Rising populations and urbanization are transforming into increased demand for livestock products, particularly in developing countries. The world will need more meat and more milk and in order to meet these demands, huge quantities of feed resources will be required. However, there is a substantial deficit of energy feeds affecting the growth and production of animals. The common method to increase energy value of ruminant diets is to provide them with fats. However, higher level of fats in the diet could prove toxic to rumen microbes and affect fibre digestibility, which ultimately results in reducing the feed intake and lowering animal production. These negative effects of fat supplementation can easily be overcome by feeding ruminants with specifically designed fats called rumen protected fats. In order to evaluate the efficacy of rumen protected fats (RPF), three different types of protected fats were examined in sheep. The results suggested that different types of protected fats have no unfavourable influences on the ruminal fermentation and productive parameters. Therefore, prilled fat, prilled fat with lecithin and calcium soaps did not improve animal performance as compared to the diet without protected fats in Dorper sheep.p < 0.05). RPF had no effect on concentrations of ammonia nitrogen, total volatile fatty acids and total bacterial population. The concentrations of rumen total saturated fatty acids, unsaturated fatty acids, total n − 3, total n − 6, unsaturated fatty acids:saturated fatty acids and polyunsaturated fatty acids:saturated fatty acids differed (p < 0.05) among the treatments with RPF supplementation. Hence supplementation of different types of protected fats did not influence animal performance in Dorper sheep.Rumen protected fats (RPF) are known to improve animal performance without affecting rumen metabolism in sheep. However, comparative effects of prilled fat, prilled fat with lecithin and calcium soap have not been fully studied. Hence this experiment was planned using 36 male Dorper sheep in a completely randomized design in four treatment groups. The diets included: Basal diet (70:30 concentrate to rice straw) with no added RPF as a control (CON), basal diet plus prilled fat (PF), basal diet plus prilled fat with lecithin (PFL) and basal diet plus calcium soap (CaS). The trial lasted 90 days following two weeks adaptation period. The body weights, average daily gain and gain to feed ratio were not affected by treatments. The intake and digestibilities of dry matter, organic matter, crude protein and neutral detergent fibre were not affected, while those for ether extract and crude fibre differed ( Rumen protected fats (RPF) are considered as insoluble fats because they are protected from microbial fermentation and biohydrogenation and remain insoluble at normal rumen pH. Thus, RPF that escape rumen fermentation are then utilized as a source of energy when absorbed through the small intestine. The fibre digestibility can be improved in high fat supplemented diets by feeding RPF . MoreoveThe studies conducted on prilled fat reported that its supplementation improved the digestibility of dry matter and organic matter . It did The supplementation of calcium soap improves fibre digestibility in the fat added diets by forming insoluble soaps and protects the fatty acids from rumen degradation . Ngidi eSalinas et al.  reportedThe experiment was carried out at Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia following the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Universiti Putra Malaysia . Thirty-six male Dorper sheep about 18 months of age with an average initial body weight 24.66 ± 0.76 kg (mean ± SE), kept in individual wooden pens measuring 1.2 m–1 m each, built inside a shed with slatted floor 0.5 m above ground with feeding and drinking facilities were fed the experimental diet for 90 days including last 10 days for digestibility trial. A two-week adaptation period was allowed before the experiment. The animals were drenched against parasites prior to the commencement of the trial.The experiment was conducted using completely randomized design (CRD) and animals were randomly allocated to four treatment groups containing nine animals in each group. The random selection was done in Microsoft Excel using tag number of animals. The rumen protected fats (RPF)  were obtProximate analysis  of the diets, refusal feed and the faecal samples was determined according to the protocol of Association of Official Analytical Chemists (AOAC) . NeutralThe body weight of each animal was recorded on the first day of the experiment before morning feeding and every 15 days thereafter, for the 90 days experimental period to determine body weight changes.Five animals from each treatment group were transferred to the metabolic cages provided with feeding and drinking facilities for digestibility trial. The animals were given three days as adaptation period followed by 10 days for faeces collection. The diets were fed twice every day at 0800 h and 1700 h with access to clean drinking water ad-libitum. The refusal feed was collected to calculate feed intake. Complete faecal samples were collected every day from each animal, weighed for 10 consecutive days and about 250 g samples were dried in an oven at 65 °C for 48 h to determine the DM contents. The faecal samples were ground to pass through 1 mm sieve and stored at −20 °C for proximate analysis.The digestibility coefficient was calculated using following formula:All the animals were slaughtered at Universiti Putra Malaysia abattoir by the Halal procedure, the gastrointestinal tract of each animal was removed and about 100 mL of rumen liquor was collected by sampling the rumen contents from the ventral, caudal and central areas of the rumen, pooled together and filtered using four layers of cheesecloth. The pH of rumen fluid was determined immediately after collection with Mettler-Toledo pH meter (Mettler-Toledo (M) Sdn Bhd, Shah Alam, Malaysia). Then 2 mL of 25% metaphosphoric acid was added to rumen liquor to stop the fermentation, snap-frozen in liquid nitrogen and kept at −80 °C until further analysis.The frozen rumen liquor was thawed and centrifuged at 10,000 g at 4 °C for 20 min, and supernatant was collected to determine the concentrations of volatile fatty acids (VFA) The supernatant (0.5 mL) was then mixed with 0.5 mL 20 mM methyl n-valeric acid  and was analyzed by gas chromatograph (Hewlett Packard) 6890 GC system  with bonded phase fused silica capillary column  equipped with a flame ionization detector (FID). The authentic standards of acetic, propionic, butyric, isobutyric, valeric, isovaleric and 4-methyl-n-valeric acids  were used to compare and identify the peaks.3-N) was determined using the colourimetric method described by Solorzano [Ammonia nitrogen (NHolorzano . A standv/v) mixture by using the protocol of Folch et al. [3) as per the procedure of Association of Official Analytical Chemists (AOAC) [Fatty acids from the rumen liquor were extracted in chloroform: methanol  . Heneico® Fast DNA Stool Mini Kit  according to manufacturer’s instructions. The quantitative real-time PCR was performed using the BioRad CFX96 Touch Real-Time PCR Detection System  by ultra-clear cap strips. The qPCR reaction was performed with the total volume of 25 µL, using the iQTMSYBR Green Supermix (BioRad). The reaction consisted of 12.5 µL SYBR Green Supermix, 1 µL of each reverse primer, 1 µL of forward primer, 2 µL of extracted DNA samples and 8.5 µL of DNAse-free water. The q-PCR conditions applied to each well were initially incubated at 94 °C for 5 min and then 40 cycles at 94 °C for 20 sec for denaturation followed by annealing temperature for 30 sec, and extended to 72 °C for 20 sec [The DNA was extracted from rumen fluid using the QIAampr 20 sec . The priStatistical analysis of the data obtained was performed using generalized linear model (GLM) procedure of the SAS software 9.4 . All data were analysed by the least-squares means method using the GLM procedures of SAS. Significantly different means were then further separated using Tukey HSD test. The microbial population data were checked for normality using the UNIVARIATE procedure of SAS software 9.4 . All statistical tests were conducted at 95% confidence level.p > 0.05) among the treatment groups (The body weight (BW), total weight gain, average daily gain (ADG), dry matter intake (DMI) and gain to feed ratio of the animals fed with the four dietary treatments were not affected (t groups .p < 0.05) across the treatment groups with supplementation of RPF (The intake and digestibilities of dry matter (DM), organic matter (OM), crude protein (CP), neutral detergent fibre (NDF) and acid detergent fibre (ADF) were similar. On the other hand, intake and digestibility values for ether extract (EE) and crude fibre (CF) were different (n of RPF .p < 0.05) across the treatments being less acidic 6.83, in the diet PFL and more acidic 6.50, in the CaS diet. The concentration of ammonia nitrogen (NH3-N) ranged from 14.94 mM for CaS to 16.55 mM for PFL but was not different across the treatments. Total VFA concentration was similar across the treatment groups. VFA molar proportions acetate, isobutyrate and acetate to propionate ratio were different (p < 0.05) while propionate and butyrate were similar among the treatments (The pH of rumen fluid was different (eatments .p < 0.05) in the populations of Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens within treatments. The supplementation of RPF increased the population of F. succinogenes and R. albus significantly as compared to the diet without RPF while the population of R. flavefaciens and total cellulolytic bacteria was found significantly lowest in the diet CaS. The population of total protozoa was different (p < 0.05) being lowest in PF and highest in CaS. The population of total methanogens was not different  by supplementation of different RPF. The concentrations of the total saturated fatty acids (SFA), total unsaturated fatty acids (UFA), total monounsaturated fatty acids (MUFA), total polyunsaturated fatty acids (PUFA), total ∑n − 3, total ∑n − 6, UFA:SFA and PUFA:SFA differ significantly among the treatments in response to supplementation of RPF  as compared to control when they added 20 and 40 g of RPF of rice bran oil to the experimental diets. No differences in average daily gain (ADG) and gain to feed ratio found among experimental diets in the current study could be ascribed to the similar energy and protein levels of the experimental diets (p = 0.03) feed conversion ratio (FCR) than the control diet. Contrary to the present study, Bhatt and Sahoo [Initial and final body weights and total weight gain in the present study were similar in all the treatment groups. Similar findings were reported by Haddad and Younis , when thal diets . Haddad nd Sahoo  reportedThe poor feed efficiency values in the current study