Bee pollen represents one of the most complete natural foods playing an important role in the diet for its health qualities and therapeutic properties. This work aimed to characterize a Tuscan bee pollen by evaluating its phytochemical profile and the in vitro and ex vivo antioxidant activities. The isolation and taxonomic and functional characterization of yeasts in the sample has been also conducted. Finally, the pollen anti-inflammatory potential has been assessed on a TNFα-inflamed human colorectal adenocarcinoma cell line (HT-29). Our results highlighted a good phytochemical composition in terms of polyphenols, flavonoids, flavonols, monomeric anthocyanins, and carotenoids. In addition, we detected good antioxidant activity and radical scavenging capacity by in vitro and ex vivo assays, as well as good antioxidant activity by isolated yeasts. Data showed no cytotoxic effects of bee pollen extracts, with average viability values >80% at each tested dose. Moreover, TNFα treatment did not affect HT-29 viability while upregulating IL-8, COX-2, and ICAM-1 gene expression, otherwise reduced by both doses of bee pollen. In conclusion, our sample represents an interesting functional food and a potential probiotic product, having high phytochemical compound levels and good antioxidant activities, as well as anti-inflammatory effects on the TNFα-inflamed HT-29 cell line. For centuries, apicultural products have been used in phytotherapy and alternative medicine and played an important role in the human diet for their health qualities and nutritional implications ,2,3,4,5.Apis mellifera) from flowering plants anthers using beehives equipped with bottom-fitted pollen traps so that honeybees lose pollen loads before entering  × 100. Data are the mean ± SD of two independent experiments. Trolox was used as the standard. The 2,2-diphenyl1-1-picrylhydrazyl (DPPH) radical scavenging ability of seven yeast strains  Y3, Y5, Y6, Y7, and Y9) was determined using the method described by Ciafardini and Zullo , with so·+) scavenging activity of bee pollen extract was determined according to Lopez-Martinez et al.  × 100, where Ai is the initial absorbance and Af is the final absorbance. The ABTS·+ scavenging activity was expressed as Trolox equivalent antioxidant capacity (TEAC) using a standard curve of Trolox (5–1000 µg/mL). The bee pollen extract concentration corresponding to 50% of ABTS·+ radical inhibition  was also determined.The 2-2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical cation ] × 100, where AS is the absorbance of the sample and AC is the absorbance of the DPPH solution. The bee pollen extract concentration corresponding to 50% of DPPH• radical inhibition  was also determined.The DPPHi et al. . The antex 485 nm and λem 514 nm using a VictorTM X3 Multilabel Plate Reader . Trolox was used as the standard, and results were expressed as ORAC units (µmol TE/g FW). The oxygen radical absorbance capacity (ORAC) of bee pollen extract was determined as described by Gabriele et al. . AAPH wa3+) to ferrous iron (Fe2+) was evaluated by the FRAP assay according to Colosimo et al. [3·6H2O at a ratio of 10:1:1). After 30 min of incubation at room temperature, the absorbance was measured at 593 nm . Results were expressed as Fe2+ equivalents (µM), using a standard curve of FeSO4·7H2O (15.62–2000 μM), and as μmol Fe2+/g FW.The ability of bee pollen extract to reduce ferric iron  after 15 min of incubation. The results were expressed as mg EDTA equivalent (EDTAE)/g FW using a standard curve of EDTA (3.125–75 µg/mL). A decrease in absorbance is linked to an increase in Fe2+ chelating capacity.The ability of aqueous bee pollen extract to chelate Fes et al.  with somg for 20 min at 4 °C to remove the buffy coat and plasma. The erythrocytes were then washed twice with PBS pH 7.4.Erythrocytes were collected from healthy blood donors upon informed consent for the use of residual blood for research purposes according to the Italian regulations and, in particular, the regulations of “Fondazione G. Monasterio CNR-Regione Toscana”. Blood was collected in EDTA-treated tubes and centrifuged at 2300× ex 485 nm and λem 535 nm . Results were expressed as follows: CAA unit = 100 − (∫SA/∫CA) × 100, where ∫SA is the integrated area of the sample curve and ∫CA is the integrated area of the control curve.The cellular antioxidant activity (CAA) of ethanolic bee pollen extract (BE 25 and 50 µg/mL) was assessed ex vivo on oxidized red blood cells according to the CAA-RBC method described by Frassinetti et al. [The anti-hemolytic effects of ethanolic bee pollen extracts (BE 25 and 50 µg/mL) were evaluated on oxidized human erythrocytes as described by Frassinetti et al. [The human colonic adenocarcinoma cell (HT-29) line  was grown, as previously reported . All tre® Total RNA Kit I  and reverse-transcribed using the iScript™ Advanced cDNA Synthesis Kit . A quantitative Real-Time PCR was performed using the SsoFast™ EvaGreen® Supermix  in a CFX Connect Real-Time PCR Detection System . IL-8 (C-X-C motif chemokine ligand 8), COX-2 (prostaglandin-endoperoxide synthase 2), ICAM-1 (intercellular adhesion molecule-1), and β-actin gene primers were previously described by Gabriele et al. [−ΔΔCT relative quantification method and data are expressed as a fold-change of expression levels compared to the control.Total RNA was isolated from HT-29 cells using the E.Z.N.A.e et al. . The genp-value lower than 0.05 was considered statistically significant.The results were expressed as mean ± standard deviation (SD) of at least three replicates. A statistical analysis was performed using GraphPad Prism, version 5.00 . Data were analyzed by one-way analysis of variance (ANOVA) with Bonferroni’s or Dunnett’s Multiple Comparison test and a Castanea sativa sp. was the most representative (88.8%), followed by Hedera helix sp. (4.2%), Rubus ulmifolius Schott sp. (1.7%), and others (5.3%). Each pollen load owned a homogeneous and monospecific pollen content. Considering the high prevalence of Castanea pollen grains (>45%), we can classify our sample as a monofloral bee pollen type according to what was reported by Alimoglu et al. [The bee pollen sample from Bagni di Lucca  is composed of more botanical species differently distributed in the analyzed sample. The u et al. .Several natural polyphenols can be found in plant-based foods, contributing to the organoleptic properties of plants/foods, such as color, odor, flavor, astringency, bitterness, and oxidative stability ,29,30. GAccording to the literature, phenolic compounds are one of the molecular classes most representative in bee pollen samples ,2,9,31. The sample herein analyzed was screened for the total polyphenol, flavonoid, flavonol, monomeric anthocyanin, and carotenoid content, and the results are listed in Castanea (24.75 ± 0.78 mg GAE/g FW), Cistus (21.19 ± 0.24 mg GAE/g FW), and Viburnum (99%) (20.15 ± 0.15 mg GAE/g FW) previously analyzed by Gabriele et al. and Barbieri et al. [Cistus ladaniferus), obtained values between 0.87 and 1.46 g GAE/100 g, corresponding to less than half of the polyphenols found in our sample. Furthermore, the flavonoid findings are in agreement with some literature data with values in the range of 0.3–79.21 mg QE/g FW [Asteraceae, Fabaceae, Campanulaceae, Cistaceae, and Rosaceae), analyzed by Gercek et al. [Castanea (4.77 ± 0.09 mg QE/g FW), Cistus (4.93 ± 0.05 mg QE/g FW), and Rubus (2.52 ± 0.14 mg QE/g FW) Tuscan bee pollens collected in Massa Macinaia (LU) [The bee pollen extract from Bagni di Lucca contained good levels of polyphenols. The present findings agreed with the literature data reporting values ranging from approximately 5 and 213.2 mg GAE/g FW ,37,38,39i et al. . Moreovei et al. , while S QE/g FW ,33,37,39k et al. , had an Cistus (57.19 ± 5.84 mg C3GE/L) and Rubus bee pollen (53.44 ± 2.36 mg C3GE/L), while it is lower than Castanea bee pollen (77.37 ± 2.55 mg C3GE/L) analyzed in the same study.Monomeric anthocyanins are part of the pigments responsible for the purple, red, and blue coloration of fruits, vegetables, and flowers, and exert several bioactivities such as anti-inflammatory effects . The antBananeira (15.85 μg/g FW) and Pyririca Branca bee pollens (12.38 μg/g FW) was similar to that obtained for our sample. According to the literature, the average carotenoid content determined in various bee pollens is placed in a range between 1.38 μg/g FW and 425.32 μg/g FW [Lastly, carotenoids are plant pigments responsible, together with flavonoids, for the characteristic yellow/orange color of bee pollen . Caroten μg/g FW ,44. Starmerella bombicola (Y1), Candida magnoliae (Y2), Metschnikowia sp. , Aureobasidium sp. (Y4 and Y5), Pichia guilliermondii (Y6), Rhodotorula sp. (Y7), and Curvibasidium sp. (Y9).According to the NCBI blast database, the ten isolated yeasts were taxonomically related to seven different genera/species , namely Starmerella (Candida) bombicola, Starmerella (Candida) magnoliae, and Curvibasidium cygneicollum respectively, strains Y3, Y8, and Y10 are all grouped in a clade hosting different Metschnikowia sp. species, despite which the most represented is Metschnikowia pulcherrima. The same can be assessed for strains Y4 and Y5, both included in an Aureobasidium sp. clade with two different species: A. pullulans and A. melanogenum. According to NCBI Blast, Y6 is closely related to Pichia guilliermondii (Meyerozyma guillermondii-Candida Guillermondi), but the phylogenetic tree attributes this strain to a clade composed of different Pichia sp./Meyerozyma sp. species, with a strong dominance of species P. guillermondi. The same observation can be performed for the Y7 Rhodotorula sp.