Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms.

Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC-MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1ß1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix.

Migration-related land use dynamics in increasingly hybrid peri-urban space: insights from two agricultural communities in Bolivia.

This article investigates the impact of transnational labor migration on agriculture in urbanizing communities in Bolivia. Previous research shows that the characteristics of rural out-migration communities affect whether agricultural practices are intensified and improved. Using a mixed methods approach, two closely located peri-urban communities with distinct prerequisites for agricultural development are analyzed. This study shows weak migration-induced changes in agriculture and concludes that transnational migration does not necessarily accelerate an ongoing urbanization process. It shows that remittances function to maintain farming for subsistence and as a secondary livelihood activity. However, major investments in agricultural intensification are not attractive due to the communities' proximity to the main cities. This article highlights the need for nuanced conceptualization when studying migration-driven agricultural change in hybrid peri-urban spaces.

Este artículo investiga el impacto de la migración laboral transnacional en la agricultura de comunidades en proceso de urbanización en Bolivia. Investigaciones anteriores muestran que las características de las comunidades rurales de donde provienen los migrantes inciden sobre la intensificación y mejora de las prácticas agrícolas. Utilizando un enfoque de métodos mixtos, se analizan dos comunidades periurbanas, geográficamente cercanas, y con prerrequisitos diferentes para el desarrollo agrícola. Este estudio muestra cambios débiles en la agricultura inducidos por la migración y concluye que la migración transnacional no necesariamente acelera el proceso creciente de urbanización. Muestra también que las remesas permiten mantener la agricultura como actividad secundaria para la subsistencia. Sin embargo, grandes inversiones en la intensificación agrícola no son atractivas, debido a la proximidad de las comunidades a las ciudades principales. Este artículo destaca la necesidad de una conceptualización matizada al momento de estudiar el cambio agrícola impulsado por la migración en espacios híbridos periurbanos.

Patient-controlled analgesia with remifentanil versus alternative parenteral methods for pain management in labour.

