A C-terminal cysteine residue is required for peptide-based inhibition of the NGF/TrkA interaction at nM concentrations:implications for peptide-based analgesics

Inhibition of the NGF/TrkA interaction presents an interesting alternative to the use of non-steroidal anti-inflammatories and/or opioids for the control of inflammatory, chronic and neuropathic pain. Most prominent of the current approaches to this therapy is the antibody Tanezumab, which is a late-stage development humanized monoclonal antibody that targets NGF. We sought to determine whether peptides might similarly inhibit the NGF/TrkA interaction and so serve as future therapeutic leads. Starting from two peptides that inhibit the NGF/TrkA interaction, we sought to eliminate a cysteine residue close to the C-terminal of both sequences, by an approach of mutagenic analysis and saturation mutagenesis of mutable residues. Elimination of cysteine from a therapeutic lead is desirable to circumvent manufacturing difficulties resulting from oxidation. Our analyses determined that the cysteine residue is not required for NGF binding, but is essential for inhibition of the NGF/TrkA interaction at pharmacologically relevant peptide concentrations. We conclude that a cysteine residue is required within potential peptide-based therapeutic leads and hypothesise that these peptides likely act as dimers, mirroring the dimeric structure of the TrkA receptor

Three Dimensional Structure of the MqsR:MqsA Complex: A Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties

One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an α/β fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state

Targeting Angiogenesis-Dependent Calcified Neoplasms Using Combined Polymer Therapeutics

There is an immense clinical need for novel therapeutics for the treatment of angiogenesis-dependent calcified neoplasms such as osteosarcomas and bone metastases. We developed a new therapeutic strategy to target bone metastases and calcified neoplasms using combined polymer-bound angiogenesis inhibitors. Using an advanced "living polymerization" technique, the reversible addition-fragmentation chain transfer (RAFT), we conjugated the aminobisphosphonate alendronate (ALN), and the potent anti-angiogenic agent TNP-470 with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer through a Glycine-Glycine-Proline-Norleucine linker, cleaved by cathepsin K, a cysteine protease overexpressed at resorption sites in bone tissues. In this approach, dual targeting is achieved. Passive accumulation is possible due to the increase in molecular weight following polymer conjugation of the drugs, thus extravasating from the tumor leaky vessels and not from normal healthy vessels. Active targeting to the calcified tissues is achieved by ALN's affinity to bone mineral.The anti-angiogenic and antitumor potency of HPMA copolymer-ALN-TNP-470 conjugate was evaluated both in vitro and in vivo. We show that free and conjugated ALN-TNP-470 have synergistic anti-angiogenic and antitumor activity by inhibiting proliferation, migration and capillary-like tube formation of endothelial and human osteosarcoma cells in vitro. Evaluation of anti-angiogenic, antitumor activity and body distribution of HPMA copolymer-ALN-TNP-470 conjugate was performed on severe combined immunodeficiency (SCID) male mice inoculated with mCherry-labeled MG-63-Ras human osteosarcoma and by modified Miles permeability assay. Our targeted bi-specific conjugate reduced VEGF-induced vascular hyperpermeability by 92% and remarkably inhibited osteosarcoma growth in mice by 96%.This is the first report to describe a new concept of a narrowly-dispersed combined polymer therapeutic designed to target both tumor and endothelial compartments of bone metastases and calcified neoplasms at a single administration. This new approach of co-delivery of two synergistic drugs may have clinical utility as a potential therapy for angiogenesis-dependent cancers such as osteosarcoma and bone metastases

EPIdemiology of Surgery-Associated Acute Kidney Injury (EPIS-AKI) : Study protocol for a multicentre, observational trial

More than 300 million surgical procedures are performed each year. Acute kidney injury (AKI) is a common complication after major surgery and is associated with adverse short-term and long-term outcomes. However, there is a large variation in the incidence of reported AKI rates. The establishment of an accurate epidemiology of surgery-associated AKI is important for healthcare policy, quality initiatives, clinical trials, as well as for improving guidelines. The objective of the Epidemiology of Surgery-associated Acute Kidney Injury (EPIS-AKI) trial is to prospectively evaluate the epidemiology of AKI after major surgery using the latest Kidney Disease: Improving Global Outcomes (KDIGO) consensus definition of AKI. EPIS-AKI is an international prospective, observational, multicentre cohort study including 10 000 patients undergoing major surgery who are subsequently admitted to the ICU or a similar high dependency unit. The primary endpoint is the incidence of AKI within 72 hours after surgery according to the KDIGO criteria. Secondary endpoints include use of renal replacement therapy (RRT), mortality during ICU and hospital stay, length of ICU and hospital stay and major adverse kidney events (combined endpoint consisting of persistent renal dysfunction, RRT and mortality) at day 90. Further, we will evaluate preoperative and intraoperative risk factors affecting the incidence of postoperative AKI. In an add-on analysis, we will assess urinary biomarkers for early detection of AKI. EPIS-AKI has been approved by the leading Ethics Committee of the Medical Council North Rhine-Westphalia, of the Westphalian Wilhelms-University Münster and the corresponding Ethics Committee at each participating site. Results will be disseminated widely and published in peer-reviewed journals, presented at conferences and used to design further AKI-related trials. Trial registration number NCT04165369