–related strain, which is taxonomically close to both the R. glutinis and R. babjevae species.The phylogenetic tree reconstruction  allowed Rhodotorula sp. and Starmerella (Candida) magnoliae are common inhabitants of bee pollen [Pichia (Candida) guillermondi [Metschnikowia (Candida) rancensis, Metschnikowia cf. pulcherrima, and Starmerella (Candida) magnoliae in bee pollen as well, thus again confirming our findings. At the same time, Starmerella (Candida) bombicola has been isolated and identified within bee bread in different stages of maturation and from bumblebee honey samples [Aureobasidium melanogenum has been retrieved as an important component of bee bread pollen, especially because it showed high potential as a bee probiotic after fermentation with bee pollen [According to the literature, e pollen , as welllermondi . Intereslermondi , reveale samples . Furthere pollen . These f• antiradical activity, with the highest DPPH• radical inhibition observed in Y3 (38.02 ± 0.82%), followed by Y9 (32.32 ± 1.21%), and the lowest one was detected in Y1 (22.63 ± 0.95%). Despite the correction with the OD600, a similar pattern of inhibition was observed with the exclusion of the Y7 strain (23.06 ± 0.61%), which showed better DPPH• radical scavenging activity. Compared to the antiradical activity of Trolox (6.4–64 µM), showing values ranging from 12.8 to 71.8% DPPH• inhibition, yeast strains showed good antioxidant activity within the interval values of 16 µM Trolox (19.6%) and 32 µM Trolox (44.9%). The DPPH antiradical activity (%) found in this work is in line with those found by Ciafardini and Zullo in yeasts isolated from virgin olive oil; indeed, the authors found values ranging from 28.50 ± 2.12% in strain Candida diddensiae 1922 and 83.50 ± 0.71% in strain Nakazawaea wickerhamii 1885 [Yeast strains  were analyzed from a functional point of view through the DPPH assay. As shown in mii 1885 . DespiteBee pollen can be considered a functional food due to its rich phytochemical profile and excellent nutraceutical properties, capable of exerting beneficial effects on human health. In fact, bee pollen has been used for years as a food supplement owing to its antioxidant molecules such as carotenoids and phenolic compounds . 2+-metal chelating activity  showed inhibition values of 86.8 ± 0.5%. According to Leja et al., the present findings suggest a remarkable anti-radical capacity of our sample [50 values reported in the literature is 0.01–7.99 mg/mL, and the EC50 value obtained for our sample, is comparable to or lower than other described pollens, indicating a powerful ability to inhibit DPPH• radicals [50 values between 0.4 and 7.9 mg/mL of extract higher than that observed for our sample [50 than that of other Tuscan bee pollens collected in Massa Macinaia (LU), mainly Castanea (215.2 μg/mL), Cistus (224 μg/mL), and Rubus (641.3 μg/mL) [On the other hand, the ability of our bee pollen sample to inhibit 50% of DPPHradicals ,54,55,56r sample . Similarubus 641. μg/mL [33 μg/mL) .2+/g FW, is higher than those obtained by Barbieri et al. [2+/g, and by Saral et al., who analyzed Turkish bee pollen samples having values ranging from 8.69 and 84.89 μmol Fe2+/g [2+-metal chelating ability of aqueous bee pollen extract was also assessed , and comparable to those obtained by Barbieri et al. for Rubus (90%) and Eucalyptus (96%) samples (50 µg/mL) [Castanea and Cistus bee pollen and higher erythrocytes lysis protection than the Rubus one [The results relative to the erythrocytes’ antioxidant protection against oxidative stress by bee pollen extract are shown in 0 µg/mL) ,34. Addiubus one .Inflammation is a complex endogenous process through which our body responds to tissue and cellular damage caused by pathogens, chemicals, and physiological events related to different disorders and pathologies such as tumors, allergies, arthritis, and diabetes ,61. InflIn this study, we evaluated the ability of bee pollen samples from Bagni di Lucca to exert anti-inflammatory effects on the HT-29 cell line exposed for 4 and 24 h to TNF-α, used to induce inflammation. First, to identify the optimal treatment condition and notice possible cytotoxic effects, we performed a toxicity curve using increasing concentrations (1–200 µg/mL) of ethanolic bee pollen extract (BE). The findings demonstrated that HT-29 cell viability was not affected by any doses of bee pollen extract (data not shown).Furthermore, after 1 h pretreatment with or without BE 10 and 50 µg/mL, HT-29 cells were exposed for 4 and 24 h with or without 25 ng/mL TNF-α. As shown in Comparing the two treatments, after 4 h of exposure to TNF-α, an acute inflammatory response was observed with a gene up-regulation of 2 to 4-fold higher than that observed after 24 h treatment, justifying the lower protective effect of bee pollen at both doses; however, the reduced protection herein observed may also be due to the reduced treatment time which may not be sufficient to activate and provide an adequate cellular anti-inflammatory response.Cistus sp. bee pollen extract to inhibit COX-2 activity and NO production after inducing, through an injection of carrageenan (1%), the formation of edema on the paw of some rats [Brassica campestris L., Nelumbo nucifera Gaertn., and Camellia sinensis L.) on RAW 264.7 cells, treated with LPS to induce NO production [The anti-inflammatory effect of bee pollen has been investigated sufficiently. Among these, Maruyama et al. demonstrated in vivo the ability of the oral administration of ethanol ome rats . MoreoveBesides modulating the activity of enzymes  involved in the inflammatory process, polyphenols can decrease the arachidonic acid, prostaglandins, leukotrienes, and nitric oxide production, and regulate the cellular activity of mast cells, lymphocytes, macrophages, and leukocytes . TherefoStarmerella bombicola, Candida magnoliae, Metschnikowia sp., Aureobasidium sp., Pichia guilliermondii, Rhodotorula sp., and Curvibasidium sp. Interestingly, all of them exhibited DPPH• antiradical activity. In conclusion, the bee pollen from Bagni di Lucca represents an interesting functional food and a potential probiotic product able to exert beneficial effects on human health. The high nutritional value and the remarkable phytochemical content make bee pollen one of the most studied bee products. In this work, the bee pollen from Bagni di Lucca  was characterized from a nutraceutical point of view, evaluating its phytochemical profile, the in vitro and ex vivo antioxidant activities, as well as its anti-inflammatory effects on TNFα-inflamed HT-29 cells. Our results showed good bio-functional activities and a good phytochemical profile of bee pollen, being rich in polyphenols, flavonoids, flavonols, monomeric anthocyanins, and carotenoids. Furthermore, our bee pollen exhibited good in vitro antioxidant activity, good erythrocytes protection from oxidative stress, as well as a fair anti-inflammatory effect. A total of ten yeasts belonging to seven different genera/species were isolated as well:


To determine if the Meibomian Gland (MG) secretion quality is associated with symptoms of ocular discomfort, hours of Video Display Terminals (VDT) use, eyelid margin abnormalities, conjunctival hyperemia, and Meibomian Gland Loss Area (MGLA) in a sample of university students.An online survey that included an Ocular Surface Disease Index (OSDI) questionnaire and an extra question about hours of VDT use recruited an initial sample of 183 participants. Only 120 participants that fulfilled the inclusion criteria were scheduled for a battery of ocular surface and MG specific exam. The tests include: 1) meibometry, 2) slit lamp exploration of eyelid margin abnormalities , MG secretion quality and conjunctival hyperemia, and 3) Meibography.Significant positive correlations between the MG secretion quality and eyelid margin hyperemia, MG orifices plugging, MGLA, nasal conjunctival hyperemia, and temporal conjunctival hyperemia  were found. Multivariate regression found association between OSDI with hours of VDT use , and eyelid hyperemia . A statistical association between MG secretion quality and eyelid margin hyperemia, MG orifices plugging, MGLA and conjunctival hyperemia  were found. Multivariate regression found association between MG secretion quality with MG orifices plugging  and meibometry .Participants with higher values in MG secretion quality have higher values in eyelid margin hyperemia, MG plugging, MGLA, and conjunctival hyperemia. No direct relationship between MG secretion quality and hours of VDT use or OSDI were found. The coronavirus disease 2019 (COVID-19) pandemic has caused many lifestyle changes in the worldwide population . The num“How many hours do you use video electronic displays during a day?”)  age 22.5 ± 2.32 years) who met the inclusion criteria were scheduled for the study session. All tests were performed in a single appointment. These tests included: meibometry, eyelid margin abnormalities, quality of the MG secretion, conjunctival hyperemia, and meibography were measured. To avoid any possible alteration, less invasive tests were performed first  –19. ThisAn exhaustive ocular surface examination under a Topcon SL-D4  slit lamp with a video camera DC-4 attached was performed on every participant. Videos of each participants’ ocular surface were recorded for the posterior analysis by the second masked observer. Ocular surface exploration examined the eyelid abnormalities, quality of the MG secretion and conjunctival hyperemia. Different settings were used on the slit lamp for the thorough examination of each ocular structure.For the eyelid margin observation, white diffuse light and 16 × magnification were used on the slit lamp. A video of the lower eyelid was recorded, and the following parameters were analyzed: eyelid margin irregularity, hyperemia, and plugging of the MG orifices. Eyelid abnormalities were categorized using the Arita et al. grading To stimulate the MG secretion, digital pressure was applied for 30 s along the length of the lower eyelid where the MG are located . For vidA total of two videos of the bulbar conjunctiva were captured on the slit lamp with 16 × magnification and diffuse white light . One vidMeibography images were captured by the Keratograph 5 M topographer  which uses an infrared camera for the MG in-vivo observation . MGLA wap < 0.05 was used for all statistical tests. Data normality was checked using the Kolmogorov–Smirnov test; all parameters were not normally distributed , except the meibometry  ) of 2 (1–3) and a distribution of 52 (43.3%) participants were grade 1 , 36 (30.0%) grade 2 (Mild group), 20 (16.7%) grade 3 (Moderate group), and 12 (10.0%) grade 4 (Severe group). Hours of VDT use showed a median (IQR) of 2 (1–3) and a distribution of 40 (33.3%) participants were in the < 6 h/day group, 40 (33.3%) were in the 6–8 h/day group, and 40 (33.3%) were in the > 8 h/day group. Meibometry showed a mean (± SD) value of 283.88 ± 128.36 MU. Eyelid margin irregularity showed a median (IQR) of 0 (0–0) and a distribution of 100 (83.3%) participants were grade 0, and 20 (16.7%) grade 1. Eye lid margin hyperemia showed a median (IQR) of 1 (0–2) and a distribution of 41 (34.2%) participants were grade 0, 43 (35.8%) grade 1, 30 (25.0%) grade 2, and 6 (5.0%) grade 3. Plugging of the MG orifices showed a median (IQR) of 0 (0–1) and a distribution of 80 (66.7%) participants were grade 0, 30 (25.0%) grade 1, 6 (5.0%) grade 2, and 4 (3.3%) grade 3. Quality of the MG secretion showed a median (IQR) of 0 (0–1) and a distribution of 63 (52.5%) participants were grade 0, 46 (38.3%) grade 1, 7 (5.8%) grade 2, and 4 (3.3%) grade 3. Nasal conjunctival hyperemia showed a median (IQR) of 2 (2–3) and a distribution of 13 (10.8%) participants were grade 1, 67 (55.8%) grade 2, 37 (30.8%) grade 3, and 3 (2.5%) grade 4. Temporal conjunctival hyperemia showed a median (IQR) of 2 (2–3) and a distribution of 20 (16.7%) participants were grade 1, 69 (57.5%) grade 2, 29 (24.2%) grade 3, and 2 (1.7%) grade 4. MGLA showed a median (IQR) of 2 (2–2) and a distribution of 19 (15.8%) participants who were grade 1, 76 (63.3%) grade 3, 22 (18.3%) grade 3, and 3 (2.5%) grade 4.r = 0.235, p = 0.010), and eyelid margin hyperemia . A statistically significant correlation was found between the quality of the MG secretion, eyelid margin hyperemia , plugging of the MG orifices , nasal conjunctival hyperemia , temporal conjunctival hyperemia , and MGLA . Eyelid margin irregularity showed a statistical correlation with eyelid margin hyperemia  between plugging of the MG orifices . Also, plugging of the MG orifices was significantly correlated with nasal conjunctival hyperemia , and temporal conjunctival hyperemia .Table B = 0.316, p = 0.007) and higher values of eyelid margin hyperemia . No other studied parameter showed any association , MGLA , nasal  and temporal  conjunctival hyperemia. No other parameter showed statistical association with the female or male gender.Table p = 0.125). Additionally, differences by pairs were performed and no statistical differences were found .The mean ± SD values of meibometry for each MG secretion quality grade were measured as: grade 0 (299.83 ± 131.48 MU), grade 1 (280.93 ± 125.12 MU), grade 2 (203.81 ± 123.34 MU), and grade 3 (206.83 ± 51.38 MU). No statistical differences were found in meibometry values between the MG secretion quality grades , and hours of VDT use . Although, a statistical association between eyelid margin hyperemia  and plugging of the MG orifices  was found; when these parameters vary the MG secretion quality grade varied too. No statistical relationship was found between the quality of the MG secretion and eyelid margin irregularity . Contingency Table p = 0.023), and temporal . MGLA grade significantly varied as the quality of the MG secretion varied .The relationship between the quality of the MG secretion and OSDI, hours of VDT use, eyelid margin abnormalities, conjunctival hyperemia, and MGLA are reported as a contingency table Table . No statB = 0.295, p = 0.004) and lower values of meibometry . No other studied parameter showed any association (Table Multivariate regression showed that the quality of the MG secretion was significantly associated with higher values of the MG orifice plugging (coefficients on Table .Table 6MThe number of hours using VDT by university students during the COVID-19 pandemic increased substantially and could influence their ocular surface health , 3. In fAlthough the main objective of this research was to analyze if the quality of the MG secretion was associated with other ocular surface parameters, results showed a positive correlation between VDT use and ocular symptomatology as well as multivariate regression showed an influence of the hours of VDT use in the DED symptomatology. Similar results to the present study were observed by García-Ayuso et al. who studA normal blink rate and complete blinking could be factors that do not allow the obstruction of the MG orifices by the meibum accumulated over the eyelid border, with the consequent alteration of the quality of the MG secretion . The preAlso, a significant relationship between nasal and temporal conjunctival hyperemia with quality of the MG secretion was found by both, Spearman Rho correlation and Fisher’s exact test. Participants with thicker MG secretion showed higher values of conjunctival hyperemia, which could be due to an inefficient evaporative function of the thicker meibum because of its difficulty to be spread over the ocular surface. To the authors’ knowledge, there are no other studies that evaluated conjunctival hyperemia and its relationship with the quality of the MG secretion and plugging of the MG orifices.No differences were reported in meibometry values between the quality of the MG secretion grades. This could be because the meibometry technique only measures the quantity of lipids present on the matt synthetic tape and not their quality , 18. It In conclusion, participants with higher values in the quality of the MG secretion have higher values in eyelid margin hyperemia, plugging of the MG orifices, MGLA, nasal conjunctival hyperemia, and temporal conjunctival hyperemia. No direct relationship between the quality of the MG secretion and hours of VDT use, or OSDI was found. Participants that spend long hours using VDT had higher values in OSDI, which can be explained by the impact of VDT in complete blinking, responsible for spreading the MG secretion over the ocular surface to avoid tear evaporation. To support the hypothesis proposed on how to complete blinking and VDT use affect the eyelid border and MGs, other parameters like blink rates, tear film break-up time, ocular protection index, and tear film lipid layer interference patterns should be measured in future research.


Apoa5 mRNA. Consistently, in vitro studies indicated that olanzapine increased medium triglyceride levels and decreased medium apoA5 levels in a dose-dependent manner in human HepG2 cells and primary mouse hepatocytes. Whereas the olanzapine treatment increased hepatic apoA5 protein in vitro, without effects on hepatic APOA5 mRNA. Of note, olanzapine increased the co-localization between apoA5 protein and accumulated lipid droplets in hepatocytes, as opposed to at the hepatocellular plasma membrane, in mouse liver as demonstrated by fluorescence staining. Therefore, our study indicated that short-term use of olanzapine induced hypertriglyceridemia due to defects of sorting and secretion of hepatic apoA5.Long-term use of olanzapine, an antipsychotic drug, induces hypertriglyceridemia, resulting in a higher risk of cardiovascular disease. However, the effects and underlying mechanisms of short-term use of olanzapine on circulating triglyceride levels remain poorly understood. Here, the role of apolipoprotein A5 (apoA5), a regulator of triglyceride metabolism, was investigated in olanzapine-induced hypertriglyceridemia. Our multi-center clinical study recruited 36 schizophrenia patients who received short-term (8 weeks) of olanzapine. Besides, female C57BL/6J mice were treated with olanzapine (3 mg/kg/day versus 6 mg/kg/day) for 6 weeks. We demonstrated that short-term use of olanzapine increased plasma triglyceride and decreased plasma apoA5 levels in the patients and mice, with a negative correlation between the two factors. However, no obesity was observed in the patients and mice. Interestingly, olanzapine increased hepatic apoA5 protein in the mice, without significant changes in hepatic  To date, the effects of short-term use of olanzapine on lipid metabolism remain unknown. Besides, it is also unclear whether olanzapine-induced hypertriglyceridemia results from the effects of this drug on lipid-related mediators. Therefore, this study aimed to determine the effects and related mechanism of short-term use of olanzapine on plasma triglyceride levels.Olanzapine, an atypical antipsychotic drug, is among the most effective therapies for schizophrenia patients because of its broad-spectrum efficacy and low risk of extrapyramidal symptoms . HoweverIn this study, the role of apolipoprotein A5 (apoA5) in the development of olanzapine-induced hypertriglyceridemia was investigated. ApoA5 is a negative regulator of plasma triglyceride levels, that is, specifically synthesized by the liver . After bClinicalTrials.gov NCT03451734) was conducted at The Second Xiangya Hospital Central South University, China, from January 2018 to June 2019. Schizophrenia was diagnosed according to the criteria established by the Diagnostic and Statistical Manual of Mental Disorders-Fifth Edition (DSM-5)  . PatientThe exclusion criteria were as follows: 1) current or prior DSM-IV diagnosis of other psychiatric disorders defined in the DSM-5; 2) pregnancy or lactating; 3) history of alcohol, cigarette, or substance use; 4) known medical conditions that might affect metabolism; and 5) history of diabetes, hypertension, cardiovascular diseases, endocrine diseases, lipid disorders, and/or serious chronic diseases, such as heart failure and liver or kidney dysfunction.This study was performed in accordance with the 1964 Declaration of Helsinki and its later amendments  and was The participants underwent an 8-week treatment program. The intervention was the intake of olanzapine (15–20 mg/day) monotherapy, administered daily at 8:00 p.m. The initial dose of olanzapine was 5 mg/day, which was then titrated to 15 mg/day to 20 mg/day during the first week.All participants underwent a clinical evaluation with scheduled follow-ups at weeks 4 and 8. The baseline assessments included demographics, comprehensive