterase-1 as a Polysorbate-Degrading Host Cell Protein in a Monoclonal Antibody Formulation Using Activity-Based Protein Profiling.

Polysorbate (PS) degradation in monoclonal antibody (mAb) formulations poses a significant challenge in the biopharmaceutical industry. PS maintains protein stability during drug product's shelf life but is vulnerable to breakdown by low-abundance residual host cell proteins (HCPs), particularly hydrolytic enzymes such as lipases and esterases. In this study, we used activity-based protein profiling (ABPP) coupled with mass spectrometry to identify acyl-protein thioesterase-1 (APT-1) as a polysorbate-degrading HCP in one case of mAb formulation with stability problems. We validated the role of APT1 by matching the polysorbate degradation fingerprint in the mAb formulation with that of a recombinant APT1 protein. Furthermore, we found an agreement between APT1 levels and PS degradation rates in the mAb formulation, and we successfully halted PS degradation using APT1-specific inhibitors ML348 and ML211. APT1 was found to co-purify with a specific mAb via hitchhiking mechanism. Our work provides a streamlined approach to identifying critical HCPs in PS degradation, supporting quality-by-design principles in pharmaceutical development.

Efficacy, safety, and patient satisfaction with oral semaglutide: first single-centre clinical experience.

OBJECTIVE: To explore the effects of oral semaglutide on glycaemic parameters, body weight, and satisfaction in the first recipient patients with type 2 diabetes mellitus in Slovenia, in a real-world clinical practice setting. METHODS: The first consecutive adult patients with type 2 diabetes who were eligible for oral semaglutide treatment were included in this prospective, open-label interventional study. Patients received increasing doses of oral semaglutide and were evaluated at inclusion, at 1 month, then 3-5 months after starting treatment. Fasting blood glucose, glycosylated haemoglobin (HbA1c), body weight, patient satisfaction with oral semaglutide treatment (using the validated Treatment Satisfaction Questionnaire for Medication), and adverse effects, were analysed. Statistical analyses were performed using one-way analysis of variance, and, when significant interactions were found, Bonferroni post-hoc test. A P-value <0.05 was considered statistically significant. RESULTS: Twenty patients (11 male: 9 female; mean age, 59.9 ± 1.5 years; mean diabetes duration, 8.5 ± 1.4 years) were included. Oral semaglutide (7 and 14 mg) significantly decreased HbA1c (from 9.4 ± 0.3% to 8.2 ± 0.2% and 7.8 ± 0.2%, respectively) and fasting plasma glucose (from 11.2 ± 0.5 mmol/L to 9.2 ± 0.7 mmol/L and 8.9 ± 0.4 mmol/L, respectively). Oral semaglutide (14 mg) significantly decreased body weight (from 100.9 ± 2.7 kg to 92.7 ± 2.4 kg). Patients reported that treatment was easy to use and expressed high global satisfaction. Mild and transient, mostly gastrointestinal, adverse effects were reported in 10 patients. CONCLUSIONS: Oral semaglutide, the first oral glucagon-like peptide 1 receptor agonist, was effective and safe, and associated with high patient satisfaction, in its first recipients in Slovenia. The results are important for daily clinical practice involving patients with type 2 diabetes, however, due to the small study population, lack of placebo control, and short exposure to oral semaglutide, the effectiveness of oral semaglutide in clinical practice requires further investigation.

Health-Related Quality of Life Assessment in Older Patients with Type 1 and Type 2 Diabetes.