could be attributed to the adult animals in the present study as the age of the animals was about 18 months thus the effect of RPF could not be seen obviously when it is offered during the finishing period. Park et al.  reportedSupplementation of RPF did not influence daily feed, DM, OM, NDF, ADF and ME intakes. The outcome of the current study is in agreement with Mudgal et al.  who founIn contrast to the current study, Haddad and Younis  found a The intake of ME was not affected by the supplementation of RPF. These findings are supported by Schauff et al.  and HaddSupplementation of RPF did not influence DM, OM, CP, NDF and ADF digestibilities in Dorper sheep. This is consistent with studies conducted by Naik et al. , who fouThe reduction in DM digestibility in diets with RPF compared to the diet without RPF is consistent with most findings ,8,48 whiThe present study also contradicts with Hightshoe et al.  who repoThe EE and CF digestibilities differ significantly among treatment groups in the present study. Animals fed PFL and PF had the highest EE and CF digestibilities. These results could be attributed to the emulsifying properties and lipid digestion enhancement from lecithin in PFL. Hence, lipids are rendered insoluble in the rumen and absorbed easily after emulsification. The increase in EE digestibility can be ascribed to high-quality fat inclusion in the diets . AnotherConversely, CaS yielded the poorest result for EE digestibility. This could be because of the FA composition, as CaS contained the highest concentration of unsaturated C18 FA and the lowest amount of saturated C18 FA. Alexander et al.  reported3-N [The increased digestibility of CP in the PFL and CaS is in agreement with findings of Bhatt et al.  who foun3-N  and lowe3-N . There iThe apparent digestibility of ADF in the PFL and CaS was significantly affected by supplementation of RPF when compared to the diet without RPF (CON). Naik et al.  also rep3-N, total VFA and molar proportions of VFA are consistent with a study reporting that rumen pH, NH3-N and VFA levels were not affected adversely by the supplementation of calcium soaps up to five percent of the DM in lactating cows [The similar rumen fermentation parameters including NHing cows .p < 0.05) across the treatment groups, and are in agreement with the findings reported by Cheng et al. [The rumen pH values in the present study are different  with supplementation of RPF among the treatment groups. Similar findings were observed by the studies conducted by [3-N levels [3-N and protozoa number follow a similar trend [3-N, total VFA, molar proportions propionate and butyrate indicate that all RPF were inert in the rumen, hence had no major influence on rumen fermentation when supplemented at current levels.The concentration of NHucted by ,47,53,57ar trend . The simDiet is one of the key factors that influence rumen microbial populations ,67. The The population of total bacteria did not differ significantly in the present study, which could be attributed to similar concentrate diets in all the treatment groups and is supported by the findings of rumen fermentation profile.p < 0.05) in rumen protozoa population was observed among the treatments being the maximum increase seen in the CaS diet. Similar to the findings of the present study, Ca soap supplementation increased protozoa population in cull ewes [Ruminococcus albus is explained by the fact that the reduction of protozoa in the rumen often results in greater proliferation of bacteria and greater passage of bacterial nitrogen N out of the rumen [The significant difference  in PF, PFL and CaS compared to CON diet. This reduction of C18:0 with RPF supplementation shows that the RPF were inert in nature, therefore, could not go under complete BH by rumen microbes as compared to CON which gave the highest concentration of C18:0. Another reason for higher C18:0 concentrations in the rumen digesta of the sheep fed diet without RPF could be that CON contained higher amount of oleic acid (C18:1n − 9), linoleic acid (C18:2n − 6) and linolenic acid (C18:3n − 3) comparative to RPF diets, thus higher intake of these FA caused higher C18:0 concentration in rumen of sheep fed CON diet due to BH process [The fatty acids released in the rumen are not absorbed by the rumen microbes, and are passed to the abomasum then the small intestine, which is the primary site for absorption of the fatty acids . The mai process .cis-9 trans-11 and C18:1 trans 11, this could be the result of incomplete BH of C18:2n − 6, C18:3n − 3 and C18:1n − 9. Similar findings were reported by several studies that incomplete BH of C18:2n − 6, C18:3n − 3 and C18:1n − 9 FA yielded CLA cis-9 trans-11, CLA cis-12 trans-10 and C18:1 trans 11 intermediate isomers [n – 6 ÷ n − 3 ratio, similarly in the present study there was the highest concentration of C18:0 in CON which had lower n – 6 ÷ n − 3 ratio. These findings could be because of the differences in the nature of dietary FA used, which in the present study was RPF. There were no differences found in concentrations of CLA among the diets with RPF supplementation. This could be because the source of fat was protected fat. Gulati et al. [The lowest concentration of C18:0 was observed in PF with the higher concentrations of BH intermediates CLA  isomers ,82,83. T isomers  who founi et al.  performeThe second highest concentration of FA found in rumen digesta of sheep was palmitic acid (C16:0) because the dietary treatments contained higher amounts of C16:0 and all RPF also had more than 70 percent of C16:0 concentration. The concentration of C16:0 in rumen digesta of sheep fed with rumen protected fat was higher in PF, PFL and CaS in comparison to CON; this was expected firstly because of the chemical composition of the diets which were high in C16:0, and secondly, because of the inertness of RPF.cis-9 trans-11 and CLA trans-10 cis-12 were not different significantly (p > 0.05) throughout the treatment groups. The lowest concentration of CLA cis-9 trans-11 was observed in CON. Similarly, Schmidely et al. [cis-9 trans-11 and CLA trans-10 cis-12 in goats fed 45 g/day of calcium salts of palm oil and 45 g/day of lipid-encapsulated conjugated linoleic acids. However, the factors that affect the concentration of CLA isomers are the fat content and polyunsaturated FA concentration of the diet, the percentage of concentrate and interaction between these two factors [The concentrations of CLA y et al.  reported factors ,87.The findings of the current study indicate that PF, PFL and CaS supplementation did not have a negative impact on body weight changes, nutrient intake, digestibility and rumen fermentation parameters. However, the RPF diets did not show significant differences when compared to the CON diet. Therefore, supplementation of different types of protected fats did not influence animal performance in Dorper sheep.


The low affinity oriC-DnaA-ATP interactions are required for the orisome’s mechanical functions, and may also play a role in timing of new rounds of DNA synthesis. To further examine this possibility, we constructed chromosomal oriCs with equal preference for DnaA-ADP or DnaA-ATP at one or more low affinity recognition sites, thereby lowering the DnaA-ATP requirement for orisome assembly, and measured the effect of the mutations on cell cycle timing of DNA synthesis. Under slow growth conditions, mutation of any one of the six low affinity DnaA-ATP sites in chromosomal oriC resulted in initiation earlier in the cell cycle, but the shift was not equivalent for every recognition site. Mutation of τ2 caused a greater change in initiation age, suggesting its occupation by DnaA-ATP is a temporal bottleneck during orisome assembly. In contrast, during rapid growth, all origins with a single mutated site displayed wild-type initiation timing. Based on these observations, we propose that E. coli uses two different, DnaA-ATP-dependent initiation timing mechanisms; a slow growth timer that is directly coupled to individual site occupation, and a fast growth timer comprising DnaA-ATP and additional factors that regulate DnaA access to oriC. Analysis of origins with paired mutated sites suggests that Fis is an important component of the fast growth timing mechanism.Although the mechanisms that precisely time initiation of chromosome replication in bacteria remain unclear, most clock models are based on accumulation of the active initiator protein, DnaA-ATP. During each cell division cycle, sufficient DnaA-ATP must become available to interact with a distinct set of low affinity recognition sites in the unique chromosomal replication origin,  In cells, DnaA is bound to either ATP or ADP, and DnaA-ATP is considered to be the active form of the initiator based on n assays . MultiplE. coli oriC, there are 11 DnaA binding sites within the 190 base pairs that are adjacent to the DNA Unwinding Element (DUE) , reviewed in Figure 1A). All low affinity DnaA recognition sites in oriC are incapable of binding DnaA directly  . Mutations in oriC were confirmed by sequence analysis (sequencing primer was 5′-CTCAACTTTGTCGGCTTGAG) and plasmids were introduced into DH5α by transformation selecting for ampicillin resistance.The positions of mutated DnaA recognition sites are shown in ,852 bp) , which coriC were amplified from plasmids using primers near gidA (5′-CACGGCCACCGCTGTAATTAT) and mioC (5′-ATCCCATACTTTTCCACAGG). The fragments were introduced by electroporation into ACL402 , nad::Tn10, ΔoriC::cat-sacB) following induction of RED gene expression, as described in sacB cassette from pK03  escribed . DnaA waescribed . Primer MG1655 was grown for at least five generations in exponential growth, in minimal media containing 0.1% uracil supplemented with 0.1% glucose plus 0.1% casamino acids; 0.1% glucose; 0.2% glycerol; or 0.4% succinate. Samples were removed from the cultures, pelleted, and flash frozen in liquid nitrogen three times to lyse the cells. Protein concentrations in each sample were quantified using the MicroBCA assay (Pierce) and then equal amounts of protein (5 mg) were resolved on 12% PAGE/SDS gels. The proteins were transferred to nitrocellulose, and DnaA and Fis on the blots were revealed using anti-DnaA and anti-Fis antibodies with detection by chemiluminescence (Bio-Rad ImunoStar kit). Assays were done in triplicate, with representative scans shown in Supplementary Figure oriC strains (MG1655 derivatives) were grown in minimal salts media , as well as pairs of sites in oriC specifically chosen for their location . Since we wished to retain the low affinity attribute while allowing DnaA-ADP binding, the I3, C2, C3, I2 I1, and τ2 sites were each individually mutated to sequences identical or similar to R5M. The sequence of the mutated sites is shown in Figure 1B. To verify that the mutations relieved the DnaA-ATP preference, but did not alter overall site affinity, binding of DnaA-ATP or DnaA-ADP to each site was evaluated