Drosophila using a Go/No-Go task that measures inhibition capacity. Here, we report that inhibition capacity declines with aging in wild-type flies, which is mitigated by lessening acetylcholine breakdown and augmented by reducing acetylcholine biosynthesis. We identified the mushroom body (MB) γ neurons as a chief neural site for acetylcholine’s contribution to the aging-associated inhibitory control deficit. In addition, we found that the MB output neurons MBON-γ2α’1 having dendrites at the MB γ2 and α’1 lobes and axons projecting to the superior medial protocerebrum and the crepine is critical for sustained movement suppression per se. This study reveals, for the first time, the central role of acetylcholine in the aging-associated loss of inhibitory control and provides a framework for further mechanistic studies.Inhibitory control is a key executive function that limits unnecessary thoughts and actions, enabling an organism to appropriately execute goal-driven behaviors. The efficiency of this inhibitory capacity declines with normal aging or in neurodegenerative dementias similar to memory or other cognitive functions. Acetylcholine signaling is crucial for executive function and also diminishes with aging. Acetylcholine’s contribution to the aging- or dementia-related decline in inhibitory control, however, remains elusive. We addressed this in  Consistently, the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine administration in rats and the nAChR beta2 knockout in mice impair attention performance in the 5-choice serial reaction time task (5-CSRTT)3, substantiating a crucial role of acetylcholine in attention. The role of acetylcholine in inhibitory control, however, seems rather complex. In healthy non-smokers, acute nicotine administration improves behavioral inhibition in the stop signal task4. Nicotine treatment in rats, on the other hand, impairs inhibitory action and choice when tested in the 5- or 3-CSRTT, go/no-go task and systemic delayed reward task6. The inhibitory effect of nicotine is blocked by mecamylamine pretreatment6 or the infusion of the alpha4beta2 nAChR antagonist dihydro-beta-erythroidine (DHbetaE) in the infralimbic cortex5. Moreover, the DHbetaE infusion5 or systemic mecamylamine administration2 without nicotine administration is sufficient to suppress impulsive action, suggesting pro-impulsive function of endogenous acetylcholine in rats, which is not in line with the findings in human subjects. It seems of great value to study an additional model organism for deeper understanding of the mechanism by which acetylcholine regulates inhibitory response control.Acetylcholine is important for executive function in human subjects and rodents. In healthy non-smokers, for example, acute nicotine administration enhances attention when tested in the spatial attentional resource allocation and rapid visual information processing tasks11. Executive dysfunctions, in particular impaired inhibitory control, represent prominent symptoms of early-stage Alzheimer’s disease, frontotemporal dementia and other dementias16; however, there is no information on the role of acetylcholine in inhibitory control dysfunction associated with normal aging or dementias. To address this knowledge gap, we developed a simple behavioral paradigm to study a fundamental form of inhibitory response control in Drosophila melanogaster. Here, we show that acetylcholine significantly contributes to the aging-dependent deficit in inhibitory control and its major neural site is the mushroom body neurons.Acetylcholine neurotransmission declines with normal aging and the decline is highly augmented in Alzheimer’s disease and related dementias such as frontotemporal dementia and Lewy body dementiaCanton-S (CS). The following fly strains were obtained from the Bloomington Drosophila Stock Center : Acec00215 (BDSC #10026), ChATMI08244 (BDSC #55439), ChATMI04508 (BDSC #37817), OK107-GAL4 (BDSC #854), MB010B-GAL4 (BDSC #68293), 201y-GAL4 (BDSC #4440), MB009B-GAL4 (BDSC #68292), c739-GAL4 (BDSC #7362), MB008B-GAL4 (BDSC #68291), c305a-GAL4 (BDSC #30829), MB005B-GAL4 (BDSC #68306), MB077B-GAL4 (BDSC #68283), MB051B-GAL4 (BDSC #68275), MB018B-GAL4 (BDSC #68296), MB082C-GAL4 (BDSC #68286), MB543B-GAL4 (BDSC #68335), MB027B-GAL4 (BDSC #68301), MB549C-GAL4 (BDSC #68373), MB542B-GAL4 (BDSC #68372), GH146-GAL4 (BDSC #30026) and UAS-ChATRNAi (BDSC #25856); pBDP-GAL4 from David Anderson ; NP225-GAL4 from Andreas Thum ; MB247-GAL4 from Scott Waddell ; and NP1131-GAL4 from Josh Dubnau .The wild-type strain used in this study is 17. Prior to behavioral tests, flies were sorted into a group of 13 per food vial (representing n = 1) and were then left to rest for 2 day in the light-, temperature-, and humidity- controlled incubator before behavioral or immunohistochemical analyses. Both males and females were examined separately but there was no sex difference thus combined data are presented.Flies were raised on a standard cornmeal/sucrose/yeast/agar medium at 25° C with 50% relative humidity under a 12 h light/12 h dark cycle. Flies were collected under carbon dioxide within two days after eclosion and were housed in mixed sex groups. For aging, all fly strains were transferred to fresh food every 2–3 day until the ages of 4 day, 2 week and 4 week. These ages were selected to examine flies at young (4 day), mid (2 week) and early old (4 week) ages. The 4-wk old allows capturing the brain and behavioral changes associated with early-stage dementia and potentially distinguishing them from those occurring during normal agingFlies were gently transferred into a rectangular plexiglass chamber (60 mm L × 60 mm W × 15 mm H) connected to filtered air and acclimated to the chamber for 10 min. The 10 L/min airflow was delivered to the chamber for 10 min. The chamber was video recorded to monitor fly movements before and after airflow. Videos were analyzed manually to score the number of flying events or using the Viewer3 tracking software  that allows tracking and measuring the average speed of individual flies in mm/s per fly. Raw data were transported to Excel  and the number of the movements exceeding 60 mm/s (flying event) that we defined as loss of inhibition events (LIE) was scored per fly per min. To quantitively compare different conditions , we analyzed LIE numbers in 1 min bins for the entire 10 min of the No-Go phase and compared the 1 min bin having the highest LIE counts. All behavioral experiments were performed blind to the experimenter. The control and experimental groups were tested in the same experimental session in a randomized manner. Multiple independent sets of flies obtained from independent crosses were used for behavioral experiments.19 with several modifications. Fly brains were dissected in ice-cold 1X phosphate buffered saline (1X PBS), fixed in 4% paraformaldehyde in 1X PBS for 3 h at 4 °C. The fixed brains were washed with 1X PBS once for 10 min, washed twice with 1X PBHT  for 10 min each, solubilized with 1% Triton X-100 in 1X PBHT for 1 h at room temperature, blocked with 5% normal goat serum (NGS) in 1X PBHT for 16–18 h at 4 °C and incubated with the 1X PBHT containing 1:1000 anti-ChAT antibody  and 5% NGS for 48 h at 4 °C. After two 10 min washes at room temperature and a 16–24 h wash at 4 °C with 1X PBHT, the brains were incubated with the Alexa Flour 488 conjugated goat anti-mouse IgG  for 48 h at 4 °C followed by three 20 min washes with 1X PBHT and three 10 min washes with 0.12 M Tris–HCl, pH 7.2. The brains were then mounted in the Vectashield mounting medium  for imaging with the 40X oil immersion objective in the LSM700 confocal microscope . Pixel resolution was set at 2048 × 2048. Optical sections were made at 1 μm thickness and 50 sections were stacked for the representative images in Fig. Total/AreaTotal] per brain. The fold change of the fluorescent intensities in the ChAT/+ mutant brains from those in the control Canton-S brains was used for data presentation.Immunostaining was performed as previously describedTo measure acetylcholine, we utilized a commercially available Choline/Acetylcholine assay kit . Briefly, 50 frozen fly heads per condition (genotype and age) were homogenized 50 μl of the Choline Assay Buffer (1 fly head per 1 μl) on ice with the KONTES Micro Tissue Grinder  for 30 to 45 s. The homogenates were transferred to fresh tubes, centrifuged at 14,000 rpm for 10 min at 4 °C, and subjected to the Choline/Acetylcholine assay per the manufacture’s instruction. Multiple independent sets of fly heads  obtained from independent crosses were used for acetylcholine quantification.t test for two groups or by ANOVA followed by post hoc Tukey’s multiple-comparison for three or more groups or Dunnett’s test to compare experimental groups with a control group. Significant difference among the groups under comparison was determined using an α level of 0.05 in all analyses. All raw data files are available on request.All statistical analyses were performed using the Minitab software  or JMP . Raw data were analyzed using the Anderson–Darling goodness-of-fit test for distribution and are reported as mean + SEM. Normally distributed data were analyzed by either two-tailed Student’s 21. A GNG task measures action restraint, requiring subjects to withhold a motor response to a “no-go” signal 22. To assess whether inhibitory control declines with aging in Drosophila similar to humans, we developed a Drosophila version of a GNG test, in which a group of 13 flies placed in a chamber were video-tracked to quantify their movements. Without salient stimuli, the wild-type Canton-S (CS) flies freely move around. Upon introduction of strong airflow, flies halt movements presumably to keep their behavior under control  inappropriate in a given context (strong “wind”), and we postulate that it represents a fundamental form of inhibitory response control. We tested the female and male CS flies at three different ages 4 day (young), 2 week (mid), and 4 week (early old). Regardless sex and age, the CS flies exhibited robust movement suppression under strong airflow with infrequent instances of flying behavior, which is an impulsive act resulting from loss of movement inhibition  at shorter latency for LIE , indicating that aging diminishes inhibitory capacity. We did not observe any difference in LIE between females and males at all ages thus the data obtained from both sexes are combined and presented in Fig. Inhibitory control confers the ability to suppress inappropriate actions or thoughts and is a key executive function supporting flexible and goal-directed behaviors. This capacity declines with aging in human subjects, for example when tested in the go/no-go (GNG)8 but this has not been demonstrated in flies. To test whether diminished acetylcholine neurotransmission is responsible for the aging-associated increase in LIE, we examined the Acec00215/+ flies with the heterozygous mutation in acetylcholinesterase  that breaks down acetylcholine and is a target for the first line medication to treat Alzheimer’s disease. The Acec00215 allele is a severe hypomorph or null since it has a piggyBac insertion in the 5’ non-coding region of the gene and homozygous lethal25. We found that the heterozygous Ace mutation rescued fully at 2 week and partly at 4 week the movement suppression deficit  as well as the LIE latency at 4 week . Consistently, the Acec00215/+ flies showed higher acetylcholine levels compared to CS at all ages tested and both genotypes have declining acetylcholine levels with aging . This finding supports that diminished acetylcholine neurotransmission accounts for inhibitory control decline in normal aging, and the GNG test is well suited to study aging-related changes in inhibitory control in flies.It is known in human subjects and rodents that acetylcholine neurotransmission modestly declines with agingDrosophila has a single gene for ChAT as in humans25. We examined two mutant alleles ChATMI04508 and ChATMI08244 