The samples were stored in darkness at different temperatures  for 8 weeks. The results showed that reducing the alcohol content and storage temperature caused turbidity to increase. The samples prepared from rye distillate were characterized by significantly lower turbidity than those produced from plum distillate. The highest increase in turbidity in comparison to the controls was observed in the samples with 40% v/v alcohol content stored at a temperature of −18 °C. Storage of the rye and plum distillates samples at different temperatures resulted in changes to the concentrations of volatile compounds, i.e., lower levels of acetaldehyde and higher alcohols, and increased content of esters. However, the alcohol content and storage temperature had no statistically significant effect on methanol concentration.The purpose of this study was to investigate the effects of alcohol by volume (ABV) and storage temperature on changes in the clarity of rye and plum distillates, and their content of volatile compounds. Distillates with initial ABVs of 93.26%  Producing spirit drinks typically involves distilling naturally fermented products , maceration or similar processes, and the addition of ingredients, such as flavoring agents or sugars [Spirit drinks are defined by regulation  2019/787 [Grain spirits, including whisky and fruit spirits are made from raw spirits/distillates, which are not purified by rectification but rather aged in wooden barrels, in which their organoleptic qualities are allowed to develop naturally through particular chemical reactions .For instance, Central and Eastern European countries have a long tradition of producing plum distillate-based drinks, known as slivovitz. Due to the different production methods used, especially with regard to fermentation and distillation, plum alcohols can vary greatly from region to region. Sádecká et al.  used thev/v [The most popular spirit drink in Poland, Russia, and Scandinavia is vodka . Vodka iv/v . In Polav/v .Given that the origin of the raw material influences the sensory quality of the rectified spirits that are used to produce vodka, Ziólkowska and Jeleń  comparedApart from ethyl alcohol and water, spirit beverages contain different concentrations of fermentation by-products, depending on the type of spirit used, such as aldehydes, ketones, acids, alcohols, and esters ,5.v/v are stored under refrigerated conditions, undesirable turbidity, referred to as haze or cloudiness, may appear [The volatile compounds in a spirit beverage determine its specific aroma and flavor. When spirit beverages with an alcohol content of less than 45% y appear . This phy appear ,10. Fatty appear . As a ruy appear .To obtain spirit drinks of the highest quality, it is necessary to control the physicochemical composition and organoleptic properties of the ingredients, i.e., spirit and water. Therefore, agricultural distillates are submitted to processes such as rectification and/or filtration . In a prThe essential requirement for the water used in spirit drinks that it should meet the requirements for potable water . The quaMetals, such as aluminum, calcium, cadmium, copper, iron, lead, and potassium can also contribute to the formation of turbidity in spirit drinks. Contact with machines, weighing, bottling, and aging can cause metal ions to migrate into a beverage. Higher concentrations of metals may result in greater turbidity in spirits. Unfortunately, the turbidity may only appear after some time, so it is not possible to state immediately after the production of vodka, whether the product is characterized by high physical and chemical stability .In order to remove impurities causing cloudiness, spirit drinks are filtered through activated carbon, a strongly adsorbent and hydrophobic material . Thanks The aim of the present study was to evaluate the effects of the concentration of ethyl alcohol and storage temperature on changes in the clarity of rye and plum distillates (raw spirits), as well as on the qualitative and quantitative composition of volatile compounds in the prepared samples. Determining the most favorable storage conditions for distillates from various raw materials could help to simplify filtration processes.v/v) was sourced from the agricultural distillery Zbig-Rol . Plum distillate (ABV of 82.03% v/v) was provided by the company +H2O . Deionized water was produced using the Simplicity® Ultrapure Water System, 18.2 MΏ·cm resistivity (25 °C) at 0.5 L/min, Merck Millipore .The raw materials used were distillates produced on an industrial scale by the Polish companies. Rye distillate , and an HP-5 MS capillary column with dimensions of 30 m × 0.25 mm × 0.25 μm .Gas chromatograph (GC) operating parameters were as follows: dispenser temperature—250 °C, sample volume—1 μL, direct injection split—1:40, GC oven temperature program—from 35 °C (6 min) to 100 °C at a rate of 5 °C/min, and then increased to 230 °C at a rate of 10 °C/min (hold time: 2 min), flow rate of the carrier gas (helium) through the column—1 mL/min.m/z, 59 m/z, 42 m/z).1-propanol .Ethyl acetate .2-methyl-1-propanol .3-methyl-1-butanol .2-methyl-1-butanol .Isoamyl acetate .Ethyl hexanoate .Ethyl octanoate .Ethyl decanoate .Ethyl tetradecanoate .Acetaldehyde diethyl acetal (monitored ions: 43 Mass detector (MS) operating parameters were as follows: ion source temperature—230 °C, transfer line temperature—250 °C, ionization energy—70 eV, the mass range during qualitative analysis in the SCAN mode was 20–230 u, dwell time—15 ms. Quantitative analysis was performed in the SIM mode:A 7890A gas chromatograph with a flame ionization detector (FID) was used to determine the content of methanol and acetaldehyde. An HP-INNOWax capillary column with dimensions of 30 m × 0.25 mm × 0.25 µm  was used to separate the compounds.Gas chromatograph (GC) operating parameters were as follows: dispenser and detector temperature—250 °C, sample volume—1 μL, direct injection split—1:50, GC oven temperature program—from 40 °C (4 min) to 195 °C at a rate of 10 °C/min (hold time—2 min), flow rate of the carrier gas (helium) through the column—1.5 mL/min.Quantitative analysis was performed using Agilent MassHunter software  and the external calibration method.p = 0.05 to verify statistical differences.All samples were prepared and analyzed in triplicate. Statistical analyses were performed using Statistica 10 software . The results are expressed as average ± SD. The results were evaluated using analysis of variance (ANOVA) followed by Tukey’s post-hoc test with a significance level of The results of turbidity measurements before storage, and after 4 and 8 weeks of storage, are shown in v/v (control samples). Measurements after 4 weeks of storage showed that turbidity had increased to 0.02 FTU in samples with an ABV of 40% v/v stored at temperatures of 8 °C, 0 °C, and −18 °C. In a sample with the same ABV but stored at 20 °C, the turbidity value was 0.01 FTU. After a further 4 weeks, an increase in turbidity was observed in all samples with alcohol contents of 40 and 50% v/v. The highest increases in turbidity, to 0.55 and 0.59 FTU, were observed in samples with an ABV of 40% v/v, stored at 0 °C, and −18 °C, respectively. These changes in turbidity were statistically significant (p < 0.05) compared to the control samples. In the case of rye distillate with an ABV of 70% v/v, no turbidity was noticed after 8 weeks of storage, regardless of the temperature.No turbidity was observed before storage in the case of rye distillate samples with alcohol contents of 40, 50, and 70% v/v was 0.02 FTU. This was most probably due to the higher (p < 0.05) content of volatile compounds in the plum distillate in comparison to the rye distillate. Volatile compounds in the spirit give the final product its specific character. However, when the alcohol content is less than 45% v/v, they can cause turbidity, especially during storage [The turbidity in the control samples prepared from plum distillate with ABV of 40%  storage .v/v. After 4 weeks of storage, the highest turbidity, ranging from 1.37 FTU (at 20 °C) to 5.41 FTU (at −18 °C) was found in the samples with an ABV of 40% v/v (p < 0.05). The samples with alcohol contents of 50 and 70% v/v stored at 8 °C showed very low turbidity. However, the changes were not statistically significant (p > 0.05). After 8 weeks of storage, the turbidity increased in all plum distillate samples, except for two, which had ABVs of 50 and 70% v/v, stored at 20 °C. The highest increase in a turbidity in comparison to the controls was observed in the sample with 40% v/v alcohol content, stored at a temperature of −18 °C. Turbidity may be associated with a decrease in the solubility of volatile compounds, such as higher alcohols and fatty acid esters , especially at low temperatures [No turbidity was observed in the control samples with alcohol contents of 50 and 70% eratures . MoreoveFTU at −1 °C was feratures  and withFTU at −1 °C was feratures .v/v were monitored over 8 weeks of storage at different temperatures. The results are shown in Changes in the qualitative and quantitative composition of volatile fermentation by-products in the samples of rye and plum distillates with ABVs of 40, 50, and 70% p < 0.05) in rye distillate  (v/v)   than in v) (v/v) . Changesv/v) (v/v)  is not subjected to regulations. The concentrations of methanol in the rye and plum distillates remained at similar levels to the control samples, regardless of the ABV and the temperature. These variables were found to have no statistically significant effect on the methanol concentration (p > 0.05).One of the undesirable compounds in spirits is methanol, which is liberated from pectic substances by enzymatic degradation under the influence of a specific pectolytic enzyme, pectin methylesterase, particularly during ripening and fermentation processes. Methanol does not directly affect the flavor of the distillate; however, it is subjected to restrictive controls due to its high toxicity ,24. Methv/v)  than thev) (v/v) . The higSaccharomyces cerevisiae yeast is to be expected, because these microorganisms produce higher alcohols as byproducts of the decarboxylation and deamination of amino acids during the fermentation process. The most representative higher alcohols are 1-propanol, 2-methylpropanol, 3-methylbutanol, and 2-methylbutanol [Agricultural distillates  contain a relatively large group of compounds known as higher alcohols (fusels), which are present in high concentrations. The presence of these compounds in spirits obtained by ethanol fermentation with the participation of lbutanol .v/v. The content of 1-propanol increased in two samples of the plum distillate, one with an alcoholic strength of 50% v/v (stored at 20 °C) and the other with an ABV of 70% v/v (stored at –18 °C) to 127.84 mg/L alcohol 100% v/v and 128.52 mg/L alcohol 100% v/v, respectively. Moreover, it was noticed that in the samples of rye distillate with an ABV of 70% v/v, at all storage temperatures the decrease in propanol content was low (p > 0.05). This was in contrast to samples with a 40 or 50% v/v alcohol content, in which the changes after storage were greater and statistically significant (p < 0.05) (v/v) than those produced industrially  [v/v [v/v.The samples prepared from rye distillate were characterized by a higher initial content of 1-propanol , compare < 0.05) . A previ/v) [v/v  reportedv/v, whereas in the samples of plum distillate it was 875.14 mg/L alcohol 100% v/v. A reduction in the content of 2-methylpropanol was observed in all tested samples, depending on their ABVs and the storage temperatures. In both types of distillates and for all tested ABVs, after 4 weeks of storage, lower temperatures resulted in a greater decrease in the concentration of isobutyl alcohol. However, after the next 4 weeks, no correlation was found between the changes in 2-methylpropanol content and the storage conditions. The changes in the concentration of 2-methylpropanol after 8 weeks of storage were statistically significant (p < 0.05), in relation to the results after 4 weeks, except in the case of rye distillates with alcohol contents of 40 and 50% v/v stored at 20 °C, and for plum distillate, with an alcohol content of 40% v/v stored at 0 °C or 8 °C  was present in high concentrations in the tested distillates. In the samples of rye spirit, the content of 2-methylpropanol was 699.09 mg/L alcohol 100%  or 8 °C .v/v, whereas in the samples of plum distillate it was 672.28 mg/L alcohol 100% v/v. The content of 3-methylbutanol was higher, and in the rye distillate samples amounted to 481.46 mg/L alcohol 100% v/v, whereas in the plum distillate samples, it was measured at 3098.98 mg/L alcohol 100% v/v. Regardless of the storage temperature, the greatest decrease in the concentration of 2-methylbutanol after 8 weeks was noted in the samples with an ABV of 70% v/v. In the samples of rye distillate, the concentration of these compounds reduced by approximately 47%, whereas in the samples prepared from plum distillate it fell by about 20% in relation to the control samples. In comparison, the differences in the concentration of 3-methylbutanol were much smaller. The content of isoamyl alcohol in the rye distillate samples decreased by a maximum of about 8%, and that in the plum spirits from about 9% to 17%. In the case of the rye distillate-based samples, there was a greater reduction in the isoamyl alcohol content for lower ABVs. The opposite relationship was observed in the case of plum distillate-based samples. The greatest changes in the isoamyl alcohol content occurred in the trials with an alcohol content of 70% v/v and decreased with lower ABVs. The decrease in optically active amyl alcohol concentration was statistically significant (p < 0.05) for all samples prepared from both distillates. For isoamyl alcohol, statistically significant changes occurred in all the samples, except for two samples of rye distillate with an alcoholic strength of 50% v/v, stored at 20 °C and −18 °C.Amyl alcohols, i.e., 3-methylbutanol  and 2-methylbutanol  are predominant higher alcohols. They usually constitute at least half of the total amount of this group of volatile compounds . A much v/v, after the first 4 weeks at all storage temperatures, the concentration of this compound increased more than threefold, from 97.51 mg/L alcohol 100% v/v to 337.39−363.20 mg/L alcohol 100% v/v. However, after the next 4 weeks, it increased by not more than a further 5% (p > 0.05). An increase in the concentration of acetaldehyde diethyl acetal was also observed in the samples of rye distillate, but it was significantly lower compared to the plum distillate. In the samples of rye distillate, the content of this compound before storage was 0.75 mg/L alcohol 100% v/v. After 4 weeks, it increased on average to values of around 1 mg/L alcohol 100% v/v. After the next 4 weeks, it did not exceed 1.8 mg/L alcohol 100% v/v. A relatively large increase in the content of acetaldehyde diethyl acetal was observed only in the sample of rye distillate with 70% v/v alcohol content, stored at 20 °C. This finally amounted to 5.22 mg/L alcohol 100% v/v. Each change in the concentration of acetaldehyde diethyl acetal was statistically significant (p < 0.05). The ABV and storage temperature was found to have no influence on the content of acetaldehyde diethyl acetal in the remaining samples of rye distillate  compared to the rye distillate . The largest differences were observed in the case of ethyl acetate, the concentration of which was approximately three times lower  in the rye distillate than in the plum distillate . Such differences result from the high concentration of esters in plum fruits, which may be transferred to the spirit during the distillation process. Literature data [The decrease in the concentration of higher alcohols in the tested distillates was most probably the result of reactions between alcohols and acids as well as between alcohols and aldehydes, resulting in the formation of esters and acetals, respectively . This hyure data  confirm v/v, regardless of the storage temperature. After 8 weeks, it increased by 30% compared to the control sample. In the samples with an alcohol content of 40% v/v, the increase in ethyl acetate content was on average 14%, whereas in the samples with an ABV of 50% v/v it increased by an average of 24% (p < 0.05) . Exceptionally, the plum distillate sample with an alcoholic strength of 40% v/v stored at 20 °C, showed no statistically significant changes (p > 0.05) in the concentration of ethyl acetate compared to the control sample.In all samples, with all tested ABVs and at all temperatures, the ester content increased over the 8-weeks storage period. In the samples of rye distillate, the greatest increase in the ethyl acetate content was noticed in the samples with an ABV of 70%  < 0.05) . The ethv/v in the rye distillate and 1.82 mg/L alcohol 100% v/v in the plum distillate. An increase in the alcoholic strength of the samples caused a rise in the concentration of isoamyl acetate, regardless of the storage temperature. In the samples with an alcohol content of 40% v/v, the concentration of this compound increased by 16% (rye distillate) and 22% (plum distillate), whereas in the samples with 50% v/v it rose by 39% (rye distillate) and 47% (plum distillate). The increase was greatest in the samples with an ABV of 70% v/v, reaching 66% (rye distillate) and 74% (plum distillate) relative to the control samples. Generally, the content of isoamyl acetate was higher in the samples prepared from plum distillate, and after 8 weeks, it increased (p < 0.05) from 1.82 mg/L alcohol 100% v/v to 1.88–3.36 mg/L alcohol 100% v/v. In the samples prepared from rye spirit, after 8 weeks of storage the concentration of isoamyl acetate increased from 0.52 mg/L alcohol 100% v/v to 0.59–0.91 mg/L alcohol 100% v/v (p < 0.05).Isoamyl acetate, which forms as a result of isoamyl alcohol acetyltransferase activity , was prev/v and 2.37 mg/L alcohol 100% v/v, respectively. In the rye distillate, their concentrations were 0.17 and 0.08 mg/L alcohol 100% v/v, respectively. In all the tested samples of the plum and rye distillates, an increase in the content of both esters was observed after 8-weeks storage. The samples of rye distillate stored at 20 °C were characterized by the largest increase in the concentration of ethyl caproate, regardless of their alcoholic strength. For example, in the rye distillate with an alcohol content of 70% v/v the concentration of ethyl caproate was 0.31 mg/L alcohol 100% v/v, whereas in the sample with an ABV of 50% v/v it was 0.27 mg/L alcohol 100% v/v. In turn, for the samples of rye distillate with 40% v/v the concentration of ethyl caproate was 0.22 mg/L alcohol 100% v/v. Moreover, in the samples with an ABV of 40% v/v stored at 0 °C or −18 °C, the content of ethyl caproate was the same as that determined in the sample stored at 20 °C. All changes in the ethyl caproate concentrations observed during the storage of the plum and rye distillates samples were statistically significant (p < 0.05). In the case of plum spirit, the dynamics of the increase in the concentration of ethyl caproate correlated with the ABVs of the tested samples. After 8 weeks of storage, the concentrations of ethyl caproate were within the following ranges: 0.69–1.16 mg/L alcohol 100% v/v (40% v/v); 1.18–1.40 mg/L alcohol 100% v/v (50% v/v); 1.46–1.79 mg/L alcohol 100% v/v (70% v/v). Only in the plum distillate sample with an ABV of 40% v/v, stored at −18 °C, did storage not significantly affect (p > 0.05) the content of ethyl caproate.Higher concentrations of ethyl hexanoate (ethyl caproate) and ethyl octanoate (ethyl caprylate) were also determined in the plum distillate than in the rye distillate . The conp > 0.05). However, over the next 4 weeks, a ten-fold increase (p < 0.05) in the concentration of ethyl octanoate was observed. Storage temperature had no significant effect on the concentration of ethyl octanoate. The samples with the same start ABVs stored at all temperature variants ultimately showed similar ethyl octanoate contents. In the samples of rye distillate with an alcoholic strength of 40% v/v, the ethyl octanoate contents were in the range of 6.36–6.39 mg/L alcohol 100% v/v. An increase in ABV to 50% v/v resulted in concentrations of ethyl octanoate in the range of 5.11–5.12 mg/L alcohol 100% v/v, whereas for the samples with 70% v/v the levels were in the range of 3.66–3.71 mg/L alcohol 100% v/v. After 8 weeks of storage, an approximately threefold increase in the concentration of ethyl octanoate was observed in the plum distillate, compared to the controls. It was also noticed that as the storage temperature was lowered, the intensity of ethyl octanoate synthesis decreased, regardless of the ABVs of the tested samples. Only in the samples with an ABV of 50% v/v stored at 0 °C and −18 °C were the concentrations of ethyl octanoate similar, at 9.18 mg/L alcohol 100% v/v and 8.95 mg/L alcohol 100% v/v, respectively.The content of ethyl octanoate (ethyl caprylate) in the rye distillate samples after 8-weeks of storage was noticeably higher in the samples with a lower alcoholic strength. Within the first 4 weeks, changes in the concentration of ethyl octanoate were very small and statistically insignificant  after storage, compared to the control samples. The concentrations of these compounds increased more sharply when the ABVs