using dimethyl sulfate (DMS) footprinting . DnaA was pre-incubated with nucleotide, and then bound to supercoiled oriC plasmid prior to DMS treatment and primer extension. Sites that are occupied by DnaA are revealed by distinctive changes to the DMS modification pattern resolved on sequencing gels. Specifically, the G4 of the 9 mer consensus 5′-TGTGGATAA (or variations of this sequence) becomes hypersensitive, and the G2 less sensitive to DMS , causing darker and lighter bands, respectively. The changes in band intensity increase as all of the plasmid molecules in the treatment population become occupied. It should be noted that not all binding sites have guanosines at both positions. For evaluation of site affinity, two concentrations of DnaA-ATP were used: 40 nM, which is insufficient for full occupation of all low affinity sites, and 100 nM, which previous studies have shown to be a saturating concentration, resulting in complete DnaA-ATP binding to all oriC copies in the reaction mix , the pattern of DnaA-ATP binding is consistent with previous studies showing R1, R2, and R4 as higher affinity sites fully occupied at 40 nM DnaA, and the remaining sites being fully occupied only at the higher concentration. The DnaA-ATP binding pattern is similar for origins containing the mutated sites, indicating that the mutations do not alter any sites’ affinity for DnaA-ATP. However, when DnaA-ADP is used in the reaction, only the mutant origin is fully occupied by 100 nM DnaA-ADP ; in contrast, binding of 100 nM DnaA-ADP binding to I3, C2, C3, I2 I1, and τ2 in wild type oriC gives a DMS modification pattern similar to that seen with 40 nM DnaA-ATP . This growth rate allows us to examine the simplest initiation scenario, in which only one oriC copy per cell must be activated, since the generation time of E. coli growing in this media should be greater than the time needed to complete chromosome replication and divide (∼60 min), so exponentially growing cells will contain only one copy of oriC prior to the time of initiation , and initiated ∼36 min after cell division (Table 1). Although previous studies report that cells growing slowly with acetate as the carbon source display rifampicin resistant initiations . The conversion of the τ2 site to τ2ADP caused a greater change in timing (Table 1), suggesting that filling this site might represent a rate-limiting step in slow growth orisome formation.To evaluate initiation timing, cells carrying the mutated tiations , we do ntiations . InteresoriC’s right half (I3/C3), left half (τ2/I2) or each half , because there is evidence that DnaA assembled onto the left and right halves of oriC play different roles in initiation . The effect of mutating two sites in the right sub-assembly (I3ADP and C3ADP) did not change timing appreciably from that obtained for cells containing these mutations individually . In contrast, placing two mutations (τ2ADP and I2ADP) in the left sub-assembly shifted the time of initiation much more than either of the single mutations . Initiation timing in cells harboring a mutated site in both left and right assemblies appeared to be determined largely by the left half mutation, with the τ2ADP/I3ADP mutant cells initiating slightly earlier that the I2ADP/I3ADP cells , consistent with the idea that τ2 plays a greater timing role than I2. These results remain consistent with models suggesting that the right and left sub-assemblies are not equivalent , and initiated ∼3 min after division. When cells containing origins carrying single site mutations were examined, we found that initiation timing was identical to that observed in cells with wild type oriC , in contrast to the shift in timing seen at the slower growth rate. These results confirm previous reports . As was done in the studies of slow growing cells, the double mutants were selected to allow examination of left and right sub-assemblies . Interestingly, changing both τ2 and I2 in the left half of oriC was sufficient to perturb initiation , but the alteration in timing was more complex than the simple shift to earlier times seen in slower growing cells. While most of the cells in the population had 4 origins, odd numbers of origins were also detected, indicating that initiations in these cells were no longer synchronous . The number of origins per cell was also slightly increased (Table 2). A similar pattern of asynchronous initiations was seen when τ2 was mutated in the left sub-assembly and I3 was mutated in the right sub-assembly . However, origins carrying both the I2ADP (left) and I3ADP (right) mutations showed essentially normal initiation timing , as was previously reported . These results reveal a clear difference in the response to changes in the left and right halves of oriC. One possible cause of this difference is the role of the right DnaA sub-assembly in displacing Fis .Unlike the slight over-initiation seen in cells with origins carrying τ2cing Fis . While fE. coli cell cycle is whether low affinity DnaA recognition sites in oriC are components of the timing mechanism. There are several possible roles for recognition sites in a clock model based on the availability of DnaA-ATP. In one simple scenario, each low affinity recognition site would require an equivalent amount of time to become occupied. Alternatively, the timer might be based on site occupation, but sites could play a non-equivalent role, i.e., some sites would require more or less time to become occupied than others. A third possibility is that despite a requirement to fill sites with DnaA-ATP during orisome assembly, some other factor acts as the initiation timer by restricting or facilitating DnaA-ATP access to oriC, thus setting the DnaA-ATP level for site occupation and origin activation. Since previous DnaA over-expression studies both support and oppose DnaA’s role in initiation timing  and τ2 to allow cross-strand DnaA donation . It is possible that some structural aspect of this orisome assembly step could impede DnaA-ATP access to τ2. It is also possible that cross-strand interactions between DnaA-ATP bound to τ2 and R1 are required to stabilize an origin configuration that promotes DnaA binding to the unwound DUE . In this model, Fis serves as both a negative and positive regulator during orisome assembly, negatively regulating IHF-dependent orisome assembly steps, but positively regulating synchronous initiation by ensuring enough DnaA-ATP accumulates to fire all oriC copies . This is the expected result if the mutations resulted in Fis displacement at lower DnaA-ATP levels than would be required for synchronous initiations at all oriC copies. Interestingly, there is more than one Fis recognition site in the right half of oriC  form of the initiator for most of the steps required for orisome assembly, as long as DnaA-ADP can access the appropriate recognition sites.Finally, it is worth noting that the change in initiation age that we observe for our mutant ng cells , the avaPR, JG, and AL planned the experiments. PR, JG, AA, and TR performed the experiments. PR, JG, and AL analyzed data. JG and AL wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Amplified Arctic warming and its relevance to mid-latitude cooling in winter have been intensively studied. Observational evidence has shown strong connections between decreasing sea ice and cooling over the Siberian/East Asian regions. However, the robustness of such connections remains a matter of discussion because modeling studies have shown divergent and controversial results. Here, we report a set of general circulation model experiments specifically designed to extract memory effects of land processes that can amplify sea ice–climate impacts. The results show that sea ice–induced cooling anomalies over the Eurasian continent are memorized in the snow amount and soil temperature fields, and they reemerge in the following winters to enhance negative Arctic Oscillation-like anomalies. The contribution from this memory effect is similar in magnitude to the direct effect of sea ice loss. The results emphasize the essential role of land processes in understanding and evaluating the Arctic–mid-latitude climate linkage. The connection between Arctic sea ice loss and mid-latitude cooling in Eurasia has been widely debated. Here, model experiments reveal that the persistence of sea ice loss-related snow and soil temperature anomalies in Eurasia may lead to further cooling in the following winters. This contrast between Arctic warming and mid-latitude cooling can be placed in the framework of the negative phase of the Arctic Oscillation (AO)/North Atlantic Oscillation (NAO). Observation-based studies have suggested a strong connection between the rapid decline of Arctic sea ice and wintertime mid-latitude cooling over the Siberian/East Asian regions in association with the negative AO/NAO5. However, the winter AO/NAO index has shown a positive trend after the strongest negative AO/NAO year of 2010, while Arctic sea ice has remained at a continuously low level. Some modeling studies have provided supporting evidence for this connection and the importance of Arctic sea ice loss8, whereas others have argued that Arctic sea ice loss has only small impacts on mid-latitude climate11. Therefore, large uncertainty exists among simulation models/studies13, while the underestimation and/or model dependency of the sea ice impacts are suggested as a possible cause15.Acceleration of warming over the Arctic region, a symbol of progressing global warming, has serious local and remote impacts on society and the environment. In the past decade, severe winters have affected densely populated areas in the Northern Hemisphere, such as North America, Europe, and East Asia16. Once sea ice is reduced significantly, stratosphere–troposphere coupling plays a key role in connecting sea ice variations to AO/NAO-like circulation anomalies on intra-seasonal timescales19. A possible role of land processes has also been considered. The relevance of anomalies in autumn snow cover extent over the Eurasian continent to winter negative AO/NAO via a stratospheric pathway is well documented20. Furthermore, a land process with a longer memory than the atmosphere could affect the atmosphere on a timescale beyond a season21. Nevertheless, there are no studies that have examined the memory effect of land processes on the current Arctic climate.Atmosphere–ocean coupling and stratosphere–troposphere coupling processes have been studied extensively in connection with Arctic climate changes. Ice-albedo feedback in the atmosphere–ice–ocean coupling process is essential in Arctic amplification, providing a long-term memory effect from the large heat capacity of the ocean and the sensitivity of the surface energy balance to albedo23.This study specifically examined a possible role of the memory effect arising from land processes, with the working hypothesis in which the negative AO–like pattern activated by Arctic