containing the gene trap MiMIC vector26 . When subjected to the GNG test, both heterozygous mutants showed dysfunctional movement suppression in an aging-dependent manner . Specifically, the heterozygous ChAT mutants at all ages showed robust movement suppression when strong air was introduced . At the age of 4 day, there was no difference in LIEs of the ChATMI04508/+ and ChATMI08244/+ mutants compared to that of CS ; however, there was significant increases in the LIEs of the ChATMI04508/+ and ChATMI08244/+ flies at the ages of 2 week  and 4 week  compared to those of CS. Also, the LIE onsets of ChATMI04508/+ and ChATMI08244/+ flies were significantly earlier than CS at 2 week  and 4 week  but not at 4 day . Together, these data indicate that the flies with reduced ChAT expression have augmented sensitivity to aging in movement suppression deficit, indicating the critical role of acetylcholine in the aging-dependent loss of inhibitory control.To directly address the role of acetylcholine in inhibitory control, we used the flies defective in ChAT. ChAT EC 2.3.1.6) is the biosynthetic enzyme for acetylcholine, which aids in the transfer of an acetyl group in Acetyl-CoA to choline, and .3.1.6 isChAT/+ mutant flies carrying the heterozygous Ace mutation27 (i.e. ChATMI04508/Acec00215) along with the flies with the heterozygous mutation only in ChAT or Ace and CS as controls. When subjected to the GNG test, there was no differences in LIEs of all genotypes at the age of 4 day as expected . Notably, the double heterozygous ChATMI04508/Acec00215 mutants exhibited the LIEs comparable to those of CS and Acec00215/+ but significantly different from that of ChATMI04508/+ at the ages of 2 week  and 4 week . Likewise, the double heterozygous ChATMI04508/Acec00215 mutants exhibited the latency to the first LIE comparable to those of CS and Acec00215/+ but significantly delayed than that of ChATMI04508/+ at the age of 2 week . At the age of 4 week, the ChATMI04508/Acec00215 mutants showed the latency comparable to that of Acec00215/+ but significantly delayed than those of ChATMI04508/+ as well as CS . Together, the heterozygous Acec00215 mutation fully suppressed the ChATMI04508/+ mutant’s LIE and latency phenotypes. In line with these results, the acetylcholine levels in the double heterozygous ChATMI04508/Acec00215 were indistinguishable from those of CS at all ages and they were significantly higher than those of ChATMI04508/+ . This indicates that the acetylcholine deficiency is responsible for the aging-associated deficit in inhibitory control.We reasoned that dampened cholinergic transmission causes the dysfunctional inhibitory control of the ChAT mutant. Should it be the case, we should be able to rescue the phenotype by reducing the amount of the acetylcholine degradation enzyme Ace. We tested this notion by examining the 28 and their axons project to most brain areas -mediated ChAT knockdown (KD). When ChAT was knocked down in all MB neurons (via the OK107- or MB010B-GAL4 driver), two MB substructures γ and α/β (via MB247- or 201y-GAL4) or γ (via NP1131- or MB009B-GAL4) but not in α/β (via c739- or MB008B-GAL4) or α’/β’ (via c305a- or MB005B-GAL4) neurons led to the aging-dependent increase in LIEs . These data indicate that the MB γ cholinergic neurotransmission is important for the aging-related LIEs.Acetylcholine neurons consist of approximately 58% of the central brain31. ChAT KD in the MBON-γ2α’1 (via MB077B- or MB051B-GAL4) but not in other cholinergic MBONs, namely MBON-α’2 (via MB018B-GAL4), MBON-α3 (via MB082C-GAL4), MBON-α’1 (via MB543B-GAL4), MBON-α’3ap and -α’3 m (via MB027B-GAL4), MBON-α2sc (via MB549C-GAL4), and MBON-α2p3p (via MB542B-GAL4), resulted in significant LIEs in all ages  tested . Thus, the MBON-γ2α’1 cholinergic transmission is critical for inhibitory control. The MBN receive input from the projection neurons (PN) and the PN are cholinergic20. We found that ChAT KD in the PN (via NP225- or GH146-GAL4) had no effect on movement inhibition at all ages under test . This suggests that the PN cholinergic transmission is dispensable for inhibitory control.Eight types of the mushroom body output neurons (MBON) are known to be cholinergic and their dendrites are mapped to the γ2α’1, α2sc, α2p3p, α3, α’1, α’2, α’3ap and α’3 m lobes36. Mechanistic studies of inhibition have been done on neurodevelopmental disorders and addiction whereas limited studies have been reported on neurodegenerative dementias. In the report, we show that inhibition capacity declines with aging in wild-type flies, which is halted by the mutation in the acetylcholine breakdown enzyme Ace and augmented by the decreased acetylcholine biosynthesis in the γ MB neurons.Inhibition or inhibitory control is a fundamental brain function and is critical for fitness and survival. Poor inhibition is associated with many brain disorders including attention deficit hyperactivity disorder, autism spectrum disorder, addiction and neurodegenerative disorders such as Alzheimer’s disease and frontotemporal dementiaDrosophila and they typically entail startle responses to olfactory, visual or mechanosensory stimuli in which increases in locomotor activity or movements represent the expected behavioral outcome40. In the startle-induced negative-geotaxis/climbing assay, for example, flies exhibit rapid climbing movements when tapped down. Older flies in this assay exhibit slower or less negative-geotaxis movements than young flies42. The GNG test used in our study measures movement suppression (no or decreased movements) in the presence of strong airflow or predator sound (unpublished data), and we show that aging causes poor suppression represented by hyperkinetic or flying behavior. Reduced or lack of motor activity of aged flies in the startle-induced negative-geotaxis assay could be confounded by general slowness of behavior related to weakened muscle or other physiological changes. The GNG test is beneficial to uncover aging-related changes in neural control of movements, in particular those leading to impulsive acts.Motor behaviors have been studied in 43 but all mutant mice defective in the individual subunits of nAChR and muscarinic acetylcholine receptor (mAChR) except for α3 are viable to adulthood44. The mice lacking the α7 nAChR subunit or the mice with the conditional knockout of M4 mAChR in the D1 receptor expressing neurons46 show significant increases in impulsive acts, supporting our finding on the acetylcholine’s role in inhibitory control. The genetic knockout studies are very insightful however no human diseases identified to date involves total absence of acetylcholine production nor signaling. It is conceivable that acetylcholine signaling via α7 nAChR and M4 mAChR may decline with aging, which in turn contributes to aging-associated inhibitory control dysfunction. The role of acetylcholine signaling in aging is unknown in Drosophila. The only related studies are those demonstrating either reduced or overexpressed vesicular acetylcholine transporter dramatically reduces lifespan of flies, suggesting that acetylcholine is important for longevity48. Our approach to employ the heterozygous ChAT mutant offers a useful strategy to identify the molecular and cellular players crucial for inhibitory control that is sensitive to aging, a key risk factor for dementia.The genetic mutation in the rate-limiting enzyme for acetylcholine biosynthesis ChAT in mice causes lethality at birth52. Our study points to the cholinergic transmission in the γ MB being responsible for aging-related loss of inhibitory control. We have previously shown that the γ neurons, upon receiving dopaminergic and octopaminergic inputs, form appetitive and aversive memory, modulate courtship motivation and develop behavioral sensitization to the disinhibition effect of ethanol55. The γ independently with α’/β’ neurons are also shown to modulate startle-induced negative geotaxis or climbing behavior in which ectopic activation of their activities inhibits the locomotor reactivity to startle but ectopic inhibition of their synaptic outputs has no effect30. Since the ChAT RNAi used in this study would dampen cholinergic output from the γ neurons, the neural controls for startle-induced negative geotaxis and movement suppression in the GNG task are likely independent. The study by Martin et al.29 shows that the MB ablation or inhibition of γ and/or α/β synaptic output causes basal hyperactivity. While it seems possible that higher basal activity could be associated with more frequent loss of movement suppression, we do not see significant difference in basal activities of the flies with and without ChAT KD at 4 weeks (data not shown). Thus, the neural control of the stimulus-induced movement suppression is distinct from that of basal activity. Together, our study is the first identifying progressive loss of acetylcholine and inhibitory control with aging in which γ cholinergic neurotransmission plays a key role.The MB is a key neural site processing olfactory behaviors as well as non-olfactory brain functions31. Our study uncovers only 1 type of the cholinergic MBONs—MBON-γ2α’1 (2 neurons per hemisphere) receiving inputs from the MB γ2 and α’1 lobes and sending outputs to the crepine and the superior medial protocerebrum (Fig. ts leads to loss of inhibitory control. MBON-γ2α’1 activity is shown to be crucial for various aspects of appetitive and aversive memory60, courtship memory61, sleep promotion63, fat accumulation64, food seeking65 and attraction/positive valence31. Regarding stimulus-induced motor behaviors, ectopic activation of MBON-γ2α’1 causes inhibition of proboscis extension response to sucrose but ectopic inhibition has no effect66. The MBONs important for startle-induced negative geotaxis include the cholinergic MBON-α2sc and α’330 but they are not important for movement suppression in the present study. Together, our study reveals a novel role for MBON-γ2α’1 in sustained inhibitory control per se. It remains to be determined whether this function requires the input from the γ MB lobes or dopamine/octopamine neurons or both, and how this MBON with only 2 neurons in each hemisphere accommodates so many functions.The MB axons convey information to 21 types of MBONs among which 8 types are cholinergicrum Fig. D—contrib


