Biorecognition of Escherichia coli K88 adhesin for glycated porcine albumin

Escherichia coli (E. coli) that expresses galactose-reactive lectins, like K88 adhesin, causes high mortality among piglets. Carbohydrates that compete for adhesion could serve as an alternative for disease prevention. Porcine serum albumin (PSA) was modified by non-enzymatic glycation with lactose to produce PSA-Lac or PSA-Glc β (1–4) Gal, as confirmed by reduction of available free amino groups, increased molecular mass and by Ricinus communis lectin recognition. E. coli K88 binds to PSA-Lac treatments containing three and four lactoses, respectively. In addition, PSA-Lac partially inhibited K88 strain adherence to mucins. These results suggest that neoglycoconjugates obtained by non-enzymatic glycation of proteins may serve in the prophylaxis of piglets’ diarrhea.     

Experimental Infection of Newborn Piglets and Weanling Pigs with Haemolytic E. Coli,Strain A1, Serotype 0149:K91:H10

The first outbreak in Denmark of enteritis in newborn piglets due to infection with E. coli serotype 0149:K91:H10 was demonstrated in the autumn 1966 (Knox & Dam 1970). Since then this serotype has spread rapidly and is to-day responsible for the vast majority of cases of enteric E. eoli infection in newborn piglets, while among pigs at weaning age it occurs with much the same frequency as haemolytic E. coli serotypes 08: K87, 0138:K81, and 0141:K85 ab.     

Characteristics of the Jejunal Microbiota in 35-Day-Old Saba and Landrace Piglets

The balanced microbiological system is a significant hallmark of piglet health. One of the crucial factors affecting intestinal microbiota is the host’s genetics. This study explored the difference in the diversity of jejunal microbiota between Saba (SB) and Landrace (LA) piglets. Nine Saba and nine Landrace piglets were fed with sow’s milk until day 35. Jejunal contents were harvested for 16S rRNA sequencing. The birth weight, body weight, and average daily gain of Saba piglets were lower than those of Landrace piglets (p < 0.01). Firmicutes were the main phylum in Saba and Landrace piglets, and the Saba piglets had a higher (p < 0.05) abundance of Bacteroidetes compared with Landrace piglets. The two most abundant genera were Lactobacilli and Clostridium XI in the jejunum of Landrace and Saba piglets. Compared with Landrace piglets, the Saba piglets had significantly lower (p < 0.05) abundance of Veillonella, Streptococcus, and Saccharibacteria genera incertae sedis. The functional prediction showed that “d-glutamine and d-glutamate metabolism” and “one carbon pool by folate” pathways were enriched in Saba piglets, while “limonene and pinene degradation”, “tryptophan metabolism”, and “sulfur relay system” pathways were enriched in Landrace piglets. In summary, the growth performance was higher for Landrace piglets compared with Saba piglets due to their genetic characteristics. The rich diversity and fewer infection-associated taxa were observed in Saba piglets, partially accounting for their higher adaptability to environmental perturbations than Landrace piglets. Furthermore, different pig breeds may regulate their health through different metabolic pathways.Key words: Saba piglets, Landrace piglets, jejunal content, the 16S rRNA gene, diversity     

The Way Humans Behave Modulates the Emotional State of Piglets

The emotional state can influence decision-making under ambiguity. Cognitive bias tests (CBT) proved to be a promising indicator of the affective valence of animals in a context of farm animal welfare. Although it is well-known that humans can influence the intensity of fear and reactions of animals, research on cognitive bias often focusses on housing and management conditions and neglects the role of humans on emotional states of animals. The present study aimed at investigating whether humans can modulate the emotional state of weaned piglets. Fifty-four piglets received a chronic experience with humans: gentle (GEN), rough (ROU) or minimal contact (MIN). Simultaneously, they were individually trained on a go/no-go task to discriminate a positive auditory cue, associated with food reward in a trough, from a negative one, associated with punishments (e.g. water spray). Independently of the treatment (P = 0.82), 59% of piglets completed the training. Successfully trained piglets were then subjected to CBT, including ambiguous cues in presence or absence of a human observer. As hypothesized, GEN piglets showed a positive judgement bias, as shown by their higher percentage of go responses following an ambiguous cue compared to ROU (P = 0.03) and MIN (P = 0.02) piglets, whereas ROU and MIN piglets did not differ (P > 0.10). The presence of an observer during CBT did not modulate the percentage of go responses following an ambiguous cue (P > 0.10). However, regardless of the treatment, piglets spent less time in contact with the trough following positive cues during CBT in which the observer was present than absent (P < 0.0001). This study originally demonstrates that the nature of a chronic experience with humans can induce a judgement bias indicating that the emotional state of farm animals such as piglets can be affected by the way humans interact with them.     