sea ice loss causes significant anomalies at the surface level that are memorized in snow and soil and can persist for several months or more than a year. The memorized anomalies in turn re-emerge at the surface in later years, affecting the atmosphere and amplifying the atmospheric response related to Arctic sea ice loss. To examine this hypothesis and quantify the contribution of the memory effect, we developed a procedure to extract the memory effect in the atmospheric general circulation model (AGCM) framework Fig. . As descFirst, we examined the winter (December–February) averaged anomalies in the Northern Hemisphere. In the upper tropospheric geopotential height field (Z300), the sea ice effect from Arctic sea ice loss appears as positive anomalies over the Arctic Ocean and negative anomalies over East Asia, a combination that resembles the negative phase of AO Fig. , sea iceIt should be noted that the responses are strongly nonlinear  of heating through the memory effect arising from land processes. The anomalous meridional circulation also acts to cool the mid-latitudes by −0.76 W m−2, in which the sea ice and memory effects contribute −0.41 and −0.35 W m−2, respectively.We also compared the column-wise heating rate caused by atmospheric heat transport with the turbulent heat flux anomaly over the Arctic Table . The aveAs described in the previous sections, anomalous wave activity induced by the memory effect appears to originate from the mid-latitudes Figs. 4b. Here27. The memory effect in soil temperature produces persistent cold anomalies over the Eurasian continent that weaken in the summer to autumn and strengthen in the late winter  are stored in the soil as a memorized anomaly that may modulate the climate several years after the initial impact. In experiments such as AMIP-type simulations, these types of extra sea ice impacts might disturb sea ice-induced memories . Insights gained from this study with regard to land processes and their role in climate memory can be used to improve the design and interpretation of the results of future studies on Arctic climate change.Multi-model intercomparisons are currently being conducted to evaluate the Arctic amplification occurring in the current climate and in future projections /Optimum interpolation (OI) SST data set35 as the boundary condition  and 56 vertical levels up to the 60 km model top. AFES incorporates a land surface model, minimal advanced treatments of surface interaction and runoff (MATSIRO)ICE Fig. . Similarrun Fig. .†–HICE] + [LICE–HICE†])/2 (sea ice). If the anomalies due to the sea ice effect explain most of the total effect, the LICE† and HICE† states should be close to the LICE and HICE states, respectively, indicating there is no memory effect beyond the annual cycle. Therefore, the residual of the total minus the seasonal effects is regarded as the memory effect that persists from previous years (memory). In the proposed framework, because the atmospheric internal variation generally does not persist for longer than a few weeks, the memory effect is achieved only through land processes. Thus, this experimental design is suitable for the purpose of this study.Using 100-year average outputs from each run, the atmospheric response to the total effect of Arctic sea ice loss was estimated as (LICE–HICE) . The atmospheric response that develops during summer to the following winter as a direct effect of sea ice loss was estimated as  applied to the daily mean dynamical field was used to diagnose changes in the atmospheric general circulation in the meridional plane, which is indicative of heat transport from the mid-latitudes to the polar regionSupplementary InformationPeer Review File


Pyruvate kinase (PK), as one of the key enzymes for glycolysis, can encode four different subtypes from two groups of genes, although the M2 subtype PKM2 is expressed mainly during embryonic development in normal humans, and is closely related to tissue repair and regeneration, with the deepening of research, the role of PKM2 in tumor tissue has received increasing attention. PKM2 can be aggregated into tetrameric and dimeric forms, PKM2 in the dimer state can enter the nuclear to regulate gene expression, the transformation between them can play an important role in tumor cell energy supply, epithelial–mesenchymal transition (EMT), invasion and metastasis and cell proliferation. We will use the switching effect of PKM2 in glucose metabolism as the entry point to expand and enrich the Warburg effect. In addition, PKM2 can also regulate each other with various proteins by phosphorylation, acetylation and other modifications, mediate the different intracellular localization of PKM2 and then exert specific biological functions. In this paper, we will illustrate each of these points.The online version of this article (10.1186/s13578-019-0317-8) contains supplementary material, which is available to authorized users. At the beginning of the twentieth century, German scientist Warburg discovered that in tumor tissues, even if oxygen is sufficient, malignant tumor cells still undergo active glucose glycolysis, the metabolic characteristic of this aerobic glycolysis is called Warburg effect . And it + and REDOX equivalents to the subsequent electron transport chain, but provides tumor cells such as Glutamine, Proline, Ornithine, Lysine, Methionine and other non-essential amino acids which tumor cells biosynthesis required for  and oxidative phosphorylation (OXPHOS) to completely decompose glucose into carbon dioxide and water, and when consumes oxygen, the cell itself is supplied with a large amount of ATP at the same time . WarburgPOST-Warburg effect, in glucose metabolism, tumor cells divide glucose metabolism into three separate parts of glycolysis, tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS). The effect of PKM2 is currently considered to be the interception of glucose metabolism and the metabolic pathway is transferred to the pentose phosphate pathway (PPP), the uronic acid pathway (UAP), the polyol pathway (PYP), etc. for the synthesis of the subsequent five-carbon ribose and non-essential amino acids. The TCA circle is backed up by fatty acid metabolism and amino acid metabolism, and its main purpose is to provide raw materials for the synthesis of non-essential amino acids, and the secondary purpose is to supply REDOX equivalents . AlthougAlthough PKM1, PKM2, PKL, and PKR all have a tetrameric form and pyruvate kinase activity, only PKM2 has both a dimeric form and a tetrameric form. In Fig. The transition between PKM2 dimers and tetramers is allosterically regulated by endogenous and exogenous activators and inhibitors. PKM2 has PK enzyme activity only when it serves as a tetramer. PKM2 is activated by the glycolytic intermediate products named fructose 1,6-bisphosphate (FBP). It can also be activated by the allosteric effects of serine and succinylaminoimidazolecarboxamide ribose-50 phosphate SDH succinate dehydrogenase (SAICAR) , 55. TheIt is now accepted that the PKM2 in the tetrameric state has an allosteric regulatory domain within its spatial structure, forming a pattern similar to the seesaw, when some allosteric regulators are inserted into the spatial structure involved in PKM2 allosteric regulation. After the domain Fig. a, the teThere are four PK subtypes encoded by two genes in mammals which was shown in Fig. In addition to the different cleavage patterns, more and more studies have shown that the expression of PKM2 is also closely related to MicroRNAs (miR/miRNA), Long non-coding RNAs (LncR/LncRNA), etc., in non-coding RNA families, among which miRNAs are a class of short-chain non-coding RNAs, and are bound to the seed region at the ‘-UTR end of mRNAs and conduct functions, therefore affect protein synthesis and folding . Some scThe genes encoding pyruvate kinase can be divided into two types: PKLR and PKM. PKLR binds to the coding gene through a tissue-specific promoter, encoding two subtypes of PKL and PKR (green for PKR and yellow for PKL). PKM encodes PKM1 and PKM2 subtypes by alternative splicing of mutually exclusive exon 9 and 10, and a high expression level of PTB, hnRNP 1 and hnRNP A1/A2 are required for during the cleavage process of exon 9 of PKM2, while the cleavage process of exon 10 of PKM1 is not required. Transcription factor SRSF3 also plays an important role in the conversion of PKM2 and PKM1. Each pyruvate kinase subtype has a different tissue expression pattern , ultraviolet radiation and other factors, and in the form of dimers to play a role in protein kinase activity in the nucleus of a variety of transcription factors and thus affect a variety of signaling pathways to promote tumor development , 115. ReThe phosphorylated PKM2 can protect mitochondrial from oxidative stress. At the beginning of the period, ROS can oxidize the Cys358 site of PKM2 when tumor cells are exposed to oxidative stress damage. PKM2 acts as a phosphorylase to phosphorylate its substrate . PKM2 caPKM2 phosphorylates PAK2 directly on Ser20, Ser141 (phosphorylated but weakly acting) and Ser192/197. PKM2-mediated phosphorylation of Ser192/197 is essential for maintaining PAK2 activity in PDAC cells, promotes HSP90 association with PKM2-PAK2 complex, which prevents the degradation of ubiquitin and protease of PAK2, and ultimately mediates tumor cell invasion, metastasis and cell proliferation .After binding to SAICAR, PKM2 phosphorylates the Thr202, Tyr204 and ERK2 Thr202 of ERK1, which in turn phosphorylates the Ser37 of PKM2. This cascaded phosphorylation pattern can form an activation loop and activate the ERK/MAPK signal pathway .PKM2 phosphorylates tyrosine at position Tyr333 on β-catenin, and phosphorylation of this site is necessary for the binding of β-catenin to the PKM2 Tyr site, the binding of PKM2-β-catenin (Tyr333p) can promote the proliferation and development of tumor cells .PKM2 can phosphorylate Thr11 of histone H3, which regulates the transcription of MYC and CCND1, and promotes the G1-S phase transition of the cell proliferation cycle, as well as Y705 phosphorylating stat3. Phosphorylation of STAT3 can promote the production of CCND1, which can enhance the activity of mitogen activated protein kinase (MAPK) pathway .PKM2 can phosphorylate Tyr118 and phosphorylate Bub3 Tyr27 sites of myosin light chain 2 (MLC2). Phosphorylation of MLC2 can also enhance the activity of mitogen activated protein kinase (MAPK) pathway, and phosphorylated Bub3 can enhance the activity of EGFR pathway .PKM2 can promote the serine at position 202/203 of protein kinase B substrate l (AKT1S1) binding to 14-3-3 to activate the mechanistic target of rapamycin complex l (mTORC l) signal pathway, which will promote tumor growth .In the nucleus of bone marrow cells of leukemia patients, In the nucleus of bone marrow cells of leukemia patients, the researchers found that PKM2 can phosphorylate and activate certain transcription factors, thereby