The article discusses the effect of modification of the plasticizing system of a single-screw extruder on selected physicochemical properties of rapeseed straw and buckwheat straw. A TS-45 single-screw extruder  with an L/D = 12 plasticizing system was used for the process. The shredded straws were moistened to four moisture levels: 20, 25, 30 and 35% dry matter. Three different rotational speeds of the extruder screw were applied for the test cycle: 70, 90 and 110 rpm. The following characteristics were determined for the extrusion-cooking process: efficiency and specific mechanical energy. Selected physical properties were determined for the extrudates obtained in the process: water absorption index (WAI), water solubility index (WSI), bulk density, and the efficiency of cumulative biogas and cumulative methane production expressed on dry mass, fresh mass, and fresh organic mass basis. It has been proved that the modification of the plasticizing system had a significant impact on the course of the process and the tested physicochemical properties. An important factor confirming the correctness of the modification is the increase in biogas efficiency. After modification, the highest yield of cumulative biogas from the fresh mass was 12.94% higher than in the sample processed before modification. A developing society needs energy to sustain its growth. Growing consumerism, technological progress, volatile geopolitical situations, challenges posed by the supply of fossil fuels, continuous population growth, production of foodstuffs and other items of everyday life—all these drive up the global energy demand . EnvironBiogas is a product of anaerobic digestion. Adequate hydration and temperature cause the development of bacteria that feed upon organic matter. The product of this process is combustible biogas, also known as mud/marsh gas . AgriculAccording to the available literature ,16, therThe aim of the research was to determine the influence of modification of the plasticizing system on the course of the extrusion-cooking process and on the physicochemical properties of selected pretreated straws as well as the effect of pretreatment on biogas efficiency during methane fermentation.As the main raw materials, rapeseed straw and buckwheat straw were used. The rape straw used in the tests was qualitatively assessed by 4.76% ash content, 19.41% lignin content, 41.33% cellulose content, 30.57% hemicellulose content, 42.47% carbon content and 0.51% nitrogen content. In the case of buckwheat straw, there was 5.73% ash content, 19.94% carbon content, 36.54% cellulose content, 28.26% hemicellulose content, 37.28% carbon content and 1.17% nitrogen content . The stuA TS-45 single-screw extruder  with an L/D = 12 plasticizing system was used for the process. A standard and a prototype screw were used. It was equipped with an additional section of beaters to ensure better compaction of the processed raw material before its molding phase at the extruder head .During the treatment of straw, the processing efficiency and specific mechanical energy consumption of the extrusion-cooking process were determined, and the influence of process variables on selected physical and chemical properties of lignocellulosic raw materials was tested with the application of two different plasticizing systems (before and after modification). The followed tests also determined the effect of extruder screw modification  and process variables on the biogas efficiency of tested pretreated straws .−1),Q—process efficiency (kg hm—mass obtained during measurement (kg),t—measurement time (h).The testing of the efficiency of the extrusion-cooking process focused on determining mass obtained over a specific time for all the raw materials used and at pre-set process parameters. The measurements were performed three times for each test series. The result was the mean value of all the measurements. The processing efficiency was determined with the formula:−1),SME—specific mechanical energy (kWh kgn—extruder screw speed (rpm),N—maximum extruder screw speed (rpm),L—motor load (%),P—power (kW),−1).Q—process efficiency  calculated according to the following formula :(2)SME= −1),WAI—water absorption index (g gs—dry sample mass (g),mz—gel mass (g).mThe processed straw samples obtained through extrusion were ground with a laboratory grinder  into particles with a diameter not greater than 0.8 mm. A suspension was prepared from a sample of 0.7 g and 7 mL of distilled water by continuous mixing for 20 min. The suspension was centrifuged at 15,000 rpm for 10 min in a laboratory centrifuge . Filtrate was collected from the obtained gel, and next, the gel was weighed . The watWSI—water solubility index (%),s—vessel mass after drying (g),mps—vessel mass before drying (g),mpp—sample mass (g).mThe filtrate obtained during the measurement of the WAI was dried at 130 °C until total water evaporation . The wat3 vessel  and then by weighing the vessel with the content. Based on the obtained mass, the bulk density was calculated as follows [u—sample mass (kg),m3).v—vessel volume ρu=muThe study was conducted at the Laboratory of Ecotechnologies of the Poznan University of Life Sciences (PULS). Biogas efficiency was investigated in the methane fermentation process under mesophilic conditions (the most popular technology in Europe) in three replications with the use of proprietary biofermentors  Figure .3 were filled with inoculum (with a dose of microorganisms from an operating biogas plant) and with rapeseed and buckwheat straws processed under various conditions of the extrusion-cooking process. The content of organic dry matter in the inoculum ranged from 1.5 to 2%. Dry mass and organic dry mass were measured prior to testing. After that, the substrates were placed in an airtight reactor for fermentation. The vessels with samples were immersed in water at a controlled temperature (about 39 °C), which simulated the actual operating conditions of biogas installations. The volume and qualitative composition of generated gases were measured every 24 h. The fermentation process was stopped when the daily biogas production lowered below 1% of the total amount of biogas produced. The samples were tested in three replications. The biogas yield was measured (m3 Mg−1) relative to fresh mass (rapeseed and buckwheat straws), dry mass, and dry organic mass, as described by Dach et al. [The pretreated with the extrusion-cooking samples were analyzed for biogas efficiency in accordance with the generally recognized standards, namely DIN 38414/S8 and VDI 4630 . The tesh et al. .The data obtained during the tests were archived and analyzed statistically using Microsoft Office Professional Plus 2019  and Statistica 13.3 . The RSM method with surface approximation was used to investigate the dependence of specific results on variable process parameters.The similarity of the screw systems (before and after modification) was analyzed using the principal component analysis (PCA) with Statistica 13.3. The PCA data matrix for the statistical analyses of both types of extrudates results had 11 columns and 24 rows. The input matrix was scaled automatically. The correct number of main components obtained in the analysis was determined based on Cattell’s criterion.The results shown below demonstrate the impact of modification of the plasticizing system of a single-screw extruder and of process variables on the extrusion-cooking and selected physicochemical properties of rapeseed straw and buckwheat straw. The results are presented both in tables and figures.The efficiency of the extrusion-cooking process depends on many factors: the rotational speed of the extruder screw, the type and moisture content of the raw material mixture used in the process, or the configuration of the extruder screws . The cha−1) was reported for straw extruded at 25% initial moisture and the screw rotational speed of 110 rpm. The lowest efficiency of rapeseed straw (5.38 kg h−1) was reported for one extruded at the screw speed of 70 rpm and at a moisture content of 35%. A considerably higher efficiency was measured in extrusion trials at 25 and 30% moisture levels than for 20 and 35%. The initial increase in the moisture content from 20 to 25% resulted in a significant surge in efficiency. However, when the moisture level kept increasing to 30%, the efficiency began to drop. Similar extrusion tests were carried out with maize straw [−1, which is close to the effect achieved when using a rapeseed straw at a 30% level of moisture and the screw speed of 90 rpm (19.44 kg h−1).−1) was reported at 20% moisture and at the screw speed of 90 rpm during processing. The lowest efficiency for this input material (−1) was observed at 25% moisture and the screw speed of 110 rpm. It has been observed that as the moisture level increases, the efficiency of the extrusion-cooking process of the buckwheat straw decreases. Depending on the level of moisture, the highest efficiency was obtained at different rotational speeds of the extruder screw. The process with the raw material moistened up to 25% showed the highest efficiency at the screw speed of 70 rpm (8.08 kg h−1), while at 30% (8.24 kg h−1) and 35% (4.96 kg h−1) moisture, the maximum efficiency was recorded at 110 rpm.In the case of buckwheat straw, the highest efficiency  was recorded for samples processed at the screw speed of 70 rpm and at 20% initial moisture. The higher efficiency (apart from at the moisture content of 20%) was seen in samples processed at the highest rotational speed. The maximum result (17.68 kg h−1) was recorded when applying a 30% moisture level. Compared with the standard (non-modified) screw, the modification of the plasticizing system increased the extrusion-cooking efficiency across the entire range of applied process variables.−1) was recorded at the moisture level of 25% and the screw speed of 90 rpm. The highest efficiency (20.16 kg h−1) was measured during processing a sample with 35% initial moisture and at the screw speed of 110 rpm. Much higher efficiency results were recorded in samples processed at the moisture content of 30 and 35%; when the standard screw was used, the maximum efficiency values were recorded for samples processed with 25 and 30% moisture.In the case of rapeseed straw extrusion-cooking A, much mThe extrusion-cooking process is energy intensive. The appropriate selection of the parameters of the extrusion-cooking process, in particular the type of and the moisture content in extruded raw material mixtures, significantly affects the volume of energy consumed by the process. Modifying the moisture content in the mixture by even 1–2% may increase the energy intensity of the process by up to several dozen percent . The eco−1) was calculated for samples pretreated at 25% moisture and the screw speed of 90 rpm. The highest SME value (0.686 kWh kg−1) was obtained in samples extruded at 35% moisture and the screw speed of 90 rpm.The energy consumption of the extrusion-cooking of rapeseed and buckwheat straws reveals the relationship between the moisture content and the rotational speed of the extruder screw . For rap−1) for samples processed at 20% moisture and the screw speed of 70 rpm. The highest SME value (1.432 kWh kg−1) was calculated in samples processed at 25% moisture and the screw speed of 110 rpm. Roye [−1. Moreover, in their work, Pardhi et al. [−1. In both cases, the energy consumption measurements presented by the mentioned authors showed lower values than obtained during the pretreatment of the tested straws.In the case of extrusion-cooking of buckwheat straw B, at thevalue 1.4 kWh kg−1i et al.  shared a−1) was obtained in the case of a sample extruded at 25% moisture and the screw speed of 70 rpm. The highest SME was reported in samples processed at the screw speed of 110 rpm across the entire range of the moisture content, and the top energy consumption value (0.474 kWh kg−1) was calculated for a sample processed at a 20% level of moisture.−1) was calculated for a sample processed at 25% moisture and at the screw speed of 90 rpm. The lowest result (0.223 kWh kg−1) was reported at the moisture level of 35% and the screw speed of 70 rpm.In the case of rapeseed straw A, pretre−1 grass, while the slow one required 0.140 ± 0.009 kWh kg−1 grass, which is much less than compared to the tested straws. When using the standard screw, the energy requirements of the rapeseed straw extrusion-cooking process were higher for the samples processed at the top level of moisture (35%); in other cases (20–30%), higher values were reported for the samples processed with the prototype screw. In the case of buckwheat straw, the modification of the plasticizing system caused a decline in energy consumption of extrusion-cooking. Only when using the lowest process variables (20% moisture and 70 rpm), higher SME were recorded compared to the standard screw. In the work of Khor et al. , they st−1 for buckwheat and 7.730 g g−1 for rapeseed.The substrates used in agricultural biogas plants are exposed to various treatments aimed at facilitating the methane fermentation process. One of such techniques is extrusion-cooking, which destructs the lignocellulosic structures and, as a result, enhances water absorption . The WAI−1) for extruded rapeseed straw was recorded at the screw speed of 110 rpm and at 35% moisture. On the other hand, the lowest WAI (4.261 g g−1) was reported for the screw speed of 70 rpm and a 30% level of raw material moisturization.