of the samples and the storage temperature were higher. As a consequence, the increases in the concentrations of both esters were the largest in the samples stored at 20 °C.The presence of ethyl decanoate (caprate) and ethyl myristate was also noted in the plum distillate samples, whereas in the rye distillate these esters were not detected. The decanoate concentration in the plum distillate control sample was 7.09 mg/L alcohol 100% observed . Changesv/v, the storage temperature was found to have no effect on the changes in the concentration of ethyl caprate. In the samples with ABVs of 50 and 70% v/v, an analogous correlation was observed to that for ethyl caprylate. As the storage temperature decreased, the intensity of the increase in the ethyl caprate content decreased. This dependence of changes in the concentration of esters on storage temperature also applied to ethyl tetradecanoate (ethyl myristate).In the case of the plum distillate with an alcoholic strength of 40% v/v. The highest turbidity was observed in the samples with an ABV of 40% v/v, whereas higher alcohol content  had a positive effect on the preservation of the clarity of the tested samples. No significant changes in methanol concentration were observed over the 8-week storage period in the rye and plum distillates diluted to ABVs of 40, 50, 70% v/v. However, higher ABVs resulted in a more intense decrease in acetaldehyde concentration. No relationship was found between the reduction in acetaldehyde and the storage temperature. Lower concentrations of acetaldehyde were connected with an increase in the concentration of acetaldehyde diethyl acetal. Especially in the samples with ASBVs of 40 and 50% v/v, this increase was correlated with storage temperature. A decrease in the concentration of higher alcohols was also noted, as a result of the reaction of alcohols with acids. This resulted in an increase in the concentrations of esters in the tested distillates. The increases in the concentrations of esters were positively correlated to the alcohol concentration of the tested samples. An inverse relationship was observed only for ethyl octanoate in the samples of rye distillate. No strict correlation was found between the storage temperature and the concentrations of esters determined in the distillates.In this study, we investigated how ethyl alcohol content and storage conditions influence the clarity and chemical composition of spirit beverages. The richer composition of the plum distillate compared to the rye distillate was reflected by the appearance of higher turbidity during storage of the samples with ABVs of 40, 50, and 70% The results of this study show that the quality of spirit beverages is not only shaped at the production stage, but can also be modified by the storage conditions, as evidenced by the observed changes in the chemical compositions of the tested samples. Our results may be useful for determining the conditions for the preparation of spirit beverages prior to final filtration and bottling. Selection of the appropriate conditions for the storage of distillates could help to avoid the formation of undesirable turbidity, while also maintaining the profile of desirable volatile compounds from the raw plant materials.


The novel material possessing good flexibility could exhibit superior antibacterial property over sulfate undoped species (Ag3PO4/PAN electrospun nanofibers). FESEM, XRD, FTIR, XPS and DRS were applied to characterize the morphology, phase structure, bonding configuration, elemental composition, and optical properties of the as fabricated samples. FESEM characterization confirmed the successful incorporation of SO42−-Ag3PO4 nanoparticles on PAN electrospun nanofibers. The doping of SO42− ions into Ag3PO4 crystal lattice by replacing PO43− ions can provide sufficient electron-hole separation capability to the SO42−-Ag3PO4/PAN heterojunction to generate reactive oxygen species (ROS) under visible light irradiation and enhances its antibacterial performance. Finally, we hope this work may offer a new paradigm to design and fabricate other types of flexible self-supporting negative-ions-doped heterojunction nanofibers using electrospinning technique for bactericidal applications. Heterojunction nanofibers of PAN decorated with sulfate doped Ag Thus, the produced ROS are responsible for the bactericidal activity of semiconductor materials but metal-based materials act as bactericidal agents without light assistance [3PO4) has drawn considerable attention as an antibacterial semiconductor material in recent years [3PO4 as a photocatalyst is limited due to its poor chemical stability, when used without any sacrificial reagent in aqueous solution [Bacteria constitute large group of single celled microorganisms that thrive in diverse environment. Along with the beneficial importance of bacteria, they also have many harmful effects on the human body, since some bacteria are the source of many serious diseases. Human activities including unimproved sanitation in developing areas, improper disposal of household waste, inadequate excreta disposal, and washing clothes near natural water sources are leading to an increasing risk of microbial contamination. Harmful and often drug-resistant bacteria, which are widespread in the environment can cause severe and deadly infections ,2,3. Hensistance ,8,9,10. nt years ,12. It isolution ,14. Furtsolution ,16, fabrsolution ,18, carbsolution ,20, non-solution ) and dopsolution ,23,24. 3PO4 through cations doping into Ag3PO4 crystal lattice [3PO4 doped with suitable anions has been rarely reported. However, recently published report demonstrated that sulfur-doped Ag3PO4 can improve its photocatalytic activity on the basis of hybrid density-functional calculation [3PO4 crystal lattice is not so easy by post-treatment process due to the strong P–O bond. Consequently, SO42− might be a suitable doping ion into Ag3PO4 crystal lattice by replacing PO43− since SO42− has smaller radius (0.218 nm) than that of PO43− (0.230 nm) [Semiconductor material doped with suitable ions could be an effective approach to design a photocatalyst with enhanced activity and stability . Doping  lattice ,27. But culation  but the .230 nm) ,30. The .230 nm) ,32,33,34.230 nm) ,36. PAN .230 nm) . 42−-doped Ag3PO4 nanoparticles and PAN electrospun nanofibers, we report the fabrication of the heterojunction of SO42−-Ag3PO4/PAN electrospun nanofibers with enhanced antibacterial performance. To the best of our knowledge, such work has not been reported so far. For the convenience of description, different samples were hereinafter referred to as: pure PAN corresponding to pure PAN electrospun nanofibers, AP/PAN corresponding to sulfate undoped Ag3PO4/PAN electrospun nanofibers, and S-AP/PAN corresponding to SO42−-doped Ag3PO4/PAN heterojunction nanofibers.Hence, to understand the combination of respective properties of SO42− doping on Ag3PO4 nanoparticles were investigated by XRD , (200), (210), (211), (220), (310), (222), (320), (321), (400), (411), (420), (421) and (332) of Ag3PO4, respectively (JCPDS card No: 06-0505). Besides, broad peaks centered at about 17° and 20°–23° in the composite nanofibers were assigned to the (100) and (110) crystallographic planes of PAN polymer, respectively [42− into Ag3PO4 crystal lattice was investigated from the magnified XRD patterns of both samples , 0, (321), samples b. As shoof PO43− ,38. Ther2HPO4/PAN electrospun nanofibers  of PAN. Similarly, characteristics bands obtained in the regions 1220–1270 cm−1, 1350–1380 cm−1, 1450–1460 cm−1 and 2870–2931 cm−1 are assigned to the aliphatic CH group vibrations of different modes in the methylene group of PAN [−1 and 3400–3500 cm−1, indicating the presence of physically absorbed water molecules [−1 and 981 cm−1 could be assigned to the molecular vibration of PO43− [42− in S-AP/PAN, which locates at about 983 cm−1 [43−. Hence, all the results obtained from FTIR analyses indicated the formation SO42−-doped Ag3PO4 nanoparticles immobilized on PAN nanofibers.The FTIR spectra of three different samples are presented in p of PAN . In all of PO43− ,41. But 42−-undoped/doped Ag3PO4 nanoparticles on the surface of PAN nanofibers, both the samples displayed obvious visible light absorption behavior, signifying their visible light absorption properties. The optical band gap energies (Eg) of S-AP/PAN and AP/PAN were estimated using a plot of (αhν)1/2 versus energy (hν) [42− doping.In order to determine the light absorption behavior, UV-vis diffusive reflectance spectra (DRS) of different samples were measured and results are plotted in rgy (hν) . As depi6+ [42− into the Ag3PO4 crystal lattice could occur during the synthesis process. The binding energies corresponding to P 2p and Ag 3d peaks of S-AP/PAN were found shifted to higher values compared to that of AP/PAN  leaving behind holes in the valence band (VB). During this process, photoexcited electrons in CB react with dissolved oxygen in solution to produce O2•− radicals, which further react with H+ to produce OH• radicals. Similarly, photogenerated holes also react with H2O to produce OH• radicals. Hence, thus produced OH• radicals are responsible for photocatalytic antibacterial behavior of SO42−-Ag3PO4/PAN heterojunction nanofibers [• radicals assist the peroxidation of lipids in the outer cell wall of bacteria and damage cell organelles including DNA leading to cell death [42− ion plays an important role in trapping and transferring electrons. SO42−-Ag3PO4 can receive additional electrons due to the presence of more valence electrons in S compared to P. Therefore, doping of SO42− into Ag3PO4 crystal lattice can enhance the concentration of photogenerated charges and inhibit the recombination of electron-hole pairs, thereby increasing the production of ROS, which is believed to be the main reason for the enhanced antibacterial activity of SO42−-Ag3PO4 [The enhanced antibacterial performance of SOnofibers . It is bll death ,51. Most−-Ag3PO4 ,52,53. 