Paurodontella, P. composticola n. sp., collected from Nazar Abad City, Alborz Province, Iran, is described and illustrated. The new species has a body length of 803–1053 μm  and 620 and 739 μm . The cuticle is weakly annulated with four lateral lines. Cephalic region is annulated and continuous with body contour. The stylet is 8.0 to 9.0 μm long with asymmetrical knobs. Esophageal basal bulb is present with a small posterior extension projecting into the intestine. Excretory pore is situated at the level of esophageal basal bulb region. Post-uterine sac is 5 to 8 μm long and uterus is without diverticulum. Tails of both sexes are similar, short and sub-cylindrical. Males have 24 to 25 μm long bursa leptoderan and spicules 24 or 25 µm long. A non-branching oviduct is present to form a uterine diverticulum; the new species is closely related to five known species of the genus, namely P. asymmetrica, P. balochistanica, P. densa, P. iranica and P. niger. It most closely resembles P. iranica, but differs from it morphologically by a shorter stem-like extension projecting into lumen of intestine and male with sub-cylindrical tail vs conoid. In addition to morphological comparisons, the molecular phylogenetic analyses based on 733 bp of the partial sequence of 28S D2/D3 expansion segments of the large subunit rDNA gene (LSU) revealed this as a new species.A new species of the genus  Paurodontella  or present only in rudimentary form, simple vulval lips and bursa not being leptoderan . Specimens for light microscopy were killed by gentle heat, fixed in a solution of 4% formaldehyde + 2% glycerol and transferred to anhydrous glycerin, according to Specimens of 2O, 2 µl 10× PCR buffer and 2 µl proteinase K (600 µg/ml)  and crushed for 2 min with a micro-homogeniser, Vibro Mixer . The tubes were incubated at 65°C for 1 hr, then at 95°C for 10 min. In total, 1 µl of extracted DNA was transferred to an Eppendorf tube containing 2.5 µl 10× NH4 reaction buffer, 0.75 µl MgCl2 (50 mM), 0.25 µl dNTPs mixture (10 mM each), 0.75 µl of each primer (10 mM), 0.2 µl BIOTAQ DNA Polymerase  and ddH2O with a final volume of 25 µl. The 28 S D2/D3 was amplified using forward primer D2A (5′-ACA AGT ACC GTG AGG GAA AGT TG-3′) and reverse primer D3B (5′-TCG GAA GGA ACC AGC TAC TA-3′)  .PCR cycle conditions were as follows: one cycle at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing temperature of 55°C for 45 s, extension at 72°C for 3 min, and finally one cycle at 72°C for 10 min. PCR products were purified after amplification using ExoSAP-IT , quantified using a Nanodrop spectrophotometer  and used for direct sequencing in both directions using the same PCR primers. The resulting products were purified and run on a DNA multicapillary sequencer , using the BigDye Terminator Sequencing Kit v.3.1 , at the Stab Vida sequencing facilities . The newly obtained sequence was submitted to the GenBank database under accession number MH427864 for partial 28S D2/D3 rDNA.Purodontella composticola n. sp. was compared with those of other nematode species available in GenBank using the BLAST homology search program. Our newly obtained DNA sequence was edited with ChromasPro1.5 2003-2009  and aligned using ClustalW . All available species of Paurodontella and some other Hexatylina species from GenBank were also selected for phylogenetic analysis .The molecular sequence of analysis . Outgrouanalysis . The basanalysis  based onanalysis . Bayesiaanalysis , runninganalysis  using thPaurodontella composticola n. sp.–3V = 87 to 91%. Reproductive system is prodelphic, with an outstretched gonad. Vagina is muscular, directed forward, and 8 to 9 μm long. Crustaformeria is composed of 8 to 10 rows. The oviduct is non-branching so as to form a uterine diverticulum. Spermatheca is spherical with round sperm cells 2.2 to 3.2 μm in diam.; in young females, it is empty. Oocytes are present in a single and two rows at the proximal end. Ovary is reflexed at the tip in some individuals, in young females, it is outstretched. A short PUS is present. The tail is short, sub-cylindrical with broadly rounded tip, ventrally curved. Phasmids are not observed.They are free-living. The body is slender or semi-obese, slightly ventrally curved after fixation. Cuticle is finely annulated, 1.3 to 2.2 μm wide at mid-body. The lateral field is marked by four incisures; outer incisures strongly crenate and wider, inner weakly crenate. Cephalic region is wide, rounded, annulated and continuous with body contour, 2.5 to 3.0 μm high, 6.0 to 7.0 μm wide. Labial plate is flattened. Stylet is delicate with asymmetrical knobs, with the subventral knobs being larger than the dorsal. Stylet length is almost equal to the head width; conus is occupying 36 to 46% of the total stylet length. Deirids are present at the level of excretory pore. The dorsal gland orifice is 1.5 to 2.5 μm posterior to stylet knobs. Esophageal corpus is a cylindrical tube with a long and fusiform median bulb, without valvular apparatus; isthmus is cylindrical and slender, encircled by nerve ring. The excretory pore is located at the level of the basal esophageal bulb region, located at 116 to 144 μm far from anterior end. Esophageal basal bulb is present with small posterior extension, 6.0 to 8.0 μm long, projecting into the intestine. Hemizonoids are distinct, situated 5.0 to 7.0 μm anterior to the excretory pore. Vulva is near anus as a transverse slit, posterior, They are rarely found . They are very similar to females in general morphology, except for the reproductive system. Cuticle is present with fine annulation, about 1.5 to 1.8 μm apart. The tail is conical, with rounded tip without mucron, arcuate after fixation. Testis is single, outstretched. Spermatocytes are arranged in double rows. Spicule is cephalate, ventrally arcuate. Gubernaculum is simple. Bursa is annulated, leptoderan.Not found.Females and males are found in compost from a few places where mushrooms failed to grow in Nazar Abad City, Alborz Province, Iran, during August 2017. .Holotype female, two paratype females and two paratype males (Slides PCS001 and PCS002) were deposited in Nematode Collection of Department of Plant Protection, College of Agricultural and Natural Resources, University of Tehran, Karaj, Iran. Two female paratypes were deposited in National Nematode Collection of the Department of Nematology, Iranian Research Institute of Plant protection, Tehran, Iran. Two paratype females were deposited in USDA Nematode Collection, Beltsville, MD, USA.The species epithet refers to Latin name of compost, the origin of species, where the type specimens were collected.Paurodontella composticola n. sp. is an amphimictic species characterized by the following characters: a large body length of 803 to 1,053 μm  and 620,739 μm , lateral fields marked by four incisures, stylet 8 to 9 μm long, stylet with minute and asymmetrical basal knobs, a short stem-like extension projecting into lumen of intestine, excretory pore being located at the level of esophageal basal bulb region ca 116 to 144 μm from anterior end in females, the absence of a uterine diverticulum branch, V = 87 to 91, the presence of a short PUS, sub-cylindrical tail, rare male with 24 to 25 μm long spicules and leptoderan bursa.Paurodontella can be divided into those with/without a uterine diverticulum. Due to the presence of a relatively long body, short PUS, shape of posterior body and a non-branching oviduct to form uterine diverticulum, the new species P. composticola n. sp. is closely related to five known species in the genus: P. asymmetrica, P. balochistanica, P. densa, P. iranica and P. niger.According to Paurodontella asymmetrica, the new species has a shorter stylet, 8 to 9 vs 13 to 14 μm, a longer body length, 803 to 1,053 vs 330 to 380 μm, a greater a-ratio, 18.6 to 31.5 vs 16 to 19, a sub-cylindrical tail vs conical tail with pointed end, a higher c-ratio, 17.8 to 32 vs 10 to 11, more posteriorly located vulva, V = 87.5 to 91.5 vs 81 to 83, and presence of male in population vs absence. It differs from P. balochistanica by having a shorter stylet, 8.0 to 9.0 vs 10 to 11 μm, with asymmetrical knobs vs symmetrical, a lower a-ratio, 18.6 to 31.5 vs 33 to 34, lateral field with four incisures wherein outer incisures crenate and inner smooth vs lateral field with four smooth incisures, sub-cylindrical tail vs conoid tail with a terminal mucron, higher c-ratio, 17.8 to 32 vs 17.1 to 17.8, more posteriorly located vulva, V = 87.5 to 91.5 vs 88.0 to 88.6, and presence of males vs absence. It differs from P. densa by having a longer body, 803 to 1053 vs 400 μm, labial plate flattened vs rounded, PUS present vs absent, lateral field with four vs six lines, posteriorly located vulva, V = 87.5 to 91.5 vs 82, sub-cylindrical tail vs subacute terminus. It differs from P. niger by having a longer body, 803 to 1053 vs 400 μm, lateral field with four incisures vs six incisures, more posteriorly located vulva, V = 87.5 to 91.5 vs 80, and sub-cylindrical tail vs conical with pointed terminus tail. The new species most closely resembles P. iranica but differs from it by having an esophageal basal bulb with a short stem-like extension, 6.0 to 8.0 μm, vs a long stem-like extension, 16 to 35 μm, into the lumen of intestine and male tail sub-cylindrical vs conical with rounded tip.Compared to Paurodontella is close to other members of the family Paurodontidae, including species of genera Abursanema , Misticius , Paurodontus .Due to the presence of non-valvate median esophageal bulb and basal bulb with an extension projecting into the intestine, ursanema , Bealiusrodontus , and PauPaurodontella composticola n. sp. can be distinguished from Abursanema by having a stylet with basal knobs (vs stylet without knobs) and the presence of bursa (vs absence or rudimentary). The new species differs from Bealius by having an esophageal basal bulb with a stem-like extension projecting into the intestine ventrally  and leptoderan bursa vs peloderan. If differs from Misticius by having four incisures in lateral field  and excretory pore opening near the basal bulb level (vs near the stylet base). Genus Paurodontella can be distinguished from Paurodontus by having the stylet with asymmetrical basal knobs , and by the presence of a rudimentary PUS (vs prominent). It can also be distinguished from Paurodontoides by having a rudimentary PUS (vs prominent) and having a leptoderan (vs peloderan) bursa.Paurodontella composticola n. sp. specimens yielded a single fragment of approximately 800 bp based on gel size.The amplification of the partial 28S D2/D3 rDNA gene sequence from Paurodontella composticola n. sp. with other nematode species, an edited long partial sequence of 733 bp of 28S rDNA with accession number MH427864 was used. The BLAST search revealed that the sequence is unique with no highly matched sequence available in GenBank; only 93% identity was achieved with P. iranica with accession number of KP642168. When the new species was compared for the 28S D2/D3 region of P. iranica, they shared 677 identical nucleotides (93.01%), 27 insertions/deletions (3%) and 1 indel over 731 total characters. The phylogenetic tree generated from 28S D2/D3 alignment by BI analysis under the TVM+I+G model is presented in Cephalenchus nemoralis  (KU723248) showed P. composticola n. sp. in a monophyletic clade with P. iranica (KP642168) with 100% support. This clade is close to a highly supported (99%) monophyletic clade, including species of the subfamily Paurodontinae (Paurodontoides and Paurodontella) and Sphaerulariinae (Abursanema and Sphaerularia), revealing the polyphyly of these genera based on limited species available in GenBank. This is supported by the morphological similarities of these taxa, mostly in the shape of the esophagus . Paurodontella persica is not in a monophyletic clade, which revealed that their morphological similarity is convergence in evolution and they are not a monophyletic group. According to Sphaerularia (Sphaerulariidae) and the new species (as a member of Paurodontidae) were clustered together with 70% of posterior probability, our phylogenetic study based on partial 28S D2/D3 appears to support this opinion. This finding is supported by previous studies as well  and basal pharyngeal bulb extension (shorter in Paurodontus) are the main morphological characters to separate these genera. Paurodontella composticola n. sp. is unique within the genus by having a stylet with asymmetrical knobs, a short esophageal basal bulb extension and a uterine diverticulum through a non-branching oviduct. However, the importance of these features for separating species in Paurodontella has to be reinforced by molecular studies on more species of the genus.an bursa . Handoo Paurodontella are clustered in this study, is also seen with several genus such Abursanema (Paurodontinae), Deladenus  are in separate clades, distantly related to each other. This polyphyly, regarding how the new species and current species of eladenus  (Neotyleowardula  (Allantozovinema  (Rubzovishkinema  (Sphaerushkinema . Likewisshkinema .In conclusion, due to the very limited number of Hexatylina species molecularly characterized, the relationships among subfamilies Paurodontinae and Sphaerulariinae are still not clearly resolved, despite of the sister relationship of members. Further evidence using valuable morphological diagnostic characters and molecular studies with different markers is needed to confirm.