inducing fusion of BCR-ABL genes. In the cytoplasm of these cells, it is characterized by the dissociation of PKM2 tetramer, the formation of dimers, and finally promote the accelerated formation of leukemia in patients .A mode of action similar to the last article 7 still exists in ETV6–NTRK3. PKM2 can phosphorylate and activate certain transcription factors, thereby inducing fusion of Est variant 6 and neurotrophic tyrosine kinase receptor. The result may lead to malignant transformation of some neurogenic tumors. PKM2 in the cytoplasmic dimer form promotes the formation of the Warburg effect .P53 and PKM2 in the nucleus of the cells can phosphorylate each other to form a cascade loop. Although the location of the PKM2 phosphorylated P53 modification site has not been specifically demonstrated, P53 has been shown to phosphorylate the Ser37 site of PKM2. When the tumor cells are under stress, this pattern is activated to protect against external stress in the form of EMT .The phosphorylated PKM2 can increase its nuclear localization and promote cell proliferation .PKM2 phoThe tyrosine residues Tyr 83, Tyr105, Tyr148, Tyr175, Tyr370, Tyr390 of PKM2 can be directly phosphorylated by fibroblast growth factor receptor 1 (FGFR1), and the binding of phosphorylated PKM2 to FBP inhibits the presence of the tetrameric form of PKM2, results in reducing its PK activity .It has been reported that tripartite motif containing 35 (TRIM35) directly binds to PKM2 to inhibit PKM2 Tyr105 phosphorylation, thereby increasing enzyme activity .ERK1/2 also has phosphorylase activity, which specifically phosphorylates the Ser37 site of PKM2 without phosphorylating PKM1, which provides a binding motif for PKM2 that interacts with peptidyl-proline isomerases, and binding to the transcript of the mitotic gene A1 (PIN1) mediates the entry of PKM2 into the nucleus . When PKGlycogen synthase kinase 3β (GSK-3β) could phosphorylate Thr-328 of PKM2, and phosphorylation of the PKM2 Thr-328 site is essential for maintaining PKM2 stability and regulating biological functions of glycolysis, mitochondrial respiration, proliferation and apoptosis. HSP90 also has a function to stabilize the PKM2–GSK3β complex in the HSP family .In the cytoplasm of tumor cells, proviral insertion in murine 1ymphomas 2 (PIM2) can also phosphorylate the Thr454 site of PKM2, to mediates PKM2-dependent anaerobic glycolysis, and maintains mitochondrial function in tumor cells .Jun N-terminal kinase 1 (JNK1) phosphorylates the PKM2 threonine residue Thr365 to inhibit PKM2 enzyme activity .PKM2 can be phosphorylated by Fms-related tyrosine kinase 3 (FLT3), Janus kinase 2 (JAK2), protein kinase C (PKC), and the modified PKM2 will exist as a dimer. Promote anaerobic glycolysis and cell proliferation of tumor cells , 133.In addition to phosphorylation of these known sites, there are also some protein molecules with phosphorylation, but the site is not yet clear. Such as A-RAF, after phosphorylation of PKM2, which could regulate the composition ratio between PKM2 dimer and tetramer, and then regulate the glucose metabolism of tumor cells . PKM2 biPKM2 not only can act as a phosphorylase to phosphorylate the substrate, but also can be phosphorylated by other phosphorylases at specific sites. The phosphorylation of its tyrosine residue Tyr105 has been found in a variety of solid tumors in humans. For example,In addition to phosphorylation, proteins such as protein tyrosine phosphatase 1B (PTP1B) inhibit the phosphorylation of PKM2, and PTP1B inhibits the biological activity of PKM2 by phosphorylating the Tyr105 and Tyr148 site of PKM2 .2 consumption and CO2 production  were utilized to identify the enrichment of target genes by DAVID (http://david.abcc.ncifcrf.gov/). The enrichment map of annotation analysis was drawn using Cytoscape (version 3.3.1) (http://www.cytoscape.org/cy3.html). GO: Gene Ontology. DAVID: Database for Annotation, Visualization and Integrated Discovery.Additional file 4: Fig. S3. The significantly KEGG pathways identified by KOBAS. Using the 30 genes predicted by MEM, 34 different KEGG pathways can be enriched by KOBAS (http://kobas.cbi.pku.edu.cn/). These signaling pathways can be roughly divided into six broad categories. The results were generated using the visualization tool in R (version 3.5.3). KOBAS: KO-Based Annotation System. KEGG: Kyoto Encyclopedia of Genes and Genomes. MEM: Multi Experiment Matrix.Additional file 5: Fig. S4. The functional protein association network enriched by STRING. PKM2 and its related proteins can not only constitute a large regulatory network to affect the energy metabolism of tumor cells, but also can form many small regulatory networks to affect different biological activities. The data used was derived from the 100 genes predicted by the MEM database by STRING (https://string-db.org/), which are highly correlated with PKM2.


In high-speed precision machining, thermal deformation caused by temperature rise affects the accuracy stability of the machine tool to a significant extent. In order to reduce the thermal deformation of ball screws and improve the accuracy, a new adaptive method based on carbon fiber reinforced plastics (CFRP) was proposed in this study and the thermal deformation of ball screws was determined. By using the sequential coupling method, the thermal–structural coupling analysis of a ball screw was conducted based on the finite element method (FEM). The analysis results were verified through a comparison with the experimental results. Based on the verification, an FE model of the improved ball screw was established to study its thermal characteristics. The key design parameters of the improved ball screw were optimized based on the Kriging model and genetic algorithm (GA). The thermal reduction effect of the improved ball screw was validated through the experimental results. The results indicate that the adaptive method proposed in this research is effective in reducing the thermal deformation of ball screws. Ball screws with high efficiency, precision, and stiffness have become important transmission mechanisms of the servo feed system used for transforming rotational motion into linear motion . With thDetermination of the thermal deformation of ball screws is an important prerequisite for reducing such unfavorable effects. Shi and Mei et al. established a thermal deformation model of the ball screw according to experimental data by considering the time and nut position based on the multiple regression, fuzzy serial, and linear regression methods ,7. Yang Thermal deformation compensation of the ball screw is an effective approach to reduce the error in machine tools. Some scholars established the temperature field and thermal deformation model of machine tools based on thermal modal analysis  or tempeIn summary, there are two major strategies for the compensation of thermal deformation in a ball screw, namely, an active control system and a hollow (liquid or air cooled) ball screw. An active control system requires a complex system and the speed of the nut is much greater than the heat transfer speed, which can lead to failure of the compensation system. Hollow and liquid-cooled ball screws have a good compensation effect ,24, but The contents of this paper are arranged as follows. In In order to reduce the thermal deformation of ball screws, an improved ball screw was designed by utilizing the negative thermal expansion coefficient of CFRP in the axial direction, as illustrated in The design at the end is to ensure that the CFRP is in a tension state by adjusting the nut for pretension when it is in operation. The end caps are tightly connected by a series of bolts. When the ball screw feeding system is assembled, nuts and bearings in the ball screws are assembled first, then the CFRP is mounted, and finally the end cap is installed, as shown in In order to check the effect and obtain better performance, thermal deformation of the ordinary ball screw and improved ball screw were simulated and compared, and the FE modeling was validated by experimental results. On that basis, the key design parameters were optimized.A ball screw can be approximated as a typical thermodynamic system, and the relevant thermodynamic parameters such as heat generation and heat transfer coefficient of the screw surface can be obtained from the literature ,28,29,30There are two typical methods for evaluating the thermal deformation of ball screws based on the FE method. The first is the sequential coupling method where the interaction between the temperature field and the structural field of ball screw is neglected. The results of the thermal analysis are directly transferred into the structural field as a temperature load, and the thermal deformation is obtained using the structural analysis. In the second method, the interaction between the two physical fields is considered. According to previous research , there iIn order to illustrate the FE modeling of ball screws, a certain type of ball screw was investigated. Parameters of the ball screw are given in During geometric modeling, complex features of the ball screw such as the raceway were ignored, and the screw was simplified to a shaft, thereby reducing the demand for computing resources and time . The FE t, the heat flow is applied in the blue region, while at t + Δt, the applied region moves a distance toward the nut moving direction. The distance can be evaluated by vt, where v is the velocity of the nut movement. When the region moves to the end, it will move back until the thermal balance is achieved. The trace of nut motion is shown in Thermal parameters of the ball screw are determined as shown in The steady-state temperature field, shown in During the structural solution, the bearing close to the motor in the feeding system restrains the axial deformation of the ball screw, while the bearing in the opposite direction provides the freedom for the axial deformation. Therefore, based on its working conditions, the ball screw is considered to be a simply supported beam. Based on the sequential coupling method, the displacement field of the ball screw was obtained as shown in In order to verify the FE model of the ball screw, the experiment was carried out on the ball screw test table, as shown in The experiment was carried out without applying any axial load and only the preload was considered. The rotational speed was 1000 r/min. The thermal imager measured the temperature of the ball screw once every 10 minutes. The experiment was conducted until the ball screw on the test table attained thermal balance. The steady-state field of the ball screw experiment is shown in The comparison between the simulation results and experimental results is given in di = 25 mm, thickness of the layer b = 2.5 mm, and volume fraction of carbon fiber cm = 65%. High modulus carbon fiber (M55J type) was used as the reinforcement of CFRP. The parameters of carbon fiber are shown