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Background: The kallikrein kinin system (KKS) is an established pharmacological target for the treatment and prevention of attacks in hereditary angioedema (HAE). Proteolytic activities of FXIIa and single-chain Factor XII (FXII) zymogen contribute to KKS activation and thereby may play roles in both initiating and propagating HAE attacks. In this report, we investigated the effects of potent small molecule FXIIa inhibitors on FXIIa and single chain FXII enzymatic activities, KKS activation, and angioedema in mice. Methods: We examined the effects of 29 structurally distinct FXIIa inhibitors on enzymatic activities of FXIIa and a mutant single chain FXII with R334A, R343A and R353A substitutions (rFXII-T), that does not undergo zymogen conversion to FXIIa, using kinetic fluorogenic substrate assays. We examined the effects of a representative FXIIa inhibitor, KV998086, on KKS activation and both carrageenan- and captopril-induced angioedema in mice. Results: FXIIa inhibitors designed to target its catalytic domain also potently inhibited the enzymatic activity of rFXII-T and the pIC 50 s of these compounds linearly correlated for rFXIIa and rFXII-T ( R 2 = 0.93). KV998086, a potent oral FXIIa inhibitor (IC 50 = 7.2 nM) inhibited dextran sulfate (DXS)-stimulated generation of plasma kallikrein and FXIIa, and the cleavage of high molecular weight kininogen (HK) in human plasma. KV998086 also inhibited rFXII-T mediated HK cleavage ( p < 0.005) in plasma from FXII knockout mice supplemented with rFXII-T and stimulated with polyphosphate or DXS. Orally administered KV998086 protected mice from 1) captopril-induced Evans blue leakage in colon and laryngotracheal tissues and 2) blocked carrageenan-induced plasma HK consumption and paw edema. Conclusion: These findings show that small molecule FXIIa inhibitors, designed to target its active site, also inhibit the enzymatic activity of FXII zymogen. Combined inhibition of FXII zymogen and FXIIa may thereby suppress both the initiation and amplification of KKS activation that contribute to hereditary angioedema attacks and other FXII-mediated diseases., Competing Interests: AC, NM, HE, DL, NB, SP, SH, and EF are employees of KalVista Pharmaceuticals. ED was an employee of KalVista Pharmaceuticals at the time of the study. DG has received consultants fees from Anthos Therapeutics, Bristol-Myers Squibb, Ionis, and Janssen., (Copyright © 2023 Clermont, Murugesan, Edwards, Lee, Bayliss, Duckworth, Pethen, Hampton, Gailani and Feener.)

Hereditary angioedema (HAE) is a rare genetic disorder in which patients experience sudden onset of swelling in various locations of the body. HAE is associated with uncontrolled plasma kallikrein (PKa) enzyme activity and generation of the potent inflammatory mediator, bradykinin, resulting in episodic attacks of angioedema. Herein, we disclose the discovery and optimization of novel small molecule PKa inhibitors. Starting from molecules containing highly basic P1 groups, which typically bind to an aspartic acid residue (Asp189) in the serine protease S1 pocket, we identified novel P1 binding groups likely to have greater potential for oral-drug-like properties. The optimization of P4 and the central core together with the particularly favorable properties of 3-fluoro-4-methoxypyridine P1 led to the development of sebetralstat, a potent, selective, orally bioavailable PKa inhibitor in phase 3 for on-demand treatment of HAE attacks.

The structures and band gaps of copper-zinc-tin selenosulfides (CZTSSe) are investigated for a range of anion compositions through experimental analysis and complementary first-principles simulations. The band gap was found to be extremely sensitive to the Sn-anion bond length, with an almost linear correlation with the average Sn-anion bond length in the mixed anion phase Cu 2 ZnSn(S x Se 1- x ) 4 . Therefore, an accurate prediction of band gaps using first-principles methods requires the accurate reproduction of the experimental bond lengths. This is challenging for many widely used approaches that are suitable for large supercells. The HSE06 functional was found to predict the structure and band gap in good agreement with the experiment but is computationally expensive for large supercells. It was shown that a geometry optimization with the MS2 meta-GGA functional followed by a single point calculation of electronic properties using HSE06 is a reasonable compromise for modeling larger supercells that are often unavoidable in the study of point and extended defects.

Competing Interests: Competing interests: None declared.