Type 1 (T1D) and type 2 diabetes (T2D) are determinants of health-related outcomes including health-related quality of life (HRQOL). We aimed to determine differences in HRQOL between older adults with T1D and T2D and specific factors influencing HRQOL in this age group. This study used a cross-sectional design with 56 age- and HbA1c-matched T1D and T2D patients (aged 68.9 ± 7.8 years; 55% had T2D). We employed several validated questionnaires (Short Form-36 (SF-36) and the EuroQol-5 Dimensions/Visual Analog Scale (VAS)) to investigate the relationships between HRQOL domains and diabetes type, glycemic control, complications, and comorbidities. T1D was associated with better self-reported general health (assessed with the SF-36 general health domain (p = 0.048) and the EuroQol-5 VAS (p = 0.002), whereas no significant differences in the other SF-36 domains, self-reported diabetes distress, anxiety, or depression were found. Most HRQOL domains were not associated with HbA1c or the presence of diabetes complications. The most significant reduction in HRQOL was experienced by patients with higher BMIs, irrespective of the diabetes type. The obtained HRQOL data could be used in clinical settings for evidence-based patient education focused on specific subgroups of patients, as well as in national healthcare policies, e.g., interventions designed to alleviate obesity.

Effectiveness of a comprehensive telemedicine intervention replacing standard care in gestational diabetes: a randomized controlled trial.

AIMS: Telemedicine improves glycemic and perinatal outcomes when used as an adjunct to standard care in gestational diabetes (GDM). Little is known about its effectiveness when used instead of standard care. We aimed to compare the outcomes of telemedicine care and the standard care in women with GDM. METHODS: In a single-center, parallel, randomized controlled trial, women were randomized to: (1) a telemedicine group, sending glucose readings via an application installed on a smartphone and monthly individual video calls replacing on-site visits or (2) standard care group with routine monthly on-site visits. The primary outcome was the effectiveness of glycemic control. The secondary outcomes were gestational weight gain (GWG) and perinatal data, including birth weight, gestational age, the incidence of the offspring large for gestational age, preterm birth, preeclampsia and cesarean section. RESULTS: A total of 106 women were randomized to the telemedicine (n = 54) and the standard care group (n = 52). The telemedicine group demonstrated less postprandial measurements above the glycemic target (10.4% [3.9-17.9] vs. 14.6% [6.5-27.1]; p = 0.015), together with lower average postprandial glucose (5.6 ± 0.3 vs. 5.9 ± 0.4; p = 0.004). Percentage of cesarean section was lower in the telemedicine group (9 (17.3%) vs. 18 (35.3%); p = 0.038). CONCLUSIONS: Telemedicine offers an effective alternative to delivering care to women with GDM. Trial registration NCT05521893, ClinicalTrials.gov Identifier URL: https://www. CLINICALTRIALS: gov/ct2/show/NCT05521893?term=NCT05521893&draw=2&rank=1.

Ara h 2-specific IgE epitope-like peptides inhibit the binding of IgE to Ara h 2 and suppress lgE-dependent effector cell activation.

BACKGROUND: Clinical and experimental analyses indicate a pathognomonic role for allergen IgE crosslinking through epitope-paratope interactions as a major initial step in the cascade leading to effector cell activation and clinical manifestations of lgE-mediated food allergies. We aimed to undertake the initial development and assessment of Ara h 2-specific IgE epitope-like peptides that can bind to allergen-specific IgE paratopes and suppress effector cell activation. METHODS: We performed biopanning, screening, IgE binding, selection and mapping of peptides. We generated synthetic peptides for use in all functional experiments. ImmunoCAP inhibition, basophil and mast cell activation tests, with LAD2 cells, a human mast cell line were performed. Twenty-six children or young adults who had peanut allergy were studied. RESULTS: We identified and selected three linear peptides (DHPRFNRDNDVA, DHPRYGP and DHPRFST), and immunoblot analyses revealed binding to lgE from peanut-allergic individuals. The peptide sequences were aligned to the disordered region corresponding to the loop between helices 2 and 3 of Ara h 2, and conformational mapping showed that the peptides match the surface of Ara h 2 and h 6 but not other peanut allergens. In ImmunoCAP inhibition experiments, the peptides significantly inhibit the binding of IgE to Ara h 2 (p < .001). In basophil and mast cell activation tests, the peptides significantly suppressed Ara h 2-induced effector cell activation (p < .05) and increased the half-maximal Ara h 2 effective concentration (p < .05). Binding of the peptides to specific IgEs did not induce activation of basophils or mast cells. CONCLUSIONS: These studies show that the indicated peptides reduce the allergenic activity of Ara h 2 and suppress lgE-dependent basophil and mast cell activation. These observations may suggest a novel therapeutic strategy for food allergy based on epitope-paratop blocking.