BACKGROUND: Multiple analgesic strategies for pain relief during labour are available. Recently remifentanil, a short-acting opioid, has recently been used as an alternative analgesic due to its unique pharmacological properties. OBJECTIVES: To systematically assess the effectiveness of remifentanil intravenous patient-controlled analgesia (PCA) for labour pain, along with any potential harms to the mother and the newborn. SEARCH METHODS: We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (9 December 2015), ClinicalTrials.gov, the WHO International Clinical Trials Registry Platform (ICTRP), handsearched congress abstracts (November 2015), and reference lists of retrieved studies. SELECTION CRITERIA: Randomised controlled trials (RCTs) and cluster-randomised trials comparing remifentanil (PCA) with another opioid (intravenous (IV)/intramuscular (IM)), or with another opioid (PCA), or with epidural analgesia, or with remifentanil (continuous IV), or with remifentanil (PCA, different regimen), or with inhalational analgesia, or with placebo/no treatment in all women in labour including high-risk groups with planned vaginal delivery. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trials for inclusion, extracted data, and appraised study quality.We contacted study authors for additional information other than incomplete outcome data. We performed random-effects meta-analysis.To reduce the risk of random error in meta-analysis we performed trial sequential analysis. We included total zero event trials and used a constant continuity correction of 0.01 (ccc 0.01) for meta-analysis. We applied the Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) approach to assess the quality of evidence. MAIN RESULTS: Twenty RCTs with 3569 women were included. Of those, 10 trials (2983 participants) compared remifentanil (PCA) to an epidural, four trials (216 participants) to another opioid (IV/IM), three trials (215 participants) to another opioid (PCA), two trials (135 participants) to remifentanil (continuous IV), and one trial (20 participants) to remifentanil (PCA, different regimen). No trials were identified for the remaining comparisons.Methodological quality of studies was moderate to poor. We assessed risk of bias as high for blinding issues and incomplete outcome data in 65% and 45% of the included studies, respectively.There is evidence of effect that women in the remifentanil (PCA) group were more satisfied with pain relief than women in the other opioids (IV/IM) group (standardised mean difference (SMD) 2.11, 95% confidence interval (CI) 0.72 to 3.49, four trials, very low-quality evidence), and that women were less satisfied compared to women in the epidural group (SMD -0.22, 95% CI -0.40 to -0.04, seven trials, very low-quality evidence).There is evidence of effect that remifentanil (PCA) provided stronger pain relief at one hour than other opioids administered IV/IM (SMD -1.58, 95% CI -2.69 to -0.48, three trials, very low-quality evidence) or via PCA (SMD -0.51, 95% CI -1.01 to -0.00, three trials, very low-quality evidence). Pain intensity was higher in the remifentanil (PCA) group compared to the epidural group (SMD 0.57, 95% CI 0.31 to 0.84, six trials, low-quality evidence).Data were limited on safety aspects for both the women and the newborns. Only one study analysed maternal apnoea in a comparison of remifentanil (PCA) versus epidural and reported that half of the women in the remifentanil and none in the epidural group had an apnoea (very low-quality evidence). There is no evidence of effect that remifentanil (PCA) was associated with an increased risk for maternal respiratory depression when compared to epidural analgesia (RR 0.91, 95% CI 0.51 to 1.62, ccc 0.01, three trials, low-quality evidence) and no reliable conclusion might be reached compared to remifentanil (continuous IV) (all study arms included zero events, two trials, low-quality evidence). In one trial of remifentanil (PCA) versus another opioid (IM) three out of 18 women in the remifentanil and none out of 18 in the control group had a respiratory depression (very low-quality evidence).There is no evidence of effect that remifentanil (PCA) was associated with an increased risk for newborns with Apgar scores less than seven at five minutes compared to epidural analgesia (RR 1.26, 95% CI 0.62 to 2.57, ccc 0.01, five trials, low-quality evidence) and no reliable conclusion might be reached compared to another opioid (IV) and compared to remifentanil (PCA, different regimen) both with zero events in all study arms (one trial, very-low quality evidence). In one trial of remifentanil (PCA) versus another opioid (PCA) none out of nine newborns in the remifentanil and three out of eight in the opioid (PCA) group had Apgar scores less than seven (very-low quality evidence).There is evidence that remifentanil (PCA) was associated with a lower risk for the requirement of additional analgesia when compared to other opioids (IV/IM) (RR 0.57, 95% CI 0.40 to 0.81, three trials, moderate-quality evidence) and that it was associated with a higher risk compared to epidural analgesia (RR 9.27, 95% CI 3.73 to 23.03, ccc 0.01, six trials, moderate-quality evidence). There is no evidence of effect that remifentanil (PCA) reduced the requirement for additional analgesia compared to other opioids (PCA) (RR 0.76, 95% CI 0.45 to 1.28, three trials, low-quality evidence).There is evidence that there was no difference in the risk for caesarean delivery between remifentanil (PCA) and other opioids (IV/IM) (RR 0.63, 95% CI 0.30 to 1.32, ccc 0.01, four trials, low-quality evidence) and epidural analgesia (RR 1.0, 95% CI 0.82 to 1.22, ccc 0.01, nine trials, moderate-quality evidence), respectively. Pooled meta-analysis revealed an increased risk for caesarean section under remifentanil (PCA) compared to other opioids (PCA) (RR 2.78, 95% CI 0.99 to 7.82, two trials, very low-quality evidence). However, a wide range of clinically relevant and non-relevant treatment effects is compatible with this result. AUTHORS' CONCLUSIONS: Based on the current systematic review, there is mostly low-quality evidence to inform practice and future research may significantly alter the current situation. The quality of evidence is mainly limited by poor quality of the studies, inconsistency, and imprecision. More research is needed on maternal and neonatal safety outcomes (maternal apnoea and respiratory depression, Apgar score) and on the optimal mode and regimen of remifentanil administration to provide highest efficacy with reasonable adverse effects for mothers and their newborns.