−1) was observed in a sample pretreated at 35% moisture and the screw speed of 70 rpm. The lowest value (4.64 g g−1) was reported in the extrudate processed with a moisture content of 25% and the extruder screw speed of 70 rpm. Similar results were obtained by Żelaziński [−1 and for maize kernels 4.66 g g−1.For the extrusion of buckwheat straw B, the to−1) was recorded for a sample processed at the screw speed of 90 rpm and at 30% moisture. The lowest measurement result (4.696 g g−1) was obtained for a sample processed at the moisture level of 20% and the screw speed of 110 rpm.−1) was obtained for samples processed at the moisture level of 25% and the screw speed of 90 rpm. The highest measured value (6.948 g g−1) of the WAI was also seen for the same screw speed and 35% moisture.In the case of buckwheat straw B, a highWhen the prototype screw was used, in most cases, buckwheat straw showed an increase in the WAI compared to the processing with the use of the standard screw. The WAI dropped only in two trials (at 90 rpm and 25% MC and at 70 rpm and 35% MC). For rapeseed straw, the modification of the plasticizing system increased the WAI at 30% of the moisture content across the entire range of screw speeds; the same was reported for trials at 70 rpm and 25% moisture; as regards 35% moisture, the WAI grew at the screw speeds of 70 and 90 rpm. Another vital parameter is the water solubility index (WSI). A higher WSI proves the destruction of lignocellulose structures in the extrusion-cooking process, which is also related to the higher biogas efficiency of the substrate. The WSI determines to what extent the processed material can dissolve in water, i.e., it shows the number of soluble particles after moisturizing. A more compact material absorbs water to a lesser degree, which makes the solubility index lower. Pretreatment causes the material to deconstruct into smaller particles, thus reducing the density of the structure, which, in turn, accelerates the process of methane fermentation . The WSIWhen measuring the WSI of rapeseed straw extrudates obtained with the standard plasticizing system A, at theIn the case of buckwheat straw extrudates B, the hiIn the case of buckwheat straw B, only aIn the case of extruded buckwheat straw, the use of the modified plasticizing system (prototype) increased the WSI in samples processed at 30 and 35% moisture (across the entire range of the screw speeds applied) and at 20% moisture and the screw speeds of 90 and 110 rpm. As regards extruded rapeseed straw, the same trend was noted for samples processed with the use of 20% of moisture content (across the entire range of the screw speeds applied) and for samples extruded at 70 rpm and 30% moisture and 110 rpm and 25 and 30% moisture. −3 for buckwheat and 105 kg m−3 for rapeseed.Another part of the research was aimed to determine the bulk density (BD) of the tested extrudates. The influence of process variables  on the BD of produced extrudates was measured. The use of the most preferred pretreatment method may help reduce the size of storage areas for bulk materials. The thermal processes occurring during the pretreatment make the raw material sterile and allow its storage for a longer period with no harm to quality. A higher level of BD may suggest a higher density of the material structure. It may also be true of WAI and WSI, thus having an impact on the efficiency of methane fermentation ,36. The −3) was recorded for samples prepared at the screw speed of 70 rpm and the moisture content of 25%. The lowest BD (68 kg m−3) was noted in samples obtained at the screw speed of 110 rpm and the moisture content of 20%.For rapeseed straw processed with the standard plasticizing system A, the sc−3) was noted in samples pretreated at the moisture content of 20% and the extruder screw speed of 110 rpm. Along with the rising level of moisture, the BD gradually decreases across the entire range of rotational speeds. The lowest BD values (105 kg m−3) were recorded in samples obtained at the screw speeds of 90 and 110 rpm and 35% moisture.As regards buckwheat straw extrudates B, the hi−3) was recorded for a sample processed at the screw speed of 110 rpm and at 35% moisture. The lowest value (60 kg m−3) was noted in a sample obtained at the screw speed of 110 rpm and the moisture content of 20%.During the measurements of the BD, higher values were recorded when buckwheat straw was processed using the prototype extruder screw B. For th−3) were noted for samples extruded at 35% moisture and at the screw speeds of 70 and 110 rpm while the lowest (46 kg m−3) was observed in a sample obtained at the screw speed of 110 rpm and the moisture content of 30%.In the case of rapeseed straw A, the hi−3 (rye straw and rape cake) and 521.4 kg m−3 (rye straw and soya bean cake), i.e., more than twice as high as in the case of the tested of the buckwheat straw and rapeseed straw extrudates described in this paper. The use of the modified plasticizing system reduced BD in both tested raw materials. Only in buckwheat straw processed at the screw speed of 110 rpm and at 35% moisture was a decrease in the BD noted, unlike with the samples processed using the prototype screw. As Kraszkiewicz et al.  reportedThe extrusion-cooking process is among the raw material pretreatment methods used in biogas plants. This paper focuses on lignocellulosic raw materials. During the extrusion-cooking of these raw materials, lignin bonds are broken, and cellulose and hemicelluloses are released. The surface of active biomass grows, followed by its initial hydrolysis, which results in a higher biogas yield. The extrusion-cooking process should be preceded by proper preparation of the raw material used. The moisture content and the degree of fragmentation (grinding) are key factors. Finer grinding allows a more effective mixing of the raw material, which makes it more homogeneous. Proper selection of the moisture level significantly affects the extrusion-cooking process. Too low a level of moisture may cause the accumulation of raw material on the cylinder walls and the extruder screw. This, in turn, may lead to the overheating of the raw material and, consequently, interrupting of the process. Thus, the right moisture content of raw material enables its processing, regardless of the structure .The efficiency of biogas production from the extruded rapeseed and buckwheat straws was studied at the last stage of the research. This study was aimed to perform the final comparison of the impact of modification of the plasticizing system on biogas efficiency and demonstrate that extrusion-cooking should be the preferred method of pretreatment of these raw materials if potentially used in agricultural biogas plants. The methane content in biogas in all the tested samples was at a similar level: for extruded rapeseed straw , the conIn extruded buckwheat straw , pretreaGenerally, for fresh mass, the production of cumulative biogas ranged from 95.99% (when extruded at 90 rpm and 30% MC) to 110.30% (at 110 rpm and 25% MC) (in both cases for rapeseed straw), and of methane from 97.26%  to 111.57% . The higher biogas productivity for dry mass ranged from 95.85%  to 111.34%  and in the case of methane, from 96.97%  to 111.65% . In the case of dry organic mass, these values for biogas ranged from 95.43%  to 110.82% , and in the case of cumulative methane from 97.46%  to 111.74% .3 Mg−1) and methane yield (209.90 m3 Mg−1) from fresh mass were higher than in the silage obtained from pretreated maize straw. According to the author [3 Mg−1 of biogas and 205.68 m3 Mg−1 of methane.Modification of the plasticizing system caused a decrease in the cumulative methane value  comparede author , the topIn extruded buckwheat straw pretreated by modifying the plasticizing system, there was an increase in the production of biogas and methane compared with the control sample (not subjected to the extrusion process) across the entire range of tests . As in tIn general, for the samples (rapeseed and buckwheat) processed after modification of the plasticizing system, and given the results for the control samples for fresh mass, the cumulative production of biogas ranged from 99.43  to 123.24% (buckwheat processed at 110 rpm and 25% MC), and methane from 92.22  to 115.98% . The cumulative production for dry mass ranged from 97.43  to 116.77%  of biogas and from 90.36  to 109.81%  of methane. As regards dry organic mass, the range was from 97.24  to 116.69%  of biogas and from 90.18  to 109.72%  of methane. The determination of biogas and methane yields, when calculated on fresh matter, dry organic matter or in total solids and volatile solids, is necessary for the comparison of energetic potential between substrates ,39.The obtained results were used for the principal component analysis (PCA) before and after modification of the extruder plasticizing system and objects were located within the first two components. Presentation of all the results in the form of a graph  offers aApproximately 71% A of all The following conclusions were drawn based on the conducted tests and the analysis of the results. The results in individual tests differed depending on the raw material used. Not only may this be attributed to the structure of the tested raw material, but it may also suggest the need to select appropriate process parameters for a specific material. In the case of buckwheat straw, the use of a prototype extruder screw increased the efficiency of the extrusion-cooking process and reduced its energy consumption. In the case of rapeseed straw, modification of the plasticizing system raised the energy requirement and reduced the efficiency of the process compared to when a standard screw was used. The opposite trend was reported only when 35% of the moisture content was applied.When the prototype screw was used, in most cases, extruded buckwheat straw showed an increase in the WAI compared to the processing with the use of the standard screw. For rapeseed straw, the WAI index was noted to depend on the moisture level of the raw material. In the case of 20 and 25% of the moisture content, a higher WAI was observed in samples processed with the standard screw; and when moisture was raised, a higher WAI was noted in samples processed with the prototype screw.In the case of extruded buckwheat straw, the use of the modified plasticizing system (prototype) increased the WSI in samples processed at 30 and 35% moisture (across the entire range of the screw speeds applied). In extruded rapeseed straw, this trend was observed for samples processed at a moisture level of 20%.The use of the modified plasticizing system reduced BD in both tested raw materials. Only in buckwheat straw processed at the screw speed of 110 rpm and at 35% moisture was a decrease in the BD noted, unlike with the samples processed using the prototype screw.In extruded buckwheat straw (obtained in the modified plasticizing system), there was an increase in the production of biogas and methane compared with the control sample (not subjected to the extrusion process) across the entire range of tests. For samples processed with the standard screw, the best yield of biogas across the entire range of tests was obtained for the sample processed at the screw speed of 70 rpm and 35% of the moisture content. Rapeseed straw processed with the prototype screw produced the highest biogas yield at the screw speed of 90 rpm applied for pretreatment. When testing rapeseed straw processed with the use of the standard screw, the top measured values were reported in samples processed at the maximum rotational speed of the extruder screw. In both cases, the best results were found in samples processed with a moisture level of 25%.In future research attempts, more attention should be paid to the proper selection of process variables for specific raw materials. It would be reasonable to carry out a comparative analysis using various extruder plastification unit configurations (L/D) or screw lengths, as well as measuring the influence of modification of the plasticizing system on the extrusion-cooking process stability and on methane efficiency.