in To study the reduction effect of the new design, FE analysis of the improved ball screw was performed. According to the method described in di, b, and cm. The diameter of the hole affects the heat transfer coefficient; the thickness of CFRP layer affects the heat dissipation and contraction, while the volume fraction of the fiber affects the elastic modulus and thermal expansion coefficient of the composite.The key design parameters of the improved ball screw are L16 (43) with three factors and four levels was employed to generate the samples, as shown in A Kriging model was established to make the optimization more efficient. The orthogonal table di had the greatest effect on the thermal deformation of the improved ball screw. The effect of layer thickness on the thermal deformation of the improved ball screw was slightly greater than that of the fiber volume fraction. The results of the range analysis are shown in The Kriging model is established according to the samples , and theThere are two important indicators to evaluate the Kriging model . The avei=1,2, …n), where y and n is the number of samples.The average error is derived as To validate the Kriging model, eight samples were randomly selected and calculated by the FE method, as listed in th verification sample. This is because the gradient at this sample  was much greater than that at other samples in the real response surface. The Kriging model was based on orthogonal designed samples, which resulted in the greater error at this point . Therefore, it can be concluded that the Kriging model developed in this study can be implemented to predict the thermal deformation of the improved ball screws instead of the FE method. The thermal deformation calculated from the Kriging model and FE method is shown in The optimization of the design parameters for the improved ball screw was implemented based on genetic algorithm (GA)  with theThe optimized results are shown in In order to verify the effectiveness of the method, a fundamental experiment was carried out. A hollow shaft with CFRP pasted inside was used to simulate the improved ball screw. A simplified shaft with the same diameter and length was also manufactured to simulate the ordinary ball screw and compare the temperature and thermal deformation with the improved design. As it is difficult to measure and control the preload, it was difficult to determine the heat generation at the nut. In this research, the shaft was heated by heating sheets to simulate the heat generation when the nut moves, and the heat power was adjusted by the supply voltage. As shown in Two working conditions were considered in this research, namely, the supply voltage of 20 V and 30 V. The temperature and thermal deformation of the two specimens were tested and compared. When the temperature rise was less than 0.5 °C in 10 minutes, it was considered that the system had reached thermal balance. The temperature rise comparison of the specimens is shown in The improved design obviously reduced the temperature rise and thermal deformation according to the experimental results. CFRP contracted the screw in the axial direction with a rise in temperature due to the negative thermal expansion coefficient, which reduces the thermal deformation. A detailed design was proposed for reducing the thermal deformation of ball screws adaptively based on the negative thermal expansion coefficient of CFRP. The effectiveness of this proposed method was theoretically and experimentally validated. The following conclusions can be drawn from this study.(1) A new adaptive method based on CFRP was proposed to reduce the thermal deformation in ball screws. It was demonstrated that the adaptive method is effective in reducing the temperature and thermal deformation of the ball screw.(2) The temperature and thermal deformation of ordinary ball screw and improved design were solved by FEM. The optimization of the key parameters of the improved ball screw was also conducted. The effectiveness of the adaptive method was validated by theoretical analysis.(3) Based on the experimental study, the adaptive method was verified. The thermal deformation was reduced by as much as 70.1%, which indicates that the adaptive method proposed in this research is effective in reducing the thermal deformation of ball screws.A fundamental experiment on the ball screw model was conducted in this research. The proposed method will be validated under actual working conditions at the next stage of this research.


Polarstern in summer 2012 (2 August-29 September). Nine transects were performed for the first time in deep-sea areas previously fully covered by ice, four of them in the Nansen Basin (3571-4066m) and five in the Amundsen Basin (4041-4384m). At seven of these stations benthic Agassiz trawls were taken near the camera tracks for species identification. Observed Arctic deep-sea megafauna was largely endemic. Several taxa showed a substantially greater depth or geographical range than previously assumed. Variations in the composition and structure of megabenthic communities were analysed and linked to several environmental variables, including state of the sea ice and phytodetritus supply to the seafloor. Three different types of communities were identified based on species dominating the biomass. Among these species were the actiniarian Bathyphellia margaritacea and the holothurians Elpidia heckeri and Kolga hyalina. Variations in megafaunal abundance were first of all related to the proximity to the marginal ice zone. Stations located closer to this zone were characterized by relatively high densities and biomass of B. margaritacea. Food supply was higher at these stations, as suggested by enhanced concentrations of pigments, organic carbon, bacterial cell abundances and nutrients in the sediments. Fully ice-covered stations closer to the North Pole and partially under multi-year ice were characterized by lower concentrations of the same biogeochemical indicators for food supply. These stations nevertheless hosted relatively high density and biomass of the holothurians E. heckeri or K. hyalina, which were observed to feed on large food falls of the sea-ice colonial diatom Melosira arctica. The link between the community structure of megafauna and the extent and condition of the Central Arctic sea-ice cover suggests that future climate changes may substantially affect deep ocean biodiversity.Quantitative camera surveys of benthic megafauna were carried out during the expedition ARK-XXVII/3 to the Eastern Central Arctic Basins with the research icebreaker  We did not use trawl data for quantitative analysis. There are difficulties with the estimation of the seafloor area sampled by trawl, because the exact bottom contact time is not known. Also trawl sampling is inefficient in catching mobile epibenthic organisms because they escape trawls. When caught in the trawl, organisms are often destroyed by massive amount of sediment, or washed through the net when they are small. On the other hand, the trawl collects infauna that is not visible on images. Number of taxa was calculated at each station separately based on images and trawl samples, and together based on images and trawls.2) and Simpson’s diversity (1-λ).Quantitative analysis was performed only for image data. Mean taxa densities (±standard deviation) and total megafauna density (±standard deviation) were calculated for each transect. Densities of each taxa were calculated per image and then average density per transect was obtained. Relative contributions (%) of the most abundant taxa to the total density were estimated. Following diversity indices were applied to describe megafauna assemblages: Pielou’s evenness (J'), Shannon–Wiener diversity (H')  was generated. Contribution of taxa to similarity and dissimilarity of different groups of stations was calculated using the SIMPER subroutine of PRIMER v6 .2 and the total megafauna biomass per m2 were estimated for each transect. Mean biomass (preserved wet weight) was calculated based on the wet weight of preserved individuals sampled by trawls. Average wet weight for the entire taxon identified to the lowest possible taxonomical level in a trawl was calculated and applied to all individuals of that taxon on image transects, regardless of visual size. For taxa with insufficient trawl data the biomass was estimated using the biomass data of taxonomically similar taxa or taxa with similar body shape.Mean taxa biomass per mKolga hyalina and Elpidia heckeri and the ophiuroid Ophiostriatus striatus associated with fresh and detrital algae aggregations was calculated for each transect in order to assess the contribution of algae to the nutrition of those species.Percentage of holothurians Spearman's rank correlation was calculated between the environmental/sediment parameters and community characteristics or taxa densities/biomass. A canonical correspondence analysis (CCA) was performed at the station scale to estimate the contribution of environmental and sediment parameters to megafauna structure (DOC and TDN were not included because they were not measured at stations 1 and 5)  based onBiogeographic distribution patterns were evaluated for 43 species and 55 genera .2 of the sea floor .Six following taxa were abundant and common at all transects: the actiniarian  hyalina . Eleven Thirty taxa were identified to the species or genus level, 16 to family; others were assigned to higher taxa. Number of taxa belonging to major taxonomical groups are presented in doi.pangaea.de/10.1594/PANGAEA.896638.Taxa richness was the highest at the ice-covered station 6 in the vicinity of Gakkel Ridge and the lowest at the multiyear-ice station 7 close to the North Pole . StationBathyphellia margaritacea. At stations 7 and 9 the holothurian Elpidia heckeri and the polychaete Macellicephalinae gen. sp.5 were dominant. Station 6 was dominated by the holothurians Kolga hyalina and E. heckeri. The highest dominance of a single taxon was at the MIZ station 4 (78%) and at the ice-covered station 9 (71%). Accordingly, evenness was the lowest at station 4 and the highest at the MIZ station 1 and ice-covered station 8. Diversity was the lowest at stations 4 and 9 and the highest at the MIZ station 3  . Two gro-2. The highest biomass (based on estimations for image data) was at the ice-covered station 6 near Gakkel Ridge. High values were also obtained at the MIZ stations 2, 3 and 5. The lowest biomass was found at the ice-covered station 8  the holothurian Elpidia heckeri and 3) the holothurian Kolga hyalina. B. margaritacea dominated at the MIZ stations 1, 2, 3, 4, 5 and at the ice-covered station 8. K. hyalina was dominant at the ice-covered station 6. E. heckeri dominated at the ice-covered stations 7 and 9.Taxa dominating the biomass  are shown in 2. At four stations  the algal falls were mostly fresh  to 10±1% (station 8) . PatchesOphiostriatus striatus and the holothurians Kolga hyalina