Continuous intravenous perioperative lidocaine infusion for postoperative pain and recovery.

BACKGROUND: The management of postoperative pain and recovery is still unsatisfactory in clinical practice. Opioids used for postoperative analgesia are frequently associated with adverse effects including nausea and constipation. These adverse effects prevent smooth postoperative recovery. On the other hand not all patients may be suited to, and take benefit from, epidural analgesia used to enhance postoperative recovery. The non-opioid lidocaine was investigated in several studies for its use in multi-modal management strategies to reduce postoperative pain and enhance recovery. OBJECTIVES: The aim of this review was to assess the effects (benefits and risks) of perioperative intravenous lidocaine infusion compared to placebo/no treatment or compared to epidural analgesia on postoperative pain and recovery in adults undergoing various surgical procedures. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL, Issue 5 2014), MEDLINE (January 1966 to May 2014), EMBASE (1980 to May 2014), CINAHL (1982 to May 2014), and reference lists of articles. We searched the trial registry database ClinicalTrials.gov, contacted researchers in the field, and handsearched journals and congress proceedings. We did not apply any language restrictions. SELECTION CRITERIA: We included randomized controlled trials comparing the effect of continuous perioperative intravenous lidocaine infusion either with placebo, or no treatment, or with epidural analgesia in adults undergoing elective or urgent surgery under general anaesthesia. The intravenous lidocaine infusion must have been started intraoperatively prior to incision and continued at least until the end of surgery. DATA COLLECTION AND ANALYSIS: Trial quality was independently assessed by two authors according to the methodological procedures specified by the Cochrane Collaboration. Data were extracted by two independent authors. We collected trial data on postoperative pain, recovery of gastrointestinal function, length of hospital stay, postoperative nausea and vomiting (PONV), opioid consumption, patient satisfaction, surgical complication rates, and adverse effects of the intervention. MAIN RESULTS: We included 45 trials involving 2802 participants. Two trials compared intravenous lidocaine versus epidural analgesia. In all the remaining trials placebo or no treatment was used as a comparator. Trials involved participants undergoing open abdominal (12), laparoscopic abdominal (13), or various other surgical procedures (20).The risk of bias was low with respect to selection bias (random sequence generation), performance bias, attrition bias, and detection bias in more than 50% of the included studies. For allocation concealment and selective reporting the quality assessment yielded low risk of bias for only approximately 20% of the included studies.We found evidence of effect for intravenous lidocaine on the reduction of postoperative pain (visual analogue scale, 0 to 10 cm) compared to placebo or no treatment at 'early time points (one to four hours)' (mean difference (MD) -0.84 cm, 95% confidence interval (CI) -1.10 to -0.59; low-quality evidence) and at 'intermediate time points (24 hours)' (MD -0.34 cm, 95% CI -0.57 to -0.11; low-quality evidence) after surgery. However, no evidence of effect was found for lidocaine to reduce pain at 'late time points (48 hours)' (MD -0.22 cm, 95% CI -0.47 to 0.03; low-quality evidence). Pain reduction was most obvious at 'early time points' in participants undergoing laparoscopic abdominal surgery (MD -1.14, 95% CI -1.51 to -0.78; low-quality evidence) and open abdominal surgery (MD -0.72, 95% CI -0.96 to -0.47; moderate-quality evidence). No evidence of effect was found for lidocaine to reduce pain in participants undergoing all other surgeries (MD -0.30, 95% CI -0.89 to 0.28; low-quality evidence). Quality of evidence is limited due to inconsistency and indirectness (small trial sizes).Evidence of effect was found for lidocaine on gastrointestinal recovery regarding the reduction of the time to first flatus (MD -5.49 hours, 95% CI -7.97 to -3.00; low-quality evidence), time to first bowel movement (MD -6.12 hours, 95% CI -7.36 to -4.89; low-quality evidence), and the risk of paralytic ileus (risk ratio (RR) 0.38, 95% CI 0.15 to 0.99; low-quality evidence). However, no evidence of effect was found for lidocaine on shortening the time to first defaecation (MD -9.52 hours, 95% CI -23.24 to 4.19; very low-quality evidence).Furthermore, we found evidence of positive effects for lidocaine administration on secondary outcomes such as reduction of length of hospital stay, postoperative nausea, intraoperative and postoperative opioid requirements. There was limited data on the effect of IV lidocaine on adverse effects (e.g. death, arrhythmias, other heart rate disorders or signs of lidocaine toxicity) compared to placebo treatment as only a limited number of studies systematically analysed the occurrence of adverse effects of the lidocaine intervention.The comparison of intravenous lidocaine versus epidural analgesia revealed no evidence of effect for lidocaine on relevant outcomes. However, the results have to be considered with caution due to imprecision of the effect estimates. AUTHORS' CONCLUSIONS: There is low to moderate evidence that this intervention, when compared to placebo, has an impact on pain scores, especially in the early postoperative phase, and on postoperative nausea. There is limited evidence that this has further impact on other relevant clinical outcomes, such as gastrointestinal recovery, length of hospital stay, and opioid requirements. So far there is a scarcity of studies that have systematically assessed the incidence of adverse effects; the optimal dose; timing (including the duration of the administration); and the effects when compared with epidural anaesthesia.

