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IDENTIFICATION OF AMPC Β-LACTAMASE-PRODUCING CLINICAL ISOLATES OF ESCHERICHIA COLI

Objective: Indiscriminate use of β-lactam antibiotics has resulted in the emergence of β-lactamase enzymes. AmpC β-lactamases, in particular, confer resistance to penicillin, first-, second-, and third-generation cephalosporins as well as monobactams and are responsible for antibiotic resistance in nosocomial pathogens. Therefore, this study was undertaken to screen nosocomial Escherichia coli isolates for the presence and characterization of AmpC β-lactamases. The study also envisaged on the detection of inducible AmpC β-lactamases and extended-spectrum β-lactamases (ESBLs) in AmpC β-lactamase-producing E. coli. Methods: A total of 102 clinical isolates of E. coli, were subjected to cefoxitin screening, and screen-positive isolates were further subjected to inhibitorbased detection method, phenotypic confirmatory test, disc antagonism test, polymerase chain reaction (PCR), and isoelectric focusing (IEF). Results: In this study, 33% of E. coli were resistant to cefoxitin, of which 35% were found to be positive for AmpC β-lactamase by inhibitor-based phenotypic test. Of the AmpC-positive isolates, 83% were positive for ESBLs, whereas 25% were producing inducible AmpC β-lactamases. PCR and IEF showed CIT and EBC types of AmpC β-lactamases present in the tested isolates. Conclusion: Our study showed the presence of inducible AmpC enzymes and ESBLs in E. coli isolates and PCR identified more isolates to be AmpC producers.

INTRODUCTION
There has been an implausible rise in the incidence of antibiotic resistance, more importantly to cephalosporins in bacterial pathogens [1,2]. Of the various resistance mechanisms, production of β-lactamases is the most widespread. AmpC β-lactamases are clinically significant as they confer resistance to a broad spectrum of antibiotics such as penicillin, first-, second-, and third-generation cephalosporins and monobactams such as aztreonam and are not inhibited by commercially available β-lactamase inhibitors [3,4]. Escherichia coli is a commensal and opportunistic pathogen which can be responsible for intestinal as well as urinary tract infections, and hence, is targets for antibiotic therapies [5]. This can lead, in some cases to a high degree of antibiotic resistance. AmpC β-lactamases are encoded by ampC genes which can be chromosomally or plasmid mediated [6]. In E. coli, ampC is expressed constitutively, but at low level, mutations in the ampC promoter/attenuator region can result in constitutive overexpression [7]. Plasmid-mediated ampC genes are also reported in E. coli, which are expressed constitutively in high levels and are also induced by β-lactams such as ACT-1 β-lactamases [6]. Hence, plasmid-mediated AmpC β-lactamases have considerable clinical significance [8]. Moreover, clinical isolates possessing plasmids encoding AmpC enzymes often are resistant to multiple antibiotics hence, leaving few therapeutic options.
Taking into consideration the significance of AmpC β-lactamases and coexistence of extended-spectrum β-lactamases (ESBLs) in AmpC β-lactamase-producing isolates in clinical world, this study was undertaken to identify and characterize different types of AmpC β-lactamases in E. coli. The present study compared different phenotypic methods with a genotypic method polymerase chain reaction (PCR) to detect AmpC β-lactamases in clinical isolates of E. coli.

Bacterial isolates
A total of 102 isolates of E. coli recovered from a variety of clinical specimens were collected from two different hospitals of Delhi during the period from April 2003 to December 2005. All the isolates were identified by standard biochemical methods. E. coli ATCC 25922 was included as a negative control in all the tests described, whereas E. coli C600 pMG230 and Klebsiella pneumoniae 48188 were used as positive controls in all the experiments.

Screening for AmpC production
The isolates were tested for antimicrobial susceptibility to cefoxitin by Kirby-Bauer disc diffusion method [9]. The inhibition zone sizes were interpreted as per the CLSI (formerly NCCLS) guidelines [10]. All the isolates with an inhibition zone diameter of <18 mm were selected.

Detection of AmpC β-lactamases
The screened positive isolates were tested by an inhibitor (boronic acid)-based detection method to detect AmpC-producing E. coli [11]. Briefly, a stock solution of boronic acid was prepared by dissolving 120 mg of phenylboronic acid (Sigma-Aldrich, St. Louis, USA) in 3 ml dimethyl sulfoxide, to which 3 ml of sterile distilled water was added. Discs were prepared by dispensing 20 µl of the stock solution on 30 µg of cefotetan discs. These were dried for 30 min and used immediately or stored in airtight vials with a desiccant at 4°C. This test was performed by inoculating Mueller-Hinton Agar (MHA) plate with test isolates by the standard disc diffusion method (CLSI/NCCLS) and placing a disc containing 30 µg of cefotetan along with a disc containing 30 µg of cefotetan and 400 µg of boronic acid. The inoculated plates were incubated overnight at 37°C. Organism showing an increase of 5 mm of zone diameter around the disc containing cefotetan and boronic acid as

Gupta et al.
compared to that of zone diameter around the disc of cefotetan alone was considered as AmpC producer.

Detection of inducible AmpC β-lactamases
Disc antagonism test (DAT) was carried out as described by Yan et al., 2002, [12]. Briefly, a disc of cefoxitin (30 µg) was kept in the center of the inoculated (test isolate) MHA plate and discs of ceftazidime (CAZ), cefpodoxime, and cefotaxime (CTX) were placed around it in a way that each of these was 15 mm apart. The plates were incubated overnight at 37°C. Blunting of the zone of inhibition around any of the cephalosporin discs adjacent to the cefoxitin disc was considered as a positive result.

Phenotypic confirmatory test (PCT) for detection of ESBLs
This test was performed by the disc diffusion technique as recommended by CLSI (NCCLS). A ≥5 mm increase in the zone diameter for either CAZ or CTX tested in combination with clavulanic acid (CLA), versus its zone when tested alone, confirmed an ESBL-producing organism.
All the media and antibiotic discs used for phenotypic tests were procured from Becton, Dickinson & Co., USA.

DNA extraction
A few (2-3) colonies of the test isolates were inoculated in 5 ml Luria Bertani broth (Becton, Dickinson & Co., USA) and incubated overnight at 37°C. Boiled lysate of each of the cultures was prepared by boiling the culture at 100°C for 10 min and collecting the supernatant after centrifugation at 9838× g for 5 min. The supernatant was used as a source of template DNA.

Detection of ampC genes by multiplex PCR
Detection of plasmid-mediated AmpC β-lactamase genes (ampC) in all the screened positive isolates was carried out by PCR using the method described by Pérez-Pérez and Hanson 2002, [13]. Family-specific primers, as shown in Table 1, were used to detect different families/ types of AmpC β-lactamases in the isolates. Each 50 µl PCR mixture contained 0.2 mM of each dNTPs (Sigma-Aldrich Chemical Pvt. Ltd, Bengaluru, India), 0.5 µM of forward and reverse primers (Sigma-Aldrich Chemical Pvt. Ltd, Bengaluru, India), 1X reaction buffer (New England Biolabs Inc., USA) with 2 mM of MgCl 2 (New England Biolabs Inc., USA), 1.25 U of Taq polymerase (New England Biolabs Inc., USA), and 2 µl of DNA. Nuclease-free water was used in all the reactions and as a negative control. PCR was carried out in a PTC-100™ Programmable thermal cycler (MJ Research Inc.) using the following conditions: 94°C for 3 min, 94°C for 30 s, 64°C for 30 s, and 72°C for 1 min for 30 cycles followed by a final extension at 72°C for 7 min. PCR products were visualized on a 2% ultrapure agarose (Sigma-Aldrich, St. Louis, USA) gel stained with ethidium bromide (Sigma-Aldrich, St. Louis, USA, 10 mgL −1 ).

Nucleotide sequence analysis of ampC genes
Nucleotide sequencing of PCR amplicons was undertaken on both strands using respective PCR-specific primers (Table 1) by dideoxychain termination method using the services of the commercial vendor (Link Biotech, India). Sequence analysis and comparisons were performed using nucleotide BLAST, and sequences were compared against the database of ampC genes present at the NCBI server (http://www.ncbi. nlm.nih.gov).

Detection and characterization of AmpC β-lactamases by isoelectric focusing (IEF)
Characterization of AmpC β-lactamases was carried out by IEF, following the procedure of Matthew et al., 1975, [14] with few modifications. Briefly, crude enzyme extract from test isolates was prepared by subjecting the cell pellet from overnight grown cultures to 20 cycles of freezing at −20°C for 30 min and thawing at 37°C for 15 min. Nitrocefin sensitivity test was performed by loading 50 µl of nitrocefin solution and 10 µl of crude extract of the test organism into a 96-well microtiter plate. The rate of color change was graded on a scale of 5-1. For the score of 5, 1 µl of crude enzyme extract, and for a score of 1, 20 µl of crude enzyme extract were loaded on commercially prepared polyacrylamide gels with a pH range of 3.5-10. Gel electrophoresis was carried out using on a LKB Bromma 2117 Multiphor II electrophoresis unit (Ampholine PAG plate; GE Healthcare Uppsala, Sweden). The gel was run at 1500V, 50mA, and 30W for 90 min. The bands were analyzed by placing a filter paper soaked in nitrocefin and placing it over the gel for 10 min.

