It was important to elucidate the mutagenic potential of C. albicans biofilms and the expression of genes related to ACH and ethanol. Mutagenic levels of ACH were produced by control biofilms during incubation in D-glucose at neutral pH. No ACH was produced by biofilms exposed to HICA during D-glucose incubation at acidic pH. At neutral pH, the ACH levels were significantly lower compared to control conditions and below the mutagenic level. At neutral pH, ACH production from ethanol or Dglucose by mature biofilms was decreased similarly by both HICA and caspofungin. Interestingly, although caspofungin inhibited ACH production by early biofilms more than HICA, it resulted in the highest ACH levels at acidic pH during D-glucose incubation. This is relevant as a normal western diet is often rich in D-glucose and the 100 mM concentration used in this study is equivalent to 18 g/l found commonly in food and beverages. In the presence of ethanol, ACH levels produced by all biofilms exposed to HICA or caspofungin were generally lower than in control biofilms. The ethanol concentration of 11 mM used in this study is found in saliva after drinking 0.5 g of alcohol per kg body weight, which is equivalent to 3 glasses of wine for an 80 kg male and thus can be considered clinically relevant. Our results on biofilms support earlier studies on planktonic cell cultures. Biofilms exposed to RPMI and ethanol produced high levels of ACH from ethanol, well above the mutagenic level, and approaching the results obtained with planktonic cultures. It is relevant to point out that the highest ACH levels were produced at later stages of biofilm growth, as would be established in niches of the human body. HICA was most active against C. albicans biofilms at acidic pH, in line with previous studies where planktonic cultures were used. The main focus of the present study was to investigate the anti-biofilm efficacy of HICA against various comparators whereby it was critical to grow the biofilms under standardised conditions before treatment at acidic and neutral conditions. Therefore, the biofilms were not pre-grown at acidic conditions, which can be considered a limitation: there are multiple sites that are physiologically acidic and pH can have an effect on C. albicans morphology. On the other hand, acidic pH may favour fungal over bacterial growth as seen in Candida esophagitis, vulvovaginal candidosis and chronic wounds. Interestingly, the final metabolic activity and biomass of untreated control biofilms after 24 h exposure to pH 5.2 and 7.4 were comparable in the present study. A recent report suggested that composition of the medium has a major effect on biofilm architecture, expression profiles and antifungal susceptibility. In RPMI medium, the D-glucose concentration of 2.0 g/l is higher than the physiological concentration normally found in the human body.

The potential energy calculations on resting and open states of zfP2X4 receptors revealed that AR1induced allosteric changes led to energy releases during conformational transition between the resting and open states. In contrast, MD simulations together with PCA analysis showed that AR2 and 3 can only induce outward and upward movements of head domain, respectively. Therefore, AR2 and AR3 would not trigger downstream allosteric changes. These observations provided a conceivable explanation for why only AR1 rather than AR2 and 3 is able to trigger channel activation of P2X4 receptors. The DGbind together with the downstream allosteric changes-induced energy releases may enable AR1 to efficiently overcome the energy barrier for the channel gating. Meanwhile, based on the observation that conformational fluctuations of head domain, especially the downward motion, greatly enhance the preference of ABP for AR1, we proposed that the downward motion of head domain facilitated by AR1 may further increase the preference of ABP for AR1. It looks like an induced-fit/positive feedback mechanism when AR1 occurs. Taken together, the contact between ATP and site S1 promoted downward motion of head domain correlated well with conformational changes of ABP and subsequently increased preference of ABP for AR1. Once ATP comes in contact with site S1, it will initiate an ‘induced-fit’/positive-feedback mechanism, with the increased free energy release and the downstream allosteric changes-induced energy releases acting as the driving force. In contrast, AR2 and 3 are not capable of facilitating the downward motion of head domain and therefore, would not evoke such positive feedback. The comparisons between closed and open structures, and the equilibrated averaged structure after MD simulations would not only facilitate studying ligand-recognition of P2X4 receptors, but also will provide some clues to understanding of channel gating mechanism. The movements observed in MD simulations exhibited high a similarity to bound-ATP induced conformational changes of ABP in open crystal structure. For instance, the structural rearrangements of K70, K68, R289 and R143, and closing movements of loop139-146 and loop169-183. However, the rightward movement of loop169-183 and the down movement of head domain during MD simulations were more evident than those of ATP-bound open structure. On the contrary, the upward movement of head domain in MD data was not as obvious as that of open structure. These observations confirmed the crucial role of inherent dynamics of head domain in both AR and channel gating of P2X4 receptors. However, the complex dynamics in the process of channel gating well beyond the inherent dynamics of head domain, during which bound-ATP evoked coordinated movements of multiple domains, especially for the head and dorsal fin domains, are crucial for this process.