and Elpidia heckeri. At stations with abundant K. hyalina , 10–40% of specimens of this holothurian were associated with algal falls. Maximum values were recorded at stations with mostly fresh (St. 3) or moderately degraded (St. 5) algae. Percent of specimens of another holothurian, E. heckeri, associated with algae falls was similar: 10–44%, they dominated at St.7 with mostly fresh algae. Percentage of individuals of the ophiuroid O. striatus associated with algae falls was lower: about 5% at stations 2 and 3.Three benthic species endemic to the Arctic were observed feeding on algae aggregations: the ophiuroid Hyalopomatus claparedii and the actiniarian Bathyphellia margaritacea . All of these stations, except 8, were associated with the ice margin and the first-year ice and were characterized by relatively higher concentrations of biogeochemical indicators for food supply. All of these variables showed lower concentrations at stations 6, 7 and 9 located at some distance from the ice edge, and at least partially under thicker multi-year ice and 100% ice cover. They showed a higher densities of the holothurians Elpidia heckeri (stations 9 and 7) and Kolga hyalina (station 6).Potential individual effects of single environmental factors on megafauna community composition and structure were evaluated. Megafauna communities are known to depend on food supply, here indicated by measured concentrations of chlorophyll a in sediments, as a proxy for the typical background phytodetritus sedimentation, or by the visually detected algal falls as a proxy for the specific 2012 melt-out of ice algae . The latElpidia heckeri and Kolga hyalina feeding on the algal falls.However, the effect of the algal coverage as a percent of the total transect area and freshness of algal aggregations was not statistically significantly related to total community structure and taxa abundance/biomass . None ofIn total 2131 specimens of benthic organisms were encountered in the trawl catches from 7 stations.Bathyphellia margaritacea, the ophiuroid Ophiostriatus striatus, the holothurian Kolga hyalina, the polychaete Anobothrus lauberi and the bryozoan Nolella cf. dilatata. The list of identified taxa with abundance and biomass values at each station can be downloaded from doi.pangaea.de/10.1594/PANGAEA.896627 and doi.pangaea.de/10.1594/PANGAEA.896629 correspondingly. Numerous fragments of dead sponges Caulophacus arcticus with epifaunal hydrozoans and bryozoans were registered at all stations. Most diverse taxa were polychaetes (9 families), amphipods  and sponges (6 families).Fifty one taxa were recorded in trawl samples (32 taxa in the Amundsen Basin and 39 taxa in the Nansen Basin). Nineteen taxa were unique for the Nansen Basin and 12 for the Amundsen Basin. Taxa numbers per trawl varied from 8 (St. 7) to 24 (St. 2). Most abundant in trawl samples were the actiniarian doi.pangaea.de/10.1594/PANGAEA.896618). 31 species was registered only in trawls; 38 only on images.20 taxa were found both in trawl samples and on images  compared to images (3 taxa). Also, Macellicephalin polychaetes were diverse and abundant on images, whereas in trawl samples only one species was found.Kolga hyalina, Elpidia heckeri, Bathyphellia margaritacea and Ophiostriatus striatus) were quantitatively underrepresented in trawl catches . This was also the case with mobile organisms  caught in very small numbers, potentially due to their ability to escape trawls. At the same time the number of epifaunal hydrozoans, bryozoans and sponges, inhabitants of hard substrata such as stalks of the sponge Caulophacus, was underestimated based on images. The infaunal polychaetes caught in trawls were not observed by the OFOS. Altogether, total megafauna density in trawl samples (0.1–0.6 ind m-2) was much lower compared to values based on images (0.9–7.8 ind m-2).The most abundant epifaunal species recorded by camera  to 56 (St. 6). Number of taxa belonging to major taxonomic groups registered on images and trawls is given in Bathyphellia margaritacea, Kolga hyalina, Elpidia heckeri and Onisimus leucopis) in this study were all Arctic endemics.Seven generally accepted regions of the ocean were used as a reference in the biogeographic analysis: Arctic Ocean, North Atlantic, South Atlantic, Indian Ocean, North Pacific, South Pacific and Antarctic. Taxa present in all seven regions were classified as cosmopolitan. Endemic species are defined as those that were not reported outside the Arctic Ocean. The Greenland-Scotland Ridge separates the deep Arctic from Atlantic, and shelves of the Chukchi and Bering Seas separate Arctic Basin from the Pacific . Forty tTubularia regalis, the bryozoan Eucratea loricate and the asteroid Tylaster wyllei. Known depth was also increased for the polychaete Hyalopomatus claparedi and the decapod Bythocaris curvirostris. Cnidarian Bouillonia sp., Echiurida fam. indet. and Enteropneusta fam. indet. were for the first time recorded in the Central Arctic. The cnidarian Oceanactis bursifera was found in our study the first time since the description of this species in 2000.Our findings expanded the bathymetrical or geographical range of 9 taxa  had shifted northwards due to an unprecedented sea-ice minimum. Total biomass was negatively correlated with sea ice coverage and distance from the ice edge. In earlier studies of pan-Arctic deep seas it was shown that the density and biomass of mega-, macro- and meiofauna increased in the marginal ice zone , 93, 94.This substantial effect of ice-cover on community composition appears to be mostly related to variations in food supply. Several biogeochemical variables corresponding to the input of phytodetrital material on the seafloor demonstrate higher fluxes closer to the MIZ than at the northernmost stations , 53. BecHyalopomatus claparedii and the actiniarian Bathyphellia margaritacea. On the other hand, the abundance of holothurians Elpidia heckeri and Kolga hyalina declined with increasing food supply. Notably the dominant species differ in their trophic modes: actiniarians and polychaetes are suspension feeders, whereas holothurians are deposit feeders. Significance of correlations increased when the biomass values were used instead of abundance.Relationship between the composition and structure of megafauna communities and the observed biogeochemical indicators of food supply was not linear. A tendency of increasing abundance with increasing food supply was shown for the polychaete Depth also had an effect on biomass in our study. Though stations in our study were located in the abyssal zone (from 3400 m to 4400 m) we observed a biomass decrease with increasing depth fitting the globally predicted trend , 96. MegThe present study also revealed a negative correlation between the number of taxa and the sea ice coverage, the latter increasing towards the North Pole. Earlier Wƚodarska-Kowalczuk et al.  demonstrRelatively high proportions of suspension feeding anthozoans at HAUSGARTEN , in CanaIn the present study unidentified swimming macellicephalin polychaetes were relatively abundant at depths 3570–4380 m. Dominance of unidentified swimming polychaetes was previously reported from the Canada Basin based on ROV observations (2800 m) .Elpidia heckeri dominated the abundance at depths 5333–5404 m and Kolga hyalina at 2609–2629 m . This feature may help species of Kolga to respond rapidly to seasonal accumulations of organic matter at the seafloor. To fully understand the role of the ice-algae supply to the abyssal seafloor in the Arctic and the effect of this phenomenon on the abyssal benthic life, further studies are required.In our study the elpidiid ore June . High deModels predict continuing rapid warming of climate in the Northern Hemisphere causing the reduction of the sea-ice extent and thickness, including ice-free summers in the Arctic Ocean , 106. DiVariations in the megabenthos community structure between stations in our study may reflect short and long-term variations in phytodetritus flux to the seafloor. This flux is likely to depend on distance from the sea-ice margin and ice thickness. Hence, it can be expected that further warming and sea ice retreat will affect carbon flux to the abyssal and thereby the biodiversity and distribution of Arctic fauna. Unfortunately, no quantitative baseline data are available for the Central Arctic megabenthos before 2000. Assessment of epibenthic megafauna of the HAUSGARTEN area from 2000 to 2012 showed significant changes in relative abundances of megafauna species that were related to variations in food supply with time, apparently linked to dynamics in sea ice cover and hydrography , 20.Present study emphasizes the need for accumulating quantitative data on seasonal and annual variations of the Central Arctic megabenthos, to detect ecosystem changes in the deep-sea. According to our comparison of image-based and trawl-based data, it seems more useful for long-term monitoring to use imaging along standard transects: image data allow for a more correct assessment of monitored species density. Present work contributes to baseline data for the Nansen and Amundsen Basins, and to the first assessment of deep-sea benthos responses to sea-ice algae food falls.Bathyphellia margaritacea, 2) the holothurian Elpidia heckeri and 3) the holothurian Kolga hyalina. Variations in megafaunal abundance were first of all related to the proximity to the marginal ice zone. Stations closer to the ice margin under first-year ice were characterized by relatively high densities and biomasses of B. margaritacea and relatively high food supply to the seafloor indicated by several biogeochemical variables. Stations located closer to the North Pole under the multi-year ice showed relatively low food supply, but relatively high densities and biomasses of holothurians E. heckeri and K. hyalina feeding on fresh algal falls of the colonial sea-ice diatom Melosira arctica. In case extensive algal food falls to the seafloor become regular as a result of increasingly frequent sea-ice melt events, the abundance of mobile deposit-feeding megafauna, such as elpidiid holothurians and ophiuroids, in the abyssal Central Arctic may rise significantly. Our data provide a baseline for long-term monitoring of the seafloor of the changing deep-sea Arctic Ocean.Our study on the composition and structure of megabenthos communities in different areas of the Eastern Central Arctic Basin combining quantitative photographic surveys with trawl sampling provides quantitative information of the dominant megafauna of the ice-covered basins and key factors structuring the distribution of abyssal megafauna in the Central Arctic. Three types of megafauna communities were distinguished: dominated by 1) the actiniarian S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableSample methods, location, depth and previously known distribution and depth range are shown (DOI) .(PDF)Click here for additional data file.


