Experimental Evidence for a Metallohydrolase Mechanism in Which the Nucleophile Is Not Delivered by a Metal Ion: EPR Spectrokinetic and Structural Studies of Aminopeptidase from \u3cem\u3eVibrio proteolyticus\u3c/em\u3e

Metallohydrolases catalyse some of the most important reactions in biology and are targets for numerous chemotherapeutic agents designed to combat bacterial infectivity, antibiotic resistance, HIV infectivity, tumour growth, angiogenesis and immune disorders. Rational design of inhibitors of these enzymes with chemotherapeutic potential relies on detailed knowledge of the catalytic mechanism. The roles of the catalytic transition ions in these enzymes have long been assumed to include the activation and delivery of a nucleophilic hydroxy moiety. In the present study, catalytic intermediates in the hydrolysis of L-leucyl-L-leucyl-L-leucine by Vibrio proteolyticus aminopeptidase were characterized in spectrokinetic and structural studies. Rapid-freeze-quench EPR studies of reaction products of L-leucyl-L-leucyl-L-leucine and Co(II)-substituted aminopeptidase, and comparison of the EPR data with those from structurally characterized complexes of aminopeptidase with inhibitors, indicated the formation of a catalytically competent post-Michaelis pre-transition state intermediate with a structure analogous to that of the inhibited complex with bestatin. The X-ray crystal structure of an aminopeptidase–L-leucyl-L-leucyl-L-leucine complex was also analogous to that of the bestatin complex. In these structures, no water/hydroxy group was observed bound to the essential metal ion. However, a water/hydroxy group was clearly identified that was bound to the metal-ligating oxygen atom of Glu152. This water/hydroxy group is proposed as a candidate for the active nucleophile in a novel metallohydrolase mechanism that shares features of the catalytic mechanisms of aspartic proteases and of B2 metallo-b-lactamases. Preliminary studies on site-directed variants are consistent with the proposal. Other features of the structure suggest roles for the dinuclear centre in geometrically and electrophilically activating the substrate

Phase Mixing of Alfvén Waves Near a 2D Magnetic Null Point

The propagation of linear Alfvén wave pulses in an inhomogeneous plasma near a 2D coronal null point is investigated. When a uniform plasma density is considered, it is seen that an initially planar Alfvén wavefront remains planar, despite the varying equilibrium Alfvén speed, and that all the wave collects at the separatrices. Thus, in the non-ideal case, these Alfvénic disturbances preferentially dissipate their energy at these locations. For a non-uniform equilibrium density, it is found that the Alfvén wavefront is significantly distorted away from the initially planar geometry, inviting the possibility of dissipation due to phase mixing. Despite this however, we conclude that for the Alfvén wave, current density accumulation and preferential heating still primarily occur at the separatrices, even when an extremely non-uniform density profile is considered

Altered brain connectivity in sudden unexpected death in epilepsy (SUDEP) revealed using resting-state fMRI.

The circumstances surrounding SUDEP suggest autonomic or respiratory collapse, implying central failure of regulation or recovery. Characterisation of the communication among brain areas mediating such processes may shed light on mechanisms and noninvasively indicate risk. We used rs-fMRI to examine network properties among brain structures in people with epilepsy who suffered SUDEP (n = 8) over an 8-year follow-up period, compared with matched high- and low-risk subjects (n = 16/group) who did not suffer SUDEP during that period, and a group of healthy controls (n = 16). Network analysis was employed to explore connectivity within a 'regulatory-subnetwork' of brain regions involved in autonomic and respiratory regulation, and over the whole-brain. Modularity, the extent of network organization into separate modules, was significantly reduced in the regulatory-subnetwork, and the whole-brain, in SUDEP and high-risk. Increased participation, a local measure of inter-modular belonging, was evident in SUDEP and high-risk groups, particularly among thalamic structures. The medial prefrontal thalamus was increased in SUDEP compared with all other control groups, including high-risk. Patterns of hub topology were similar in SUDEP and high-risk, but were more extensive in low-risk patients, who displayed greater hub prevalence and a radical reorganization of hubs in the subnetwork. SUDEP is associated with reduced functional organization among cortical and sub-cortical brain regions mediating autonomic and respiratory regulation. Living high-risk subjects demonstrated similar patterns, suggesting such network measures may provide prospective risk-indicating value, though a crucial difference between SUDEP and high-risk was altered connectivity of the medial thalamus in SUDEP, which was also elevated compared with all sub-groups. Disturbed thalamic connectivity may reflect a potential non-invasive marker of elevated SUDEP risk

Differential temporal expression of milk miRNA during the lactation cycle of the marsupial tammar wallaby (Macropus eugenii)

Lactation is a key aspect of mammalian evolution for adaptation of various reproductive strategies along different mammalian lineages. Marsupials, such as tammar wallaby, adopted a short gestation and a relatively long lactation cycle, the newborn is immature at birth and significant development occurs postnatally during lactation. Continuous changes of tammar milk composition may contribute to development and immune protection of pouch young. Here, in order to address the putative contribution of newly identified secretory milk miRNA in these processes, high throughput sequencing of miRNAs collected from tammar milk at different time points of lactation was conducted. A comparative analysis was performed to find distribution of miRNA in milk and blood serum of lactating wallaby

