Glaucoma filtration surgery is regularly performed for the treatment of glaucoma and trabeculectomy is often regarded as the ‘gold standard' glaucoma operation. The biggest risk of failure of the operation is bleb scarring. The advent of antifibrotic agents, such as mitomycin C (MMC) and 5-fluorouracil (5FU) has vastly prolonged the longevity of the bleb, but concerns remain regarding the potential increase in postoperative complications. More selective therapeutic targets have therefore been explored. One of these is vascular endothelial growth factor (VEGF) inhibition. VEGF inhibition has a role not only in subconjunctival angiogenesis inhibition but also it has direct anti-fibrotic properties. Newer pharmacological compounds and materials have also been developed in recent years in attempt to modulate the wound healing in different ways after glaucoma surgery. These include physical barriers to scarring and vehicles for sustained release of pharmacological agents, and early promising results have been demonstrated. This two-part review will provide a discussion of the application of anti-fibrotic agents in glaucoma filtration surgery and evaluate the newer agents that have been developed.How to cite this article: Fan Gaskin JC, Nguyen DQ, Ang GS, O'Connor J, Crowston JG. Wound Healing Modulation in Glau coma Filtration Surgery–Conventional Practices and New Pers pectives: Antivascular Endothelial Growth Factor and Novel Agents (Part II). J Curr Glaucoma Pract 2014;8(2):46-53. However, these substances have also been associated with the potential for increased complication rate of the filtering procedure due to their nonspecific cito-cidal activity, resulting in loss of the conjunctival barrier that can lead to thin-walled avascular blebs with an elevated risk of hypotony and infection. Consequently, alternative, safer forms of wound healing modulation have been sought and, in particular, agents with more specific and targeted activity. Antivascular endothelial growth factor (anti-VEGF) has been identified as a potential solution due to its more selective mode of action. Part II of this series will examine the evidence behind the use of anti-VEGF in trabe culectomy as well as discuss the newer agents/materials being developed.Trabeculectomy was introduced by Cairns in 1968 and remains the most commonly performed procedure for the treat ment of glaucoma.3 assessed the presence of VEGF in trabeculectomy blebs and reported VEGF(165) and VEGF(121) to be predominantly associated with blood vessel growth, and VEGF(189) to be involved in fibrosis. Lopilly Park et al4 analyzed the levels of VEGF in both the aqueous humor and Tenon's tissue in patients with primary open angle glaucoma (POAG) and correlated the levels with their outcome following glaucoma surgery. They identified a significant association between the VEGF level in Tenon's tissue at the time of glaucoma surgery with the 1-year intraocular pressure (IOP) and the ultimate success of the operation. Therefore by reducing angiogenesis and subsequent Inflammation and collagen deposition, VEGF inhibition may have a beneficial impact on glaucoma surgical outcome.Due to the nonspecific inhibitory activity and continued risk of failure associated with antifibrotic agents, other forms of more selective wound modulation with acceptable side effects have been explored in recent years. Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis throughout the body. Different VEGF isoforms may be responsible for differential roles in ocular wound healing. Van Bergen et al5 Delayed wound healing is also a known complication of systemic bevacizumab, such as the wound dehiscence seen after colo-rectal surgery.6 In an early case report, Coote et al7 demonstrated significantly reduced bleb vascularity following a single subconjunctival injection of bevacizumab in an eye that had recently undergone cataract surgery 3 months post-MMC trabeculectomy.Bevacizumab is a recombinant humanized monoclonal immuno globulin antibody of nonselective VEGF. In a cell culture model, bevacizumab demonstrated anti-fibrotic pro perties in a number of ways, including disrup tion of fibro blast proliferation, inhibition of collagen contracti-bility and induction of fibroblast cell death.Unfortunately, the number of studies comparing the efficacy of bevacizumab with conventional antifibrotic agents is small, limited in sample size and often with conficting conclusions.8 conducted a prospective, randomized study assessing the efficacy of subconjunctival bevacizumab  at the end of trabeculectomy compared with MMC-augmented trabeculectomy  and demonstrated significantly lower IOP control postoperatively in the MMC group compared to the bevacizumab group . The MMC group also had a higher cumulative probability of total success, although this was not statistically significant.Niforushan et al9 performed a similar pilot study in patients under going combined phacotrabeculectomy by randomizing 38 patients into three groups: (1) conventional single-site phacotrabeculectomy with 0.3 mg/ml intraoperative MMC for 2 to 3 minutes; (2) single-site phacotrabeculectomy with three subconjunctival bevacizumab injections (1.25 mg/ 0.05 ml) performed immediately prior to surgery, immediately following surgery, and on the seventh postoperative day, and (3) single-site phacotrabeculectomy with bevaci zumab-soaked sponge (1.25 mg/0.05 ml) applied intra operatively for 3 minutes to the scleral bed. They reported subconjunctival bevacizumab to be equally effective in reducing intraocular pressure compared with MMC, only with a better safety profile. However, bevacizumab soaked in a sponge appeared to have no advantages over MMC.Sengupta et al10 analyzed the efficacy of subconjunctival 5FU compared to both subconjunctival and intravitreal bevacizumab in moderating bleb size, vascularity, and IOP postoperatively in an animal model. They observed greater bleb length, width and height in the subconjunctival bevacizumab group compared to the other two groups. Eyes in the subconjunctival bevacizumab group also demons trated significantly lower IOP values compared to the subconjunctival 5FU group, but not compared to the intravitreal bevacizumab group. Inflammation, neovas-cularization and fibrosis were correspondingly less in the subconjunctival bevacizumab group on histological analysis of the blebs.When comparing bevacizumab with 5FU, a recent study by Ozgonul et al11 reported no significant differences between groups of patients who received four repeated subconjunctival bevacizumab injections (1.25 mg) immediately before and after trabeculectomy and patients who underwent 5FU augmented trabeculectomy without postoperative bevacizumab injections. Simsek et al12 com pared subconjunctival bevacizumab (1.25 mg/0.1 ml) and 5FU (5 mg/0.2 ml) in needle revision of failed trabe-culectomy blebs and reported 5FU to be more effective than bevacizumab application.Jurkowska-Dudzinska et al13 reported that combined bevaci-zumab and 5FU offered superior anti-fibrotic effect over mono therapy using either agent, and at the same time prolonging bleb survival. In a randomized controlled trial, Chua et al14 obser ved a trend for increased central bleb avas cularity after a single post operative combined injection of bevaci zumab (1.25 mg/0.05 ml) and 5FU (7.5 mg/0.15 ml), compared to 5FU (7.5 mg/0.15 ml) alone. Freiberg et al15 compared postoperative subconjunctival 5FU (5 mg/ 0.5 ml) injections with or without one single injection of subconjunctival bevacizumab  following MMC-augmented trabeculectomy in 61 patients. They demonst rated a significantly lower nu mber of 5FU injections if bevacizumab was also administered. Whilst there was no difference in mean postoperative IOPs between the two groups, significantly fewer patients in the bevaci zumab group required bleb needling in the follow-up period (mean 25 ± 19 months).There is evidence that bevacizumab may act in synergy with 5FU. In a rabbit model of glaucoma fl tra-tion surgery, How et alThere is currently no conclusive evidence as to the optimal method of delivery of anti-VEGF therapy, with the majority of published studies evaluating either subcon-junctival or intravitreal injection.In recent years, many alternative agents and materials have been investigated for the purpose of wound modulation in glaucoma filtration surgery. In particular, research has focused on novel pharmacological compounds to modulate wound healing, physical barriers to limit scleral-conjunctival fibrosis, and better drug delivery of wound modulating agents. This section will provide a discussion of some of these agents. Most of these agents have only been analyzed through one or two studies and are still in the laboratory phase of development but do demonstrate some promise.Anti-Placental Growth Factor (Anti-PIGF)1617 Researchers have hypothesised that upregulation of placental growth factor may account for the persistent Inflammatory response postfiltration surgery that cannot be inhibited by either selective or nonselective anti-VEGF.20 Placental growth factor (PIGF) is a VEGF-homologue that binds solely to VEGF-R1.21 It is notinvolve dinphy siological angiogenesis and acts only on pat hological angiogenic and in fammatory processes.2223 In a multifaceted study conducted by Van Bergen et al,24 the authors demonstrated that PIGF was upregulated by 40% in the aqueous humor of human eyes with glaucoma compared to nonglaucomatous eyes; this upregulation was locally produced as the increased PIGF was not demonstrated in the serum of the same patients. The authors also reported a further enhanced upregulation of PIGF if intravitreal bevacizumab was administered. In a mouse model of glaucoma filtration surgery, the authors reported improved surgical outcome with increasing bleb survival and bleb area following intracameral injection of anti-PIGF. This was associated with a significant reduction in cellular proliferation, Inflammation and angiogenesis during the first postoperative days after surgery, and with a decrease in collagen deposition at the later stages. They hypothesized that PIGF may be a potential therapeutic target for glaucoma surgery, and proposed that a combination of optimal-dosing anti-PIGF with suboptimal-dosing anti-VEGF may be more effective at reducing scar formation compared to monotherapy with either agent alone.Although anti-VEGF has been demonstrated to improve surgical outcomes, the improvement is thought to occur mostly as a result of inhibition of angiogenesis rather than a ny significant anti-Inflammatory response. Infiximab25 and it is increasingly employed to treat ocular inflammatory conditions.2627 Turgut et al28 investigated the efficacy of post-trabeculectomy topical infiximab eye drops  compared with (1) intraoperative MMC 0.4 mg/ml for 3 minutes, (2) trabeculectomy with postoperative saline drops (four times a day for 14 days) (sham group), and (3) no treatment (control group) in 28 rabbits. At day 14, the rewere higher mean fibroblast and mononuclear cell num bers as well as higher intensities of immunofuorescent staining with transforming growth factor-β (TGF-β), fibroblast growth factor-β (FGF-β), and platelet-derived growth factor (PDGF) in the sham group compared to the control group. The mean Inflammatory cell counts and immunostaining intensities TGF-β, FGF-β, a nd PDGF were lower in bot h the MMC group and infiximab group compared to the sham group, but there was no significant difference between the MMC group and infiximab group.Tumor ne crosis factor alpha (TNF- α) ha s also been identi fe d as a potential target for wound healing modulation in glaucoma filtration surgery. It is a local paracrine and autocrine regulator for low-density leukocyte and endothelial cells. It stimulates mononuclear phagocytes as well as other cell types that produce various proInflammatory cytokines and chemokines, and induces migration of polymorphic nuclear leukocytes. Infiximab is a mouse/human chimeric monoclonal IgG1 antibody against TNF-α. It binds to TNF-α and reduces