medical history, physical examination, anthropometric measurements (weight and height), and positive and negative syndrome scale (PANSS) scores. The baseline laboratory tests included assessments of fasting lipid and glucose levels, liver and renal function, and complete blood count, as well as electrocardiography. At each follow-up visit, all baseline clinical evaluations, including physical examination, anthropometric measurements, and laboratory tests, were repeated. The Treatment Emergent Symptom Scale (TESS)  was usedThe primary outcomes were changes in fasting plasma triglyceride and apoA5 levels. Plasma apoA5 levels were determined using ELISA kits . Blood glucose and lipids, including triglycerides, total cholesterol (TC), LDL-C, and HDL-C, were detected using an automatic biochemical analyzer.The secondary outcomes were the following: 1) increased levels of other lipids, including TC, LDL-C, and HDL-C; 2) changes in fasting glucose levels and body mass index (BMI); and 3) psychopathological symptoms measured by PANSS scoring. BMI was calculated as the weight in kilograms divided by the square of the height in meters.p-value was used to indicate statistically significant differences, with a p < 0.05 considered significant. The GraphPad Prism statistical software  and SPSS software  were used for statistical analyses of all data in this study.All continuous variables with normal distributions are expressed as the mean ± standard error of the mean (SEM). Changes in parameters, such as BMI, plasma lipid levels, and apoA5 levels, were defined as the amount of their alterations from the baseline to the endpoint . Continuous variables were analyzed using analysis of variance (ANOVA). Pearson’s analysis was used for the correlation test. A ad libitum access to food and water. The mice were fed a regular chow diet containing 4.5% fat (0.02% cholesterol) throughout the experimental period. All animal procedures were conducted in the Department of Laboratory Animals in Central South University and performed in accordance with the National Institute Health Guide for the Care and Use of Laboratory Animals. The entire experimental procedure was approved by the Experimental Animal Ethics Committee of The Second Xiangya Hospital, Central South University.A total of 21 female C57BL/6J mice (8-weeks old) were purchased from Hunan Stryker Jingda Animal Co., Ltd. . All animals were housed under standard conditions in individually ventilated cages and an artificial 12/12 h light/dark cycle (lights on: 8:00 a.m.) at 20–25°C and humidity of 45 ± 10%. Mice had n = 7) received 3 mg/kg olanzapine  daily through gavage for 6 weeks, whereas the high-dose group received 6 mg/kg olanzapine for 6 weeks. Olanzapine was dissolved in 10% dimethyl sulfoxide (DMSO), and therefore the control group was maintained on a regular diet with 0.01 ml/g DMSO daily through gavage. The dosages of olanzapine used in this study referenced previous studies . Plasma apoA5 levels were determined using ELISA kits .The body weights of all animals were assessed weekly during the study. At week 6, animals were fasted for 4 h and then anesthetized with pentobarbital according to AVMA Guidelines for the Euthanasia of Animals: 2020 Edition . Liver sad libitum from 9 a.m. on the experimental day. Before the test of blood glucose, all the mice were weighted. The required volume of 20% glucose solution (1.5 g of glucose/kg body mass) for IP injection was calculated and recorded as follows: VGTT (μL) = 10 X body weight (g). The initial blood glucose levels of mice were assessed 2 h prior to the GTT. During the GTT, blood glucose levels were obtained at 15, 30, 60, 90, and 120 min after the intraperitoneal dose of glucose. Blood samples were drawn from the tail veins and analyzed using a contour glucometer .At the end of the study, all animals underwent a GTT. Briefly, mice were fasted for 6 h, with only water provided Frozen liver samples were cut into 6- to 8-µm-thick sections and subjected to histologic examination. Hematoxylin and eosin (H&E) staining was performed on formalin-fixed, paraffin-embedded sections using a standard protocol as described previously . Oil redFor this study, two cell lines were selected. The primary mouse hepatocyte was chosen because of its involvement in the metabolism of olanzapine. The hepatoma cell line HepG2 (ATCC HB-8065)  was selected because of the intense lipid metabolism of this tissue .2 and Liberase was performed for 10 min. All solutions were heated to 37°C. Hepatocytes were filtered through a gauze mesh and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 1 U/mL penicillin, and 1 mg/ml streptomycin. Cells were plated in 6-well plates and incubated in a humidified atmosphere containing 5% CO2 at 37°C. The human hepatoma cell line HepG2 was also cultured in DMEM in an atmosphere containing 5% CO2 at 37°C and was passaged every 3 days.Primary mouse hepatocytes were isolated and purified from C57BL/6J mice by using a modified two-step perfusion method described in previous studies . BrieflyBoth the HepG2 cells and primary mouse hepatocytes were divided into 4 groups: olanzapine 25 µmol/L group, olanzapine 50 µmol/L group, olanzapine 100 µmol/L group, and the control group as described previously . In the All the cultured medium stated in 2.3.2. was collected. The triglyceride levels of the medium were determined subsequently in the Clinical Laboratory of the second Xiangya hospital using Spotchem EZ SP 4430 . The corresponding treated hepatocytes were collected, and the protein levels were measured by BCA protein assay kit  for the normalization of triglyceride levels in the medium.To probe the relationship between apoA5 and lipid droplets in hepatocytes, lipid droplets were isolated by sucrose gradient centrifugation as described previously . The priThe effects of olanzapine administration on apoA5 protein levels in mice and hepatocytes were determined by Western blotting. The cultured primary mouse hepatocytes and HepG2 cells were washed thrice with ice-cold phosphate-buffered saline (PBS), and radioimmunoprecipitation assay (RIPA) lysis buffer  containing 1% 0.5 mm phenylmethanesulfonyl fluoride (PMSF) (v/v) was added, after which the cells were incubated at 4°C for 30 min. The cells were then collected and centrifuged at 13,000 g for 15 min at 4°C after being mixed with a pipette. The liver tissues were homogenized in the RIPA lysis buffer  containing 1% 0.5 mm PMSF (v/v) at 100 mg/ml. Liver tissues were then incubated for 30 min, and the homogenate was centrifuged at 13,000 rpm for 15 min at 4°C, after which the supernatant was saved as a protein extract. Protein levels were quantified using a BCA protein assay kit . Protein extracts were mixed with loading buffer and PBS, followed by incubation at 95°C for 5 min to allow degeneration. Protein extracts were then separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. To visualize protein samples and to confirm the complete and equivalent transfer of protein samples extracted from the medium, 0.1% Ponceau S  was applied. After blocking with 5% skimmed milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies . Samples were then incubated with secondary goat anti-mouse and anti-rabbit antibodies . The bound complexes were detected using Pierce ECL Western blotting substrate  and quantified using a ChemiDoc XRS + system .ApoA5/APOA5 mRNA level was measured by qPCR. Total RNA was extracted from mouse liver tissues, primary mouse hepatocytes, and HepG2 cells using an RNA extraction kit  according to manufacturer instructions. First-strand cDNA was synthesized using the Revert Aid first-strand cDNA synthesis kit . qPCR was performed using SYBR Green select master mix , with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as an endogenous control. Primers were synthesized by Tsingke Biological Technology . Primer sequences are shown in To further explore the effects of olanzapine on hepatic apoA5 metabolism, p < 0.05.All statistical analyses were performed using GraphPad Prism software (GraphPad Software) and statistical package IBM SPSS Statistics software (SPSS). Data were expressed as the mean ± SEM. Parametric analyses were conducted using Dunnett’s t test, one-way ANOVA, simple linear regression, Pearson Correlation coefficient. Non-parametric analyses were performed using Wilcoxon rank sum test, Kruskal-Wallis H test, and Spearman’s rank correlation coefficient. When the repeated measured data were normally distributed, the assumptions of repeated measures ANOVA, also called sphericity, would be conducted. Once the assumption accepted, the data would be analyzed by One-way Repeated Measures ANOVA. Otherwise, Geisser-Greenhouse correction would be used to correct for violations of the assumption. Multiple stepwise regression analysis was conducted to screen and eliminate the variables causing multicollinearity. Differences were considered statistically significant at a p = 0.032, p = 0.013, p = 0.0008, week 4, p = 0.028, week 8, p = 0.0664, week 4, p = 0.2359, week 8, p = 0.8571, week 4, p = 0.3767, week 8, p = 0.001) and week 8 (p = 0.028) after olanzapine treatment  in their first psychotic episode were enrolled in this study. All participants completed the 8-week study. As shown in reatment .2 but remained within the normal range (18.5–24 kg/m2)  . Plasma 4 kg/m2) , whereas4 kg/m2) . Meanwhi4 kg/m2)  or 8-wee4 kg/m2)  of olanz4 kg/m2)  or 8-wee4 kg/m2) , either.p < 0.05). Moreover, the results demonstrated that olanzapine administration dose-dependently decreased plasma apoA5 levels in mice   .p < 0.05) (ApoA5 mRNA level among the three groups (p > 0.05)  . Interes > 0.05) .in vivo, we detected the effects of olanzapine on triglyceride levels, apoA5 protein levels, and APOA5/ApoA5 mRNA expression in human and mouse hepatocytes in vitro. In line with the findings in vivo, olanzapine intervention elevated the medium triglyceride levels in a dose-dependent manner  in this study. Consistently, no significant weight gain was observed in olanzapine-treated mice. The stepwise multiple regression analysis showed