Detection of ampC genes
All the screened positive isolates were subjected to multiplex PCR. Of the 34 screened positive isolates of E. coli, 16 were found to show ampC genes of amplified products of 462 bp (CIT type) and 302 bp (EBC), respectively (Fig. 1). Of these 16 PCR-positive isolates, 13 harbored ampC genes of CIT family (EC 2, 3, 4, 10,11,14,15,20,24,26,29,32, and 34) and 3 (EC 1, 19, and 22) of EBC family ( Table 4). None of the tested isolates harbored genes for MOX, ACC, DHA, and FOX families. CIT-type ampC gene was found to be predominant in the PCR-positive isolates (Table 4). Nucleotide sequencing of the CIT family amplicons was found to have 100% homology to CMY-2 and EBC family amplicons to have 100% homology to ACT-1-type AmpC β-lactamases. PCR detected four additional isolates: EC 14, 15, 32, and 34 to produce AmpC β-lactamases as compared to phenotypic test, and all these isolates produced CITtype AmpC enzyme ( Table 3).   (Fig. 2 and Tables 3 and 5). Interestingly, in 12/34 isolates of E. coli, bands of pI 5.4 could be detected, which did not correspond to any of the known AmpC types (data not shown). In addition, 6 of the isolates showed the presence of multiple β-lactamase bands (data not shown).
Our study showed similar results (35% by phenotypic test) to some of the researchers reporting a high incidence of AmpC β-lactamases in E. coli, indicating a rising trend of resistance, which should be a cause of concern.    Amoxicillin-CLA combination is usually given in treating infection caused by β-lactamase-producing isolates because CLA is known to be an active inhibitor of ESBLs. Since CLA can act as an inducer for AmpC β-lactamases, it becomes imperative to check for the presence of inducible AmpC β-lactamases in the clinical isolates. Studies have reported the presence of inducible AmpC β-lactamases in 4-21% of E. coli isolates [25,29]. Our study showed similar results with 16.6% of the AmpC-positive isolates to be inducible; however, more number of isolates need to be tested to show an increasing trend. Hence, testing for the presence of inducible AmpC producers becomes important, as there can be a therapeutic failure if antibiotics inducing the enzymes are administered for treating the infection empirically.
A multiplex PCR which distinguished plasmid-mediated AmpC enzymes from chromosomally mediated and also differentiated between six different families of plasmid-mediated AmpC β-lactamases was used in this study. A number of workers have used PCR to detect the presence of ampC genes E. coli and have reported PCR to be more sensitive than phenotypic methods to detect AmpC-producing isolates [4,[30][31][32]. Our study showed the presence of ampC gene for plasmid-mediated AmpC enzymes in 47% of the screen-positive isolates of E. coli and was found to detect four additional AmpC producers than inhibitor-based method.
In the present study, a high percentage (81.2%) of the PCR-positive isolates of E. coli were shown to produce CIT type of AmpC β-lactamases, followed by 18.75% producing EBC type. Our study corroborates the findings of other studies, showing a high percentage (88%) of E. coli isolates to produce CIT type of AmpC enzymes [28,33,34]. However, there are studies which reported a lower prevalence, 11%, 34%, and 56%, respectively, of the E. coli isolates to be producing CIT-type AmpC β-lactamases [4,35,36]. The presence of EBC family or ACT-1 subtype of AmpC enzymes in E. coli has been reported in many studies; however, EBC-type AmpC β-lactamases was found to be more prevalent in Klebsiella spp. [37][38][39][40]. In the present study, we found ~19% of the inducible AmpC-producing E. coli isolates to be of EBC (ACT-1) type. In our study, inducible AmpC β-lactamases were detected by DAT, IEF as well as PCR.
The inhibitor-based phenotypic test detected less number of isolates to be AmpC positive as compared to the PCR, and this suggests that PCR should be used as the gold standard method for identification of beta-lactamases. However, more number of isolates need to be tested to confirm this.
Detection and characterization of β-lactamases employing IEF were for the first time carried out by Matthew et al. in 1975 [14]. Subsequently, IEF was used for characterizing AmpC β-lactamases in a number of bacterial pathogens [12,[41][42][43]. In the present study, β-lactamase bands of pI values >8.4, corresponding to CIT type, bands of pI value 8.4 corresponding to EBC type, bands of pI values ranging between 6.3-7.2 corresponding to FOX/DHA, and bands of pI value 7.6 corresponding to ACC type AmpC enzymes were observed. These were further confirmed by PCR to be the respective AmpC enzymes except ACC and FOX or DHA types. According to our PCR results, none of the E. coli isolates harbored ampC genes for ACC and DHA families. In comparison to PCR, IEF detected less isolates to produce CIT type but extraisolates to produce ACC and FOX-/DHA-type AmpC β-lactamases. Moreover, bands of pI value 5.4 were detected which did not correspond to any of the known AmpC β-lactamases. This suggests that there may be a possibility that these pI bands correspond to some ESBLs and that some of the tested isolates coproduced ESBLs. It was found that 83% of the AmpCproducing isolates in this study were also producing ESBLs. Many studies have shown the presence of ESBLs along with AmpC enzymes which also showed bands of different pI values [24,27,[44][45][46][47]. In this study, there was one isolate, EC 22, which was found to be positive for inducible AmpC β-lactamases, coproduced ESBL and was found to harbor genes for ACT-1 subtype of AmpC β-lactamase. This coexistence of multiple enzymes in clinical isolates may restrict the therapeutic options available for treating cases infected with such pathogens.

CONCLUSION
Our findings may suggest that there is a need to initiate steps to rationalize the use of the β-lactams and also to have a more vigorous surveillance for detection of such resistance in clinical isolates both at the hospital as well as at the community level. However, a larger set of E. coli isolates along with other nosocomial pathogens need to be tested to have more substantiate data. This constitutes the part of our future studies.


extfieldsofstudy: Chemistry Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Glycolysis and glutaminolysis cooperatively control T cell function by limiting metabolite supply to N-glycosylation

Rapidly proliferating cells switch from oxidative phosphorylation to aerobic glycolysis plus glutaminolysis, markedly increasing glucose and glutamine catabolism. Although Otto Warburg first described aerobic glycolysis in cancer cells >90 years ago, the primary purpose of this metabolic switch remains controversial. The hexosamine biosynthetic pathway requires glucose and glutamine for de novo synthesis of UDP-GlcNAc, a sugar-nucleotide that inhibits receptor endocytosis and signaling by promoting N-acetylglucosamine branching of Asn (N)-linked glycans. Here, we report that aerobic glycolysis and glutaminolysis co-operatively reduce UDP-GlcNAc biosynthesis and N-glycan branching in mouse T cell blasts by starving the hexosamine pathway of glucose and glutamine. This drives growth and pro-inflammatory TH17 over anti-inflammatory-induced T regulatory (iTreg) differentiation, the latter by promoting endocytic loss of IL-2 receptor-α (CD25). Thus, a primary function of aerobic glycolysis and glutaminolysis is to co-operatively limit metabolite supply to N-glycan biosynthesis, an activity with widespread implications for autoimmunity and cancer. DOI: http://dx.doi.org/10.7554/eLife.21330.001

Introduction
Under anaerobic conditions oxidative phosphorylation is blocked, forcing cells to generate ATP by converting glucose to lactate via glycolysis. This is a highly inefficient use of glucose, generating only 2 ATP per glucose instead of 36-38 ATP per glucose with complete oxidation. Yet under aerobic conditions, rapidly dividing cells such as T cells paradoxically switch their metabolism from oxidative phosphorylation to aerobic glycolysis and glutaminolysis, where glucose is fermented to lactate despite the presence of oxygen (Wang et al., 1976;Warburg et al., 1927;Warburg et al., 1924) and primary carbon flux into the tricarboxylic acid cycle (TCA cycle) is switched from glucose to glutamine (DeBerardinis et al., 2007). The function of this metabolic switch has remained obscure since Otto Warburg first reported that cancer cells ferment glucose to lactate despite the presence of oxygen (aerobic glycolysis) over 90 years ago (Warburg et al., 1927;Warburg et al., 1924). Glucose metabolites generated by glycolysis are precursors for nucleotide, amino acid and lipid biosynthetic pathways, leading some to suggest that increased glucose uptake may serve to promote the biomass required for rapid cell division (Lunt and Vander Heiden, 2011;Vander Heiden et al., 2009). However, these biosynthetic pathways account for <10% of the increase in glucose uptake induced by the Warburg effect (DeBerardinis et al., 2007;Hume et al., 1978), implying other critical functions. Alternatively, constraining oxidative phosphorylation in rapidly dividing cells may serve to limit cellular damage from reactive oxygen species (Le et al., 2010). This hypothesis is questioned by the observation that genetically blocking lactate production in lung cancer does not activate oxidative phosphorylation in vivo yet still induces disease regression (Xie et al., 2014).
In contrast to cancer, T cells provide an excellent physiologic model to investigate the normal function of aerobic glycolysis and glutaminolysis. Indeed, aerobic glycolysis and glutaminolysis have recently been suggested to promote pro-inflammatory IFNg synthesis and T H 1 differentiation (Chang et al., 2013;Gubser et al., 2013;Klysz et al., 2015), respectively. Aerobic glycolysis also promotes anti-tumor T cell activity and differentiation into pro-inflammatory T helper 17 (T H 17) over anti-inflammatory T regulatory (Treg) cells (Ho et al., 2015;Michalek et al., 2011;Shi et al., 2011). Several of these studies implicated specific pathway enzymes or metabolites, such as GAPDH, aketoglutarate and phosphoenolpyruvate, for control of IFNg translation, T H 1 differentiation and intracellular Ca 2+ flux, respectively. However, these mechanisms are not easily generalizable to other cell types and/or do not consider the global consequences of metabolite shifts or the combined effects of aerobic glycolysis and glutaminolysis.
Branching in N-glycans depends on UDP-GlcNAc biosynthesis via the hexosamine pathway (Dennis et al., 2009;Grigorian et al., 2011Grigorian et al., , 2007Lau et al., 2007;Mkhikian et al., 2016). De novo synthesis of UDP-GlcNAc, the sugar-nucleotide donor substrate required by the N-glycan branching Golgi enzymes Mgat1, 2, 4 and 5, begins with the conversion of fructose-6-phosphate to glucosamine-6-phosphate by the rate-limiting enzyme glutamine-fructose-6-phosphate transaminase (GFPT, Figure 1A). Fructose-6-phosphate is derived from glucose by the action of hexokinase/glucokinase (HK) followed by glucose-6-phosphate isomerase (GPI) and is the critical entry step into glycolysis via the key regulatory enzyme phosphofructokinase 1 (PFK1) ( Figure 1A). GFPT also requires glutamine as an amine donor to generate glucosamine-6-phosphate from fructose-6-phosphate. Thus, the hexosamine pathway may be in direct competition with glycolysis and glutaminolysis for fructose-6-phosphate and glutamine, respectively. Here, we test the hypothesis that aerobic glycolysis and glutaminolysis cooperatively starve the hexosamine pathway of both glucose and glutamine, thereby reducing de novo UDP-GlcNAc biosynthesis and N-glycan branching, the latter altering cell function.

T H 17 cytokines lower UDP-GlcNAc biosynthesis and N-glycan branching
To initially explore our hypothesis, we investigated whether aerobic glycolysis regulates branching during the differentiation of T cells into pro-inflammatory T H 17 cells. Induction of T H 17 differentiation strongly induces aerobic glycolysis, but when glycolysis is inhibited, cell fate is switched from pro-inflammatory T H 17 to anti-inflammatory iTreg cells by an unknown mechanism (Shi et al., 2011). If aerobic glycolysis starves the hexosamine pathway of fructose-6-phosphate, T H 17 cytokines are expected to decrease branching via reduced biosynthesis of UDP-GlcNAc. To evaluate this possibility, we performed flow cytometry with L-PHA (Phaseolus vulgaris, leukoagglutinin), a plant lectin that binds specifically to b1,6 GlcNAc-branched N-glycans made by the Mgat5 enzyme and serves as a gauge of overall branching (Cummings and Kornfeld, 1982;Demetriou et al., 2001;Zhou et al., 2014). Strikingly, T H 17 inducing cytokines (TGFb+IL-6+IL-23) acted synergistically to markedly reduce L-PHA binding in activated T cells by~3-4 fold, despite each cytokine individually having As only a~20% change in branching is sufficient to alter T cell growth Mkhikian et al., 2011;Zhou et al., 2014), the reduction induced by T H 17 inducing cytokines is highly significant. Although branching was reduced, mRNA expression of the Golgi branching genes Mgat1 and Mgat5 were unchanged or increased, consistent with reduced UDP-GlcNAc supply being primarily responsible for lowering branching ( Figure 1-figure supplement 1C). Indeed, while T cell activation markedly increases protein expression of GFPT1 as well as the critical glycolytic enzymes HK1, GPI and PFK1 isoenzymes (liver, platelet and muscle), GFPT1 is uniquely and specifically down-regulated by T H 17 cytokines ( Figure 1C). GFPT2 is an isoenzyme of GFPT1 but is not detectable by Western blot in T cells (data not shown). As GFPT1 and the three PFK1 isoenzymes all utilize fructose-6-phosphate, the reduction in GFPT1 induced by T H 17 cytokines should favor glucose flux into glycolysis over the hexosamine pathway. Indeed, UDP-GlcNAc production is reduced by T H 17 cytokines ( Figure 1D, Figure 1-figure supplement 1D). Together, these data demonstrate that T H 17 cytokines reduce UDP-GlcNAc production, branching and GFPT1 expression, the rate-limiting enzyme for entry of fructose-6-phosphate into the hexosamine pathway.