Impact of general injection anaesthesia and analgesia on post-castration behaviour and teat order of piglets

Injection anaesthesia with a combination of ketamine and azaperone (K/A) is discussed as a painless alternative to commonly used non-anaesthetized castration. To protect anaesthetized piglets from being crushed, they have to be separated from the sow for 3 h following castration. The aim of this study was to test if this separation and the different treatments would affect short-term behaviour after castration (3 to 6 h after castration) as well as weight gain. Piglets were 5 to 7 days old. Treatment Group 1 received a combination of anaesthesia and analgesia (n = 29, ketamine: 25 mg/kg BW; azaperone: 2 mg/kg BW; meloxicam: 0.4 mg/kg BW), Group 2 received only analgesia (n = 24) and Group 3 received no medication (n = 29). Behaviour and suckling order were compared for a 3 h period the day before castration and after castration. A significantly higher number of teats used by anaesthetized piglets (P = 0.004) suggests a decrease in suckling order stability. There were significant treatment effects between all three groups in the time spent at the sow's teat, with an increase in Group 2 (+69%), decrease in Group 1 (−28%), whereas the control Group 3 (+2%) almost remained unchanged. The anaesthetized piglets showed an increase in the time spent active away from the sow after castration of almost 200% (Groups 2 and 3: ∼50%, P < 0.001). However, no significant treatment effect was seen for weight gain. The results suggest that analgesia has an effect on behaviour, perhaps due to less post-castration pain. This advantage is not apparent for animals receiving additional anaesthesia, probably because of impaired coordination. Although the behavioural changes did not affect weight gain significantly, a decrease in suckling order stability indicates a certain degree of stress due to fighting over teat positions as a consequence of separation. Thus, post-castration behaviour must be taken into account when evaluating alternative castration methods.     

The growth performance,intestinal digestive and absorptive capabilities in piglets with different lengths of small intestines

The small intestine is an important digestive organ and plays a vital role in the life of a pig. We tested the hypothesis that the length of the small intestine is related to growth performance and intestinal functions of piglets. A total of 60 piglets (Duroc × Landrace × Yorkshire), weaned at day 21, were fed an identical diet during a 28-day trial. At the end of the study, all piglets were sacrificed, dissected and grouped according to small intestine lengths (SILs), either short small intestine (SSI), middle small intestine (MSI) or long small intestine (LSI), respectively. Positive relationships between SIL and BW, average daily gain (ADG), average daily feed intake (ADFI) and gain-to-feed ratios (G : F) were observed. Final BW, ADG, ADFI and G : F significantly increased (P < 0.05) in MSI and LSI piglets compared with SSI piglets. Short small intestine and MSI had greater jejunal mucosa sucrase and alkaline phosphatase activities (P < 0.05) than LSI piglets. The mRNA level of solute carrier family 2 member 2 (Slc2a2) in the jejunal mucosa of SSI piglets was the greatest. The MSI piglets had a greater (P < 0.05) ileal villus height than other piglets and greater (P < 0.05) villus height-to-crypt depth ratios than LSI piglets. However, the LSI piglets had a greater (P < 0.05) ileal crypt depth than SSI piglets. No significant differences in duodenal, jejunal, caecal and colonic morphologies were detected among the groups. Moreover, luminal acetate, propionate, butyrate and total short-chain fatty acid contents were greater (P < 0.05) in SSI and MSI piglets than those in LSI piglets. In addition, there was greater serum glucose concentration in MSI piglets than other piglets. Serum albumin concentration in SSI piglets was the lowest. In conclusion, these results indicate that SIL was significantly positively associated with growth performance, and in terms of intestinal morphology and mucosal digestive enzyme activity, the piglets with a medium length of small intestine have better digestion and absorption properties.     