2HPO4.2H2O), Silver nitrate (AgNO3), Sodium sulfate (Na2SO4), were purchased from Sigma-Aldrich, USA. N, N-dimethylformamide (DMF) purchased from Daejung Chemicals, South Korea. All the chemicals were used as received without further purification.Polyacrylonitrile , Disodium hydrogen phosphate dihydrate  containing 0.01 M of Na2SO4 for 30 min. The color of nanofibers mat was changed to yellow indicating the formation of SO42−-Ag3PO4 nanoparticles immobilized on PAN nanofibers surface. For comparison, sulfate undoped Ag3PO4/PAN nanofiber mat was also fabricated without adding Na2SO4 under similar conditions. The resulting nanofibers mats were washed with copious amount of deionized water and dried at 60 °C for 6 h before characterization. The schematic for SO42−-Ag3PO4/PAN heterojunction nanofibers fabrication is illustrated in 0.1 g of Na42−-doped/undoped samples was recorded using X-ray photoelectron spectroscopy .Information about phase and crystallinity of the samples were investigated using X-ray diffractometer  with Cu Kα (λ = 1.540 Å) radiation over Bragg angles ranging from 10° to 80°. Morphological structure and distribution of nanoparticles on the surface of nanofibers were characterized using field emission scanning electron microscope  equipped with energy dispersive X-ray spectroscopy (EDS) to analyze the elemental composition of samples. Prior to characterization, samples were prepared by coating with platinum for 180 s using a Pt-coater . Bonding configuration of the polymer with nanoparticles was characterized with the help of Fourier-transform infrared  using attenuated total reflectance mode (ATR). Light absorption behavior of all samples was investigated from Uv-vis diffusive reflectance spectra (DSR) obtained from UV-vis spectrophotometer . Furthermore, the surface element composition analysis of SOE. coli (ATCC 25922) and Gram positive S. aureus (ATCC 4330) bacterial strains. Both disk diffusion and bacterial colony count tests were performed to determine growth suppressing action of pure PAN, AP/PAN and S-AP/PAN under day light conditions. In the disk diffusion test, Tryptic soya agar was used to perform the disk diffusion method to evaluate the antibacterial activity of the samples according to the previously described procedure [E. coli and S. aureus culture. A 100 µl cell suspension was plated in each tested petri dish and 5 mm disk of every samples were carefully dropped on top of the culture plates. All petri dishes were incubated at 37 °C for 12 h under day light and after incubation the zone of inhibitions were measured using ImageJ software. For comparison, the disk diffusion test was performed to evaluate the antibacterial activity of pure PAN and S-AP/PAN towards E. coli and S. aureus in dark conditions. The antibacterial effects of Pure PAN, AP/PAN and S-AP/PAN were further determined via bacterial colony count test on tryptic soya agar plates according to previous method with modification [E. coli and S. aureus cell suspension were spread on each tested petri dish. Culture dishes were incubated at 37 °C for 12 h and the bacterial colonies were counted after incubation. All experiments were performed in triplicates, and statistical analysis was carried out using SPSS software. The antibacterial efficacy of the samples was assessed by using Gram negative rocedure . Individfication . The frap < 0.05 was considered as significant and represented as * p < 0.05, ** p < 0.01 and *** p < 0.001.All statistical data were expressed as the mean ± s. d., as indicated. Statistical significance was performed by one-way ANOVA using SPSS software . p < 0.03PO4 nanoparticles supported on PAN electrospun nanofibers was fabricated using the electrospinning technique followed by the ion exchange method. The antibacterial activity of the fabricated material was investigated on E. coli (Gram negative) and S. aureus (Gram positive) bacteria by disc diffusion and bacterial colony count methods under visible light. Experimental results showed that the flexible SO42−-Ag3PO4/PAN heterojunction nanofibers (S-AP/PAN) can work as a bactericidal material with enhanced activity. This was the first time that a heterojunction of sulfate-doped Ag


Glioblastoma (GBM) is highly invasive and the deadliest brain tumor in adults. It is characterized by inter-tumor and intra-tumor heterogeneity, short patient survival, and lack of effective treatment. Prognosis and therapy selection is driven by molecular data from gene transcription, genetic alterations and DNA methylation. The four GBM molecular subtypes are proneural, neural, classical, and mesenchymal. More effective personalized therapy heavily depends on higher resolution molecular subtype signatures, combined with gene therapy, immunotherapy and organoid technology. In this review, we summarize the principal GBM molecular classifications that guide diagnosis, prognosis, and therapeutic recommendations. The World Health Organization (WHO) defines adult diffuse gliomas into grade II and grade III astrocytic tumors, grade II and III oligodendrogliomas, and grade IV glioblastomas . GlioblaHistomorphology ambiguity and tumor heterogeneity pose challenges to GBM diagnosis, prognosis and treatment. Histologic diagnosis often varies among clinicians and limits diagnostic reproducibility. GBM histologically and genetically show significant inter-tumoral and intra-tumoral heterogeneity, differing mutations, and indistinct phenotypic and epigenetic states reflect genomic instability that leads to varying therapy choices and clinical outcomes . MoleculMolecular-based diagnosis, patient stratification, and personalized treatment are increasingly important. The ISN Haarlem recommends “hierarchical diagnosis with histological classification, WHO classification, and molecular information for comprehensive diagnosis” . In 2016Multi-omics studies from the landscape of GBM in the Cancer Genome Atlas Research Network (TCGA), the Chinese Glioma Genome Atlas (CGGA), and other databases, together reveal the complicated genetic profile of GBM . These aIn this paper, we summarize the process of GBM classification based on transcription levels, genetic alterations, and DNA methylation. We also describe the molecular characteristics of each category, and the relationship between different classification methods. Finally, we provide the current guiding strategy for diagnosis and treatment.Deciphering GBM heterogeneity and complexity is the key to understanding it’s progression and creating effective therapies. Some important and aberrant molecular events drive GBM malignant transformation, highlighting the importance of molecular classification. First, GBM has a wide variety of chromosomal changes, including amplification in chromosome 4 , Chr.7 , Chr.12 , and deletion in Chr.10 (PTEN). Notably, some GBM patients have simultaneous gain of Chr.19 and 20 .Second, the TCGA GBM project describes somatic genome changes based on multidimensional and comprehensive features that show significant mutations in GBM, including TP53 (34.4%), EGFR (32.6%), PTEN (32%), NF1 , PIK3CA , PIK3R1 , RB1 , SPTA1 , ATRX (6%), IDH1 (5.2%), KEL , PDGFRA , and GABRA6  .Lastly, DNA methylation is a key factor when measuring heterogeneity and stratification of GBM patients. Epigenetic modifications of GBM is related to biological characteristics and are considered therapeutic targets . DNA metWith the recent development of technology and classification algorithms, GBM is divided into different subtypes based on transcription profiles, genetic alterations, and DNA methylation. This allows targeted therapy based on molecular characteristics of subclasses. For example, clinicians can target the mesenchymal subtype from transcription subtypes in GBM via inhibition of diacylglycerol kinase alpha. In doing so, patients with MGMT methylation had a more robust response to temozolomide . The TCGGBM classification based on gene expression profiles initially used microarray technology, then large-scale high-throughput next-generation sequencing technology. The molecular map of GBM is shown in In the 1990s, scientists acquired data from techniques like PCR, allele analysis, and first-generation sequencing to analyze gliomas. They found a variety of molecular markers of different types and grades, but the landscape was not clear . Indeed,In a groundbreaking study, Phillips et al. classified three GBM subtypes: Proneural, Proliferative and Mesenchymal . ProneurThe above studies demonstrated tumors often cluster in groups that display heterogeneity, highlighting the weaknesses of conventional diagnosis. With the advent of large-scale, high-throughput, next-generation sequencing methods, and with algorithms in machine learning, complex tumor data is becoming more precise.In 2017, Using cancer genome data from the TCGA GBM project and classification from Recent studies offer new insights into GBM classification based on transcription. Teo et al. validated three robust GBM-subtypes: Proneural/Neural, Classical, and Mesenchymal across six different datasets . This waIn recent years, large-scale genomic studies have revealed many mutations in tumor suppressor genes and oncogenes, and significantly improved our understanding of GBM. Specifically, mutated IDH, PTEN and EGFR are related to patient survival and can be used as indicators of patient classification.The identification of the IDH mutation is an important contribution to the molecular pathology of GBM. In 2008, In 2016, the WHO divided it into two: IDH mutation and IDH wild type . IDH wilMultiple studies have confirmed that IDH mutations have prognosis and predictive value . CompareIn IDH1 wild type GBM, the median survival rate of patients with CDK4/MDM2 co-amplification is 6.6 months after diagnosis, while the median survival rate of patients without an CDK4/MDM2 co-amplification is 12.7 months . The TEREGFR amplification is usually accompanied by EGFR mutation, the most frequent being EGFRvIII . Under nThe PTEN protein catalyzes the dephosphorylation of 3’ phosphorylation of the inositol ring in PIP3  to produce PIP2 . The dephosphorylation is critical because it inhibits the AKT signaling pathway. The PI3K/AKT pathway is normally dormant in differentiated and quiescent cells, but when activated, the cell cycle modulation leads to cancer. The deficiency of PTEN mainly plays the role of lipid phosphatase through the PI3K/AKT pathway . TherefoIn addition to the genes above, other genetic mutations also drive GBM development. However, the mutation or deletion of a single gene may not serve to classify GBM independently. The combination of aberrant events related to survival may be a more effective classifier. Data suggest combination of two or three genes provide a robust classifier to diagnostic analysis for clinical applications . ClassifEpigenetic changes are common markers of human cancers, including GBM . DNA metBrennan et al. used large-scale methylated sequencing data to classify GBM, divided into six categories based on the expression level of DNA methylation, including Cluster M1 to Cluster M6, in which Cluster M5 was G-CIMP subtype . ClusterRecently, DNA methylation provides a framework for understanding GBM and guiding a therapeutic strategy. It has offered more molecular biomarkers for each subtype and suggested more targets for treatment. Methylation is a powerful complement to classification based on genetic alterations and transcription, making GBM classification