Stress and burnout in anaesthesia: a real world problem?

PURPOSE OF REVIEW: Stress and burnout have arisen as relevant problems to the healthcare workers and the health systems themselves. As anaesthesiologists work in stressful areas of the hospitals, we are exposed to these problems. In this review, we summarize recent studies regarding stress and burnout, exploring possible solutions. RECENT FINDINGS: Almost 50% of anaesthesiologists scored positive for some of the burnout domains in different surveys, with one-third reporting high levels. The management of this problem not only needs an institutional but also an individual approach. Some recent randomized clinical trials and longitudinal studies suggest that there is some benefit of using several techniques to control stress, although benefits are still modest and most of the studies are based on transversal surveys and self-reported questionnaires. SUMMARY: There is a lack of definitive evidence to guide the management of stress and burnout in medical professions in general and in anaesthesiology in particular. Longitudinal and interventional studies are needed to better determine ways of action. In the meantime, creating a positive work climate and institutional support as well as promoting control over one's job and the autonomy of employees are the most recommended strategies. Workers should also learn how to cope with stressors and practice personal strategies of wellness and resilience to fight against burnout.

Molecular mechanism of alpha2beta1 integrin interaction with human echovirus 1.

Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of alpha2beta1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a 'closed' than an activated 'open' form of the alpha2I domain. Furthermore, a mutation E336A in the alpha2 subunit, which inactivated alpha2beta1 as a collagen receptor, enhanced alpha2beta1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of alpha2beta1. This was supported by the fact that the integrin clustering by EV1 did not activate the p38 MAP kinase pathway, a signalling pathway that was shown to be dependent on E336-related conformational changes in alpha2beta1. Furthermore, the mutation E336A did neither prevent EV1 induced and alpha2beta1 mediated protein kinase C activation nor EV1 internalization. Thus, in its entry strategy EV1 seems to rely on the activation of signalling pathways that are dependent on alpha2beta1 clustering, but do not require the conformational regulation of the receptor.

Embigin deficiency leads to delayed embryonic lung development and high neonatal mortality in mice.

Embigin (Gp70), a receptor for fibronectin and an ancillary protein for monocarboxylate transporters, is known to regulate stem cell niches in sebaceous gland and bone marrow. Here, we show that embigin expression is at high level during early mouse embryogenesis and that embigin is essential for lung development. Markedly increased neonatal mortality of Emb-/- mice can be explained by the compromised lung maturation: in Emb-/- mice (E17.5) the number and the size of the small airways and distal airspace are significantly smaller, there are fewer ATI and ATII cells, and the alkaline phosphatase activity in amniotic fluid is lower. Emb-/- lungs show less peripheral branching already at E12.5, and embigin is highly expressed in lung primordium. Thus, embigin function is essential at early pseudoglandular stage or even earlier. Furthermore, our RNA-seq analysis and Ki67 staining results support the idea that the development of Emb-/- lungs is rather delayed than defected.