Intracellular location of NRl-plasmid DNA inEscherichia coli minicells

Intracellular location of plasmid NR1 (M = 58 Mg/mol, stringent control of replication, 1–2 copies perEscherichia coli chromosomal equivalent) was studied and compared with that of plasmid R6KΔ1 (M = 21 Mg/mol, relaxed control of replication, 10–15 copies perE. coli chromosomal equivalent), both inE. coli minicells. Considerable difference in relative distribution of molecules of these two plasmid DNA’s between the cytoplasm and the membrane fraction was found when components of the corresponding minicell lyzates were fractionated by sedimentation in a double-linear gradient of caesium chlorid and sucrose. Also the difference in relative numbers of NR1 DNA and R6KΔ1 DNA molecules stably associated with the membrane of minicells, determined by electron-microscopic examination of the fractions containing plasmid DNA-membrane complexes, was evaluated as statistically significant. The association of NR1 DNA molecules withE. coli minicell membrane was found to be a much more frequent event than such association of R6KΔ1 molecules. The absolute amount of plasmid DNA associated with membrane in a single minicell corresponds to one molecule for both NR1 and R6KAΔ1.     

Exopolysaccharides Synthesized by Lactobacillus reuteri Protect against Enterotoxigenic Escherichia coli in Piglets

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in piglets; ETEC cells colonize the intestinal mucosa with adhesins and deliver toxins that cause fluid loss. This study determined the antiadhesive properties of bacterial exopolysaccharides (reuteran and levan) and related glycans (dextran and inulin) in a small intestinal segment perfusion (SISP) model. The SISP model used 10 jejunal segments from 5-week-old piglets. Five segments were infected with ETEC expressing K88 fimbriae (ETEC K88), while five segments were treated with saline. Every two segments (ETEC and non-ETEC infected) were infused with 65 ml of 10 g liter⁻¹ of glycans or saline (control) for 8 h. High-resolution melting-curve (HRM) quantitative PCR (qPCR) indicated that E. coli is the dominant bacterium in infected segments, while other bacteria were predominant in noninfected segments. Infection by ETEC K88 was also verified by qPCR; gene copy numbers of K88 fimbriae and the heat-labile toxin (LT) in mucosal scrapings and outflow fluid of infected segments were significantly higher than those in noninfected segments. Genes coding for K88 fimbriae and LT were also detected in noninfected segments. LT amplicons from infected and noninfected segments were 99% identical over 481 bp, demonstrating the presence of autochthonous ETEC K88. All glycans reduced fluid loss caused by ETEC K88 infection. Reuteran tended (P = 0.06) to decrease ETEC K88 levels in mucosal scraping sample, as judged by qPCR. Fluorescent in situ hybridization analysis demonstrated that reuteran significantly (P = 0.012) decreased levels of adherent ETEC K88. Overall, reuteran may prevent piglet diarrhea by reducing adhesion of ETEC K88.     

The opsonic activity of complement to rough strains ofEscherichia coli in vivo in newborn precolostral pigs

The participation of complement of newborn precolostral piglets (in the absence of antibody) in the opsonization of roughEscherichia coli was investigated using blood clearance test. It was found that the complement inhibitor-yeast mannan inhibits thein vivo blood clearance when injected i. venously into newborn precolostral piglets. This inhibition takes place at the serum level and not by saturation of RES cells by mannan: sera from mannan-treated piglets are not bactericidal in anin vitro bactericidal assay against roughEscherichia coli; secondly, the presence of antibody removes the blockade of phagocytosis by mannan indicating that the RES cells are functionally capable to perform phagocytosis. On the basis of our findings we postulate that in newborn precolostral piglets in which antibody is not present in their sera, complement alone (or with cooperation with other unknown serum component) acts as an opsonin on roughEscherichia coli and is responsible for the blood clearance rate of these bacteria.     