both lymphocyte migration and production of proInflammatory cytokines. Its use in systemic inflammatory disorders is well-documented,Trastuzumab29 Trastuzumab is a humanized monoclonal antibody against the extracellular domain of the human epidermal growth factor 2 (HER2). It is most widely used in the treatment of overexpression of HER2 in early or metastatic breast cancer, although its mechanism of action is not completely understood.30 When compared to trabe-culectomy combined with intrascleral application of MMC (0.4 mg/ml for 3 minutes), trabeculectomy with a subcon-junctival injection of trastuzumab (1.2 mg/0.1 ml) showed comparable numbers of mononuclear cells and fibroblasts in the filtration site on histology (p > 0.05). Similarly, the immunostaining intensities of TGF-β, FGF-β, and PDGF were also comparable between the two groups (p > 0.05).The same investigators also assessed the effects of subcon-junctivally-administered trastuzumab in the rabbit model of filtration surgery.Connexin43 Antisense Oligodeoxynucleotides (Cxn43 AsODN)31 They play a role in in fammation, 32 cell migra-tion33 and tissue contraction.34 Gap junctions have a complex structure: Six connexin protein subunits oligomerize to form a hemichannel called a connexon; two connexons from neighboring cells dock to form a complete intercellular junction channel. Multiple intercellular channels cluster together to form gap junctions with the number of channels in a plaque varying considerably.31 Connexin43 (Cxn43) is one of the most ubiquitous and studied isoforms.3536 Connexin43 antisense oligodeoxynucleotides (Cxn43 AsODN) has been demonstrated to cause a transient reduc tion in Cxn43 protein expression and improve cutaneous wound healing. Coutinho et al37 reported faster wound closure and smaller scar formation following partial-thickness burn wounds in mice after application of Cxn43 AsODN. Qiu et al38 also demonstrated reduced Inflammation, both macroscopically and microscopically, in addition to improved wound healing following one application of Cxn43 AsODN in both incisional and excisional cutaneous wounds in rats. Deva et al39 compared the histological outcome of trabeculectomy combined with a subconjunctival injection of Cxn43 AsODN in pluronic gel with an injection balanced salt solution or pluronic gel into the formed bleb of rabbit eyes. They observed that eyes that received Cxn43 AsODN had reduced Cx43 upregulation at 8 and 24 hours post-injection, which led to less myofibroblast upregulation at days 5 and 21 and reduced scarring at day 21 compared to controls.Gap junctions are structures that allow direct signalling between cells.Physical Barriers40 In addition to acting as a physical barrier, amniotic membrane also has additional anti-Inflammatory and anti-fibrotic properties, which may further reduce the risk of scar formation. Trabe cu-lectomy combined with amniotic membrane trans plantation and MMC has shown very promising outcomes in terms of IOP control and complications for eyes with refractory glaucoma4142 and eyes considered at high risk of surgical failure.43 However, other studies, including two random ized clinical trials, assessing primary trabeculectomy with amniotic membrane vs primary trabeculectomy alone (without the use of MMC or 5FU) for POAG have demons-t rated no sign ifica nt benefit in IOP cont rol at the 1 and 2-year time points.46 These differences in reported out comes may partly be due to the different techniques of amniotic membrane placement as well as the use of antimeta bolites. Nevertheless, the evidence base for the use of amniotic membrane in glaucoma filtration surgery currently remains inconclusive.The concept of using amniotic membrane during glaucoma filtration surgery was first described in 1998.49 It is a nonabsorbable material that reduces tissue adhesion and fibrous cap sule thick ness. However, a ra ndomized clinical trial found no significant benefit in IOP control or complication rates in primary trabeculectomy with or without expanded polytetra fuoroethylene membrane placed underneath the scleral fap.50Expanded polytetrafuoroethylene (Gore-Tex) membrane has been assessed in glaucoma filtration surgery with some promising results.5152 When compared to Interceed, an oxidated regenerated cellulose matrix, Sepraflm demonstrated no significant difference in IOP or bleb appearance but unfortunately, neither seemed to be able to significantly suppress the wound healing reaction following trabeculectomy.53 Correspondingly, when Interceed was evaluated in trabeculectomy alongside Surgical, another oxidated regenerated cellulose matrix, both had similar outcomes in terms of IOP, bleb appearance and lack of effect on fibroblast proliferation.54 However, a more promising option may be trabeculectomy with a honeycomb-patterned biodegradable film composed of poly(L-lactide-co-ε-caprolactone). This has been demonstrated to produce comparable IOP reduction and bleb survival to MMC-en hance d t r abeculectomy, but wit hout the associated complications.55Various other flms and materials have also been evaluated for their ability to form a physical barrier and pre vent adhesions between the sclera and the conjunctiva, mostly in rabbit models. Sepraflm is a biodegradable solid flm composed of hyaluronic acid and carboxymethyl cellulose. Studies of trabeculectomy have shown Sepraflm to reduce adhesions around the conjunctiva and sclera resulting in more prominent bleb formation and lower mean IOP compared to without Seprafilm.5657 OloGen degrades naturally within 90 to 180 days of implantation.OloGen is a bioengineered, biodegradable, porous collagen-glycosaminoglycans matrix implant that is placed on top of the scleral fap and under Tenon's capsule during trabeculectomy. It was developed as a means of providing controlled resistance between the anterior chamber and the subconjunctival space in the early postoperative period. In addition, its porous structure encourages conjunctival fibroblasts and myofibroblasts to grow into the pores and secrete connective tissue in the form of a loose matrix. As a result, it reduces scar formation and wound contraction and eliminates the need for adjunctive antimetabolites.56 significantly lower IOP was seen up to 28 days in eyes that underwent trabeculectomy with OloGen implant compared to unaugmented trabeculectomy. Histological analysis of the surgical site demonstrated loose, randomized collagen fibers in the conjunctival bleb and an unhealed scleral tunnel in the OloGen group compared to dense collagen deposition in the subconjunctival space in the control group. In a pilot study,58 40 eyes of 30 patients were randomized to either trabeculectomy with OloGen implant or unaugmented trabeculectomy alone and followed for 6 months. There was no statistically significant difference in IOP control between the two groups at 6 months: the mean IOP was comparable between the two groups , and 90% from each group achieved complete success of achieving IOP ≤ 21 mm Hg without medication. Although not statistically significant, one eye from the OloGen group developed endophthalmitis 10 days after initial surgery and required vitreoret inalinte rvention . Five eyes from the control group and two eyes from the OloGen group developed bleb encystment at 1 month post-trabeculectomy and all patients required treatment with subconjunctival 5FU injections postoperatively (number of injections ranged 2-7).In the rabbit model,57 In 39 eyes of 33 subjects, Senthill et al59 observed lower IOP in the MMC group at 6 months compared to the OloGen group, but no significant difference at 12 or 24 months. On the contrary, Rosentreter et al60 demonstrated significantly lower IOP in MMC-augmented trabeculectomy compared to OloGen-augmented trabe culectomy in 20 eyes of 20 patients at 1 year; 100% of eyes in the MMC group achieved complete success of IOP ≤18 mm Hg and only 50% of eyes in OloGen groups achieved complete success at 1 year. However, they also reported more avascularity in the MMC-augmented blebs than the OloGen-treated blebs in this study.60Subsequently, several randomized controlled trials have compared MMC-augmented trabeculectomy with OloGen-implanted trabeculectomy with varying results. In the largest RCT evaluating 40 eyes of 40 patients, Cillion et al found no significant difference in terms of IOP control and complications between the two groups at up to 24 months' follow-up.6162The use of OloGen implantation has also extended to phacotrabeculectomies and Ex-Press shunts, again with varying reported results when compared to MMC.63 In this study, one patient developed implant exposure, one developed a corneal dellen adjacent to the implant, and two patients required topical hy poten sive medication at the end of t he follow-up per iod.63Another biodegradable implant composed of collagen glycosaminoglycan matrices (CGM) has been introduced for the treatment of post-trabeculectomy hypotony. The implant is inserted subconjunct ivally t h roug h an incision adjacent to the bleb and placed over the scleral fap. In a pilot study of 12 eyes, IOP increased from a mean of 4 to 10 mm Hg after a naver age follow-up of 6 months. Drug Delivery of Current Pharmacological AgentsAlternative methods of delivering antimetabolites in a sus tained yet safe manner was discussed in Part I of this review. In this section, the focus will be on novel forms of drug delivery of other pharmacological agents.64 In canine trabeculectomy, implantation of a gelatin hydrogel chymase inhibitor combination reduced cell proliferation and better maintained IOP at 12 weeks compared to controls.65 Other novel hydrogels include a bioactive self-assembled peptide that can be used either alone or loaded with 5FU in glaucoma filtration surgery.6667 These hydrogel-compounds are still in the experimental phase.As previously discussed in Part I, various hydrogels have been evaluated in animal models for their ability to provide slow, sustained release of incorporated wound healing modulators. In addition to anti-fibrotic agents, hydrogels have also been developed to incorporate other phar macological agents for use in animal trabeculectomy models. Carbopol 980 hydrogel loaded with paclitaxel demonstrated anti-fibrotic effects on rabbit conjunctival wound healing that was similar to that of MMC alone.68 Compared to the rabbit eyes with non-bevacizumab-loaded polyurethane implants, the eyes with bevacizumab-loaded implants did not demonstrate significant differences in bleb area in vivo or collagen density ex vivo. However, there was a significantly lower number of VEGF-expressing fibroblasts in the bevacizumab group.A new bevacizumab-loaded polyurethane subconjunctival implant has been developed in a rabbit model designed to be inserted adjacent to the filtration site on the episclera.69 Two randomized clinical trials evaluated the benefit of subconjunctival hyaluronate in glaucoma surgery: the first at the conclusion of trabeculectomy,70 and the second when performed in conjunction with bleb needling for failing trabeculectomy blebs.71 Lopes et al reported no significant difference in success rates when comparing subconjunctival sodium hyaluronate 2.3% against balanced salt solution at the conclusion of trabeculectomy after a mean follow-up of 12 months.70 Naravanaswamy et al compared a 5FU and hyaluronate 2.3% mixture vs 5FU alone in bleb needling of failing blebs and found no significant differences in outcomes at 3 months although the 5FU alone group required repeat needling more frequently.71 Aretrospective case series with longer follow-up of 12 months found of bleb needling with subconjunctival sodium hyaluronate 1.4% and 5FU to be effective for reducing intraocular pressure in the medium term with few serious complications.72The interest in sodium hyaluronate stems from its utility in sustained drug delivery as well as its in herent anti fibrotic properties and ability to act as a physical spacer. When loaded with dexamethasone, it allowed extended release of the steroid at concentrations sufficient for antiproliferative activity over a period of nearly 2 days.in vitro; subconjunctival implantation reduced postoperative Inflammation and prolonged bleb survival in rabbit trabeculectomy.73 A sirolimus sustained-delivery flm composed of polyactioglyconic acid polymer also showed similar anti-Inflammatory effects after filtration surgery in a rabbit model.74Another approach that has received keen research interest is microflms loaded with pharma cological agents. Microflms composed of poly  and loaded with pred nisolone acetate provi ded steady, sust ained release of prednisolone Glaucoma filtration surgery has an indisputable place in the management of glaucoma but efforts to minimize com-pli cations associated with antifibrotic use remain ongoing. Significant research is being conducted on newer, alternative forms of trabeculectomy wound modu lation. Agents such as anti-VEGF and other novel pharmacological compounds have shown promise, but require confirmation and refinement in application through larger, prospective clinical trials. It is an exciting evolving era for glaucoma wound modulation, and undoubtedly the humble trabeculectomy will be undergoing a facelift in the near future.