N-glycan branching induces a cell fate switch from T H 17 to iTreg
Next, we examined whether TGFb+IL-6+IL-23 induced reductions in UDP-GlcNAc and branching was required for T H 17 differentiation. To test this hypothesis, we bypassed the effects of GFPT1 competition for fructose-6-phosphate by exploiting the hexosamine salvage pathway, where N-acetylglucosamine (GlcNAc) is used to generate UDP-GlcNAc directly ( Figure 1A) Lau et al., 2007). GlcNAc is metabolically inert within cells and does not enter glycolysis, the TCA cycle or the pentose phosphate pathway (Wellen et al., 2010). Supplementing T cells with GlcNAc reversed the reduction in branching induced by T H 17 cytokines and markedly inhibited T H 17 differentiation ( Figure 1E,F). Remarkably, GlcNAc supplementation not only blocked T H 17 differentiation but also induced a cell fate switch to iTreg cells, despite the presence of T H 17-inducing cytokines ( Figure 1F). The mannosidase I inhibitor kifunensine ( Figure 1A) blocks branching (Figure 1figure supplement 1E) and reversed the effects of GlcNAc supplementation, confirming that raising UDP-GlcNAc levels with GlcNAc supplementation blocked T H 17 and promoted iTreg differentiation by restoring branching (Figure 1-figure supplement 1F). Oral delivery of GlcNAc to mice with Experimental Autoimmune Encephalomyelitis, a model of multiple sclerosis, blocked disease progression, raised branching in T cells and suppressed T H 17 in vivo .
To confirm this result genetically, we utilized the tet-on system to generate a mouse with inducible expression of the Golgi branching enzyme Mgat5 (ROSA rtTA Mgat5 tet ). Indeed, directly raising branching via transgenic over-expression of Mgat5 also induced a cell fate switch from T H 17 to iTreg cells despite T H 17-inducing cytokines ( Figure 1G, Figure 1-figure supplement 2A). The magnitude of this change was less than that of GlcNAc supplementation, consistent with reduced de novo synthesis of UDP-GlcNAc by aerobic glycolysis primarily limiting branching. Directly inhibiting branching should have the opposite effect of raising branching and indeed, blocking branching by culturing cells with kifunensine or by inducing deficiency of the branching enzymes Mgat1 (via doxycycline treatment of Mgat1 f/f tetO-Cre + ROSA rtTA mice, Figure 1-figure supplement 2B) (Zhou et al., 2014) or Mgat5 (Mgat5 -/-) (Demetriou et al., 2001) all significantly enhanced T H 17 induction ( Figure 1H, Figure 1-figure supplements 1F and 2C). These data confirm that reduced UDP-GlcNAc production and branching induced by T H 17 cytokines is required for T H 17 differentiation and serves to block iTreg differentiation.

N-glycan branching promotes IL-2Ra signaling to drive iTreg over T H 17 differentiation
Interleukin-2 (IL-2) suppresses T H 17 and promotes iTreg differentiation Laurence et al., 2007). As branching promotes surface retention of multiple receptors (Dennis et al., 2009), we investigated whether reduced branching limits IL-2 signaling via decreased surface retention of the IL-2 receptor. Blocking branching during T H 17 induction via Mgat1 deletion markedly reduced surface expression and retention of CD25, the high-affinity alpha subunit of the IL-2 receptor (  significantly altered by GlcNAc or kifunensine (Figure 2-figure supplement 1E). The IL-2 receptor signals via STAT5 and this is markedly reduced by T H 17 cytokines ( Figure 2D). GlcNAc supplementation restored pSTAT5 signaling despite T H 17 conditions ( Figure 2D). Sequestering endogenous IL-2 with anti-IL-2 antibody blocked the ability of GlcNAc to switch cell fate from T H 17 to iTreg, suggesting that IL-2 is required for branching to promote iTreg over T H 17 differentiation ( Figure 2E Aerobic glycolysis drives T H 17 over iTreg differentiation by lowering N-glycan branching Next, we investigated whether aerobic glycolysis was required for T H 17 differentiation. Branching promotes surface retention of glucose transporters (Lau et al., 2007;Ohtsubo et al., 2005). Consistent with this, glucose uptake under T H 17-inducing conditions was enhanced by GlcNAc and inhibited by kifunensine, as measured by intracellular staining for the glucose analog 2-NBDG (Figure 3figure supplement 1A). This suggests that GlcNAc supplementation does not directly inhibit aerobic glycolysis. Indeed, under T H 17 inducing conditions, GlcNAc supplementation did not significantly alter the glycolytic state of the cells prior to differentiation, as measured by the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) at day 2 of culture ( Figure 3A, Figure 3figure supplement 1B). This suggests that catabolism of fructose-6-phosphate to lactate is not necessary to drive T H 17 differentiation. To confirm this, we directly limited glucose flux into glycolysis by forcing oxidative phosphorylation using either the glycolytic inhibitor rapamycin or media supplemented with galactose instead of glucose (Chang et al., 2013). Rapamycin inhibits mTOR (mechanistic Target of Rapamycin) signaling via binding to mTOR complex 1, thereby reducing HIF-1a expression (Land and Tee, 2007), a transcription factor that up-regulates glycolysis enzymes including HK, PGM and PFK1 (Semenza et al., 1994). Galactose is much more slowly converted to pyruvate than glucose, forcing cells to up-regulate oxidative phosphorylation at the expense of glycolysis (Chang et al., 2013). Rapamycin and galactose both lowered aerobic glycolysis, enhanced branching and drove iTreg over T H 17 differentiation ( Figure 3B-G). Importantly, the latter was reversed with Mgat1 deficiency or kifunensine ( Figure 3F,G, Figure 3-figure supplement 1C,D), demonstrating that the increase in branching induced by blocking glycolysis was necessary to switch cell fate from T H 17 to iTreg. Rapamycin had little effect on GFPT1 levels, while galactose marginally increased GFPT1, consistent with glycolysis largely acting by metabolically limiting flux of fructose-6-phosphate to the hexosamine/branching pathway rather than altering enzyme expression ( Figure 3E). Importantly, kifunensine did not reverse the inhibition of aerobic glycolysis induced by rapamycin and galactose ( Figure 3B,C), establishing that T H 17 differentiation does not require aerobic glycolysis beyond its effects on branching. 2-Deoxy-D-glucose is another commonly used inhibitor of glycolysis; however, this molecule is also 2-deoxy-D-mannose and inhibits N-glycan biosynthesis via incorporation of 2-deoxy-D-mannose into the lipid-linked oligosaccharide precursor (Zhang et al., 2014). Indeed, 2-deoxy-D-glucose/2-deoxy-D-mannose reduced branching and promoted T H 17 differentiation (Figure 3-figure supplement 1F,G), consistent with branching being dominant over aerobic glycolysis in controlling T H 17 differentiation. Taken together, these data demonstrate that a primary function of aerobic glycolysis during T H 17 differentiation is to lower branching by starving the hexosamine pathway of fructose-6-phosphate substrate availability.
Oxidative phosphorylation drives iTreg differentiation by enhancing N-glycan branching iTreg cells induced by TGFb alone are non-glycolytic and utilize lipid oxidation for ATP production (Michalek et al., 2011;Shi et al., 2011). Based on our model that glycolysis and the hexosamine pathway compete for fructose-6-phosphate, lipid oxidation should favor flux of fructose-6-phosphate into the hexosamine pathway over glycolysis and thereby increase UDP-GlcNAc, branching and iTreg differentiation via IL-2/CD25/STAT5 signaling. Indeed, abolishing branching via Mgat1 deficiency virtually eliminated iTreg differentiation induced by TGFb ( Figure 4A, Figure 4-figure supplement 1A). iTreg differentiation was also inhibited by Mgat5 deficiency and kifunensine ( Figure 4B,C), while raising branching with GlcNAc had the opposite effect ( Figure 4C, Figure 4-figure supplement 1B). Kifunensine reversed the effects of GlcNAc, confirming GlcNAc promoted iTreg by raising branching ( Figure 4C). Treatment with etomoxir (ETX), a specific inhibitor of fatty acid oxidation (Michalek et al., 2011), should up-regulate glycolysis and thereby limit flux of fructose-6-phosphate into the hexosamine pathway. Indeed, etomoxir decreased branching, CD25 surface expression and iTreg generation; phenotypes reversed by GlcNAc supplementation (Figure 4D-F). Taken together, these data confirm that oxidative phosphorylation promotes iTreg differentiation by limiting utilization of fructose-6-phosphate by glycolysis and thereby promoting flux of fructose-6-phosphate into the hexosamine pathway.

Aerobic glycolysis promotes T cell growth by lowering N-glycan branching
Branching inhibits T cell growth by reducing T cell receptor clustering/signaling and promoting surface retention of the growth inhibitor CTLA-4 (Demetriou et al., 2001;Lau et al., 2007;Mkhikian et al., 2011). In the absence of polarizing cytokines, TCR stimulation also increases glucose uptake and induces aerobic glycolysis. However, in contrast to T H 17 cells, GFPT1 is markedly up-regulated in neutral T cell blasts ( Figure 1C). Thus, increased GFPT1 should be better at competing with PFK1 for the increased supply of fructose-6-phosphate to promote UPD-GlcNAc production. Coupled with increased expression of multiple Golgi branching enzymes in T cell blasts (Chen et al., 2009), this should promote branching in neutral T cell blasts relative to resting cells.  Indeed, we previously observed increased UDP-GlcNAc production and branching in non-polarized T cell blasts relative to resting naive T cells (Lau et al., 2007). However, inhibiting aerobic glycolysis under neutral conditions should further drive fructose-6-phosphate into the hexosamine pathway to promote branching and limit growth of T cell blasts. Indeed, rapamycin and galactose enhanced branching and inhibited T cell growth, effects reversed with the Golgi branching inhibitor kifunensine ( Figure 5A-C, Figure 5-figure supplement 1). This indicates that like T H 17 differentiation, aerobic glycolysis promotes T cell growth by limiting supply of fructose-6-phosphate to the hexosamine pathway.

Glutaminolysis drives T H 17 over iTreg differentiation by lowering N-glycan branching
Aerobic glycolysis is frequently accompanied by glutaminolysis, where glutamine is metabolized to glutamate by the enzyme glutaminase (DeBerardinis et al., 2007). GFPT1 absolutely requires glutamine as an amine donor to convert fructose-6-phosphate to glucosamine-6-phosphate, the first step of the de novo hexosamine pathway ( Figure 1A). This suggests that glutaminolysis may reduce availability of glutamine to the hexosamine pathway, thereby lowering branching to drive T H 17 over iTreg differentiation. To examine this possibility, we investigated whether excess exogenous glutamine can reverse the reduction in branching induced by T H 17 cytokines and thereby drive iTreg over T H 17 differentiation. Indeed, under T H 17 conditions excess glutamine raised branching, markedly inhibited T H 17 differentiation and induced a cell fate switch to iTreg ( Figure 6A,B, Figure 6-figure supplement 1A). These phenotypes were reversed by blocking branching with kifunensine, confirming that excess glutamine acted by raising branching. Similarly, increasing glutamine under iTreginducing conditions raised branching and increased iTreg generation in the absence but not in the presence of kifunensine ( Figure 6C,D, Figure 6-figure supplement 1B). Blocking glutaminase activity with 6-diazo-5-oxo-L-norleucine (DON) or azaserine should increase glutamine supply to GFPT1. Indeed, both glutaminase inhibitors enhanced branching and induced a cell fate switch from T H 17 to iTreg ( Figure 6E-J). Klysz et al. reported that glutamine deprivation drives iTreg over T H 1 differentiation by reducing glutamine catabolism to a-ketoglutarate, a conclusion suggested by the ability of the a-ketoglutarate analog dimethyl-2-oxoglutarate (DMK) to restore T H 1 over iTreg differentiation in the presence of glutamine deprivation (Klysz et al., 2015). In contrast, we observed that DMK did not reverse the effects of glutaminase inhibition by DON on branching or T H 17 induction ( Figure 6E,G). However, DMK did induce a partial rescue of iTreg generation ( Figure 6I). Moreover, blocking branching with kifunensine only partially reversed the effects of DON and Azaserine on T H 17 versus iTreg generation ( Figure 6G-J). Together, these data indicate that glutamine availability regulates branching to affect T H 17 versus iTreg differentiation, but other factors such as catabolism of glutamine to a-ketoglutarate likely also play a role.