Novel α2ß1 integrin inhibitors reveal that integrin binding to collagen under shear stress conditions does not require receptor preactivation.

The interaction between α2ß1 integrin (GPIa/IIa, VLA-2) and vascular collagen is one of the initiating events in thrombus formation. Here, we describe two structurally similar sulfonamide derivatives, BTT-3033 and BTT-3034, and show that, under static conditions, they have an almost identical effect on α2-expressing CHO cell adhesion to collagen I, but only BTT-3033 blocks platelet attachment under flow (90 dynes/cm(2)). Differential scanning fluorimetry showed that both molecules bind to the α2I domain of the recombinant α2 subunit. To further study integrin binding mechanism(s) of the two sulfonamides, we created an α2 Y285F mutant containing a substitution near the metal ion-dependent adhesion site motif in the α2I domain. The action of BTT-3033, unlike that of BTT-3034, was dependent on Tyr-285. In static conditions BTT-3034, but not BTT-3033, inhibited collagen binding by an α2 variant carrying a conformationally activating E318W mutation. Conversely, in under flow conditions (90 dynes/cm(2)) BTT-3033, but not BTT-3034, inhibited collagen binding by an α2 variant expressing E336A loss-of-function mutation. Thus, the binding sites for BTT-3033 and BTT-3034 are differentially available in distinct integrin conformations. Therefore, these sulfonamides can be used to study the biological role of different functional stages of α2ß1. Furthermore, only the inhibitor that recognized the non-activated conformation of α2ß1 integrin under shear stress conditions effectively blocked platelet adhesion, suggesting that the initial interaction between integrin and collagen takes place prior to receptor activation.

Must we press on until a young mother dies? Remifentanil patient controlled analgesia in labour may not be suited as a "poor man's epidural".

BACKGROUND: The epidural route is still considered the gold standard for labour analgesia, although it is not without serious consequences when incorrect placement goes unrecognized, e.g. in case of intravascular, intrathecal and subdural placements. Until now there has not been a viable alternative to epidural analgesia especially in view of the neonatal outcome and the need for respiratory support when long-acting opioids are used via the parenteral route. Pethidine and meptazinol are far from ideal having been described as providing rather sedation than analgesia, affecting the cardiotocograph (CTG), causing fetal acidosis and having active metabolites with prolonged half-lives especially in the neonate. Despite these obvious shortcomings, intramuscular and intravenously administered pethidine and comparable substances are still frequently used in delivery units. Since the end of the 90 ths remifentanil administered in a patient-controlled mode (PCA) had been reported as a useful alternative for labour analgesia in those women who either don't want, can't have or don't need epidural analgesia. DISCUSSION: In view of the need for conversion to central neuraxial blocks and the analgesic effect remifentanil has been demonstrated to be superior to pethidine. Despite being less effective in terms of the resulting pain scores, clinical studies suggest that the satisfaction with analgesia may be comparable to that obtained with epidural analgesia. Owing to this fact, remifentanil has gained a place in modern labour analgesia in many institutions. However, the fact that remifentanil may cause harm should not be forgotten when the use of this potent mu-agonist is considered for the use in labouring women. In the setting of one-to-one midwifery care, appropriate monitoring and providing that enough experience exists with this potent opioid and the treatment of potential complications, remifentanil PCA is a useful option in addition to epidural analgesia and other central neuraxial blocks. Already described serious consequences should remind us not refer to remifentanil PCA as a "poor man's epidural" and to safely administer remifentanil with an appropriate indication. SUMMARY: Therefore, the authors conclude that economic considerations and potential cost-savings in conjunction with remifentanil PCA may not be appropriate main endpoints when studying this valuable method for labour analgesia.