Pax-C intron [PaxC]) could be useful for increasing the resolution of coral spat identification in ecological studies. One hundred and fifty settlement plates were emplaced at nine sites on the fringing reefs of Kenting National Park in southern Taiwan between April 2011 and September 2012. A total of 248 living coral spats and juveniles  were detected on the plates with the aid of fluorescent light and collected for molecular analyses. Using the COI DNA barcoding technique, 90.3% (224/248) of coral spats were successfully identified into six genera, including Acropora, Isopora, Montipora, Pocillopora, Porites, and Pavona. PaxC further separated I. cuneata and I. palifera of Isopora from Acropora, and ORF successfully identified the species of Pocillopora (except P. meandrina and P. eydouxi). Moreover, other cnidarian species such as actinarians, zoanthids, and Millepora species were visually found using fluorescence and identified by COI DNA barcoding. This combination of existing approaches greatly improved the taxonomic resolution of early coral life stages, which to date has been mainly limited to the family level based on skeletal identification. Overall, this study suggests important improvements for the identification of coral recruits in ecological studies.The identification of coral recruits has been problematic due to a lack of definitive morphological characters being available for higher taxonomic resolution. In this study, we tested whether fluorescent detection of coral recruits used in combinations of different DNA-barcoding markers (cytochrome oxidase I gene [COI], open reading frame [ORF], and nuclear  Coral recruitment, defined as the number of coral larvae entering the adult population, plays a critical role in the resilience of coral populations e.g., settlement plates) have been widely used to study scleractinian coral recruitment in the past three decades e.g., strong waves) Artificial substrates  gene has been widely used in diverse applications of DNA barcoding in animals Stylophorab gene to increase the taxonomic resolution of coral recruits in damaged reef sites in southeast Florida. In the Pacific region, only Suzuki et al. Acropora species recruits in the Ryukyu Archipelago using two-step molecular sorting with mitochondrial and nuclear markers.DNA barcoding is a taxonomic method that uses a short genetic marker in an organism's DNA to identify it as belonging to a particular species Here, we describe diversity in coral recruits collected from plates deposited during April 2011 to September 2012 at different sites around Kenting National Park, Taiwan. We combined fluorescent censusing and DNA barcoding techniques to determine if those tools can improve the resolution of coral recruit identification. Finally, we recommend a combination of molecular markers to use in future studies to significantly increase our knowledge of critical ecological processes like coral recruitment.This study was conducted with permission of the Kenting National Park authorities, Taiwan. Samples were collected as part of long-term ecological monitoring research with permit nos. 1002901146 and 1010001032 for the years 2011 and 2012, respectively. All collecting procedures were done with proper precautions for minimizing impacts to reefs.This study was conducted along the coast of Kenting National Park (KNP), Taiwan . FringinTerra cotta plates 12.5×13×1 cm) were deposited at every site, fastened to 10-cm stainless steel expansion bolts imbedded into natural substrates at ∼5 m depths with a pneumatic drill, with bolts separated by distances of 20–50 cm. Plates were secured in horizontal positions with two stainless steel washers and a hex nut about 3–5 cm above the substrate that was available for coral recruitment in close proximity to adult colonies. Plates with different deposition/collection dates as well as time spent underwater were pooled for this study to maximize the diversity of coral recruits by integrating the wide variety of pre- and post-settlement processes at the different study sites. Settlement plates were deployed for short-term (4 to 7 months) and long-term (4 to 16.5 months) surveys. The first deployment of plates was conducted just before the coral spawning season, which extends from May to September in southern Taiwan (some brooder species can reproduce year round) ×1 cm werPlates collected were placed individually in Ziploc bags underwater, transported to the laboratory, and processed as soon as possible to detect and sample coral recruits.i.e., spats surviving post-settlement mortality and having a chance to join the population. Live spats were spotted under excited blue light using long-pass (wavelength >500 nm) barrier filter glasses (Night Sea VG1) in a darkened room. Fluorescent and normal images of coral spats were photographed under blue and white light for confirmation of coral spats or other fluorescing organisms using a digital Canon Kiss 4 camera with a 100 mm macro lens. Photographic lighting was provided by a dive light (Night Sea BW-1) by toggling between white light without a lens filter and blue light excitation with a long-pass lens filter (Night Sea BB67) mounted on the lens. Spats were then removed from plates and fixed separately in 1.5 mL Eppendorf tubes containing 100% ethanol for further molecular analysis. The above workflow was processed within 6–12 hours after plates were collected from the sea on each retrieval day. The time spent by two people inspecting each plate was 30 min on average (10–120 min) depending on how many recruits were found.Coral spats were defined as individuals that successfully settled and survived by the time of sampling. This corresponds to effective recruitment; 2O, with a final centrifugation at 13,000 rpm for 3 min. PCR amplification was performed in a 50 µl total volume with a final concentration of 1X Buffer , 0.1 mM dNTP, 0.1 mM of each primer, 3 mM MgCl2, and 0.5 U Taq DNA Polymerase (Invitrogen). The COI primers used were described by Folmer et al. Acropora/Isopora species were further subjected to a second PCR reaction with the Pax-C intron PaxC_intron-FP1: 5′-TCC AGA GCA GTT AGA GAT GCT GG-3′ and PaxC_intron-RP1: 5′-GGC GAT TTG AGA ACC AAA CCT GTA-3′. Coral recruits identified by COI as Pocillopora were subsequently examined by PCR using the ORF region DNA extraction was conducted using a modified high-salt method www.barcodeoflife.org) were retrieved and compared to the 224 sequences  from this study. Because size differences in the scleractinian DNA COI database introduced a significant number of gaps, short or incomplete sequences were removed from the analysis in order to use only sequences with >500 bp. Sequences were aligned using CLUSTAL W with default settings. After a model test, neighbor-joining (NJ) cluster analysis was conducted with 1000 bootstrap replications using MEGA 5.2 Acropora/Isopora samples  were aligned and trimmed to a length of 335 bp to compare with known Isopora sequences from adult colonies and Acropora sequences downloaded from NCBI. The sequences of ORF from Pocillopora spats  were aligned with sequences from DNA sequences were assembled using SeqMan  and then sequence alignments and analyses were conducted using MEGA v5.2 A total of 150 out of 159 plates were successfully retrieved from the nine sites between August 2011 and September 2012 . In totaUnder blue light excitation, most of the coral spats emitted a green fluorescence against a contrasting red or orange chlorophyll fluorescence from algae . Ascidia2  appear to be the most difficult to phylotype, with only 86.7% (157/181) of them being successfully identified. All larger spats were successfully phylotyped  were identified based on COI phylogenetic analysis (Pocillopora (n  =  183) represented 81.7% of identified coral spats, with Porites  and Acropora  as the second- and third-most abundant groups in our samples (Pavona (n  =  3) was detected in TS and WLT, whereas Isopora (n  =  7) and Montipora (n  =  1) were found only in TZB and WLT, respectively , as observed in Porites) exhibit very low fluorescence. Additionally, the use of white light may avoid sampling other organisms presenting highly similar fluorescence levels, including other cnidarians, polychaetes, ascidians, and some algae.Fluorescence censusing has significantly improved our ability to detect coral recruits In the last three decades, the analysis of the coral recruit diversity has relied mainly upon the microscopic examination of skeletal characters after soft tissues have been removed Montipora, Isopora, and Pavona recruits are the first reliable records of these genera on settlement plates around Taiwan. If present in our study, we should also have been able to identify Seriatopora and Stylophora recruits, as COI sequences have recently shown their efficiency in delineating both genera While some studies show limitations to the use of DNA barcoding techniques on corals, they provide useful tools for documenting diversity at least to the generic level. However, and surprisingly, only a few studies to date have tried to use these techniques to identify coral recruits Isopora and Pocillopora. PaxC has been previously used to delineate Acropora from Isopora by using Isopora cuneata as an outgroup Isopora cuneata and Isopora palifera with a 100% bootstrap value. Schmidt-Roach et al. Pocillopora damicornis  of coral spats remained unidentified after DNA extraction, PCR amplification, and sequencing, we believe this value can be reduced further by improving the molecular analysis process. Because DNA extraction requires good quality DNA for accurate analyses, improving coral spat preservation after field collection and adapting the DNA extraction protocol to these tiny and degraded organisms could result in better yields. Our use of a high-salt extraction technique may not have been the best method for this biological material. Alternative DNA extraction protocols (such as the DNA extraction kit, organic extraction, and the whole genome amplification kit (REPLI-g, Qiagen, Low recruitment at OL was probably due to the low fecundity or low survivorship under the influence of warm water discharges from a nuclear power plant , which hBy combining the use of existing tools, this report provides an original approach for precisely measuring effective coral recruitment on reefs. Further research based on this work may consider post-settlement mortality, which is usually disregarded in most studies on coral recruitment. In the future, finding additional molecular markers that are better suited for the identification of coral spat to genus and species is expected to compensate for historically lower levels of taxonomic resolution in previous research on early coral life stages and allow a more precise understanding of their role in the resilience of coral reef communities.