Discussion
It has been over 90 years since Otto Warburg's seminal discovery that cancer cells switch their metabolism from oxidative phosphorylation to aerobic glycolysis (Warburg et al., 1927;Warburg et al., 1924). Rapidly proliferating mammalian cells paradoxically stop the complete oxidation of glucose and instead ferment glucose to lactate while using glutamine rather than glucose to supply the TCA cycle. This extracts 18-fold less energy per glucose molecule while markedly increasing catabolism of glutamine, changes that should limit glucose and glutamine flux into other pathways. Indeed, our data support a model where a primary function of aerobic glycolysis and glutaminolysis is to co-operatively limit the flux of glucose and glutamine into the hexosamine pathway, thereby inhibiting N-glycan branching and associated down-stream phenotypes.
In T cells, reduced branching induced by aerobic glycolysis and glutaminolysis promotes proinflammatory T H 17 differentiation over anti-inflammatory iTreg differentiation. Restoring branching and UDP-GlcNAc levels via salvage of N-acetylglucosamine (GlcNAc) into the hexosamine pathway does not alter the glycolytic state of the cell, yet switches cell fate from T H 17 to iTreg cells by blocking endocytic loss of IL-2 receptor-a (CD25). Forcing oxidative phosphorylation raises branching to drive iTreg over T H 17 differentiation. Blocking the increase in branching reverses the latter phenotypes, demonstrating that aerobic glycolysis is not essential for T cell growth/differentiation control beyond regulation of branching. We conclude that metabolic switches between oxidative phosphorylation and aerobic glycolysis plus glutaminolysis primarily target hexosamine/N-glycosylation via altered glucose and glutamine flux and as a consequence, influence T cell differentiation and growth.
GlcNAc has recently been reported to promote glucose, glutamine, and fatty acid uptake in liver and/or adipose tissue via branching induced increases in surface retention of metabolite transporters (Ryczko et al., 2016). Similarly, we observed that modifying branching in T cells with GlcNAc or kifunensine altered glucose uptake. However, GlcNAc or kifunensine did not acutely alter the metabolic state of the T cell, as measured by the oxygen consumption rate and extracellular acidification rate. Consistent with this, we have previously shown that lowering branching does not alter UDP-GlcNAc levels in T cells (Mkhikian et al., 2016). GlcNAc does raise UDP-GlcNAc levels, which may impact other glycan pathways such as O-glycans, O-GlcNAc or CMP-sialic scid, in addition to N-glycan branching. However, the effects of GlcNAc on T cell differentiation were reversed by directly blocking Golgi branching activity, confirming that the effects of raised UDP-GlcNAc were predominantly through branching rather than other pathways. Moreover, blocking the effects of the metabolic modulators rapamycin, galactose or etoximer on branching reversed their effects on T cell differentiation despite not reversing effects on metabolism. In this manner, T cell differentiation can be decoupled from the metabolic state of the cell by manipulating branching. However, our data does not exclude metabolically driven changes in other pathways and indeed, the effects of glutaminolysis on branching appear to only partially explain the phenotype.
The competition between PFK1 and GFPT for fructose-6-phosphate is essential for determining the extent of glucose flux into the hexosamine/branching pathways. During T H 17 differentiation, GFPT1 is down-regulated and PFK1 dominates, restricting glucose flux into the hexosamine pathway and lowering branching relative to neutral T cell blasts. In contrast, GFPT1 is up-regulated in neutral T cell blasts, allowing increases in glucose uptake to raise de novo UDP-GlcNAc production and branching relative to resting cells (Lau et al., 2007); however, aerobic glycolysis still functions to limit the extent of glucose flux into the hexosamine pathway and thereby functions to promote growth.
The Warburg effect was first described in cancer cells, where branching and Mgat5 are pathologically over-expressed and serve as essential drivers of growth, motility and metastasis (Granovsky et al., 2000;Lau et al., 2007;Partridge et al., 2004). N-glycan branching and the hexosamine pathway promote epithelial to mesenchymal transition and tumor growth by regulating surface expression of receptor tyrosine kinases and TGFb receptors (Demetriou et al., 1995;Lau et al., 2007;Partridge et al., 2004). While oncogenes such as Kras have been reported to turn on hexosamine and N-glycan pathway genes (Ying et al., 2012), the functional connection between aerobic glycolysis and hexosamine/branching in cancer has not been established. GFPT expression is elevated in a number of cancers (Itkonen et al., 2013;Ying et al., 2012) (R. Zhou and M. Demetriou, unpublished data), where it is expected to promote flux of glucose into the hexosamine pathway and branching. Our data in T cells suggest that although increased GFPT and MGAT5 expression in cancer drive UDP-GlcNAc and branching, aerobic glycolysis is expected to do the opposite and limit UDP-GlcNAc expression and branching. The latter would inhibit tumor growth and metastasis, but this phenotype may be masked by up-regulation of GFPT and/or MGAT5. This would parallel our results in neutral T cell blasts, where aerobic glycolysis limits branching yet elevated GFPT and MGAT5 act downstream to increase branching. The functional consequences of aerobic glycolysis and glutaminolysis on branching in cancer merit further investigation.
The ability of GlcNAc to promote iTreg while blocking T H 17 differentiation further validates its potential as a therapeutic for autoimmune disorders. We have previously shown that GlcNAc limits T cell activation/growth and when provided orally to mice, inhibits experimental autoimmune encephalomyelitis, a mouse model of Multiple Sclerosis (MS), as well as autoimmune diabetes in the Non Obese Diabetic mouse model . GlcNAc has also been given orally (3-6 g/day) to children with refractory inflammatory bowel disease for~2 years, with 8 of 12 showing clinical improvement without reported toxicities and/or side effects (Salvatore et al., 2000). We have recently observed that serum levels of endogenous GlcNAc are markedly reduced in patients with the progressive form of MS and correlate with clinical disability and imaging measures of neurodegeneration (Alexander Brandt and Michael Demetriou, unpublished data). A pilot study of lowdose oral GlcNAc in MS (3 g/day) increased serum GlcNAc levels and branching in T cells (Barbara Newton and Michael Demetriou, unpublished data). As GlcNAc is a dietary supplement that is for sale 'over the counter' in the US, these data suggest that GlcNAc may serve as a safe and inexpensive therapeutic for MS patients and potentially other autoimmune diseases.
In summary, our data brings new clarity to the function of aerobic glycolysis and glutaminolysis and has widespread implications for the role of metabolism in disease states as diverse as autoimmunity and cancer.

Mice
Mgat5-deficient and doxycycline-inducible Mgat1-deficient mice (Mgat1 f/f tetO-Cre + ROSA rtTA ) were previously described (Demetriou et al., 2001;Zhou et al., 2014). To delete Mgat1 in peripheral T cells, 2 mg/ml doxycycline plus 1% sucrose was provided in the drinking water to Mgat1 f/f tetO-Cre + -ROSA rtTA mice for !3 weeks. To generate doxycycline-inducible Mgat5 over-expression mice (Mgat5 tetO ROSA rtTA ), the cDNA encoding the mouse Mgat5 (a gift from James Dennis) was subcloned into the transgene expression vector pTRE-Tight (Clontech, Mountain View, CA), containing a tet-regulated promoter controlling the expression of the subcloned Mgat5 cDNA (Mgat5 tetO ). The transgene was microinjected into fertilized C57BL/6 mouse eggs and placed into pseudo-pregnant females. Founder mice were bred with C57BL/6 mice and transgenic offspring were identified by PCR using a PCR primer pair targeting the Tet promoter and Mgat5 transgene (left -CGAGG TAGGCGTGTACGG, right -AGACCGGTTTCCAACAACCT). Transgenic-positive mice were then crossed with ROSA rtTA to generate Mgat5 tetO ROSA rtTA mice and detected using primers as per Jackson Laboratory (stock 006965). To overexpress Mgat5 in mouse splenic CD4 + T cells, doxycycline was added to cell culture at 4 mg/ml. All the mice were on the C57BL/6 background.

Analysis of cell metabolism
Cells were harvested after 48 hr of culture and plated on 24-well XF cell culture micro-plates coated with Cell-Tak (Corning, Corning, NY) at a concentration of 22.4 mg/ml in non-buffered RPMI 1640 (containing either 10 mM glucose or 10 mM galactose, 2 mM L-glutamine and 1 mM sodium pyruvate) (Sigma) at a density of 7.5 Â 10 5 cells per well. Using two-step plating, microplates were incubated for 10 min with 100 ml of cells in non-buffered media at 37˚C in a non-CO 2 incubator. Then, 400 ml of non-buffered media was added to each well and incubated for an additional 20 min. The Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR) were measured using an XF24 Extracellular Flux Analyzer (Seahorse Biosciences/Agilent Technologies, Santa Clara, CA) under basal conditions.

Flow cytometry and intracellular staining
Flow cytometric analysis was performed as previously described (Demetriou et al., 2001;Grigorian et al., 2007;Zhou et al., 2014). Monoclonal antibodies specific to the following mouse antigens (and labeled with the indicated fluorescent markers) were purchased from eBioscience: IL-17 PE (eBio17B7), Foxp3 APC (Fjk-16s), CD4 PrpCy5.5 (RM4-5), CD25 PE (PC61.5). In addition, FITC L-PHA, FITC E-PHA and FITC ConA (Vector Labs, Burlingame, CA) was used to stain cells. For galectin-3 binding, recombinant mouse galectin-3 (R&D systems) was labeled using an Alexa Fluor 488 protein labeling kit (ThermoFisher Scientific). Cells were stained for flow cytometry using 3 mg labeled galectin-3 per test. Staining was carried out for 30 min at room temperature followed by one wash and fixation with paraformaldehyde. For the lactose control samples, 50 mM lactose was included in all steps. For intracellular staining, cells were stimulated at 37˚C for 5 hr with phorbol 12myristate 13-acetate (50 ng/ml, Sigma-Aldrich) and ionomycin (750 ng/ml, Sigma-Aldrich) in the presence of GolgiPlug (1 ml per 1 ml cell culture) (BD Biosciences) and stained using the Foxp3 Transcription Factor Fixation/Permeabilization Kit (eBioscience) according to the manufacturer's instruction. Flow cytometry experiments were performed with BD FACS Calibur, LSR II and Attune Acoustic Focusing Cytometer.