Structure of collagen receptor integrin α(1)I domain carrying the activating mutation E317A.

We have analyzed the structure and function of the integrin α(1)I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α(1)I C139S/E317A was a higher avidity collagen binder than α(1)I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α(1)I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α(2)I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α(2)I structure, has not changed its position in the activated α(1)I variant. During the integrin activation, Glu(335) on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the ß(1) subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu(335). This indicates that the stabilization of helix 7 into its downward position is not required if the α(1) MIDAS is already open. To conclude, the activated α(1)I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg(287)-Glu(317) ion pair has just broken during the integrin activation.

The role of the maleimide ring system on the structure-activity relationship of showdomycin.

Showdomycin produced by Streptomyces showdoensis ATCC 15227 is a C-nucleoside microbial natural product with antimicrobial and cytotoxic properties. The unique feature of showdomycin in comparison to other nucleosides is its maleimide base moiety, which has the distinct ability to alkylate nucleophilic thiol groups by a Michael addition reaction. In order to understand structure-activity relationships of showdomycin, we synthesized a series of derivatives with modifications in the maleimide ring at the site of alkylation to moderate its reactivity. The showdomycin congeners were designed to retain the planarity of the base ring system to allow Watson-Crick base pairing and preserve the nucleosidic character of the compounds. Consequently, we synthesized triphosphates of showdomycin derivatives and tested their activity against RNA polymerases. Bromo, methylthio, and ethylthio derivatives of showdomycin were incorporated into RNA by bacterial and mitochondrial RNA polymerases and somewhat less efficiently by the eukaryotic RNA polymerase II. Showdomycin derivatives acted as uridine mimics and delayed further extension of the RNA chain by multi-subunit, but not mitochondrial RNA polymerases. Bioactivity profiling indicated that the mechanism of action of ethylthioshowdomycin was altered, with approximately 4-fold reduction in both cytotoxicity against human embryonic kidney cells and antibacterial activity against Escherichia coli. In addition, the ethylthio derivative was not inactivated by medium components or influenced by addition of uridine in contrast to showdomycin. The results explain how both the maleimide ring and the nucleoside nature contribute to the bioactivity of showdomycin and demonstrates for the first time that the two activities can be separated.

Embigin is a fibronectin receptor that affects sebaceous gland differentiation and metabolism.

Stem cell renewal and differentiation are regulated by interactions with the niche. Although multiple cell populations have been identified in distinct anatomical compartments, little is known about niche-specific molecular factors. Using skin as a model system and combining single-cell RNA-seq data analysis, immunofluorescence, and transgenic mouse models, we show that the transmembrane protein embigin is specifically expressed in the sebaceous gland and that the number of embigin-expressing cells is negatively regulated by Wnt. The loss of embigin promotes exit from the progenitor compartment and progression toward differentiation, and also compromises lipid metabolism. Embigin modulates sebaceous niche architecture by affecting extracellular matrix organization and basolateral targeting of monocarboxylate transport. We discover through ligand screening that embigin is a direct fibronectin receptor, binding to the N-terminal fibronectin domain without impairing integrin function. Our results solve the long-standing question of how embigin regulates cell adhesion and demonstrate a mechanism that couples adhesion and metabolism.

Fluorescent small molecule probe to modulate and explore α2ß1 integrin function.

Collagen binding integrins are an important family of cell surface receptors that mediate bidirectionally signals between the interior of the cell and the extracellular matrix. The protein-protein interactions between cells and collagen are necessary for many physiological functions, but also promote diseases. For example, the interaction of α2ß1 integrin and collagen has been shown to have an important role in thrombus formation and cancer spread. The fact that the discovery of small molecules that can block such protein-protein interactions is highly challenging has significantly hindered the discovery of pharmaceutical agents to treat these diseases. Here, we present a rationally designed novel fluorescent molecule that can be synthesized in just a few minutes from commercially available starting materials. This molecule blocks the protein-protein interaction between α2ß1 integrin and collagen, and due to its fluorescent properties, it can be employed in wide variety of biological applications.