no significant correlation between BMI and plasma triglyceride elevation in patients and mice. Further, reduction of plasma apoA5 levels was observed in schizophrenia patients and mice after short-term treatment of olanzapine. More importantly, we showed that olanzapine directly disturbed hepatocyte apoA5 metabolism, resulting in decreased plasma apoA5 levels and increased triglyceride levels. Together, these data suggested that apoA5 plays an important role in olanzapine-induced hypertriglyceridemia.For a long time, dyslipidemia after long-term use of olanzapine has mostly been attributed to weight gain associated with olanzapine-induced diet and lifestyle changes . HoweverAPOA5/Apoa5 mRNA expressions were observed in the liver of mice and hepatocytes in vitro after olanzapine administration, albeit with a significant increase in hepatic apoA5 protein levels in vivo and in vitro. Together, our study identified an unexpected paradox of olanzapine on apoA5 metabolism, i.e., olanzapine induced reduction in plasma apoA5 levels, increase in hepatic apoA5 levels, and no change to the expression of hepatic APOA5 gene. As stated above, apoA5 is a protein specifically synthesized and secreted by the liver  in a breast-fed infant resulted in changes in the signal peptide. The mutated apoA5 was intracellularly missorted to lipid droplets and not secreted, leading to undetectable apoA5 plasma levels, and consequently severe hypertriglyceridemia -Golgi secretion pathway . The oveeridemia . Based oeridemia . Due to in vitro  secretion . Indeed,pression . These din vivo and in vitro data established that mRNA levels of apoA5 remained unaffected, short-term use of olanzapine significantly inhibited apoA5 secretion and redirected the trafficking of apoA5 to the surface of cytosolic lipid droplets, and reduced plasma apoA5 levels led to hypertriglyceridemia. Namely, olanzapine intervention did not directly affect the transcription of apoA5. It may impair the apoA5 sorting and secretion process from hepatocytes to plasma at the post-translation level, in which the dysfunctional signal peptide of apoA5 may be involved (In summary, our involved .Here, we proposed a hypothesis for the role of apoA5 in hypertriglyceridemia induced by short-term use of olanzapine . Physiolin vitro and in vivo had determined a novel perspective to explain lipid metabolism disorders in clinical schizophrenia patients treated with olanzapine and provided a promising therapeutic target for the future.Our current work on short-term use of olanzapine APOA5/ApoA5 knockdown tests can also be carried out in vivo and in vitro to observe the changes of plasma and medium triglycerides in olanzapine intervention. In addition, the mechanism of apoA5 secretion disorder was not further explored in this study.Both the clinical and animal experiments were short-term studies, and the changes of plasma apoA5 and triglycerides should be observed over a longer intervention period in further research. What’s more,


Aggregatibacter actinomycetemcomitans. This Gram-negative, capnophilic, facultative anaerobe is well recognized as a causative agent leading to periodontal diseases, as well as seriously systemic infections including endocarditis. A. actinomycetemcomitans has also evolved mechanisms against complement-mediated phagocytosis and resiliently survives in serum-rich in vivo environments, i.e., inflamed periodontal pockets and blood circulations. This bacterium, however, demonstrated increasing sensitivity to human serum, when being infected by a pseudolysogenic bacteriophage S1249, which switched to the lytic state as a response to human serum. Concomitantly, the pyruvate dehydrogenase complex (PDHc), which is composed of multiple copies of three enzymes  and oxidatively decarboxylates pyruvate to acetyl-CoA available for tricarboxylic acid (TCA) cycle, was found up-regulated 10-fold in the bacterial lysogen after human serum exposure. The data clearly indicated that certain human serum components induced phage virion replication and egress, resulting in bacterial lysis. Phage manipulation of bacterial ATP production through regulation of PDHc, a gatekeeper linking glycolysis to TCA cycle through aerobic respiration, suggests that a more efficient energy production and delivery system is required for phage progeny replication and release in this in vivo environment. Insights into bacteriophage regulation of bacterial fitness in a mimic in vivo condition will provide alternative strategies to control bacterial infection, in addition to antibiotics.Antimicrobial resistance is rising as a major global public health threat and antibiotic resistance genes are widely spread among species, including human oral pathogens, e.g.,  Aggregatibacter actinomycetemcomitans [A. actinomycetemcomitans is well recognized for expressing multiple virulence factors that exacerbate the progression of both oral and systemic infections, including bundle fimbriae [Antimicrobial resistance is rising as a major global public health threat , and antcomitans ,4, with comitans  and the comitans ,7. This comitans , and caucomitans . Periodocomitans ,11. A. afimbriae ,13, non-fimbriae ,16,17,18fimbriae ,20, two fimbriae ,22 and cfimbriae , and at fimbriae ,26,27,28fimbriae ,30,31,32A. actinomycetemcomitans to human serum [We, however, have identified a pseudolysogenic phage S1249, which increases the sensitivity of an serum ,33,34,35an serum ,33,35. Ban serum , as wellan serum . TherefoIn this study, we used a newly generated lysogenic strain of phage S1249 as a tool to investigate how in vivo growth environment may affect the life cycle determination of the phage. We focused on bacterial host membrane-associated protein changes during the phage life cycle switch from the lysogenic to the lytic state after exposure to human serum. Blue native polyacrylamide gel electrophoresis (BN-PAGE) and the liquid chromatography/mass spectrometry (LC/MS) analyses clearly demonstrated an over 10-fold increase of detected protein of the pyruvate dehydrogenase complex (PDHc), which is composed of pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoyl dehydrogenase (E3), in the lysogen grown in the presence of human serum. Importantly, the detected PDHc level peaked approximately the same time when virions released. PDHc is the central gatekeeper in glucose metabolism in both mammals and Gram-negative bacteria , which lA. actinomycetemcomitans is a capnophilic, facultative anaerobe and requires 5% to 10% carbon dioxide for its growth. This periodontal pathogen can produce energy through either aerobic respiration at the presence of oxygen, e.g., occasional presence on the surface of oral mucosa; or anaerobic fermentation using fumarate as an electron acceptor, e.g., in the periodontal pocket, which allows itself to adapt to different in vivo habitats [habitats ,41. The A. actinomycetemcomitans was routinely recovered from the freezer using TSBYE agar, containing 3% trypticase soy broth, 0.6% yeast extract, and 1.5% agar  and incubated statically in a 37 °C incubator with 5% humidified carbon dioxide atmosphere for each experiment. The phage-naïve A. actinomycetemcomitans strain IDH84 of serotype c [All bacterial and bacteriophage strains were stored in a −80 °C freezer. rotype c ,35 and iAggregatibacter phage S1249 was isolated from its originally identified clinical lysogenic strain D11S-1 [2 atmosphere using TSBYE was diluted in 100 mL, grown for two doubling-time (6 h), and exposed to 0.5 μg/mL mitomycin C from Streptomyces caespitosus  for 30 min. Bacterial cells were then pelleted at 8000× g, 4 °C for 10 min, washed twice with fresh TSBYE broth, resuspended in the same volume of TSBYE broth. After 12 h recovering at 37 °C with 5% humidified CO2 atmosphere for virion replication and egress, the culture suspension was centrifuged at 16,000× g, 4 °C for 30 min, and the supernatant was collected, filtered with 0.22 μm to remove any contaminated bacteria, and ultra-centrifuged at 100,000× g, 4 °C for 1 h to collect phages. The isolated phage was resuspended and stored in phage suspension medium , confirmed without bacterial contamination by culture, and aliquots were kept at −80 °C before use [n D11S-1 ,35,42, u495 = 0.2), which was equivalent to 5 × 108 cells/mL, grown for one doubling-time (3 h). Each strain was prepared in duplicates: one was exposed to 0.5 μg/mL mitomycin C for 30 min , and the second sample was used as a negative control without exposure to mitomycin C . After induction, cells were pelleted using 8000× g at 4 °C for 10 min, washed twice with fresh TSBYE broth, resuspended in the same volume of TSBYE broth, incubated at 37 °C with 5% humidified CO2 atmosphere. The recovery of bacteria was monitored by measuring OD495 values. Data were collected from triplicate experiments, analyzed using ANOVA through GraphPad Prism 9 and, p < 0.05 was considered statistically significant.The phage-naïve strain IDH84 and the lysogenized strain IDH84/S1249 were recovered from −80 °C using TSBYE agar plates. A single colony of each strain was inoculated into 10 mL TSBYE broth and grown overnight. The overnight culture of each strain will be diluted using fresh TSBYE and adjusted to the same optical density  in volume, and TSBYE mixed with 50% heat-inactivated, pooled, human male, type AB serum . The bottom layers were prepared with a final concentration of 1.5% agar, and approximately 6 mL of medium was poured into a petri dish plate (100 mm × 15 mm), kept warm in a 37 °C incubator with 5% humidified CO9 colony forming units (CFUs)/mL. Stationary phase bacterial cells were diluted in 1:10, grown one doubling time (3 h). A total of 300 μL of bacteria in the early logarithmic phase with a concentration of 4.0 × 108 CFUs/mL in TSBYE was used for each plaque assay. The isolated phage S1249 was diluted in phage suspension medium [3 plaque forming units (PFUs)/mL. A total of 300 μL bacterial suspension (~1.2 × 108 CFUs) in TSBYE and 100 μL phage suspension (~2.0 × 102 PFUs) were mixed first and preincubated at room temperature for 30 min for phage absorption. For preparing