Visual stimuli quickly activate a broad network of brain areas that often show reciprocal structural connections between them. Activity at short latencies (<100 ms) is thought to represent a feed-forward activation of widespread cortical areas, but fast activation combined with reciprocal connectivity between areas in principle allows for two-way, recurrent interactions to occur at short latencies after stimulus onset. Here we combined EEG source-imaging and Granger-causal modeling with high temporal resolution to investigate whether recurrent and top-down interactions between visual and attentional brain areas can be identified and distinguished at short latencies in humans. We investigated the directed interactions between widespread occipital, parietal and frontal areas that we localized within participants using fMRI. The connectivity results showed two-way interactions between area MT and V1 already at short latencies. In addition, the results suggested a large role for lateral parietal cortex in coordinating visual activity that may be understood as an ongoing top-down allocation of attentional resources. Our results support the notion that indirect pathways allow early, evoked driving from MT to V1 to highlight spatial locations of motion transients, while influence from parietal areas is continuously exerted around stimulus onset, presumably reflecting task-related attentional processes. Visual cortex consists of numerous hierarchically organized areas that form a densely connected network. Anatomical projections between these areas are often reciprocal: when a lower-level area projects to a higher-level one, a projection back to the lower-level area often exists1Quickly after stimulus onset, visual activity propagates from primary visual cortex (V1) to widespread occipital, parietal and frontal areas, like the frontal eye fields (FEF)34591011In addition to recurrent processing between visual areas, the parietal cortex and FEF are known to exert top-down influences that help flexibly adapt behavior to changing task demands and context101314161718Effects of top-down influence on visual processing are well-documented by experiments in which participants view identical stimuli under changing task demands. Activity in motion- and color-sensitive areas like MT and V4 depends on whether participants attend to stimulus motion or color, at latencies as early as 100 ms after stimulus onset20212425Top-down influence has thus been well studied by manipulating task-demands, but it is less clear what directed interactions from higher level areas take place in the absence of explicit, stimulus-related task demands in early visual processing. Here we use EEG and electrical source imaging to investigate the dynamics of recurrent interactions between visual areas and of top-down influence from attentional areas in human observers. We presented task-irrelevant checkerboards in the lower left or right visual field, which elicit well-characterized activation patterns in the EEG that can be accurately localized using EEG source imaging57To investigate the dynamics of two-way interactions in visual processing, directed connectivity measures with high temporal resolution are needed. Granger-causal modeling is an established statistical method to estimate the strength of directed interactions between brain areas29313233353637We hypothesized that by combining EEG source imaging and Granger-causal modeling in human observers, feedback and top-down influence can be identified and distinguished at short latencies. We first localized a network of 12 ROIs in each observer that included areas in occipital, parietal and frontal cortex for each observer using fMRI. We then applied wPDC to time-series of estimated current densities obtained from separate EEG measurements to identify the major drivers at the C1 and N1 peak latencies and determine whether they selectively targeted other areas. The results showed the expected bottom-up driving in the first 100 ms (i.e. from V1 to higher-level areas like MT and LOC), but in addition, we found fast recurrent interactions between MT and V1 in response to stimulation and a large, ongoing influence from parietal cortex already around stimulus onset.We briefly presented checkerboard stimuli in the lower left or right visual field while participants detected a color change at the center of the screen. Visual evoked potentials on the scalp time-locked to checkerboard onset showed typical C1 and N1 components over the hemisphere contralateral to checkerboard presentation, peaking at 76 and 146 ms after stimulus onset .Twelve ROIs were successfully identified for each individual participant using data recorded in a separate fMRI session. The ROI locations are illustrated in To obtain activity estimates for each ROI from the EEG signal we used a distributed linear inverse solution (WMN) based on individual headmodels, thus taking into account each participant’s unique anatomy in the forward model 40d = 0.13) and beta band , see 5d = 0.24).The C1 component is to a large extent generated by activity in lower-level visual areas of the contralateral hemisphere56To analyze the total influence from each ROI we summed its outgoing functional connectivity strengths (wPDC) to all other ROIs. This summed driving reflects the total influence of the ROI on the network at a given time and frequency. At the C1 peak latency, V1, MT and LOC showed larger summed driving in the beta band for contralateral than ipsilateral stimulation. This stimulus effect selectively occurred around the peak latency of summed driving from these areas, coinciding with the peak latency of the C1 component ,b.Overall, lateral intraparietal cortex (LIP) in both hemispheres showed the largest summed driving at the C1 peak latency ,c, and tWe next analyzed whether the areas showing a stimulus effect showed specific driving toward other ROIs. We found that the main targets of contralateral V1 (cV1) were cLOC and cMT, in line with a feed-forward information flow from V1 toward higher-level visual areas . The maiAt the N1 peak latency stimulus effects were most pronounced in the alpha band . GrangerThe main directions of influence from cV1, cLOC and cLIP are shown in To study the dynamics of recurrent interactions and top-down influence in early visual processing of human observers we combined EEG source imaging and Granger-causal modeling in a network of twelve ROIs that included visual and attentional areas in occipital, parietal and frontal cortex. The directed functional connectivity results at the C1 peak latency showed a pattern of driving largely in line with feed-forward information flows from V1 and MT. However, recurrent driving from MT toward V1 was also observed at short latencies. In addition, the results suggest that LIP is an important network driver at both the C1 and N1 peak latencies, particularly in the alpha band.Previous studies using directed functional connectivity measures have shown evidence for top-down interactions in visual processing. Early PET and fMRI studies used structural equation modeling to show a role for directed interactions from lateral frontal areas in visual processing43These past studies, however, have been unable to provide a dynamic characterization of top-down influence because they used fMRI or PET recordings that have insufficient temporal resolution, or analyzed interactions across time. Here, we used Granger-causal modeling with high temporal resolution to investigate two-way interactions in visual processing.At the C1 latency, stimulus effects in the PSD were largest in the beta band, whereas at the N1 latency, stimulus effects appeared in the lower alpha band . Activit4748495051Connectivity results around the C1 latency showed larger summed driving from V1, MT and LOC for contralateral stimuli. MT is activated quickly after stimulation, in line with known bottom-up spread of activity in the contralateral hemisphere. Activity in LOC can also occur at short latencies38The main targets of cV1 at the C1 latency were cLOC and cMT, in line with a feed-forward spread of activity from V1 toward higher-level visual areas ,e. At th3The results also showed a fast two-way interaction between cV1 and cMT around the C1 peak latency (76 ms after stimulus onset). Direct interactions between MT and V1 are possible through monosynaptic, reciprocal projections112How could driving from MT to V1 arise at these short latencies? In primates, initial MT activation can co-occur with the initial activation of V13125657Evidence for the importance of fast MT–V1 interactions in human comes from a TMS study that showed that interference with V1 can abolish phosphene percepts elicited by TMS over MT, but only when TMS over V1 follows the TMS over MT at short latencies6010We found a second instance of feedback influence around the C1 latency in the driving from cLOC toward iV1 . This drThe fast interactions described here support the view that even simple, task-irrelevant visual stimuli evoke fast, two-way interactions between multiple areas91054In addition to specific recurrent interactions between visual areas, our findings also demonstrate interactions involving LIP and FEF, whose activity is known to reflect attentional processes131410The results identified surprisingly strong and ongoing driving from LIP, already at stimulus onset. LIP is a key area of the dorsal attention network for top-down control of visual attentionIn addition, LIP driving did not change with stimulus location and was strongest in the alpha band. LIP plays a role in spatial attention which are known to manifest in the alpha bandOur results, LIP appears to modulate activity in various, broadly distributed areas, in line with its widespread structural connectivityThe dominance of LIP over time  suggestsIn sum, our directed connectivity results distinguished a pattern of fast feed-forward driving in visual processes that co-exists with top-down driving from MT onto V1 at short latencies. LIP appears to play a considerable role in evoked visual responses through ongoing driving that most-likely