Scirpus rhizosphere, and ecological drift and homogeneous selection processes were more important in the Phragmites rhizosphere. Regarding the different effects of community assembly processes on abundant and rare microbes, abundant microbes were controlled more by homogeneous selection. In contrast, rare microbes were controlled more by ecological drift, dispersal limitation, and heterogeneous selection, especially bacteria. This is potentially caused by the low growth rates or the intermediate niche breadths of rare microbes under the ice. Our findings suggest the high diversity of microbial communities under the ice, which deepens our understanding of various ecological processes of community assembly across stages and reveals the distinct effects of community assembly processes on abundant and rare microbes at the bin level.The rhizosphere soil microbial community under ice exhibits higher diversity and community turnover in the ice-covered stage. The mechanisms by which community assembly processes shape those patterns are poorly understood in high-latitude wetlands. Based on the 16S rRNA gene and ITS sequencing data, we determined the diversity patterns for the rhizosphere microbial community of two plant species in a seasonally ice-covered wetland, during the ice-covered and ice-free stages. The ecological processes of the community assembly were inferred using the null model at the phylogenetic bins (taxonomic groups divided according to phylogenetic relationships) level. Different effects of ecological processes on rare and abundant microbial sub-communities (defined by the relative abundance of bins) and bins were further analyzed. We found that bacterial and fungal communities had higher alpha and gamma diversity under the ice. During the ice-free stage, the dissimilarity of fungal communities decreased sharply, and the spatial variation disappeared. For the bacterial community, homogeneous selection, dispersal limitation, and ecological processes (undominated processes) were the main processes, and they remained relatively stable across all stages. For the fungal community, during the ice-covered stage, dispersal limitation was the dominant process. In contrast, during the ice-free stage, ecological drift processes were more important in the  It is increasingly recognized that microorganisms can exist and grow under the ice  on bacterial communities under the ice has been studied, specifically relating to dissolved oxygen  the diversity patterns of microbial communities across ice-covered and ice-free stages; (2) the relative importance and the potential drivers of different community assembly processes across ice-covered and ice-free stages; (3) the effects of community assembly processes on different relative abundance microbes . The research was carried out in Momoge wetland, a seasonal ice-covered wetland located in Northeast China. Rhizosphere soil samples were collected throughout the ice-covered and ice-free stages. Microbial communities were characterized using the amplicon sequencing of the 16S rRNA gene  and ITS . Diversity was calculated across different ice-covered stages, and the relative importance of different ecological processes was calculated using the “iCAMP” framework. The relationship between relative abundance and different ecological processes was also analyzed. We hypothesized that (1) the diversity of bacterial and fungal communities would be higher under the ice, (2) the selection processes would be influenced by the species of plants, and the dispersal limitation processes would dominate microbial communities during ice-covered stages, and (3) the rare microbial taxa would be more controlled by ecological drift than selection.Our study was conducted at the Momoge National Nature Reserve , located in northeastern China. As an important hydrology node in the Nenjiang River basin Meng, , the locPhragmites australis (Cav.) Trin. ex Steud. and Scirpus mucronatus Linn.) as sample plots (0.5 × 0.5 m). In each sample plot, we sampled plant rhizosphere soil (root around 1 cm); three similar cores of rhizosphere soils were collected and mixed as one sample. Each soil sample was divided into four parts: one was used for DNA extraction and sequencing (~5 g), one for soil moisture measurements, one was freeze-dried for subsequent soil chemistry analysis, and one was stored at 4°C as a standby soil. Rhizosphere soil for DNA extraction and sequencing was saved at −20°C during transportation and stored at −80°C.We chose three sites connected by surface water but over three kilometers apart as our sample sites (30 × 30 m) . Within Scirpus mucronatus Linn. in Site 2 at 25 cm water depth), amounting to 68 mixed rhizosphere soil samples for all four stages.To reflect the dynamic change of the microbial communities, we set up three sample stages in the Momoge wetlands during the winter  for bacteria, and the ITS2 region was amplified using a specific primer (ITS3-2024F and ITS4-2409R) for fungi. All polymerase chain reactions (PCR) contained 15 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs), 0.2 μM of forward and reverse primers, and ~10 ng of template DNA. PCR conditions were 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, 50°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. The PCR products were mixed with the same volume of IX loading buffer (contained SYB green). The mixed PCR products underwent gel electrophoresis on 2% agarose gel for detection and were purified with a Qiagen Gel Extraction Kit . The library quality was assessed on the Qubit@2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina NovaSeq PE250 platform, and 250 bp paired-end reads were generated. Paired-end reads were assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. DNA extraction and sequencing were then performed at the Tianjin Sequencing Center and Clinical Lab .Paired-end reads were merged using FLASH    rather than the entire community  data. To calculate community assembly processes at the bin level, the relationship between the relative importance of different ecological processes in governing each bin (Pkτ) and the relative abundance of each bin were analyzed by Spearman's rank correlation. The relative abundance of all bins (explanatory variables), the value of which was equal to zero, was deleted.To explore the relationship between microbial rarity and community assembly processes, bacterial and fungal communities were divided into rare, intermediate, and abundant sub-communities according to the relative abundance of phylogenetic bins. In previous studies, the sub-communities were divided according to the relative of OTUs  (p < 0.05), and was more significant than the diversity pattern of the bacteria communities. The gamma diversity of the bacterial community during the ice-covered stage was also greater than that during the ice-free stage (31.1–35.9%). Similarly, the gamma diversity of the fungal community during the ice-covered stage was more than that during the ice-free stage (82.4–100.4%). In the other plant rhizosphere habitats, most of the alpha and gamma diversity of microbial communities did not show a significant difference during the same stage and showed a similar change between different stages. Only in the first partially ice-covered stage (October 2020) did the alpha diversity of bacterial communities differ significantly between the two rhizosphere habitats.The alpha diversity of the bacterial communities during the ice-covered stage was greater than that during the ice-free stage  . Similarp < 0.05) during all stages, with no exceptions. However, the dissimilarity of the fungal communities showed different patterns during the ice-free stage and other stages. During entirely and partially ice-covered stages, the dissimilarity of the fungal communities was significantly lower in intra-sites than in inter-sites (p < 0.05). During the ice-free stage, the dissimilarity of the fungal communities showed no significant difference either in intra-sites or in inter-sites. The dissimilarity of the fungal communities during the ice-free stage was significantly lower than at any other stage (p < 0.05).To assess the influence of geographical distance on the microbial communities, the community dissimilarity  between intra-sites and inter-sites was calculated and tested using the Wilcoxon signed-rank test . The disTo reveal the community assembly mechanism during different ice-covered stages, ecological processes and their relative importance in the microbial communities were calculated . The resThe turnover of different ecological processes during various ice-covered stages for the bacterial communities was minor. In contrast, for the fungal communities, there was a distinct shift between different stages. From entirely and partially ice-covered stages to the ice-free stage, the main ecological processes of the fungal community assembly changed from dispersal limitation (from 62.5–68.3 to 12.1 %) to homogeneous selection (from 10.8–18.6 to 28.5%) and ecological drift (from 14.0–20.5 to 58.3%). This means that during entirely and partially ice-covered stages, the fungal communities tended to be heterogeneous between different samples  and that during the ice-free stage, they tended to be homogeneous (homogeneous selection). Therefore, the community assembly processes of fungi were more influenced by the ice-covered environment or seasons than the community assembly processes of bacteria.Phragmites  Trin. ex Steud.), the proportion of homogeneous selection processes increased in both bacterial and fungal communities during the ice-free stage. In contrast, in the rhizosphere habitat of Scirpus (Scirpus mucronatus Linn.), the proportion of different ecological processes in the bacterial communities during various ice-covered stages was stable. However, the ecological drift process increased to the highest magnitude in the fungal communities during the ice-free stage.The turnover of different ecological processes was also different in the different plant rhizospheres. In the rhizosphere habitat of To explore the relationship between microbe rarity and the ecological processes of community assembly, the impact of the ecological processes on different microbial subcommunities  was calculated . During To verify the pattern of relative abundance and the ecological processes of microbial bins, Spearman's rank correlation was employed to test the relationship between the relative abundance of microbial bins and the relative importance of different ecological processes in governing each bin .p < 0.05) to the abundance of each bin. This means that if a microorganism belongs to an abundant microbial subcommunity , it tends to be influenced by certain community assembly processes rather than others. For bacterial communities, the homogeneous selection tended to exert control more on a relatively high abundance microbe rather than on a low abundance microbe . Moreover, the homogeneous selection, ecological drift, dispersal limitation, and heterogeneous selection processes tended to have more of an influence on low abundance than on high abundance bacteria. Compared to bacteria, the relationship between the fungal community assembly processes and the relative abundance had a similar direction but lower strength. Moreover, there was a significant correlation between the relative importance of ecological drift and heterogeneous selection processes for each OTU.Interestingly, this result shows that ecological processes affect microbial bins of varying relative abundance in a different way for both bacterial and fungal communities. The relative importance of most processes for each bin was significantly correlated  repository, accession number JM provided concept, designed methodology, performed the field sampling, conducted the data analyses and wrote the manuscript. KM performed the field sampling and reviewed this manuscript. JL supervised this research and reviewed this manuscript. KM, JL, and NC assisted to methodology designing. All authors contributed to the article and approved the submitted version.This research was supported by the National Key Research and Development Program of China (No. 2016YFC0500402).