Endocytosis assay
Endocytosis assay was assessed as previously described (Mkhikian et al., 2011;Zhou et al., 2014). Briefly, splenocytes were stained with anti-CD25, resuspended in complete RPMI 1640 medium, incubated in a 37˚C incubator for 2.5 hr, washed in FACS buffer or acid-wash buffer (150 mM NaCl and 20 mM HCl, pH 1.7) for 3 min at 25˚C and then fixed before analyzing by flow cytometry. The acid wash removes surface-bound antibody and the MFI of acid-washed cells is divided by the MFI of FACS buffer washed cells to determine internalized antibody.

UDP-GlcNAc measurement by LC-MS/MS
Purified splenic CD4 + T cells activated for 72 hr in the presence or absence of T H 17-inducing conditions were washed twice with ice-cold 1x PBS and counted. 15 Â 10 6 cells were pelleted in 1.5 ml Eppendorf tubes by centrifuging at 350 g for 7 min at 4˚C. Metabolites were extracted from the pellets by the addition of 1 ml of ice-cold solution of 40% acetonitrile, 40% methanol and 20% water. The tubes were vortexed for 1 hr at 4˚C and spun down at 14,000 rpm for 10 min at 4˚C (Eppendorf, Germany). The supernatant was transferred to fresh tubes and evaporated to dryness in an Eppendorf Vacufuge at 30˚C (Eppendorf, Germany). The dry samples were stored at À80˚C until analyzed, at which point they were reconstituted in 100 ml of LC-MS grade water. The samples were run on a Waters (Micromass) Quattro Premier XE LC-MS/MS machine in negative mode with Water:Acetonitrile (+0.2% Acetic Acid and 5 mM Ammonium Acetate) solvent. UPLC with C18 reverse phase column was employed. Area was converted to UDP-GlcNAc concentration using a standard curve simultaneously generated with purified UDP-GlcNAc (Sigma).


extfieldsofstudy: Biology Medicine

text:
Sequence variations in estrogen receptor 1 and 2 genes and their association with egg production traits in Chinese Dagu chickens

Estrogen receptors α (ESR1) and β (ESR2) play central roles in folliculogenesis and therefore in reproductive biology. In the present study, two single nucleotide polymorphisms (SNPs) were identified in the ESR1 and ESR2 genes using PCR-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing. One of the identified SNPs, a T1101C transition located within exon 4 of the ESR1 gene, was significantly associated with hen-housed egg production (HHEP) at 30, 43, 57 and 66 weeks of age (P<0.05), and egg weight (EW) at 30 weeks (P<0.05). Another SNP, a G1755A transition leading to a non-synonymous substitution (valine 459-to-isoleucine) located within exon 8 of the ESR2 gene, was also markedly correlated with the HHEP at 30, 43, 57 and 66 weeks of age (P<0.05), and EW at 30 weeks (P<0.05). A greater proportion of the additive variance was explained by the SNPs for most of the associated egg production traits (>1%). Furthermore, the results of the combined genotype-based association analysis supported the finding that the two SNPs were associated with the traits under a study. Taken together, our findings suggest that the two sequence variations in the ESR1 and ESR2 genes may provide promising genetic markers for the early selection and prediction of advantageous phenotypes in chicken breeding.

One of the major objectives of selection in genetic breeding programs in laying hens is to improve egg productivity [43]; however, most egg production traits in chicken, including those in hen-houses, egg weight and age of sexual maturity are inherited as polygenic traits, with low to moderate heritability, leading to difficulty in the estimation of the level of genetic improvement [5,30,33,43]. Many modern chicken breeds have been generated by conventional breeding methods (such as family selection assisted by self-selection), with improved egg production traits, a key focus. However, the high costs and low effectiveness of the conventional breeding approach in chicken using pedigree breeding and selection, has been disappointing for poultry breeders and researchers. Fortunately, with recent progresses in molecular biology technologies, breeding for target traits can be fast-tracked by the utilization of marker-assisted selection (MAS), focusing on improving egg productivity in chickens. A candidate gene approach is a cost-effective means of investigating associations between gene polymorphisms and quantitative trait loci that are responsible for variations in traits of interest [26,33,35,42,47]. This approach enables more efficient breeding selection for egg production traits in chickens. Therefore, identification and application of potential candidate genes and associated genotypes can have significant economic implications and have become increasingly important in poultry breeding programs [27,44,48]. However, more candidate genes and single nucleotide polymorphisms (SNPs) are required for MAS in chicken breeding. Currently, accumulating evidence has confirmed that estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2) genes are involved in ovarian follicular development, and ovulation in chicken and mice [13,14,18,19], with a potential impact on egg production traits in laying hens.
Estrogen is essential for folliculogenesis, with independent functions attributed to each of the two estrogen receptors [14]. Estrogen plays a pivotal role as an intrafollicular modulator by stimulating granulosa cell proliferation and facilitating the differentiative actions of FSH and LH on these cells [13,34]. Estrogen receptors (ERs) are ligand-activated transcription factors, which are stabilized in the cytosol and translocate to the nucleus upon ligand binding and dimerization in addition to their doi: 10.1292/jvms.  involvement of membrane ER-centered signaling triggered by estrogen [28,32,40]. The expression of ESR1 (ER alpha) and ESR2 (ER beta) mRNA was examined in the ovaries of laying hens; the expression of ESR1 was higher than that of ESR2 [22]. The ESR1 gene is located on chromosome 3 and contains eight exons encoding a 589-amino acid protein (ERα) [23]. Previous studies on mice with targeted disruption in the ERα gene have indicated the presence of other estrogen binding elements, given that ovarian follicular growth appears to be unperturbed up to the secondary and antral stages [12]. In quails, the marked expression of ERα mRNA in the granulosa layer of the largest follicle may be related to the role of estrogens in cell proliferation and protein synthesis in the oviduct [21]. The results of recent studies revealed that adult female mutant mice that lack ERα in the neurons during the neonatal period fail to exhibit estrous cycles or negative feedback [10], whereas the differential expression of the ESR1 gene in chicken is involved in asymmetric ovarian development [19]. In addition, a recent study demonstrated that variants in the ESR1 gene, revealed using SNP analysis, were associated with laying traits in quails [41].
The chicken ESR2 gene, located on chromosome 5, has been cloned and found to comprise of eight exons encoding a 472-amino acid protein (ERβ), which plays a predominant role in estrogen activity in the chicken ovary [13,23]. ERβ, expressed predominantly by the ovarian granulosa cells, is required for antrum formation, preovulatory follicle maturation and ovulation in follicle development [16,20]. However, ERα and ERβ play different roles in folliculogenesis [13], where the proliferative action of estrogen is transmitted preferentially via ERα, whereas the differentiative effects of estrogen are mediated principally by ERβ [6,7]. Ovulatory defects have been linked to polymorphisms in human ERβ [39]. Thus, these data indicate that sequence mutations in ESR1 and ESR2 may have a great influence on chicken ovarian development and egg laying performance. Recent studies have shown that a polymorphism in the first intron of the ESR1 gene was associated with egg laying traits (such as the onset of egg laying, egg production at the age of 300 days and egg-laying sequence) in Guizhou local blue-shell chickens [9,50]. Accordingly, chicken ESR1 and ESR2 were proposed as candidate genes for egg production traits.
The Chinese Dagu chicken is an important poultry resource that is widely distributed in the northeast area of China; however, characterizations of genetic polymorphisms within the ESR1 and ESR2 encoding regions, and their possible correlation with egg production traits in Chinese breeds have been poorly investigated. In the present study, we detected two sequence variations in the coding regions of the ESR1 and ESR2 genes using the PCR-SSCP method and sequencing analysis. Associations between the newly identified single nucleotide polymorphisms (SNPs) and egg production traits were explored in local Chinese Dagu hens, and additive genetic effects of the SNP genotypes on the correlated traits were also evaluated. The purpose of this study was to determine a potential genetic marker to assist in the improvement of egg productivity in chicken breeding.

Chicken and trait measurements
Chinese Dagu chickens were produced by the College of Animal Science and Technology of Jilin Agricultural University. This chicken breed is an important indigenous chicken genetic resource in China [8], which originated and is mainly distributed in Northeast China. These birds are adapted to cold weather conditions (the minimum temperature in winter is below −30°C outdoors) in the Northern-East area, with some excellent economic traits, such as high meat and egg qualities, relatively strong disease resistance and high egg productivity [8,33]. In this experiment, 360 Dagu hens were sampled. They were hatched at the same time and raised in layered batteries under the same rearing conditions, which included free access to water and feed in accordance with the nutrient requirements of local Chinese chicken breeds and ration standard (including the Dagu chicken) established and published by the Chinese government, NY/T 33-2004, China. Approaching 16 weeks of age, the birds were reared in individual cages under a constantly maintained condition. As previously reported [33], the birds were exposed to a 16L:8D photoperiod, with lights on at 5:00 am. With the onset of egg-laying, all eggs were collected and recorded daily, with egg weights determined one day each week. Body weight was recorded following feed and water restrictions at 30 and 43 weeks of age, with the individual laying performance calculated. Egg production traits examined in this study included hen-housed egg production (egg laying number) at 30, 43, 57 and 66 weeks of age, and egg weight and body weight were determined at 30 and 43 weeks of age. All animal experiments were performed in accordance with laws of the People's Republic of China regarding animal protection.

DNA extraction and PCR amplification
At 300 days of age, peripheral blood was sampled from individuals of the 360 Chinese Dagu hens via the wing vein. Genomic DNA was extracted using a standard phenol-chloroform method, and DNA purity was examined using 1% agarose gel electrophoresis and ultraviolet-spectrophotometry, with final concentrations between 2 and 10 ng/µl detected. Primers for PCR were designed according to the published ESR1 (GenBank accession No. NM_205183.2) and ESR2 (Accession No. NM_204794.2) mRNA sequences in chicken. The primer pairs (Table 1) utilized to amplify fragments of the ESR1 and ESR2 genes were screened after examination, from a clutch of primers that cover almost the whole coding region of each candidate gene.
PCR was performed in a total volume of 50 µl, containing 25 µl of 2X Taq Master Mix (CWBIO, Beijing, China), 1 µl each primer (100 nM), 1 µl template DNA (25 to 50 ng) and 22 µl RNase-free Water. The PCR conditions were as follows: 94°C for 2 min, followed by 35 cycles at 94°C for 30 sec for denaturing, 55°C (57°C) for 30 sec for annealing (see Table 1), 72°C for 30 sec for extension and a final extension at 72°C for 2 min. products were then cloned into the Promega pGEM-T easy vector for sequencing commercially, according to the methods published by Sambrook and Russell [36]. For each sampled bird, two independent PCRs were conducted, with sequences analyzed using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) to confirm the amplification of the expected chicken ESR1 and ESR2 gene fragments. The obtained sequences were then aligned using DNAMAN software version 6.0 (LynnonBiosoft, San Ramon, CA, U.S.A.) to identify nucleotide variations.