the top soft agar for the phage plaque assay, 3 mL of 45 °C prewarmed TSBYE with 0.7% agar and preincubated 400 μL mixture of bacteria and phages were combined by gentle and quick tilting the tube for a few times, before spreading evenly onto the bottom agar [2 for 24–48 h, after the top soft agar completely solidified. Plaques formed on three different bottom agars were analyzed using ANOVA through GraphPad Prism Version 9.5.0 (525), Boston, MA, USA and, p < 0.05 was considered statistically significant.A phage naïve serotype c strain of A. actinomycetemcomitans IDH84  was chosn medium  with a ctom agar ,44. Platg, 4 °C for 40 min. The collected supernatant samples containing DDM-solubilized membrane protein complexes were loaded into a 3–12% Bis-Tris gradient gel and separated by running the BN-PAGE  at 150 V, 4 °C for 1 h, followed by 250 V for 90 min. Proteins separated by BN-PAGE were fixed with 50% methanol, 10% acetic acid for 10 min, further stained with colloidal blue stain  with agitation for 3 h, and de-stained in deionized water for 16 h. Bands of interest were excised for liquid chromatography/mass spectrometry (LC/MS) analysis. All BN-PAGE analyses were based on samples collected from duplicate experiments.Bacteria were recovered from −80 °C using TSBYE agar plates, and a single colony was inoculated into 10 mL fresh TSBYE broth, grown overnight, and bacteria were inoculated from TSBYE agar to liquid TSBYE culture medium using polystyrene culture tubes without serum and grown overnight, and the cells were subsequently exposed to fresh TSBYE media with and without 50% human serum. Cells were collected every 3 h for protein biochemical analysis. The collected bacterial samples were washed twice with sterile phosphate-buffered saline  at pH 7.4, and re-suspended in proper volume of PBS. Approximately equal numbers of bacterial cells from each sampling were treated with a mild, non-ionic detergent, n-dodecyl-β-D-maltoside  to solubilize bacterial membrane proteins in their native forms ,46. Cell4HCO3 and acetonitrile, treated with 10 mM dithiothreitol (DTT) in 100 mM NH4HCO3 for reducing disulfide bonds, alkylated using 55 mM iodoacetamide in 100 mM NH4HCO3, and digested with trypsin. Final yielded peptides were extracted using 5% formic acid. Samples were analyzed using Thermo Q Exactive Plus mass spectrometer . Obtained MS spectra were searched against the databases of A. actinomycetemcomitans strain D11S-1 (https://www.ncbi.nlm.nih.gov/nuccore/CP001733.2 accessed on 27 June 2016) and Aggregatibacter phage S1249 (https://www.ncbi.nlm.nih.gov/nuccore/GQ866233.1 accessed on 31 January 2014), as well as Homo sapiens (https://www.uniprot.org/proteomes/UP000005640 accessed in 1 July 2014) using Proteome Discoverer 2.5 . The measurement accuracy used ±10 ppm peptide for internal calibration and 0.02 Da for MS/MS tolerance. Carboxymethylation of cysteines was set as a fixed modification and oxidation of methionine was set as a dynamic modification. Up to two missed tryptic peptide cleavages were considered, and three maximum dynamic modifications were allowed per peptide. The fixed value peptide spectrum match (PSM) validator node is included in the workflow to limit the false positive rate lower than 1%.Identified protein bands showing differences in the lysogenic strain, particularly in the growth condition of human serum, based on the BN-PAGE analysis were excised for the LC/MS analysis, following an in-gel trypsin digestion protocol. Briefly, each excised protein band was cut into 1 mm cubes, destained using 25 mM NHThe obtained data from Plasma desorption (PD) were consolidated using Scaffold . Identified peptides were only considered if they reached >95.0% probability with at least one identified peptide, based on the peptide prophet algorithm with Scaffold delta-mass correction ,48. Protcsp) and glutamyl-tRNA synthetase (gltX). In the pseudolysogenic state, phage DNA does not integrate into bacterial chromosome and forms a circular DNA structure in the cells [Our earlier work has demonstrated that phage S1249 actively switches between truly lysogenic and pseudolysogenic states in its bacterial hosts . In the he cells ,42. Theshe cells .p < 0.05)  . In cont < 0.05) .Further examination of the lysogen IDH84/S1249 after mitomycin C induction using uranyl acetate negatively stained TEM demonstrated that the phage entered lytic cycle . Fifteenp < 0.01). The phage plaque assay data lent additional support to our thesis that the life cycle switch of this phage occurs in the presence of certain human serum components.Our early study demonstrated that the growth of generated lysogen IDH84/S1249 was significantly inhibited in the presence of human serum, showing 63% decrease in cell viability after 22 h exposure to human serum . In addi1), 700 kilodalton , 680 kDa (B3), 600 kDa (B4), 400 kDa (B5), 300 kDa (B6), 200 kDa (B7), and 100 kDa (B8) showing differences in the lysogen when being exposed to human serum, were excised for LC/MS analyses. The top five detected bacterial proteins in highest abundance based on total spectrum counts (PSMs) averaged from two independent experiments are described in Our early investigation indicated that the phage DNA detection in the supernatant significantly increased 6 h after human serum exposure , which iAmong those most abundant identified bacterial proteins based on PSMs , over 501_T6, B1_H6; B2_T6, B2_H6, B2_CT6, B2_CH6; B8_T6, B8_H6), migrated at three positions , associated with two different strains , and grown in two different conditions  were excised for semi-quantitative LC/MS analyses described in Further analyses of the membrane proteins of the lysogen (IDH84/S1249) and its isogenic, phage-naïve strain (IDH84) demonstrated that significantly more proteins were released from the lysogenic strain . Eight d1_H6) compared to without serum (1_T6). The E1, E2, and E3 enzymes function consequently for oxidative decarboxylation of pyruvate, which generates acetyl-CoA, CO2, and NADH (H+), a gateway position in glucose metabolism to generate energy through aerobic expiration. In contrast, there was no obvious protein bands detected from the phage-naïve strain IDH84 at the same position of the BN-PAGE gel (1_H6) compared to the same lysogen grown without serum (1_T6).Among those identified proteins, the multi-enzyme complex PDHc, composed of E1, E2, and E3, was detected with approximately 10-fold increase from the DDM-solubilized membrane sample of the lysogen, when grown with human serum : B1_H6 cut serum : B1_T6. PAGE gel : IDH84 aPAGE gel : B1_H6 cut serum : B1_T6.2_CH6) or without human serum (2_CT6). In stark contrast, the GroEL detected from the lysogen demonstrated approximately 5-fold in the TSBYE (2_T6) and more than 7-fold increase in the presence of human serum (2_H6), compared to the phage-naïve strain . The co-isolated proteins with GroEL were also different between the phage-naïve strain and its isogenic lysogenic strain  compared to grown in TSBYE without serum (8_T6).In addition to PDHc and GroEL, the elongation factors EF-Ts and EF-Tu, which are functioning in the peptide elongation during protein biosynthesis, were also found two-fold increase in the lysogen, when the lysogenic bacterial cells were grown in human serum : B8_H6 cut serum : B8_T6.1 protein band at the position of 1.5 mDa and B2 band at the position of ~700 kDa, which are described in 1 position, representing 0, 3, 6, and 9 h exposures to human serum labelled as B1_T0/H0, B1_H3, B1_H6, and B1_H9, and five gel slices at the B2 position representing 0, 3, 6, 9, and 12 h exposures to human serum labelled as B2_T0/H0, B2_H3, B2_H6, B2_H9, and B2_H12, were collected for semi-quantitative LC/MS analysis . Up-regulation of PDHc indicates activation of aerobic respiration, leading to 18 times more ATP production compared to anaerobic respiration [The protein profiles of the lysogenic strain solubilized by 1% DDM were in stark difference, compared to its isogenic, phage-naïve strain . Serial piration , which sEscherichia coli conducted by Carey et al. [A. actinomycetemcomitans (aceE-aceF-lpdA with an upstream 393-bp intergenic region (IGR). In silico analysis of the IGR sequence demonstrated cognate −10 and −35 box sequences for the regular house keeping σ70 binding, which are located at approximately the positions of −325 and −299, as well as potential alternative transcription factor binding site located close to the position of −150 , human serum exposure of the lysogen also stimulated up regulation of phage regulatory factors , includiFurthermore, chaperone proteins, including GroEL Hsp60), Dnak (Hsp70), and heat shock protein 90 (Hsp90), were found up-regulated in the lysogen . These m, Dnak (HAs an essential element for phage virion replication, GroEL was detected from the lysogen approximately 5-fold higher, compared to its isogenic, phage-naïve strain, and exposure to human serum, but not equine serum, further boosted the membrane-associated protein level of GroEL, but not the phage-naïve strain. The highly ATP-dependent chaperonin GroEL showed co-isolated with PDHc , which iIn terms of what specific human serum components that might contribute to the phage life cycle switch and virions egress, we observed significantly increased numbers of apolipoproteins, which included the high-density lipoprotein ApoA-I and the low-density lipoprotein ApoB-100, and immunoglobulins including IGHG1 and IGHM detected from the membrane of the lysogen, particularly during the early stage when phages started to egress . NeverthTogether, the data here suggest that human serum induces phage S1249 to enter lytic cycle, which leads to bacterial lysis. The up-regulation of PDHc leads to a more efficient ATP production and may be required for the phage remaining in the lytic state in a serum-rich in vivo environment. Nonetheless, additional investigation is necessary to understand regulatory pathways of phage mediated bacterial PDHc activation in this capnophilic, facultative anaerobe in vivo, as well as human serum components that potentially participate in this regulation.