Moreover, in the experimental chicken model, the strip test demonstrated consistency in detecting IBV with RT-PCR gene detection. Taken together, this antigen detection strip has the potential to serve as an on-farm rapid test for IBV; therefore, it may facilitate surveillance and control of the disease.Avian infectious bronchitis virus (IBV) causes considerable economic losses in the poultry industry worldwide, including Taiwan. IBV is among the most important pathogens in chickens, and it spreads rapidly among flocks. In addition to dozens of known serotypes, new viral variants have emerged due to the viral evolution and antigenic variation in IBVs. Therefore, the development of a sensitive, specific, and easily performed assay is crucial for the rapid detection and surveillance of IBV infections. A rapid and simple immunochromatographic strip (ICS) was developed in this study by employing monoclonal antibodies against spike and nucleocapsid proteins of IBV as the tracer and the capture antibody. The ICS showed high specificity in detecting IBV antigens, including several IBV genotypes and novel variants, as opposed to three other common avian respiratory viruses. The detection limit of the strip reached 10 Gammacoronavirus, family Coronaviridae. IBV can infect the respiratory and urogenital systems in growing chickens, causing high mortality, slowing down growth, and reducing egg production in the flock [Avian infectious bronchitis virus (IBV) is a positive-sense single-stranded RNA virus belonging to the genus he flock . Therefohe flock . IBV strhe flock ,4. Becauhe flock . Due to he flock .Molecular studies reported that IBV contains four major structural proteins: The spike (S) glycoprotein, the phosphorylated nucleocapsid protein (N), the envelope protein (E) and the membrane glycoprotein (M). The S glycoprotein is believed to be related to the host range, and it can be post-translationally cleaved into S1 and S2 subunits . The S1 Monoclonal antibodies (mAbs), with the characteristic of high specificity, simple purification, and stable sources, are broadly utilized in the field and research as a reliably powerful tool for grouping and differentiating virus serotypes. The antigenic characterization of IBV strains by using mAbs directed against different epitopes on the S, N and M proteins could provide valuable information regarding antigenic relationships and variations ,14,15. MThe immunochromatographic strip (ICS) test, or lateral flow assay, is an antibody-based technique, referring to the migration of antigen-antibody complexes through a filter matrix such as nitrocellulose strips ,22. DuriA total of five IBV-specific mAbs  were obtained through a series of subclonings and screenings in this study. The cross-reactivity of mAbs against IBV strains was assessed through Western blot. Of the five mAbs, 2296-4 was observed to interact with the spike protein subunit (approximately 90 kDa) of IBVs, including two locally dominant genotypes (TW-I and TW-II) and the Mass type H120 A. Four oThe optimal pH value of gold particles and the concentration of mAbs for the adsorption of colloidal gold conjugation were determined by coagulation curves. The colloidal gold solution was adjusted to four different pH values between five and eight, physiological conditions for antibodies. Different concentrations of mAbs were added to each colloidal gold solution. Each point represented the absorbance at OD 580 nm after an additional 10% NaCl was added. The lower OD of the mAb-gold conjugate indicates the dispersed gold nanoparticle distribution in the solution as small particles have higher light transmittance, thereby indicating that the antibodies sufficiently stabilize the gold nanoparticles in the given pH environment. As indicated in 5.5 EID50) of AIV, NDV, ILTV and IBV, were used to test the specificity of the assembled ICS. The test line was found to respond to IBV only but not to other respiratory diseases’ antigens  at 104.6 EID50, 2992/02 (TW-CN variant) at 104.8 EID50 and IBV 2296/95 (TW-II) at 104.8 EID50.The schematic illustration of ICS is indicated in antigens A. The ICantigens B. MoroveTo validate the use of ICS in clinical settings, a total of 18 throat and cloacal swab samples were collected from IBV 2992/02-infected chickens at one, three and five days post-infection. RT-PCR was used simultaneously as a reference test. Results indicated that ICS successfully detected viral antigens from throat swabs sampled on one (3/3), three (2/3) and five (3/3) days post-infection (dpi), with highly consistent results from RT-PCR . For cloMany highly contagious viral diseases can affect the respiratory system of poultry, such as Newcastle disease, avian influenza, infectious laryngotracheitis and infectious bronchitis. Making a differential diagnosis of these diseases in the field is difficult, because affected chickens exhibit similar clinical signs: Respiratory distress and watery discharge from eyes and nostrils. Therefore, the rapid and accurate diagnosis of the disease facilitates disease management. Avian infectious bronchitis has posed a threat in the poultry industry worldwide. Even though protection schemes have been applied by means of a live attenuated IBV vaccine, low levels of cross-protection have been observed, ,27. In s4.5 to 104.8 EID50. The results of our study were slightly better than those reported for the liquid-phase blocking ELISA in Lougovskaia et al. [5.8 EID50. The nature of mAbs developed in this study is specific to the IBV among avian respiratory pathogens. Furthermore, by employing broadly-reactive mAbs of IBV, this described ICS displayed cross-reactivity against multiple types of IBVs, including two local Taiwan genotypes, recombinant variants, and the Mass type strain. Additional studies should be taken to ensure an antigen–antibody reaction against some other common strains worldwide; for example, Arkansas, Conn, JMK or QX-IBV.In this study, we successfully developed an ICS rapid test, which can be completed within 10 min to identify the IBV infection. To the best of our knowledge, this is the first report of IBV antigen detection by using an ICS. The results of comparing the reference RT-PCR test with our ICS implied that the ICS could detect antigens as early as 1 dpi from throat swab samples and 5 dpi from cloacal swab samples. The detection limit of the ICS strip was approximately 10a et al.  with theBased on the five mAbs we have obtained, the performance serving the capture antibody or detection antibody among several pairs of the mAbs were examined. Several different tracers were produced as described in the methods section. Various 1:1 combinations of the mAb mixture were also prepared, consisting of one mixture of both anti-N mAbs and one of anti-N and anti-S mAbs, with a total of 17 combinations tested. Both the tracers and test line mixtures were applied to the ICS. A positive IBV TW-I type AF was used as an antigen, and SPF AF was used as a negative control to determine the detection capacity of each combination. It was found that the use of mAb LK-10a and LK2-13a in the ICS generated non-specific signals and thus was ruled out as an option. In the preparation of the tracer, the stability of the mAb-gold conjugate is critical for optimizing the ICS strip’s performance. In this study, an mAb concentration of 15 μg/mL with colloidal gold solution at pH 8 exhibited the most stable condition in coagulation curves. As depicted in TEM images, the tracers were thoroughly dispersed in the solution, showing a unimodal particle distribution with an average size of 40 nm.In summary, the ICS developed in this study was a rapid and simple method for detecting IBV antigens. Moreover, mAbs employed on the strip and the colloidal gold-mAb tracer were ideal for identifying various IBV genotypes. The ICS has the potential to be employed as a screening tool for diagnosing IBV infections in the poultry industry. Further studies evaluating the sensitivity and specificity of ICS detection using the field samples are warranted.The care and use of experimental animals were approved by the Institute Animal Care and Use Committee, National Taiwan University . All animal experiments were conducted in accordance with approved guidelines.50). For viral antigens used in Western blots, the AF containing the virus was further concentrated and sucrose-gradient-purified as previously described [IBV strains 2575/98 (TW-I), 2296/95 (TW-II), 3575/08 (TW-I), 2992/02 (TW-China variant), and H120 (Mass) were propagated in 10-day-old SPF chicken embryos through the allantoic route as previously described , NDV strescribed .7.4 EID50/mL), and hybridoma cells were produced as previously described [Female BALB/c mice  were intraperitoneally immunized with 1 mL of IBV 2575/98 (10escribed . PositivOne μg of IBV 2575/98 (TW-I), 2296/95 (TW-II), and H120 viral antigens were mixed with an equal volume of SDS-PAGE sample buffer  and boiled for 5 min. The protein solution was separated on 10% SDS-PAGE. Protein gels were transferred onto a 0.45-μm nitrocellulose membrane . After the transfer, the membrane was soaked in blocking buffer (5% skim milk in PBS) at room temperature for 1 h, and the hybridoma supernatant diluted 1:5 in blocking buffer was then added. After three washes, the membrane was incubated with peroxidase-conjugated goat anti-mouse IgG (H+L)  diluted 1:2000 in blocking buffer at room temperature for 1 h. Following three further washes, protein blots were detected using a TMB membrane peroxidase substrate .2O mixture. OD absorptions at each pH and mAb concentration were obtained in triplicate. The optimal pH value of gold particles and the concentration of mAbs for stabilizing colloidal gold particles were determined.Coagulation curves were analyzed as previously described ,34. Briew/v) bovine serum albumin (BSA) to block the excess reactivity of colloidal gold. After centrifugation at 4500× g for 45 min, the mAb-colloidal gold pellet was washed with 2 mM borax buffer containing 0.1% PEG-20000 (pH 9.0) twice and then resuspended in 1 mL of 2 mM borax buffer containing 0.1% PEG-20000 and 10% sucrose (pH 9.0).The mAb-colloidal gold conjugate (tracer) was prepared as previously described  with modw/v) BSA for 1 h. The grid was then stained with 6-nm gold-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) diluted in blocking solution (1:20) for 1 h and washed three times in PBS. Particles were visualized under a 200-kV high-resolution transmission electron microscope .For immunogold labelling of the mAb tracer, eight μL of tracer sample was deposited onto a glow-discharged carbon-coated grid for 2 min and fixed with 4% paraformaldehyde for 5 min. Following PBS washing, the grid was blocked with 1%  at one, three and five dpi. The swabs were clarified by centrifuging at 3000× g for 20 min and stored at −80 °C until further testing with ICS and RT-PCR.Two-week-old SPF chickens  were intranasally infected with IBV 2992/02  at a dosViral RNA was extracted from swab samples by using the QIAamp Viral RNA kit  as per the manufacturer’s instructions. Subsequently, a 25 μL reaction was set up consisting of 2.5 μL of template RNA, 0.5 μL of 2.5 mM dNTP, 2.5 μL of 10× buffer, 0.25 μL each of forward and reverse primers (50 μM)  for targeting the N protein gene, 0.2 μL of RNase inhibitor , 0.2 μL of RealTaq DNA polymerase , 0.1 μL of MMLV (Invitrogen), and 18.5 μL of RNase-free water. Reverse transcription was performed at 40 °C for 30 min, followed by initial denaturation at 95 °C for 5 min. Then, 35 cycles were applied with 95 °C for 30 s, 55 °C for 30 s and 72 °C for 45 s. The final extension stage was performed at a temperature of 72 °C for 12 min and then maintained at 4 °C. PCR products were analyzed through the agarose gel electrophoresis.