reflects task-related processes. Our results suggest that vision is best viewed as a fast, highly interactive processes distributed among cortical areas. That we were able to separate fast feedback and top-down attentional processing in human by combining EEG source imaging and Granger-causal modeling is a promising basis for future studies in which stimulus and tasks properties are independently varied to better understand the functional roles of feedforward, recurrent and top-down processing.Sixteen right-handed participants took part in the experiment  with good visual acuity , as measured by the Freiburg visual acuity testThe checkerboard stimulus was a circular wedge (70 degrees of arc), offset by 2 degrees from the center of the screen and subtending 6 degrees of visual angle. Polarity changes (black/white) where spaced 3 cycles per degree radially, and 12 cycles per degree angularly .Object images came from the publicly available SVLO databaseThe same stimuli were used in the fMRI and the EEG session. Stimuli were back-projected onto a screen during fMRI recordings, and presented on a CRT monitor during EEG recordings. Stimulus generation, presentation and timing was controlled with PsychoPy software70We used MRI to acquire structural brain images and to functionally localize bilateral regions of interest (ROIs) that respond to different aspects of visual processing, namely primary visual areas (V1/2), lateral occipital cortex (LOC), frontal eye fields (FEF), lateral intraparietal area (LIP) and the middle temporal motion area (MT).Participants were scanned on a whole-body Tim Trio system , at the Brain and Behaviour Laboratory at the University of Geneva, with a radio-frequency (RF) body transmitter and a 32-channel receiver head coil. Each scanning session consisted of an initial localizer scan, enabling appropriate centering of the field of view for subsequent scans. Three T2*-weighted echo-planar-imaging (EPI) functional runs  were acquired, followed by a T1-weighted structural image . The three functional localizer runs are described below.In the localizer run for V1/2, participants were required to fixate a central dot and to indicate with a button-press (using their right index finger) when they detected a change in its color. A block design was employed in which checkerboard stimuli were presented successively in the lower left and right visual field. Checkerboards remained on screen for between 15 and 23 seconds (7–11 TRs) while their contrast polarity was flipped at random intervals (100–300 ms). Every pair of left-then-right checkerboards was followed by a period of variable duration (13–21 s), during which only the fixation dot remained on screen. A total of fifteen blocks were presented, with a total duration of 4.5 min (130 TRs) that remained identical over participants.To localize LOC we presented object and scrambled stimuli in the center of the screen for 300 ms, ISI varied randomly (200–500 ms). Participants were instructed to press a button when a target picture (a red flag) was presented. This decoy task was employed to keep participants alert and focused on the content of the images. Stimulus presentation was blocked with blocks lengths varying between 15 and 23 seconds (7–11 TRs), and each block of stimulation was followed by a period of variable duration (10–21 s) during which only the fixation dot remained on screen. A total of twenty blocks were presented, with a fixed total duration of 6.5 min (165 TRs).FEF, LIP area MT were localized using a saccadic pursuit taskAnalysis and preprocessing was carried out using SPM 8, running in Matlab R2010b . Preprocessing for each subject included: 1) rigid realignment of each EPI volume to the first in the session, 2) co-registration of the structural volume to the mean EPI image, and 3) spatial smoothing using a Gaussian kernel of 8 mm.Fixed-effects analyses were carried out separately for each localizer, using a general linear model for each participant in which every scan was coded for condition, and null events were left unmodeled. The analysis was carried out in the participants’ native space in order to permit comparability of the single-subject fMRI results with the single-subject EEG results. In order to allow identification of functional activations according to existing templates, and to make the reporting of results comparable with the existing literature, the normalization parameters warping each participant’s T1 scan to MNI template space were calculated.V1/V2, MT, FEF and LIP were localized using statistical contrasts with baseline. LOC was localized by a statistical contrast between images of objects and phase-scrambled versions of those objects. This localizer also separately identified fusiform gyrus (FG) in each subject.The 12 ROIs were successfully localized in each participant. Where possible, a statistical threshold of p < 0.05, corrected for familywise-error was applied. Otherwise, the peaks were identified at a threshold of p < 0.001 uncorrected  or p < 0.01 .EEG was recorded on a separate day in an electrically shielded room with a 256 channel EGI Geodesic setup . Recordings were referenced against the Cz electrode, the impedances were kept below 50 kΩ, data were digitized at 1000 Hz.Checkerboard patterns were presented for 200 ms in the lower left or right visual field (200 repetitions per location) with a random presentation order and randomly varying inter-stimulus intervals (500–1200 ms).Participants sat at 1 m from the screen and were instructed to maintain fixation on a small disc  placed centrally on the screen during the entire recording block. Recording blocks lasted about 4 minutes, breaks were offered in between.To help maintain attention on the center of the screen participants were instructed to press a button when the fixation point occasionally changed to red. Color changes of the fixation point were correctly detected in 93% of cases (range across participants 68–100%), showing good task-compliance. For three participants intermittent response box failures precluded the collection of behavioral data during EEG recordings. In those cases we verified task-compliance immediately after the recording block.Individual electrode positions were digitized in 3D with a photogrammetry system . Off-line, recordings were DC-corrected, high-pass filtered at 0.1 Hz and divided into epochs between −100 and 400 ms around stimulus onset. We excluded the cheek and lower neck electrodes for the further analysis, keeping 204 electrodes. Epochs were visually inspected and those with muscle or eye-movement artifacts and/or amplitudes over 75 μV were excluded. On average 163 Left and 163 Right stimulation epochs per participant were kept for further analysis. Noisy channels were identified and interpolated using 3D spherical splinesp-value, as obtained by the fMRI localizers. To calculate individual lead fields we used realistic, analytical head models constructed from individual MRIs using a manifold of locally adapted spheres4075We derived current density time-series for each epoch using a distributed linear inverse solution (weighted minimum norm), implemented in the freely available Cartool softwareDirected connectivity between the ROIs was estimated using Granger-causal modeling of z-scored single trial data in the 1–100 Hz range29303928For each ROI we summed the outgoing wPDC strengths toward the other 11 ROIs as a measure of the total driving influence on the network.The analyses of the EEG and connectivity data aimed to limit the number of statistical tests by selecting a small number of ROIs from the more than 5000 possible source-points. In addition, we used a data-driven approach to restrict testing to the C1 and N1 peak latencies, as determined by the grand-average ERP , and to At the C1 and N1 latency we determined whether responses to contralateral stimuli were increased with respect to those of ipsilateral stimuli for each frequency band, using a non-parametric bootstrapping approach (n = 10000) to avoid unwarranted assumptions of normality. This analysis was carried out on normalized individual PSD responses, using the same data used for spectral scaling of the PDC. Subtracting ipsilateral from contralateral responses assures that the stimulus-driven response is isolated. Ipsilateral activity provides a good control because activity is not expected in the ipsilateral hemisphere until later on. It also controls against the limited spatial resolution  of inverse solutions and provides a simple alternative to computationally intensive and competing surrogate data approaches3180d with pooled s.d.s in the denominatorAt the C1 and N1 peak latency we bootstrapped 95% confidence intervals (CI) of the difference between evoked responses contra- and ipsilateral to stimulation. If the lower bound of the 95% CI exceeds zero, the contralateral response was deemed to exceed that of the ipsilateral response, and the null hypothesis of equal amplitudes for contra- and ipsilateral stimuli rejected. We calculated effect sizes using Cohen’s In the thus selected frequency bands we determined whether the summed wPDC from each contralateral ROI exceeded that of the corresponding ipsilateral ROI by bootstrapping 95% CIs of the difference between their summed wPDC values.For the ROIs that showed increased driving for contralateral stimuli we calculated the average wPDC to all other ROIs and their 95% CIs. This way we explored the strengths of driving toward the other ROIs and determined whether specific ROIs were targeted, thus refraining from arbitrary testing between wPDC strengths toward the 11 other ROIs and from comparing all possible connections in strength without theoretical motivation. The 95% confidence interval provides a useful estimate of the variability in the sample and the range in which the true values are most likely to fallFor connectivity plots, we adapted functions from the eConnectome toolboxHow to cite this article: Plomp, G. et al. Early recurrence and ongoing parietal driving during elementary visual processing. Sci. Rep.5, 18733; doi: 10.1038/srep18733 (2015).


MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs  whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated  Quiescent hepatic stellate cells (qHSCs) are the major cell type responsible for the intrahepatic uptake, storage and secretion of vitamin A (retinoids). Moreover, they are the main regulators of the extracellular matrix (ECM) turnover within the space of Disse by expressing ECM proteins  and by modulating the expression of metalloproteinases and their inhibitors. In response to fibrogenic stimuli HSCs activate and acquire a myofibroblast-like phenotype which is characterized by an aberrant production and deposition of ECM proteins potentially leading to liver fibrosis and resulting in the disruption of the vascular architecture12in vivo and in vitro56COL1A1 and ACTA2 in primary HSCs. Furthermore, several miRNAs down-regulated during HSC activation have been suggested to play an anti-fibrogenic role9891011miRNAs are small non-coding RNAs of approximately 22 nucleotides that act as post-transcriptional gene expression regulators by degrading target mRNAs through perfect complementarity of the miRNA with the 3’ untranslated region (3’UTR) of mRNA or by inhibiting translation if an imperfect base pairing match existsBy integrating miRNA and gene-expression data obtained through the analysis of human qHSCs and their respective culture-activated counterparts we identified relevant miRNAs involved in the maintenance of the quiescent phenotype of human HSCs. Moreover, the integrative analysis allowed us to identify miRNA-mRNA interaction networks potentially involved in HSC quiescence and activation. Finally, we have identified a panel of miRNAs with predicted target genes associated with HSC activation, and thus with a potential role in the repression of activation.MiRNA and mRNA expression profiles were performed using HSCs and liver sinusoidal endothelial cells (LSECs) isolated from 4 cadaveric donors and 2 samples of hepatocytes obtained from two different donors. The protocol and conducted experiments were approved by the ethical committees of St-Luc Hospital and faculty of Medicine of Université Catholique de Louvain. An agreement from the Belgian Ministry of Health was obtained for the hepatocytes and hepatic stem cell bank. MiRNA expression validation was performed on liver samples of 14 healthy subjects and 15 patients with cirrhosis with superimposed alcoholic hepatitis (AH). The healthy controls were selected as previously described+CD45− cells. Purified populations of qHSCs and LSECs were immediately used for total cell RNA and miRNA extraction.Human liver cells were isolated from the left liver segment of healthy donors up to 12 hours after clamping using a two-step perfusion technique2.Homogeneous populations of aHSCs were obtained as previously describedSPARCL1 and ATP1B2) and activation  markers as well as the general HSC (PDGFRB), LSEC (CD32b), macrophage (F4/80) and hepatocyte  markers.The purity of the FACS sorted cell populations and culture-activated HSCs was assessed by quantitative real time PCR for HSC quiescence  were used and isolations were performed as described previously16Male BALB/c mice (aged 9–10 weeks) were used. The experimental procedures were approved by the institutional Animal Care and Use Committee of the Vrije Universiteit Brussel, permit number 12-212-2, and National Institutes of Health principles of laboratory animal care  were followed.4 50 μl /100 g body weight in mineral oil; both from Sigma) twice per week for 2 to 8 weeks or by common BDL (3–10 days). For common BDL, a peritoneal incision was made and the common bile duct was ligated and disrupted. Next, the abdominal cavity was closed with sutures and the animals were allowed to recover. At the end of the procedure, HSCs were isolated by FACS, based on UV-positivity. Isolated cells were not cultured, but immediately used for mRNA and miRNA analysis.Chronic liver injury was induced by repeated intraperitoneal injections of carbon tetrachloride  following a deviation of the standard protocol as recommended by the manufacturer and using 1.5 volumes of 100% ethanol before applying the sample into the column. Total RNA concentration and quality control was assessed using the RNA 6000 pico kit .Taqman human miRNA Array (card A and B)  was used to profile the expression of 758 miRNAs in 4 quiescent and 4 activated HSCs by PCR amplification and real time analysis. Each miRNA array was used in conjunction with pre-defined pools of primers for each array . Due to the small quantity of starting material (30 ng each sample), miRNA cDNA samples were pre-amplified by PCR  prior to loading on the array. The abundance of each miRNA in a total RNA sample was normalized to the level of the RNU6 expression (computation of –ΔCt). Fold changes were computed from the means of each group and statistical significance was established at p ≤ 0.05 (student t-test). A total of 13 mRNA samples, 4 qHSC together with their respective activated HSC samples, 2 hepatocyte samples and 3 LSEC samples were analyzed for gene expression profiling using Affymetrix HG-U219 genechips . Raw data are made publically available on the NCBI Gene Expression Omnibus database, with accession number GSE67664.http://mircomb.sourceforge.net). This software package allowed us to identify potential relevant miRNAs regulating target genes differentially expressed in qHSCs by first identifying differentially expressed genes and microRNAs, secondly calculating significant microRNA and mRNA correlations and third screening databases for known and predicted miRNA-mRNA target pairs. The first step consisted in the identification of differentially expressed genes and miRNAs in qHSCs.In order to identify potential functional interactions of miRNAs with their target genes, we integrated gene and miRNA expression profiles obtained from both miRNA and mRNA arrays by using miRComb-R package .Secondly, we classified mRNAs more highly expressed in aHSCs by using a cut-off of 2-fold higher expression than in hepatocytes and LSECs; again the number of genes in this category was reduced by selecting only those mRNAs that are significantly upregulated between qHSCs and aHSCs.For the miRNAs we also used the 2-FC criteria for selecting those that are up-regulated during culture induced activation and a 1.5-FC cut-off was used for the miRNAs down-regulated during HSC activation.In a second phase of the integration procedure we identified those miRNA-gene pairs showing negative correlation between miRNA and mRNAs. To accomplish this, Pearson correlation of the expressions of all possible combinations of deregulated mRNAs vs deregulated miRNAs were computed. Multiple testing correction was performed in order to reduce the number of false positive correlations and a final cut-off was set to FDR  <0.05. Computed correlations are shown in ® Advantage qPCR premix (Takara). mRNA levels were determined by quantitative real time PCR on an ABI 7900HT cycler (Applied Biosystems) using SYBR green master mix (Life technologies). Individual gene and miRNA expression was normalized to GAPDH or U6 expression, respectively. Relative expression was calculated using the comparative Ct method (2−ΔΔCt). Gene specific primers were produced by Integrated DNA Technologies . Primer sequences used are listed in Expression of mature miRNAs was assessed by reverse transcription using Mir-X miRNA First-strand Synthesis Kit  according to the manufacturer’s instructions and qPCR reaction was performed by using SYBR2 and humidity. LX2 cells were transfected with 50 nM of mirVanaTM miRNA Inhibitor for miR-21 and miR-100 and mirVanaTM miRNA mimic for miR-192 (Life Technologies) using JetPRIME®  according to the manufacturer’s recommendations. mirVanaTMmiRNA mimic and mirVanaTMmiRNA inhibitor negative control (Life Technologies) were used as a calibrator sample to evaluate miRNA and target gene relative expressions. 24 hours after transfection, cells were harvested and immediately used for miRNA and mRNA expression analysis.In order to validate relevant miRNA-mRNA interactions resulting from the integrative analysis, miR-21 and miR-100 expression were knocked down and miR-192 was over-expressed in a human HSC cell line (LX2) (kindly provided by Dr. Friedman). LX2 cells were grown in DMEM containing 10% FBS at 37 °C with 5% CO2, 48 hours after miRNA mimic transfection cells were incubated with EdU for 48 hours prior to fixation and staining of the cells at day 5 of the culture. The ratio of total cells and EdU-incorporated cells was calculated.Cell proliferation was assessed in mouse HSCs after 24 hours mimic miR-192 transfection (Life Technologies). Cell proliferation was measured as active DNA synthesis with the Click-iT EdU Cell Proliferation Assay Kit , following the manufacturer’s instructions as described beforeCell migration of mouse primary HSCs was determined by a trans-well migration assay as described beforeFreshly isolated, primary mouse HSCs were seeded at a density of 15.000 cells/cm2 and transfected with miR-192 or control mimic 24 h after plating. On day 2, transfected cells were serum-starved overnight and subsequently exposed to 10 ng/ml human recombinant TGFβ1 (R&D) for 48 h, prior to being lyzed for RNA extraction.GraphPad Prism v4.0.0  and SPSS V.14.0 for Windows  were used for statistical analysis. Data in the figures are expressed as means ± SEM. Differences among groups were tested for statistical significance by Student t-test or Mann Whitney U test when appropriate. Ns = not significant p ≥0.05, *p <0.05, **p <0.01, ***p < 0.001.+CD32−CD45−) and LSECs (CD32+CD45−) were purified from the resulting non-parenchymal cell fraction by FACS-sorting. Moreover, qHSCs isolated by density gradient from the same patients were cultured until passage four to obtain pure populations of aHSCs. To assess the purity of the different cell populations we measured the expression of specific markers for each liver cell type. qHSC and LSEC sorted fractions were highly enriched in PDGFRB and CD32b respectively and no significant expression of albumin was detected , in the qHSC-enriched nycodenz fraction and in culture-activated HSCs  were found to be highly expressed in qHSCs and significantly down regulated upon in vitro activation (p < 0.05). In contrast, 212 (82%) miRNAs barely expressed in qHSCs were significantly up-regulated during activation  as well as actin cytoskeleton signaling  pathways were found enriched in this set of target genes. These results indicate that qHSCs express a set of miRNAs that may be responsible, at least in part, for maintaining the quiescent phenotype by repressing genes involved in HSC activation. In order to investigate whether miRNAs expressed in qHSCs were targeting/inhibiting the activation process we performed an Although HSCs are the major cell type responsible for liver fibrogenesis, they represent only 8% of the total liver cells. In order to explore if miRNAs dysregulated in HSCs could be detected in human liver we assessed the expression of selected miRNAs in cirrhotic (n = 15) and healthy (n = 14) human liver tissues. MiR-192 was found significantly down regulated  in cirrhotic livers compared to healthy livers indicating that this miRNA, which we have identified as highly expressed in qHSC by the miRNA array, could also be detected in healthy