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The M2 proton channel aids in the exit of mature influenza viral particles from the host plasma membrane through its ability to stabilize regions of high negative Gaussian curvature (NGC) that occur at the neck of budding virions. The channels are homo-tetramers that contain a cytoplasm-facing amphipathic helix (AH) that is necessary and sufficient for NGC generation; however, constructs containing the transmembrane spanning helix, which facilitates tetramerization, exhibit enhanced curvature generation. Here, we used all-atom molecular dynamics (MD) simulations to explore the conformational dynamics of M2 channels in lipid bilayers revealing that the AH is dynamic, quickly breaking the fourfold symmetry observed in most structures. Next, we carried out MD simulations with the protein restrained in four- and twofold symmetric conformations to determine the impact on the membrane shape. While each pattern was distinct, all configurations induced pronounced curvature in the outer leaflet, while conversely, the inner leaflets showed minimal curvature and significant lipid tilt around the AHs. The MD-generated profiles at the protein-membrane interface were then extracted and used as boundary conditions in a continuum elastic membrane model to calculate the membrane-bending energy of each conformation embedded in different membrane surfaces characteristic of a budding virus. The calculations show that all three M2 conformations are stabilized in inward-budding, concave spherical caps and destabilized in outward-budding, convex spherical caps, the latter reminiscent of a budding virus. One of the C2-broken symmetry conformations is stabilized by 4 kT in NGC surfaces with the minimum energy conformation occurring at a curvature corresponding to 33 nm radii. In total, our work provides atomistic insight into the curvature sensing capabilities of M2 channels and how enrichment in the nascent viral particle depends on protein shape and membrane geometry., Competing Interests: JL, CH, FM, AN, MG No competing interests declared, (© 2024, Lincoff, Helsell, Marcoline et al.)

(1) Background: Patent foramen ovale (PFO) is a congenital abnormality present in up to 25% of the general population, and it is a relevant cause of cryptogenic stroke. We applied the hospital-based HTA model (AdHopHTA) to conduct a multidimensional assessment of NobleStitch EL, an innovative suture-mediated PFO closure device. We compared it to Amplatzer PFO Occluder (APO) to provide evidence to inform technologies' governance in hospital settings. (2) Methods: For each AdHopHTA dimension we: systematically retrieved available evidence from the literature applying the PRISMA guidelines and then analyzed original clinical and cost data of a NobleStitch EL device at San Raffaele research hospital in Milan (Italy). The economic dimension was analyzed through activity-based costing and a cost analysis. We conducted semi-structured interviews with selected healthcare professionals to explore the organizational, legal, social, and ethical impact. (3) Results: A single study was included for the NobleStitch EL, with 10 for APO. Both literature data and original data showed comparable safety. Efficacy data analysis found that the PFO closure was at 89% for NobleStitch EL vs. 89-97% for APO. APO has a better impact on the budget and minor process costs. Consulted experts reported that the organizational impact of NobleStitch EL in the short and the long run as null, albeit a better impact under the social and the ethical aspects. (4) Conclusion: We suggest that there is inadequate evidence to conclude the relative efficacy of NobleStitch EL as compared to APO. Nevertheless, this report shows a good safety profile and higher costs for NobleStitch EL, with no organizational or legal impact. Further studies in selected population are recommended.

Mitochondria generate heat due to H + leak (I H ) across their inner membrane 1 . I H results from the action of long-chain fatty acids on uncoupling protein 1 (UCP1) in brown fat 2-6 and ADP/ATP carrier (AAC) in other tissues 1,7-9 , but the underlying mechanism is poorly understood. As evidence of pharmacological activators of I H through UCP1 and AAC is lacking, I H is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) 10,11 . Although protonophores show potential in combating obesity, diabetes and fatty liver in animal models 12-14 , their clinical potential for treating human disease is limited due to indiscriminately increasing H + conductance across all biological membranes 10,11 and adverse side effects 15 . Here we report the direct measurement of I H induced by DNP, FCCP and other common protonophores and find that it is dependent on AAC and UCP1. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain fatty acids, and find that they overlap with the putative ADP/ATP-binding site. We also develop a mathematical model that proposes a mechanism of uncoupler-dependent I H through AAC. Thus, common protonophoric uncouplers are synthetic activators of I H through AAC and UCP1, paving the way for the development of new and more specific activators of these two central mediators of mitochondrial bioenergetics., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Consistent buckling distortions of a large membrane patch (200 × 200 Å) are observed during molecular dynamics (MD) simulations using the Monte-Carlo (MC) barostat in combination with a hard Lennard-Jones (LJ) cutoff. The buckling behavior is independent of both the simulation engine and the force field but requires the MC barostat-hard LJ cutoff combination. Similar simulations of a smaller patch (90 × 90 Å) do not show buckling, but do show a small, systematic reduction in the surface area accompanied by ~1 Å thickening suggestive of compression. We show that a mismatch in the way potentials and forces are handled in the dynamical equations versus the MC barostat results in a compressive load on the membrane. Moreover, a straightforward application of elasticity theory reveals that a minimal compression of the linear dimensions of the membrane, inversely proportional to the edge length, is required for buckling, explaining this differential behavior. We recommend always using LJ force or potential-switching when the MC barostat is employed to avoid undesirable membrane deformations., (© 2021 Wiley Periodicals LLC.)

Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg 2+ to the sensor domain of an E. coli two component histidine kinase (HK), PhoQ, modulates its cytoplasmic kinase domain. We use cysteine-crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ's sensor and autokinase domains in a set of over 30 mutants. Strikingly, conservative single-site mutations distant from the sensor or catalytic site strongly influence PhoQ's ligand-sensitivity as well as the magnitude and direction of the signal. Data from 35 mutants are explained by a semi-empirical three-domain model in which the sensor, intervening HAMP, and catalytic domains can adopt kinase-promoting or inhibiting conformations that are in allosteric communication. The catalytic and sensor domains intrinsically favor a constitutively 'kinase-on' conformation, while the HAMP domain favors the 'off' state; when coupled, they create a bistable system responsive to physiological concentrations of Mg 2+ . Mutations alter signaling by locally modulating domain intrinsic equilibrium constants and interdomain couplings. Our model suggests signals transmit via interdomain allostery rather than propagation of a single concerted conformational change, explaining the diversity of signaling structural transitions observed in individual HK domains., Competing Interests: BM, NP, AN, TL, WD No competing interests declared, KM is an employee of Codexis Inc. The author declares that no other competing interests exist, (© 2021, Mensa et al.)

Leak currents, defined as voltage and time independent flows of ions across cell membranes, are central to cellular electrical excitability control. The K 2P (KCNK) potassium channel class comprises an ion channel family that produces potassium leak currents that oppose excitation and stabilize the resting membrane potential in cells in the brain, cardiovascular system, immune system, and sensory organs. Due to their widespread tissue distribution, K 2P s contribute to many physiological and pathophysiological processes including anesthesia, pain, arrythmias, ischemia, hypertension, migraine, intraocular pressure regulation, and lung injury responses. Structural studies of six homomeric K 2P s have established the basic architecture of this channel family, revealed key moving parts involved in K 2P function, uncovered the importance of asymmetric pinching and dilation motions in the K 2P selectivity filter (SF) C-type gate, and defined two K 2P structural classes based on the absence or presence of an intracellular gate. Further, a series of structures characterizing K 2P :modulator interactions have revealed a striking polysite pharmacology housed within a relatively modestly sized (~70 kDa) channel. Binding sites for small molecules or lipids that control channel function are found at every layer of the channel structure, starting from its extracellular side through the portion that interacts with the membrane bilayer inner leaflet. This framework provides the basis for understanding how gating cues sensed by different channel parts control function and how small molecules and lipids modulate K 2P activity. Such knowledge should catalyze development of new K 2P modulators to probe function and treat a wide range of disorders., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