Combination of Experimental and Bioinformatic Approaches for Identification of Immunologically Relevant Protein-Peptide Interactions.

Protein-peptide interactions are an essential player in cellular processes and, thus, of great interest as potential therapeutic agents. However, identifying the protein's interacting surface has been shown to be a challenging task. Here, we present a methodology for protein-peptide interaction identification, implementing phage panning, next-generation sequencing and bioinformatic analysis. One of the uses of this methodology is identification of allergen epitopes, especially suitable for globular inhaled and venom allergens, where their binding capability is determined by the allergen's conformation, meaning their interaction cannot be properly studied when denatured. A Ph.D. commercial system based on the M13 phage vector was used for the panning process. Utilization of various bioinformatic tools, such as PuLSE, SAROTUP, MEME, Hammock and Pepitope, allowed us to evaluate a large amount of obtained data. Using the described methodology, we identified three peptide clusters representing potential epitopes on the major wasp venom allergen Ves v 5.

No Indices of Increased Type 2 Diabetes Risk in Individuals with Reactive Postprandial Hypoglycemia.

Reactive postprandial hypoglycemia (RPH) is an understudied condition that lacks clinical definition, knowledge of future health implications, and an understanding of precise underlying mechanisms. Therefore, our study aimed to assess the glycemic response after glucose ingestion in individuals several years after the initial evaluation of RPH and to compare glucose regulation in individuals with RPH vs. healthy volunteers. We assessed the inter- and intra-individual differences in glucose, insulin, and C-peptide concentrations during 5-h oral glucose tolerance tests (OGTTs); the surrogate markers of insulin resistance (HOMA-IR and Matsuda index); and beta-cell function (distribution index and insulinogenic index). The study included 29 subjects with RPH (all females, aged 39 (28, 46) years) and 11 sex-, age-, and body mass index (BMI)-matched controls. No biochemical deterioration of beta-cell secretory capacity and no progression to dysglycemia after 6.4 ± 4.2 years of follow-up were detected. RPH subjects were not insulin resistant, and their insulin sensitivity did not deteriorate. RPH subjects exhibited no differences in concentrations or in the shape of the glucose-insulin curves during the 5-h OGTTs compared to age- and BMI-matched controls. No increased incident type 2 diabetes risk indices were evident in individuals with RPH. This dictates the need for further research to investigate the magnitude of future diabetes risk in individuals experiencing RPH.

Empagliflozin-Metformin Combination Has Antioxidative and Anti-Inflammatory Properties that Correlate with Vascular Protection in Adults with Type 1 Diabetes.

Methods: 40 individuals with type 1 diabetes (average age of 44.7 ± 2.5 years) were randomized into four groups: (1) control (placebo), (2) empagliflozin 25 mg daily, (3) metformin 2000 mg daily, and (4) empagliflozin-metformin combination (25 mg and 2000 mg daily, respectively). At inclusion and after 12 weeks of treatment, the blood samples were collected, and the oxidative stress (total antioxidative status (TAS), superoxide dismutase (SOD), glutathione peroxidase (GPx), uric acid, advanced oxidation protein products (AOPP), advanced glycosylation end products ((AGE) and isoprostane), and inflammation (C-reactive protein (CRP) and interleukin-6 (IL-6)) parameters were determined. Results: The empagliflozin-metformin combination increased levels of the antioxidants (TAS, SOD, and GPx up to 1.1-fold; P < 0.01), decreased the levels of prooxidants (AOPP and isoprostanes up to 1.2-fold, P < 0.01; AGE up to 1.5-fold, P < 0.01), and decreased inflammatory parameters (up to 1.5-fold, CRP P < 0.01; IL-6 P < 0.001). Antioxidative action was associated with the improvement in arterial function (obtained in the previous study) in the empagliflozin-metformin combination group. Conclusion: Empagliflozin-metformin combination has strong antioxidative and anti-inflammatory capacity, in adults with type 1 diabetes that is greater than that for the individual drugs. Its antioxidative activity at least partially explains the improvement in arterial function. Therefore, it appears that the combination provides the most powerful vascular protection.