tissue . On the MiR-192 has a high number of predicted target genes (n = 28) in qHSCs, displays a significant reduction in expression between healthy and cirrhotic human liver tissue and it has not been previously reported to be associated with HSC or liver fibrosis. For those reasons, we selected miR-192 for further functional characterization by modulating its expression in human and mouse HSCs  and 6. APLAU and COL5A1, as well as a modest but significant reduction of the activation marker LOX and an increase in SPARCL1, a novel quiescence marker identified in the gene expression array. On the contrary, an inhibition of the endogenous miR-100 expression level enhanced the expression of its two predicted target genes, i.e. BTG2 and SPARCL1, but also caused a ~20% reduction of the basal expression of pro-fibrogenic genes such as COL1A1 and LOX. Accordingly, miR-21 inhibition resulted in the reduction of miR-21 target genes expression (BTG2 and SPRY1) and also in a significant decrease of ACTA2  and chronic CClogenesis . In ordetivation . Additiotivation . We firs in HSCs . Next we in HSCs . Taken tIn the present study we provide the first miRNA expression profile in human HSCs. Moreover, to identify key miRNAs involved in the maintenance of the quiescent phenotype and explore the pathways that might be regulated by miRNA, we have combined gene and miRNA expression data from human qHSCs and from corresponding aHSCs. Our study identified a reduced panel of miRNAs highly expressed in qHSCs which are predicted to target a high number of genes known to be involved in the activation process. Moreover, this study provides basic information on miRNAs in HSCs and forms the basis for further studies deciphering the role of miRNAs in liver fibrosis.in vitro from the same individuals from which quiescent cells were isolated. The use of culture-activated HSCs allowed us to minimize the variability existing among patients with chronic liver diseases due to etiology, stage of disease development or sample collection procedure.HSCs have been extensively studied due to their important role during liver fibrogenesis212219The expression level of a miRNA provides limited information about its role. miRNAs are non-coding regulators of gene expression and therefore, their role depends on their level of expression but also the expression of their target genes2712in vitro HSC activation. Actually, approximately 80% of the differentially expressed miRNAs (212/259) were found expressed at low levels in the qHSCs and up-regulated in aHSCs. By contrast, the transcriptome analysis showed a more balanced ratio of up and down-regulated genes during activation. In order to understand the relevance of these findings, we merged the miRNA and gene expression profiles to reveal the potential miRNA-target interactions.An interesting observation of the present study is the consistent up-regulation of miRNAs during COL5A1,COL4A5, COL8A1, COL9A3), LOX, TGFβ or IL-6120in silico functional analysis of these targeted mRNAs showing their association with important pathways regulating HSC activation. These results may indicate that miRNAs highly expressed in qHSCs could be acting as suppressors of HSC activation, thereby preventing HSC activation towards myofibroblast-like cells. This would suggest that HSC quiescence is an actively maintained state. This finding has a potential physiological and clinical relevance as it suggests that strategies aiming at preventing HSC activation could be based on mimicking the function of these miRNAs expressed in qHSCs.One of the most important findings of this study is the higher number of target genes assigned by the computational integrative analysis to miRNAs expressed in quiescent cells compared to those measured in their activated counterparts. Importantly, miRNAs expressed in qHSCs targeted genes involved in HSC activation and liver fibrosis such as different collagen types  of all up-regulated genes during activation and some of these miRNAs have been previously reported to be associated with activation of HSCs. For instance, miR-150 has been shown to be down-regulated in HSCs isolated from rat fibrotic liversin vitro and in vivo activation. As found in human, HSCs derived from healthy mice presented significant higher levels of miR-192 compared to the HSCs isolated from fibrotic mice and its expression was rapidly reduced upon induction of fibrosis in vivo. In the present study, we also provide evidence for the role of miR-192 in the regulation of HSC activation by showing that overexpression of miR-192 suppresses TGFβ1-induced up-regulation of classical activation genes, i.e. Acta2, Col1a1 and Lox. Additionally, we find that miR-192 overexpression significantly repressed the proliferation and migratory potential of primary mouse HSCs, both key functional properties acquired during HSC activation. Moreover, miR-192 mimics reduced the expression of key activation markers in a human HSC cell line. Although the modulation of individual activation markers by miR-192 mimic may be modest, we provide evidence that miR-192 has a functional effect on HSCs. Particularly, these findings indicate that miR-192 can actively promote a quiescent phenotype in fibrogenic conditions and suggest potential therapeutic value for the modulation of miR-192 expression.Among all miRNAs identified in qHSCs, miR-192 was selected for further functional assays in human and murine HSCs. The selection was based on its enrichment in quiescent HSC and healthy liver tissue, the high number of predicted targets, the loss of expression in cirrhotic patients as well as the novelty of miR-192 in the context of liver fibrosis. We first studied whether miR-192 could be considered as a quiescent-specific miRNA during both SPARCL1) is the gene that is targeted by the highest number of miRNAs (10 miRNAs up-regulated in aHSC are predicted to target SPARCL1) (vs quiescent HSC gene expression we found that SPARCL1 was strongly (fold change = −40) downregulated during HSC activation and this infra-expression was also validated by qPCR. SPARCL1 is a protein member of the matricellular SPARC family of proteins, a diverse group of proteins that modulate cell interaction with the extracellular milieu. SPARCL1 is the only SPARC family member, in addition to SPARC itself, which can bind to fibrilar collagens and influence collagen fibril architecture32in vitro assays showed that mir-100 inhibition resulted in SPARCL1 up-regulation and in a significant reduction of COL1A1 and LOX expression levels.Our data also provides information regarding the miRNAs that may be playing a role during HSC activation. Interestingly, the bioinformatic analysis assigned a small number of targets to the miRNAs up-regulated in activated state. Among all miRNAs found highly expressed in aHSCs, miR-100 is the miRNA with the highest number of predicted target genes. Interestingly, according to the mRNA-miRNA integrative analysis performed, SPARCL1-like (SPARCL1) . Moreovein vivo and in vitro activation of qHSCs and that overexpression of miR-192 exerts a functional role during the activation program by reducing activation, migration and proliferation of HSCs. Further studies are needed to evaluate whether the identified miRNAs have a coordinated functional role in the maintenance of the quiescent phenotype and the repression of HSC activation.In conclusion, by integrating miRNA and mRNA expression data obtained from quiescent and activated human HSCs we have identified a panel of miRNAs highly expressed in qHSCs compared to aHSCs. These miRNAs are predicted to target over 25% of all up-regulated genes following HSC activation and therefore might have a common functional role in repressing HSC activation. Among all miRNAs included in this panel we have shown that miR-192 is down-regulated both during How to cite this article: Coll, M. et al. Integrative Mirna and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells. Sci. Rep.5, 11549; doi: 10.1038/srep11549 (2015).


Listeria monocytogenes to specific host genotypes and for exploring the potential of FTIR spectroscopy to gain novel insights into the host-pathogen interaction. Three different mouse genotypes, showing different susceptibility to L. monocytogenes infections, were challenged with L. monocytogenes and re-isolated bacteria were subjected to FTIR spectroscopy. The bacteria from mice with different survival characteristics showed distinct IR spectral patterns, reflecting specific changes in the backbone conformation and the hydrogen-bonding pattern of the protein secondary structure in the bacterial cell. Coupling FTIR spectroscopy with chemometrics allowed us to link bacterial metabolic fingerprints with host infection susceptibility and to decipher longtime memory effects of the host on the bacteria. After prolonged cultivation of host-passaged bacteria under standard laboratory conditions, the host's imprint on bacterial metabolism vanished, which suggests a revertible metabolic adaptation of bacteria to host environment and loss of host environment triggered memory effects over time. In summary, our work demonstrates the potential and power of FTIR spectroscopy to be used as a fast, simple and highly discriminatory tool to investigate the mechanism of bacterial host adaptation on a macromolar and metabolic level.Bacterial pathogens are known for their wide range of strategies to specifically adapt to host environments and infection sites. An in-depth understanding of these adaptation mechanisms is crucial for the development of effective therapeutics and new prevention measures. In this study, we assessed the suitability of Fourier Transform Infrared (FTIR) spectroscopy for monitoring metabolic adaptations of the bacterial pathogen  Listeria monocytogenes is a food-borne human pathogen that has become one of the most useful model organisms for the study of host–pathogen interactions and bacterial adaptation to mammalian hosts in vitro culturing of L. monocytogenes leads to a reduction of its infective capacity and the loss of virulence, bacteria are regularly passaged in vivo in mice to maintain the virulence state. Although this is a commonly used laboratory practice, the impact of mouse lineage selection on bacterial physiology, virulence and metabolism is largely unknown. We therefore aimed to employ Fourier-transform infrared (FTIR) spectroscopy, a metabolic fingerprinting technique, to decipher the influence of the host genotype on the passaged pathogen.in vitro−1) in Pseudomonas aeruginosa and pronounced alterations in the polysaccharide region (1200–900 cm−1) were shown for Salmonella enterica grown under acid and alkaline stress as well as for Bordetella pertussis grown in biofilm compared to planktonic growth FTIR is a vibrational spectroscopic technique with high discriminatory power. Due to its high throughput capacities at low operation costs it is an ideal tool for screening purposes. The FTIR spectroscopic patterns reflect the overall biochemical composition of living cells. By the stretching and bending vibrations of all functional groups of the cellular constituents including polysaccharides, proteins, nucleic acids and fatty acids, unique metabolic fingerprints are generated In the present study we demonstrate the potential of chemometric-assisted FTIR spectroscopy for monitoring