K 2P potassium channels regulate cellular excitability using their selectivity filter (C-type) gate. C-type gating mechanisms, best characterized in homotetrameric potassium channels, remain controversial and are attributed to selectivity filter pinching, dilation, or subtle structural changes. The extent to which such mechanisms control C-type gating of innately heterodimeric K 2P s is unknown. Here, combining K 2P 2.1 (TREK-1) x-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and electrophysiology, we uncover unprecedented, asymmetric, potassium-dependent conformational changes that underlie K 2P C-type gating. These asymmetric order-disorder transitions, enabled by the K 2P heterodimeric architecture, encompass pinching and dilation, disrupt the S1 and S2 ion binding sites, require the uniquely long K 2P SF2-M4 loop and conserved "M3 glutamate network," and are suppressed by the K 2P C-type gate activator ML335. These findings demonstrate that two distinct C-type gating mechanisms can operate in one channel and underscore the SF2-M4 loop as a target for K 2P channel modulator development., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl 2 , Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The ultrasensitive, ultrastable bacterial chemosensory array of Escherichia coli and Salmonella typhimurium is representative of the large, conserved family of sensory arrays that control the cellular chemotaxis of motile bacteria and Archaea. The core framework of the membrane-bound array is a lattice assembled from three components: a transmembrane receptor, a cytoplasmic His kinase (CheA), and a cytoplasmic adaptor protein (CheW). Structural studies in the field have revealed the global architecture of the array and complexes between specific components, but much remains to be learned about the essential protein-protein interfaces that define array structure and transmit signals between components. This study has focused on the structure, function, and on-off switching of a key contact between the kinase and adaptor proteins in the working, membrane-bound array. Specifically, the study addressed interface 1 in the putative kinase-adaptor ring where subdomain 1 of the kinase regulatory domain contacts subdomain 2 of the adaptor protein. Two independent approaches, disulfide mapping and site-directed Trp and Ala mutagenesis, were employed (i) to test the structural model of interface 1 and (ii) to investigate its functional roles in both stable kinase incorporation and receptor-regulated kinase on-off switching. Studies were conducted in functional, membrane-bound arrays or in live cells. The findings reveal that crystal structures of binary and ternary complexes accurately depict the native interface in its kinase-activating on state. Furthermore, the findings indicate that at least part of the interface becomes less closely packed in its kinase-inhibiting off state. Together, the evidence shows the interface has a dual structural and signaling function that is crucial for incorporation of the stable kinase into the array, for kinase activation in the array on state, and likely for attractant-triggered kinase on-off switching. A model is presented that describes the concerted transmission of a conformational signal among the receptor, the kinase regulatory domain, and the adaptor protein. In principle, this signal could spread out into the surrounding array via the kinase-adaptor ring, employing a series of alternating frozen-dynamic transitions that transmit low-energy attractant signals long distances.

The therapeutic efficacy of 5-fluorouracil (5-FUra; 0.6 mg/day x 5 days) + leucovorin (LV; 1.8 mg/day x 5 days) and of 131I-labeled MN-14 anticarcinoembryonic antigen IgG (275 microCi single dose) was evaluated in size-matched (0.3-0.7 cm3) s.c. LoVo, HT-29, DLD-1, HCT-15, LS174T, and MOSER, GW-39, and WidR human colonic tumors. These lines express varying amounts of carcinoembryonic antigen and exhibit varying degrees of in vitro responsiveness to 5-FUra. Unlike radioimmunotherapy (RAIT), multiple cycles of chemotherapy were feasible over a 3-week period. However, no therapeutic advantage to a second cycle of 5-FUra/LV administration was found. Therefore, it is reasonable to compare single cycles of both treatment modalities. RAIT was statistically more effective in 5 of 8 tumor lines (LoVo, LS174T, MOSER, WidR, and GW-39). In 1 other line (DLD-1), RAIT was marginally more efficacious, but tumors responded well to both therapies. The lack of a statistical difference between the 2 modalities of treatment may indicate that the efficacy of the 2 treatments is equivalent, or the relatively large variability within the treatment groups may have prevented significance given the number of animals evaluated. RAIT and 5-FUra/LV were equally efficacious in the HT-29 and the HCT-15 tumor lines. Of the 5 xenografts that responded better to RAIT, 3 lines (LS174T, GW-39, and WidR) demonstrated a greater percentage of tumors responding over a 5- to 6-week period. The other 3 lines (LoVo, MOSER, and DLD-1) exhibited a similar percent of tumors responding to both therapies, but a greater growth inhibition in those RAIT-treated tumors that responded. In vitro responsiveness to 5-FUra/LV did not directly correlate with in vivo responsiveness (r2 = -0.664), since LS174T and LoVo tumors, with rapid growth rates (0.05-0.36 cm3/day), were not highly responsive to therapy. Growth inhibition from RAIT also did not correlate with total tumor carcinoembryonic antigen content (r2 = 0.003), an observation that may be due to additional variables, such as accessibility of antigen and innate radiosensitivity of the tumor. RAIT was most effective in the fastest growing tumor lines (LS174T, GW-39, MOSER, WidR, and LoVo). These preclinical results suggest an advantage to radioantibody therapy over one of the most commonly used forms of chemotherapy to treat colorectal cancer. These studies also highlight the need to establish criteria that will enable the selection of therapeutic modalities in patients.