Effect of Oral Semaglutide on Cardiovascular Parameters and Their Mechanisms in Patients with Type 2 Diabetes: Rationale and Design of the Semaglutide Anti-Atherosclerotic Mechanisms of Action Study (SAMAS).

INTRODUCTION: Type 2 diabetes (T2D) management has reached a point where not only optimal glycaemic control is necessary, but also additional interventions with proven cardiovascular risk reduction benefit. Subcutaneous semaglutide has been shown to provide cardiovascular protection, but its use may be limited by its injection formulation. To overcome this limitation, an oral semaglutide tablet has been developed, which could potentially be of the same value as its injection counterpart, but in a much wider group of patients with T2D, thereby allowing for broader cardiovascular risk reduction in this vulnerable patient population. METHODS: A total of 100 consecutive patients with T2D and a disease duration of up to 10 years, without manifest cardiovascular disease, who are treated with metformin (± sulphonylurea) and optimal cardioprotective therapy, will be recruited in a single-blinded, randomized trial named "Semaglutide Anti-atherosclerotic Mechanisms of Action Study (SAMAS)." After 1:1 randomization, patients will receive either oral semaglutide 14 mg daily or placebo for 1 year. The primary outcome comprises changes in atherosclerosis-related structural and functional characteristics of the arterial wall, namely: reduction of the carotid intima-media thickness, improvement of endothelial function and decrease in arterial stiffness. Secondary outcomes are changes in atherogenic small dense low-density lipoproteins, glucose control (HbA1c) and inflammatory markers (hsCRP). Possible correlations between primary endpoints and changes in lipids, HbA1c and high-sensitivity C-reactive protein will be sought. DISCUSSION: This is the first study to investigate the direct and indirect anti-atherosclerotic mechanisms of oral semaglutide. The results are expected to confirm the position of oral semaglutide in the multifactorial management of T2D with an emphasis on cardiovascular disease prevention. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05147896.

Treating Arterial Ageing in Patients with Diabetes: From Mechanisms to Effective Drugs.

Diabetes mellitus is a major healthcare problem. It is not only characterized by hyperglycemia and chronic complications, but in longer lasting diabetes and a longer living population, it is also associated with accelerated arterial ageing, which importantly contributes to cardiovascular complications. The accelerated arterial ageing in patients with diabetes should be considered separately from arterial ageing in patients without diabetes. Basic and clinical research have allowed better insight into the mechanisms of arterial ageing. In a simplified mechanistic way, it could be considered that the three tightly connected cornerstone characteristics of arterial ageing in patients with diabetes are: phenotypic presentation as endothelial dysfunction and arterial stiffness, and the underlying basic ageing-facilitating mechanism represented as the impaired expression of genetic longevity pathways. Currently, specific drugs for preventing/treating arterial ageing are not available. Therefore, we aimed to review the capacity of available drugs, particularly antidiabetic drugs, to interfere with the arterial ageing process. In the near future, these characteristics could help to guide therapy in patients with diabetes. Overall, it appears that arterial ageing could become a new target in diabetes. The expanding knowledge regarding the capability of antidiabetic drugs and other available drugs to inhibit/delay arterial aging is therefore essential.

Bee Venom Immunotherapy: Current Status and Future Directions.