The spatial-numerical association of response codes (SNARC) effect refers to the phenomenon that responses involving small numbers are faster with the left hand and responses involving large numbers are faster with the right hand. Previous studies have investigated the mechanism of the SNARC effect only when the time sequence is induced by centrally presented successive numbers. No study has investigated the mechanism of the SNARC effect when the spatial sequence is induced. Given that the induction of a spatial sequence together with a time sequence provides a new temporary reference for the serial order to be coded in working memory (WM), it would be interesting to examine the SNARC effect when both the time sequence and spatial sequence are induced. Therefore, a novel priming paradigm that simultaneously induced the time sequence and spatial sequence was employed in the present study to investigate the mechanism of the SNARC effect. Specifically, the time sequence and spatial sequence were induced by presenting a series of self-paced successive numbers, centrally or in a left-to-right or right-to-left direction, on the screen. Following the presentation of successive numbers, the probe number was centrally presented on the screen and university students were asked to distinguish to which time sequence or spatial sequence the probe number belonged by pressing a specified key of a qwerty keyboard. The results indicated that (1) the SNARC effect simultaneously appeared in the processing of the number magnitude and time sequence when only the time sequence was induced. (2) The SNARC effect disappeared in the processing of the time sequence; however, the SNARC effect coexisted in the processing of the numerical magnitude and spatial sequence when the spatial sequence was induced and participants performed a time sequence relevant task. (3) The SNARC effect coexisted in the processing of the numerical magnitude, time sequence, and spatial sequence when the spatial sequence was induced and participants performed a spatial sequence relevant task. Based on these results, we conclude that whether the SNARC effect coexists in the processing of the numerical magnitude, the time sequence and spatial sequence were influenced by the spatial sequence and relevant task. The results further support the mental whiteboard hypothesis and extended the WM account. Implications for theories on the SNARC effect were discussed. When participants were instructed to perform the task of classifying random Arabic numbers presented on a screen by pressing the left or right key of a keyboard with their left or right hand, they responded faster to small numbers with the left hand and faster to large numbers with the right hand, regardless of the classification standard that was set . This phenomenon is referred to as the spatial numerical association of response codes (SNARC) effect, and it was first identified by It was argued that the numbers were represented on the MNL from left to right according to the numerical magnitude . This MNAlthough the MNL account was largely supported, researchers reported that the SNARC effect could be reversed or disappeared . For exaAlthough the LTM-dependent MNL plays an important role in the origination of the SNARC effect, recent studies have indicated that the serial order in WM could also activate the SNARC effect . For exaHow is serial order processed within WM? According to the mental whiteboard hypothesis, in the processing of serial order, each item in a sequence is bound to a specific position marker. The conjunctions will be recalled in a later retrieval task . AbrahamSerial order consists of both a time sequence (on a time dimension) and a spatial sequence (on a space dimension). Previous studies have investigated the mechanism of the SNARC effect only when the time sequence was induced by centrally presented successive numbers . No studThe current study consisted of three main experiments. The first experiment adopted a task-relevant time sequence, in which the participants were required to judge the time sequence  of a probe digit when the time sequence was induced by a centrally presented series of self-paced successive numbers that consisted of five Arabic numbers on a computer screen. The purpose of Experiment 1 was to replicate the results of the previous research to examine the feasibility of our novel paradigm. Previous research has indicated that the SNARC effect coexists in the processing of both numerical magnitude and serial order in go tasks . Thus, wThe second experiment was designed to investigate whether the SNARC effect could occur in the processing of the numerical magnitude, time sequence, and spatial sequence simultaneously when participants were instructed to judge which time sequence  the probe numbers belong to following the presentation of successive numbers with a left-to-right or right-to-left presentation direction. It should be noted that the successive numbers were presented from left to right or right to left  on the computer screen to induce both the time sequence and spatial sequence. Moreover, it is possible that the time sequence and spatial sequence might correspond to and confound each other in a single presentation direction. For example, in the presentation of the successive numbers 1–2–5–8–9 from the left to right direction, 1, 2 belong to before 5 in the time sequence corresponding to belong to left in the spatial sequence if only from the left to right direction was used. Thus, the reverse direction was also employed to control the confounding effect of a single direction presentation. Given the involvement of the spatial sequence could provide a new temporary reference for serial order coding in WM, we expect the WM-related SNARC effect would be affected when both the time sequence and spatial sequence were induced.The third experiment was designed to further determine whether the SNARC effect occurs in parallel in the processing of the numerical magnitude, time sequence, and spatial sequence when participants were instructed to determine which spatial sequence the probe number belonged to instead of the time sequence used in the second experiment.SD = 3 years, with a range from 18 to 29 years). All participants were right-handed and had normal or corrected-to-normal vision. They all exclusively used a left-to-right reading/writing direction. Participation in the study was completely voluntary. Informed consent was obtained prior to starting the experiment. Students who participated received bonus research marks toward their final grade in a psychology course. None of the students had recently participated in similar experiments. The experimental protocol was approved by the ethics committee of the Shanghai psychological society.The participants included 26 university students  from Northeast Normal University, China. The average age was 23.12  printed in Times New Roman font (72 in size). The five digits were presented in various sequences at the rate of 1 digit per second. The stimuli were presented on a 19” computer screen running at 1024 × 768 resolution, with a refresh rate of 60 Hz.Figure 1). The respondents had 3 s to respond to each digit.The experiment was compiled with E-prime1.0 and was composed of two stages, including an initiating stage and a classification stage. In the initiating stage, a series of successive numbers that consisted of five Arabic numbers  were continuously presented in the center of the computer screen at the frequency of one number per second in black type against a white background. Participants were instructed to memorize all digits in the correct order. A numerical sequence of five numbers was selected for the experiment because the adult memory capacity is 7 ± 2 blocks . In the The successive numbers presented sequence was arranged as follows: 1–2–5–8–9; 2–8–5–9–1; 8–9–5–1–2; and 9–1–5–2–8. Among the five digits, five was always presented as the third number, whereas the remaining four numbers were presented in various sequences. Each of the remaining four numbers was presented in every sequential position with an equal frequency to eliminate interference caused by a difference in the numerical magnitude in each position of the sequential position.The experiment was composed of two blocks. In the first block, the subjects were instructed to press the left key with the left index finger on the qwerty keyboard in response to digits before five in the memory sequence or the right key with their right index finger in response to digits after five in the memory sequence. The second block was the opposite . The left and right keys corresponded to the “F” and “J” keys of the qwerty keyboard and were covered with stickers to prevent a potential confound caused by letters because letters of the alphabet are spatially coded similarly to numbers . The twoM = 3.89%, range: 0.6–7.5%). Moreover, there was no speed–accuracy trade-off because the correlation between the reaction time and error was positive over all trials, r(26) = 0.48, p < 0.05. Therefore, no further error rate analyses were conducted. The mean response times (RTs)  were analyzed in an analysis of variance (ANOVA) with 2 [numerical magnitude: small  vs. large ] × 2 (time sequence: before 5 vs. after 5) × 2 (side of response: left key vs. right key) as within subject factors.The overall error rate of the experiment was very low  = 5.80, p < 0.05, η2 = 0.188. A simple effect analysis showed that small numbers (596 ± 24.48 ms) were responded to faster than large numbers (684 ± 28.78 ms) with the left key, F = 13.29, p < 0.01, η2 = 0.347. By contrast, large numbers (608 ± 23.20 ms) were responded to faster than small numbers (669 ± 26.67 ms) with the right key, F = 5.84, p < 0.05, η2 = 0.189, which indicates the SNARC effect was identified in the processing of the numerical magnitude . Moreover, there was a significant interaction between the time sequence and side of the key, F = 7.25, p < 0.05, η2 = 0.225. A simple effect analysis showed that digits before five (637 ± 24.02 ms) were responded to slightly faster than digits after five (643 ± 24.41 ms) with the left key, F = 0.41, p > 0.05, η2 = 0.016. By contrast, digits after five (624 ± 20.78 ms) were responded to faster than digits before five (653 ± 23.52 ms) with the right key, F = 7.30, p < 0.05, η2 = 0.226, which indicates the SNARC effect was identified in the processing of the time sequence . In addition, a significant interaction was identified between numerical magnitude and time sequence, F = 12.17, p < 0.01, η2 = 0.327. A simple effect analysis showed a consistent effect that small numbers (631 ± 24.60 ms) were responded to slightly faster than large numbers (634 ± 24.46 ms) in the time sequence of before five, F = 0.05, p > 0.05, η2 = 0.002, whereas large numbers (633 ± 22.23 ms) were responded to faster than small numbers (659 ± 23.34 ms) in the time sequence of after five, F = 13.34, p < 0.01, η2 = 0.348. No significant interaction was identified among the numerical magnitude, time sequence, and side of response. In conclusion, the results of the first experiment indicated the SNARC effect appears simultaneously in the processing of the numerical magnitude and time sequence. This result replicated the results of previous studies  and instructed subjects to judge whether the probe number centrally presented