more comprehensive.Early attempts to identify specific tumor subtypes generally focused only on gene expression patterns. But biological processes are not so simply regulated. Omics data have helped identify clusters of tumors with similar characteristics, including genotypic and epigenetic regulation. Many studies have found that molecular subtypes classified at different levels are related and overlapped, as exampled from the four transcription-based subtypes from Recently, some studies have shown that subtype migration and molecular changes occur in recurrent GBM, highlighting the need for further research . The recTranscription-based molecular subtypes are also associated with tumor recurrence. For example, Wang et al. found that two-thirds of patients with primary GBM switched transcriptional subtype after recurrence. Importantly, the Mesenchymal subtype was the most stable primary GBM subtype . TherefoStudies focused on recurrent GBM have shown molecular composition and molecular subtypes of tumors evolve in response to radiotherapy and targeted therapy, therefore molecular signatures guiding treatment protocols may improve patient survival . HoweverTreatment corresponding to tumor subtypes is an effective strategy to avoid the obstacles caused by molecular heterogeneity . Chen etFurther evidence of molecularly guided treatment comes from Sandmann et al. that showed a 4.3 month increase in median survival with the addition of bevacizumab for IDH1 wild-type GBM in the proneural subgroup . The IDHTemozolomide is an oral alkylation agent. The main mechanism of temozolomide arrests the cell cycle at G2/M checkpoint, which leads to apoptosis of cancer cells . The stuDue to the heterogeneity of GBM, individualized treatment based on specific tumor subtype is clearly a more effective clinical strategy. Gene mutations in TP53, IDH-1 and PDGFR-A in Proneural subtype; mutations or amplification of EGFR gene in Classical subtype; NF-1 gene mutations in Mesenchymal subtype; and the expression of neural markers in Neural subtype are promising therapeutic targets. Several studies have shown that targeting these molecules improves treatment. For example, Sang et al. found the efficacy of SHP099, a potent, selective, and oral SHP-2 inhibitor for treating GBM with activated PDGFR-A signaling; and Liu et al. proved that the third-generation EGFR inhibitor osimertinib overcomes primary resistance by continuously blocking ERK signaling in GBM .Due to subtype migration and molecular changes after recurrence, molecular evaluation of patients must be performed prior to chemotherapy. Patients undergoing surgical resection must undergo immunohistochemical studies to determine various predictors, such as MGMT methylation, to assist in treatment planning.Gene therapy aims to introduce genetic material into cells to compensate for abnormal genes or to make beneficial proteins. If a mutated gene causes a necessary protein loss, gene therapy can introduce a normal gene to supplement the protein’s function. Gene therapy is the delivery of a gene through a vector to a cell. Viruses are often used as vectors because they can deliver new genes by infecting cells. These viruses are modified so that when they are used in humans, they do not cause disease. Adenovirus (AAV) vectors have been used to inject directly into GBM cells in the brain to express tumor-killing genes. Crommentujin et al. demonstrated the AAV9 vector, which produces the anticancer agent sTRAIL, killed up to 60% of GBM cells in mouse models and transfected cell lines . AAV9 viImmunotherapy offers the promise of a sustained antitumor immunity that is pathway independent and has the potential to amplify antigens to boost immune responses. Peptide vaccines, such as EGFRvIII found in Classical GBM subtypes, can trigger immunity to GBM tumor cells expressing EGFRvIII. In a phase II trial involving 18 patients, EGFRvIII patients showed an overall survival of 26 months, compared with only 15 months for the control group . The vacGlioblastoma organs can be an effective model for rapid testing of personalized treatment strategies. The models allow researchers to reconstruct key features of a patient’s diseased brain to help paint a clearer picture of the cancer and then allow researchers to explore the best ways to treat it. Researchers have successfully transplanted eight glioblastoma organoids (GMOs) samples into brains of adult mice, administering standard care and targeted therapy to GBOs, including clinical trial drugs and chimeric antigen receptor T (CAR-T) cell immunotherapy . For eacThe unique and highly reproducible molecular changes discovered in recent years have begun to elucidate the diversity of GBM and contribute to the more effective classification of tumors. These studies provide insights into how to improve current treatment strategies. GBM genomics, transcription, and epigenetic features reveal critical molecular changes that may lead to pathologic disease progression. Large-scale analysis, like the TCGA project, confirm that GBM is a heterogenetic tumor at the molecular level that can be subdivided into different subtypes according to the molecular pathogenesis and biological entities of “driving factor” lesions. Although these comprehensive studies provide useful insights into the characteristics and classification of tumors, their limitations need to be considered when drawing conclusions. Some prognostic markers have appeared in these studies, and there is still a great need to identify true predictive markers to improve the treatment process of personalized care. In addition, the intra-tumoral heterogeneity of GBM needs to be further classified by single-cell sequencing technology to obtain a more complete and more precise inter-tumoral and intra-tumoral classification. We still need large-scale animal experiments and human clinical verification to improve treatment response and survival time among the different subtypes.Through these molecular-level studies, we can further improve the molecular detection methods, guide the targeted therapy based on molecular classification, and form a set of accurate GBM molecular therapy manuals that can improve patient outcome.QX and PZ contributed to the conception and design of manuscript. PZ drafted the manuscript. QX provided useful comments and suggestions. LD and QX revised the manuscript. All authors reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) demonstrated good glycemic efficacy in patients with type 2 diabetes mellitus (T2DM) recent years, whereas studies on GLP-1 RAs’ biliary effects were limited. Therefore, we aimed to assess the effect of exenatide on bile acids (BAs) and investigate the role of BAs in the glycemic control effect of exenatide.Thirty-eight newly diagnosed T2DM participants without glucose-lowering drugs intake were recruited. Plasma total bile acids in fasting state (FTBAs) and other parameters were tested at baseline. Then exenatide were applied to the T2DM participants for 12 weeks. FTBAs and glycemic parameters were measured again after exenatide treatment, and correlation analysis between changes of FTBAs and glycemic parameters were conducted to investigate the role of BAs in the glycemic control effect of exenatide.P = 0.954) compared with healthy subjects. After 12-week exenatide treatment for the T2DM patients, FTBAs were decreased from 3.84 ± 2.06 μmol/L to 3.06 ± 1.27 μmol/L (P < 0.01). The correlation analysis showed that changes of FTBAs was positively correlated with changes of FPG .The baseline FTBAs level of T2DM patients had no significance (3.84 ± 2.06 vs. 3.87 ± 2.89, Our results demonstrated a decreased FTBAs level after exenatide treatment for 12 weeks, without the interference of metformin and other glucose-lowering drugs. The reduction of FTBAs might not exert a positive role in the glycemic control effect of exenatide.NCT04303819. Registered in March 11, 2020 - Retrospectively registered.Trial registration number:  Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) demonstrated good glycemic efficacy in type 2 diabetes mellitus (T2DM) patients recent years  . ChangesP-value < 0.05 was considered statistically significant.Variables which were normally distributed, such as age, BMI, TC, LDL-C, HDL-C, glucose, FINS, C-peptide, HbA1c and FTBAs, were analyzed by Student’s t test and expressed as means ± SD. Variables which were not normally distributed, such as TG, HOMA-IR and HOMA-B, were analyzed by the Mann–Whitney U test or Wilcoxon test, and expressed as medians . Correlations between changes of FTBAs and glycemic parameters were measured using the Pearson correlation technique. All analyses were performed using SPSS 22.0 , and a two-side P = 0.954).Baseline characteristics were presented in Table P < 0.01)      Fig. a. We didTo avoid the interference of metformin and other glucose-lowering drugs on BAs, newly diagnosed T2DM participants without glucose-lowering drugs intake history were recruited. As well known, several drugs may affect the enterohepatic circulation of BAs, namely metformin, inhibitors of the apical sodium-dependent bile acid transporter (ASBT) , 19 and Our results showed that baseline FTBAs of newly diagnosed T2DM patients did not differ from healthy subjects. This was consistent with Andersen E’s study, in which they examined BAs’ kinetics in 15 normal glycemic controls and 22 diet-treated T2DM patients and found no difference . These iP<0.01. The pharmacokinetics of exenatide twice a day are dose proportional, with maximum serum concentrations after a single subcutaneous dose of 2.5 or 5 μg of 56 or 85 pg/mL, respectively, and the area under the concentration–time curve of 159 and 340 pg∙h/mL [After 12-week exenatide treatment for the overweight newly diagnosed T2DM patients group, FTBAs were decreased from 3.84 ± 2.06 to 3.06 ± 1.27 μmol/L, with a significance of  pg∙h/mL . It was  pg∙h/mL . The posThrough the correlation analysis, we got the result that the decrease of FTBAs was positively correlated with the improvement of FPG. Accumulating data suggested that BAs improved metabolism through activation of FXR and TGR5 –30. FXR The limitation of our study included: 1) this study was a pilot exploration on exenatide’s biliary effects. More detection about changes of BAs and their metabolites in different prandial phases could reveal more; 2) the sample size was small. More statistical significance might appear if the sample size was enlarged.In conclusion, our results demonstrated a decreased FTBAs level after exenatide treatment for 12 weeks, without interference of metformin and other glucose-lowering drugs. The reduction of FTBAs might not exert a positive role in the glycemic control effect of exenatide.