Additionally, the repeatability from fiber to fiber and the intra-day and inter-day reproducibility were determined to be 1.4–6.0, 4.7–10.1, and 0.9–9.7%, respectively. The nanofiber adsorption capacity was found to be 670–720 pg/g for CBs at an initial concentration of 400 pg mL−1. A satisfactory recovery of 80–106% was attained when the suggested method’s application for detecting chlorobenzenes (CBs) in tap water, river water, sewage water, and industrial water was assessed.Chlorobenzenes (CBs) are persistent and potentially have a carcinogenic effect on mammals. Thus, the determination of CBs is essential for human health. Hence, in this study, novel polyurethane–polysulfone/calix[4]arene  nanofibers were synthesized using an electrospinning approach over in-situ coating on a stainless-steel wire. The nanosorbent was comprehensively characterized using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR) techniques. The SEM analysis depicted the nanofiber’s unique morphology and size distribution in the range of 50–200 nm. To determine the levels of 1,2,4-trichlorobenzene, 1,2,3-trichlorobenzene, and 1,2,3,4-tetrachlorobenzene in water samples, freshly prepared nanosorbent was employed using headspace-solid phase microextraction (HS-SPME) in combination with gas chromatography micro electron capture detector (GC-µECD). Other calixarenes, such as sulfonated calix[4]arene, p-tert-calixarene, and calix[6]arene were also examined, and among the fabricated sorbents, the PU–PSU/calix[4]arene showed the highest efficiency. The key variables of the procedure, including ionic strength, extraction temperature, extraction duration, and desorption conditions were examined. Under optimal conditions, the LOD (0.1–1.0 pg mL Consequently, introducing an effective analytical technique for precisely monitoring CBs in aqueous media is of prime importance and the most challenging issue of the modern era.Over the past few years, chlorinated volatile organic compounds have attracted substantial attention because they can cause environmental water contamination, which seriously affects the environment and health of human beings . The chlA review of the relevant literature revealed the importance of sample preparation techniques, such as the extraction and preconcentration of CBs, for their subsequent determination, alongside advances in modern analytical tools. For extraction, scientists have turned to many different methods, including liquid–liquid extraction, solid-phase extraction, liquid-phase microextraction, dispersive liquid–liquid microextraction, vortex-assisted liquid–liquid microextraction, single drop microextraction, and magnetic headspace adsorptive extraction. Solid-phase microextraction (SPME) ,7 is oneIn alkaline conditions, formaldehyde and para-substituted phenols can be cyclically condensed to generate calixarenes, which are macrocyclic ligands. The calixarene framework as a host–guest platform provides space for accepting various analytes . They prA suitable platform is required to separate, extract, and enrich target analytes. Nanofibers and nanostructured materials consist of all these properties owing to their high surface-to-volume ratio and many active sites for adsorption . RecentlThis research was conducted to fabricate a novel electrospun PU–PSU/C4A nanofiber to be used as a coating for HS-SPME to extract CB compounds from water samples. Accordingly, various calix[4]arene derivatives were electrospun on the surface of a stainless steel wire and examined for headspace preconcentration of CBs under the best experimental conditions. The presence of functionalized calixarenes in the structure of nanofiber provides an appropriate force interactive with CBs, including electrostatic and π-π interactions. To our knowledge, PU–PSU/calix[4]arene has never been synthesized and used for extracting the chosen CBs as model compounds from water media.−1 and stored at a temperature of 4 °C until their subsequent usage. Stock standard solutions of CBs were prepared, and working standard solutions were prepared daily before the extraction process.Polyurethane (PU) and polysulfone (PSU) were purchased from Bayer Company . The Merck Company  provided the methanol, 1,2,4-trichlorobenzene , 1,2,3-trichlorobenzene , 1,2,3,4-tetrachlorobenzene , and N, N-dimethylformamide (DMF). The standard stock solutions of CBs were produced in methanol at a concentration of 2000 mg L−1, and nitrogen with a purity of 99.999% and a flow rate of 30 mL min−1 were employed as a carrier gas and makeup gas, respectively. 3 min of splitless injection were used to inject the sample. Temperature settings for the injector and detector were 170 °C and 290 °C, respectively. Initially, the oven was set to preheat to 70 °C for 2 min, then heated to 170 °C at a rate of 40 °C per minute for 2 min, and finally heated to 200 °C at a rate of 20 °C per minute for 5 min.A gas chromatograph manufactured by Agilent  was utilized to determine the composition of the extracted analytes. This particular model was equipped with a µ-ECD detector, an HP-5 fused silica capillary column (30 m × 0.32 mm × 0.25 µm), and a split/splitless injection port. Helium with a purity of 99.999% and a flow rate of 1 mL minElectrospinning was carried out using a spinal needle attached to a rotating motor (to act as a collector), a syringe pump, and a direct current high voltage power source. The SPME syringe consisted of two spinal needles, an internal needle, G27 as an SPME coated needle, an external needle, and G22 as an SPME barrel.−1 range.Micrographs of nanofibers were recorded using a Zeiss DSM-960 scanning electron microscope (SEM) . The functional groups of produced nanosorbent were monitored using Fourier transform infrared spectroscopy, with an Equinox 55 FTIR–A.T.R. spectrometer  in the 400–4000 cmThe four calixarene derivatives , includi−1 and the electrospinning voltage of 15.5 kV was used. The SPME fiber was then placed in the GC inlet and heated to 150 °C for 1 h as the final step in the procedure.After that, the solution was drawn into a syringe with a capacity of 2 mL and mounted on the syringe pump set. At a distance of 10 cm from the tip of the syringe needle, electrospun nanofibers were collected for 8 min. The flow rate of 0.15 mL h−1 of each CP, and 1 g of sodium chloride. The mixture was stirred for 5 min. Afterward, the solution was sealed using a polytetrafluoroethylene septum and an open-top aluminum cap. Next, the sample solution’s headspace was allowed to interact with the fiber for a total of 5.5 min. At the end of the process, the fiber was removed, then placed into the inlet of the GC to undergo thermal desorption of the adsorbed analytes at a temperature of 170 °C for 3 min.At first, the PU–PSU/calix[4]arene was placed in the GC inlet and heated to 150 °C for 1 h. After that, a vial with a capacity of 10 mL was filled with 5 mL of the sample solution, which contained 100 ng mLPU–PSU nanofibers were treated with calix[4]arene, calix[6]arene, sulfonated calix[4]arene, and p-tert-butyl-calix[4]arene to see how each type of calixarene affected the extraction efficiency of the fiber. The process described in −1 (C-H), 1580 and 1500 cm−1 (aromatic ring), 1160 cm−1 (S=O of sulfone), 1250 and 1090 cm−1 (C-O), and 850 cm−1 (C-S) in −1 (O-H and N-H), 2955 cm−1 (C-H), 1726 cm−1 (C=O), 1582 and 1563 cm−1 (C-N stretching and N-H bending), 1125 cm−1 (C-O), and 1068 cm−1 (C-N) arene was fabricated and used as an effective SPME fiber coating in the headspace extraction of CBs in aquatic samples. The large surface area and porous structure of the nanofibrous PU–PSU/calix[4]arene mat provided fast adsorption of the analytes (5.5 min). The π-π stacking interactions are possibly the main reason for adsorbing CB molecules that diffused to the nanofibers/calixarene mat. The method is eco-friendly since it requires no organic solvents in the extraction and analysis steps. Moreover, the low LOD (0.1–1 pg mL


The limits of detection and quantification were 0.34 and 1.12 μmol L−1, respectively. Besides, the accuracy of the proposed electrode was assessed by determining dopamine in artificial cerebrospinal fluid, obtaining recovery values ranging from 98.7 to 102.4%. The selectivity was also evaluated by dopamine detection against several interferent species, demonstrating good precision and promising application for the proposed method. Furthermore, DFT-based electronic structure calculations were also conducted to help the interpretation. The dominant dopamine species were determined according to the experimental conditions, and their interaction with the iron vanadate photoanode was proposed. The improved light-induced DOP detection was likewise evaluated regarding the charge transfer process.This study reports a facile approach for constructing low-cost and remarkable electroactivity iron vanadate (Fe-V-O) semiconductor material to be used as a photoelectrochemical sensor for dopamine detection. The structure and morphology of the iron vanadate obtained by the Successive Ionic Adsorption and Reaction process were critically characterized, and the photoelectrochemical characterization showed a high photoelectroactivity of the photoanode in visible light irradiation. Under best conditions, dopamine was detected by chronoamperometry at +0.35 V vs. Ag/AgCl, achieving two linear response ranges (between 1.21 and 30.32 μmol L At the same time, the monoclinic structure shows the strongest peaks at 12.4°, 17.4°, 21.7°, 22.7°, 23.8°, and 26.1°. These correspond to the [0 0 2], [1 1 −2], [1 1 −3], [0 2 2], [2 1 0], and [0 1 4] crystal planes, respectively. Furthermore, there are no peaks regarding iron oxide structures, such as Fe2O3 and Fe3O4.Infrared spectra were obtained for the powder and film samples, as shown in  and IV) a. In rege–O bond ,46,47,48Eg), diffuse reflectance spectroscopy was used. Eg value was obtained from the Wood–Tauc model, according to Equations (1) and (2) —Fmaia, 99.0%), uric acid , L-ascorbic acid , dopamine hydrochloride , D-glucose , and urea , were of analytical grade and used without prior purification. Besides, the Prolopa® BD (levodopa + benserazide hydrochloride) (Roche) medicine was also used. The solutions were prepared using ultrapure water, with resistivity higher than 18.00 MΩ cm, obtained from a water purificator system (Elga model USF CE). The reagents were weighed using an analytical balance (Shimadzu AY 220). An oven (Brasdonto Model 5) and a muffle furnace (Edgcon 1P) were used for the heat treatment. The ITO glass conductive substrate (tin-doped indium oxide) used was purchased from Zhuhai Kaivo Optoelectronic Technology Co., Ltd. Company  .Ammonium metavanadate  was prepared by dissolving 25 × 10−3 mol L−1 of FeCl3.6H2O in 50 mL deionized water at 25 °C, resulting in an orange color solution. Both solutions were maintained under magnetic stirring for 30 min. The deposition sequence was made as follows: immersion of the conductive substrate (1.0 cm2) in 10 mL of solution A, followed by immersion of the substrate in 10 mL of