Genotyping by PCR-SSCP and reconstruction of combined genotypes
To determine ESR1 and ESR2 gene polymorphisms, the confirmed PCR products were further analyzed using the singlestrand conformation polymorphism (SSCP) assay, as previously described [33]. Briefly, each 10-µl PCR product was mixed with 6 µl loading dye (0.025% bromophenol blue, 0.025% xylene cyanol, 98% deionized formamide, 2% glycerin and 10 mM ethylenediaminetetraacetic acid [EDTA]). Samples were denatured at 99°C for 10 min, cooled rapidly on ice and then loaded on 10% polymerized gels (acrylamide:bisacrylamide, 39:1; 0.75 mm thickness) 16 × 18 cm in size. Electrophoresis was carried out at 110 V for 5 hr in 1X tris-borate-EDTA (TBE) buffer at 20°C under an air-conditioning unit. After silver staining, the gels were examined under upper white by gel photography system (GeneSnap from SynGene). To avoid false positive/negative results due to artificial manipulation in the experiment, each sample was confirmed by repeated examinations. Combined genotypes were reconstructed based on the genotyping data obtained from all 360 birds using the PHASE program [38].

Polymorphism evaluation
Frequencies of genotypes and alleles at each SNP site were calculated, with each polymorphism evaluated for Hardy-Weinberg equilibrium using a Pearson's goodness-of-fit chi-square test (degree of freedom=1). Gene homozygosity (Ho), heterozygosity (He), effective number of alleles (Ne) and polymorphism information content (PIC) were statistically analyzed using the POPGENE v. 1.32 software [46].

Marker-trait association analysis
Associations of single polymorphisms and combined genotypes with laying performance traits were evaluated using the General Linear Model (GLM) procedure in SPSS 18.0. The following linear mixed effects model was used in the analysis: Y ijk = µ + L i + G j + F k + e ijk where, Y ijk is the phenotypic value of the target trait, such as hen-housed egg production and egg weight, µ is the population mean, L i is the fixed effect of the line, G j is the fixed effect of the SNP genotype or combined genotype, F k is the random effect of the family, and e ijk is the residual. Type III sum of squares was used in each test. Values were considered significant at P<0.05 and presented as least square means ± standard errors (SE).

Predicted SNP genotype effects
For the SNP(s) that showed significant association with the egg-laying traits, differences between the means of each genotype and allelic frequencies were used to estimate additive effects [17]. The percentage of additive genetic variance (%V j ) explained by the SNPs was determined using the following formula: %V j = 100p j q j α j ^ 2 / V g where p and q are the allele frequencies for the jth SNP estimated across the entire population; α j is the estimated additive effect of the jth SNP (allele substitution effect) on the trait under analysis; and V g is the restricted maximum likelihood (REML) estimate of the (poly-) genetic variance for the trait.

Confirmation of the amplified nucleotide sequence
Two targeted fragments were amplified using screened primers specific for Chinese Dagu hens, in which a 121-bp PCR amplicon for ESR1 and a 103-bp fragment for ESR2 were obtained, respectively. The purified PCR products were then cloned into the Promega pGEM-T easy vector for sequencing. Alignments of the amplified sequences with the corresponding template sequences (GenBank No. NM_205183.2 for ESR1 and NM_204794.2 for ESR2 gene) were accomplished using the BLAST software provided by the NCBI server. Following confirmation of primer specificity, whereby the sizes of the PCR amplicons corresponded to the expected sequences of the candidate genes (Table 1), the cloned PCR products were identified by aligning with the direct genomic PCR products from the same chicken. No more than two allelic sequences were observed for all examined birds, confirming that the primer pairs specifically amplified the targeted genes.

Genotyping by PCR-SSCP and reconstruction of combined genotypes
Genotyping results obtained by PCR-SSCP analysis revealed that the TT and CT genotypes were classified for the ESR1 genomic fragment (Fig. 1), and the GG and GA genotypes, for the ESR2 sequence within the Dagu hen population (Fig. 2). The combined genotype was reconstruction based on these genotype data, and four combined genotypes (H1, H2, H3 and H4) were determined among the 360 birds sampled ( Table 6). The combined genotype present at the highest frequency was H1 (TTGG; 0.56), with H2 (TTGA) being the next most frequent (0.27), followed by H3 (CTGG; 0.14) and H4 (CTGA; 0.03).

Polymorphism of the target sequences
Two single nucleotide polymorphisms (SNPs) were identified, corresponding to the PCR-SSCP banding patterns of the ESR1 and ESR2 genes following sequence alignment. In the ESR1 gene, a T/C transition at nucleotide position 1,101 of exon 4 was found and named SNP T1101C. However, this T/C transition leads to a synonymous substitution (alanine 301 to alanine). For this SNP, the hens sampled were typed as either TT or CT based upon the SSCP banding pattern. In the ESR2 gene fragment, a G/A transition at nucleotide position 1,755 was detected in exon 8 and named SNP G1755A. This transition results in a non-synonymous substitution of valine 459 to isoleucine with birds categorized as having GG and GA genotypes following PCR-SSCP analysis.

Frequencies of genotype and alleles at the SNP locus
Genotype and allelic frequencies of SNPs at the ESR1 and ESR2 loci in the Dagu chicken population are shown in Table 2. At SNP T1101C of the ESR1 gene (designated as the E1 locus), the frequency of allele T was significantly higher than that of allele C, with the frequency of genotype TT being higher than that of genotype CT in this population. At SNP G1755A of the ESR2 (E2 locus), the frequency of allele G was higher than that of allele A, with the frequency of genotype GG being higher than that of genotype GA. Polymorphisms at both SNP loci were evaluated and were found to exhibit significant genetic disequilibrium between the T and C alleles of ESR1 and the G and A alleles of ESR2 (P<0.05).
As shown in Table 3, gene homozygosity (Ho) was higher than gene heterozygosity (He) for both E1 locus and E2 locus, with

Association of the SNP genotypes with laying performance and the predicted SNP genotype effects
Results of the association analysis between SNPs and egg production traits are shown in Table 4. The SNP T1101C genotype TT was significantly associated with higher HHEP at 30, 43, 57 and 66 weeks of age (P<0.05), and with the lower EW at 30 weeks (P<0.05). Moreover, the SNP G1755A genotype GG was also markedly associated with the higher HHEP at 30, 43, 57 and 66 weeks of age (P<0.05), and with the smaller EW at 30 weeks (P<0.05). Furthermore, a large percentage of the additive variance was explained by the SNP for its significant association with the traits (>1%), respectively (Table 5). However, no significant difference was observed between the TT and CT or between the GG and GA genotypes regarding the BW at 30 and 43 weeks (P>0.05) or EW at 43 weeks (P>0.05).
As shown in Table 6, the combined genotype H1 was found to be significantly correlated with the highest HHEP at 30, 43, 57 and 66 weeks of age (P<0.05), but with lower BW and EW at 30 and 43 weeks (P<0.05). In contrast, the H2 type was notably associated with the higher HHEP at 30, 43, 57 and 66 weeks of age (P<0.05), and with the smaller EW at 30 and 43 weeks (P<0.05). However, H4 was found to be significantly associated with the lowest HHEP at 30, 43, 57 and 66 weeks of age (P<0.05), and with higher BW and EW at 30 and 43 weeks (P<0.05).

dISCUSSIoN
Egg-laying performance is one of the most important economic traits in the poultry industry; improvement in egg production traits is a key goal of breeding programs and has been attracting increasing interest. However, most of the egg production traits in chicken, are inherited polygenically, with low to moderate heritability [5,30,43]; moreover, they obtained only from sexually mature females. This makes genetic improvements in males more difficult to estimate using traditional methods, which depend on a breeding value assessment. To explore methods that are more effective than the traditional breeding approach, many SNP detection techniques, including the PCP-SSCP genotyping analysis, have been established and are currently utilized. Nevertheless, because  egg production traits are polygenically inherited, more associated target genes and favorable alleles are required for egg-laying improvement. Therefore, the ESR1 and ESR2 genes were screened and used to study genetic associations with egg production traits.
To explore the potential association between the ESR1 and ESR2 genes with the egg production traits, including BW, HHEP and EW, we first examined SNPs in the coding region of the ESR1 and ESR2 genes in the Chinese Dagu chicken population, in which the main breeding goal focused on HHEP trait improvement. In the present study, two SNPs in the chicken ESR1 and ESR2 fragments were identified; the frequency of allele T at the E1 locus was higher than that of allele C, and the frequency of genotype TT was higher than that of genotype CT. Moreover, genotype CC was missing at the E1 locus. For the E2 locus, the frequency of allele G was significantly higher than that of allele A and the frequency of genotype GG was higher than that of genotype GA, and the homologous AA genotype was missing. This phenomenon may be explained by several factors: (i) alleles T at E1 or G at E2 may be tightly linked with either an advantageous allele or with an artificially selected economically favorable trait, such as a higher HHEP and/or EW, as demonstrated above. As a result, the levels of homozygotes with genotype TT or GG were either promoted under natural selection pressures or were artificially selected for favorable agricultural attributes; (ii) both the SNPs were confirmed to be under genetic disequilibrium in this study. This was due to either allele T or G being predominant under artificial and/or natural genetic selection pressure, thereby becoming more common than other alleles in this population; (iii) the egg production traits of the local Dagu populations sampled in this study have been developed with the aim of enhancing early sexual maturity and egg laying number traits for the last six generations. During Dagu chicken breeding, the T or G alleles may be coincidently linked with one or more of the selected breeding traits, thereby presenting a possible explanation for the higher allelic frequencies; (iv) the number of birds examined in each population may have been insufficient to demonstrate the true event, and therefore, an extreme allele frequency was estimated as a result.
Previous studies have demonstrated that N e and PIC are important genetic parameters that indicate the level of intra-population genetic variation [1,29]. The results of the present study show that the two SNPs exhibited low polymorphism, with the mean PIC value being lower than 0.25; however, allele homozygosity was higher than 0.7, indicating that the predominant allele had been subjected to selection pressure. Continuous selection aiming to obtain a favorable trait may lead to an increase in allelic frequencies; simultaneously, it brought about Hardy-Weinberg disequilibrium at the linked allele locus. This result may be partially explained by the following data on the trait association analyses.
Age at first egg is a critical factor for egg-laying performance in poultry. For the Dagu hens sampled here, the average age at first egg was 30 weeks, indicating the sexual maturity of the entire chicken population. Therefore, the BW, HHEP and EW in 30-week-old Dagu hens were qualified in this study. Based upon the genotype-based association analysis, the newly identified TT genotype at the E1 locus and GG genotype at the E2 locus were found to be significantly associated with higher HHEP at 30, 43, 57 and 66 weeks of age, and with the smaller EW at 30 weeks of age under a study (P<0.05), respectively. This was coincident with the negative correlation observed between HHEP and EW. Furthermore, to analyze the genetic effects of the SNPs on the associated traits, including HHEP and EW, and considering that additive variance is an important component of genetic variance in the expression of traits related to egg production [3,5,17], the percentage of additive genetic variance explained by the SNPs was estimated in this study. The results of the present study indicated that a larger proportion of variance was explained by these markers (>1%), especially for the phenotypes including HHEP at 43, 57 and 66 weeks of age by the ESR1 locus and HHEP at 66 weeks of age by the ESR2 locus. Although a relatively smaller proportion of variance (<1%) was explained by these markers for the phenotypes, including the HHEP and EW at 30 weeks of age, the effects of allele substitution on the egg production traits were not neglected.
In the present study, four combined genotypes (H1-H4) were detected, and the association analysis of combined genotypes showed that ESR1 and ESR2 polymorphisms are significantly associated with egg production traits in Dagu chickens. Moreover, the results of the combined genotype-based association analysis were consistent with the significant effect detected by the genotype-based association analysis. The combined genotype H1, which carried the favorable T and G SNP alleles, was significantly associated with the highest HHEP at 30, 43, 57 and 66 weeks of age, and with the lower EW at 30 and 43 weeks of age (P<0.05). Interestingly, each of the alleles (T and G) was coincidentally found to be predominant at the locus; this may be due to the artificial selection for favorable HHEP traits in the last six generations. Collectively, these results support a strong association between the two variations in ESR1 and ESR2 with the egg production traits, via some functional genetic mechanisms.
The biological actions of estrogens are mediated via two distinct intranuclear estrogen receptor proteins, ERα and ERβ [11,14]. The carboxyl-terminal ligand-binding domains (LBDs) of ERs are conserved, as suggested by the similar affinities of the two ERs for 17β-estradiol [25], which is encoded by exon 8 of the ER gene. The amino-terminal domain of ERβ is shorter than that of ERα, but is well conserved between rat, mouse and human ERβ, suggesting an evolutionary constraint and a functional importance [14]. A single Tyr 443-to-Asn substitution within LBD leads to changes in the dimerization and transcription activation functions of ERβ [41,49]. In the present study, SNP G1755A of the ESR2 gene resulted in a Val 459-to-Ile mutation within the LBD and was found to be associated with variation in egg production traits, indicating that changes in the LBD of ERβ may directly affect the folliculogenesis or follicular growth and development in the ovaries of chicken, and consequently, influence phenotype (egg production traits). This SNP within the ESR2 gene might represent a promising molecular marker for the early selection of individuals or families with favorable phenotypes in chicken breeding.
In addition, variations in the ESR1 genes have been associated with the natural defense of eggs against bacterial penetration by increasing cuticle deposition in chickens [4]. These findings are useful for the further investigation of possible genetic effects of ESR1 and ESR2 gene polymorphisms, and isotype-selective ESR agonists on the physiology of folliculogenesis, follicular growth and ovarian functional transformation [20]. Additionally, in some cases, these variations indicate abnormal estrogen secretion or deficiency [37]. In recent years, accumulating evidence has indicated that the role of estrogen receptor sequence variants is associated with bone mineral density and affects estradiol levels [15,24,31]. This increases the risk of HBV-related acute liver failure [45] and endometrial cancer in humans [2]. Hence, the results of the present study are also likely to be of considerable veterinary, clinical and biological importance.
Taken together, our data reinforce the conclusion that the two sequence variations in the ESR1 and ESR2 genes are significantly associated with HHEP and EW in Chinese Dagu chickens, and represent promising genetic markers for the early selection and prediction of advantageous phenotypes in chicken breeding programs. Additionally, these data also suggest that polymorphisms of the ESR1 and ESR2 genes might be further explored as indicators for relevant diseases or for the occurrence of ovarian dysfunction.