Cell lysis is the most important first step for molecular biology and diagnostic testing. Recently, microfluidic systems have attracted considerable attention due to advantages associated with automation, integration and miniaturization, especially in resource-limited settings. In this work, novel centrifugal microfluidic platforms with new configurations for chemical cell lysis are presented. The developed systems employ passive form of pneumatic and inertial forces for effective mixing of lysis reagents and cell samples as well as precise fluidic control. Characterizations of the developed Lab-on-a-Discs (LoaDs) have been conducted with dyed deionized (DI) waters and white blood cells (WBCs) to demonstrate the suitability of the proposed systems in terms of mixing, fluidic control and chemical cell lysis. By making comparison between the results of a well-established manual protocol for chemical cell lysis and the proposed chemical cell lysis discs, it has been proved that the developed systems are capable of realizing automated cell lysis with high throughput in terms of proper values of average DNA yield (ranging from 20.6 to 29.8 ng/µl) and purity (ranging from 1.873 to 1.907) as well as suitability of the released DNA for polymerase chain reaction (PCR). By considering the manual chemical lysis protocol as a reference, the efficiency of the LoaDs has been determined 95.5% and 91% for 10 min and 5 min lysis time, respectively. The developed LoaDs provide simple, efficient, and fully automated chemical cell lysis units, which can be easily integrated into operational on-disc elements to obtain sample-to answer settings systems. Recently, Lab-on-a-Disc (LoaD), as a relatively new type of microfluidic systems, has gained a considerable attention due to its unique features. In these systems a configuration, consisting of chambers and channels, is mounted on the surface of a disc, and the liquid is transferred through it by means of rotational forces generated by the centrifugation. The rotational forces can be harnessed for pumping and liquid handling operations. In addition, system-innate centrifugal force field decreases the number of required physical connections or pumps used in a typical microfluidic platform. Thus, the unique features result in greater complexity, sensitivity, enhanced robustness and usability of LoaD platforms compared to typical paper-based or lateral flow platforms. Collectively, LoaD platforms can be potential candidates in terms of point-of-care or point-of-use applications3.Microfluidic technologies have paved the way for miniaturization and automation of analytical protocols, enabling development of integrated point-of-care (POC) devices for early-stage diagnosis4. While numerous LoaDs for various applications have been proposed, the integration of simple and efficient on-disc cell lysis has remained a challenge in the design and fabrication of sample preparation unit of LoaD platforms6.Cell lysis is a process, in which cell membranes are disrupted with the aim of obtaining intercellular components such as DNA, RNA, proteins, and other bio-substances for further analysis. Therefore, cell lysis is considered as one of the primary steps of sample preparation in biomedical and biological applications7. However, these methods may have some downsides, including additional instrumentation and probable damage of macromoleculars9. In addition, the design and fabrication of these platforms can be challenging10. Regarding the thermal methods, the microfluidic platform should be equipped with relevant accessories for heat generation, which make the system more complex. Also heating up to high temperature can cause damage to facilities or exposed biological elements11. Another practical method is chemical cell lysis, in which cell membranes are disrupted by adding genuine reagents to the samples. This type of cell lysis is widely being used in protocols of the most commercial DNA extraction kits. Based on the protocols of these kits, proper mixing and subsequent incubation of lysis reagents and cell sample are performed for a certain time11. Thus, for implementing the chemical lysis method on centrifugal microfluidic platforms, design and fabrication of units for efficient mixing as well as precise fluidic control of samples are required. All these requirements can be readily achieved by directly designing the units on a LoaD, independent of complicated and external accessories. Thus, chemical cell lysis approach, in comparison with other types of lysis methods, has the highest potential to be integrated into LoaD platforms14.According to previous researches, mechanical, thermal, and chemical approaches have been employed to perform cell lysis in centrifugal microfluidic platforms. Commonly in mechanical cell lysis, friction and collision between cells and beads or magnets are used to break the cell membrane13. Also, other types of automated cell lysis LoaD platforms were proposed, utilizing an integration of thermal and chemical approaches. In these studies, laser modules were used to provide desire temperature in the lysis chamber and trigger valving system. Consequently, the systems rely heavily upon external accessories, which may imposes an additional cost/complexity on performing cell lysis protocol 15.Up to now, several innovative ideas have been applied to realize on-disc chemical cell lysis. Recently, buoyancy driven bubble was employed for mixing a cell sample and lysis reagents to perform chemical lysis on a LoaD platform. Although high cell lysis efficiency has been reported for the system, and the it has a novel mechanism and configuration, complexity of design and fabrication of the system may hamper further developments and integration of the proposed lysis unit into other on-disc units for achieving a sample-to-answer system12. In contrast to straightforward mixing process in macroscale, the typically low flow rates and small spatial dimensions in microfluidic environments make mixing process a challenging task. Particularly, by nature, centrifugation promotes the segregation of fluids and particles with different densities, resulting in more difficult mixing operation on LoaD. Numerous mixing concepts have been applied on LoaD platform recently, including applying magnetic fields to stir liquids by magnetic beads’ movement17, convective flow induction by shake mode method16, liquid mixing using the moment of inertia of the liquids18, supply of external pneumatic pressure to agitate samples19, implementation of a reciprocating flow between mixing chamber and a compression chamber20, making the liquid streams of interest flow through specifically designed microchannels such as a serpentine channel21, employing highly elastic properties of embedded layers of latex to mix different liquids in a single chamber22, and employing buoyancy driven bubble mixing similar to macroscopic bubble columns23. As mentioned before, reliable and robust valving system is necessary for handling the reagents and processed samples on LoaDs. The valves which have been developed on centrifugal microfluidics platforms can be classified into passive and active systems, according to their reliance on external accessories. Passive valves are usually preferable due to the simplicity, being cost-effective and employing only the intrinsic centrifugation to release or retain a certain amount of liquid24. Siphon valves and non-siphon valves are the major subcategories of passive valves. Sharp pinning edge25 or hydrophobic coating26 are usually implemented in non-siphon valves to raise the contact angle to more than 90°. The most significant issue in designing siphon valves is engineering siphon priming. Siphon priming has been realized in varied approaches so far including hydrophilic surface treatment of siphon microchannels27, inducing Euler forces by applying high rotational acceleration28, driving the sample liquid into the siphon by creating a locally vacuum29, and using pneumatic action through deceleration from high to low rotational frequencies30. Furthermore, it seems that siphon valves triggered by pneumatic and inertial forces can be more reliable in addition to being independent of pretreatment32.In order to carry out on-disc chemical cell lysis, effective mixing and valving systems play the most significant roles. In the proper on-disc mixer, target cells and lysis reagents should be agitated effectively, and also the reagents are taken enough time to completely disrupt the cells’ membrane and other barriers to fully obtain intercellular substances. Subsequently, suitable valving systems should precisely control fluid flow and collect the lysate in a separate chamber for further analysisThe aim of this work is to design and fabricate efficient and simple centrifugal microfluidic platforms for carrying out chemical cell lysis in a robust and automated manner. Here, for development of the chemical cell lysis discs, pneumatic and inertial forces, as two pragmatic approaches have been employed. Due to easy implementation and high efficiency of pneumatic and inertial forces, they have been applied to design passive form of mixers and valves with new configurations, which allow for effective mixing of cell sample and lysing reagents, as well as precise control of the lysate. Prior to conducting chemical cell lysis, the performance of the developed mixing and valving systems was measured. Subsequently, on-disc chemical cell lysis was conducted using a sample containing white blood cells (WBCs) and commercial lysis buffers. Finally, the lysis efficiency were measured through several relevant tests including, quantitative measurement of yields and purity of the eluted DNA samples, DNA gel electrophoresis and Polymerase chain reaction (PCR).33. Furthermore, to increase hydrophilicity of the microchannels and improve optical contrast of acquired images, the microchannels can be backed by an additional PSA layer 32. Figures 3.In the present work, the centrifugal microfluidic discs are comprised of three layers of Poly methyl methacrylate  with 1.5 mm thickness, bonded together using three layers of Pressure Sensitive Adhesive  with 60 μm thickness. SOLIDWORKS software was used to design all pattern of elements, including microchannels, chambers, loading holes and air vents. As shown in Figs. The principal concept behind the design of disc 1 is to implement pneumatic energy for obtaining effective mixing and precise fluidic control of the samples, in a manner that the system is simple and independent of any external