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The complement system plays a critical role in the innate immune response, acting as a first line of defense against invading pathogens. However, dysregulation of the complement system is implicated in the pathogenesis of numerous diseases, ranging from Alzheimer's to age-related macular degeneration and rare blood disorders. As such, complement inhibitors have enormous potential to alleviate disease burden. While a few complement inhibitors are in clinical use, there is still a significant unmet medical need for the discovery and development of novel inhibitors to treat patients suffering from disorders of the complement system. A key hurdle in the development of complement inhibitors has been the determination of their mechanism of action. Progression along the complement cascade involves the formation of numerous multimeric protein complexes, creating the potential for inhibitors to act at multiple nodes in the pathway. This is especially true for molecules that target the central component C3 and its fragment C3b, which serve a dual role as a substrate for the C3 convertases and as a scaffolding protein in both the C3 and C5 convertases. Here, we report a step-by-step in vitro reconstitution of the complement alternative pathway using bio-layer interferometry. By physically uncoupling each step in the pathway, we were able to determine the kinetic signature of inhibitors that act at single steps in the pathway and delineate the full mechanism of action of known and novel C3 inhibitors. The method could have utility in drug discovery and further elucidating the biochemistry of the complement system., Competing Interests: Conflict of 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

Purpose: To determine the rate of increase in serum bupivacaine concentration during continuous extrapleural infusion., Methods: After thoracotomy for lobectomy under general anaesthesia, nine patients had an extrapleural catheter inserted, before chest closure, in a costovertebral gutter constructed surgically by lifting the parietal pleural. Bupivacaine 0.5% with epinephrine 1:200.000 was injected through the catheter as 0.3 ml.kg-1 bolus followed by 0.1 ml.kg-1.hr-1 for five days. Serum bupivacaine (free and total), albumin, alpha-1 acid glycoprotein concentrations were measured 15 min after injection and at 24 hr intervals for five days. Bupivacaine concentrations were determined by column liquid chromatography using solid phase extraction. Serum alpha-1 acid glycoprotein concentration was determined by nephelometry on QM 300 protein analyzer. Serum albumin concentration was determined by bromocresol green dye binding procedure on Hitachi 717 Autoanalyzer., Results: A continuous elevation in total serum bupivacaine was observed, with an average value of 0.75 microgram.ml-1 on day 1 to 2.77 micrograms.ml-1 on day 4 (P < 0.05). There was no increase in postoperative free serum bupivacaine concentration; average value of 177 pcg.ml-1 on day 1 and 249 pcg.ml-1 on day 4 (P = 0.92). Postoperative serum alpha-1 acid glycoprotein concentration showed a steady rise with an average value of 0.94 microgram.ml-1 on day 1 and 1.47 micrograms.ml-1 on day 4 (P < 0.05). No change was observed in post-operative serum albumin with an average value of 31.4 g.l-1 on day 1 and 31.3 g.l-1 on day 4., Conclusion: Continuous extrapleural infusion of bupivacaine over five days after thoracotomy is associated with a steady increase in total serum bupivacaine concentration and no elevation in free serum bupivacaine concentration.

In this study, the cardiac output of 11 children (5 days to 18 yr of age) was measured following cardiopulmonary bypass. Standard dye dilution cardiac outputs and an indirect Fick-CO2 technique were simultaneously determined. A rebreathing maneuver is described to estimate the oxygenated mixed venous PCO2 (PvCO2). This estimate of PvCO2 was combined with the end tidal PCO2 and CO2 production to give a non-invasive Fick estimate of the cardiac output. Non-invasive cardiac outputs correlated well with those measured by dye-dilution (r2 = 0.94) over a range of cardiac outputs from 490-7280 ml/min. All non-invasive determinations were within 15% of the line of identity, and within 22% of averaged replicate dye-dilution values. Our results agree with previous comparisons of indirect Fick-CO2 methods with invasive determinations of cardiac output. These data suggest that the indirect Fick-CO2 technique offers a rapid, non-invasive alternative to invasive monitoring of cardiac output.

In this study we identified the precipitate formed when pancuronium bromide (pH 3.9) and sodium thiopentone (pH 10.6) are combined in vitro. A precipitate formed when sufficient pancuronium was added to thiopentone to decrease the pH below 9.25. The precipitate was isolated, redissolved in alkaline solution and identified as thiopentone using ultraviolet spectrophotometry. Pancuronium was soluble in water in the pH range of 2.0 to 12.0, whereas thiopentone was soluble in water only at a pH greater than 9.90. We conclude that thiopentone precipitates when combined with pancuronium because of a pH-dependent decrease in the solubility of thiopentone, rather than a chemical interaction.

Planaria which have cannibalized untrained planaria exposed to photic stimuli only, handling only, or previous conditioning, all require significantly fewer trials in a conditioning situation than naive planaria.