Stochastic make-to-stock inventory deployment problem: an endosymbiotic psychoclonal algorithm based approach

Integrated steel manufacturers (ISMs) have no specific product, they just produce finished product from the ore. This enhances the uncertainty prevailing in the ISM regarding the nature of the finished product and significant demand by customers. At present low cost mini-mills are giving firm competition to ISMs in terms of cost, and this has compelled the ISM industry to target customers who want exotic products and faster reliable deliveries. To meet this objective, ISMs are exploring the option of satisfying part of their demand by converting strategically placed products, this helps in increasing the variability of product produced by the ISM in a short lead time. In this paper the authors have proposed a new hybrid evolutionary algorithm named endosymbiotic-psychoclonal (ESPC) to decide what and how much to stock as a semi-product in inventory. In the proposed theory, the ability of previously proposed psychoclonal algorithms to exploit the search space has been increased by making antibodies and antigen more co-operative interacting species. The efficacy of the proposed algorithm has been tested on randomly generated datasets and the results compared with other evolutionary algorithms such as genetic algorithms (GA) and simulated annealing (SA). The comparison of ESPC with GA and SA proves the superiority of the proposed algorithm both in terms of quality of the solution obtained and convergence time required to reach the optimal/near optimal value of the solution

An IoE Blockchain-Based Network Knowledge Management Model for Resilient Disaster Frameworks

The disaster area is a constantly changing environment, which can make it challenging to distribute supplies effectively. The lack of accurate information about the required goods and potential bottlenecks in the distribution process can be detrimental. The success of a response network is dependent on collaboration, coordination, sovereignty, and equal distribution of relief resources. To facilitate these interactions and improve knowledge of supply chain operations, a reliable and dynamic logistic system is essential. This study proposes the integration of blockchain technology, the Internet of Things (IoT), and the Internet of Everything (IoE) into the disaster management structure. The proposed disaster response model aims to reduce response times and ensure the secure and timely distribution of goods. The hyper-connected disaster supply network is modeled through a concrete implementation on the Network Simulation (NS2) platform. The simulation results demonstrate that the proposed method yields significant improvements in several key performance metrics. Specifically, it achieved more than a 30% improvement in the successful migration of tasks, a 17% reduction in errors, a 15% reduction in delays, and a 9% reduction in energy consumption

Assay strategies for the discovery and validation of therapeutics targeting <i>Brugia pahangi</i> Hsp90

The chemotherapy of lymphatic filariasis relies upon drugs such as diethylcarbamazine and ivermectin that largely target the microfilarial stages of the parasite, necessitating continued treatment over the long reproductive life span of the adult worm. The identification of compounds that target adult worms has been a long-term goal of WHO. Here we describe a fluorescence polarization assay for the identification of compounds that target Hsp90 in adult filarial worms. The assay was originally developed to identify inhibitors of Hsp90 in tumor cells, and relies upon the ability of small molecules to inhibit the binding of fluorescently labelled geldanamycin to Hsp90. We demonstrate that the assay works well with soluble extracts of Brugia, while extracts of the free-living nematode C. elegans fail to bind the probe, in agreement with data from other experiments. The assay was validated using known inhibitors of Hsp90 that compete with geldanamycin for binding to Hsp90, including members of the synthetic purine-scaffold series of compounds. The efficacy of some of these compounds against adult worms was confirmed in vitro. Moreover, the assay is sufficiently sensitive to differentiate between binding of purine-scaffold compounds to human and Brugia Hsp90. The assay is suitable for high-throughput screening and provides the first example of a format with the potential to identify novel inhibitors of Hsp90 in filarial worms and in other parasitic species where Hsp90 may be a target

Late periorbital haemorrhage following functional endoscopic sinus surgery: a caution for potential day case surgery

BACKGROUND: Orbital complications following functional endoscopic sinus surgery (FESS) are fortunately rare. They are usually easily and rapidly recognizable. CASE PRESENTATION: We present an unusual case of a forty-five year old woman who underwent routine FESS and was not packed nasally after the procedure. Six hours later she started bleeding and nasal packs were inserted. She soon developed unilateral periorbital bruising and within hours her condition had worsened so much that the viability of the eye was thrown into question. She underwent medial and lateral canthotomies and made an uneventful post-operative recovery. CONCLUSION: This rare case demonstrates that late, brisk post-operative bleeding can occur after FESS with potentially catastrophic consequences. Clinicians should be aware that discharging patients after FESS too early may lead to medico-legal problems

Anomaly Equations and Intersection Theory

Six-dimensional supergravity theories with N=(1,0) supersymmetry must satisfy anomaly equations. These equations come from demanding the cancellation of gravitational, gauge and mixed anomalies. The anomaly equations have implications for the geometrical data of Calabi-Yau threefolds, since F-theory compactified on an elliptically fibered Calabi-Yau threefold with a section generates a consistent six-dimensional N=(1,0) supergravity theory. In this paper, we show that the anomaly equations can be summarized by three intersection theory identities. In the process we also identify the geometric counterpart of the anomaly coefficients---in particular, those of the abelian gauge groups---that govern the low-energy dynamics of the theory. We discuss the results in the context of investigating string universality in six dimensions.Comment: 29 pages + appendices, 8 figures; v2: minor corrections, references added; v3: minor corrections, reference adde