Followed by the mutation analysis based on the high-throughput sequencing. Although the phenotypes were simply classified into FR and FL subgroups, the relationship between 4000 EcFbFP mutants to their phenotypes was determined in a single run of highthroughput sequencing. Using the depth of the enriched mutations in the FR and FL libraries, we characterized the mutations that are sensitive and tolerant to the loss of EcFbFP function. These data were used to calculate the positional effects of mutations on EcFbFP function. Evaluation of the highly sensitive positions among the FMNbinding identified all the residues that display direct bond formation with the FMN cofactor. However, for hydrophobic interaction of FMN with amino acid residues in the EcFbFP active site, six residues were defined as highly tolerant to mutation. For example, in position L82, the function retained mutation of L82M was highly enriched, while average mutation count in a single mutation was 26.5 for FR library. One possible reason is that the docking of FMN to EcFbFP is not highly specific. FMN binds to the LOV domain of YtvA as a chromophore and becomes covalently linked to the conserved cysteine residue on blue-light activation, thereby activating the signaling cascade of stress s response in B. subtilis. The structure of the LOV domain in YtvA, thus, contains a strong potential for conformational changes upon activation of the photocycle. However, in EcFbFP, only the LOV domain of YtvA is isolated and covalent bond forming cysteine residue, Cys62 is substituted for alanine, which resulted in photo-cycle inhibition. Thus, FMN cofactor is non-covalently bound to the pocket of EcFbFP, and the STAS domain is not linked to the Ja helix. The dynamic potential of EcFbFP for conformational change indicates that the FMN-binding pocket of EcFbFP is not completely rigid and spatially compact. The homo-dimerization of YtvA-LOV domain is mediated by the hydrophobic residues located at Ab,B b,H b, and Ib, and our mutational analysis of FR library agrees with the hydrophobic interaction of homo-dimerization of EcFbFP. Among the 25 highly sensitive positions of EcFbFP, only a single position, V120 located at the Ib sheet, was found to be highly sensitive. Although we speculated that the homo-dimerization is achieved by a cooperative interaction of hydrophobic residues, our analysis suggests that Ib sheet may have a higher sensitivity toward homo-dimer stabilization. The added weight of Ib sheet in homo-dimerization is also supported by the fact that Hb-Ib hairpin moves toward the dimer interface upon light absorption. In addition to residues located at the FMN-binding pocket and hydrophobic homo-dimerization sites, highly sensitive residues were located at loops and turns. Conformational change in YtvA-LOV is initiated from the FMN-binding pocket and leads to the movement of Ea and Ja helices in a scissor-like motion.