Bee venom immunotherapy is the main treatment option for bee sting allergy. Its major limitations are the high percentage of allergic side effects and long duration, which are driving the development of novel therapeutic modalities. Three general approaches have been evaluated including the use of hypoallergenic allergen derivatives, adjunctive therapy, and alternative delivery routes. This article reviews preclinical and clinical evidence on the therapeutic potential of these new therapies. Among hypoallergenic derivatives, hybrid allergens showed a markedly reduced IgE reactivity in mouse models. Whether they will offer therapeutic benefit over extract, it is still not known since clinical trials have not been carried out yet. T cell epitope peptides have proven effective in small clinical trials. Major histocompatibility complex class II restriction was circumvented by using long overlapping or promiscuous T cell epitope peptides. However, the T cell-mediated late-phase adverse events have been reported with both short and longer peptides. Application of mimotopes could potentially overcome both T cell- and IgE-mediated adverse events. During this evolution of vaccine, there has been a gain in safety. The efficacy was further improved with the use of Toll-like receptor-activating adjuvants and delivery systems. In murine models, the association of allergen Api m 1 with cytosine-guanosine rich oligonucleotides stimulated strong T-helper type-1 response, whereas its encapsulation into microbubbles protected mice against allergen challenge. An intralymphatic administration of low-dose vaccine has shown the potential to decrease treatment from 5 years to only 12 weeks. Bigger clinical trials are needed to follow up on these results.

Acceptability of Continuous Glucose Monitoring in Elderly Diabetes Patients Using Multiple Daily Insulin Injections.

Background: Integrative diabetes care is lifelong and encompasses patient-reported outcome measures (PROMs). Understanding older adults' perceptions of continuous glucose monitoring (CGM) benefits and potential annoyances is important to assist with introducing it in this population. The aim of this study was to investigate PROMs and effectiveness of CGM introduction in elderly multiple daily injection (MDI) users with well-controlled diabetes. Methods: MDI-treated elderly (n = 25, mean age 67.6 ± 1.2 years, HbA1c = 7.1% ± 0.2%, 56% type 1 diabetes) were instructed to use a CGM device. PROMs were measured by questionnaires. CGM-recorded glycemic control metrics (time in range [TIR], time in hypoglycemia, coefficient of variation [CV]) were compared during blinded CGM and real-time CGM. Results: Satisfaction with CGM use was high; with perceived advantages as "very common" (4.22 out of 5) and annoyances as "modest" (1.82 out of 5). In total 95% of participants expressed improved sense of security with CGM use, 68% reported of improved sleep quality, and 82% were willing to use a CGM device after finishing the study protocol. CGM introduction did not impose additional diabetes-related distress (measured by the Problem Areas in Diabetes questionnaire). Significant improvements in TIR (3.9-10.0 mmol/L) (66.3% ± 2.6% vs. 76.9% ± 3.0%; P < 0.001), time in hypoglycemia (9.6% ± 2.1% vs. 5.2% ± 1.1%; P = 0.041), as well as reduced glycemic variability (%CV) (37.3 ± 11.1 vs. 32.9 ± 6.3; P < 0.001) were observed. Conclusion: Introduction of CGM in elderly patients with well-controlled diabetes resulted in high satisfaction without imposing additional diabetes distress. Furthermore, an added benefit in glucose control with stabilizing glycemia in target range was proven.

Expression of Longevity Genes Induced by a Low-Dose Fluvastatin and Valsartan Combination with the Potential to Prevent/Treat "Aging-Related Disorders".

The incidence of aging-related disorders may be decreased through strategies influencing the expression of longevity genes. Although numerous approaches have been suggested, no effective, safe, and easily applicable approach is yet available. Efficacy of low-dose fluvastatin and valsartan, separately or in combination, on the expression of the longevity genes in middle-aged males, was assessed. Stored blood samples from 130 apparently healthy middle-aged males treated with fluvastatin (10 mg daily), valsartan (20 mg daily), fluvastatin-valsartan combination (10 and 20 mg, respectively), and placebo (control) were analyzed. They were taken before and after 30 days of treatment and, additionally, five months after treatment discontinuation. The expression of the following longevity genes was assessed: SIRT1, PRKAA, KLOTHO, NFE2L2, mTOR, and NF-κB. Treatment with fluvastatin and valsartan in combination significantly increased the expression of SIRT1 (1.8-fold; p < 0.0001), PRKAA (1.5-fold; p = 0.262) and KLOTHO (1.7-fold; p < 0.0001), but not NFE2L2, mTOR and NF-κB. Both fluvastatin and valsartan alone significantly, but to a lesser extent, increased the expression of SIRT1, and did not influence the expression of other genes. Five months after treatment discontinuation, genes expression decreased to the basal levels. In addition, analysis with previously obtained results revealed significant correlation between SIRT1 and both increased telomerase activity and improved arterial wall characteristics. We showed that low-dose fluvastatin and valsartan, separately and in combination, substantially increase expression of SIRT1, PRKAA, and KLOTHO genes, which may be attributed to their so far unreported pleiotropic beneficial effects. This approach could be used for prevention of ageing (and longevity genes)-related disorders.

