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Neuromuscular blocking agents (NMBAs) relax skeletal muscles to facilitate surgeries and ease intubation but can lead to adverse reactions, including complications because of postoperative residual neuromuscular blockade (rNMB) and, in rare cases, anaphylaxis. Both adverse reactions vary between types of NMBAs, with rocuronium, a widely used nondepolarizing NMBA, inducing one of the longest rNMB durations and highest anaphylaxis incidences. rNMB induced by rocuronium can be reversed by the synthetic γ-cyclodextrin sugammadex. However, in rare cases, sugammadex can provoke anaphylaxis. Thus, additional therapeutic options are needed. Rocuronium-induced anaphylaxis is proposed to rely on preexisting rocuronium-binding antibodies. To understand the pathogenesis of rocuronium-induced anaphylaxis and to identify potential therapeutics, we investigated the memory B cell antibody repertoire of patients with suspected hypersensitivity to rocuronium. We identified polyclonal antibody repertoires with a high diversity among V(D)J genes without evidence of clonal groups. When recombinantly expressed, these antibodies demonstrated specificity and low affinity for rocuronium without cross-reactivity for other NMBAs. Moreover, when these antibodies were expressed as human immunoglobulin E (IgE), they triggered human mast cell activation and passive systemic anaphylaxis in transgenic mice, although their affinities were insufficient to serve as reversal agents. Rocuronium-specific, high-affinity antibodies were thus isolated from rocuronium-immunized mice. The highest-affinity antibody was able to reverse rocuronium-induced neuromuscular blockade in nonhuman primates with kinetics comparable to that of sugammadex. Together, these data support the hypothesis that antibodies cause anaphylactic reactions to rocuronium and pave the way for improved diagnostics and neuromuscular blockade reversal agents. Editor's summary: Neuromuscular blocking agents (NMBAs) such as rocuronium are used during anesthesia to assist with surgeries and intubations. Although these agents are effective, they are not without risk. Patients can develop postoperative residual neuromuscular blockade, which can be treated with an NMBA reversal agent, and life-threatening anaphylaxis can occur in rare cases. Here, Dejoux et al. isolated and characterized memory B cells from three individuals who developed reactions to NMBA treatment and had serum antibodies to rocuronium. The authors found that IgE antibodies generated from these memory B cells were sufficient to induce anaphylaxis in mice treated with rocuronium, although they were too low affinity to be used as reversal agents. This led the authors to immunize mice with rocuronium to generate antibodies of higher affinity, which were able to reverse neuromuscular blockade in rocuronium-treated nonhuman primates. Together, these data explain why these three patients had a reaction and offer a therapeutic approach to reversing neuromuscular blockade. —Courtney Malo [ABSTRACT FROM AUTHOR]

Cell migration modes can vary, depending on a number of environmental and intracellular factors. The high motility of the pathogenic amoeba Entamoeba histolytica is a decisive factor in its ability to cross the human colonic barrier. We used quantitative live imaging techniques to study the migration of this parasite on fibronectin, a key tissue component. Entamoeba histolytica amoebae on fibronectin contain abundant podosome-like structures. By using a laminar flow chamber, we determined that the adhesion forces generated on fibronectin were twice those on non-coated glass. When migrating on fibronectin, elongated amoeboid cells converted into fan-shaped cells characterized by the presence of a dorsal column of F-actin and a broad cytoplasmic extension at the front. The fan shape depended on the Arp2/3 complex, and the amoebae moved laterally and more slowly. Intracellular measurements of physical variables related to fluid dynamics revealed that cytoplasmic pressure gradients were weaker within fan-shaped cells; hence, actomyosin motors might be less involved in driving the cell body forward. We also found that the Rho-associated coiled-coil containing protein kinase regulated podosome dynamics. We conclude that E. histolytica spontaneously changes its migration mode as a function of the substrate composition. This adaptive ability might favour E. histolytica's invasion of human colonic tissue. By combining microfluidic experiments, mechanical modelling, and image analysis, our work also introduces a computational pipeline for the study of cell migration. Author summary: Even though we know increasingly well how cells move in tissues, the question of whether parasites modify their migration as a function of the host tissue environment requires further investigation. Cell motility is triggered by environmental stimuli, and the migration mode depends on adhesion, activated enzymatic pathways, and changes in the actin cytoskeleton dynamics and thus cell morphology. The protozoan intestinal parasitic Entamoeba histolytica is the causative agent of amoebiasis (a major food- and water-borne disease) in humans. During invasion of the colon, E. histolytica degrades the mucus layer, adheres to and removes the epithelium, progresses along the fibronectin-rich basement membrane towards the crypts of Lieberkühn, and penetrates the mucosa. This invasive process raises the question of how the parasite moves in a varying intestinal environment. E. histolytica is known to display an amoeboid migration mode and binds fibronectin through podosome adhesion structures. We hypothesized that fibronectin triggers signalling pathways that influence the trophozoite's migration. We found that E. histolytica can migrate with a non-amoeboid mode on fibronectin-coated glass. Our finding suggests that for tissue invasion, E. histolytica's migration mode is modulated to suit the environment. [ABSTRACT FROM AUTHOR]