Impact of litter size,supplementary milk replacer and housing on the body composition of piglets from hyper-prolific sows at weaning

The modern hyper-prolific sow gives birth to 17 live-born piglets on average. An alternative strategy to nurse sows and artificial rearing may be providing milk replacer while letting all the piglets stay with their dam. However, milk replacer is of lower nutritional quality than sow milk and may reduce the body fat content of piglets who use milk replacer to compensate for low suckling success due to competition at the udder. Therefore, the aim of this study was to investigate the body composition at weaning of two random sow-reared piglets per litter from 93 litters by using the deuterium oxide dilution technique. The piglets were part of large study with a 2×2×2 factorial design of either 14 or 17 piglets from day 1 (LS: LS14/LS17) with or without access to milk replacer (MILK: -MILK/+MILK) and reared by crated or loose-housed sows (HOUSING: CRATE/ LOOSE). From behavioral observations day 21 in +MILK, piglets were divided according to their frequency of drinking milk replacer and suckling (Nutrition Source). Increasing LS from 14 to 17 reduced the average daily gain from 258 to 228 g/d and body fat % from 14.4 to 12.7% (P<0.01). In a two-way interaction between LS and HOUSNG, the body fat percentage was lower (P=0.04) and the water percentage tended to be higher (P=0.07) in LS17 CRATE compared to the other treatments (i.e. LS17 LOOSE, LS14 CRATE and LOOSE). There was no effect of MILK on piglet composition day 28 (P>0.1). In +MILK, the Nutrition Source affected piglet body composition (P<0.05) as piglets with low suckling frequency (LOW) had lower body fat and higher water content compared to piglets who had high suckling frequency (SUCKLE). Unexpectedly, drinking milk replacer in addition to suckling (MIXED) did not increase piglet body fat content. Relying mainly on milk replacer (CUP) caused body fat and water contents to be intermediate to piglets with high (SUCKLE and MIXED) and low suckling frequency (LOW). In conclusion, LS had a clear impact on piglet growth and body composition at weaning. In contrast, supplementation of milk and housing had only negligible impact on litter performance. Some individual piglets that had low frequency of sow milk intake benefitted from milk supplementation. Loose housing appeared to benefit piglet body fat at weaning but this was due to a greater piglet mortality.     

Behavioural responses of piglets to different types of music

Music or other background sounds are often played in barns as environmental enrichment for animals on farms or to mask sudden disruptive noises. Previous studies looking at the effects of this practice on non-human animal well-being and productivity have found contradictory results. However, there is still a lack of discussion on whether piglets have the ability to distinguish different types of music. In this study, we exposed piglets to different music conditions to investigate whether the piglets preferred certain music types, in which case those types would have the potential to be used as environmental enrichment. In total, 30 piglets were tested for music type preference to determine whether growing pigs respond differently to different types of music. We used music from two families of instruments (S: string, W: wind) and with two tempos (S: slow, 65 beats/min (bpm); F: fast, 200 bpm), providing four music-type combinations (SS: string-slow; SF: string-fast; WS: wind-slow; WF: wind-fast). The piglets were given a choice between two chambers, one with no music and the other with one of the four types of music, and their behaviour was observed. The results showed that SS and WF music significantly increased residence time (P<0.01) compared with the other music conditions. Compared with the control group (with no music), the different music conditions led to different behavioural responses, where SS music significantly increased lying (P<0.01) and exploration behaviour (P<0.01); SF music significantly increased tail-wagging behaviour (P<0.01); WS music significantly increased exploration (P<0.01); and WF music significantly increased walking, lying, standing and exploration (all P<0.01). The results also showed that musical instruments and tempo had little effect on most of the behaviours. Fast-tempo music significantly increased walking (P=0.02), standing (P<0.01) and tail wagging (P=0.04) compared with slow-tempo music. In conclusion, the results of this experiment show that piglets are more sensitive to tempo than to musical instruments in their response to musical stimulation and seem to prefer SS and WF music to the other two types. The results also suggest a need for further research on the effect of music types on animals.     