Layered two-anion compounds are of interest for their diverse electronic properties. The modular nature of their layered structures offers opportunities for the construction of complex stackings used to introduce or tune functionality, but the accessible layer combinations are limited by the crystal chemistries of the available anions. We present a layered three-anion material, Bi 4 O 4 Cu 1.7 Se 2.7 Cl 0.3 , which adopts a new structure type composed of alternately stacked BiOCuSe and Bi 2 O 2 Se-like units. This structure is accessed by inclusion of three chemically distinct anions, which are accommodated by aliovalently substituted Bi 2 O 2 Se 0.7 Cl 0.3 blocks coupled to Cu-deficient Bi 2 O 2 Cu 1.7 Se 2 blocks, producing a formal charge modulation along the stacking direction. The hypothetical parent phase Bi 4 O 4 Cu 2 Se 3 is unstable with respect to its charge-neutral stoichiometric building blocks. The complex layer stacking confers excellent thermal properties upon Bi 4 O 4 Cu 1.7 Se 2.7 Cl 0.3 : a room-temperature thermal conductivity (κ) of 0.4(1) W/mK was measured on a pellet with preferred crystallite orientation along the stacking axis, with perpendicular measurement indicating it is also highly anisotropic. This κ value lies in the ultralow regime and is smaller than those of both BiOCuSe and Bi 2 O 2 Se. Bi 4 O 4 Cu 1.7 Se 2.7 Cl 0.3 behaves like a charge-balanced semiconductor with a narrow band gap. The chemical diversity offered by the additional anion allows the integration of two common structural units in a single phase by the simultaneous and coupled creation of charge-balancing defects in each of the units.

The selective synthesis of 2,6-trans-tetrahydropyran derivatives employing the rhodium catalysed addition of organoboron reagents to dihydropyranone templates, derived from a zinc-catalysed hetero-Diels-Alder reaction, is reported. The addition of both arylboronic acids and potassium alkenyltrifluoroborates have been accomplished in high yields using commercially-available [Rh(cod)(OH)]2 catalyst. The selective formation of the 2,6-trans-tetrahydropyran stereoisomer is consistent with a mechanism involving alkene association and carbometalation on the less hindered face of the dihydropyranone.

The ECG is a test that is used frequently in the acute setting. It has a significant impact on decisions regarding patient discharge and further investigations. On a single day in the ambulatory emergency care setting two patients presented with chest pain. The ECG findings were abnormal, but also out of context with the clinical findings. On close inspection of the ECG machine it was identified that although all leads attached to the patient were in the correct position, the two cables connecting the leads to the machine had been reversed. Had the error not been discovered promptly there was the potential that further, more harmful investigations would have been performed. These cases highlight that although the ECG is a simple and non-invasive investigation it should be an adjunct to clinical working diagnosis., (2014 BMJ Publishing Group Ltd.)

Rising sea temperatures cause mass coral bleaching and threaten reefs worldwide. We show how maps of variations in thermal stress can be used to help manage reefs for climate change. We map proxies of chronic and acute thermal stress and develop evidence-based hypotheses for the future response of corals to each stress regime. We then incorporate spatially realistic predictions of larval connectivity among reefs of the Bahamas and apply novel reserve design algorithms to create reserve networks for a changing climate. We show that scales of larval dispersal are large enough to connect reefs from desirable thermal stress regimes into a reserve network. Critically, we find that reserve designs differ according to the anticipated scope for phenotypic and genetic adaptation in corals, which remains uncertain. Attempts to provide a complete reserve design that hedged against different evolutionary outcomes achieved limited success, which emphasises the importance of considering the scope for adaptation explicitly. Nonetheless, 15% of reserve locations were selected under all evolutionary scenarios, making them a high priority for early designation. Our approach allows new insights into coral holobiont adaptation to be integrated directly into an adaptive approach to management., (© 2010 Blackwell Publishing Ltd/CNRS.)

In mammalian cells, cytotoxic stress triggers several signaling cascades that converge in the phosphorylation of translation initiation factor 2alpha, shuttling of nuclear RNA-binding proteins such as TIA-1 to the cytoplasm, and aggregation of most cellular mRNAs into TIA-1-containing stress granules (SGs). As a result, protein synthesis is greatly impaired. Here we describe different dynamics of endogenous transcripts according to their cellular location, in response to stress. While cytosolic mRNAs aggregate into SGs, endoplasmic reticulum (ER) -bound transcripts escape sequestration. This has been specifically demonstrated using the multidrug resistance transporter gene (MDR1) as a model and showing that chimeric RNA constructs can be directed to the cytosol or tethered to the ER depending on the nature of the chimera, in response to stress. In addition, polysome profile analyses indicate that, on stress, ribosomes do not disengage from ER-associated transcripts (puromycin insensitive) and recover their translation status faster than SG-targeted cytosolic mRNAs once the stress is lifted. These findings have important implications for cell survival given that many membrane proteins, which are translated at the ER, have important roles in detoxification.

The rhodium catalysed conjugate addition of organometallics to activated alkenes is a powerful synthetic tool for establishing new carbon-carbon bonds often with high stereoselectivity. The introduction of a practical, efficient method for introducing functionalised aryl and alkenyl fragments with predictable stereocontrol has caught the attention of synthetic chemists and emerging examples are growing in number and complexity. In this tutorial review, we document notable advances in the application of rhodium catalysed conjugate addition processes within the context of synthesis of complex molecules and intermediates in drug discovery. The chosen examples illustrate important issues regarding scope, selectivity and reactivity that will help guide the selection of appropriate donor and acceptor to achieve the desired carbon-carbon bond construction when planning new synthetic routes.