The precise mechanism leading to profound immunodeficiency of HIV-infected patients is still only partially understood. Here, we show that more than 80% of [CD4.sup.+] T cells from HIV-infected patients have morphological abnormalities. Their membranes exhibited numerous large abnormal membrane microdomains (aMMDs), which trap and inactivate physiological receptors, such as that for IL-7. In patient plasma, we identified phospholipase A2 group IB (PLA2G1B) as the key molecule responsible for the formation of aMMDs. At physiological concentrations, PLA2G1B synergized with the HIV gp41 envelope protein, which appears to be a driver that targets PLA2G1B to the [CD4.sup.+] T cell surface. The PLA2G1B/gp41 pair induced [CD4.sup.+] T cell unresponsiveness (anergy). At high concentrations in vitro, PLA2G1B acted alone, independently of gp41, and inhibited the IL-2, IL-4, and IL-7 responses, as well as TCR-mediated activation and proliferation, of [CD4.sup.+] T cells. PLA2G1B also decreased [CD4.sup.+] T cell survival in vitro, likely playing a role in CD4 lymphopenia in conjunction with its induced IL-7 receptor defects. The effects on [CD4.sup.+] T cell anergy could be blocked by a PLA2G1B- specific neutralizing mAb in vitro and in vivo. The PLA2G1B/gp41 pair constitutes what we believe is a new mechanism of immune dysfunction and a compelling target for boosting immune responses in HIV-infected patients., Introduction [CD4.sup.+] lymphocytes play a critical role in the severe immunodeficiency that characterizes HIV-infected patients. Although fewer than 0.5% of blood [CD4.sup.+] T cells are infected, almost all are dysfunctional. [...]

Polyamines cause inward rectification of (Kir) [K.sup.+] channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they. preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSE[A.sup.+] modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter. KEY WORDS: inward rectifier * spermine * diamine * rectification * selectivity filter

Isothermal titration calorimetry and X-ray crystallography are used to determine the structural and thermodynamic consequences associated with constraining the pTyr residue of the pYEE Iigand for the Src homology two domain of the Src kinase. It is observed that drop in enthalpy with the conformationally constrained peptide mimic arises from sources other than changes in buried surface areas, though the exact origin of the differences remain unclear.

Research has been conducted on the PapG, the adhesin at the tip of the P pilus which mediates the uropathogenic Escherichia coli attachment to the human kidney uroepithelium. The crystal structures of the PapG receptor binding domain's binary complex bound to the globoside have been investigated and the results are reported.

Research has been conducted on the Na (super)+ -binding properties of factor X. The characterization of these properties is described.

A combination of two-dimensional nuclear magnetic resonance spectroscopy and molecular simulation was used to derive the solution structure of the nonpalindromic dodecanucleotide homopyrimidine:homopurine. The dodecamer sequence in question was located within the HIV-1 envelope sequence. All four Gs in two hypermutants were substituted by A. The solution structure derived resulted in a set of low-energy B-DNA conformations satisfying the nuclear Overhauser effect data.

In this work on the design and studies of luciferins related to the blue‐hued coelenterazine, the synthesis of heterocyclic analogues susceptible to produce a photon, possibly at a different wavelength, is undertaken. Here, the synthesis of O‐acetylated derivatives of imidazo[1,2‐b]pyridazin‐3(5 H)‐one, imidazo[2,1‐f][1,2,4]triazin‐7(1 H)‐one, imidazo[1,2‐a]pyridin‐3‐ol, imidazo[1,2‐a]quinoxalin‐1(5 H)‐one, benzo[f]imidazo[1,2‐a]quinoxalin‐3(11 H)‐one, imidazo[1′,2′:1,6]pyrazino[2,3‐c]quinolin‐3(11 H)‐one, and 5,11‐dihydro‐3 H‐chromeno[4,3‐e]imidazo[1,2‐a]pyrazin‐3‐one is described thanks to extensive use of the Buchwald–Hartwig N‐arylation reaction. The acidic hydrolysis of these derivatives then gave solutions of the corresponding luciferin analogues, which were studied. Not too unexpectedly, even if these were "dressed" with substituents found in actual substrates of the nanoKAZ/NanoLuc luciferase, no bioluminescence was observed with these compounds. However, in a phosphate buffer, all produced a light signal, by chemiluminescence, with extensive variations in their respective intensity and this could be increased by adding a quaternary ammonium salt in the buffer. This aspect was actually instrumental to determine the emission spectra of many of these luciferin analogues. [ABSTRACT FROM AUTHOR]