on the screen in the classification stage was presented before five or after five in the initiating stage to investigate whether the SNARC effect can coexist in the numerical magnitude, time sequence, and spatial sequence.SD = 2.75 years, with a range from 18 to 28 years). All participants were right-handed and had normal or corrected-to-normal vision. They all exclusively used a left-to-right reading/writing direction. Participation in this study was completely voluntary. Informed consent was obtained prior to starting the experiment. Students who participated received bonus research marks toward their final grade in a psychology course. None of the participants had recently participated in a similar experiment. The experimental protocol was approved by the ethics committee of the Shanghai psychological society.The participants included 48 university students  from Northeast Normal University, China. The average age was 21.79 . The horizontal spatial position of each number was at 20, 35, 50, 65, and 80% of the screen width, respectively . The task of this experiment was to judge the time sequence; specifically, participants were instructed to judge whether the probe number centrally presented on the screen in the classification stage was presented before five or after five in the initiating stage.M = 3.11%, range: 0.39–10.94%). Moreover, there was no speed–accuracy trade-off because of an indistinctively positive correlation between the reaction time and error over all trials, r(48) = 0.27, p > 0.05. Thus, we did not further analyze the error rate in this experiment. The mean RTs  were analyzed via an ANOVA with 2 [numerical magnitude: small  vs. large ] × 2 (time sequence: before 5 vs. after 5) × 2  × 2 (side of response: left key vs. right key) as within subject factors.The overall error rate was very low  = 48.55, p < 0.001, η2 = 0.508, in which digits presented before 5 (617 ± 20.97 ms) were responded to faster than after 5 (675 ± 24.17 ms), which suggests a serial scanning strategy. There was a significant interaction between the numerical magnitude and side of response, F = 4.64, p < 0.05, η2 = 0.09. A simple effect analysis showed a classical SNARC effect exhibited in the processing of the numerical magnitude, in which small numbers (642 ± 22.78 ms) were responded to slightly faster than large numbers (653 ± 22.85 ms) with the left key, F = 2.04, p > 0.05, η2 = 0.042, and large numbers (639 ± 21.66 ms) were responded to faster than small numbers (651 ± 23.31 ms) with the right key, F = 4.98, p < 0.05, η2 = 0.096 . There was a significant interaction between the spatial sequence and side of response, F = 8.44, p < 0.01, η2 = 0.152. A simple effect analysis showed that digits presented on the left of the screen (641 ± 22.42 ms) were responded to faster than digits presented on the right of the screen (654 ± 22.89 ms) with the left key, F = 5.47, p < 0.05, η2 = 0.104, and digits presented on the right of the screen (638 ± 22.34 ms) were responded to faster than digits presented on the left of the screen (652 ± 22.82 ms) with the right key, F = 4.19, p < 0.05, η2 = 0.082; these findings indicate the SNARC effect was exhibited in the processing of the spatial position . A significant interaction was also identified between the numerical magnitude and time sequence, F = 27.66, p < 0.001, η2 = 0.37. A simple effect analysis showed a consistent effect that numbers smaller than five (605 ± 20.52 ms) were responded to faster than numbers larger than five (688 ± 25.79 ms) in the time sequence of before five, F = 61.26, p < 0.001, η2 = 0.566. By contrast, numbers larger than five (629 ± 21.70 ms) were responded to faster than numbers smaller than five (663 ± 22.97 ms) in the time sequence of after five, F = 16.18, p < 0.001, η2 = 0.256. No other main effects or significant interaction effects were identified in this experiment.There was a significant main effect in the time sequence, F = 48.55, p < 0.001, η2 = 0.508, in which digits presented before five (617 ± 20.97 ms) were responded to faster than after five (675 ± 24.17 ms), which suggests a serial scanning strategy. There was a significant interaction between numerical magnitude and side of response, F = 4.64, p < 0.05, η2 = 0.09. A simple effect analysis showed a classical SNARC effect exhibited in the processing of the numerical magnitude, in which small numbers (642 ± 22.78 ms) were responded to slightly faster than large numbers (653 ± 22.85 ms) with the left key, F = 2.04, p > 0.05, η2 = 0.042, and large numbers (639 ± 21.66 ms) were responded to faster than small numbers (651 ± 23.31 ms) with the right key, F = 4.98, p < 0.05, η2 = 0.096. There was a significant interaction between numerical magnitude and time sequence, F = 27.66, p < 0.001, η2 = 0.37. A simple effect analysis showed a consistent effect that numbers smaller than five (605 ± 20.52 ms) were responded to faster than numbers larger than five (688 ± 25.79 ms) in the time sequence of before five, F = 61.26, p < 0.001, η2 = 0.566. By contrast, numbers larger than five (629 ± 21.70 ms) were responded to faster than numbers smaller than five (663 ± 22.97 ms) in the time sequence of after five, F = 16.18, p < 0.001, η2 = 0.256. Moreover, there was a significant interaction among time sequence, side of response, and presentation direction, F = 8.44, p < 0.01, η2 = 0.152. A simple effect analysis indicated that there was no interaction between time sequence and side of response when successive numbers were presented from left to right, F = 0.08, p = 0.779, η2 = 0.002, or when successive numbers were presented from right to left, F = 1.66, p = 0.204, η2 = 0.034. However, the results indicated that there was a significant interaction between time sequence and presentation direction when responding to probe numbers with the left key, F = 4.19, p < 0.05, η2 = 0.104, or when responding to probe numbers with the right key, F = 5.47, p < 0.05, η2 = 0.082. A simple effect analysis showed that the digits presented before five (613 ± 20.94 ms) were responded to faster than its presented after five (675 ± 27.60 ms) with the left key in left–right condition, F = 11.26, p < 0.01, η2 = 0.193, but the digits presented before five (632 ± 22.29 ms) were responded to slightly faster than its presented after five (669 ± 26.95 ms) with the left key in right–left condition, F = 3.47, p = 0.069, η2 = 0.069. The digits presented before five (618 ± 22.23 ms) were responded to faster than its presented after five (672 ± 23.57 ms) with the right key in left–right condition, F = 10.28, p < 0.01, η2 = 0.179, the digits presented before five (605 ± 24.60 ms) were responded to faster than its presented after five (686 ± 26.46 ms) with the right key in right–left condition, F = 18.46, p < 0.001, η2 = 0.282. No other main effects or interaction effects were identified. In addition, to test whether the influence of numerical magnitude obscured the chance of finding a time sequence SNARC effect depending on presentation direction, for example, it is possible that the numerical SNARC would be present when presented successive numbers from left to right, overruling the time sequence SNARC, while in the right-to-left presentation direction, the numerical SNARC disappears, creating more chance of the time sequence SNARC to emerge. We further analyzed the SNARC effect of time sequence in each level of numerical magnitude and presentation direction. The results indicated that the SNARC effect of time sequence disappeared both in left–right condition and right–left condition, regardless of the numerical magnitude. Based on these results, we can eliminate the possibilities that the SNARC effect disappeared in the processing of the time sequence because analysis together with the left–right condition led to the effects that may cancel each other out and because of the influence of numerical magnitude.The SNARC effect was not identified in the processing of the time sequence in Experiment 2. It is possible that the time sequence coded in WM is coded according to the spatial direction during encoding because the use of space in WM is very flexible . As suchTherefore, in Experiment 2, we conclude that the SNARC effect could not coexist in the numerical magnitude, time sequence, and spatial sequence; however, it coexisted in the numerical magnitude and spatial sequence when subjects were instructed to judge whether the digit centrally presented on the screen in the classification stage was presented before five or after five in the initiating stage.In Experiment 2, we determined that the SNARC effect cannot coexist in numerical magnitude, time sequence, and spatial sequence when participants were instructed to judge whether the digit centrally presented on the screen in the classification stage was presented before five or after five in the initiating stage. The third experiment was designed to further investigate whether the SNARC effect coexists in the numerical magnitude, time sequence, and spatial sequence when participants are instructed to determine the spatial sequence instead of the time sequence used in Experiment 2.SD = 3.14 years, with a range from 16 to 30 years). All participants were right-handed and had normal or corrected-to-normal vision. They all exclusively used to a left-to-right reading/writing direction. Participation in the study was completely voluntary. Informed consent was obtained prior to starting the experiment. Students who participated received bonus research marks toward their final grade in a psychology course. None of the students had recently participated in similar experiments. The experiment protocol was approved by the ethics committee of the Shanghai psychological society.The participants included 46 university students  from Northeast Normal University, China. The average age was 22.05 . The horizontal spatial position of each number was at 20, 35, 50, 65, and 80% of the screen width, respectively . Participants were instructed to judge whether the digit centrally presented on the screen in the classification stage was presented on the left or right space in the initiating stage in this experiment.M = 4.24%, range: 0.78–11.33%). Moreover, there was no speed–accuracy trade-off because of an indistinctively positive correlation between the reaction time and error over all trials, r(46) = 0.07, p > 0.05. Thus, we did not further analyze the error rate in this experiment. The mean RTs  were analyzed via an ANOVA with 2 [numerical magnitude: small  vs. large ] × 2 (time sequence: before 5 vs. after 5) × 2  × 2 (side of response: left key vs. right key) as within subject factors.The overall error rate was very low  = 18.30, p < 0.001, η2 = 0.289, in which large numbers (601 ± 22.78 ms) were responded to faster than small numbers (621 ± 24.59 ms). There was a significant interaction between the numerical magnitude and side of response, F = 5.88, p < 0.05, η2 = 0.116. A simple effect analysis showed a classical SNARC effect exhibited in the processing of the numerical magnitude, in which small numbers were responded to slightly faster with the left key (616 ± 24.77 ms) than the right key (625 ± 24.94 ms), F = 1.78, p > 0.05, η2 = 0.038, and large numbers were responded to faster with the right key (594 ± 22.32 ms) than the left key (609 ± 23.76 ms), F = 4.64, p < 0.05, η2 = 0.093 . There was a significant interaction