Detection and identification of pathotypes of verocytotoxigenic Escherichia coli isolated from weaned piglets using gene probes for seven E. coli toxins

Seventy verocytotoxigenic (VTEC) and sixty-three non VTEC haemolytic Escherichia coli isolated from recently weaned piglets were examined by the colony hybridization assay using gene probes for three verocytotoxins: Edema disease principle (EDP) and Shiga-like toxins I and II (SLTI and SLTII). The results with the EDP and SLTII probes were identical. All VTEC hybridized with these two probes, while non VTEC did not. All 133 E. coli were negative for the SLTI probe. Hybridization of the plasmid content of 14 VTEC did not show any evidence for plasmid localization of the genes coding for the EDP. The 70 VTEC were also assayed with gene probes for heat-stable (STaP, STb) and heat-labile (LT, LTIIa) enterotoxins. Only the STb probe was hybridized by 36 of them. Most STb-positive isolates belonged to serotype O141: K85 biotypes 9 and 13 PC.     

Effect of oral polyamine supplementation pre-weaning on piglet growth and intestinal characteristics

A high proportion of piglets fail to adapt to the changing composition of their diet at weaning, resulting in weight loss and increased susceptibility to pathogens. Polyamines are present in sow milk and promote neonatal maturation of the gut. We hypothesised that oral spermine and spermidine supplementation before weaning would increase piglet growth and promote gastrointestinal development at weaning. In Experiment One, one pair of liveweight (LW)-matched piglets per litter from first and third lactation sows received 2 ml of a 0 (Control) or 463 nmol/ml spermine solution at 14, 16, 18, 20 and 22 days of age (n=6 piglets/treatment per parity). Villus height and crypt depth in the duodenum and jejunum were measured at weaning (day 23 postpartum). In Experiment Two, piglets suckling 18 first and 18 third lactation sows were used. Within each litter, piglets received 2 ml of either water (Control), 463 nmol/ml spermine solution or 2013 nmol/ml spermidine solution at 14, 16, 18, 22 and 24 days of age (n=54 piglets/treatment per sow parity). Piglets were weighed individually at 14, 18, 24 (weaning) and 61 days of age. In Experiment One, oral spermine supplementation resulted in a 41% increase in villus height, a 21% decrease in crypt depth and 79% decrease in the villus height : crypt depth ratio compared with control piglets (P<0.01). In Experiment Two, spermine and spermidine-supplemented piglets suckling first lactation sows grew faster (P<0.05) between days 14 and 18 postpartum than control piglets: 0.230±0.011 and 0.227±0.012 v. 0.183±0.012 kg/day, respectively. Spermine supplementation tended (P<0.1) to increase piglet LW gain from weaning to day 37 post-weaning compared with control piglets (0.373±0.009 v. 0.341±0.010 kg/day). In conclusion, spermine supplementation increased villus height at weaning, and appears to have the potential to improve the pre- and post-weaning growth of conventionally weaned piglets.     

Lactobacillus succession in the piglet digestive tract demonstrated by plasmid profiling

Plasmid profiling was used to distinguish strains of lactobacilli inhabiting the digestive tract of piglets and the feces of sows. Fifteen plasmid profile types were detected among 328 isolates of lactobacilli. Plasmid profiling of lactobacilli permitted the following conclusions to be made: the maternal feces were a major source of lactobacilli colonizing the piglet digestive tract; the lactobacillus population of the gastric region of the piglet digestive tract was composed of lactobacillus strains different from those present in the rectal population; and a lactobacillus succession was observed in the digestive tract of piglets drawn from a single litter, and one plasmid profile type became dominant in the gastric region of these animals.     