solution B, and ending with the washing in 10 mL of deionized water. For each solution, the electrode was maintained for 5 min. Therefore, one layer was obtained. This procedure was repeated five, ten, and fifteen times to obtain electrodes with different layers , based on the research group  and an a−1 (6.0 s as counting time). To better understand some XRD parameters, the Rietveld refinement method was carried out . The UV–Vis Shimadzu UV-2600 equipment  was used to estimate the band gap energy. The diffuse reflectance spectrum was obtained directly from the solid sample and the electrodes. Besides, the sample’s morphology was evaluated using the scanning electron microscope, model Quanta 200 produced by Philips/FEI Company , applying a voltage of 25 kV, and the secondary electrons to generate the images.The physical characterization was carried out by X-ray diffraction (XRD), Fourier-transform infrared spectrophotometer (FTIR spectroscopy), diffuse reflectance, and scanning electron microscopy (SEM). Powder XRD data were obtained at room temperature on a PANalytical X′Pert PRO MPD diffractometer  equipped with Cu-Kα radiation (1.54060 Å). The 2θ scan ranged between 5° and 80° with a scan step of 0.04° s−1 KCl), a platinum wire 10 cm, and a working electrode, the ITO/FeVO(I), -(II), -(III), were used as reference, auxiliary, and working electrode, respectively. A visible light source (35 W Xe lamp—Vision HIDlamp) was positioned 15 cm away from the electrochemical cell. Photoelectrochemical and electrochemical characterizations were conducted by cyclic voltammetry (CV) curves, chronoamperometry, and electrochemical impedance spectroscopy (EIS), using 10 mL of Na2SO4 0.1 mol L−1, as the electrolytic solution. The procedures were controlled by the PGSTAT204 Autolab potentiostat/galvanostat , using free NOVA 2.1.5 software. The CVs were obtained in the potential range between 0 and +1.0 V (vs. Ag/AgCl), in the absence and light presence (20 mV s−1). Chronoamperometry curves and EIS measurements were obtained under different applied potentials ), under presence and absence of light. The Nyquist plots were obtained in the frequency range between 0.05 and 100 kHz, with an AC amplitude of 20 mV, and using Na2SO4 as an electrolytic solution. Furthermore, the electroactive surface area was also determined using the CV procedure and the Randles–Sevcik equation [The photoelectrochemical characterization was performed in a classical electrochemical quartz cell (20 mL), with only one compartment. An Ag/AgCl electrode  and chronoamperometry techniques were used. DPVs voltammograms were obtained in the potential range between 0.20 and +0.45 V (vs. Ag/AgCl), with a modulation amplitude of 0.05 V, a step of 4 mV, and a modulation time of 0.05 s . Chrono® BD) was used in the ITO/FeVO(I) electrode accuracy test. Firstly, a tablet (0.2742 g) was macerated, and a stock solution of approximately 1.33 mmol L−1 was prepared. For the standard addition method, 150 μL of levodopa diluted solution was added into the electrochemical cell, followed by successive addition of dopamine (5–25 μmol L−1). The photocurrent density was monitored at +0.35 V (vs. Ag/AgCl).The commercial drug sample of levodopa + benserazide hydrochloride (Prolopa0 to DOP4) [o-quinone and DOPaminochrome) [0, which were obtained via molecular dynamics (MD) calculations at high temperature, as described in ref. [Theoretical studies were conducted for dopamine at distinct redox, protonation, and states (DOPto DOP4) , includiochrome)   in the powder and film form (onto indium tin oxide (ITO) glass electrode surface). The material was critically characterized through X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), diffuse reflectance, and scanning electron microscopy (SEM) techniques. Under visible light, the photoanode with five layers (ITO/FeVO(I) electrode) showed the best photoelectrochemical response, such as higher photocurrent density, shorter response time, and lowest charge transfer resistance between the electrodes with 10 and 15 layers. Furthermore, the novel ITO/FeVO(I) sensor exhibited remarkable performance in dopamine detection. The reproducibility, selectivity, and accuracy were also evaluated, which indicated a good electrode performance with acceptable precision and no significant matrix interference effects. DFT-based electronic structure calculations were also employed to understand the electrode behavior in the presence of dopamine and other organic compounds. The improved light-induced DOP detection in terms of charge transfer processes was also discussed. Thus, from what was addressed, these results highlight the potential application of the assembled ITO/FeVO(I) platform as an alternative device for the dopamine photoelectrooxidation reaction.The present manuscript shows a simple, efficient, and low-cost method of detecting dopamine using a photoelectrochemical platform. The SILAR process obtained the iron vanadate semiconductor material (FeVO


Solanum spp. fruits. A two-factor field experiment with four different Solanum spp. species  and three ripening stages was conducted over two growing seasons (2020–2021). The fatty acid composition of the Solanum fruits was characterized using gas chromatography with a flame ionization detection. The results show that PUFAs are the dominant type of fatty acid in Solanum fruits, followed by MUFAs and SFAs. Overall, the highest PUFA contents were observed in S. nigrum fruits in the ripening stage I, and the highest MUFA and SFA contents were observed in S. melanocerasum fruits during ripening stages I and II, respectively.This study aimed to determine the effects of the ripening stage and species on the contents of saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids in  Solanum belongs to the Solanaceae family, which are also known as wonderberry or sunberry .,23.SolanIn the field experiment, the seedlings were planted in the soil. The seeds were sown in nurseries in March, and the strongest seedlings were transferred to the field in the 3rd ten-day period of May. Before planting, the soil surface was covered with a black agro film; holes were cut in the film, and the seedlings were then planted in these holes. A drip irrigation system was installed under the agro film; the watering rate was 1 L per hour, as needed, considering the meteorological conditions.2, including the protective zone.Experimental plots were arranged in randomized blocks with four replicates for each treatment. Each replication consisted of four seedlings of each species. Each experimental plot was 7.5 m long and 1.5 m wide. The entire experimental area was 148 mFruits were randomly collected for analysis from July 1st through to the first frost, according to the ripening stage. In each experimental year, the harvest dates depended on the meteorological conditions and the fruit ripening stages, which were visually assessed. For the laboratory analyses, 30–40 fruit samples were randomly harvested from each block of each treatment for preparing a 1.5 kg composite fruit sample.The fruits were washed using tap water, dried, and stored at −34 °C. The samples were lyophilized for 24 h using a Freeze–Drying Plant Sublimator 3′4′5 . After lyophilization, the fruits were milled  and stored in airtight containers at 5 °C in the dark until the chemical analysis.−1) was determined using the Kjeldahl method.Soil samples were taken in spring from the arable layer (0–20 cm depth) using an agrochemical auger. The soil samples were air-dried in open plastic boxes, homogenized, and sieved through a 1 mm mesh sieve. The agrochemical analyses of the experimental soil were conducted at the Laboratory of Analyses of Vytautas Magnus University Agriculture Academy. The soil samples were analyzed for the pH, KCl, and phosphorus, potassium, and total nitrogen contents. The soil’s pH was measured according to the potentiometric method using a pH-meter in 1 N of KCl extract . The ava2O = 78.5–102.2 mg kg−1), low phosphorus status (P2O5 = 45.9–69.3 mg kg−1), and 1.25% total nitrogen content.The experimental field soil was characterized by the acidity (pH = 4.16), medium potassium status , C8:0 (caprylic acid), C10:0 (capric acid), C11:0 (undecylic acid), C12:0 (lauric acid), C13:0 (tridecylic acid), C14:0 (myristic acid), C15:0 (pentadecylic acid), C16:0 , C17:0 (margaric acid), C18:0 (stearic acid), C20:0 (arachidic acid), C21:0 (heneicosylic acid), C22:0 (behenic acid), C23:0 (tricosylic acid), C24:0 (lignoceric acid), C14:1 (myristoleic acid), C15:1 (pentadecenoic acid), C16:1 , C17:1 (methyl heptadecanoate acid), C18:1 (oleic acid), C20:1 (gondoic acid), C22:1 (erucic acid), C18:3 (linolenic acid), C18:2 (linoleic acid), C20:5 (eicosapentaenoic acid), C20:3 (dihomo linolenic acid), C20:2 (eicosadienoic acid), C20:4 (arachidonic acid), C22:6 (cervonic acid), and C23:2 (tricosanoic acid).The analysis conditions were as follows. Column temperature: 60 °C for 2 min, 200 °C/min up to 230 °C, holding for 45 min, evaporator temperature 250 °C, flame ionization detector temperature 270 °C, gas carriers, and nitrogen. Fatty acid set “supelco 37 component” was used to identify the fatty acids in the “FAME mix”. The tetradecadiene and hexadecadiene fatty acids were identified via interpolation.The preparation of the samples was as follows: 15 ± 0.01 g of each sample was weighed, poured into 25 mL of chromatographically clean hexane, and then the fat was extracted by stirring for 1 h. Next, 4 mL of the extract was poured into a test tube, 200 µL of 2 mol/L KOH solution was added, and the mixture was then centrifuged and left for 30 min for exfoliation. A 2 mL aliquot was taken from each prepared sample and poured into the bottle of the automatic feeding system. An automatic syringe removed 1 µL, which was injected into the chromatographic evaporator. A chromatographic analysis was performed.Solanum spp. fatty acid contents were processed using Microsoft®® Excel®® 2016 MSO software and the STATISTICA 10  package. The reliability of the results was evaluated using a two-way analysis of variance (ANOVA software package). The statistical significance of the differences between the means was estimated using Fisher’s LSD test (p < 0.05).The data for the S. melanocerasum fruits in ripening stage I, and oleic acids were the most abundant among monounsaturated fatty acids. The highest amount of saturated fatty acids (10.323%) of all the tested samples was observed for S. melanocerasum fruits during ripening stage II. Stearic acid and palmitic acid were the two predominant SFAs.Our results demonstrate that the quality and quantity of the fatty acids in all of the investigated fruits is dependent on the species and ripening stage. The polyunsaturated fatty acids were the most prevalent type, with the contents ranging from 40.753% to 63.740% in all of the investigated species during their ripening. Linoleic acid was the most abundant polyunsaturated fatty acid. Significantly, the highest total amount (49.532%) of monounsaturated fatty acids was identified in