accessories. As shown in Fig. Similar to disc 1, the main function of disc 2 is to conduct efficient mixing and precise fluidic control, while the system is simple and independent of any external accessories. However, the general concept behind the design of this centrifugal microfluidic system is implementation of inertial force. As shown in Fig. 20.In this study, combinations of mixing and valving units have been developed in form of new configurations by employing pneumatic (disc1) and inertial (disc 2) forces. Image processing have been done to assess the mixing efficiency and valving performance. In order to highlight mixing process, yellow and blue colors were used to make two different dyed deionized (DI) water samples. The image acquisition of the liquid during the process was processed using ImageJ software for obtaining the color intensity histogram of the mixed liquid in various steps of the mixing process. In addition, the standard deviation (δ), the square root of the variance, was used for evaluating the dispersion of the data values. Standard deviation represents how much the data points differ from the mean value. Therefore, the less value of this parameter indicates the higher mixing efficiency34. Next, the separated pellets were dissolved in 100 μl of phosphate-buffered saline (WBCs-PBS) in order to be prepared for being loaded in the inlet chamber 2. In the meantime, 200 μl and 10 μl of the lysis reagent  and proteinase K, respectively, were loaded in inlet chamber 1. After lysate recovery from the collection chamber with a pipette, it was manually processed, according to the standard protocol of the used DNA extraction kit to obtain purified DNA. To compare the performance of the developed lysis discs with the manual protocol, as the gold-standard, the cell lysis experiments were carried out concurrently using the both proposed discs and the conventional method15. The manual lysis procedure includes vortexing the WBCs-PBS and the lysis reagents for 20 s at the highest setting followed by incubation of the mixture for 600 s.WBCs can be potential candidates as the target cells for our lysis tests because of two reasons. First, they can be readily obtained through simple procedures from whole blood. Second, WBCs contain DNA in their nucleus, which its release can be used as a benchmark for measuring the cell lysis efficiency. To prepare the sample for the cell lysis tests, WBC pellets were isolated from 200 μl of fresh human whole blood based on typical protocolsTo compare the performance of our chemical cell lysis discs with the conventional manual approach, the released DNAs in the cell lysates, obtained from both manual and automated methods, were purified, and using spectrophotometry , the absorbance at 260 and 280 nm were measured to determine purity and yield (ng/µl) of the DNA. Moreover, the efficiency of the developed chemical cell lysis discs were calculated by considering the manual protocol as reference yield. The used formula has been presented below.Furthermore, to check the integrity of the extracted DNA and show the quality of the lysis process in the proposed LoaD, gel electrophoresis and PCR  were run for the eluted DNA in the both methods. Briefly, five µl of the eluted DNA samples, obtained from manual and automated lysis methods (disc1 and disc2), were electrophoresed on 1% (w/v) Agarose gel with 1X TAE running buffer . Then, imaging under UV light using Gel-Doc system  was carried out on DNA staining by Ethidium bromide. Moreover, the PCR amplification of human mitochondrial Cytochrome C oxidase subunit I (COI) gene was carried out by thermal cycler . PCR was performed as below; reaction mixture contained 2 µl of template DNA (50 ng), 1 µl of forward primer (LCO1490: 5′-GGTCAACAAATCATAAAGATATTGG-3′) and 1 µl of reverse primer (HCO2198: 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (10 PM), 12.5 µl of 2× master mix (Thermo fisher scientific) and 9.5 µl of deionized water. Fragment amplification was done by initial denaturation at 94 °C for 3 min followed by 5 cycles of 94 °C for 45 s, 45 °C for 90 s, and 72 °C for 60 s, 25 cycles of 94 °C for 45 s, 51 °C for 90 s, and 72 °C for 60 s, final extension at 72 °C for 5 min. Five µl of PCR products was electrophoresed on 1% (w/v) Agarose gel with 1X TAE running buffer , PCR product staining by Ethidium bromide and imaging under UV light with Gel-Doc system (Bio-Rad-USA).To measure the performance of the mixing and valving systems of the discs, the developed discs were run for dyed deionized (DI) water. However, owing to the fact that in our designs the only driving force for handling liquids are rotational forces, one of the main challenges is to obtain an optimized spin protocol for the discs. Regarding disc 1, in which pneumatic force is implemented in a passive form, four steps have been determined to realize its complete function Fig. . In step1 and H2 , the standard deviation values of the mixed liquids after each rotation cycle has been plotted in Fig. In terms of disc 2, in which inertial force is implemented in a passive form, the loaded samples in the inlet chambers are released to mixing chambers by starting disc spinning as shown in Fig. The effect of moment of inertia in mixing process is demonstrated in Fig. As shown in Figs. Figure S5 demonstrates the function of the developed inertial siphon valves, which can transfer the processed sample through three cycles, which can vary depending on the value of deceleration.Collectively, the evaluations prove that disc 2 is superior to disc 1 in terms of performing mixing. However, due to the different mechanism of pneumatic and inertial siphon valves, the latter probably cannot transfer the whole processed sample and it is more likely that an insignificant amount of liquid remains in the mixing chamber of disc 2. In addition, generally inertial siphon valve requires higher levels of deceleration (more than threefold) for its function compared to pneumatic siphon valve. In our work the value of deceleration needed for inertial siphon valve is more than three times as high as the value required for the pneumatic siphon valve. But, it is notable that this ratio can vary depending on the physical characteristics of the valves.13. However, some flaws in configuration of the disc's elements, including chambers and microchannels, brought about relatively ineffective cell lysis. The relative lower efficiency of on-disc chemical cell lysis using pneumatic and rotational forces may stem from two main origins. The former is most likely due to the low intensity of mixing in some configurations. For example, the Euler force is not strong enough to bring about a sloshing of the liquids and the internal fluid flows are not adequate to cause rigorous mixing of cell sample and lysis reagent. The latter would be sedimentation of cells, which should be lysed, in both chambers and the connecting microfluidic channel due to high rotational frequencies. Therefore, in the present work, some modifications have been made to conventional disc architectures in order to create more effective agitation and eliminate sedimentation effect. Regarding employing rigorous mixing, the image processing has demonstrated that desirable mixing can be obtained through effective implementation of inertial and pneumatic forces in passive forms. Moreover, to reduce the possible sedimentation effect, connecting channels between mixing chambers and also between collection and mixing chambers should connect the elements from the points located in the relative higher radial distance , more time is needed for effective interactions between the WBCs and lysis reagents. Therefore, the operation time of the discs should be extended for lysate preparation. Approximately 500 and 100 s as extended discs’ operation time were considered for the mixing process and collectively transferring and incubation of lysate in collection chamber respectively. Thus, WBCs-PBS and the lysis reagents were mixed for 500 s, followed by 100 s for transferring and incubation of lysate. If any cells remain intact in mixing process, they are lysed during incubation at low frequency (300 rpm) in the collection chamber. Figures According to the used DNA extraction protocol, the required volume of cell sample (100 µl of PBS-WBC) and lysis reagents (190 µl of lysis buffer and 10 µl of proteinase K) match the volume capacity of type B of disc 1 and also disc 2. Therefore, the chemical cell lysis was performed by means of these two types of discs.35. It is obvious that the quantities of purity and yield of the extracted DNA are indicative of the performance of the suggested chemical cell lysis discs. Indeed, proper values of the DNA purity, ranging from 1.8 to 2, and cell lysis efficiency  can demonstrate capability of the proposed systems to effectively perform chemical cell lysis35. In addition, to show the importance of the required time for interaction between lysis reagents and WBCs, the 10 min lysis time was halved to 5 min, and same quantitative measurements were performed for this case. To confirm reproducibility, 7 and 5 replicate experiments were conducted for 10 and 5 min lysis time, respectively , it can be clearly seen that there are insignificant differences between the performances of the developed discs for automated chemical cell lysis. This observation shows that the both centrifugal microfluidic platforms (disc 1 and disc 2) are able to provide an effective interaction between WBCs and lysis reagents for cell disruption for the duration of 10 or 5 min.To evaluate the integrity of the extracted DNA in the lysate, gel electrophoresis was performed on the eluted DNA samples, obtained from manual and automated lysis methods (disc 1 and disc 2). As can be seen in Fig. To assess the clinical applicability of the developed chemical cell lysis discs, a PCR amplification and visualization was conducted for the purified DNA, obtained from on-disc and manual lysis protocols Fig. . Due to Generally, in addition to cell lysis automation on LoaD platforms, the developed systems showed comparable results with the conventional commercially chemical cell lysis protocols. It is worth noting that the modifications do not add complexity to the design and fabrication of the discs while enhance the loss in DNA yields.Here, in an application oriented way, two different centrifugal microfluidic platforms have been developed for performing automated chemical cell lysis. Pneumatic and inertial forces, in passive forms, have been employed for carrying out mixing cell samples