Syndecan-1 supports integrin alpha2beta1-mediated adhesion to collagen.

Several different receptor molecules act in concert to regulate cell adhesion. Among these are cell-surface proteoglycans and integrins, which collaborate extensively in mediating binding of cells to extracellular matrix molecules fibronectin and vitronectin. However, very little is known about possible functional synergism between proteoglycans and integrins during adhesion of cells to collagen, although collagen is the most abundant protein in the human body. Here we show that cell-surface heparan sulphate proteoglycans (HSPGs) support integrin alpha2beta1-mediated adhesion to collagen. Cells made devoid of HSPGs either by genetic means or by enzymatic digestions were unable to adhere to collagen via alpha2beta1 integrin. HSPG-deficient cells also displayed impaired spreading and actin organization on collagen. Among different HSPG molecules syndecan-1 was found to play an important role in supporting alpha2beta1 integrin-mediated adhesion. Using overexpression and knock-down experiments we demonstrated that syndecan-1, but not syndecan-2 or -4, enhanced binding of alpha2beta1 to collagen. Moreover, syndecan-1 co-localized with alpha2beta1 integrin and contributed to proper organization of cortical actin. Finally, crosstalk between syndecan-1 and alpha2beta1 integrin was found to enhance the transcription of matrix metalloproteinase-1 in response to collagen binding. Our findings thus suggest that a previously unknown link between integrin alpha2beta1 and syndecan-1 is important in regulating cell adhesion to collagen and in triggering integrin downstream signalling.

TPA primes alpha2beta1 integrins for cell adhesion.

Integrin avidity is regulated by changes in the conformation of the heterodimer and cluster formation. We measured cell adhesion by integrin alpha2beta1 (CHO-alpha2) to collagen at short contact times (0.5-60s) by single cell force spectroscopy (SCFS). The adhesion increased rapidly with contact time and was further strengthened by the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) and integrin activator. TPA also improved the strength of adhesive units. Furthermore, changes in membrane nanotube properties indicated better coupling of integrins to the cell cytoskeleton. We conclude that in addition to increasing integrin avidity TPA strengthens integrin-cytoskeletal linkage.

Integrin α2ß1 decelerates proliferation, but promotes survival and invasion of prostate cancer cells.

High expression level of integrin α2ß1 is a hallmark of prostate cancer stem cell like cells. The role of this collagen receptor is controversial since it is down regulated in poorly differentiated carcinomas, but concomitantly proposed to promote metastasis. Here, we show that docetaxel resistant DU145 prostate cancer cells express high levels of α2ß1 and that α2ß1High subpopulation of DU145 cells proliferates slower than the cells representing α2ß1Low subpopulation. To further study this initial observation we used Crispr/Cas9 technology to create an α2ß1 negative DU145 cell line. Furthermore, we performed rescue experiment by transfecting α2 knockout cells with vector carrying α2 cDNA or with an empty vector for appropriate control. When these two cell lines were compared, α2ß1 positive cells proliferated slower, were more resistant to docetaxel and also migrated more effectively on collagen and invaded faster through matrigel or collagen. Integrin α2ß1 was demonstrated to be a positive regulator of p38 MAPK phosphorylation and a selective p38 inhibitor (SB203580) promoted proliferation and inhibited invasion. Effects of α2ß1 integrin on the global gene expression pattern of DU145 cells in spheroid cultures were studied by RNA sequencing. Integrin α2ß1 was shown to regulate several cancer progression related genes, most notably matrix metalloproteinase-1 (MMP-1), a recognized invasion promoting protein. To conclude, the fact that α2ß1 decelerates cell proliferation may explain the dominance of α2ß1 negative/low cells in primary sites of poorly differentiated carcinomas, while the critical role of α2ß1 integrin in invasion stresses the importance of this adhesion receptor in cancer dissemination.