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Biological systems operate in constant communication through shared components and feedback from changes in the environment. Casein kinase 1 (CK1) is a family of protein kinases that functions in diverse biological pathways and its regulation is beginning to be understood. The several isoforms of CK1 take part in key steps of processes including protein translation, cell-cell interactions, synaptic dopaminergic signaling and circadian rhythms. While CK1 mutations are rarely the primary drivers of disease, the kinases are often found to play an accessory role in metabolic disorders and cancers. In these settings, the dysregulation of CK1 coincides with increased disease severity. Among kinases, CK1 is unique in that its substrate specificity changes dramatically with its own phosphorylation state. Understanding the process that governs CK1 substrate selection is thus useful in identifying its role in various ailments. An illustrative example is the PERIOD2 (PER2) phosphoswitch, where CK1δ/ε kinase activity can be varied between three different substrate motifs to regulate the circadian clock. CK1δ and CK1ε are members of a family of serine/threonine kinases that are highly conserved among eukaryotes. In humans, this group of kinases is encoded by six distinct genes, CSNK1A/D/E/G1/G2/G3 . Of these isoforms, CK1δ and CK1ε are the most closely related, sharing over 96% sequence identity in the kinase body, and have very similar carboxyterminal extensions of ∼124 amino acids. CK1δ and CK1ε bind to and phosphorylate many of the same substrates, but with some subtle and important differences. The major regulation of CK1δ/ε comes from their C-terminal tails. These CK1δ/ε C-terminal tails are differentially phosphorylated and dephosphorylated, and it has been long known that this tail phosphorylation regulates kinase activity . ImportaCK1 is best known for its preference to phosphorylate acidic substrates. The first-identified CK1 targets are either preceded by acidic residues, or primed by a phosphorylated serine or threonine. The acidic or phosphorylated amino acid is usually three residues upstream of a CK1 target serine/threonine . Repeatsin vitro and in animal models. The modelling of how CK1δ/ε selects sites on PER2 could provide a better understanding of how CK1 phosphorylate its other substrates. Here we review selected literature describing the phosphoswitch mechanism of PER2 and highlight several diseases where a similar CK1 substrate selection mechanism might be involved.Interest in targeting CK1 for therapeutic intervention of sleep disorders, cancer and drug addiction has grown over the years. Multiple CK1 inhibitors have recently been developed and tested D. melanogaster, they identified mutations in a single gene that altered both the time-of-day when flies would emerge from their pupae and locomotor rhythms in adult flies. This gene was named Period (Per) and the rise and fall of its mRNA and protein abundance across the day results in the periodicity of animal clocks. Price, Kloss then identified that the gene Doubletime (dbt), encoding a CK1 orthologue in Drosophila, regulated Per degradation via direct phosphorylation  by CK1 may contribute to tumor metastasis in pancreatic and breast cancers. Under normal conditions, CK1 helps maintain cell-cell interfaces in part by phosphorylating Cx-43, a key structural protein of gap junctions (GJ). These structures facilitate cell-cell communication by allowing the exchange of small molecules and ions through their pore-like hemichannels. GJs are also known to transduce signals from the cell membrane to alter transcriptional programs. This includes signals that promote extravasation, cell migration, differentiation, and apoptosis. Therefore, Cx-43 can both prevent and promote tumorigenesis depending on its regulation.CK1A, reduced cell migration in wound healing assays. Correspondingly, serine to glutamic acid substitutions to generate phosphomimetic Cx-43 at these sites caused increased migration  , a form of cancer known for its aggressive metastatic ability. In 2021, Solan et al., showed that in a KRASon (EMT) . KRASG12in vivo and ex vivo. Although CX-43 phosphorylation was not directly assessed in this model, they noted repression of junctional proteins in these breast cancer models, making CX-43 an interesting candidate for further study.CK1-enhanced GJ activity may be a feature of other highly metastatic tumors. Rosenberg et al. found that suppressing CK1δ activity with the potent CK1δ/ε inhibitor Sr-3029 reduced the tumorigenicity of Triple Negative Breast Cancer (TNBC) cells . In the There is growing interest in the use of translation inhibition as a therapy for cancer. Due to its critical role in cell growth, translation is a tightly regulated process. Most of this control takes place at the translation initiation phase and is regulated by the heterotrimeric Elongation Initiation Factor 4a Complex (EIF4A). Elongation Initiation Factor 4e (EIF4E) is a rate-limiting component of the EIF4A complex. It functions to bind the m7g cap of mRNA to recruit it to the ribosome for translation. Suppressing translation is thus accomplished through the sequestration of EIF4E by 4 E-binding protein 1 (4EBP1). 4EBP1 phosphorylation releases EIF4E to drive translation, so the kinases that phosphorylate 4EBP1 are of great interest. While initial studies on focused on the mTOR and PIK3-regulated phosphorylation, more recent studies have found that CK1δ/ε also phosphorylates 4EBP1 on a distinct set of regulatory sites, also leading to EIF4E release , 2001.. How might the activity of CK1 on 4EBP1 be regulated? One possibility is via AMP-activated protein kinase (AMPK). AMPK is a cellular energy sensor that shuts down protein translation when intracellular ATP stores are depleted. AMPK has been shown to activate CK1ε by phosphorylating its regulatory tail on S389 . Based oMYC proto-oncogene encodes a transcription factor that influences the expression of around 15% of total human transcripts. Under normal circumstances, MYC is under tight transcriptional and translational regulation, and MYC protein is quickly degraded to prevent its accumulation. However, MYC overexpression is detected in a majority of cancers and correlates with poor treatment outcomes. MYC gene amplification and translocation cancers are particularly aggressive and challenging to treat, owing to the undruggable nature of Myc protein (50 = 22 nM) and CK1ε (IC50 = 6 uM) dual kinase inhibitor, umbralisib (TGR-1202). Umbralisib gave promising results in reducing viability of multiple MYC-driven lymphomas. It was found to out-perform idelalisib, the best-in-class approved PI3Kδ inhibitor. Umbralisib treatment greatly reduced C-MYC protein levels by effectively blocking the release of EIF4E from 4EBP1. This effect was reproduced when the lymphoma cells were treated with idelalisib in combination with the CK1ε specific inhibitor PF4800567, showing that co-inhibition of PI3KD and CK1ε was required to achieve potency. Interestingly, Maharaj et al., showed that the CK1ε inhibitory ability of umbralisib contributes to the safety profile of the drug .While CK1 is rarely the primary driver of disease, elevated CK1δ/ε has been associated with highly malignant cancers . One pot protein . As part protein . Thus, Dthe drug . This waBryja and coworkers recently demonstrated the potential of CK1 inhibition in Chronic Lymphocytic Leukemia (CLL) . TreatmeMYC, PPARγ translation is also tightly regulated by highly stable secondary structures in its 5’ UTR (Could CK1δ/ε regulate translation of other important genes? A growing body of research has identified CK1 as a contributing factor to various metabolic disorders. One example is its role in promoting the maturation of pre-adipocytes in white adipose tissue (WAT) via EIF4E-dependent translation. This proliferation of WAT heightens the risk of disease by enhancing hunger signals, inducing insulin resistance, raising basal inflammation and altering sleep behavior. While PPARγ and C/EBPs transcription factors are considered the master regulators of adipocyte differentiation, they are in turn regulated by the cellular energy sensor, AMPK . As withs 5’ UTR . Thus, as 5’ UTR . CK1 inhs 5’ UTR , an effe50 = 1.24 uM) as the kinase most sensitive to Orobol treatment. However, Orobol also inhibited MAP4K5 (IC50 = 1.48 uM), which also has the potential to affect 4EBP1 phosphorylation via the mTOR pathway , which in turn activates CREB through phosphorylation. Dephosphorylation of CREB by Protein Phosphatase 1 (PP1) attenuates this pathway. Another target of PKA is Dopamine- and cAMP-Regulated Neuronal Phosphoprotein of 32kDa, DARPP-32. PKA phosphorylates DARPP-32 on Threonine-34, turning it into a potent inhibitor of Protein Phosphatase 1 (PP1), thus strengthening CREB-dependent transcription. Activation of metabotropic glutamate receptors (mGluR), which are known to enhance dopaminergic signaling, decreases phosphorylation of CK1 C-terminal tail by activation of calcineurin. This increases CK1 activity on S137 of DARPP-32, which protects dephosphorylation of T-34 from Calcineurin, further strengthening suppression of PP1. DARPP-350 = 14 nM, CK1ε IC50 = 7.7 nM), a dual CK1δ/ε inhibitor, and this blocked MA-induced locomotor activity leading to the conclusion that CK1 promoted drug sensitivity (50 = 32 nM) enhanced MA-induced locomotor activity in the MA sensitive mice, suggesting that CK1ε acts to attenuate drug addiction. The differences in substrate preference between CK1δ and CK1ε may account for this contradictory finding. Supporting this theory, Wager et al. observed a suppressive effect on drug-seeking behavior in rats treated with PF50067399 , a brain-penetrant dual CK1δ/ε inhibitor  is a neurodegenerative disease characterized by a progressive decline in cognitive functions such as memory, speech, and mood regulation. While the cause of AD remains unknown, disease severity is correlated with the accumulation of hyperphosphorylated Tau protein and amyloid-ß plaques (Aß) which impairs proper neuronal function . The faiAltered regulation of CK1δ/ε activity is associated with many diseases including sleep disorders, metabolic syndrome, Alzheimer Disease and cancer . HoweverCasein Kinase 1 is a key regulator of the circadian clock, determining the duration of each biological day through the phosphorylation of at least three unique PER2 domains. The ability of CK1 to choose between these three potential phosphorylation sites is a combination of several regulatory mechanisms. Firstly, the various CK1 isoforms have differing propensities for carboxyterminal tail modification. Secondly, CK1 preference for the substrates are influenced by modifications to its carboxyterminal tail. Finally, the ratio of expression between the various CK1 isoforms (and the CK1δ splice variants) also plays a role. These mechanisms may extend to other disease-relevant substrates including Cx-43, 4EBP1 and DARPP-32. Unlocking the ability to switch CK1 activity between substrates potentially enables the precise modulation of these CK1 target proteins, offering new avenues of therapy for their respective diseases.