The therapeutic potential of radiolabeled antibodies is usually evaluated in experimental animal models bearing s.c. xenografts. We have established a micrometastatic model of the GW-39 human colonic carcinoma in the nude mouse lung (J. Natl. Cancer Inst., 83: 627-632, 1991) and presented preliminary findings on the efficacy of a 131I-anticarcinoembryonic antigen (CEA) antibody in this model. We now extend our observations on the use of radioiodinated labeled monoclonal antibodies (MAbs) to treat multiple small tumor nodules. Biodistribution and dosimetry analysis was performed for intact and F(ab')2 of NP-4 anti-CEA IgG, Mu-9 anti-colon-specific antigen IgG, isotype-matched irrelevant anti-AFP IgG, and intact MAb 34A anti-lung endothelial IgG antibody. Comparisons were made for rad dose delivered to small s.c. tumors, normal lung, lung with tumor nodules, and isolated tumor nodules. Survival curves were generated for tumor-bearing animals treated 1, 7, or 14 days after tumor cell implantation with these antibodies using the maximal tolerated dose for intact antibodies (275 microCi) and for F(ab')2 fragments (1.2 mCi). The studies established the following observations: (a) in contrast to previous results in a bulky tumor model in hamsters, intact antibodies are more therapeutic than MAb fragments for both NP-4 and Mu-9; (b) tumor nodule size, even on the microscopic level, affects therapeutic outcome; antibodies were more effective when administered 7 days postimplantation (mean nodule diameter, 150 microns) compared with treatment 14 days postimplantation (mean nodule diameter, 750 microns); (c) administration of radioiodinated Mu-9 was exquisitely effective on single avascular tumor cells that had seeded in lung; irrelevant antibody was minimally radiotoxic; (d) as in the bulky disease model, the anti-colon-specific antigen p antibody delivers a higher rad dose than the anti-CEA antibody and is significantly more therapeutic in the micrometastasis model; (e) a higher affinity anti-CEA antibody (MN-14) recognizing the same epitope on CEA as NP-4 was equally therapeutic; (f) the use of MAb directed against the lung endothelium was not as therapeutic as a tumor-associated antibody; and (g) all tumor-associated antibodies were more efficacious than administration of the maximal tolerated dose of 5-fluorouracil and leucovorin in this human tumor-xenograft model. These results provide further support for the use of radioimmunotherapy in the handling of minimal disease, probably as part of an adjuvant treatment regimen.

Background and Methods: The development of a multifactorial coagulopathy after massive transfusion is a well-recognized clinical problem that is almost always accompanied by hypothermia. The purpose of this study was to investigate the isolated effect of alterations of temperature on the integrity of the coagulation cascade. Prothrombin times and partial thromboplastin times were each performed 15 times on samples of pooled normal plasma at the temperatures of 37 degrees C, 34 degrees C, 31 degrees C, and 28 degrees C, as well as 39 degrees C and 41 degrees C., Results: Mean prothrombin time results increased from 11.8 +/- 0.3 (SD) secs at 37 degrees C to 12.9 +/- 0.5, 14.2 +/- 0.5, and 16.6 +/- 0.2 secs at 34 degrees C, 31 degrees C, and 28 degrees C, respectively (p < or = .001 for each). Partial thromboplastin time determinations increased from 36.0 +/- 0.7 (SD) secs at 37 degrees C to 39.4 +/- 1.0, 46.1 +/- 1.1, and 57.2 +/- 0.6 secs at 34 degrees C, 31 degrees C, and 28 degrees C, respectively (p < or = .001 for each). Both prothrombin time and partial thromboplastin time determinations were only minimally shortened at hyperthermic temperatures., Conclusions: The series of enzymatic reactions of the coagulation cascade are strongly inhibited by hypothermia, as demonstrated by the dramatic prolongation of prothrombin time and partial thromboplastin time tests at hypothermic deviations from normal temperature in a situation where factor levels were all known to be normal. Clinicians who deal with critically ill massively transfused hypothermic patients all recognize the inevitable appearance of a coagulopathy that has a multifactorial origin. Unless specifically considered, the contribution of hypothermia to the hemorrhagic diathesis may be overlooked since coagulation testing is performed at 37 degrees C, rather than at the patient's actual in vivo temperature.