Epitope Mapping of Major Ragweed Allergen Amb a 1.

Ragweed is a prominent cause of seasonal allergies. Thus far, information on IgE-binding sites of major allergen in ragweed pollen, Amb a 1, is very limited. A powerful experimental method to gain insights on the allergen epitopes is the selection of peptides from biological libraries that bind to anti-allergen antibodies. In this work, we aimed to map IgE epitopes of Amb a 1 using epitope-mimicking short peptides - mimotopes that were affinity-selected from phage-displayed random peptide libraries. The peptides weakly aligned with the Amb a 1 primary sequence, thus suggesting that the epitopes are conformational. When the peptides were mapped onto the surface of Amb a 1 homology model, the EpiSearch analysis predicted the location of four potential epitopic sites on surface patches centred at residues K104, S110, H214, and W312. The peptides matching to the predicted epitopes bound selectively to the IgE from pool of ragweed-allergic patients' sera and therefore represent mimetics of Amb a 1 IgE epitopes. The knowledge of IgE epitopes is a prerequisite for the rational design of molecular-based approaches to diagnosis and immunotherapy of allergic diseases.

In-vitro study of the influence of octocrylene on a selected metastatic melanoma cell line.

BACKGROUND: Octocrylene (OCT) is one of the most widespread chemical UV filters used in sunscreens and cosmetic products. Despite the use of sunscreens and personal care products over decades, melanoma as the most serious and aggressive form of skin cancer is still a cause of concern. Hence the aim of this study was to investigate any potential influence of OCT on metabolic activity, cytotoxicity and ABCB5 mRNA expression in melanoma cells. The ABCB5 transmembrane protein was tested due to its well-known role in the initiation, invasion and metastatic spread of various cancers, including melanoma. METHODS: Metastatic melanoma cell line WM-266-4 (ATCC) was incubated with selected concentrations of OCT and for different time intervals. The MTT and LDH assays to measure the cells' metabolic activity and cytotoxicity were used respectively. Target gene (ABCB5) expression was detected by quantitative real-time PCR (qRT-PCR), using TaqMan® chemistry. RESULTS: Our results suggest decreased metastatic melanoma cells' metabolic activity, increased cytotoxicity and increased ABCB5 mRNA expression (P<0.05) with longer time of exposure to OCT as compared to control cells. Accordingly, we suspect that the surviving cells are more invasive and aggressive, which might explain their microscopically observed cannibalistic activity. CONCLUSIONS: With this study, we elucidate a new promising field for further research to contribute to etiology and prevention of melanoma.

Prevention of Vascular Complications in Diabetes Mellitus Patients: Focus on the Arterial Wall.