ΔHf values of the plant-based ice cream samples varied from 144.70 J/g to 172.70 J/g. The low-fat COB ice cream stabilized with 3% COB and full-fat COB ice cream samples showed lower ΔHf values than control ice cream samples, indicating that the COB ice cream showed desired thermal properties. The COB drink may be utilized in plant-based ice cream without altering sensory qualities, and low-fat ice cream could be manufactured in the same manner to attain full-fat ice cream quality characteristics. The results of this study demonstrated that COB can be successfully used as an inexpensive raw material source in the production of full-fat and reduced-fat vegetable-based ice cream.This study aimed to investigate the potential use of cold-pressed coconut oil by-products (COB) as a low-cost alternative source for plant-based drink and ice cream production. Firstly, a plant-based drink was produced from cold-pressed coconut oil by-products (COB drink) and compared with a commercial coconut drink. The fat, protein, and zeta potential values of coconut drink obtained from COB were higher than those of the commercial samples. In addition, the particle size value of the drink obtained from COB was found to be lower than that of the commercial drink. In the second stage, full-fat and low-fat plant-based ice cream samples using COB drink were produced and compared to control ice cream samples  in terms of rheological, sensorial, and thermal properties. Rheological analysis showed that all plant-based ice cream samples indicated pseudoplastic, solid-like, and recoverable characteristics. Low-fat commercial control ice cream samples (C1) indicated the lowest K value (9.05 Pas The milk of animal origin and dairy products, such as ice cream, are among the most consumed food groups worldwide. However, in recent years, the tendency towards animal products has decreased due to allergenic effects, lactose intolerance, veganism, and ethical reasons, and the tendency toward plant-based alternatives of these products has increased ,2. For tCold-pressed oils are extracted mechanically using a screw press without the application of heat, solvents, or chemical treatments. The maximum temperature of cold-pressed oil production does not exceed 50 °C. Therefore, there is a growing trend in cold-pressed oil consumption due to their high bioactive compounds. In addition, after oil extraction, a by-product rich in protein and carbohydrates emerges . CoconutIce cream is a frozen and aerated oil-in-water (O/W) emulsion with a high amount of dairy or non-dairy fat 10–16%) combined with air bubbles, fat globules, and ice crystals % combine. HoweverThe main purpose of this study is to produce a plant-based drink and ice cream using the by-products of cold-pressed coconut oil. In this study, firstly, a plant-based drink was produced from cold-pressed coconut oil by-products (COB drink) and compared with a commercial coconut drink. In the second stage, full-fat and low-fat plant-based ice cream samples using the COB drink were produced and compared to control ice cream samples  in terms of rheological, sensorial, and thermal properties. Therefore, it was evaluated whether low-fat and full-fat ice cream could be produced from COB drink.COB was provided from ONEVA . COB was obtained after mechanical screw press process. The coconut was ground up into coconut flakes and dried at 8% moisture content. Finally, screw press was applied to extract coconut oil and obtain COB. COB and distilled water were used to produce COB drink. COB drink, powdered sugar, sunflower oil, xanthan gum (as a stabilizer), and lecithin (as an emulsifier) were used for ice cream production. Coconut drink was bought from a local market as commercial control drink . Xanthan gum and lecithin were obtained from Sigma-Aldrich .w/v) in our preliminary trial. The prepared mixture was homogenized with a homogenizer  at 10,000 rpm for 5 min. The optimum parameters of ultrasound homogenization were determined as 405.16 W and 2.52 min to decrease in particle size of plant-based drink. After the homogenization, the drink was filtered and the temperature of the drink was decreased to 10 °C and stored at 4 °C in the refrigerator. COB drink was produced by only drinking water and COB. COB was milled in a laboratory mill  and then sieved by No.140 mesh. Formulation of plant-based ice cream samples consisted of 2.5–12.5% oil, 12% sugar, 2% lecithin, and 0.35% xanthan gum (XG). To begin, XG and COB drink were weighed in accordance with the formulae established at the first stage of ice cream manufacturing. XG was gradually poured to completely dissolve it and held at 1000 rpm in a magnetic stirrer at 80 °C. Then, COB (1–3%) was added, blended, and weighted according to the low-fat ice cream recipe . Then, sugar was added sequentially. The lecithin and oil were mixed and added to all ice cream formulations. Then, the ice cream mixes were homogenized with a homogenizer  at 10,000 rpm for 3 min. Following this procedure, all ice cream mixtures were kept at 0–4 °C for two days to mature. The mixtures were then used to make ice cream with an ice cream machine . Ice cream samples were stored at −18 °C for analysis.w/v) COB in distilled water. The browning index (BI) value of COB was calculated according to the difference between the absorbance values of COB measured at 420 and 550 nm and expressed as optical density/g dry solid. Water activity (aw) is carried out with a water activity analyzer (Novasina Labtouch-Aw Water Activity Meter) at 30 °C.The dry matter content of COB was determined after the drying of samples at 105 °C for 2.5 h in an oven . Ash content of COB was determined at 550 °C for 6 h. The oil contents of COB were determined according to the Soxhlet method using hexane as a solvent. The protein content of COB was assessed by an automated nitrogen analyzer  using the Dumas method and calculated by multiplying the obtained nitrogen value (N:6.25). The pH value of the sample was determined at 25 °C using a pH meter  in a suspension of 50%  was performed by Folin–Ciocalteu method identified by Singleton and Rossi (1965) . The TPCThe oil contents of the plant drink samples were determined according to the Gerber method. The protein content of the drink samples was assessed by the Kjeldahl method (N x 6.38). The pH of the drink sample was measured with a pH meter .The fat globule size distributions and zeta potential values were measured in the drink samples using Malvern Nanosizer . The samples were diluted to 500 folds with distilled water to prevent multiple scattering effects and were homogenized by an ultrasonic water bath for 1 min . The color parameters , gray (0), and green (−), and b*: yellow (+), gray (0), and blue (−)) of the plant drink samples were measured by a colorimeter  in the CIELAB system, and the measurements were carried out in triplicate at 25 °C . The colloidal stability of the plant drink samples was determined by phase separation when stored at 4 °C . COB driThe rheological measurements  of plant-based ice cream mixes were carried out by a stress-controlled rheometer  equipped with parallel-plate configuration to shear and Peltier heating/cooling system. The probe was selected as PP50 , and the gap size was arranged as 0.5 mm. Each rheological measurement was performed three times at 25 °C.−1 and 100 s−1. The steady shear rheological data were fitted to the model parameters and were calculated using the Power law model and nonlinear regression.τ value is the shear stress (Pa), K is the consistency coefficient (Pasn), γ is the shear rate (s−1), and n is the flow behavior index. The steady shear rheological properties of ice cream mixes were assessed at the shear rate between 0.1 s−1 angular velocity (ω). The storage modulus (G′) and loss modulus (G′′) were determined depending on the angular velocity of the samples. The power law model and nonlinear regression were used to calculate the dynamic rheological parameters.ω is angular velocity value (s−1), K′, K″ are consistency coefficient values (Pasn) and n′, n″ are flow behavior index values.The dynamic rheological analysis of ice cream mixes was performed with parallel plate configuration. The linear viscoelastic region was first identified using the amplitude sweep test, which was conducted at 0.1% and 100% strain. Based on the results, the frequency sweep test was performed at the 0.1–10 Hz frequency range and 0.1–64 s−1 for variable shear rate and 0.5 s−1 for constant shear rate. In determining the values, the linear viscoelastic region was taken into account, and in the samples, the linear viscoelastic region stops at 50 s−1. During the first 100 s of the first interval, the ice cream mixes were treated to a relatively low shear rate (0.5 s−1). The determined shear rate was applied to 150 s−1 for 40 s during the 2nd time interval. By exposing the samples to the low shear rate in the 1st time interval, the 3rd time interval investigated the dynamic rheological behavior of the ice cream mixes in the second time period. There was a change in the viscoelastic solid structure (G′) of the samples. A second-order structural kinetic model was used to simulate the behavior of samples in the 3rd time period.0 is the initial storage module value (Pa) in the 3rd time interval; Ge is the storage module at the moment when the product fully recovered, in other words, the storage module (Pa) at the moment when the product is fully balanced; and k is the thixotropic velocity constant.The 3-ITT rheological characteristics of ice cream samples were calculated as 150 sThe fat globule size distributions and zeta potential values were measured in the ice cream mixes using Malvern Nanosizer  and performed, as mentioned in The color parameters of the ice cream samples were calibrated with the CR-400 Chroma Meter, Konica, Minolta, Japan color measuring device, and the measurements were carried out in parallel from three different points . L* valuIn order to determine the volume increase in the ice creams, ice cream samples were placed in the measuring cylinder, which was tared in such a way that there was no space up to a certain volume. The same process was applied to each ice cream and weighed on a precision scale. Afterward, the ice creams were allowed to melt at room temperature. Melted ice creams were filled to the same volume in the same cylinder measuring cup. Afterward, the melted ice creams were weighed with precision scales. The equation used in the calculation of the volume increase in ice creams is indicated.zero), and temperature data were collected from −20 to +50 °C in a nitrogen atmosphere at a heating rate of 1 °C/min. As a reference, an empty pan was utilized. The nitrogen gas flow rates for cooling were 50 mL/min. The onset temperatures (T0), Tf, and ΔHf of the ice formation and melting transitions were determined. The onset temperatures are defined as the point at which the tangent and baseline connect to the left of the melting peak. The steepest slope’s temperature was used to calculate freezing points. Extrapolating the baseline under the peak by linking the flat baseline before and after the melting peak and integrating the peak over the baseline yielded the enthalpy of fusion.The thermal properties of plant-based ice cream samples were measured using a differential scanning calorimeter (DSC) by A DTA-DSC operating at atmospheric pressure  . Ice creThe sensory analysis was analyzed by 10 trained panelists, 5 of whom were male and 5 were female. After the panelists were informed about the product, fifty grams of ice cream samples (n = 7) were prepared for sensory evaluation. A table was used as a sensory evaluation form. The taste criteria of the ice cream samples were determined, such as aroma, cream taste, aftertaste, gummy structure, icy structure, roughness, foreign taste, color, melting in the mouth, and general acceptability. In sensory evaluations, they were asked to score on a scale ranging from 1 to 9 points. It was evaluated with the Hedonic scale of liking ((1): dislike extremely, (2): dislike very much, (3): dislike moderately, (4): dislike slightly, (5): neither like or dislike, (6): like slightly, (7): like moderately, (8): like very much, and (9): like extremely).The statistical analysis was carried out using the Statistica software program . All the analyses were carried out in triplicate. The standard deviation and mean value were presented in all tables. ANOVA was conducted to determine the differences in all parameters of ice cream samples. Duncan’s multiple comparison tests at 95% significance level was used to determine the effect of oil and COB concentration on ice cream characteristics. The fat, protein, and zeta potential values of COB drink were higher than those of the commercial coconut drink (CC drink). On the other hand, the pH and L* values of the CC drink were higher than the COB drink. The difference in pH values of drink samples can be attributed to the chemical structures and acid compounds of the sources from which they are obtained. The difference in L* value was probably due to the dark pigments naturally found in the structure of the raw materials of the drink and/or formed during the homogenization process. In addition, the particle size value of the COB drink was found to be lower than the commercial drink, indicating that an effective homogenization process was applied during COB drink production. The physical stabilities of the drink samples were 98% and 99% for COB drink and commercial drink, respectively. These results could be an indicator of the successful production of plant drink from COB.The effect on the flow curves of the plant-based ice cream mixes was assessed using the data of the steady shear rheological characteristics . For thin. As can be observed, the K value of the samples increased significantly as the oil and COB ratio increased, indicating a more viscous nature as a result of increased apparent viscosity in ice cream mixes. C1 had the lowest K value (9.05 Pasn), whereas the COB-3 exhibited the highest K value (17.69 Pasn). The oil/fat ratio is one of the vital factors affecting the consistency of emulsion products, such as ice cream mix [ream mix . In low-32) of ice cream mixes were determined between 1502.26 µm and 4.629.67 µm. These results indicated that the use of COB could have a positive effect on the fat droplet size and zeta potential values.The zeta potential values were utilized to estimate the stability of the emulsions by measuring the level of electrical repulsion force between particles. A high zeta potential suggests strong stability, whereas a low zeta potential indicates low stability because flocculation and close contact are prevented by a low repulsive force between the droplets . TherefoThe dynamic rheological properties of ice cream mixes were investigated to evaluate the solid or liquid nature of ice cream samples. The frequency sweep test can simulate the solid/liquid behavior of ice cream samples while being chewed in the mouth , which e2 values were confirmed to be more than 0.95. As shown in The three interval thixotropy test (3-ITT) simulates the sudden shear stress/shear rate deformation of ice cream mixes and gives information about the structural recovery of ice cream mixes following abrupt deformation. 