Gestational dietary betaine supplementation suppresses hepatic expression of lipogenic genes in neonatal piglets through epigenetic and glucocorticoid receptor-dependent mechanisms

Methyl donors play critical roles in nutritional programming through epigenetic regulation of gene expression. Here we fed gestational sows with control or betaine-supplemented diets (3 g/kg) throughout the pregnancy to explore the effects of maternal methyl-donor nutrient on neonatal expression of hepatic lipogenic genes. Betaine-exposed piglets demonstrated significantly lower liver triglyceride content associated with down-regulated hepatic expression of lipogenic genes acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD) and sterol regulatory element-binding protein-1c. Moreover, s-adenosyl methionine to s-adenosyl homocysteine ratio was elevated in the liver of betaine-exposed piglets, which was accompanied by DNA hypermethylation on FAS and SCD gene promoters and more enriched repression histone mark H3K27me3 on SCD gene promoter. Furthermore, glucocorticoid receptor (GR) binding to SCD gene promoter was diminished along with reduced serum cortisol and liver GR protein content in betaine-exposed piglets. GR-mediated SCD gene regulation was confirmed in HepG2 cells in vitro. Dexamethasone (Dex) drastically increased the luciferase activity of porcine SCD promoter, while the deletion of GR response element on SCD promoter significantly attenuated Dex-mediated SCD transactivation. In addition, miR-let-7e, miR-1285 and miR-124a, which respectively target porcine SCD, ACC and GR, were significantly up-regulated in the liver of betaine-exposed piglets, being in accordance with decreased protein content of these three genes. Taken together, our results suggest that maternal dietary betaine supplementation during gestation attenuates hepatic lipogenesis in neonatal piglets via epigenetic and GR-mediated mechanisms.     

Lactobacillus succession in the piglet digestive tract demonstrated by plasmid profiling.

Plasmid profiling was used to distinguish strains of lactobacilli inhabiting the digestive tract of piglets and the feces of sows. Fifteen plasmid profile types were detected among 328 isolates of lactobacilli. Plasmid profiling of lactobacilli permitted the following conclusions to be made: the maternal feces were a major source of lactobacilli colonizing the piglet digestive tract; the lactobacillus population of the gastric region of the piglet digestive tract was composed of lactobacillus strains different from those present in the rectal population; and a lactobacillus succession was observed in the digestive tract of piglets drawn from a single litter, and one plasmid profile type became dominant in the gastric region of these animals.     

Unusual outcome of in utero infection and subsequent postnatal super-infection with different PCV2b strains

VC2002, isolated from postweaning multisystemic wasting syndrome (PMWS)-affected pig, is a mixture of two porcine circovirus genotype 2b (PCV2b) viruses, K2 and K39. Preliminary experiments disclosed short-term adverse effects of K39, but not K2, on porcine foetuses. These findings led to the hypothesis that infection of immuno-incompetent foetuses with K2 confers a status of immunotolerance, and postnatal super-infection with K39 triggers PMWS. To explore this hypothesis, nine 55-day-old foetuses were inoculated in utero (three with K2-10^4.3TCID₅₀, three with K39-10^4.3TCID₅₀ and three with medium), and foeto-pathogenicity examined. At 21 days post-inoculation (dpi), K2 did not induce pathology, whereas pathological effects of K39 were evident. Twenty-four 45-day-old foetuses were subsequently inoculated to examine the long-term effect of K2, including six with K2-high dose-10^4.3TCID₅₀, six with K2-low dose-10^2.3TCID₅₀ and 12 mock-inoculated controls. Both doses resulted in five mummified foetuses and one live-born piglet each (69dpi). K2 was recovered from all mummies. K2 and K2-specific antibodies were not detected in serum of the two live-born piglets at birth, indicating full control of K2 infection. The K2-low dose-infected piglet was immunostimulated at day 2, but not the K2-high dose-infected piglet. Both non-stimulated and stimulated K2-infected piglets were super-inoculated with K39 at day 6 or 8 (taken as 0 days post super-inoculation). Low viral replication was observed in the non-stimulated K2-K39 piglet (up to 10^3.3TCID₅₀/g; identified as K39). In contrast, viral replication was extremely high in the stimulated K2-K39 piglet (up to 10^5.6TCID₅₀/g) and identified as K2, indicating that K2 infection is controlled during foetal life, but emerges after birth upon immunostimulation. However, none of the piglets showed any signs of PMWS.     

Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells

Background

We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments.

Results

K14 IBs were electroporated into SW13 cells grown in culture together with a ??reporter?? plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5.

Conclusions

Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells.