text:
Angiogenesis and bleeding disorders in FNAIT.

Angiogenesis is a physiological process essential for embryo/fetal growth, wound healing, and repair of the myocardium after myocardial infarction. Angiogenesis is also implicated in pathological processes such as retinopathy and tumor growth. Anti-angiogenic agents have been demonstrated to have promising therapeutic potential for inhibiting tumour growth. Earlier studies suggested that β3 integrin (i.e. αVβ3 that is formed by β3 subunit and αV subunit) expressed on angiogenic endothelial cells (ECs), is required for angiogenesis [1]; however, subsequent studies demonstrated that β3 integrin deficiency (β3−/−) did not prevent embryo/fetal growth and interestingly, enhanced pathological angiogenesis was observed in β3−/− mice [2]. It is therefore important to further study the roles of β3 integrin in angiogenesis, fetal development and related diseases.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder that occurs when maternal alloantibodies cross the placenta and target paternally derived antigens, especially GPIIbIIIa (integrin αIIbβ3) and GPIbα, on fetal/neonatal platelets. In contrast to αVβ3, integrin αIIbβ3 is composed of a β3 subunit and αIIb subunit, which are almost exclusively expressed on platelets and megakaryocytes. Half of the polymorphisms known to cause FNAIT are located on the β3 subunit. Approximately 80-90% of reported FNAIT cases are caused by antibodies targeting Human-Platelet-Antigen-1a (HPA-1a) on β3 subunit. Antibodies targeting the HPA-2a on GPIbα have also been reported. Intracranial hemorrhage (ICH), which occurs in 10-20% of affected fetuses/neonates, is a major clinical complication of FNAIT, leading to neurological impairment and death. Unfortunately, the mechanism responsible for ICH, has only been inferred but not adequately explored.

Thrombocytopenia was considered to be the cause of bleeding in FNAIT. Interestingly, mice deficient in transcription factor NF-E2, which lack circulating platelets, did not develop significant bleeding disorders in utero. More strikingly, the murine fetuses with combined deficiencies in NF-E2 and fibrinogen genes exhibited normal embryonic development and were morphologically indistinguishable from their wild-type control counterparts at 18.5 days postcoitum [3]. Therefore, it is likely that neither thrombocytopenia nor blood coagulation are crucial for the development of ICH in FNAIT. Furthermore, several animal models have demonstrated that impairment of angiogenesis may contribute to bleeding in fetuses, particularly in their brains, since the brain is one of the major sites of angiogenesis during fetal development. However, whether ICH in FNAIT is caused by impairment of angiogenesis induced by maternal anti-platelet antibodies, is unclear. This is particularly interesting since the roles of β3 integrin in angiogenesis are still controversial.

In a recent publication [4], we reported both active and passive murine models of FNAIT using β3−/− and GPIbα−/− mice, in which gene deficient female mice were bred with wild-type males to mimic human FNAIT. We found that both maternal anti-β3 and anti-GPIbα antibodies caused severe thrombocytopenia, however, ICH was only observed in anti-β3-mediated FNAIT fetuses/neonates (active model), and only anti-β3 sera-injected neonates (passive model) developed ICH. Moreover, we found that anti-β3 but not anti-GPIbα antibodies induced EC apoptosis, decreased the vessel density in the brains, and impaired the retinal vascular development. The anti-β3 antibodies also decreased the expression of several pro-angiogenic factors, increased the expression of anti-angiogenic factors, and down-regulated Akt signalling. The antibodies also inhibited proliferation and network formation of human umbilical vein endothelial cells (HUVECs) in vitro. Importantly, anti-HPA-1a IgG purified from mothers who gave birth to neonates with FNAIT, also showed similar effects on HUVECs in vitro. This study suggests that anti-platelet β3 antibodies also cross-react with αVβ3 on ECs, thereby impairing angiogenesis and contributing to ICH. The impairment of retinal vascular development may explain why some FNAIT untreated siblings suffered visual impairment in adulthood. Our study may also provide insights into the mechanisms of intrauterine growth restriction (IUGR) in FNAIT as well as the possible pathogenic role of αVβ3 integrin in tumorigenesis.

Clinical management of such devastating bleeding diatheses in fetuses/neonate remains a formidable challenge. Since our findings indicate that impairment of angiogenesis but not thrombocytopenia may be the major cause of ICH, invasive fetal platelet transfusion may not be an effective prenatal therapy. Our data demonstrated that antenatal administration of intravenous immunoglobulin (IVIG) decreased EC apoptosis, rescued brain and retinal vessel development, increased platelet counts, and ameliorated ICH, suggesting that IVIG may be an effective therapy [4]. In addition, our previous studies demonstrated that fetal (but not maternal) neonatal Fc receptor (FcRn) is required for IgG trans-placental transport [6-7]. Whether anti-FcRn is a more effective and economical therapy for FNAIT requires further clinical studies.

Aside from its expression on platelets and ECs, β3 integrin is also expressed on highly invasive trophoblasts, which are essential for normal placental vascular remodeling and development. Therefore, further studies on the effects of anti-β3 antibodies on trophoblast proliferation, differentiation, migration, and invasion, may provide insights into the mechanism of IUGR and miscarriage in FNAIT, as well as aid in the development of new therapies to manage these disorders.

Issaka Yougbare, Darko Zdravic and Heyu Ni
Angiogenesis is a physiological process essential for embryo/fetal growth, wound healing, and repair of the myocardium after myocardial infarction. Angiogenesis is also implicated in pathological processes such as retinopathy and tumor growth. Anti-angiogenic agents have been demonstrated to have promising therapeutic potential for inhibiting tumour growth. Earlier studies suggested that β3 integrin (i.e. αVβ3 that is formed by β3 subunit and αV subunit) expressed on angiogenic endothelial cells (ECs), is required for angiogenesis [1]; however, subsequent studies demonstrated that β3 integrin deficiency (β3 -/-) did not prevent embryo/fetal growth and interestingly, enhanced pathological angiogenesis was observed in β3 -/mice [2]. It is therefore important to further study the roles of β3 integrin in angiogenesis, fetal development and related diseases.
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder that occurs when maternal alloantibodies cross the placenta and target paternally derived antigens, especially GPIIbIIIa (integrin αIIbβ3) and GPIbα, on fetal/neonatal platelets. In contrast to αVβ3, integrin αIIbβ3 is composed of a β3 subunit and αIIb subunit, which are almost exclusively expressed on platelets and megakaryocytes. Half of the polymorphisms known to cause FNAIT are located on the β3 subunit. Approximately 80-90% of reported FNAIT cases are caused by antibodies targeting Human-Platelet-Antigen-1a (HPA-1a) on β3 subunit. Antibodies targeting the HPA-2a on GPIbα have also been reported. Intracranial hemorrhage (ICH), which occurs in 10-20% of affected fetuses/neonates, is a major clinical complication of FNAIT, leading to neurological impairment and death. Unfortunately, the mechanism responsible for ICH, has only been inferred but not adequately explored.
Thrombocytopenia was considered to be the cause of bleeding in FNAIT. Interestingly, mice deficient in transcription factor NF-E2, which lack circulating platelets, did not develop significant bleeding disorders in utero. More strikingly, the murine fetuses with combined deficiencies in NF-E2 and fibrinogen genes exhibited normal embryonic development and were morphologically indistinguishable from their wild-type control counterparts at 18.5 days postcoitum [3]. Therefore, it is likely that neither thrombocytopenia nor blood coagulation are crucial for the development of ICH in FNAIT. Furthermore, several animal models have demonstrated that impairment of angiogenesis may contribute to bleeding in fetuses, particularly in their brains, since the brain is one of the major sites of angiogenesis during fetal development. However, whether ICH in FNAIT is caused by impairment of angiogenesis induced by maternal anti-platelet antibodies, is unclear. This is particularly interesting since the roles of β3 integrin in angiogenesis are still controversial.
In a recent publication [4], we reported both active and passive murine models of FNAIT using β3 -/and GPIbα -/mice, in which gene deficient female mice were bred with wild-type males to mimic human FNAIT. We found that both maternal anti-β3 and anti-GPIbα antibodies caused severe thrombocytopenia, however, ICH was only observed in anti-β3-mediated FNAIT fetuses/ neonates (active model), and only anti-β3 sera-injected neonates (passive model) developed ICH. Moreover, we found that anti-β3 but not anti-GPIbα antibodies induced EC apoptosis, decreased the vessel density in the brains, and impaired the retinal vascular development. The anti-β3 antibodies also decreased the expression of several pro-angiogenic factors, increased the expression of anti-angiogenic factors, and down-regulated Akt signalling. The antibodies also inhibited proliferation and network formation of human umbilical vein endothelial cells (HUVECs) in vitro. Importantly, anti-HPA-1a IgG purified from mothers who gave birth to neonates with FNAIT, also showed similar effects on HUVECs in vitro. This study suggests that anti-platelet β3 antibodies also cross-react with αVβ3 on ECs, thereby impairing angiogenesis and contributing to ICH. The impairment of retinal vascular development may explain why some FNAIT untreated siblings suffered visual impairment in adulthood. Our study may also provide insights into the mechanisms of intrauterine growth restriction (IUGR) in FNAIT as well as the possible pathogenic role of αVβ3 integrin in tumorigenesis.
Clinical management of such devastating bleeding diatheses in fetuses/neonate remains a formidable challenge. Since our findings indicate that impairment of angiogenesis but not thrombocytopenia may be the major cause of ICH, invasive fetal platelet transfusion may not be an effective prenatal therapy. Our data demonstrated that antenatal administration of intravenous immunoglobulin (IVIG) decreased EC apoptosis, rescued brain and retinal vessel development, increased platelet counts, and ameliorated ICH, suggesting that IVIG may be an effective therapy [4]. In addition, our previous studies demonstrated that fetal (but not maternal) neonatal Fc receptor (FcRn) is required for IgG trans-placental transport [6][7]. Whether Editorial www.impactjournals.com/oncotarget anti-FcRn is a more effective and economical therapy for FNAIT requires further clinical studies.
Aside from its expression on platelets and ECs, β3 integrin is also expressed on highly invasive trophoblasts, which are essential for normal placental vascular remodeling and development. Therefore, further studies on the effects of anti-β3 antibodies on trophoblast proliferation, differentiation, migration, and invasion, may provide insights into the mechanism of IUGR and miscarriage in FNAIT, as well as aid in the development of new therapies to manage these disorders.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy:
Biology
Medicine
Agricultural And Food Sciences

text:
Probiotic potential of Enterococcus faecalis strains isolated from meconium

107 bacterial isolates with Gram positive staining and negative catalase activity, presumably assumed as lactic acid bacteria, were isolated from samples of meconium of 6 donors at Roubaix hospital, in the north of France. All these bacterial isolates were identified by MALDI-TOF mass spectrometry as Enterococcus faecalis. However, only six isolates among which E. faecalis 14, E. faecalis 28, E. faecalis 90, E. faecalis 97, and E. faecalis 101 (obtained from donor 3), and E. faecalis 93 (obtained from donor 5) were active against some Gram-negative bacteria and Gram-positive bacteria , through production of lactic acid, and bacteriocin like inhibitory substances. The identification of these isolates was confirmed by 16rDNA sequencing and their genetic relatedness was established by REP-PCR and pulsed field gel electrophoresis methods. Importantly, the aforementioned antagonistic isolates were sensitive to various classes of antibiotics tested, exhibited high scores of coaggregation and hydrophobicity, and were not hemolytic. Taken together, these properties render these strains as potential candidates for probiotic applications.

Introduction
Human microbiome is undergoing dynamic changes in bacterial content in the gut during pregnancy and development of childhood (Dominguez-Bello et al., 2010). In early childhood, the presence of pathogenic species, or absence of beneficial ones, leads to adverse effects such as initiation of preterm birth (DiGiulio et al., 2008), and development of asthma, allergy, and autism (Hong et al., 2010;Johansson et al., 2011;Wang et al., 2011). Microbes colonizing the gastrointestinal tract of the infant are originated from the mother and surrounding environment during the birth and shortly thereafter (Mshvildadze et al., 2010). The fetus, as well as the intrauterine environment, have been considered sterile, but the presence of microbes was reported in amniotic fluid (Hitti et al., 1987), fetal membranes (Steel et al., 2005), and meconium (Gosalbes et al., 2013). Meconium is the first intestinal discharge of newborns, which consists of a viscous, sticky, dark green substance. It might contain materials ingested during time that the infant has spent in the uterus; these could include epithelial cells, mucus, bile acid, blood, lanugo, and amniotic fluid (Cleary and Wiswell, 1998;Gosalbes et al., 2013). Newborns seem to acquire their first microbiota at birth and maternal vaginal or skin bacteria colonize newborns delivered vaginally or by caesarian section (C-section; Alicea-Serrano et al., 2013). Hu et al. (2013) confirmed that meconium is not sterile and does contain diversified microbiota. Moles et al. (2013) characterized the microbiota from meconium and fecal samples obtained during the first 3 weeks of life from 14 donors. They showed that bacilli and other Firmicutes were important in meconium whilst Proteobacteria dominated in the fecal samples based on molecular method. Staphylococcus predominated in meconium while Enterococcus and Gram-negative bacteria (GNB) were more abundant in fecal samples (Moles et al., 2013). Makino et al. (2013) showed that several Bifidobacterium strains transmitted from the mother are colonizing the infant's intestine shortly after birth. Thus, the gut of infant might contain different species such as Enterococci, Bifidobacteria, and Lactobacilli that protect mucus of infants from pathogenic species through production of inhibitory substances including hydrogen peroxide, organic acids, and bacteriocins (Vizoso-Pinto et al., 2006;Rodríguez et al., 2012). Enterococci, particularly Enterococcus faecalis and E. faecium are involved in the reduction or prevention of gastro intestinal tract infections (Franz et al., 2011). Enterococci belong to the group of LAB, which is known to produce lactic acid as the end product of sugar fermentation, and antimicrobials, which are active against pathogens including Staphylococcus aureus and Listeria monocytogenes (Campos et al., 2006). Further, many strains of E. faecalis produce bacteriocins named enterocins, a family of safe (Belguesmia et al., 2011), and ribosomally synthesized antimicrobial peptides (AMP; Drider and Rebuffat, 2011). This study aimed at studying and taking advantage of the LAB isolated from meconium sampled at Roubaix hospital in the North of France.

Materials and Methods
Preparation of Samples, Isolation, and Identification of Lactic acid Bacteria Samples of fresh meconium were collected, in sterile dry plastic containers, from 6 donors (newborn infants), at Roubaix hospital in the North of France. Samples of meconium were stored at 4 • C until to be processed. One gram of meconium was resuspended in 9 ml of 0.9% (w/v) of sterile saline solution, and was serially diluted from 10 −1 to 10 −6 . One ml of each dilution was poured onto Petri dishes and melt MRS (de Man-Rogosa-Sharpe) agar medium (Biokar, France;de Man et al., 1960) was poured gently. After agar solidification at room temperature, plates were incubated under 5% CO 2 at 37 • C for 24-48 h. The grown colonies were Gram-stained and checked for catalase activity. Presumptive LAB strains were selected, maintained at −80 • C in MRS with 25% glycerol as stock culture, until use. The presumptive LAB isolates were identified by the VITEK MS v2.0 MALDI-TOF mass spectrometry according to manufacturer's instructions.

Antimicrobial Activity
The antibacterial activity was assessed against the GNB and Gram-positive bacteria (GPB) listed in Table 1, using the well known agar diffusion test (Naghmouchi et al., 2006). The cell free supernatants (CFS) used for antibacterial activity measurement were obtained by centrifuging (9000 × g, 10 min, 4 • C), overnight cultures of E. faecalis grown at 37 • C for 18-24 h, on MRS broth. Wells were performed in solid agar and 50 μl of each CFS or neutralized cell-free supernatant (NCFS; pH6.5) were poured into the wells. The Petri plates were left at room temperature, in sterile conditions, for 1 h before incubation for 18 h at adequate temperature. After this period of incubation, the antibacterial activity was detected by observing the inhibition zones around the well containing the CFS or NCFS. Importantly, the impacts of temperature and pH variations as well as proteinase K, papain, trypsin, and α-chymotrypsin at 1 mg/ml (Sigma-Aldrich Germany) were determined toward Listeria innocua CIP 103982.
The amplicons for 16S rDNAs were purified with the pJET1Kit (Fermentas, France), and sequenced at Eurofins MWG Operon (Germany). Partial 16S rDNAs sequences were compared to that of E. faecalis ATCC 19433 using ClustalW2 software 1 .
Challenge Tests: Staphylococcus aureus ATCC 33862 vs. Antagonistic E. faecalis Enterococcus faecalis was grown in MRS and S. aureus ATCC33862 in Brain Heart Infusion (BHI; 10 5 CFU/ml) at 37 • C for 18 h. After this period, the cultures had reached 10 8 CFU/ml for E. faecalis, and 10 5 CFU/ml for S. aureus ATCC33862. Afterward, 0.1 ml of each culture was added to 20 ml of BHI containing leading to 5. 10 5 CFU/ml (E. faecalis) and 5. 10 2 CFU/ml (S. aureus ATCC33862). One ml of this coculture was withdrawn at 0, 4, 8, 12, 18, and 24 h, and was serially diluted from 10 −1 to 10 −6 , in 0.9% (w/v) of saline water. Then 1 ml of each dilution was plated on Plates dishes containing MRS agar for E. faecalis, and Chapman agar for S. aureus ATCC33862. The number of colonies was determined after 24 h of incubation at 37 • C.

Lactic Acid Quantification
Lactic acid was quantified by HPLC spectra system P1000XR (Thermo, USA). Isolates E. faecalis 28 and E. faecalis 93 were grown in MRS broth at 37 • C, samples were withdrawn after 0, 4, 8, 12, 18, and 24 h of incubation, centrifuged (10,000 ×x g,  Frontiers in Microbiology | www.frontiersin.org 10 min, 4 • C), and sterilized by filtration using Millipore filter (0.2 μm). The CFS were divided into 3 samples. Sample 1 was used to measure pH, sample 2 to assess the antibacterial activity against S. aureus ATCC33862 by the well diffusion method (Naghmouchi et al., 2006), and sample 3 to determine the concentration of lactic acid. All these experiments were performed in triplicate.

Autoaggregation Assay
Autoaggregation assays were performed according to initial protocol of Del Re et al. (2000) and modified by Al Kassaa et al. (2014). Thus, isolates of interest were cultured on MRS for 18 h at 37 • C, then cells were centrifuged (5,000 × g, 4 • C, 15 min), washed three times with phosphate saline (PBS) buffer (pH7.0) and were resuspended in sterile PBS to obtain a viable cell count of 10 8 CFU/ml. The bacterial suspension of 4 ml was mixed by vortexing for 10 s and incubated at room temperature for different time intervals (0, 1, 2, 3, 4, or 5 h). At each interval, the growth was measured at OD 600 nm . The percentage of autoaggregation was expressed as follows: Where OD 1 represents the optical density at time (1, 2, 3, 4, or 5 h) and OD 2 the data at time 0 h. All experiments were performed in triplicate.

Hydrophobicity
The hydrophobicity was determined using in vitro method to detect the bacterial adhesion to hydrocarbons (Rosenberg et al., 1980). Each antagonistic LAB isolate was grown in MRS broth at 37 • C for 18 h. The bacterial cells harvested by centrifugation (5,000 × g, 4 • C, 15 min) were washed twice with PBS (pH7.0), resuspended in the same solution and the OD 600 nm was determined. One milliliter of xylene (Fluka, Germany) was added to 3 ml of cell suspension and vortexed for 2 min after 10 min of incubation at room temperature. The aqueous phase was removed after 2 h of incubation at room temperature and the OD 600 nm was determined. The percentage of hydrophobicity was calculated using the formula given below. All experiments were performed in triplicate.

Hemolytic Activity
The hemolytic activity was determined by streaking Enterococcal isolates on Columbia blood agar supplemented with 5% (v/v) of human blood or Sheep blood. The plates were incubated at 37 • C for 48 h. The presence or absence of zones of clearing around the colonies was interpreted as β-hemolysis (positive hemolytic activity) or χ -hemolysis (negative hemolytic activity), respectively. When observed, greenish zones around the colonies were interpreted as α-hemolysis and taken as negative for the assessment of hemolytic activity (Semedo et al., 2003).

Scavenging of Lactic Acid Bacteria and Elucidation of their Antagonism
Hundred and seven LAB isolates were isolated from 6 samples of meconium newborns infants (6 donors) obtained at Roubaix hospital in the North of France. The number of LAB isolates per sample of meconium/donor was as follows: sample 1 (20 LAB isolates), sample 2 (12 LAB isolates), sample 3 (LAB 26 isolates), sample 4 (LAB 12 isolates), sample 5 (LAB 17 isolates), and sample 6 (LAB 20 isolates). In addition to their Gram positive staining, and absence of catalase activity, all these 107 LAB isolates were able to hydrolyze esculine. Importantly, all of them were identified by MALDI TOF mass spectrometry (Biomérieux, France), with high score (>99%), as E. faecalis. Among these E. faecalis species, only six isolates designed as E. faecalis 14, E. faecalis 28, E. faecalis 90, E. faecalis 93, E. faecalis 97, and E. faecalis 101 resulted to be antagonistic. Partial 16S ribosomal DNA sequences of these isolates as well as those of E. faecalis ATCC 19433 were aligned using ClustalW2 software 2 . This alignment shows a percentage of similarity higher than 99.7% to E. faecalis, confirming phylogenetic identification of our isolates. When the 16S rDNA sequence of E. coli ATCC 11229 was compared to that of enterococci, the percentage of similarity was estimated to 76.33%, which clearly is very low. These antagonistic isolates were able to produce lactic acid and bacteriocin like inhibitory substances (BLIS) and consequently to inhibit growth of GNB and GPB (Table 1). This antagonism was restricted to GPB when the pH of CFS was adjusted to 6.5 ( Table 1). The BLIS produced by the aforementioned antagonistic strains resulted to be insensitive to pH and temperature variations but sensitive to proteases. Remarkably, only E. faecalis 90 and E. faecalis 101 were active against Pseudomonas fluorescens ( Table 1).  (Figure 1).

Genetic Patterns of the Antagonistic Strains
Based on their genetic patterns and analysis of the dendrogram

Highlights on Anti-S. aureus ATCC33862 Activity
The data gathered from the challenge tests indicate that inhibition of S. aureus ATCC 33862 by E. faecalis 28 and E. faecalis 93 was due to production of lactic acid and bacteriocin. The number of S. aureus ATCC33862 cells has decreased of about 5.5 Log, 5 Log, and 2 log in presence of E. faecalis 28, E. faecalis 93, and E. faecalis ATCC29212 (Figures 2A-C).

Production of Lactic Acid and Bacteriocins like Inhibitory Substances
Enterococcus faecalis 28 and E. faecalis 93 isolates produced up to 7.06 g/l of lactic acid, after 24 h of culture. As shown on Figures 3A,B, the drop of pH observed during coculture, was correlated to increase of lactic acid production. The antistaphylococcal activity was ascribed to both lactic acid and bacteriocins, and this activity appeared to increase until 18 h of co-culture.   tetracycline, nitrofurantoin, and chloramphenicol but are resistant to clindamycin and trimethoprim-sulfamethoxazole. The data analysis was performed according to "Antibiogram committee of French Microbiology of Society" based on the European Committee on Antimicrobial Susceptibility Testing recommendations. Remarkably, these strains were not hemolytic (data not shown).

Accession Numbers
The

Discussion
This study aimed at strictly isolating LAB and particularly bacteriocinogenic LAB from meconium of newborns infants. Thus, 107 bacterial isolates were obtained from meconium of 6 donors at Roubaix Hospital in the north of France. All the bacterial isolates were identified as E. faecalis. Interestingly, Borderon et al. (1996), Adlerberth et al. (2006) showed that E. faecalis colonizes the gut of vaginally and cesarean-delivered term and preterm neonates Clinical isolates of enterococci are weakly diverse comparatively to those obtained from the environment, but E. faecalis remains the dominant species (Kuhn et al., 2003). According to Fisher and Phillips (2009), this weak diversity could be linked to the virulence factors present in this species. Isolates E. faecalis 14, E. faecalis 28, E. faecalis 90, E. faecalis 93, E. faecalis 97, and E. faecalis 101 produce inhibitory compounds that comprise lactic acid and BLIS. The concomitant production of these antimicrobials inhibited a set of GNB and GPB, including the robust methicillin-resistant S. aureus (MRSA), which is frequently encountered in hospital environment. Remarkably, this antagonism was abolished when the CFS was neutralized, highlighting thereof the role of BLIS. Vancomycin is the standard antibiotic used for the treatment of MRSA infections. Recently, Liang et al. (2014), suggested daptomycin as a reasonable alternative for treating MRSA mainly for osteoarticular infections (OAIs). The activity of these antibiotics could be potentialized with bacteriocins, which are overall safe (Belguesmia et al., 2011), ribosomally synthesized AMP (Drider and Rebuffat, 2011). Genotyping of these isolates performed by REP-PCR and PFGE (data not shown) concluded to three different strains. Percent Identity Matrix based on 16S ribosomal DNA confirms a high score of similarity of enterococci sequences and a low score of similarity with E. coli ATCC 11229 used as different bacterial taxon.
In this study, E. faecalis 28 and E. faecalis 93, obtained from two different sources, were the most actives isolates. In their presence, the counts of S. aureus ATCC33862 has drastically decreased. Further, the antagonistic isolates displayed high scores of autoaggregation and hydrophobicity. These two characteristics might be important for the interplay of these isolates with S. aureus ATCC33862. Clewell et al. (2002) reported that production of aggregation substance (Agg) on the donor cell-surface facilitates contact with recipient cell by binding to enterococcal binding substance.
Importantly, these antagonistic strains were not hemolytic and resulted to be sensitive to antibiotic usually used to     treat enterococci infections. The resistance to clindamycin and Trimethoprime/sulfamethoxzole is thought to be a species characteristic. According to the opinion of the FEEDAP panel on the updating of criteria used in the assessment of bacteria for resistance to antibiotics of human and veterinary importance, strains carrying an acquired resistance to antimicrobial(s) should not be used as feed additives, unless it can be demonstrated that it is a result of chromosomal mutation(s) (European Food Safety Authority [EFSA], 2005). This study unveiled the presence of only E. faecalis species in samples of meconium obtained from six donors at Roubaix hospital in the north of France. Off 107 E. faecalis isolates, only E. faecalis 14, E. faecalis 28, E. faecalis 90, E. faecalis 93, E. faecalis 97, and E. faecalis 101 resulted to be bacteriocinogenic. In the best of our knowledge, this is the first report dealing with bacteriocinogenic LAB from meconium. The number of antagonistic isolates is relatively low (5.60%) compared to the frequency usually reported in the literature (Nes et al., 2014). The high hydrophobicity and aggregation scores registered for the aforementioned antagonistic isolates, as well as absence of hemolytic activity and sensibility to various antibiotic render these strains as potential candidates for probiotic applications.


extfieldsofstudy: Medicine Biology

text:
Patterns of evolution of MHC class II genes of crows (Corvus) suggest trans-species polymorphism

A distinguishing characteristic of genes that code for the major histocompatibility complex (MHC) is that alleles often share more similarity between, rather than within species. There are two likely mechanisms that can explain this pattern: convergent evolution and trans-species polymorphism (TSP), in which ancient allelic lineages are maintained by balancing selection and retained by descendant species. Distinguishing between these two mechanisms has major implications in how we view adaptation of immune genes. In this study we analyzed exon 2 of the MHC class IIB in three passerine bird species in the genus Corvus: jungle crows (Corvus macrorhynchos japonensis) American crows (C. brachyrhynchos) and carrion crows (C. corone orientalis). Carrion crows and American crows are recently diverged, but allopatric, sister species, whereas carrion crows and jungle crows are more distantly related but sympatric species, and possibly share pathogens linked to MHC IIB polymorphisms. These patterns of evolutionary divergence and current geographic ranges enabled us to test for trans-species polymorphism and convergent evolution of the MHC IIB in crows. Phylogenetic reconstructions of MHC IIB sequences revealed several well supported interspecific clusters containing all three species, and there was no biased clustering of variants among the sympatric carrion crows and jungle crows. The topologies of phylogenetic trees constructed from putatively selected sites were remarkably different than those constructed from putatively neutral sites. In addition, trees constructed using non-synonymous substitutions from a continuous fragment of exon 2 had more, and generally more inclusive, supported interspecific MHC IIB variant clusters than those constructed from the same fragment using synonymous substitutions. These phylogenetic patterns suggest that recombination, especially gene conversion, has partially erased the signal of allelic ancestry in these species. While clustering of positively selected amino acids by supertyping revealed a single supertype shared by only jungle and carrion crows, a pattern consistent with convergence, the overall phylogenetic patterns we observed suggest that TSP, rather than convergence, explains the interspecific allelic similarity of MHC IIB genes in these species of crows.

INTRODUCTION
The major histocompatibility complex (MHC) is an unusual example of a functional gene complex that exhibits high levels of polymorphism. The MHC is a multigene cluster that encodes molecules that bind and present peptides to T-cells in vertebrates, initiating a cascade of immunological responses to pathogens (Janeway et al., 2005). The MHC is the most polymorphic coding gene family in vertebrate genomes (Klein, 1986) and the maintenance of this polymorphism is usually attributed to pathogen-mediated balancing selection (Sommer, 2005), although sexual selection, as well as molecular mechanisms such as recombination and gene conversion, may play important roles (Andersson & Mikko, 1995;Martinsohn et al., 1999;Zelano & Edwards, 2002).
One distinguishing characteristic of MHC genes is that alleles often share more similarity between rather than among species. Two mechanisms have been proposed to explain this "trans-specific" similarity of MHC alleles. The first explanation, called "trans-species polymorphism" (TSP), proposes that orthologous MHC allelic lineages are maintained by balancing selection, often over macro-evolutionary time-scales, and persist through speciation events (Klein et al., 1998). Strong evidence for TSP in MHC IIB genes has been found in a wide range of taxa, particularly those of mammals and fish (Klein et al., 1993;Graser et al., 1996;Garrigan & Hedrick, 2003;Lenz et al., 2013).
An alternative explanation for trans-specific MHC allele clustering is convergent or parallel evolution. The term molecular convergent evolution has been used to describe similar, but distinct phenomena (e.g., convergence between non-orthologous genes or within orthologues only) (Zhang & Kumar, 1997;Yeager & Hughes, 1999;Kriener et al., 2000), and debate exists for distinguishing molecular parallel evolution from molecular convergent evolution as well (Arendt & Reznick, 2008;Pearce, 2011). For the purpose of this study, we define convergence as any interspecific allele similarity that arises from adaptation to similar selective pressures (Gustafsson & Andersson, 1994;Hughes, 1999).
A common method used for identifying TSP, and in some cases for distinguishing TSP from convergence, is the comparison of codons thought to be under selection, such as those found in the MHC peptide binding region (PBR), with synonymous substitutions in surrounding codons or flanking introns. Alleles that are similar by decent should retain a signature of ancestry at selectively neutral sites. On the other hand, convergence is indicated if alleles are more similar at putatively selected sites than at neutral sites (Klein et al., 1998). Specifically, convergence and TSP can be tested by constructing different phylogenetic trees and comparing clustering patterns of trees constructed from non-synonymous mutations at codons likely to be under selection with trees constructed from synonymous substitutions at putatively neutral sites (Graser et al., 1996;Kupfermann et al., 1999;Kriener et al., 2000). While many studies have provided evidence for TSP in MHC IIB allelic lineages, only a few have provided evidence for convergence. One study of MHC DRB sequences in New World monkeys found that PBR amino acid motifs were shared among species but some sequences of the flanking introns sorted intra-specifically (Kriener et al., 2000). Another study that favored convergence over TSP in MHC class IIB based their conclusions on different codon usage in shared amino acids among three Musteloid species, two that were sympatric and one that was allopatric (Srithayakumar et al., 2012).
While evidence for TSP in MHC genes has been found in birds (Vincek, O'Huigin & Klein, 1997;Sato et al., 2001;Richardson & Westerdahl, 2003;Alcaide, Edwards & Negro, 2007;Burri et al., 2008;Alcaide, Lui & Edwards, 2013), few studies to date have provided convincing evidence of TSP in MHC IIB genes in passerines. The strongest evidence for TSP in passerine MHC IIB genes was shown in a study of recently diverged Darwin's finches, where several well supported interspecific allelic lineages were described (Sato et al., 2011). Well-supported interspecific clustering of variants was also reported in closely related species within the Muscicapidae family (Zagalska-Neubauer et al., 2010).
The unusually complex nature of the passerine MHC may partially explain why TSP is difficult to detect in this order. The MHC of passerines is among the most complex in terrestrial vertebrates, exhibiting highly duplicated classical MHC class I and II genes and more than ten functional class II B loci have been reported in some species. This contrasts sharply with other bird taxa such as those within the Galliformes, where one to three MHC IIB loci have been reported (Balakrishnan et al., 2010;Bollmer et al., 2010;Eimes et al., 2012;Sepil et al., 2012). It is likely that the unusually high levels of polymorphism observed in the MHC of songbirds is the result of a combination of several factors, including gene duplication, subsequent recombination (including gene conversion), and balancing selection (Hess & Edwards, 2002). Because of the potential for recombination between non-homologous loci, the role of recombination, especially gene conversion, may be exaggerated in passerines relative to other vertebrate taxa in shaping observed patterns of interspecific MHC variation between, as well as within species. The best evidence for this can be seen in phylogenetic trees of songbirds, where multi-locus MHC IIB sequences tend to cluster by species, rather than by locus (e.g., Sutton et al., 2013). This lack of trans-specific clustering of orthologous loci in passerines is thought to be due to concerted evolution as a result of gene conversion (Hess & Edwards, 2002;Edwards, Grahn & Potts, 1995).
In this study, we characterized MHC IIB loci in three closely related species of crows: jungle crow (Corvus macrorhynchos japonensis), carrion crow (C. corone orientalis), and American crow (C. brachyrhynchos). We also tested for TSP and convergence by taking advantage of the unusual arrangement of phylogeny and geographical ranges of the three species. The contemporary range of eastern carrion crows and jungle crows overlaps in East Asia (southeastern China, the Korean peninsula and the Japanese archipelago) while American crows are limited to North America (Madge & Burn, 1993;Haring et al., 2012). Mitochondrial DNA (mtDNA) phylogenies, however, place carrion crows and American crows in a monophyletic group, with jungle crows paraphyletic to these two species (Haring et al., 2012). The genus Corvus likely has a Palearctic origin, and mtDNA phylogenies suggest that carrion crows and American crows diverged in the Nearctic relatively recently; jungle crows represent a single, older lineage originating in the south-east Asian tropics (Haring et al., 2012).
Crows are suitable species for assessing TSP and convergence because their habitat, diet and social behavior are likely to render them susceptible to a diverse array of pathogens. Crows are omnivores that forage, in part, on carrion and human refuse (McGowan, 2001); scavengers, in general, could be under selection for more robust immune responses because of contact with pathogen-rich carcasses (Blount, 2003). Furthermore, all three of the Corvus species investigated in this study are found in densely human-populated areas (Ali & Ripley, 1972;Richner, 1989;McGowan, 2001;Kurosawa et al., 2003), and some data suggest that species with the ability to exploit urban areas have disproportionately strong immune responses (Moller, 2009). Finally, most crows are highly gregarious, often foraging and roosting in large communal flocks in the non-breeding season (Madge & Burn, 1993;Verbeek & Caffrey, 2002), which could promote pathogen transmission among conspecifics (Bull, 1994;Frank, 1996;Møller et al., 2001).
For this study, we assumed that the two sympatric species (carrion crows and jungle crows), which have similar habitats and co-occur in east Asia, likely share a more similar suite of pathogens with each other than with American crows. Diverse pathogen burdens are well-documented in all three species, and a comparative study of jungle crows and carrion crows revealed that ten specific helminth species were shared between the two species (Mizuno, 1984;Miller et al., 2010;Wheeler et al., 2014). There have been several surveys of intestinal parasites in American crows; however no report to date has identified these Asian helminth species in American crows, except for one anecdotal case (Jones, 1968;Hendricks, Harkema & Miller, 1969;Cawthorn, Anderson & Barker, 1980;Naderman & Pence, 1980;Mizuno, 1984;Miller et al., 2010). A strong relationship between MHC class IIB and resistance to helminth parasites has been established (Goüy de Bellocq, Charbonnel & Morand, 2008;Zhang & He, 2013). For example, Froeschke & Sommer (2012) reported both a positive and negative relationship between helminth parasites and specific MHC alleles in rodents. Thus, similar selective pressures may have acted on the MHC IIB of carrion crows and jungle crows, and a signal of convergent evolution may be detectable.
We tested for TSP among all three species of crows and convergence among carrion crows and jungle crows by analyzing a 246 bp fragment of MHC IIB exon 2 generated by 454 pyrosequencing. For the analyses, we used five different nucleotide partitions as well as supertyping, which clusters functionally equivalent amino acid sequences into units of selection (Sette & Sidney, 1999;Lund et al., 2004;Sidney et al., 2008). We also estimated species divergence times for all three species using published, as well as newly generated, mtDNA Control Region (CR) sequences.

Sample collection
We analyzed the data from 18 individuals of each species. American crows were sampled from nestlings (one bird per nest) in Yolo County, California (38 • 32 ′ N,121 • 45 ′ W) on the campus of the University of California, Davis, in May and June 2012. Collection methods were approved by the Institutional Animal Care and Use Committee of the University of California, Davis (Permit Number: 16897). Approximately 50 µL of whole blood was preserved in Queens's lysis buffer, and gDNA was extracted using a standard phenol/chloroform protocol followed by ethanol precipitation. Previously extracted gDNA from the blood of jungle crows and carrion crows was obtained from the Japanese National Museum of Nature and Science (Tsukuba, Japan) and the Yamashina Institute of Ornithology (Chiba, Japan). These samples were collected between 1994 and 2010 from the entire range of the Japanese archipelago, although most jungle crows were from the Tokyo area (16 from Tokyo, 1 each from Hokkaido and Okinawa), whereas carrion crow collection was more evenly distributed across the Japanese archipelago (6 from Kagoshima, 1 from Tsushima, 4 from Nara, 1 from Osaka and 6 from Tokyo; Fig. S1). One mL of whole blood for RNA extraction was collected from three juvenile jungle crows trapped at Ueno Zoo in Tokyo, Japan in 2012. The blood was flash frozen on site and stored at −80 • C. Jungle crow RNA was extracted from whole blood using the RNeasy Protect Animal Blood System (Qiagen, Hilden, Germany).

Amplification of MHC class IIB exon 2
In order to isolate MHC IIB exon 2, which codes for the PBR, jungle crow cDNA from two individuals (U2 and U3) was synthesized with the LongRange 2Step RT-PCR System (Qiagen, Hilden, Germany) using the manufacturer's recommended amplification protocol. MHC IIB was then targeted in the cDNA using the primers MHC05 (Miller & Lambert, 2004), which anneals within exon 1 in several passerines (positions variable), and ComaIIbex3R, which was designed by aligning the conserved regions of IIB exon 3 from several passerines (positions 19-38). These primers amplified a 359 bp cDNA fragment. We verified that the amplicon was MHC IIB by cloning the fragment from both individuals (TOPO XL One Shot; Invitrogen, Carlsbad, California, USA). Twenty-four clones from each individual were selected and whole colony PCR was performed using the TOPO vector primers M13 F and M13 R. The 20 µL PCR contained a pluck of colony cells as template, 0.625 µM of each primer, 0.5 mM of each dNTP, 1.5 mM MgCl2, 1X PCR buffer and 0.5 U of ExTaq (Takara, Mountain View, California, USA) polymerase. The cycling conditions included an initial denaturation at 94 • C for 2 min followed by 30 cycles of 94, 55 and 72 • C each for 1 min and a final extension of 72 • C for 10 min. We sequenced each amplicon in both directions using M13 primers (Applied Biosystems 3130xl; Applied Biosystems, Carlsbad, Calfornia, USA). We performed a BLAST search and identified eight sequences that aligned with known passerine MHC IIB.
We targeted intron 1 by aligning the cDNA-derived amplicons using MUSCLE in Geneious 6.1.4 (Biomatters, San Francisco, California, USA) and a reverse primer, ComaIIbex2R, was designed in a conserved region of exon 2 and paired with MHC05. Using gDNA from two jungle crows (Coma5061 and Coma7509), a 516 bp fragment extending from exon 1 to exon 2 was amplified using 50-100 ng of gDNA, 1.0 µM of each primer, 0.5 mM of each dNTP, 1.5 mM MgCl2, 1X PCR buffer and 0.5 U of ExTaq (Takara, Mountain View, California, USA) polymerase (25 µL total volume). The cycling conditions included an initial denaturation at 94 • C for 30 s followed by 30 cycles of 94 (20 s), 60 (20 s) and 72 (45 s) • C and a final extension of 72 • C for 10 min. Each of these two amplicons was cloned and 10 colonies were sequenced in each direction using the cloning and whole colony PCR protocol described above. Sequences were aligned using MUSCLE and a forward primer flanking exon 2, ComaiF2, was designed for the (100%) conserved region at position 282-304 of intron 1. Next, we isolated intron 2 by pairing ComaiF2 with ComaIIBex3R using the same individuals and PCR, cloning, and whole colony PCR conditions described above. When amplifying from exon 2 to exon 3 in jungle crows, we discovered indels that resulted in large gaps at different locations in intron 2. 454 pyrosequencing is most efficient when sequences are of equal length, and for this reason, we designed a single degenerate reverse primer, ComaEx2RA, between positions 249-268 of exon 2. Finally, we confirmed that the primers ComaiF2 and ComaEx2RA amplified MHC IIB loci in all three crow species. Primer sequences and annealing temperatures are listed in Table S1.

pyrosequencing
Fusion primers were synthesized (Fasmac, Atsugi City, Kanagawa, Japan) by ligating the standard Roche multiplex identifiers (MIDs) to both the forward and reverse MHC IIB primers and the 454 adaptor primers (the Roche "Basic Amplicon" design). In order to minimize the formation of PCR artifacts, we reduced the cycle number and primer concentration and eliminated the final extension step during amplicon generation (Lenz & Becker, 2008). PCRs contained 25-50 ngs of template, 0.5 µM of each fusion primer, 0.5 mM of each dNTP, 1.5 mM MgCl2, 1X PCR buffer, 5% DMSO and 0.5 U of ExTaq (Takara, Mountain View, California, USA) polymerase (25 µL total volume). The cycling conditions included an initial denaturation at 94 • C for 30 s followed by 28 cycles of 94 (20 s), 60 (20 s) and 72 (45 s) • C.
PCR amplicons were purified using the Agencourt AMPure XP System (Beckman Coulter, Brea, California, USA) and recovered DNA was checked for quality and primer dimers on a 1.5% agarose gel. Samples were then quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, California, USA) and pooled in equimolar amounts according to the Roche GS Junior Titanium Amplicon Library Preparation Method Manual. The quality and concentration of the pooled samples was checked on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA) and then sequenced using the Roche GS Junior Titanium Sequencing System at the Japanese National Museum of Nature and Science in Tsukuba, Japan.
jMHC is especially efficient for de-multiplexing bi-directionally sequenced double-tagged amplicons because it bins only those sequences that contain both forward and reverse primers and their associated MIDs with no ambiguous characters ("N"s) in the primers, MIDs or target sequence. Sorted FASTA files (excluding primers and MIDs) were then imported into Geneious and aligned with published MHC class IIB sequences. Sequences containing indels were removed from the data set at this time.

Bioinformatics variant validation and genotyping
Variants were accepted as "true" if they occurred in at least three reads (r > 2) in both of two independent PCRs of the same individual. The criteria of (r > 2) is derived from the estimate of a read containing at least one sequence error for a 171 bp fragment of (HLA) DRB exon 2 of ∼0.11 (Galan et al., 2010). Thus, the probability that an identical error occurs three times in the same sample is ∼10 −8 (Galan et al., 2010). At this stage, the most likely types of artefacts remaining in the data set are either single base substitutions or fragment chimeras generated during PCR. Although both of these artifacts are randomly generated, if these errors occur early in the PCR, they could be replicated and represented at relatively high frequencies in individual amplicons. To further reduce the probability of erroneously including single base substitutions, we classified sequences as true variants only when they differed by at least two nucleotide substitutions from more common variants. Galan et al. (2010) reported an average chimera frequency of 0.06 and some chimeras occurred at frequencies >0.1; thus, elimination of chimeras using frequency thresholds may erroneously inflate variant estimates. It is highly unlikely that two independent PCRs would each generate identical chimeras (where as many as 10 different variants could recombine) or single base substitution errors three times (r > 2). By validating variants with a second PCR for each individual (rather than a second PCR within or across individuals) chimeras are eliminated, yet variants that are rare in the population are not erroneously eliminated from the data set. The total number of reads that were included at this stage was then designated as "net reads" for the purposes of genotype confidence level calculations.
We used the program 'Negative Multinomial' to calculate the number of sequences that are necessary for amplifying all the variants at least three times with a confidence level of 99% (Galan et al., 2010). While the program is limited to eight possible variants, the relationship between n, net reads, r, the number of times each variant must be sampled, and m, the maximum number of variants per amplicon, is linear. Our cloned sequences indicated a maximum of 20 variants per individual which corresponds to a minimum of 175 reads for 99.9% confidence; however, this assumes equal sampling of variants. Because amplification bias is likely when co-amplifying 10 loci, we expected this bias to be reflected in over-and under-representation of some variants (Burri et al., 2014). Therefore, we increased the minimum required net reads for genotyping to 275, which represents a frequency of 1.1% (amplification bias of ∼ one order of magnitude) for a variant that has exactly three copies in an amplicon represented by 275 net reads.

Tests for selection and MHC IIB phylogenetics
For each species, we generated an alignment of validated variants, and identified the codons that have been shown to comprise the PBR of HLA in humans (Brown et al., 1993). While most MHC studies rely on the HLA PBR codons identified in humans to estimate selection, it has not been shown that these same codon positions apply universally or that selection is limited to peptide binding codons. Therefore, in order to identify specific codons that are likely under selection in crows, we performed a Wu-Kabat analysis on the amino acid alignment of verified alleles in all three species. A Wu-Kabat plot predicts amino acids that are likely subject to selection by identifying positions of high variability. Variability is calculated by dividing the number of amino acids at a position by the frequency of the most common amino acid (Wu & Kabat, 1970). For each species, we identified codons/amino acids as likely under selection if the Wu-Kabat variability metric was twice the mean of Wu-Kabat values for all sites (Wu & Kabat, 1970;Bos & Waldman, 2006). These sites are denoted WuK-PS (PS = polymorphic site). We tested for selection on the HLA PBR sites by calculating the ratios of non-synonymous (dN) and synonymous mutations (dS) using a Z test (modified Nei-Gojobori-Jukes Cantor correction) in MEGA v. 6.0 (Tamura et al., 2011). For each species, individual codons were tested for selection using the HYPHY package in MEGA. This method, known as the counting method, estimates the number of nonsynonymous and synonymous mutations that have occurred at each codon by using a maximum likelihood reconstruction of the ancestral state of each sequence. The test statistic dN-dS is used for detecting codons that have undergone selection and for positive values the probability of rejecting the null hypothesis of neutral evolution (p-value) is calculated (Suzuki & Gojobori, 1999;Pond & Frost, 2005). Significant p values are denoted as positively selected sites. This method is more computationally compatible with large data sets than empirical or hierarchal Bayesian approaches (Yang et al., 2000), yet has been shown to provide nearly identical results with a sufficient number of sequences (Pond & Frost, 2005).
We constructed nucleotide phylogenies from five different partitions of exon 2: variable sites identified by the Wu-Kabot plot (WuK-PS, 33bp), non-WuK-PS sites (213 bp), HLA PBR sites (66 bp), non-HLA PBR sites (180 bp) and the contiguous 246 bp fragment of exon 2. For each analysis, 24 different nucleotide substitution models were tested for best fit (ML) in MEGA and the Jukes-Cantor (JC) Model + G (Gamma) + I (invariant sites) was chosen based on the Bayesian Information Criterion (BIC) score.

Supertyping
Functionally equivalent MHC IIB alleles were clustered using the supertyping method described in Sepil et al. (2012), a study that also examined highly duplicated MHC loci in a passerine species. Briefly, amino acid sites identified as positively selected sites were aligned and characterized by five physicochemical descriptor variables: z1 (hydrophobicity), z2 (steric bulk), z3 (polarity), z4 and z5 (electronic effects). These descriptor variables were placed into a matrix and subjected to K-means clustering algorithm (Sandberg et al., 1998;Doytchinova & Flower, 2005). Discriminant analysis of principle components (DAPC) was used for describing clusters in the 'adegenet' package in R (Jombart, Devillard & Balloux, 2010). We performed three different supertype analyses, using three different partitions: (i) the nine positively selected sites identified in the HYPHY analysis that were shared among all three species; (ii) WuK-PS sites that were shared among all three species; and (iii) HLA PBR codons.

DNA phylogenetics and species divergence
To ensure that the mtDNA phylogenies used in Haring et al. (2012) were consistent with our populations, we tested a subset of carrion crows (N = 4), jungle crows (N = 6) and American crows (N = 4) at the same mtDNA region using the primers CR-Cor+ and Phe-Cor- (Table S1) and the same PCR conditions as Haring et al. (2012), followed by Sanger sequencing as described above. We aligned these sequences with four mtDNA sequences from each species that were used in the original phylogenetic construction by Haring et al. (2012). The Hasegawa-Kishino-Yano (HKY) model (Lenz et al., 2013) had the lowest BIC scores in the best-fit substitution model (ML). HKY ML trees with 500 bootstrap replicates were constructed in MEGA v. 6.0.
Using the mtDNA sequences, we estimated divergence times for the three crow species: the divergence of carrion crow/American crow clade from jungle crow, and the divergence between carrion crows and American crows. Divergence times to the most recent common ancestor were estimated in BEAST v. 2.0 (Bouckaert et al., 2014). We used the HKY site model and a Yule process speciation prior for the branching rates. We tested the assumption of a molecular clock using the maximum likelihood method which compares the ML value for the given topology with and without the molecular clock constraints under the HKY model (MEGA v. 6.0). We ran two separate analyses using two different mtDNA substitution rates (per site, per My) previously estimated for crows (C. macrorynchos = 0.0567555 and C. coronoides = 0.0603431) (Nabholz, Glemin & Galtier, 2009). The Markov chain Monte Carlo (MCMC) analyses were run for 10 7 generations (10,000 iteration burn in); the mean and 95% highest posterior density interval (HPD) for divergence times were calculated in Tracer v. 1.6 (Bouckaert et al., 2014).

Pyrosequencing
The goal of the first 454 run was to estimate the number of IIB loci and the minimum read number required for accurate genotyping (99.9% confidence). Six individuals from each species were sequenced and a total of 125,839 reads passed the initial 454 filter. After excluding reads of less than 270 bp, there were 76,891 reads with an average length of 340 nucleotides. Amplicons had between 8,384 and 483 initial reads (mean = 1,213) that were sorted by jMHC. These samples had an average of 376 net reads (range 171-3,211). The maximum number of variants (differing by at least 2 nucleotides) found in a single individual was 20, which suggests that crows have up to 10 IIB loci per haplotype. The required minimum read number was calculated as 211; however, we increased the cutoff to 275 net reads to account for amplification bias (see methods).

MHCIIB variation and selection
The 248 bp fragment was conserved at the final two base pair positions in all three species and was trimmed to 246 bp. The number of validated MHC IIB nucleotide variants for each species was: jungle crows = 89, carrion crows = 81 and American crows = 67. Individuals had a range of 7-20 variants, indicating that the MHC IIB in crows exhibits copy number variation (Table 1), although it is possible that the range of variant number could be explained by other phenomena, including null alleles, allele sharing across loci, homozygous vs. heterozygous loci or the variant validation methodology (where sequences with indels were excluded from the data set). Few variants were shared among species and there was no discernable pattern of allele sharing relating to phylogeny or sympatry: Four unique variants were shared among American crows and jungle crows, three among American crows and carrion crows, three among carrion crows and jungle crows and four variants were shared by all three species.
In total, we recovered 172 unique amino acid sequences among the three species (70 in jungle crows, 58 in carrion crows and 44 in American crows). We detected no species-specific motifs or motifs that were shared exclusively between two species. Within-species amino acid diversity was relatively high (0.245 ± 1.5), similar to that which we calculated for scrub jays (Aphelocoma coerulescens), another corvid species (23.3%; N = 11; NCBI (U23958-65; 72,73,75) (Edwards, Grahn & Potts, 1995). An alignment (82 sites) of common amino acid fragments from the three crow species with four other passerine species, and a falconiform species for comparison, highlights the high level of amino acid diversity found among and between species of passerines (Fig. S2).

MHC IIB phylogenies
We observed a similar pattern among the five different partitions of exon 2: MHC IIB variants formed several supported (bootstrap values ≥50), interspecific clusters in trees comparing putatively functional sites. Interspecific clusters were also observed when comparing putatively neutral sites, but there were fewer of these supported clusters, and in general they were less inclusive. This pattern was most striking in trees constructed using the contiguous 246 bp exon 2 fragment (Fig. 3). For instance, a tree that was constructed by comparing non-synonymous substitutions (Fig. 3A) shows six well supported clusters, each containing all three crow species, whereas a tree constructed by comparing synonymous substitutions of the same fragment (Fig. 3B), shows only three such clusters. Supported clustering at putatively functional sites was also more inclusive in these exon 2 trees, with 44% of all variants (105/237) forming supported interspecific clades compared to just 18% (32/237) in the tree using synonymous sites. The trees constructed from WuK-PS and non-Wuk-PS sites (Figs. S3A and S3B) as well as those constructed from the HLA PBR and non-PBR sites (Figs. S4A and S4B) displayed similar, though less distinct patterns. Among all partitions, the most common (supported) clustering was among all three species. We did not observe biased clustering of variants among the sympatric carrion crows and jungle crows; or among the more recently diverged, allopatric, American crows and carrion crows.

Supertyping
Among the three species, there were nine shared positively selected sites in exon 2. Five of these nine sites corresponded to HLA PBR sites (Fig. 1). From the 140 unique positively selected sites amino acid variants a total of eight supertypes were identified (Fig. 4). Only one supertype (Supertype 4) was limited to carrion crows and jungle crows, whereas the rest of the supertypes were shared among the three species. The supertypes generated by the WuK-PS and the HLA PBR codons were not well supported and thus excluded from our analysis.

mtDNA phylogeny and divergence estimate
The CR-Cor+ and Phe-Cor-primers amplified an 836-920 bp fragment of the mtDNA CR region in the three species of crows. The ML tree placed carrion crows and American crows in a separate clade from jungle crows with 100% bootstrap support (Fig. 2), thus confirming that the jungle crows and carrion crow populations from this study conform to the phylogeny of Haring et al. (2012). The ML-based test of the molecular clock failed to reject the null hypothesis of equal evolutionary rate throughout the tree (p = 0.85); thus we used a strict clock setting for the MCMC iterations in the BEAST program.

DISCUSSION
We observed strong interspecific clustering of MHC IIB variants in three closely related species of crows (Corvus sp.), and most of the supported clusters contained all three species; however, the clustering was much more inclusive, and better supported, when comparing non-synonymous substitutions at putatively selected sites than synonymous substitutions at putatively non-selected sites ( Fig. 3; Figs. S3 and S4). This pattern was observed in all of the phylogenies constructed from the different partitions of exon 2, but was most obvious in the trees constructed using the entire 246 fragment of exon 2 (Fig. 3). This pattern of strong clustering among functional sites is suggestive of convergent evolution (Klein, Sato & Nikolaidis , 2007). In fact, if this study been limited to the sympatric carrion crows and jungle crows, we may have erroneously concluded that convergent evolution explains these patterns; however, inclusion of sequences from the allopatric American crow, and the resulting interspecific clustering of all three species at  (Nabholz, Glemin & Galtier, 2009). Accession numbers with an asterisk are from this study; all others are from Haring et al. (2012). Coco, carrion crow; Cobr, American crow; Coma, jungle crow. Aphelocoma coerulescens (Accession number AF218919) was used as an outgroup.  10 principle components (PC) and three discriminant functions (dimensions) were retained during analyses, to describe the relationship between the clusters. The scatterplot show only the first two discriminant functions (d = 2). The bottom graph displays the bar plot of eigenvalues for the discriminant analysis. The Eigenvalues that were not retained are marked in white. The Eigenvalues that are used in the scatterplot are marked in black. The Eigenvalue that was retained for the analysis but not used in the scatterplot is marked in grey. Each allele is represented as a dot and the supertypes as ellipses.
functional sites, suggests that the pattern observed in the phylogenies is not convergence, but rather trans-species polymorphism.
Our results indicate that the MHC IIB in crows exhibits the same characteristics of the MHC IIB observed in other passerine species (Bollmer et al., 2010;Zagalska-Neubauer et al., 2010), namely, high levels of polymorphism, highly duplicated loci, probable copy number variation of loci and a strong signature of selection. Consistent with similar studies, we also observed highly polymorphic amino acid positions and detected selection at sites other than the HLA PBR (Fig. 1), which indicates that either the PBR in passerines comprises different codon positions than the human MHC or that regions other than the PBR are under selection (Anmarkrud et al., 2010;Bollmer et al., 2010;Zagalska-Neubauer et al., 2010). The Wuk-Kabot plot was a better fit to the HLA PBR than the HYPHY probability based test for selection (Fig. 1), which may indicate that such probability tests are not reliable when identifying selected sites across many duplicated loci if the relationships between these loci are unknown (e.g., orthologous versus paralogous relationships). For example, it is unlikely that the hyper-polymorphic amino acid sites at positions 6, 23, 32, 62, and 65 are not subject to selection. This highlights the importance of using a combination of different exon 2 partitions when evaluating selection or establishing putative neutral and selected sites for phylogenetic analyses in complicated systems such as the passerine MHC.
Several factors may explain the relatively high MHC IIB neutral genetic variation between allelic lineages we observed in these crow species. First, if these allelic clusters represent ancient lineages, they are likely millions of years older than the MRCA of all three species, which allows sufficient time for random mutations to be incorporated, and the likelihood that these neutral mutations persist is increased by their close association with selected sites (Klein et al., 1998;O'hUigín et al., 2000). Thus, the majority of the synonymous polymorphisms observed in neutral sites were likely inherited, rather than generated since species divergence. Other factors that may contribute to the relatively high synonymous nucleotide variation between species observed at non-selected sites relative to selected sites are the combined effects of recombination through genetic exchange and gene conversion. In a study of MHC IIB in sticklebacks (Gasterosteidae), Lenz et al. (2013) observed a similar pattern of clustering of selected sites (in this case, the HLA PBR) but a lack of clustering in the remaining sites of exon 2, a pattern similar to the one reported here. The authors reasoned that selected sites may evolve under site-specific functional constraints that maintain similar amino acid motifs, and that any new variants generated by recombination are more likely to be retained if they share this functional similarity. Intraspecific recombination and gene conversion could then erode the signal of common allelic ancestry over time.
Gene conversion is thought to explain why passerine MHC sequences often sort by species, rather than by locus, on phylogenetic trees (Hess & Edwards, 2002). Elevated rates of gene conversion have been documented in birds, and one study reported a gene conversion rate over a magnitude higher than the neutral mutation rate (Spurgin et al., 2011). It is possible that the reason why the crow MHC IIB sequences do not sort intra-specifically is that insufficient time has passed since species divergence for gene conversion to homogenize the neutral sites within exon 2. The patterns we observed were similar to those described for MHC IIB in Darwin's finches in that the sequences sorted in a manner that would be expected from a single, undifferentiated species (Sato et al., 2011). The similar results between our study and that of Sato et al. (2011) are likely due to the relatively recent divergence times of the focal taxa in both studies. In the Galapagos, Darwin finches' radiated from a founding population approximately 2.5 Ma, while the MRCA to the three crow species was estimated at approximately 900,000 years ago.
While we did not detect evidence of convergence between the sympatric carrion crow and jungle crow in the MHC IIB phylogenies, the supertype analysis revealed a pattern consistent with convergent evolution. While seven of the eight supertypes were shared among the three species, one supertype, ST-4, was exclusive to carrion crows and jungle crows, which might reflect selective pressure from a specific, shared parasite. None of the other seven supertypes were exclusive to either a single species or group of two species. This supertyping method classifies MHC II B loci into clusters based on the physio-chemical properties of the amino acids in the positively selected sites of the PBR. Several studies of primate MHC have shown that different MHC alleles bind very similar peptide motifs, suggesting significant overlap in peptide binding repertoires (Sette & Sidney, 1999;Lund et al., 2004;Sidney et al., 2008). Thus, different amino acid sequences can be functionally equivalent, and grouped together as a supertype, considered a unit of selection. Recently, Sepil et al. (2013) showed that different MHC class I supertypes confer resistance to malaria in great tits (Parus major). In our study, supertypes ST-3 and ST-4 grouped together, away from the other six supertypes, which may suggest that these groups are functionally important for a specific type of parasite, whereas the other supertypes may confer resistance to more common, generalist parasites (Fig. 4).

CONCLUSION
In the present study we describe strong evidence for trans-specific polymorphism at the MHC IIB in three species of crows. Our results are consistent with two other studies that examined MHC IIB in closely related passerine taxa and indicate that TSP in passerines may be common, but due to intra-specific gene conversion, most easily detectable among recently diverged species.
Given that the allopatric American crow and carrion crow diverged approximately 430,000 years ago, we expected to find some signal of convergence of MHC IIB genes between the sympatric jungle crow and carrion crow in the MHC IIB phylogenies. While we did find patterns consistent with convergence in the phylogenetic analyses, any convergence would have had to occur among all three species, before the MRCA split approximately 900,000 years ago. While this scenario cannot be ruled out, the results presented here are better explained by TSP.


extfieldsofstudy:
Materials Science
Medicine

s2fieldsofstudy:
Biology
Materials Science

text:
Optical regulation of cell chain

Formation of cell chains is a straightforward and efficient method to study the cell interaction. By regulating the contact sequence and interaction distance, the influence of different extracellular cues on the cell interaction can be investigated. However, it faces great challenges in stable retaining and precise regulation of cell chain, especially in cell culture with relatively low cell concentration. Here we demonstrated an optical method to realize the precise regulation of cell chain, including removing or adding a single cell, adjusting interaction distance, and changing cell contact sequence. After injecting a 980-nm wavelength laser beam into a tapered optical fiber probe (FP), a cell chain of Escherichia colis (E. colis) is formed under the optical gradient force. By manipulating another FP close to the cell chain, a targeted E. coli cell can be trapped by the FP and removed from the chain. Further, the targeted cell can be added back to the chain at different positions to change the cell contact sequence. The experiments were interpreted by numerical simulations and the impact of cell sizes and shapes on this method was analyzed.

Increasing attention has been paid to the interaction between neighboring cells because of its crucial role in ruling cell fate and function, such as cell growth 1-3 , differentiation 4-6 , intercellular signaling pathway [7][8][9] and mechanical force transduction 10,11 . The cell interaction occurs mainly through exchange of soluble signaling molecules and the direct cell-cell contact, which can vary in both time and space continuously 12,13 . Thus, it is of great importance in dynamically adjusting cell-cell interaction distance and contact sequence, and further individually investigating the spatiotemporal aspects of different extracellular cues. Recent advances in micropatterening 14,15 and microelectromechanical systems 16,17 show the invaluable performance in multi-cellular arrangements and dynamic control of tissue organization. However, the cells must be confined on an elaborated substrate which was usually not reusable in these techniques.
Recent studies have demonstrated the remarkable ability to form cell patterns by using holographic optical tweezers (HOTs) [18][19][20][21] , with significant advantages in achieving the formation of arbitrary patterns. The cells can also be removed from or added into the pattern by controlling the laser beam movements. However, for HOTs, appropriate algorithms are required for precise phase hologram calculation to realize a pattern of cells. The case becomes more complicated when manipulating targeted cells from a pattern because the movements of the laser beams for manipulation and the retaining of the other beams are both required. In addition, HOTs were usually built on an optical system consisted of dichroic mirrors, spatial light modulators and high-numerical-aperture focusing objectives. The working distance of the objectives limits the depth at which cells can be manipulated in the cell suspensions. Besides HOTs, surface plasmon-based optical tweezers (SPOTs) 22,23 have been demonstrated to form cell patterns with high trapping stability and retaining ability, especially for the sub-micro sized cells and biological molecules. However, for SPOTs, the specific patterns of cells are decided by the carefully designed lithographic substrates, making it difficult to dynamically adjust the cell-cell contact. Moreover, in SPOTs approaches, the formation of cell patterns is also limited at a specific depth of the cell suspension. Notably, formation of cell chain is an alternative approach to realize the cell-cell contact. A very simple and efficient way to form a cell chain, especially in the cell culture with relatively low concentration, is to use an optical fiber probe (FP) 24 . The method is free from the limitation on the depth of cell manipulation in the suspensions and does not require any elaborated substrates. Unfortunately, the cell contact sequence and interaction distance are fixed once the cell chain is formed, which limit the further investigation of extracellular cues on the interaction. Therefore, in this work, we present an optical method to realize the precise regulation of cell chain, including changing cell contact sequence and adjusting interaction distance. A tapered FP was used for the formation of Escherichia coli (E. coli) cell chain to realize the cell-cell contact. By using another taper FP, a targeted cell can be removed from the cell chain and then added back into it, realizing the regulation of cell chain.

Results
Schematic for the regulation of cell chain. Figure 1 schematically shows the formation and regulation process of an E. coli cell chain. Once a laser beam is injected into FP 1, E. colis are trapped one after another along the axial direction of FP 1 to form a chain under the optical gradient force (Fig. 1a) 24 . For illustration, a cell chain consisted of six trapped E. colis is shown in Fig. 1. Then FP 2 is adjusted to approach E. coli 5. After a laser beam is injected into FP 2, E. coli 5 is rotated and orientated toward the axial direction of FP 2. FP 2 is shifted until E. coli 5 is removed from the chain so that the cell contact sequence is changed from 1-2-3-4-5-6 to 1-2-3-4-6 ( Fig. 1b). Then E. coli 5 is put between the E. coli 3 and 4 by moving FP 2 (Fig. 1c). After turning off the laser in FP 2, E. coli 5 is gradually rotated and orientated along the axial direction of FP 1 (Fig. 1d), changing the cell contact sequence again (from 1-2-3-4-6 to 1-2-3-5-4-6). Figure 1e schematically shows the experiment setup. FPs 1 and 2 (see methods for fabrication) were connected to the lasers 1 and 2, respectively. The wavelength of the both lasers is 980 nm, which is weakly absorbed by most living matter 25 . More importantly, 980 nm has been shown to be a good wavelength to prevent optical damage in bacteria and mammalian cells 26 . After sheathed with a glass capillary, FPs 1 and 2 were fixed on microstages 1 and 2, respectively. The probe tips were immersed in the E. coli solution (see methods for the suspension preparation) which was dropped on a glass slide with an injector. The slide was mounted on an x-y manual translation stage (resolution: 50 nm) to achieve fine positioning and mechanical stability. An optical microscope incorporated with a charged coupled device (CCD) was used for the real time monitoring, image capturing and video recording.
Design and numerical calculation. As shown in Fig. 1, the functions of FPs 1 and 2 are forming the E. coli chain and manipulating a targeted cell, respectively. Since the optical field distributions outputted from the FPs are strongly affected by the fiber tips, a specific shape is required for each of the two FPs. The shapes were designed based on a series of field distribution simulations for different FPs.  fiber, water and E. coli, respectively 27 . The E. coli in the simulations was assumed to be a homogeneous rod (length L: 1.5 μ m; diameter D: 500 nm) with hemispherical caps. The curves in Fig. 2a, b show the normalized energy density along the fiber axial direction as indicated by the black dashed lines. The laser intensity outputted from the FPs varies with the distance to the fiber tips. It can be seen that for FPs 1 and 2, the decay speeds of the intensity with the distance are different due to the different tip shapes (Fig. 2a,b). For FP 2, the normalized energy density (along the fiber axial direction) decreased from 1 to 0.4 within 1 μ m. It is indicated that FP 2, with a much more concentrated energy density at the fiber tip, is suitable for manipulating a single cell. While for FP 1, the density decreased from 1 to 0.4 within 8 μ m, indicating that the outputted light from the tip of FP 1 decreases much more slowly and covers a much longer distance. Therefore, FP 1 can be used for forming the cell chain 24 . It should be noted the cell number in the cell chain increased with the power of FP 1. Moreover, the cell number also depended on the local cell concentration around the tip of FP 1.
Besides, the angle between FPs (θ) should be set a proper value to realize the optimal regulation of cell chain. According to the experiment results and theoretical simulation, it can be known that for θ < 30°, the taken cell by FP 2 will be easily trapped by FP 1 again, which decreases the orientation stability. While for θ > 60°, the power in FP 2 will be larger to remove the specific cell from the cell chain, which increases the photo-toxicity for cells. Thus, θ should be ranged from 30° to 60°. During the experiment, it was set to be 50°. Further, the laser power injected into FP 1 was fixed at 30 mW to retain the formation of E. coli chain. To realize single cell manipulation without affecting other E. colis in the chain, a proper value is required for the power in FP 2 (P). Thus, optical torques (T) exerted on the six E. colis in the chain were calculated as a function of P (Fig. 2c). The optical torque T is defined as 28 : where r i is the position vector pointing from the central point of E. coli to the interaction point, dF EM is the electromagnetic force element exerted on the interaction point i. F EM can be expressed as 29 : where dS is the surface element surrounding the cell, n is the surface normal vector, 〈 T M 〉 is the time-independent Maxwell stress tensor which can be derived from the simulations. Note that since the Maxwell stress tensor was used in the calculations, the obtained optical forces and torques are related to all the interaction of light with matter, i.e., the calculated torques were contributed by both the scattering torque and absorption torque. It should be pointed out that the absorption torque is very small, which is because of the weak absorption (α ≈ 0.5 cm −1 ) of biological cells for the 980 nm laser 30 . According to our calculation, the absorption torque is two orders of magnitudes smaller than the resultant torque. The optical torque along + z direction is defined as positive, under which the cell will be rotated counterclockwise. From Fig. 2c, it can be seen the absolute values of the torques |T| on the E. colis increase linearly as P increases. In the region I, as |T| is less than 1 pN·μ m, the E. colis remain oriented along the axial direction of FP 1. At P = 32 mW, the torque exerted on E. coli 6 reaches 1 pN·μ m and the E. coli starts to be rotated counterclockwise toward FP 2 (region II). After P increased to 90 mW, the torque exerted on E. coli 5 also reaches 1 pN·μ m so that E. coli 5 will also be rotated and trapped by FP 2 (region III). Meanwhile, the biological safety of this method should be also assayed since it is developed for biological study. To study the cell viability, 5% Trypan blue was added into the cell suspension during the regulation progress. For alive cell, it is able to prevent the dye from entering the cytoplasm due to the permselectivity mechanism of cell membrane. While for the dead one, the dye will penetrate the cell membrane and the whole cell appears blue. The results showed that for P < 100 mW, no staining was observed which indicated the cells survived without any detectable damages. But if P > 100 mW, the cells will be gradually stained to be blue, which indicated the cells suffered from the photo-toxicity and cannot maintain their normal function. Therefore, to ensure cell viability and a single-cell manipulation, P should be ranged from 32 to 90 mW. During the regulation progress, it was set to be 50 mW.
Trapping and removing a targeted cell. After forming a cell chain consisted of 6 E. colis by using FP 1, the regulation operation is started by injecting the 980 nm laser (P = 50 mW) into FP 2 at t = 0 s (Fig. 3a). As FP 2 approached the end of cell chain, E. coli 6 started to be counterclockwise rotated and gradually orientated along the axial direction of FP 2 with a rotation angle of 50° (Fig. 3b,c). With the shift of FP 2 along the − x direction (Fig. 3d,e), the distance between E. coli 6 and E. coli 5 was dynamically enlarged until E. coli 6 was removed from the original cell chain, with a shifting distance of 5.74 μ m (Fig. 3f). The detailed trapping and removal progress is shown by Movie S1 in the Supporting Information. To numerically interpret the above process, the optical field distribution was simulated at different azimuthal angles (θ) of E. coli 6 during the rotation process. Meanwhile, the optical torques exerted on the 6 E. colis of the chain were calculated as shown in Fig. 4a. The inset shows the energy density distribution at θ = 180°. In the region I (130° < θ < 183°), the torque exerted on E. coli 6 was positive so that E. coli 6 will be rotated counterclockwise (as indicated by the red arrow). In the region II (183° < θ < 230°), the torque of E. coli 6 was negative so that E. coli 6 will be rotated clockwise (as the Scientific RepoRts | 5:11578 | DOi: 10.1038/srep11578 blue arrow shown). At θ = 183°, the torque on E. coli 6 was zero and so E. coli 6 will be stably trapped and oriented along the axial direction of FP 2. From Fig. 4a, it is also indicated that during the rotation process of E. coli 6, the torques exerted on the other E. colis of the chain were nearly zero so that they kept trapped by FP 1. In addition, the torque and F x (the x component of F EM ) exerted on E. coli 6 were calculated with the shift of FP 2 along the − x direction, as shown in Fig. 4b. To indicate the direction of F x , + x direction is defined as positive. It can be seen that the torque was nearly zero and F x was negative (i.e., toward the FP 2) during the shift of FP 2. Therefore, E. coli 6 kept trapped by FP 2 and was shifted with FP 2. Note that E. coli 5 was also rotated a bit during the shift of FP 2 (Fig. 3e). This was because  a slight shift of FP 2 also occurred along − y direction in the experiment, increasing the positive torque exerted on E. coli 5. In addition, although Fig. 3 shows the removal of the E. coli at the end of the cell chain, a targeted E. coli at any other location of the chain can be trapped and removed from the chain with this method. For example, experiment was also performed for removing E. coli 2 in the chain of nine cells using FP 2 (see Fig. S1 in the Supporting Information).

Adding the cell back into cell chain.
To further regulate the cell chain, E. coli 6 was added back into the original chain, as shown in Fig. 5. At t = 0 s, the laser in FP 2 was turned off (Fig. 5a). E. coli 6 was gradually rotated clockwise and finally orientated along the axial direction of FP 1 (Fig. 5b-d). The detailed adding progress is presented by Movie S2 in the Supporting Information. The torques exerted on the six E. colis were also calculated at different azimuthal angles (θ) of E. coli 6 during the rotation process (Fig. 5e). It can be seen that E. coli 6 will be rotated counterclockwise and clockwise in the region I (80° < θ < 130°) and region II (130° < θ < 180°), respectively. At θ = 130°, the torque on E. coli 6 was zero so that the E. coli will be stably trapped and oriented along the direction of θ = 130°, i.e., the axial direction of FP 1.
Changing the cell contact sequence. Besides the removal and adding operations, cell contact sequence of the chain can also be regulated with this method. For illustration, cell contact sequence of a chain consisted of 10 E. colis was changed as shown in Fig. 6. As the above mentioned, although the laser power injected into FP 1 was fixed at 30 mW, the cell number of the chain varied due to the local cell concentration around the tip of FP 1. At t = 0 s, FP 2 was adjusted to approach the downside tip of E. coli 3 in the cell chain (Fig .6a). After turning on the laser in FP 2 (P 2 = 50 mW) at t = 1 s (Fig. 6b), the downside tip (indicated by the yellow dot) of E. coli 3 was trapped by FP 2 and then shifted along − x direction with the shift of FP 2, while the upside tip (indicated by the red dot) remained stationary. With a shift of 1.9 μ m along the − x direction, the angle between the axial direction of E. coil 3 and the + x direction became 50° (Fig. 6c). In this process, E. coil 3 was rotated clockwise and gradually orientated along the axial direction of FP 2 (Fig. 6d). Then it was removed from the cell chain and shifted along − x direction with a distance of 7.1 μ m. To adjust the contact sequence of cell chain, FP 2 was shifted along + y direction with a distance of ~1 μ m (Fig. 6e), followed by a shift of 9 μ m along the + x direction (Fig. 6f). After that, E. coli 3 was added back into the chain between E. coils 4 and 5 (Fig. 6g). By turning off the laser in FP 2, E. coli 3 was gradually orientated along the axial direction of FP 1 so that the contact sequence of cell chain was changed to 1-2-4-3-5-6-7-8-9-10 ( Fig. 6h,i). The detailed adjusting process is presented by Movie S3 in the Supporting Information. The optical torques on the E. colis were also calculated in the adjusting process, as shown in Fig. 7a. The torques are positive and negative in the regions I (− 50° < θ < 0°) and II (0° < θ < 50°) and E. coli 3 will be rotated counterclockwise and clockwise (as indicated by the red and blue arrows), respectively. Therefore, E. coli 3 will be stably trapped and oriented along the direction at θ = 0°, i.e., the axial direction of FP 2. The torques exerted on E. colis after turning off the laser in FP 2 were calculated as shown in Fig. 7b. The torques were positive and negative in the region I (80° < θ < 130°) and II (130° < θ < 180°), respectively. At θ = 130°, the torque exerted on E. coli 3 was zero so that E. coli 3 will be stably trapped and oriented along the axial direction of FP 1. Moreover, the trapping force and torque exerted on E. coli 3 were calculated during the shift process of FP 2, as shown in Fig. 7c. The trapping force was negative, i.e. toward the FP 2, and the torque kept nearly zero during the shift process, indicating E. coli 3 will be stably trapped and shifted with FP 2. Note that in the operation process presented above, the total rotation angle of E. coil 3 was 180° rather than 0°, i.e., the two tips of cell (indicated by yellow and red dots in Fig. 6) was inversed in the chain. Therefore, the contact tips of two neighboring cells can also be regulated by rotating a single cell in the chain with this method. For example, by rotating E. coli 3 with an angle of 180° using FP 2, the E. coli was inversed so that the tips of E. coil 3 contacted with E. colis 2 and 4 were exchanged. (see Fig. S2 in the Supporting Information).

Discussion
The experimental results and numerical analysis above have indicated that the contact sequence of the cell chain and the interaction distance between cells can be efficiently regulated by using two fiber probes. To further investigate the impact of cell sizes and shapes on this method, a series of simulations and calculations was performed. Figure 8a shows the optical torque exerted on E. coli 6 at (e,f) E. coli 3 kept trapped and shifted with FP 2 along the + y and + x direction. (g-i) After turning off laser in FP 2, E. coli 3 was added back into the cell chain between E. coils 4 and 5 and the contact sequence was changed to 1-2-4-3-5-6-7-8-9-10. The two tips of E. coil 3 were indicated by yellow and red dots. different azimuthal angles (θ) when the E. coli is rotated and trapped by FP 2. The cell length varies from 1.2 to 1.7 μ m while the diameter D of the cell is fixed at D = 0.5 μ m. It can be seen the curves of the torques exerted on E. coli 6 with different lengths exhibit similar trends and shapes, which indicates that the presented method is valid for E. colis with different lengths. Moreover, at a specific azimuthal angle, the absolute value of the torque exerted on E. coli 6 becomes larger for longer cells. This is because the optical field at the end of the cell chain consisted of longer E. colis is weaker than that of a shorter chain, which leads to a smaller resisting torque (yielded by FP 1) and consequently a larger resultant torque exerted on E. coli 6. The torques were also calculated for the E. colis with different diameters (Fig. 8b). It can be seen the curves for different E. coli diameters also exhibit similar trends and shapes, indicating the validity of this method for E. colis with different diameters. Note that for the thinner cell, e.g. D = 0.35 μ m, the resultant torque also becomes zero at θ = 225°. However, the E. coli cannot be stably oriented at θ = 225° because the E. coli at this angle is in a metastable state which will be disrupted by a perturbation. Finally, to investigate applicability of the method for cells in other shapes (e.g. spherical), the regulation of a yeast cell chain was also simulated and numerically analyzed. The simulated process is removing a targeted yeast cell (at the end of the chain) from a chain consisted of six yeast cells. In the simulations, the refractive index of yeast cells was set to be 1.40 31 . Note that as the yeast cells are in spherical shapes, rotation cannot be observed in any regulation process. Figure 8c shows the trapping forces F x on the targeted yeast cells with different diameters as functions of the shift distance of FP 2. It can be seen that although F x undergoes a fluctuation at the beginning of the shift, which is due to the outputted optical field redistribution with the targeted cell shifted, F x remains negative during the shift process, indicating that the yeast cell will keep trapped by FP 2. Therefore, the presented method can also be used for the regulation of cell chain consisted of spherical cells. Compared with the reported methods using multiple optical traps, the presented FPs approach has the advantages of easy fabrication, simple configuration, and high flexibility of manipulation. Moreover, the FPs can be precisely adjusted to manipulate the specific cells at different depths in the cell suspension. It should be pointed out that although the formation control of cells in this work is limited to a chain, more types of cell patterns are expected to be formed and regulated by employing multiple fiber arrays with the presented method.
In conclusion, an optical method has been demonstrated to realize the regulation of cell chain by using two FPs. Targeted E. coli cells were trapped by the FP and removed from the cell chain. The E. coli cells were then added back to the chain at different positions to change the cell contact sequence. Numerical simulations interpret the regulation process and indicate that the method can be used for the regulation of cell chains consisted of cell with different sizes and shapes (e.g. spherical). By incorporating the FPs into lap-on-chip platforms, the presented regulation method is expected to enable a new opportunity for the investigation of the cell growth, intercellular singling pathway, and pathogenic processes.

Methods
Fabrication of FPs. The fiber probes were fabricated through a flame-heating technique with the commercial single-mode optical fibers (connector type: FC/PC, core diameter: 9 μ m, cladding diameter: 125 μ m; Corning Inc.). Firstly, the buffer and polymer jacket were stripped off with a fiber stripper, and then the fibers were sheathed with a glass capillary (inner diameter: ~0.9 mm, wall thickness: ~0.1 mm, length: ~120 mm) to protect the fibers from breakage and warping. Secondly, the fibers were heated for approximately 1 min to reach their melting point. Then the drawing speed of about 0.5 and 0.6 mm/s were applied to the heated fibers 1 and 2, respectively. Meanwhile, the fiber diameters were decreased from 125 μ m to 5.5 and 4 μ m over a 2-mm and 2.5-mm length, respectively. Finally, the drawing speeds were increased to about 2 and 3 mm/s for fibers 1 and 2, respectively. Then the fibers were broken and FPs were fabricated with different abrupt tapered tips.