0, Ge, k, Ge/G0) are shown in e/G0. The greatest G0, Ge, Ge/G0, and k values were found in samples C1 and COB-3, indicating that sample COB-3 exhibited the most thixotropic behavior. The COB showed similar recovery characteristics with the high-oil control sample. The tendency to recover is greater the higher the thixotropic rate constant value signifies samples. These findings suggested that COB may be used to enhance the ice cream mix’s recoverable attributes.The second-order structural kinetic model’s parameters (Gp < 0.05). The highest a* value of the ice cream samples was observed with 0.32 in C3 and the lowest a* value of the C1 was observed. The a* values of ice cream samples below zero indicate that green color is more dominant than red in these samples. The effect of the oil and COB content used on the a* values of the samples were found to be significant (p < 0.05). Whereas the highest b* value was observed in COB 3, the lowest b* value was observed in the C3 sample and the b* values of the samples were found to be statistically significant (p < 0.05). It is thought that the difference between the color values of the samples, especially the L*, a*, and b* values, is due to the amount of oil and COB. When Ürkek [Consumer taste is defined by certain visual and taste criteria. As a result, color is a significant aspect of the first impression when consumers accept or reject a food product based on their sensory expectations. Color parameters  of ice cream samples were presented in en Ürkek  examinedp < 0.05) on the overrun values. The sample of the COB-C3 and COB3 containing 3% of COB and 2.5% oil showed a higher overrun value than C2 (7.5% oil). The increase in overrun was supplied so that it increases as the oil content increases and it increases further with the increase in the COB amount. Tekin et al. (2021) [The overrun values of plant-based ice cream samples were between 22.02 and 28.33% . Statist. (2021)  reported. (2021) .Overrun is related to the quantity of air introduced into the ice cream manufacturing process . Chang aonset, Tend, Tf, ΔT, and ΔHf,) associated with ice crystal melting of ice cream samples were determined by DSC. Tonset, Tend, and Tf were between −13.66 and −61.11 °C, 4.45 and 5.26 °C, and −4.60 and −3.31 °C, respectively. Melting resistance is the capacity of ice cream to withstand melting when subjected to high temperatures. The temperature triggers the creation of an endothermic peak provided by the DSC’s heating system. The amount of energy leaving the system is represented by the melting enthalpy (ΔHf), which is determined by integrating with the endothermic peak’s area. ΔHf values of the ice cream samples ranged from 144.70 J/g to 172.70 J/g. Depending on whether the water in the ice cream is free or bound, ice creams with higher frozen water will have ice formation, resulting in a reduction in enthalpy. Ice creams with lower frozen water have less melting enthalpy [f value (172.70 J/g). The increase in the liquid fraction with the decrease in the fat ratio may have caused an increase in the amount of frozen water and thus an increase in the freezing enthalpy. The samples stabilized with COB contained less oil content but showed similar thermal properties to the oily sample. The ΔHf value decreased with increasing COB content. This result can be explained by the water retention of the protein and polysaccharides in the COB and the decrease in the amount of freezable water. In general, the temperature range (ΔT) is a measure of the uniformity of ice crystal size distribution. Therefore, a narrow melting temperature range represents a more homogenous distribution of ice crystals melting at a lower temperature range [enthalpy . Therefore range . ΔT wereThe sensory parameters of plant-based ice cream samples were presented in There was no significant difference in the general acceptability between the COB-containing sample (COB 1–2–3) and control samples . This demonstrated that the color difference noticed in the low-fat ice cream samples with the addition of full-fat COB-C3 and COB had no negative impact on the ice cream’s consumability. As the consistency increases in ice cream samples, the melting resistance increases. Oil content is very important for ice cream samples because the quantity of fat aggregates was substantially linked with ice cream melting resistance. Melting resistance was found in the ice cream with the highest melting resistance. Similar to our study, Atik, Tekin Cakmak  revealedThe plant-based product consumption has increased in recent years thanks to ethical reasons, lactose intolerance, and diet preferences (vegan or vegetarian). However, plant-based drinks are more expensive compared to cow drink. Therefore, a significant option to lower the price of plant drink products may be found in the utilization of cold-pressed coconut oil by-products in the manufacturing of plant-based drinks. The oil industry may also benefit from gaining a valuable by-product identity. Based on its physicochemical and bioactive properties, COB can be preferred for successful plant drink production. The fat, protein, and zeta potential values of the COB drink were higher than those of the commercial coconut drink. All ice cream samples showed shear thinning (pseudoplastic), solid-like structure, and recoverable characters, which are expected rheological properties from conventional ice cream. In this study, it has been shown that ice cream samples produced from a COB drink can be developed without negatively impacting their sensory qualities, and sensory properties similar to full-fat ice cream can be obtained by using COB as a stabilizer in low-fat ice cream production. This study demonstrated that COB may be successfully employed in plant-based drink manufacturing and low-fat plant-based ice cream, providing a fresh viewpoint for the evaluation of this low-economic by-product.


However, the inhibitory mechanism of H2S on ROCK2 expression is still unclear. The study aimed to investigate the target and mechanism of H2S in inhibition of ROCK2. Methods: His-ROCK2wild protein was constructed, expressed, and was used for phosphorylation assay in vitro. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used to determine the potential phosphorylation sites of ROCK2. Recombinant ROCK2wild-pEGFP-N1, ROCK2T436A-pEGFP-N1, and ROCK2S575F-pEGFP-N1 plasmids were constructed and transfected into rat hippocampal neurons (RHNs). ROCK2 expression, cell viability, the release of lactate dehydrogenase (LDH), nerve-specific enolase (NSE), and Ca2+ were detected to evaluate the neuroprotective mechanism of H2S. Results: Phosphorylation at Thr436 and Ser575 of ROCK2 was observed by mass spectrometry when Polo-like kinase 1 (PLK1) and protein kinase A (PKA) were added in vitro, and NaHS significantly inhibited phosphorylation at Thr436 and Ser575. Additionally, NaHS significantly inhibited the expression of ROCK2 and recombinant proteins GFP-ROCK2, GFP-ROCK2T436A, and GFP-ROCK2S575F in transfected RHNs. Compared with empty plasmid, GFP-ROCK2T436A, and GFP-ROCK2S575F groups, NaHS significantly inhibited the release of LDH, NSE, and Ca2+ and promoted ROCK2 activity in the GFP-ROCK2wild group. Thr436 and Ser575 may be dominant sites that mediate NaHS inhibition of ROCK2 protein activity in RHNs. Compared with the empty plasmid, GFP-ROCK2T436A, and the GFP-ROCK2S575F group, NaHS had more significant inhibitory effects on hypoxia/reoxygenation (H/R) injury-induced cell viability reduction and increased LDH and NSE release in the GFP-ROCK2wild group. Conclusion: Exogenous H2S protected the RHNs against H/R injury via Thr436 and Ser575 of ROCK2. These findings suggested that Thr436 and Ser575 may be the dominant sites that mediated the effect of NaHS on protecting RHNs against H/R injury. Background: H Ischemic cerebral stroke is due to an interruption in cerebral blood flow, which is induced by thrombosis or embolism. There was an increased incidence of ischemic cerebral stroke with increasing age worldwide, and the treatment cost of ischemic cerebral stroke is the largest economic burden in East Asia . Restori2S), a novel gaseous signaling molecule, is known as a gasotransmitter in mammals in addition to nitric oxide and carbon monoxide i in each type of the transfected RHN. There was no significant difference among the inhibitions of Nif in the four types of the transfected RHNs. However, the inhibition of NaHS in the ROCK2wild-pEGFP-N1-transfected RHNs was significantly stronger than that in the empty plasmid-transfected RHNs. The inhibition of NaHS in either the ROCK2T436A-pEGFP-N1- or the ROCK2S575F-pEGFP-N1-transfected RHNs was remarkably weaker than that in the ROCK2wild-pEGFP-N1-transfected RHNs. The result indicated that Thr436 and Ser575 of ROCK2 are engaged with the inhibitory effect of H2S on [Ca2+]i in RHNs.As shown in 2 phosphorylation at related sites in the inhibitory effects of H2S on ROCK2 activation and against H/R injury in RHNs. The main findings include: (1) H2S inhibits the phosphorylation of ROCK2 at the Thr436 and Ser575 sites in vitro; (2) the Thr436 and Ser575 sites of ROCK2 mediate the inhibition of H2S on ROCK2 activation in RHNs; and (3) Thr436 and Ser575 of ROCK2 participate in the protective effect of H2S against an H/R injury via attenuating [Ca2+]i in RHNs.The present study focused on the roles of ROCK2S on the phosphorylation of ROCK2 at some sites, prokaryotic plasmid ROCK2wild-pET-32a(+) was constructed and transfected into E. colis to express His-ROCK2wild protein in the present study. By using Coomassie brilliant blue staining and Western blot assays, His-ROCK2wild protein was detected in the lysate of the E. colis and the lysate precipitation but not in the supernatant, suggesting that His-ROCK2wild protein exists in an insoluble form, which may be because ROCK2 is a macromolecular protein composed of more than 1300 amino acid residues. Phosphorylation refers to the transfer of phosphorylation of adenosine triphosphate (ATP) or GTP at the γ position to amino acid residues of substrate protein catalyzed by protein kinase. Phosphorylation regulation mainly occurs on serine, threonine, and tyrosine . In the 2wild protein in vitro. However, the phosphorylation of these three sites did not take place in the absence of PLK1 or PKA, indicating that the phosphorylation of His-ROCK2wild at the three sites cannot happen by autophosphorylation. As far as we know, the phosphorylation of ROCK2 at Ser1099 was reported previously i is a key factor with a regulatory role in a variety of cell functions. Intracellular [Ca2+]i overload plays a major role in the cerebral I/R process [2+]i was significantly inhibited by treatment with the ROCK inhibitor fasudil or Y-27632 [2+]i in each type of the transfected RHNs in the absence and presence of NaHS was detected. The result indicated that Thr436 and Ser575 of ROCK2 are engaged with the inhibitory effect of H2S on H/R-increased [Ca2+]i in RHNs. Taken together with the result of the inhibition of H2S on the activation of ROCK2, our results suggested that the inhibition of H2S on the increased [Ca2+]i may be owed to its attenuation on the phosphorylation of ROCK2 at Thr436 and Ser575. This may be related to the protection of H2S against H/R injury in RHNs.The i fluorescence intensity in the RHNs was measured using a Ca2+ imaging system fluorescence microscope.RHNs were incubated with a final concentration of 10 μM of Fluo-8 AM for 10 min at room temperature and then washed three times with PBS buffer. The i via inhibiting the phosphorylation of ROCK2 at Thr436 and Ser575 and subsequently protects RHNs from H/R injury. Our findings not only contribute to a deeper understanding on the mechanism of H2S against cerebral I/R injury but also provide potential targets for the treatment of ischemic cerebral stroke.The present study is the first attempt to explore the regulation of H