metabolic adaptations of bacterial pathogens to their hosts. Multivariate statistical analysis allowed to link host infection susceptibility with bacterial metabolic fingerprints and to follow host imprints on bacterial physiology over time.Mice were housed under specific pathogen-free conditions according to FELASA guidelines. All animal experiments were discussed and approved by the Ethics and Animal Welfare Committee of the University of Veterinary Medicine Vienna, conform to the guidelines of the national authority  as laid down in §8ff of the Animal Science and Experiments Act  and according to the guidelines of FELASA and ARRIVE. To assess the distress of the animals during infection experiments, a scoring system was established. Based on this, health status and behavior of the animals were controlled by trained staff (participants of FELASA B training course) every 3–4 h during 7 a.m. and 7 p.m. In order not to disturb the circadian rhythm of the animals there was no monitoring after 7 p.m. Human endpoint was conducted by cervical dislocation if death of the animals was to be expected during the following hours.L. monocytogenes EGDe was grown in Brain Heart Infusion broth (BHI)  at 37°C to logarithmic phase, pelleted and diluted in PBS. The infection dose was controlled by plating serial dilutions on BHI (Difco) or Oxford agar  plates. Two laboratory inbred mouse lineages 129/Sv, C57BL/6 and Tyk2-deficient (Tyk2−/−) mice on C57BL/6 background 4 cfu L. monocytogenes EGDe intraperitoneally as previously described in vitro subcultivation was directly carried out after in vivo passage in mice. Isolates were therefore cultivated at 37°C on TSA plates and transferred to a new plate every 2–3 days. Subcultivation was continued until all isolates derived from different host background formed a homogenous population as monitored and tested by PCA analysis of the spectral data.Prolonged consecutive Bacteria were grown as lawn on TSA at 37°C for 24 h. Subsequently one loopful of bacteria was scraped from the agar plate and suspended in 100 µl of sterile deionised water. 30 µl bacterial suspension was spotted on a zinc selenite (ZnSe) optical plate and dried at 40°C for 40 min −1 using the following parameters: 6 cm−1 spectral resolution, zero-filling factor 4, Blackmann-Harris 3-term apodization and 32 interferogramms were averaged with background subtraction for each spectrum. Spectral quality assessment was performed for each spectrum with thresholds for minimum (0.300) and maximum (1.300) absorbance, signal-to-noise (S/N) ratio and water vapor content.Infrared spectroscopy absorption spectra were recorded using a HTS-XT microplate adapter coupled to a Tensor 27 FTIR spectrometer . Spectral acquisition was performed in transmission mode in the spectral range of 4000 to 500 cmSpectral evaluation and processing were performed using the software OPUS 7.2 (Bruker Optics GmbH). After passing spectral quality control parameters, second derivatives of the original spectra with a 9-point Savitzky-Golay filter were further calculated to increase spectral resolution and to minimize problems with baseline shifts. Subsequent vector normalization was performed for the whole spectral range to adjust biomass variations among different sample preparations.http://cran.r-project.org) to perform PCA and LDA. Loading plots were used to define spectral regions that were important for the discrimination of the three different experimental groups. The spectral window of the ‘amide region’, dominated by the amide I and amide II bands of proteins and peptides (1800–1500 cm−1), was selected for PCA and LDA, offering the maximum information and discriminatory power for characteristic cellular macromolecules.Chemometric analysis was performed on preprocessed data employing unsupervised principal component analysis (PCA) including loading plot analysis and supervised linear discriminant analysis (LDA). LDA discriminates between the three different groups based on prior knowledge of the experimental classes by minimizing within-class variance, whereas maximizing discrimination between classes. Supervised methods, i.e. methods that use priori information on class membership have the tendency of overfitting, when the number of spectral data points relative to the number of samples is high. PCA does not use a priori knowledge and allows reduction of data complexity, while maintaining most of the original variance Analysis of variance (ANOVA)-pairwise comparison was used to test the significance of pairwise differences of mean values of weighted loadings of the three groups on the first principal component (PC1). With this variable, an ANOVA was calculated to assess significant differentiation among the three genotypes.L. monocytogenes, 129/Sv and C57BL/6 and Tyk2-deficient (Tyk2−/−) mice on C57BL/6 background were infected intraperitoneally with 5×104 cfu L. monocytogenes EGDe. As depicted in L. monocytogenes, 129/Sv are susceptible and Tyk2−/− (C57BL/6) show an intermediate phenotype L. monocytogenes was re-isolated from the spleen of three infected mice per genotype 48 hours post infection and three randomly selected colonies per animal were subjected to FTIR spectroscopy (for details see L. monocytogenes derived from the C57BL/6 mice (‘non susceptible’) was subtracted from the average spectrum of L. monocytogenes derived from 129/Sv and from the average spectrum of L. monocytogenes isolates derived from Tyk2−/− mice, respectively (the latter two mouse genetic backgrounds are both ‘susceptible’). Significant differences in the spectra generated from bacteria passaged through different host genotypes were observed within the (i) ‘protein region’ (1800–1500cm−1) and (ii) ‘mixed region’ (1500–1200 cm−1) (see −1) and amide II (1560–1530 cm−1) bands. Both the amide I and amide II protein bands comprise the backbone conformation and the hydrogen bonding pattern independent of the primary protein structure. The amide I band arises principally from the symmetric stretching vibrations of the carbonyl (C = O) functional group and indicates changes in the α– and β secondary structures. Subtraction spectra revealed specific host genotype-induced changes associated to plated sheets and β-turns (1664 cm−1), α-helical (1652 cm−1) and β-plated sheet  protein secondary structures. Additional spectral alterations at 1539 cm−1 and 1553 cm−1 could be assigned to the amide II band, which is mainly related to in-plane amine (N-H) bending coupled to amine (C-N) stretching vibrations of proteins. Considerable differences at 1405 cm−1 and 1385 cm−1 were also observed in the more complex ‘mixed region’ resulting predominately from>CH2 and>CH3 bending modes of fatty acids and proteins/peptides L. monocytogenes isolates derived from the C57BL/6 and 129/Sv mice  and amide II (1560–1530 cm−1) bands of proteins to spectral variation between intact and sublethally thermal-injured L. monocytogenes cells was described previously L. monocytogenes isolated from the mouse spleen. Especially proteins modifying the peptidoglycan chemistry seem to play a substantial role in counteracting the innate immune system To analyze the impact of different host genotypes on the metabolism of the human pathogen m−1) see . Spectra/Sv mice , black. −/− mice , red wer−/− mice , blue, aions see . It is t−1) were limited to the corresponding spectral region of 1800 to 1500 cm−1 (‘protein region’) for chemometric analysis. Multivariate statistical analysis was carried out using LDA as a supervised classification method and PCA as an unsupervised classification method to cross-validate the results. L. monocytogenes infections (susceptibly 129/Sv mice>Tyk2−/−mice>>C57BL/6 mice) correlated with changes in the bacterial metabolic fingerprints. In line with the results from the LDA, PCA analysis of the spectral data revealed a distinct and separated grouping of bacteria derived from 129/Sv and C57BL/6 mice, with the Tyk2−/− cluster interspersed between the two groups   . It is tL. monocytogenes as well as other bacterial pathogens has been studied in some detail, while far less is known about pathogen response to multiple stresses as encountered in mammalian hosts. Especially the impact of the host's genotype on the bacterial metabolism is largely unknown. The data presented here demonstrate the potential of FTIR spectroscopy for exploring metabolic adaptations of bacterial pathogens to host environments with different infection susceptibilities. Consecutive subcultivation of host-passaged bacteria under laboratory standard conditions showed that these host mediated metabolic adaptations are reversible. As shown in Bacteria generally modulate their physiology and metabolism quickly in response to stress and changes in their environment. The physiological response to a broad variety of abiotic stress factors of Escherichia coliIt is tempting to speculate that the changes in the metabolic fingerprints of host-passaged bacteria reflect a memory effect of the host on bacterial metabolism. Memory effects triggered by environmental conditions, such as sugar availability and specific gut conditions, have very recently reported for bioengineered In summary, these results prove for the first time the ability of FTIR spectroscopy to link bacterial metabolic fingerprints to host susceptibility, solely based on IR spectral data and subsequent chemometric analysis.L. monocytogenes. Chemometric analysis of bacterial spectral data revealed a link between bacterial metabolic fingerprints and host survival characteristics. Evidence is presented that host adaptive mechanisms mainly correlate with changes of the protein secondary structure on the bacterial cell and/or cell membrane, which are reversible after consecutive subcultivation under standard laboratory conditions. These results demonstrate that host-adaptation is a highly dynamic process that can be accurately and easily monitored by FTIR spectroscopy. In addition, our study pinpoints the importance of choosing adequate animal models to maintain full bacterial virulence, which is a prerequisite for subsequent investigations of L. monocytogenes pathogenicity mechanisms. The availability of appropriate tools such as FTIR spectroscopy, allowing a fast and accurate monitoring of the metabolic status of host passaged bacteria, is expected to open new avenues for studying molecular mechanisms of host–pathogen interactions. Its high-throughput capacities at low operating costs make FTIR spectroscopy to a promising and interesting screening method prior to detailed studies using targeted metabolomic and proteomic approaches. Our work provides a proof of principle and it is expected that FTIR spectroscopy is also suitable for investigating host adaptation of other bacterial pathogens.Metabolic fingerprinting by FTIR spectroscopy was successfully employed to investigate the host adaptive mechanism of S1 FigPotential host memory effects on mice passaged L. monocytogenes isolates monitored by FTIR spectroscopy. PCA on second derivative, vector-normalized FTIR spectra from L. monocytogenes isolates derived from challenged 129/Sv (X), Tyk2−/− (O) and C57BL/6 (+) mice of the second experiment (A) directly after passage in mice and (B) after consecutive sub-cultivations at 37°C on laboratory standard media for three weeks.(TIF)Click here for additional data file.