Plasma dermatan sulfate proteoglycan in a patient on chronic hemodialysis

Abstract A 68-year-old man on chronic hemodialysis for 6 years, presented with a spontaneous psoas muscle hemorrhage. Investigations showed intermittently elevated activated partial-thromboplastin time and thrombin time. Preliminary investigations suggested a heparin-like inhibitor in the patient's plasma, but no anti-Xa activity could be detected. Investigation of the ability of patient plasma to inhibit exogenous thrombin showed that most thrombin was inhibited by heparin cofactor II, in contrast to normal plasma in which most thrombin was inhibited by antithrombin III. Treatment of plasma with glycosaminoglycan-degrading enzymes suggested the presence of dermatan sulfate (DS) in patient plasma. This was confirmed in a heparin cofactor II-dependent antithrombin assay for DS that showed anticoagulant equivalent to 2.2 +/- 0.3 micrograms/mL (mean +/- SD) of porcine mucosal DS. Of this activity, approximately 90% was sensitive to enzymes that degrade DS. The glycosaminoglycan containing fraction of plasma was isolated and subjected to gel chromatography. Anticoagulant activity eluted from Sephadex G-100 (Pharmacia, Montreal, Quebec, Canada) as two peaks with Kav of 0.10 and 0.45. After treatment with base, the Kav of the higher molecular weight species was increased to 0.55. This activity was completely sensitive to enzymes that degrade DS. Thus, the active DS was present as a proteoglycan. The lower molecular weight material was not sensitive to enzymes that degrade DS or heparan sulfate and it was active in the heparin cofactor II- dependent antithrombin assay but not in an antithrombin III-dependent antithrombin assay. This activity was not degraded by heating. Subsequently, measurement of DS activity was performed in plasmas obtained from eight other patients on hemodialysis before administration of heparin that showed that all patients had DS activity present that varied from 0.05 to 0.4 microgram/mL. No enzyme-resistant activity could be shown in these patients. In summary, a circulating anticoagulant with properties of DS is present in patients requiring hemodialysis.</jats:p

Plasma dermatan sulfate proteoglycan in a patient on chronic hemodialysis

A 68-year-old man on chronic hemodialysis for 6 years, presented with a spontaneous psoas muscle hemorrhage. Investigations showed intermittently elevated activated partial-thromboplastin time and thrombin time. Preliminary investigations suggested a heparin-like inhibitor in the patient's plasma, but no anti-Xa activity could be detected. Investigation of the ability of patient plasma to inhibit exogenous thrombin showed that most thrombin was inhibited by heparin cofactor II, in contrast to normal plasma in which most thrombin was inhibited by antithrombin III. Treatment of plasma with glycosaminoglycan-degrading enzymes suggested the presence of dermatan sulfate (DS) in patient plasma. This was confirmed in a heparin cofactor II-dependent antithrombin assay for DS that showed anticoagulant equivalent to 2.2 +/- 0.3 micrograms/mL (mean +/- SD) of porcine mucosal DS. Of this activity, approximately 90% was sensitive to enzymes that degrade DS. The glycosaminoglycan containing fraction of plasma was isolated and subjected to gel chromatography. Anticoagulant activity eluted from Sephadex G-100 (Pharmacia, Montreal, Quebec, Canada) as two peaks with Kav of 0.10 and 0.45. After treatment with base, the Kav of the higher molecular weight species was increased to 0.55. This activity was completely sensitive to enzymes that degrade DS. Thus, the active DS was present as a proteoglycan. The lower molecular weight material was not sensitive to enzymes that degrade DS or heparan sulfate and it was active in the heparin cofactor II- dependent antithrombin assay but not in an antithrombin III-dependent antithrombin assay. This activity was not degraded by heating. Subsequently, measurement of DS activity was performed in plasmas obtained from eight other patients on hemodialysis before administration of heparin that showed that all patients had DS activity present that varied from 0.05 to 0.4 microgram/mL. No enzyme-resistant activity could be shown in these patients. In summary, a circulating anticoagulant with properties of DS is present in patients requiring hemodialysis.</jats:p

Energy Efficiency Maximization of Two-Time-Slot and Three-Time-Slot Two-Way Relay-Assisted Device-to-Device Underlaying Cellular Networks

The continuous development of fifth generation (5G) communication and Internet of Thing (IoT) inevitably necessitates more advanced systems that can satisfy the growing wireless data rate demand of future equipment. Device-to-Device (D2D) communication, whose performance is evaluated in terms of the overall throughput, energy efficiency (EE) and spectral efficiency (SE), is considered a promising solution for the aforementioned problem. Thereby, this paper aims at improving the performance of the D2D communication underlaying cellular networks operating on multiple bands by maximizing the EE in its uplink. Thanks to the stochastic geometry theory, it is possible to derive the closed-form expressions for the successful transmission probability (STP), the total average transmission rate (TATR), and the total average energy efficiency (TAEE) of cellular and D2D users in different time slot setting. Particularly investigated and compared in this study, there are one-hop, direct, D2D communication in two time slots (2TS), and multi-hop, indirect, D2D communication in three time slots (3TS) with an additional D2D user acting as a two-way relay to assist the communication. Moreover, an optimization problem is formulated to calculate the maximum TAEE of D2D users and the optimum transmission power of both the cellular and D2D users. Herein this optimization study, which is proven to be non-convex, the Quality of Service (QoS) is ensured as the STP on every link is considered. The herein approach is referred to as relay-assisted D2D communication which is capable of delivering a notably better QoS and lower transmission power for communication among distant D2D users.</jats:p