In Diabetes Mellitus (DM), hyperglycaemia and insulin resistance progressively lead to both microvascular and macrovascular complications. Whereas the incidence of microvascular complications is closely related to tight glycaemic control, this does not apply to macrovascular complications. Hyperglycaemia influences many interweaving molecular pathways that initially lead to increased oxidative stress, increased inflammation and endothelial dysfunction. The latter represents the initial in both types of vascular complications; it represents the "obligatory damage" in microvascular complications development and only "introductory damage" in macrovascular complications development. Other risk factors, such as arterial hypertension and dyslipidaemia, also play an important role in the progression of macrovascular complications. All these effects accumulate and lead to functional and structural arterial wall damage. In the end, all factors combined lead to the promotion of atherosclerosis and consequently major adverse cardiovascular events. If we accept the pivotal role of vascular wall impairment in the pathogenesis and progression of microvascular and macrovascular complications, treatment focused directly on the arterial wall should be one of the priorities in prevention of vascular complications in patients with DM. In this review, an innovative approach aimed at improving arterial wall dysfunction is described, which may show efficacy in clinical studies. In addition, the potential protective effects of current treatment approaches targeting the arterial wall are summarised.

Silver Fir (Abies alba) Extracts Inhibit Enzymes Involved in Blood Glucose Management and Protect against Oxidative Stress in High Glucose Environment.

The diet rich in fruits and vegetables reduces the risk of metabolic syndrome, including diabetes development by various mechanisms of action, mainly due to the presence of polyphenolic compounds. Extracts from different conifer species are known to be a rich source of various polyphenols. In the present study we elucidated the in vitro mechanism of anti-diabetic activity of silver fir (Abies alba) wood and bark extracts and compared their activity to non-coniferous sweet chestnut wood extract and standardized maritime pine bark extract. Extracts and lignans were tested for their inhibitory activity of enzymes involved in the regulation of blood glucose in vitro. The ability of extracts to protect against oxidative stress in high glucose environment was tested on mouse myoblast cell line. Silver fir wood and bark extracts were shown to be effective inhibitors of α-glucosidase, α-amylase and dipeptidyl peptidase 4, three enzymes involved in the regulation of blood glucose levels. Coniferous extracts also showed protection against oxidative stress generated in high glucose environment. Lignans, particularly pinoresinol diglucoside, isolariciresinol and secolariciresinol were shown to be important contributors of antihyperglycemic activity through inhibition of dipeptidyl peptidase 4. This corroborates previously published in vivo results on blood glucose level obtained with silver fir wood extract and supports the use of silver fir wood and bark extracts as food supplements or functional foods in borderline diabetes.

The effect of micro-sized titanium dioxide on WM-266-4 metastatic melanoma cell line.

Titanium dioxide (TiO2) is widely used as an inorganic UV-filter in cosmetic products; however, it has been classified as possibly carcinogenic to humans. While numerous studies demonstrated cytotoxic and genotoxic effects of nano-sized TiO2 in different cell lines, including human skin cells, studies investigating the effects of micro-TiO2 on human keratinocytes and melanocytes, in healthy and cancer cells, are scarce. Adenosine triphosphate (ATP) binding cassette subfamily B member 5 (ABCB5) is a plasma membrane protein known for its role in the tumorigenicity, progression, and recurrence of melanoma. Here, we investigated the effect of micro-TiO2 (average particle size ≤5 µm) on the metabolic activity, cytotoxicity and ABCB5 mRNA expression in metastatic melanoma cells. Metastatic melanoma cell line WM-266-4 was treated with different concentrations of micro-TiO2 for different incubation times to obtain dose- and time-dependent responses. Untreated WM-266-4 cells, cultured under the same conditions, were used as control. The cell metabolic activity was determined by MTT assay. Cytotoxicity of micro-TiO2 was analyzed by lactate dehydrogenase (LDH) cytotoxicity assay. The ABCB5 mRNA expression in melanoma cells was analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). After 120 hours of exposure to micro-TiO2 the metabolic activity of melanoma cells decreased, especially at the two highest micro-TiO2 concentrations. Comparably, the cytotoxicity of micro-TiO2 on melanoma cells increased after 48 and 120 hours of exposure, in a time-dependent manner. The ABCB5 mRNA expression in micro-TiO2-treated melanoma cells also decreased significantly after 24 and 48 hours, in a time-dependent manner. Overall, our results suggest inhibitory effects of micro-TiO2 on the metabolic activity and ABCB5 mRNA expression in metastatic melanoma cells, indicating its potential use as an anticancer agent.