To predict CE even in patients who are negative for troponin T. Yet, the complexity of the molecular pathways in the chain of events leading to ACS, as well as the heterogeneity in clinical presentation, imply that a single-marker strategy is most likely inferior to a multi-marker approach. Indeed, multi-marker testing adds useful prognostic information to that of individual markers, both for the prediction of early and long-term outcomes in patients with suspected ACS. However, the potential of different novel cardiac biomarkers in comparison to and combined with clinical assessment in predicting CE in individuals presenting with symptoms suggestive of ACS has not been fully elucidated. This study assessed novel sensitive biomarkers along with established clinical parameters on admission of patients with suspected ACS, for the prediction of CE defined as a composite of early coronary revascularization, myocardial infarction, and cardiovascular death within 30 days. At variance with other biomarker studies, in this analysis CE were prespecified to include immediate revascularization therapy, since in the contemporary setting of primary PCI for ACS, emergency room triage firstly aims at identifying patients in need of PCI or CABG. Acute coronary syndromes presenting as myocardial infarction may be suspected or diagnosed by ECG already at admittance, and diagnosis may be corroborated or not with biomarker testing. Most likely, those patients will undergo cardiac catheterization immediately. But in all patients with suspected ACS, identification and therapy of a culprit lesion nowadays is paramount and complementary to the clinical endpoints of myocardial infarction and cardiovascular death within 30 days, which in turn may reflect both success or failure of therapy, or a missed diagnosis in patients not having undergone coronary angiography. Importantly, this study showed that performance of single biomarkers – notably of hs-cTnT and MPO – depend on the clinical pretest probability of ACS, with hs-cTnT adding to the sensitivity in patients with low and MPO improving the specificity of patients with high clinical risk scores, respectively. Accordingly, identification of those prone to CE was best based on clinical criteria complemented by hs-cTnT based on decision tree analysis. Interestingly, prediction of CE based on the clinical TIMI risk score alone was not improved by adding hs-cTnT to the risk prediction model. Although this study was originally designed also to assess the diagnostic value of MRP 8/14 for early diagnosis of ACS, in the current setting this marker of phagocyte activation did not show sufficient discriminatory performance, notwithstanding its significant elevation in ACS. Scoring systems predicting the patients’ risk of ischemic events and cardiovascular death have been established such as the TIMI risk score for unstable angina/NSTEMI.

The CC domain-dependent homodimerization of MLA has been suggested to attract particular WRKY hetero or homo-oligomers for the purpose of downstream signaling. Whether the heterodimer of the CC/TIR domain is therefore also a critical player in downstream signaling remains an open question. However, the coexpression of the CC domains of both Pikh-1 and Pikh-2 was insufficient to induce HR, suggesting that the association of the two CC domains may be related to the formation of AvrPikh-Pikh1-Pikh2 recognition complexes rather than specifically related to a downstream signaling event. The sub-cellular localization of R proteins is relevant to their activity in resistance signalling. The tobacco R gene product N, which confers resistance against tobacco mosaic virus, is found in both the cytoplasm and the nucleus. During TMV infection, the shuttling of p50-activated N from the cytoplasm to the nucleus appears to be required for an effective defence response. As a second example, the activity of the A. thaliana SNC1 and RPS4 have also been associated with their nuclear accumulation. Strikingly, a recent study has confirmed that the enhanced defense responses mounted under conditions of ABA deficiency and high temperature is dependent on the nuclear localization of SNC1 and RPS4. In contrast, the regulation of the potato CC-NBS-LRR Rx protein requires its nucleocytoplasmic distribution, which is brought about by its CC and LRR domains and facilitated by an accessory protein. While the nuclear Rx protein may play a role in transcriptional reprogramming, the cytoplasmic form mounts a defence process against the virus. With respect to the Pik-h product, the Pikh-1 CC domain disrupted its balanced nucleocytoplasmic partitioning through its interaction with both AvrPik-h and Pikh-2. When GFP fusion proteins comprising either the full-length Pikh-1 or specific Pikh-1 domains were expressed in rice protoplasts, it was only the former which retained a balanced distribution between the cytoplasm and the nucleus. Thus, similar to the situation with Rx, the Pikh-1 CC and LRR domains may well have contrasting roles in determining the cellular distribution of the full-length Pikh-1 protein. Given that the full-length Pikh-1 protein was required for HR induction, and that it interacted with AvrPik-h or Pikh-2 in both nucleus and cytoplasm, the assumption is that Pikh-mediated resistance relies on its balanced cellular distribution. Indeed, the enforced restriction of Pikh-1 to either the nucleus or the cytoplasm blocked the AvrPikh-dependent cell death. The full-length and CC domain of Pikh-2 accumulated mostly, and the NBS and LRR domains exclusively, in the nucleus. An enhanced nuclear or cytoplasmic accumulation of Pikh-2 also compromised its HR activity. The inference is that in the expression of Pik-h resistance, Pikh-2 may interact with Pikh-1 in the nucleus to direct transcriptional reprogramming.