and lysis buffers as well as multidirectional pumping of liquids in form of siphon valves. In this work, it has been attempted to eliminate shortcomings in configurations of previous cell lysis discs. In other words, novel arrangements of elements (chambers and microchannels) have been suggested to deal with both low level of liquid agitation and sedimentation of cells, which have been major challenges for on-disc chemical cell lysis. In addition, the proposed arrangement can be a viable alternative to current complicated LoaDs platforms used for mixing and precise fluidic control. The high efficiency of mixing and valving systems was proved by means of image processing. Indeed, the both types of the developed discs after a few mixing cycles (ranging from 2 to up to 6 cycles) can make the samples fully mixed. Also, approximately whole processed sample can be transported using developed multidirectional pumping systems, which are called inertial and siphon valves.Moreover, quantitative measurements of the cell lysate, including measuring of yields and purity of the eluted DNA samples, DNA gel electrophoresis and PCR, demonstrate the suitability of the developed chemical cell lysis discs. Because, the results obtained from the developed LoaD are totally comparable with the manual lysis procedure. The purified DNA obtained from the both types of developed chemical cell lysis discs possessed high purity (ranging from 1.873 to 1.907). Nuclear lysis of WBCs, separated from human whole blood, in 10 min using disc 1 and disc 2 lead to average yield of 29.8 ng/µl and 29.9 ng/µl purified DNA respectively, and these values decrease to 21.1 ng/µl and 20.6 ng/µl by reducing lysis time to 5 min. Theses yield values can be sufficient for clinical uses. Additionally, by considering the manual protocol as a reference yield, the average efficiency of the discs are 95.5% and 91% for 10 min and 5 min respectively, which show great performance of the developed LoaDs. Collectively, although disc 2 shows superiority in terms mixing at initial time compared to disc 1, they have same lysis performance due to the required extended time for cell lysis.Therefore, all of the required measures, which should be taken into account to ensure that the developed discs are robust platforms for chemical cell lysis, have been employed in this work. Furthermore, although our systems may not be considered as real point-of-care platforms, the proposed configurations have a great potential to be easily integrated into relevant on-disc operational elements for obtaining complete sample-to-answer systems. This is because of ease of design and fabrication of the developed centrifugal platforms as well as high efficiency of the automated chemical cell lysis discs. For example, due to the fact that reagents mixing and sequential fluidic control are highly required in automated DNA extraction and purification, the developed arrangement of the inertial and pneumatic mixing and also valving systems can be employed with some simple design changes. In terms of our future work, by using obtained technical knowledge about mechanism of pneumatic and inertial systems on centrifugal microfluidic platforms, we will develop a LoaD for DNA extraction and purification from whole blood.Supplementary information


The gut microbiota is composed of a large number of different bacteria, that play a key role in the construction of a metabolic signaling network. Deepening the link between metabolic pathways of the gut microbiota and human health, it seems increasingly essential to evolutionarily define the principal technologies applied in the field and their future trends. We use a topic analysis tool, Latent Dirichlet Allocation, to extract themes as a probabilistic distribution of latent topics from literature dataset. We also use the Prophet neural network prediction tool to predict future trend of this area of study. A total of 1,271 abstracts (from 2006 to 2020) were retrieved from MEDLINE with the query on “gut microbiota” and “metabolic pathway.” Our study found 10 topics covering current research types: dietary health, inflammation and liver cancer, fatty and diabetes, microbiota community, hepatic metabolism, metabolomics-based approach and SFCAs, allergic and immune disorders, gut dysbiosis, obesity, brain reaction, and cardiovascular disease. The analysis indicates that, with the rapid development of gut microbiota research, the metabolomics-based approach and SCFAs (topic 6) and dietary health (topic 1) have more studies being reported in the last 15 years. We also conclude from the data that, three other topics could be heavily focused in the future: metabolomics-based approach and SCFAs (topic 6), obesity (topic 8) and brain reaction and cardiovascular disease (topic 10), to unravel microbial affecting human health. The microbiota—the collection of microorganisms that live within and on all mammals—provides crucial signals for the development and function of the immune system  and bibliometric analysis, our study provides specific methods for scientific researchers in this field, and furnishes more followers with intuitive understanding and disciplinary analysis in the study of intestinal diseases and metabolic pathways of intestinal microbiology. The method adopted in this paper has several advantages. The thematic model used in this paper describes and predicts the evolution of time segments, which is beneficial for scholars to track the development of research and to look for opportunities for expansion. The results beyond theoretical and framework recognition can facilitate scholars and readers to understand the evolutionary trends of research topics in the field of gut microbiota.This study evaluates the “Gut Microbiota” and “Metabolic Pathways” to explore the evolution of the research topic. The research framework is mainly embodied in two stages: the first phase is data preprocessing and topic extraction. The purpose of this stage is to represent. The intensity of each topic has time-series features. Thus, the second phase concentrates on the prediction analysis and display of the topic trend. The aim of this stage is to predict topic development and discover more possibilities for hot research content. The first stage serves as the foundation and premise of the research. The second one plays a part in the purpose and result of the study.This article explored and recovered titles, abstracts, and keywords from publications regarding “Gut Microbiota” and “Metabolic Pathways” using Web of Knowledge-MEDLINE database. The MEDLINE database is produced by the Us National Library of Medicine and covers a wide range of biomedical disciplines.We used the following keywords to extract literatures from the MEDLINE database: gut microbiota [TS] AND metabolic pathway [TS] AND “2006/01/01 [DATE]”: “2020/08/01 [DATE].” Information retrieved includes title, author, abstract, keywords, references, and journal sources. In total, 3,588 literature were obtained. We then filtered the literatures to only keep journal articles for downstream analysis, which leaves 1,651 non-duplicated literatures. Topic distributions of studies between 2006 and 2020 , suggestLatent Dirichlet allocation (LDA) was proposed by Blei in 2003, on which a layer of Dirichlet conjugating prior distribution was added based on the PLSI model Hofmann, . In addilog_perplexity and model.coherence are used to calculate the results. The lower the perplexity, the better the effect of the topic number, and the better the training subject distribution model can fit the training set data. When the number of topics increases, the perplexity of titles, and abstracts decreases gradually. The coherence of the topic tends to be stable gradually after reaching the optimal level. When K is 10, the title and abstract topic reaches it's optimum and tends to be stable  and dietary health (topic 1) have been more focused during the last 15 years. Based on our analysis, it is clear that metabolomics-based approach and SCFAs (topic 6), obesity (topic 8), brain reaction and cardiovascular disease (topic 10) will be paid more attention in the near future, in adherence with the quantitative analysis of all small molecular metabolites in cells of a biological system at a given time and under certain conditions. In the study of health and disease in gut microbes and the metabolic pathways of their metabolites, the topic of microbial communities remains rare evolved in the near future. In our opinion, this area of investigation requires a much greater commitment than that described by our study, which identified 46 studies in this area in topic 4.Our analysis in this perspective summarizes the existing evolution and predicts the future trends of researches in Metabolic Pathways of the Gut Microbiota. In the first place, the topic model used in this paper describes and predicts the evolution of time segments, which does good for scholars to track the research development and find opportunities to expand it. Second, the results go beyond the recognition of theory and framework and help scholars and readers to understand the hot spots and future topic combination in the study of gut microbiota metabolic pathways.It is noted that we also limited our analysis in the following aspects. Firstly, although we described the hot research issues and predicted the frontier research issues through natural language processing technology and bibliometric methods, there is still a dearth of standard in the natural language. Besides, the rules of language vary in different areas of medical research. So far, some basic rules can be found, but one root can express many levels of meaning, or the same meaning can be expounded in many ways. This paper tries harder to comprehend natural language based on rules, but still has a few deviations. Secondly, the conclusion of this paper does not involve to find out the research deficiencies, technical challenges, and other problems in the field of metabolic pathways of gut microbiota and its metabolites on human health and human disease. As a result, these deficiencies need to be further solved optimizing algorithms and developing professional language dictionaries or expert systems in the future for the sake of providing a more comprehensive analysis for the research problems.The original contributions presented in the study are included in the article/LN wrote the manuscript. HH contributed to the modification of the manuscript. PL also contributed to the modification of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.