s2fieldsofstudy:
Biology
Medicine
Materials Science

text:
Adhesion, proliferation and osteogenic differentiation of mesenchymal stem cells in 3D printed poly-ε-caprolactone/hydroxyapatite scaffolds combined with bone marrow clots

Mesenchymal stem cells (MSCs), a stem cell population capable of multi-lineage differentiation, bound to porous biomaterial scaffolds, are widely used for bone tissue regeneration. However, there is evidence to suggest that MSC collection from bone marrow and expansion in vitro may result in phenotypic changes including a loss of differentiation potential and cell senescence. The aim of the present study was to find a facile and efficient approach to enable MSC adhesion and proliferation to scaffolds with osteogenic differentiation. Unprocessed bone marrow blood from the condyle of the distal femur in the rabbits were added to three-dimensional (3D) printed porous poly-ε-caprolactone/hydroxyapatite (PCL/HA) scaffolds with bone marrow clots (MC) formed, using two different methods for Group A (MC enriched scaffolds) and Group B (MC combined scaffolds), and then were cultured in osteogenic medium for 4 weeks. The scaffolds were assessed macroscopically and microscopically. Scaffold bioactivity and the proliferation and osteogenic differentiation of seeded MSCs were measured. Higher cellular viability and greater cell numbers in the scaffolds at later phases of culture were observed in Group B compared with Group A. In addition, Group B was associated with greater osteoinductivity, alkaline phosphatase activity and bony nodule formation, as assessed using scanning electron microscopy. Furthermore, reverse transcription-quantitative polymerase chain reaction analysis revealed that more osteogenic differentiation was present in Group B, compared with Group A. MC combined scaffolds proved to be a highly efficient, reliable and simple novel method for MSC adhesion, proliferation and differentiation. The MC combined PCL-HA multi-scale porosity scaffold may represent a candidate for future bone regeneration studies.

Introduction
Mesenchymal stem cells (MSCs) are of particular interest for therapeutic applications in tissue engineering, as they can differentiate into a number of lineages including chondrocytes, adipocytes and osteoblasts (1). MSCs exist in very low numbers in the highly cellular and heterogeneous bone marrow, and lack unique identifying markers necessary for definitive isolation. Standard protocols for the isolation of MSCs from the bone marrow include extraction of whole bone marrow, density gradient centrifugation and expansion of the mononuclear cell fraction on tissue culture polystyrene. While this protocol is well accepted for the use of MSCs, there is evidence to suggest that this expansion phase may result in phenotypic changes in MSCs, including a loss of differentiation potential and cell senescence (2,3). The use of biomaterials for the incorporation and expansion of MSCs in a three-dimensional (3D) environment is of growing interest in the field, with several successful attempts to expand MSCs three-dimensionally with the use of micro carriers under dynamic conditions (4)(5)(6). However, these strategies do not eliminate the need for initial culture on plastic.
Marrow clots (MCs), formed by unprocessed bone marrow blood (UBMB) agglutination, provided a simple, convenient and relatively inexpensive biomimetic approach for tissue regeneration through MSC delivery and microenvironment modification inside 3D scaffolds (7,8). Our previous study demonstrated that MCs enriched with scaffolds exhibit improved biological performance in inductive culture compared with conventional MSC-seeded scaffolds. However, the MC-mediated obstruction of pores can block the transfer of nutrients and prevent stem cell homing inside 3D printed (3DP) scaffolds, and during the culture of such scaffolds, MSCs were easily washed out when changing the medium (9). A facile and efficient MC concentration technique for 3DP scaffolds needs to be identified as an optimal procedure for tissue regeneration.
Previous studies have demonstrated that poly-ε-caprolactone (PCL), a biodegradable semi-crystalline linear aliphatic polyester, exhibits good mechanical properties (10). The Food and Drug Administration has approved this material for use as a drug delivery device and suture staple, and it has been extensively investigated as a biomaterial for regenerative medicine (11,12). Hydroxyapatite (HA) has been widely investigated as an osteoconductive and inductive biomaterial for use as a porous bone substitute. PCL and HA have been blended to enhance cell proliferation and differentiation (13,14). PCL-HA scaffolds with a precisely-controlled macroarchitecture and microstructure can be fabricated using a 3DP technique for bone regeneration (15,16).
Furthermore, PCL-HA scaffolds produced by 3DP with suitable MC concentration may serve an important role in bone tissue engineering research. To address these issues, the present study involved preparation of 3DP PCL-HA scaffolds with two different MC concentration procedures, based on our previous findings (9). The MSC adhesion, proliferation and osteogenesis performance, cultured in osteogenic differentiation medium in vitro, was examined to evaluate the availability of MC concentration 3DP PCL-HA scaffolds for bone regeneration.