Small molecule designed to target metal binding site in the alpha2I domain inhibits integrin function.

Integrin alpha2beta1 is a potential target molecule in drug development. We have established "design" criteria for molecules that bind to the "closed" conformation of alpha2I domain via Mg(2+) in MIDAS (metal ion dependent adhesion site) while simultaneously forming interactions with neighboring amino acid residues. Specific tetracyclic Streptomyces products belonging to the group of aromatic polyketides fulfill our criteria and inhibit alpha2beta1 integrin. All previously described inhibitors of alphaI domain integrins act in an allosteric manner.

Integrin α2ß1 in nonactivated conformation can induce focal adhesion kinase signaling.

Conformational activation of integrins is generally required for ligand binding and cellular signalling. However, we have previously reported that the nonactivated conformation of α2ß1 integrin can also bind to large ligands, such as human echovirus 1. In this study, we show that the interaction between the nonactivated integrin and a ligand resulted in the activation of focal adhesion kinase (FAK) in a protein kinase C dependent manner. A loss-of-function mutation, α2E336A, in the α2-integrin did not prevent the activation of FAK, nor did EDTA-mediated inactivation of the integrin. Full FAK activation was observed, since phosphorylation was not only confirmed in residue Y397, but also in residues Y576/7. Furthermore, initiation of downstream signaling by paxillin phosphorylation in residue Y118 was evident, even though this activation was transient by nature, probably due to the lack of talin involvement in FAK activation and the absence of vinculin in the adhesion complexes formed by the nonactivated integrins. Altogether these results indicate that the nonactivated integrins can induce cellular signaling, but the outcome of the signaling differs from conventional integrin signaling.

Joint inflammation related citrullination of functional arginines in extracellular proteins.

We report the extent, specific sites and structural requirements of joint inflammation related citrullination in extracellular proteins. A total of 40 synovial fluid samples derived from chronically inflamed human joints were analysed by heparin-agarose fractionation and LC-MS/MS. Citrullination of 55 arginines in extracellular proteins was detected. Importantly, 20% of the sites have a characterized function related to the hallmarks of destructive joint inflammation. E.g. four arginine residues, shown here to be citrullinated, are also affected by mutations in inherited diseases causing haemolysis or blood clotting dysfunction. Citrullination of integrin ligands was selected for further studies since fibronectin R234 in isoDGR was among the most frequently citrullinated arginines in synovial fluid. Assays with synovial fibroblasts and integrin αVß3 indicated decreased affinity to the enzymatically citrullinated integrin binding sites. To conclude, our data indicate that in inflamed joints extensive citrullination affects the functional arginine residues in extracellular proteins.

Tumor promoter PMA enhances kindlin-2 and decreases vimentin recruitment into cell adhesion sites.

Phorbol diester PMA (phorbol 12-myristate 13-acetate) is a well-known promoter of tumor progression. PMA also regulates cell adhesion by several mechanisms including conformational activation of integrins and integrin clustering. Here, PMA was shown to induce lamellipodia formation and reorganization of the adhesion sites as well as actin and vimentin filaments independently of integrin preactivation. To further analyze the mechanism of PMA action, the protein composition in the α1ß1 integrin/collagen IV adhesion sites was analyzed by mass spectrometry and proteomics. In four independent experiments we observed the reduced recruitment of vimentin in relation to integrin α1 subunit. This was in full agreement with the fact that we also detected the retraction of vimentin from cell adhesions by confocal microscopy. Furthermore, the accumulation of kindlin-2 into cell adhesions was significantly increased after PMA treatment. Kindlin-2 siRNA inhibited cell spreading as well as the formation of actin fibrils and cell adhesions, but did not prevent the effect of PMA on lamellipodia formation. Thus, kindlin-2 recruitment was considered to be a consequence rather than the primary cause for the loss of connection between vimentin and the adhesion sites.