between the spatial sequence and side of response, F = 45.86, p < 0.001, η2 = 0.505. A simple effect analysis showed that digits presented on the left of the screen were responded to faster with the left key (574 ± 21.80 ms) than the right key (647 ± 28.02 ms), F = 36.14, p < 0.001, η2 = 0.445, and digits presented on the right of the screen were responded to faster with the right key (572 ± 19.96 ms) than the left key (650 ± 27.40 ms), F = 40.55, p < 0.001, η2 = 0.474, which indicates the SNARC effect was exhibited in the processing of spatial position . A significant interaction was identified between numerical magnitude and time sequence, F = 9.68, p < 0.01, η2 = 0.177. A simple effect analysis showed a consistent effect that small numbers were responded to slightly faster in a time sequence of before five (615 ± 23.64 ms) than after five (626 ± 26.52 ms), F = 1.08, p > 0.05, η2 = 0.024, whereas large numbers were responded to faster in a time sequence after five (591 ± 23.51 ms) than before five (611 ± 22.68 ms), F = 7.20, p = 0.01, η2 = 0.138. No other main effects or significant interaction effects were identified in this experiment.There was a significant main effect in the numerical magnitude, F = 45.86, p < 0.001, η2 = 0.505. A simple effect analysis showed a significant interaction between the time sequence and side of response both in left–right condition, F = 34.49, p < 0.001, η2 = 0.434, and right–left condition, F = 41.14, p < 0.001, η2 = 0.478. A further simple effect analysis indicated that when successive numbers were presented from left to right, the digits presented before five were responded to faster with the left key (562 ± 19.43 ms) than the right key (656 ± 27.21 ms), F = 42.06, p < 0.001, η2 = 0.483, and the digits presented after five were responded to faster with the right key (572 ± 24.76 ms) than the left key (626 ± 25.38 ms), F = 15.71, p < 0.001, η2 = 0.259, which suggests a classic SNARC effect in the processing of the time sequence . When successive numbers were presented from right to left, the digits presented before five were responded to faster with the right key (561 ± 18.59 ms) than the left key (674 ± 30.58 ms), F = 45.37, p < 0.001, η2 = 0.502, and the digits presented after five were responded to faster with the left key (586 ± 25.15 ms) than the right key (639 ± 29.82 ms), F = 19.06, p < 0.001, η2 = 0.298, which suggests a reversed SNARC effect in the processing of the time sequence when successive numbers were presented from right to left . In addition, to test whether the numerical magnitude influenced the chance of finding a time sequence SNARC effect depending on presentation direction, we further analyzed the SNARC effect of time sequence in each level of numerical magnitude and presentation direction. The results indicated that the classic SNARC effect of time sequence appeared in the left–right condition, and the reverse SNARC effect appeared in the right–left condition, regardless of the numerical magnitude. Therefore, in Experiment 3, we concluded that the SNARC effect coexisted in the processing of the numerical magnitude, time sequence, and spatial sequence when participants were instructed to determine the spatial sequence instead of the time sequence.Similar to Experiment 2, the SNARC effect was not exhibited in the processing of the time sequence in Experiment 3. It is possible that the SNARC effect disappeared in the processing of the time sequence because the effects may cancel each other out when analyzed together with the left–right condition or because of the strong impact of numerical magnitude and the interaction with time sequence. To test these possibilities, an ANOVA with 2 [numerical magnitude: small  vs. large ] × 2 (time sequence: before 5 vs. after 5) × 2 (presentation direction: left-to-right vs. right-to-left) × 2 (side of response: left key vs. right key) as within subject factors was conducted. There was a significant interaction among time sequence, side of response, and presentation direction, Previous studies have shown that whether the SNARC effect appears simultaneously in the processing of numerical magnitude and time sequence was mediated by the relevant task performed. Specifically, the SNARC effect could not coexist in the processing of serial order and magnitude in a go/no-go task . For exaThe numerical magnitude SNARC effect, which originated from the numerical spatial representation on the MNL in LTM, was identified throughout all three experiments in the present study through various tasks, including the task relevant time sequence and the spatial sequence. The results further support the viewpoint that the numerical magnitude SNARC effect could appear even when the numerical serial order was directly or indirectly activated . Our finIn addition, not only was the numerical magnitude SNARC effect elicited in our three experiments but also the spatial sequence SNARC effect, which relates to WM. These findings suggest that the numerical spatial representation on the MNL in LTM and the spatial sequence in WM independently elicits the SNARC effect. The reason may be that subjects were provided with a numerical position marker, which may help solve the task . Thus, tRecently, Furthermore, The SNARC effect widely exists in the processing of numerical magnitude and serial order; however, the mechanism may be different . We showIn summary, we can draw the following conclusions from this study: (1) the SNARC effect simultaneously appeared in the processing of the number magnitude and time sequence when only the time sequence was induced. (2) The SNARC effect disappeared in the processing of the time sequence; however, the SNARC effect coexisted in the processing of the numerical magnitude and spatial sequence when the spatial sequence was induced and participants performed a time sequence relevant task. (3) The SNARC effect coexisted in the processing of the numerical magnitude, time sequence, and spatial sequence when the spatial sequence was induced and participants performed a spatial sequence relevant task. Based on these results, we conclude that whether the SNARC effect coexists in the processing of the numerical magnitude, the time sequence and spatial sequence were influenced by the spatial sequence and relevant task.QW conducted the research under the guidance of WS and wrote the first draft of the manuscript. ML further modified and embellished the manuscript. JK gave strong support and help in the research.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.


Meanwhile, in our previous study, we revealed that a part of OZ is absorbed into the body and exists in its intact form. However, the comprehensive absorption profile of OZ and its metabolites  after OZ intake has not been fully elucidated yet. Therefore, in this study, we measured the concentrations of OZ, FA, and FA conjugates  in the blood of rats with the use of HPLC-MS/MS after a single oral administration of 300 µmol/kg body weight of rice bran OZ (RBOZ). As a result, intact OZ along with FA and FA conjugates existed in the blood, which implied that these constituents may all contribute to the physiological effects under OZ intake. Additionally, when an equimolar amount of FA (300 µmol/kg body weight) was administered, it was found that the absorption profile of FA was significantly different from that when RBOZ was administered. Since OZ was discovered from rice oil in 1954 ,3. In pa14C-labeled OZ and measured its radioactivity in rabbits [In order to further understand the physiological effects of OZ, it is important to reveal the abundance of OZ and its metabolites in the blood and target tissues in human and animals. To investigate the blood OZ profile, Fujiwara et al. orally administered  rabbits . The res rabbits . Taking To further prove this hypothesis, it is necessary to determine the profiles of OZ together with FA in biological samples after OZ intake. To the best of our knowledge, such information has not yet been available, except for our previous study in which we measured the plasma concentrations of OZ and FA at a single time point after administration of OZ . While iO-β-d-glucuronide disodium salt were obtained from Toronto Research Chemicals, Inc. , and FA 4-O-sulfate disodium salt was purchased from Carbosynth, Ltd. . Soybean oil was purchased from Wako Pure Chemical Industries, Ltd. All other reagents were of the highest grade available.RBOZ was received from Tsuno Food Industry Co., Ltd. . The purity of RBOZ as OZ equivalent was determined as 99.7% based on ultraviolet (UV) spectrophotometric (325 nm) analysis . Standarn = 6) and the second group was given an oral dose of FA dispersed in soybean oil (16.4 g/kg) via gastric intubation (300 µmol/kg body weight) . At 0, 0.5, 1, 3, 6, 9, and 12 h after sample administration, 440 µL of blood was collected via tail vein by capillary tubes. During the test period, all animals were given free access to distilled water. Plasma was isolated from the collected blood by centrifugation at 1000× g for 20 min at 4 °C and stored at −80 °C until use. This study was conducted according to protocols approved by the Institutional Committee for the Tohoku University Ethics Review Board (2018AgA-040).Male Sprague–Dawley rats  weighing 370–390 g were housed with free access to commercial chow diet  and distilled water in a room with controlled temperature (20–25 °C) and lighting (lights on from 08:00 to 20:00). After 1 week of acclimatization, rats were fasted for 12 h and divided into two groups: the first group was administered with RBOZ dispersed in soybean oil (32.8 g/kg) via gastric intubation (300 µmol/kg body weight)  containing 0.002% butylated hydroxytoluene was added to the diluted plasma. The mixture was subjected to centrifugation at 2000× g for 20 min at 4 °C resulting in the formation of two layers. The lower chloroform layer (lipid fraction) was collected, while the remaining upper aqueous layer was re-extracted using 1.4 mL of chloroform–methanol . The mixture was then subjected to centrifugation at 2000× g for 20 min at 4 °C to obtain two layers. The lower layer was collected and combined with the previously collected lower layer from the first extraction. The obtained total lipid extract was evaporated under nitrogen gas and re-dissolved in 1 mL of hexane–chloroform . To further purify the extract, 900 µL of the total lipid solution was loaded onto a Strata SI-1 Silica cartridge  equilibrated with 1.5 mL of hexane–chloroform . The cartridge was rinsed with 1.5 mL hexane–chloroform  and OZ was eluted with 1.5 mL of hexane–2-propanol . The eluent was evaporated under nitrogen gas, and the residue was dissolved in 200 µL of methanol. The solution was cooled on ice for 15 min and was filtered by Nanosep with Bio-Inert membrane . Finally, a 20-µL aliquot of the OZ fraction was subjected to HPLC-MS/MS analysis.Extraction of OZ was carried out based on our previous method ,13 with g for 10 min at 4 °C. The upper layer was collected, while 300 μL of 1% formic acid methanol solution was added to the remaining residue, vortexed, and subjected to centrifugation at 13,000× g for 10 min at 4 °C. The upper layer was collected and combined with the previously collected upper layer from the first extraction. Chloroform (200 µL) was added to the obtained methanol extract, evaporated under nitrogen gas, and re-dissolved in 