We evaluated the accretion of 131I-labeled NP-4 anticarcinoembryonic antigen (CEA) into 4 size-matched human colonic carcinomas grown s.c. in nude mice. Antibody uptake for LS174T and GW-39 tumors was relatively high (19 to 23% ID/g on day 3), whereas moderate uptake was seen in the Moser tumor (7.5% on day 3) and low uptake was detected in the GS-2 tumor (1.8% on day 3). Blood clearance of radioantibody was twice as fast in mice with GS-2 tumors than in mice with GW-39, LS174T or Moser tumors. Seven physiological parameters that might influence radioantibody accretion were evaluated in order to better understand the differences in observed tumor targeting: vascular volume, blood flow rate, vascular permeability, tumor antigen content, serum antigen content and complexation of radioantibody, intratumoral antigen distribution, and intracellular antigen distribution. Although marked variability in vascular physiology, antigen content and antibody complexation of the 4 tumors grown in the same host and site existed, it was insufficient to explain the differences in antibody uptake. However, intra-tumoral distribution of antigen, and sub-cellular accessibility of antigen for radioantibody were important considerations. GS-2 tumors are well differentiated and have polarized cells. CEA in GS-2 is largely inaccessible to radioantibody; most of the antigen is located in the lumen of the glands or on the apical surface of gland cells and most of the antibody distributes to the stromal region on the basolateral surface. The low antibody targeting in GS-2 could therefore be explained by restricted intra-tumor accessibility of antibody. Scatchard analysis of NP-4 binding to Moser cells under non-internalizing and internalizing conditions revealed that 90% of the antigen is found within the cell, unavailable to bind with the NP-4 antibody, which is slow to internalize. In contrast, CEA in LS174T cells was almost entirely accessible. The reduced antibody targeting to Moser xenografts might therefore, be explained by restricted antibody accessibility at the cellular level.

Antiplatelet drugs as exemplified by aspirin are used frequently to prevent stroke. Aspirin inhibits the formation of both the potent platelet aggregator, thromboxane A2 and the potent anti-aggregator, prostacyclin. Another approach to the inhibition of platelet aggregation might involve selective suppression of thromboxane formation. We report our experience in swine with UK-38,485, a drug which selectively inhibits thromboxane formation. The rationale and potential uses of UK-38,485 in the in vivo prevention of platelet aggregation and for the therapy of cerebrovascular disease are discussed.

The magnitude and kinetics of tumor uptake of a monoclonal antibody (MAb) directed against carcinoembryonic antigen (CEA) in the GW-39 human colorectal cancer xenograft differ according to the animal used (nude mouse or hamster) and the site of implantation of the tumor within the animal (cheek pouch, leg muscle, subcutaneous or liver). Several physiological factors have been evaluated in an attempt to explain these differences in radio-antibody accumulation. The following observations have been made: (1) The animal host with the slower blood clearance of radio-antibody and the higher non-tumor tissue uptake has the higher tumor uptake; (2) the xenografts with a higher blood-flow rate, vascular volume and/or vascular permeability have a higher specific radio-antibody targeting; (3) the smaller, more viable tumors take up more radio-antibody per gram than the larger tumors; and (4) tumors with higher specific antigen content accrete more radio-antibody. These results are discussed in terms of the feasibility of clinical tumor imaging and therapy with radiolabelled antibodies.

Vegetarians have an apparent diminished risk for the development of ischemic coronary heart disease. This may be secondary to dietary effects of plasma lipids and lipoproteins, but platelets, which may also play a role, have also been observed to have aberrant functions in vegetarians. We measured plasma lipid and lipoprotein levels, platelet function, platelet fatty acid levels, and platelet active prostaglandins in ten strict vegetarians (vegans), 15 lactovegetarians, and 25 age- and sex-matched omnivorous controls. The most striking observations were a highly significant rise in platelet linoleic acid concentration and a decline in platelet arachidonic acid concentration in both vegetarian subgroups as compared with omnivorous controls. Serum thromboxane and prostacyclin levels as well as results of platelet aggregation studies did not differ among the groups tested. Cholesterol levels were significantly lower in both vegetarian groups as compared with controls, but plasma high- and low-density lipoprotein levels were lower only in the vegan subgroup as compared with omnivores. If diet produces these changes in platelet fatty acid and plasma lipid levels it may contribute to the decreased risk of coronary heart disease and possibly atherosclerosis in vegetarians.

We found a small but statistically significant reduction in the linoleic acid concentration of white blood cells and platelets in MS patients. The percent linoleic acid concentration (mean +/- S.D.) in the white blood cells if 24 MS patients was 8.8 +/- 1.8% as compared with 11.4 +/- 4.9 in 24 age and sex-matched controls (p less than 0.05). Platelet levels were 8.5 +/- 2.4% and 10.6 +/- 3.8% respectively (P less than 0.05). Serum linoleic acid levels were not significantly different in the two groups. The possible role of linoleic acid in the pathogenesis of MS has yet to be defined.