Materials and methods
Animals. Female New Zealand white rabbits (n=10; age, 5-6 months old; weight, 2.0-2.5 kg; Animal Core Facility, Nanjing Medical University, Nanjing, Jiangsu, China) were used for bone marrow blood collection using a micro-fracture procedure. Prior to the experiment, all rabbits were housed at room temperature (25˚C), 60% relative humidity and with a 12-h light/dark cycle for 1 week; all animals had free access to food and water. The use of animals in the present study was approved by the Institutional Animal Experiment Committee of Nanjing Medical University (Nanjing, China), and animals were treated according to the US National Institute of Health guidelines (National Institutes of Health, Bethesda, MA, USA). All animals underwent a veterinary examination to evaluate their general health status. All experimental procedures for bone marrow blood collection were performed under anesthesia with ear vein administration of 2% pentobarbital sodium (30 mg/kg; Sigma; Merck KGaA, Darmstadt, Germany).
Scaffold design and fabrication. 3DP PCL-HA porous scaffolds were designed and manufactured as detailed in the following section. The scaffolds were 4 mm in diameter, 2 mm in thickness, had 100% pore interconnectivity and a 500-µm fiber diameter with a 0/45˚/90˚/135˚ laydown pattern of fibers for porous structure following a previously published protocol (17). In brief, PCL powder (molecular weight ~60,000, 3D Biotek, LLC, North Brunswick, NJ, USA) and HA (Plasma Biotal Ltd., Buxton, UK) were dried separately for 24 h in a vacuum oven at 120˚C and 40˚C, respectively. All chemicals used were of pharmaceutical grade, and the detailed characterization of these materials is previously described (18,19). The scaffolds were fabricated using a 60% PCL/HA (by weight) (20) composite and a 3D fused deposition modelling (FDM) micro-fabrication technology (FDM 700 system; Nanjing Songsun Medical Technology Co, Ltd., Nanjing, Jiangsu, China) according to the manufacturer's protocol. Scaffolds (n=80/group) were fabricated with 10 sampled at each time point (day 1, weeks 1, 2 and 4 of in vitro culture; Fig. 1).
Preparation of MC concentration PCL-HA scaffold. A total of 10 rabbits were randomly divided into two groups (n=5 rabbits/group) and used for subsequent experiments. UBMB collection was performed using a micro-fracture procedure bilaterally on the condyle of the distal femur in the rabbits, as previously described (7). The amount of seeded MSCs was calculated following the protocol in our previous study (7,21). For the MC enriched group (Group A), the scaffold was immersed and mixed in UBMB retrieved from one rabbit which contained heparin to prevent blood coagulation during the micro-fracture procedure. In brief, the 3DP scaffolds were immersed in UBMB for 1 h without agitation until they were fully enriched with MC, and were then incubated at 37˚C in a 5% CO 2 humidified incubator in L-Dulbecco's modified Eagle's medium (L-DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 4 days, with the medium changed every 1-2 days until clear. For the MC combined group (Group B), the scaffold was plugged into the micro-fracture hole at the condyle of the distal femur until full of UBMB, and the MC formed. The scaffolds were then cultured in L-DMEM in the same manner as those in Group A (Fig. 2). Then two groups were cultured in osteogenic medium (Gibco; Thermo Fisher Scientific Inc.), supplemented with high-glucose DMEM, 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin-streptomycin, 10 mmol/l β-glycerophosphate, 50 µM ascorbic acid and 100 nM dexamethasone, for 28 days. A total of 10 samples were tested at each time point for each group at day 1 and at weeks 1, 2 and 4 of in vitro culture.
Macro and micro-morphology observation. The macro morphology of the cultured scaffolds was observed using an inverted microscope (IM) and a digital camera. The micro-structural morphology was observed using a scanning electron microscope (SEM; JEOL, Ltd., Tokyo, Japan), as in our previous study (9). In brief, specimens were fixed in 2.5% glutaraldehyde overnight, dehydrated using a series of graded ethanol solutions, dried overnight at room temperature and gold sputtered. SEM observation was then performed at an accelerating voltage of 5 keV.
Cell viability and proliferation assessment. Cell viability was assessed using a Live/Dead Reduced Biohazard Viability/Cytotoxicity kit (Molecular Probes; Thermo Fisher Scientific, Inc.) as in our previous study (9). Samples from each group were assessed at day 1, 14 and 28. In brief, samples were washed using phosphate buffered saline, incubated in the dilute dye solution for 15 min in the dark at room temperature and then fixed in 4% glutaraldehyde for 1 h. These samples were observed and imaged using a confocal microscope (Leica T7100M Confocal Microscope, Leica Microsystems GmbH, Wetzlar, Germany). Live cells were stained green and dead cells were stained red.
A MTT cell proliferation assay kit (Roche Applied Science, Penzburg, Germany) was used to assess proliferation of the cells on the scaffolds, following the protocol supplied by the manufacturer. After 1, 7, 14 and 28 days of cell culture, 20 µl MTT reagent was added to each well of the microtiter plates containing the scaffolds, and cells were incubated for 4 h at 37˚C. After 200 µl solubilization solution (DMSO; Roche Diagnostics, Indianapolis, IN, USA) was added to each well, the plates were incubated overnight. The absorbance was measured at 595 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Osteogenic differentiation analyses of the scaffolds. The intracellular alkaline phosphatase (ALP) activity on the cultured scaffolds was compared between the two groups on days 0, 7, 14 and 28 to estimate cell differentiation. Cell lysates were tested for ALP activity using a SensoLyte™ pNPP Alkaline Phosphatase Assay kit (AnaSpec, Fremont, CA, USA) according the manufacturer's protocol. The absorbance was measured at 415 nm using a microplate reader (Bio-Rad Laboratories, Inc.) (22).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of osteogenic genes on days 14 and 28 were measured using RT-qPCR. The scaffolds were lysed using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). The samples in TRIzol were incubated at -80˚C until RNA isolation. Total RNA was isolated and reverse transcribed to cDNA using a High-Capacity cDNA Reverse Transcription  Marrow clots blended with a poly-ε-caprolactone/hydroxyapatite scaffold using two different procedures. In Group A, the scaffold was immersed in unprocessed bone marrow blood for 1 h without agitation until MC were obtained in each structure. In Group B, the scaffold was plugged into a micro-fracture hole at the condyle of the distal femur until full with unprocessed bone marrow blood and a MC formed. The scaffolds were cultured in vitro and 10 samples were analyzed at day 1, and 1, 2 and 4-week time-points. MC, marrow clots. kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed for the quantification of gene expression using the primers listed in Table I in an ABI Prism 7500 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The target genes osteopontin (OPN), osteocalcin (OCN) and core binding factor α1 (Cbfα1), were normalized against GAPDH expression. Each 20 µl reaction mix contained 10 µl 2X LightCycler ® 480 SYBR Green I Master (Roche Diagnostics, Basel, Switzerland), 10 ng modified DNA and 0.8 µl primer mix (10 pM/µl). qPCR was performed using a LightCycler ® 480 with the following cycling conditions: 95˚C of initial denaturation for 10 min followed by 40 cycles of denaturation at 95˚C for 15 sec, annealing at 60˚C for 1 min, annealing at 62˚C for 10 sec, then polymerization at 72˚C for 20 sec. A melting curve was created by cooling the products at 50˚C for 30 sec and then heating to 80˚C at a rate of 0.1˚C/sec, while simultaneously measuring the fluorescence. Data were analyzed via the comparative quantitation cycle (Cq) method (23). ΔCq values were first calculated using the formula: ΔCq=Cq Target gene -Cq GAPDH ; then the mean level of target gene (2 -ΔCq ) in all samples was determined.
Histology staining. For Alizarin Red staining, cells on scaffolds were fixed using 3.7% formaldehyde for 24 h at 37˚C and then stained with Alizarin Red S (Nanjing KeygGen Biotech Co., Ltd., Nanjing, China) for 15 min at 25˚C. Scaffolds were washed five times using deionized water to remove excess stain and then air-dried. The morphology of the stained scaffolds was assessed using a digital camera.
Statistical analysis. All statistical analysis was performed using SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. The difference between the groups was analyzed by one-way analysis of variance and a Student-Newman-Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results
Scaffold characterization. The fabricated PCL-HA scaffolds, a 100% pore interconnectivity and 500 µm fiber diameter with a 0/45˚/90˚/135˚ laydown pattern of fibers for porous structure cylinder with a 4-mm diameter and a 2-mm height, matched the design parameters as assessed through IM and SEM observations. The gross scaffold morphology and cross sectional view of the composite are presented in Fig. 1.
Observation of macro and micro-structure of the scaffold. SEM imaging revealed that the pores of the scaffolds were filled and obstructed by fibroblast-like tissue, monocytes and erythrocytes at day 1 in Groups A and B (Fig. 3). Further observation of the scaffold morphology revealed that most erythrocytes and UBMB matrix inside the scaffold pores were washed off after 1 week in normal culture medium in both groups. There was a decrease in the red coloration of the scaffolds, which was more pronounced in scaffolds in Group A. As time progressed, a gradual accumulation of extracellular matrix (ECM) was observed in both groups. Meanwhile, the density of the scaffold content gradually increased with a subsequent increase of the matrix in the pores during the culture period, as assessed using IM observations (data not shown).
Cell adhesion and proliferation. A cell viability assessment using confocal imaging of live/dead assay samples demonstrated that green-stained live cells in Group B were apparently increased at the 4-week in vitro time-point, compared with those in Group A. The pore obstruction and recanalization could also be observed in the confocal images (Fig. 4A). Cell proliferation on the scaffolds was assessed using an MTT assay (Fig. 4B). The optical density (OD) values were similar in the two groups during the first 14 days of culture. At day 28, the OD values were higher in Group B. The ratio of living cells was the same as the MTT values. Therefore, the MC combined scaffold was considered to be more effective for cell adhesion and proliferation.
Biochemical analyses of the scaffolds. ALP activity was measured to examine the osteogenic differentiation of MSCs on the scaffolds (Fig. 5A). ALP activity gradually increased with time. The ALP activity of Group B was significantly greater compared with Group A, compared at day 28 (P<0.05). SEM images of the scaffolds at day 28 (Fig. 5B) demonstrate higher mineralization in the Group B scaffolds, which corroborates with the ALP activity results. The expression levels of osteogenic genes on days 14 and 28 were measured using RT-qPCR (Fig. 5C). Group B demonstrated a higher mean expression of Cbfα1 compared with Group A on day 14. However, Cbfα1 expression in Group B on day 28 was lower than in Group A. The mRNA expression levels of OPN and OCN in both Group A and B gradually increased during in vitro culture, with the level of expression in Group B being significantly higher than in Group A ( Fig. 5C; P<0.05).
Alizarin Red S staining. When cultured in the osteogenic supplements, MSCs in both groups calcified their matrix by day 28 (Fig. 5D). Calcification is a standard marker for osteogenesis and osteogenic differentiation, and can be assessed using Alizarin Red S staining. The staining in Group B was visibly darker than in Group A. Controls using empty scaffolds without MSCs did not mineralize, indicating that the Table I. Primer nucleotide sequences for RT-PCR.

Discussion
Bone marrow cell systems include both non-hematopoietic stromal cells and hematopoietic cells. The stromal functions as a scaffold, composed of a network of cells that provide physical and functional support to the hematopoietic cells. The stromal fraction, characteristically, is able to adhere to tissue culture plastic, while the non-adherent hematopoietic cells can be readily removed from the adherent stromal cell cultures using a simple wash step (24). From the SEM and live/dead confocal images of the two groups, the present study demonstrated that the pores of the scaffolds were filled with cells and erythrocytes from the MC at day 1. During the following culture, erythrocytes and other non-adherent hematopoietic cells were gradually eluted. This may have caused the red color to fade and the initially observed decrease in the live/dead cell ratio. An increase in the live/dead cell ratio was observed in both groups at the 2-week time-point, with MSC proliferation. However, in the MC combined PCL-HA scaffolds, gradual cell proliferation was observed over time, which may have caused a higher live/dead cell ratio and a lower cell/ECM ratio during culture, compared with Group A. The present study compared two different methods of MC concentration by observing cell adhesion and proliferation. The results demonstrated more stem cell adhesion and proliferation and improved osteogenic differentiation properties in the scaffold combined with UBMB. In the MC-enriched scaffold group, heparin was used to interfere with the blood clotting process during the micro-fracture procedure. Ultimately, heparin prevented the conversion of fibrinogen into fibrin, resulting in preventing blood coagulation. However, when  clotting was not impaired, the fibrin formed bridge shaped structures both along the rods and across the pores of the scaffolds combined with BMB clots, thereby promoting cell adhesion in these scaffolds.
Previous investigations of 3D fabricated porous scaffolds have demonstrated that the formation of new tissues is greatly influenced by the pore size, porosity and the 3D structure of the scaffold (11,25). Synthetic scaffolds for bone regeneration must be a 3D structure with interconnected porosity and appropriate pore sizes to assure good vascularization, nutrient delivery and cell attachment in growth (26)(27)(28). A large pore size is necessary to deliver a sufficient number of cells, and interconnected porosity is required for the easy diffusion of nutrients. Previous studies have suggested that a small pore size improves bone growth into scaffolds and provides attachment points for osteoblasts (29). The present study fabricated a PCL/HA scaffold with macro and micro porosity for osteogenic differentiation.
In the present study, the ALP activity in Group B was significantly increased compared with Group A. Similarly, RT-qPCR demonstrated that that the expression of OCN and OPN in Group B was higher than in Group A. Cbfα1 expression was higher in Group B at day 14, but the expression of Cbfα1 in the two groups gradually decreased over time, and the expression in Group A was slightly higher than in Group B at day 28. Cbfα1 inhibits the transition of osteoblasts to osteocytes and thereby maintains osteoblastic cells at an immature stage and effectively directs the formation of immature bone. Normally, the expression of Cbfα1 is first detected in pre-osteoblasts and is upregulated in immature osteoblasts, but downregulated in mature osteoblasts (30,31). The present study demonstrated that Group B exhibited a greater osteogenetic capacity than Group A. This may be because the UBMB concentration method used in Group B had a significant effect on cell adhesion and osteogenesis compared with Group A, which was demonstrated by the cell proliferation assay and the Alizarin Red S staining. Perhaps the different concentration of growth factors in the MC also attribute to the osteogenetic difference, which requires checking in future studies.
There are some limitations to the present study. The difference between the two different coagulation methods in UBMB was not determined. Whether there is a significant change in the blood composition of the bone marrow after the addition of heparin needs to be clarified by further research. Although MC-PCL-HA demonstrated good osteogenic properties in vitro, the performance of the MC-PCL-HA scaffolds in autologous in vivo studies requires further investigation. The composition of the UBMB is complex. Though most of the non-adherent cells were eluted in culture, residual cellular constituents remained in the pores of the scaffold, which is much closer to the autogenous bone repair process. Furthermore, as an autologous procedure, MC may circumvent the barriers encountered in allograft procedures, including immune rejection, pathogen transmission, issues with packaging, storage and shipping, and difficulties in clinical adoption.
In conclusion, MC-combined scaffolds proved to be a highly efficient, reliable and simple novel method for MSC adhesion, proliferation and osteogenic differentiation. The MC-combined PCL-HA multi-scale porosity scaffold can be a candidate for future bone regeneration studies.