1 mL of 1% formic acid methanol–water . To further purify the extract, 900 µL of the mixture was loaded onto a Strata-X column  conditioned with 1% formic acid methanol solution and equilibrated with 1% formic acid aqueous solution. After loading the sample, the cartridge was rinsed with 1.5 mL of 1% formic acid aqueous solution followed by elution of FA and FA conjugates with 1.5 mL methanol–water . Chloroform (4 mL) was added to the eluent, evaporated, and the residue was dissolved in 300 µL of acetonitrile–water . Finally, a 20-µL aliquot of this solution was subjected to HPLC-MS/MS analysis.For the extraction of FA and FA conjugates, plasma (40–80 µL) was added with 400 μL of 1% formic acid methanol solution, vortexed, and subjected to centrifugation at 13,000× The HPLC-MS/MS system consisted of a Shimadzu liquid chromatography system  and a 4000 QTRAP mass spectrometer . MS/MS parameters were optimized under negative ion electrospray ionization see  section.OZ molecular species were separated using a HPLC column  at 40 °C with a flow rate of 0.5 mL/min. Gradient elution was performed using two mobile phases: A, methanol containing 1% acetic acid; and B, 2-propanol. The gradient solvent system was as follows: 0–5 min, 0% B; 5.01–7.00 min, 100% B; 7.01–10.00 min, 0% B. The standard curves of OZ molecular species were prepared in the range of 1 to 100 fmol.FA and FA conjugates were separated using a HPLC column  at 40 °C with a flow rate of 0.3 mL/min. Gradient elution was performed using two mobile phases: A, water containing 0.1% formic acid; and B, acetonitrile containing 0.1% formic acid. The gradient solvent system was as follows: 0–7 min, 10–25% B linear; 7.01–9.50 min, 100% B; 9.51–12.00 min, 10% B. Total flow was set as follows: 0–7 min, 0.3–0.4 mL/min linear; 7.01–9.50 min, 0.4 mL/min; 9.51–12.00 min, 0.3 mL/min. The standard curves of FA and FA conjugates were prepared in the range of 0.01 to 60 pmol.p < 0.05.Data are expressed as means ± standard errors (SE). Data regarding the concentration of plasma OZ molecular species and FA and FA conjugates were analyzed by one-way ANOVA followed by Tukey’s test. Differences were considered significant at OZ is a mixture of FA esters of triterpene alcohols and plant sterols. Since OZ was discovered from rice oil in 1954 . To furtPrior to administration, the molar ratio of OZ molecular species in RBOZ was determined to administer OZ accurately at a dose of 300 µmol/kg body weight. Previous studies have described CA-FA, 24MCA-FA, Camp-FA, and Sito-FA as the major OZ species in rice bran ,6, and hn = 6), and at first, the absorption profile of OZ in rat plasma was investigated using HPLC-MS/MS. The detection limit of each OZ molecular species was 1 fmol. Typical multiple reaction monitoring (MRM) chromatograms of OZ molecular species in plasma before and 9 h after RBOZ administration are shown in RBOZ (300 µmol/kg body weight) was administered to male Sprague–Dawley rats  for each OZ molecular species is shown in Interestingly, it was found that the relative proportion of OZ molecular species in plasma was different to that of RBOZ . A similar observation was made in a previous study where different plant sterols were administered to rats; the lymphatic transfer rate of plant sterols differed between plant sterol molecular species . Hence, O-sulfate (FAS) and FA 4-O-β-d-glucuronide (FAG)) after RBOZ administration were also investigated using HPLC-MS/MS. The detection limits of FA and FA conjugates were as follows: FA (0.1 pmol), FAS (0.01 pmol), and FAG (0.05 pmol). Typical MRM chromatograms of FA and its conjugates in plasma before and 6 h after RBOZ administration are shown in The absorption profiles of FA and FA conjugates  of FA and its conjugates were typically achieved at 0.5 h. This rapid increase in concentrations of FA and its conjugates reflects the initial increase (within 1 h) in FA concentrations under RBOZ administration  after a single dose administration of RBOZ were first determined at multiple time points using HPLC-MS/MS. As a result, not only FA and its conjugates but also OZ was found from plasma, suggesting that a part of OZ avoids hydrolysis to FA and remains in blood. Additionally, the blood profile after administration of an equimolar amount of FA was analyzed to compare the absorption profile of OZ with FA. The absorption profile of FA was significantly different from that when RBOZ was administered, providing insights into how OZ is metabolized to FA and its conjugates in vivo. The results of this study may help predict which active molecules  contribute to a major role in the physiological effects of OZ.


Honokiol (HK), a well-tolerated natural product, has many multiple pharmacological activities. However, its poor water solubility and low bioavailability limit its clinical application and development. The aim of this research was to prepare the solid dispersion (SD) formulation of honokiol (HK) with poloxamer-188 (PLX) as the carrier, thereby improving its solubility and oral bioavailability. Firstly, by investigating the relationship between the addition amount of the PLX and the solubility of HK, and the effects of solid dispersions with different ratios of HK–PLX on the solubility of HK, we determined that the optimum ratio of PLX to HK was (1:4). Then, the HK–PLX (1:4) SD of HK was prepared using the solvent evaporation method. The morphology of the obtained HK–PLX (1:4) SD was different from that of free HK. The HK in the HK–PLX (1:4) SD existed in amorphous form and formed intermolecular hydrogen bonds with PLX. Additionally, the solubility values of the HK–PLX (1:4) SD were about 32.43 ± 0.36 mg/mL and 34.41 ± 0.38 mg/mL in artificial gastric juice (AGJ) and in artificial intestinal juice (AIJ), respectively. Compared with free HK, the release rate and the bioavailability was also substantially improved for HK in its SD form. 3--2,5-diphenyltetrazolium bromide (MTT) assay indicated that the HK–PLX (1:4) SD showed higher inhibition of HepG2 cells than free HK. Taken together, the present study suggests that the HK–PLX (1:4) SD could become a new oral drug formulation with high bioavailability and could produce a better response for clinical applications of HK. Today, in the process of drug development, low water solubility is still a difficult problem that needs to be solved for drug candidates. Many insoluble drugs are lipid-soluble drugs with low water solubility and high permeability . So far,Magnolia officinalis [18H18O2 [Generally, the formation of intramolecular hydrogen bonds in the drug molecules reduces the polarity of the molecules, thus reducing the solubility in water. The formation of intermolecular hydrogen bonds increases the solubility of the drugs, probably because the formation of hydrogen bonds between drug molecules and water molecules accelerates the dissolution of the drugs. Honokiol (HK) is a bioactive natural compound extracted from the stem, bark, and roots of icinalis . It is a[18H18O2 . HK exis[18H18O2 . Moreove[18H18O2 ,15,16. H[18H18O2 ,18. If aHydrophilic poloxamer-188 (PLX) is a polyoxyethylene–polyoxypropylene copolymer. Due to its low melting point, high drug loading, good hydrophilicity, and good safety, PLX is not only widely used in some traditional formulations, such as suppositories, tablets, and emulsions, but also can form micelles to increase the solubility of insoluble drugs, increase the stability of drugs, and promote the absorption of drugs by the body. Additionally, it is a surfactant approved by the FDA . MoreoveCmax of the inclusion complex increased by approximately 158% and 123%, respectively, compared with free HK [Cmax increase and a 2.2-fold AUC (0–t) increase [Cmax increased by 4.06- and 3.60-fold and AUC by 6.26- and 5.83-fold in comparison to 40 and 80 mg/kg oral application of free HK, respectively [In addition, some technologies have been used to enhance the solubility and oral bioavailability of free HK. For example, Xu and coworkers prepared an inclusion complex of HK with sulfobutyl ether-β-cyclodextrin (SB-β-CD), and in vivo results showed that the AUC(0–t) and  free HK . Han andectively . NeverthIn recent years, various technologies, such as melting method , spray-dIn the present investigation, a HK–PLX solid dispersion was prepared using solvent evaporation method, and its physicochemical properties were characterized by SEM, FTIR, XRD, and DSC. Furthermore, the dissolution in vitro, the solubility and the bioavailability in vivo, and the MTT assay were also investigated and evaluated. Poloxamer-188 (PLX), hydroxypropyl methylcellulose (HPMC), polyethylene glycol (PEG) 4000, PEG 6000, and polyvinylpyrrolidone (PVP) K30 were purchased from Aladdin . Honokiol (purity = 98.5%) was obtained from Baoji Haoxiang Biotechnology Co., Ltd . Ethanol, acetonitrile, and methanol were obtained from Shandong Yuwang Industrial Co., Ltd .Initially, the HK and the PLX were dissolved in ethanol for 5 min to reach the clear organic phase, and then the solution was stirred for 30 min under ambient conditions. After 30 min, the ethanol was evaporated using a rotary evaporator  at 35 °C to obtain the SD formulation of HK with PLX (HK–PLX SD), and the obtained HK–PLX SD was then dried in a vacuum oven to remove the residual solvent. The removed ethanol could be recycled and reused. Finally, the SDs with different mass ratios of HK/PLX were obtained through the same process. The short names for as-synthesized HK SDs are as follows: HK–PLX 2:1, HK–PLX 1:1, HK–PLX 1:2, HK–PLX 1:3, HK–PLX 1:4, HK–PLX 1:5, HK–PLX 1:6, and HK–PLX 1:7. In addition, the SD formulation of the HK and other carriers (the mass ratio of HK to carriers was 1:5), including HPMC, PEG 4000, PEG 6000, and PVP K30, were also prepared via solvent evaporation method.Apparent morphology of the HK–PLX (1:4) SD and the free HK was examined by a SEM . Dry samples were fixed on the sample tables with a conductive paste and made to be electrically conductive by sputter-coating with gold using the ion sputtering coating machine. −1 by FTIR spectrophotometer . Each sample (2 mg) was accurately weighed and mixed with KBr (200 mg) and then pellets were made to perform the measurements. The structure of the HK–PLX (1:4) SD, the physical mixture (PM), and the free HK were examined in the scanning range of 400–4000 cmThe crystal forms of the HK–PLX (1:4) SD, the PM, and the free HK were tested and evaluated by an X-ray diffractometer . Each sample was measured in the range of 5–60°. Appropriate process parameters were: scanning speed 5°/min; generator current 30 mA; generator tension (voltage) 40 kV. Thermal characteristics of the HK–PLX (1:4) SD, the PM, and the free HK were detected by DSC . Samples of 4–6 mg were placed in a sealed aluminum pan and then heated from 40 °C to 400 °C in nitrogen atmosphere at a heating rate of 10 °C/min. The solvent residue of ethanol in the HK SD was inspected by an Agilent 7890A gas chromatograph . Firstly, 100 mg of HK SD powder was weighed and dissolved in acetone (1 mL), and then centrifuged at 10,000 rpm for 10 min to obtain the supernatant. Then, 5 μL of the supernatant was injected into a GC system for analysis, with a split ratio of 20:1. The detection conditions were as follows: initial temperature was kept at 40 °C for 5 min, then the temperature was raised to 100 °C for 2 min at 10 °C/min, then 