We describe here an extensive structure‐bioluminescence relationship study of a chemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, a mutated luciferase originated from the catalytic subunit of the deep‐sea shrimp Oplophorus gracilirostris. Out of the 135 O‐acetylated precursors that were prepared by using our recently reported synthesis and following their hydrolysis to give solutions of the corresponding luciferins, notable bioluminescence improvements were achieved in comparison with furimazine, which is currently amongst the best substrates of nanoKAZ/NanoLuc. For instance, the rather more lipophilic analogue 8‐(2,3‐difluorobenzyl)‐2‐((5‐methylfuran‐2‐yl)methyl)‐6‐phenylimidazo[1,2‐a]pyrazin‐3(7H)‐one provided a 1.5‐fold improvement of the total light output over a 2 h period, a close to threefold increase of the initial signal intensity and a signal‐to‐background ratio five times greater than furimazine. The kinetic parameters for the enzymatic reaction were obtained for a selection of luciferin analogues and provided unexpected insights into the luciferase activity. Most prominently, along with a general substrate‐dependent and irreversible inactivation of this enzyme, in the case of the optimized luciferin mentioned above, the consumption of 2664 molecules was found to be required for the detection of a single Relative Light Unit (RLU; a luminometer‐dependent fraction of a photon). [ABSTRACT FROM AUTHOR]

The molecular and supramolecular self-assembly mechanism of synthetic octapeptide into nanotubes in equilibrium conditions is demonstrated. The rational design of oligopeptides which could encode both molecular- and macro-scale morphological characteristics of their higher-order assemblies provide a new pathway to engineer ultrahigh resolution peptide scaffold.

A new series of alkyl tripeptide that target the four-stranded antiparallel [beta]-sheet formed by the interdigitation of the N- and C-monomer ends of each monomer is used to gain insight into the dimerization inhibition of wild-type and drug-resistant mutated HIV-1 proteases. The results showed that the alkyl tripeptides that disrupt HIV-1 protease dimerization are likely to remain active against HIV strains that are resistant to classical protease inhibitors (PIs).

Interactions of sulfhydryl reagents with introduced cysteines in the pore-forming (Kir6.2) subunits of the [K.sub.ATP] channel were examined. 2-Aminoethyl methanethiosulfonate ([MTSEA.sup.+]) failed to modify [Cd.sup.2+]-insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels. Although a single [Cd.sup.2+] ion is coordinated by L164C, four [MTSEA.sup.+] 'hits' can occur, each sequentially reducing the single-channel current. A dimeric fusion of control-Kir6.2 and L164C subunits generates [Cd.sup.2+]-insensitive channels, confirming that at least three cysteines are required for coordination, but [MTSEA.sup.+] modification of the dimer occurs in two hits. L164C channels were not modified by bromotrimethyl ammoniumbimane ([qBBr.sup.+]), even though [qBBr.sup.+] caused voltage-dependent block (as opposed to modification) that was comparable to that of [MTSEA.sup.+] or 3-(triethylammonium)propyl methanethiosulfonate ([MTSPTrEA.sup.+]), implying that [qBBr.sup.+] can also enter the inner cavity but does not modify L164C residues. The Kir channel pore structure was modeled by homology with the KcsA crystal structure. A stable conformation optimally places the four L164C side chains for coordination of a single [Cd.sup.2+] ion. Modification of these cysteines by up to four [MTSEA.sup.+] (or three [MTSPTrEA.sup.+], or two [qBBr.sup.+]) does not require widening of the cavity to accommodate the derivatives within it. However, like the KcsA crystal structure, the energy-minimized model shows a narrowing at the inner entrance, and in the Kir6.2 model this narrowing excludes all ions. To allow entry of ions as large as [MTSPTrEA.sup.+] or [qBBr.sup.+], the entrance must widen to [is greater than] 8 A, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement. inner pore | [Cd.sup.2+] | MTS reagents | subconductance

ABSTRACT Background Methods Results Conclusions Diagnosis of allergies is mostly based on the patient's clinical history and allergen provocation tests. Determination of specific IgE (sIgE) profiles can be performed to support allergy diagnosis. This is commonly done in vivo by the skin prick test or in vitro with automated systems. Several platforms exist to quantify sIgE levels, but all these methods require access to specific instruments, often delaying the test's results. The IgE luciferase‐linked immunosorbent assay (LuLISA) allows bioluminescent quantification of IgE against peanut in microliter samples, but this method awaits extension to other allergens. This study aimed to validate a new method, named LuLIPLEX, for multiplexed bioluminescent detection of sIgE against 20 major molecular allergens.Quantification of sIgE against 12 recombinant or purified food allergens and eight aeroallergens was performed by LuLIPLEX versus standard IgE detection methods (ImmunoCAP, ISAC, ALEX, or NOVEOS). Multiplexed detection of IgE against these 20 allergens was performed within 45 min using 50 μL of serum, plasma, or whole blood samples.A head‐to‐head comparison between LuLIPLEX and standard IgE detection methods showed a high correlation among all allergens tested. sIgE profiles in polysensitized subjects could be determined within 45 min in serum and plasma samples, as well as using a single drop of capillary blood.LuLIPLEX is a rapid and sensitive method to quantify sIgE levels against multiple allergens. Given that the test is very fast and can be performed on small and inexpensive luminometers, the IgE LuLIPLEX could allow point‐of‐care testing of sIgE profiles in allergic subjects. [ABSTRACT FROM AUTHOR]