240 °C at 40 °C/min, and then maintained for 2 min. In addition, both the inlet and detector temperatures were set to 250 °C. The air flow rate was 400 mL/min, the nitrogen flow rate was 25 mL/min, and the hydrogen flow rate was 30 mL/min.18 reverse-phase column . A methanol (55%), acetonitrile (20%), and water (25%) mixture was used as mobile phase. Other parameters of HPLC detection were as follows: A wavelength was 294 nm, the flow rate of the mobile phase was 1 mL/min, and the injection volume was 10 μL.HK was quantified by a Waters 1525-2489 high performance liquid chromatograph  consisting of a pump (Waters 1525 binary) and UV detector (Waters 2489 Tunable Absorbance Detector), which was equipped with a Diamonsil CY = 11326308.72X − 8679 (R2 = 0.9998). The retention time of the HK was about 10–11 min.A standard solution of HK was prepared as follows: HK (10 mg) was accurately weighed and dissolved in 10 mL methanol solution, and then the solution was diluted appropriately to obtain solutions of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.007813, and 0.003906 mg/mL. Using the concentration of HK standard solution as the abscissa and the absorbency as the y-coordinate, the linear chart was constructed, and the regression equation was Solid dispersions of HK with different polymers (the mass ratio of HK to polymer was fixed at 1:5) were also prepared by solvent evaporation method. Each solid dispersion (the content of HK was excessive) was weighed and placed in small beakers with 2 mL of deionized water, and then the beakers were sealed and placed in a water bath at 37 ± 1.0 °C for 4 h with constant stirring (100 rpm). After 4 h, the samples were taken out and centrifuged at 12,000 rpm for 10 min, and then filtered through a 0.22 μm membrane filter. The supernatant obtained was used for HPLC to determine HK concentration.The solubility of the SD of HK with PLX and the corresponding PMs in water were determined by HPLC detection. Firstly, the excess amounts of SD and PM samples  were placed in 25 mL beakers, and then 10 mL distilled water was added to each beaker. Then, the beakers were sealed and placed in a 37 ± 1.0 °C water bath for 4 h with constant stirring (100 rpm). After 4 h, the samples were taken out and centrifuged at 12,000 rpm for 10 min, and then filtered through 0.22 μm membrane filter to remove the possible residual drugs. The final supernatant was assayed for HK by the HPLC system, as described above. The equilibrium solubility studies of HK–PLX (1:4) SD, free HK, and the PM in artificial gastric juice (AGJ) (pH 1.2) were carried out and investigated with 0.4% Tween-80 and artificial intestinal juice (AIJ) (pH 6.8). Briefly, excess samples were weighed and placed in small vials containing 2 mL dissolution media. Then, each vial was placed in a 37 °C water bath and stirred for 48 h at 100 rpm. After 48 h, the samples were centrifuged at 12,000 rpm for 10 min and then filtered through 0.22 μm membrane filters. The final supernatant was diluted with methanol, and then the concentration of HK in the samples was determined by HPLC detection.The in vitro release of the HK–PLX (1:4) SD, the PM, and the free HK was analyzed in the two media mentioned above. Dissolution experiments were consistent with the sink conditions and were carried out at 37.0 ± 0.5 °C at a rotation speed of 100 rpm. Samples equivalent to 200 mg of HK were weighed and dispersed in a 250 mL beaker containing 200 mL of dissolution media. One milliliter of sample was taken out at predetermined times of 5, 10, 15, 20, 30, 45, 60, 90, 120, 240, 480, and 720 min, and the corresponding volume of dissolution medium was supplemented. The samples obtained were centrifuged at 12,000 rpm for 10 min and then filtered through 0.22 μm membrane filters to separate the excess drug. The final supernatant was diluted with methanol, and then the concentration of HK in the samples was determined by the HPLC detection.Eighteen Sprague–Dawley rats (200–220 g) were maintained at 25 ± 2 °C and 50–60% relative humidity (RH) under natural light/dark conditions for one week before the experiment. In the experiment, the rats were randomized into three different groups, administered orally with free HK, the HK–PLX (1:4) SD, or the PM (at the dose of 50 mg/kg). The experimental protocols were approved by the Institutional Animal Care and Use Committee of Harbin Medical University  on June 23, 2006.Blood samples were collected from the retro-orbital plexus at different time intervals of 0.08, 0.17, 0.25, 0.33, 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h. Samples were collected in 1.5 mL centrifuge tubes containing heparin, and were then centrifuged at 3000 rpm for 10 min. The upper plasma was taken and stored in a refrigerator at −40 °C until analysis.Plasma samples (200 μL) were placed in a clean 1.5 mL centrifugal tube. Then, 400 μL acetonitrile was added and vortexed for 30 s to precipitate plasma proteins, which was then centrifuged at 12,000 rpm for 10 min to separate the supernatant. Then, 50 mg sodium chloride was placed in a new centrifugal tube containing the supernatant. All the samples were placed in a 25 °C water bath for 10 min and then centrifuged at 12,000 rpm for 5 min. Finally, 10 μL of supernatant was analyzed for the amount of HK by HPLC method. 4 cells per well in 96-well plates and incubated for 24 h. Subsequently, the old medium was removed and the samples containing free HK and the HK–PLX (1:4) SD at different HK concentrations  were added to the plates (the samples containing free HK and the HK–PLX (1:4) SD with different HK concentration were dissolved with culture medium, and the obtained samples were filtered through a 0.45 μm pore diameter membrane, and then added in the plates). In addition, the corresponding PLX at different concentrations  was also added to the plates. After 48 h incubation, the cells were washed and the fresh medium containing MTT was added into each plate, followed by incubation for another 4 h. Thereafter, the medium was removed and the formazan crystals were solubilized with DMSO (150 µL). After mild shaking for 10 min, the absorbance value (OD) was detected by the enzyme mark analyzer instrument (detection wavelength of 490 nm and reference wavelength of 630 nm) and compared with the blank control group. Inhibition rate of the HK–PLX (1:4) SD on HepG2 cell lines was evaluated by conventional methyl thiazole tetrazolium (MTT) cell survival assay. The assay was based on the reduction of MTT by the mitochon-drial dehydrogenase of viable cells into purple formazan crystals, which were dissolved in dimethyl sulfoxide (DMSO), and absorbance was then detected by the enzyme mark analyzer instrument. Firstly, HepG2 cell lines were seeded at a density of 1 × 10t-test in the Origin software. A value of * p < 0.05 was accepted as statistical significance. For in vitro studies, 3–4 replicates were used each time. Three individual experiments were performed and results were averaged. For in vivo experiments, n = 6 was used.The data were presented as mean ± standard deviation. The statistical analysis was analyzed by using one-sample In the experiments, we investigated the effects of different polymers, namely HPMC, PEG 4000, PEG 6000, PVP K30, and PLX, as carriers on the solubility of HK, and the measured solubility of the samples was shown in In addition, the appearance of different solid dispersions  in water is illustrated in −4 M [ST = S0 + k (PT − CMC) [w/v, the solubility of HK was about 11.56 ± 0.23 mg/mL, which was about 825.7 times that of free HK. It was expected that the solubility of HK would increase linearly with the further increase of PLX content, but that was not the case. When the content of PLX was 7.5%, 9%, and 10.5% w/v, the solubility of HK was about 12.4 ± 0.15, 11.9 ± 0.24, and 12.9 ± 0.12 mg/mL, respectively, and the magnitude of solubility enhancement was inconspicuous. Additionally, considering that the presence of large doses of surfactants in pharmaceutical products might cause unpredictable side effects in vivo, the amount of PLX did not need to be added too much.The measured solubility of free HK, PMs of HK with PLX, and HK SD are shown in −4 M . As mighT − CMC) , as showp < 0.05), and was obviously higher than that of the same proportion of PMs. As could be seen, the drugs existed in the SD in amorphous form, which was the critical factor for solubility enhancement. Additionally, when the mass ratio of HK to PLX was 1:4, the solubility of the SD was about 27.59 ± 0.33 mg/mL, while when the mass of PLX increased further, the solubility of the corresponding SD changed inconspicuously. Consequently, in this paper, the HK–PLX (1:4) SD was selected and used for the following detections. Conversely, as expected, SD could enhance the solubility of HK more effectively. It was noteworthy that the solubility of the SDs in The free HK and the HK–PLX (1:4) SD were detected by SEM to observe their morphology, as shown in −1 are shown in −1 (–OH vibration), 1638 cm−1 , 1498 cm−1 (C=C aromatic stretching), 1217 and 908 cm−1 (C–O), and 989 and 825 cm−1 (C–C). The FTIR spectra of PLX (curve d) were characterized by principal absorption peaks at 2885 cm−1 , 1338 cm−1 (in-plane O–H bend), and 1106 cm−1 (C–O stretching). As for PM of HK with PLX (curve c), the spectrum basically consisted of the overlapping peaks of HK and PLX. Some characteristic absorption peaks of HK at 3303, 1638, 1497, 906, and 825 cm−1 were easy to find in PM, suggesting there was no interaction between HK and PLX. Meanwhile, these characteristic peaks of HK almost disappear in the spectrum of SD (curve b), which could be attributable to interaction between HK and PLX. Moreover, considering the chemical structures of PLX and HK, the hydrogen bonds may be formed between PLX and HK during the formulation of the HK–PLX (1:4) SD, and this interaction played a vital role in solubilizing the drug.The possible interaction between HK and PLX was determined by FTIR. The FTIR spectra of free HK, the HK–PLX (1:4) SD, PM of HK with PLX, and PLX from 4000 to 400 cmθ = 19.23° and 23.4°, and as seen in the DSC curves of PLX in The XRD and DSC results of the samples, including free HK, HK–PLX (1:4) SD, PM of HK with PLX, and PLX, are shown in In addition, the crystal structures of the SDs of HK prepared in different batches were also investigated, as shown in x) was obtained as Y = 1980.6 x + 221.13 (R2 = 0.999) over the range of 0.015625–1 mg/mL. According to the regression equation, the residual ethanol content in the HK–PLX (1:4) SD was 1733 ppm, which met the minimum standard (5000 ppm) required by the International Conference on Harmonization (ICH) and was suitable for pharmaceutical use.In this work, the ethanol with low toxicity was applied to prepare the HK–PLX (1:4) SD. p < 0.05), indicating that the HK existed as an amorphism in HK–PLX (1:4) SD and formed intermolecular hydrogen bonds with PLX. So, the HK–PLX (1:4) SD had better dissolution and bioavailability.The drug solubility and dissolution are displayed in Furthermore, the SD formulation of HK significantly improved drug dissolution compared to the drug powder. The release profiles of HK from the SD formulation compared to free HK are shown in p < 0.05). Additionally, the Cmax values of the free HK, the PM, and the HK–PLX (1:4) SD were 159.02 ± 5.65 ng/mL, 443.81 ± 9.14 ng/mL, and 1109.87 ± 7.24 ng/mL, respectively. The AUC values of the free HK, the PM, and the HK–PLX (1:4) SD were 580.45 ± 11.15 ng/mL·h, 848.34 ± 12.24 ng/mL·h, and 2558.22 ± 8.15 ng/mL·h, respectively. The results illustrated that the AUC and the Cmax values of the PM were slightly higher than those of the free HK, while the HK–PLX (1:4) SD gave significantly higher AUC and Cmax values compared to the PM and the free HK. Moreover, the value of Cmax for the HK–PLX (1:4) SD was about 6.98-fold greater than that of free HK, and the AUC(0→t) of the HK–PLX (1:4) SD was about 4.41-fold greater than that of free HK. Furthermore, the half-life (t1/2) of the HK–PLX (1:4) SD (0.37 ± 0.05 h) was shorter than that of the PM (0.51 ± 0.12 h) and the free HK (0.74 ± 0.15 h). The Tmax of the HK–PLX (1:4) SD (0.5 ± 0.08 h) was also shorter than that of the PM (0.75 ± 0.09 h) and the free HK (2 ± 0.15 h). Thus, we concluded that the HK–PLX (1:4) SD had good oral bioavailability in rats compared to the PM of HK with PLX and the free HK. Plasma concentration–time profiles and corresponding pharmacokinetic parameters of HK, PM of HK with PLX, and the HK–PLX (1:4) SD in rats are displayed in t) value in rats, which was about 2.2 times that of HK–CMCS. However, in this study, the in vivo pharmacokinetics in rats demonstrated that the AUC(0→t) value of the HK–PLX (1:4) SD preparation was approximately 4.41-fold greater than that of free HK, which indicated that the HK–PLX (1:4) SD should be the potential oral delivery system for clinical applications and could be better absorbed by the body.Furthermore, it had been reported that the honokiol nanosuspensions were prepared by the precipitation–ultrasonication method, and the honokiol existed as an amorphous form in the honokiol nanosuspensions . The phap < 0.05), which could be explained by the increased cellular uptake of the HK SD formulation.In this study, the inhibitory effect of the HK–PLX (1:4) SD on HepG2 cell lines was studied and compared with free HK. Cell viabilities of HepG2 cells after 48 h incubation with different concentrations of the HK–PLX (1:4) SD, free HK, and PLX are shown in Cmax value and the AUC(0→t) value of the HK–PLX (1:4) SD  were about 6.98- and 4.41-fold higher than those of free HK ; hence, the HK–PLX (1:4) SD had better bioavailability than free HK. MTT study showed that the HK–PLX (1:4) SD generated higher inhibition of HepG2 cells than free HK. In addition, the solvent residual of the HK–PLX (1:4) SD was suitable for pharmaceutical use. Thus, it can be concluded that the HK–PLX (1:4) SD prepared in this study significantly improved the absorption of HK in vivo, and has potential clinical application value as a new oral drug formulation.The SD formulation of HK with PLX ((1:4)) was prepared by the solvent evaporation method, and characterized by SEM, FTIR, XRD, and DSC. The SEM analysis illustrated that the SD of HK exhibited different morphology than free HK, and no HK crystals were observed in the SD. The XRD and DSC results illustrated that the HK existed in the SD in amorphous form. The FTIR spectra indicated that there might be intermolecular hydrogen bonds between HK and PLX. Moreover, the solubility and dissolution experiments in AGJ and AIJ clearly indicated that the HK–PLX (1:4) SD had the higher solubility, with values of 32.43 ± 0.36 mg/mL and 34.41 ± 0.38 mg/mL, respectively. The dissolution rate of the HK–PLX (1:4) SD was also apparently higher than free HK in AGJ and AIJ. Moreover, the


Origanum syriacum against the L3 larvae of the parasitic nematode Anisakis simplex and larvae and adults of the mosquito Culex quinquefasciatus. Tests on A. simplex were performed by standard larvicidal and penetration assays, while mosquito toxicity was assessed relying on larvicidal, tarsal contact, and fumigation tests. To shed light on the possible mode of action, we analyzed the oil impact as acetylcholinesterase (AChE) inhibitor. This oil was particularly active on L3 larvae of A. simplex, showing a LC50 of 0.087 and 0.067 mg mL−1 after 24 and 48 h treatment, respectively. O. syriacum essential oil was highly effective on both larvae and adults of C. quinquefasciatus, showing LC50 values of 32.4 mg L−1 and 28.1 µg cm−2, respectively. Its main constituent, carvacrol, achieved larvicidal LC50(90) of 29.5 and 39.2 mg L−1, while contact toxicity assays on adults had an LC50(90) of 25.5 and 35.8 µg cm−2, respectively. In fumigation assays, the LC50 was 12.1 µL L−1 after 1 h and decreased to 1.3 µL L−1 in 24 h of exposure. Similarly, the fumigation LC50 of carvacrol was 8.2 µL L−1 after 1 h of exposure, strongly decreasing to 0.8 µL L−1 after 24 h of exposure. These results support the folk usage of Lebanese oregano as an antiparasitic agent, providing new insights about its utilization for developing new effective and eco-friendly nematocidal and insecticidal products.Developing effective and eco-friendly antiparasitic drugs and insecticides is an issue of high importance nowadays. In this study, we evaluated the anthelminthic and insecticidal potential of the leaf essential oil obtained from  Anisakis, such as A. simplex Dujardin larvae, is a public health concern worldwide, particularly in Asian countries such as Japan, and in Mediterranean areas, such as Spain and Italy .i et al. . Then, li et al. . The O. O. syriacum essential oil or its major constituent, carvacrol, was formulated in 2 mL of acetone plus silicon oil (3.6 mg cm−2) and then provided on Whatman no. 1 filter paper (12 × 15 cm), testing seven doses . α-Cypermethrin (Vaztak®) was the positive control . Concerning negative controls, mosquitoes were exposed to filter paper pretreated with the same amount of acetone + silicon oil, without O. syriacum essential oil. In all controls, four groups, each composed by 20 insects, were tested. In all cases, filter paper was then dried at 22 °C for 24 h, and placed in test tubes . Each experiment was replicated three times.The theoretic fumigation exposure time needed to achieve mortality of mosquito adults was determined in a series of experiments carried out using identical methods as above (O. syriacum was performed and quantified in 96 microplates using the Ellman method [26H2O), 50 µL buffer B , and 25 µL of O. syriacum essential oil at different concentrations. The O. syriacum essential oil was diluted in DMSO and tested in triplicates over different days. Then, 25 µL 0.22 U/mL AChE was added and the absorbance was measured at 405 nm using a kinetic mode. Galantamine was tested as the positive control.AChE inhibition by the essential oil of n method  with minA. simplex larvae. LD50  for A. simplex larvicidal activity was calculated using nonlinear regression (GraphPad Prism 5). Data were subjected to analysis of variance, and mean comparison was performed by one-way ANOVA plus Scheffe’s multiple comparisons (p ≤ 0.05). Statistical analysis was performed using PASW Statistics 18. In mosquito larvicidal and adulticidal assays, when mortality in the control ranged from 1% to 20%, we corrected experimental mortality with Abbott’s formula [50(90) as well as LT50(90) related parameters detailed in p = 0.05 was used as a threshold to separate means; the IC50  of galantamine and the tested essential oil were calculated as described above for A. simplex using nonlinear regression.In anthelminthic assays, all experiments were performed in triplicates over different weeks using new  formula ; if contO. syriacum essential oil is highly effective against the filariasis vector C. quinquefasciatus and the parasite A. simplex. Notably, its bioactivity is related to the high content of carvacrol, a phenolic monoterpene. The possibility of developing effective, eco-friendly, and safe botanical insecticides with this essential oil is high. Indeed, scalability is assured by both wild and cultivated accessions of this species that occurs in several Middle East countries. Moreover, these prospects are enhanced by the fact that O. syriacum is currently grown as a commercial crop, and provided that a suitable growing technology is used, more than 4500 kg of dry mass can be obtained from one hectare, yielding about 180 kg of essential oil. Thus, the crop may provide an easily available and relatively inexpensive source of active substances for potential botanical-based drugs and insecticides, which can be further stabilized through nano- and microemulsions [The findings of our study highlighted that the mulsions  and prop


Enterococcus faecium SROD on in vitro rumen fermentation, methane concentration, and microbial population structure. Ruminal samples were collected from ruminally cannulated Holstein–Friesian cattle, and 40:60 rice straw to concentrate ratio was used as substrate. Fresh culture of E. faecium SROD at different inclusion rates  were investigated using in vitro rumen fermentation system. Addition of E. faecium SROD had a significant effect on total gas production with the greatest effect observed with 0.1% supplementation; however, there was no significant influence on pH. Supplementation of 0.1% E. faecium SROD resulted in the highest propionate (P = 0.005) but the lowest methane concentration (P = 0.001). In addition, acetate, butyrate, and total VFA concentrations in treatments were comparatively higher than control. Bioinformatics analysis revealed the predominance of the bacterial phyla Bacteroidetes and Firmicutes and the archaeal phylum Euryarchaeota. At the genus level, Prevotella (15–17%) and Methanobrevibacter (96%) dominated the bacterial and archaeal communities of the in vitro rumen fermenta, respectively. Supplementation of 0.1% E. faecium SROD resulted in the highest quantities of total bacteria and Ruminococcus flavefaciens, whereas 1.0% E. faecium SROD resulted in the highest contents of total fungi and Fibrobacter succinogenes. Overall, supplementation of 0.1% E. faecium SROD significantly increased the propionate and total volatile fatty acids concentrations but decreased the methane concentration while changing the microbial community abundance and composition.Supplementation of appropriate probiotics can improve the health and productivity of ruminants while mitigating environmental methane production. Hence, this study was conducted to determine the effects of  Probiotics are beneficial live microorganisms that are used as feed supplements for improving the intestinal microbial balance as well as growth performance in livestock. The term “probiotics” has been amended by the Food and Agriculture Organization/World Health Organization to “live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host” Fuller . AccordiBacillus, and lactic acid bacteria , which are now commonly used for human as well as animal consumption.The rumen microbiome is composed of complex and diverse groups of microorganisms, which are responsible for converting fibrous plant materials into energy used by the ruminants. These microorganisms thus play an important role in animal health and productivity, food safety, and the environment. The rumen microbial community, diversity, and quantity vary depending on the host’s dietary composition and dry matter intake. Hence, supplementation of specific probiotics to ruminants can increase microbial diversity and enhance the proportion of beneficial microbes in the community. Probiotics belong to a wide variety of yeasts, Enterococcus is one of the main genera of lactic acid bacteria, which has been used as probiotics for decades. Enterococci are ubiquitous and facultative anaerobes, which means that they can be easily cultivated and proliferate under aerobic conditions during production as well as under the anaerobic conditions found inside the rumen. Moreover, enterococci are resistant to gastric juices and bile salts   and pyrosequencing to determine these effects and gain a better general understanding of rumen microbes’ symbiosis and functions. In particular, we added E. faecium SROD at different inclusion rates to an in vitro rumen fermentation system and determined the effects on methane concentration, microbial diversity, and population structure.We previously isolated an Enterococcus faecium SROD KCCM11098P, which was previously isolated in our laboratory , and then incubated in a horizontal shaking incubator (120 rpm)  at 37 °C. E. faecium SROD was then subcultured three times on the same medium to ensure full activity. The cell growth was monitored based on an optical density value at 600 nm of approximately 1.50, which is equivalent to 7.0 × 108 colony-forming units (CFU)/mL.2HPO4, 0.45 g KH2PO4, 0.9 g (NH4)2SO4, 0.12 g CaCl2·2H20, 0.19 g MgSO4·7H2O, 1.0 g trypticase peptone , 1.0 g yeast extract , and 0.6 g cysteine HCl  was anaerobically transferred (100 mL) to 160 mL serum bottles containing the substrates. The substrates were grinded and sieved to 1 mm particle size and then 1 g dry matter of rice straw and concentrate at 40:60 ratio was put into the serum bottles. The following inocula treatments were conducted under a stream of O2-free N2: no addition , and 0.1% (T1), 0.5% (T2), and 1.0% (T3) supplementation of E. faecium SROD (7.0 × 108 colony-forming units (CFU)/mL). The bottles were then sealed and incubated at 39 °C  and rice straw (2:8 ratio) were used in this study. Three hours after morning feeding, the ruminal contents were collected, strained through four layers of surgical gauze, placed in amber bottles, maintained at 39 °C, and then immediately transported to the laboratory. Asanuma buffer used in this study was composed of (per L) 0.45 g K3–N), VFA, and molecular analyses. One millilitre of gas sample contained in vacuum tubes was used to determine the methane concentration through gas chromatography  with a thermal conductivity detector and an HP stainless packed GC Porapak Q 80/100 outer dimension 1/8 in × inner dimension 2.0 mm, length 3.05 m (10 ft) with 200 °C inlet, 200 °C detector, 40 °C oven temperature, and 3 mL/min N2 carrier gas flow. Estimation of the amount of methane produced was conducted using the formula described by Ørskov and McDonald , and the pH was determined using a Pinnacle series M530p meter . Gas samples were collected for determination of methane concentration, and the in vitro rumen fermenta was collected for ammonia–nitrogen . Then, supernatant and pellet were separated, kept in 1.5 Eppendorf tubes and stored at − 80 °C until subjected to NH3–N, VFA, and molecular analyses. The supernatant was used for determination of NH3–N  before analysis. Standards with R2 = 0.999 were prepared prior to sample analysis. The VFA concentration in mM was calculated in ppm divided by the molecular weight.Samples were measured from each of the serum bottles at different incubation times. Two 1-mL in vitro rumen fermenta from each serum bottles were immediately centrifuged after sampling at 16,609×u et al.  and Han u et al. . StandarFibrobacter succinogenes, and Ruminococcus flavefaciens were enumerated using qRT-PCR on the DNA extracted from the in vitro rumen fermenta using the primers reported in Denman and McSweeney  from the rumen pellets was extracted using a Fast-DNA spin kit (MPbio) according to the manufacturer instructions. The general bacteria, general fungi, methanogens, protozoa, cSweeney  and DenmcSweeney  (Table 1X-AC-WTT ACC GCG GCT GG-3′) and Arc21F (5′-adaptor B-GA-TCC GGT TGA TCC YGC CGG-3′)/Arc519R (5′-adaptor A-X-GA-GGT DTT ACC GCG GCK GCT G-3′) (Delong http://pyro.cme.msu.edu/)  Delong . Unique E. faecium SROD supplementation were analyzed using orthogonal polynomial coefficients to describe the functional relationships among the control and treatment levels. P ≤ 0.05 indicated statistical significance. All analyses were carried out using Statistical Analysis Systems (SAS) software version 9.4  in supplementation of 0.1% E. faecium SROD with 59.45 mL and lowest in control with 55.15 mL  with E. faecium SROD addition, and was lowest (P = 0.001) with supplementation of 0.1%, followed by 0.5% E. faecium SROD, and was highest in the control and 1.0% E. faecium SROD groups with no significant difference between them. Addition of 0.1% E. faecium SROD resulted in the lowest carbon dioxide (P = 0.053) with 3.70 mM/mL but the highest  concentrations of total VFAs and propionate with 55.40 mM and 14.15 mM, respectively (Table P < 0.001) with increasing inclusion rate of E. faecium SROD with linearly (P < 0.020) and quadratically (P < 0.043) correlation of the concentration and inclusion rate. Butyrate (P < 0.018) and total VFA (P < 0.001) concentrations were comparatively higher than control while propionate (P < 0.005) concentration was found the highest in addition of 0.1% E. faecium SROD.The volume of total gas produced was found highest , F. succinogenes (P = 0.010), and R. flavefaciens (P = 0.008). The control group had the lowest quantities of general fungi (P = 0.026) and F. succinogenes (P = 0.010) but the highest log copy numbers of methanogens (P = 0.048), which showed a significant linear decrease with increasing supplementation of E. faecium SROD. In addition, supplementation of 0.1% E. faecium SROD resulted in lower quantities of methanogen (P = 0.048) than control.Comparable quantities of general bacteria were observed among the control and treatment groups Table . BetweenThe barcoded pyrosequencing results of 24 PCR amplicons  of the 16S rRNA genes for the bacterial and archaeal communities are shown in Tables Bacteroidetes and Firmicutes  and Methanobrevibacter (96%) dominated the bacterial and archaeal communities’ composition of the in vitro rumen fermenta, respectively , followed by 0.5% E. faecium SROD (0.211%); similar relative abundances were detected with 1.0% E. faecium SROD and the control of 0.184% and 0.175%, respectively.Bioinformatics analysis revealed that the bacterial sequences were predominantly affiliated with two phyla, tes Fig. , while tly Figs.  and 3. TE. faecium SROD displays full activity after 12 h of incubation  cells per cow per day. Moreover, the increased acetate concentration observed in the E. faecium SROD-supplemented treatment groups compared to the control was comparable that observed in our previous study on E. faecium SROD  for first lactation cows and increased milk protein percentages for second and greater lactation cows. Moreover, E. faecium with yeast as DFM increased dry matter intake, milk yield, and milk protein content during the postpartum period  had comparable methane concentration to control but it tended decrease. This might be due to increase in population of R. flavefaciens, which might increase available H2 and electrons for methanogenesis.Microorganisms inhabiting in the rumen contribute directly or indirectly to dietary organic matter degradation (Wang et al. d et al.  study whAnalysis of the rumen microbiome is of great importance for understanding the microbial ecosystem at large, which could be accomplished through determination of the microbial communities and their symbiosis. To best correlate and describe the results of in vitro rumen fermentation parameters with microbial abundance and community composition at greater resolution, we utilized a new and high-throughput molecular technique. Further adoption of high-throughput techniques can lay the foundation for new advancements in ruminant production by gaining a deeper-level microbial understanding of proven nutritional strategies (McCann et al. Through calculation of the number of OTUs and the measure of species richness estimators, we estimated the diversity within samples. The Chao index estimates the richness of the diversity, while the Shannon–Weaver index takes into account the number and evenness of species present. On the other hand, the Simpson index depicts probability of the that two randomly selected individuals in the habitat will belong to the same species. In this study, the pyrosequencing data demonstrated comparable bacterial or archaeal communities, in terms of diversity, richness, number, and evenness of species, among treatments. However, higher communities, diversity, richness, number and evenness of species were observed in bacteria than in archaea.Bacteroidetes and Firmicutes were present at the highest relative abundance at the phylum level for all groups, which is consistent with the findings of Jami et al. (Bacteroidetes specialize in lignocellulose degradation and are associated with butyrate production; however, Firmicutes represent the major butyrate-producing group of microbes (Naas et al. Euryarchaeota in this study is in line with the results of Wang et al. (Thaumarchaeota, which was observed only under supplementation with 0.1% E. faecium SROD, represents a group of chemolithoautotrophic ammonia-oxidizers (Spang et al. E. faecium SROD also enhanced the growth of Anaerovibrio, Enterococcus, Lachnobacterium, unclassified Clostridiales Incertae Sedis XII, unclassified Clostridiaceae 1, unclassified Clostridiales, and unclassified Ruminococcaceae by increasing their relative abundances. The increased relative abundance of Enterococcus with increasing inclusion rates of E. faecium SROD indicates that E. faecium SROD grew well anaerobically and in symbiosis with other microbes.i et al.  and Wangi et al. . Naas eti et al.  reportedg et al. . ThaumarE. faecium SROD increased the concentrations of propionate and total VFAs but decreased the methane concentration during in vitro rumen fermentation. Also, a significant increase in butyrate concentration indicates regulatory and immunological functions in cattle. These findings were validated by the determination of the quantities of specific microbes related to the production of these components. In particular, the quantities of general fungi, F. succinogenes, and R. flavefaciens increased with the inclusion of 0.1% E. faecium SROD, while lower quantities of methanogens were observed in the treatment groups compared to the control. Using a pyrosequencing technique, we further demonstrated that supplementation of E. faecium SROD enhances the growth of Anaerovibrio, Enterococcus, Lachnobacterium, unclassified Clostridiales Incertae Sedis XII, unclassified Clostridiaceae 1, unclassified Clostridiales, and unclassified Ruminococcaceae by increasing their relative abundances.The inclusion of 0.1% E. faecium SROD is a potentially valuable feed additive for ruminal methane mitigation and to enhance the productivity of the ruminant. This will further help to reduce the use of harmful chemicals and antibiotics. To further evaluate the potential of E. faecium SROD, in vivo trial will be done to determine the growth performance, efficiency, and their effect on rumen microbiome and population of the animals as well as the methane production using Greenfeed technology.Overall, these results demonstrate that