The deteriorating health of the world's coral reefs threatens global biodiversity, ecosystem function, and the livelihoods of millions of people living in tropical coastal regions. Reefs in the Caribbean are among the most heavily affected, having experienced mass disease-induced mortality of the herbivorous urchin Diadema antillarum in 1983 and two framework-building species of coral. Declining reef health is characterized by increases in macroalgae. A critical question is whether the observed macroalgal bloom on Caribbean reefs is easily reversible. To answer this question, we must resolve whether algal-dominated reefs are an alternative stable state of the ecosystem or simply the readily reversible result of a phase change along a gradient of some environmental or ecological parameter. Here, using a fully parameterized simulation model in combination with a simple analytical model, we show that Caribbean reefs became susceptible to alternative stable states once the urchin mortality event of 1983 confined the majority of grazing to parrotfishes. We reveal dramatic hysteresis in a natural system and define critical thresholds of grazing and coral cover beyond which resilience is lost. Most grazing thresholds lie near the upper level observed for parrotfishes in nature, suggesting that reefs are highly sensitive to parrotfish exploitation. Ecosystem thresholds can be combined with stochastic models of disturbance to identify targets for the restoration of ecosystem processes. We illustrate this principle by estimating the relationship between current reef state (coral cover and grazing) and the probability that the reef will withstand moderate hurricane intensity for two decades without becoming entrained in a shift towards a stable macroalgal-dominated state. Such targets may help reef managers face the challenge of addressing global disturbance at local scales.

A model is developed to describe the interaction between a predator and two prey types located in different regions. Conditions for stability and persistence are analysed. The effects of harvesting the predators are investigated by making the predator mortality rate habitat dependent. Results demonstrate that for any given set of parameter values there is a value of the intrinsic preference of the predator for each prey type at which the system undergoes a Hopf bifurcation. Above this critical value the system evolves towards a stable equilibrium, whereas below it, stable limit cycles arise by Hopf bifurcations. Simulations demonstrate that the presence of demographic stochasticity may destabilise oscillatory populations, thereby causing population extinctions. An application of the model to the foraging behaviour of North Sea cod is described. It is shown that if the preferred prey is more productive, it is likely that the equilibrium will be stable, whereas if the less preferred prey is more productive, populations are likely to display cycles and in the stochastic case become extinct. As cod fishing mortality is increased, the point of bifurcation and region of parameter space for which the system is unstable decreases. An increased understanding of how cod behave may enable fish stocks to be managed more successfully, for example by indicating where marine reserves should be placed.

Heparin-binding EGF-like growth factor (HBEGF), which is expressed in the placenta during normal pregnancy, is down regulated in pre-eclampsia, a human pregnancy disorder associated with poor trophoblast differentiation and survival. This growth factor protects against apoptosis during stress, suggesting a role in trophoblast survival in the relatively low O(2) ( approximately 2%) environment of the first trimester conceptus. Using a well-characterized human first trimester cytotrophoblast cell line, we found that a 4-hour exposure to 2% O(2) upregulates HBEGF synthesis and secretion independently of an increase in its mRNA. Five other expressed members of the EGF family are largely unaffected. At 2% O(2), signaling via HER1 or HER4, known HBEGF receptors, is required for both HBEGF upregulation and protection against apoptosis. This positive-feedback loop is dependent on metalloproteinase-mediated cleavage and shedding of the HBEGF ectodomain. The restoration of trophoblast survival by the addition of soluble HBEGF in cultures exposed to low O(2) and metalloproteinase inhibitor suggests that the effects of HBEGF are mediated by autocrine/paracrine, rather than juxtacrine, signaling. Our results provide evidence that a post-transcriptional mechanism induced in trophoblasts by low O(2) rapidly amplifies HBEGF signaling to inhibit apoptosis. These findings have a high clinical significance, as the downregulation of HBEGF in pre-eclampsia is likely to be a contributing factor leading to the demise of trophoblasts.

The efficacy of a topical preparation containing 0.5% fusidic acid and 0.1% betamethasone-17-valerate was compared to a systemic therapy (comprising a combination of parenteral dexamethasone and oral clavulanate-potentiated amoxycillin) in the treatment of 104 dogs with acute moist dermatitis. Significant improvement was evident after seven days in both treatment groups in all clinical parameters assessed and there was no significant difference in the overall response between the two treatment groups. Staphylococcus intermedius was the most frequently isolated organism from swabs at the first visit (Day 0). No resistance to fusidic acid or clavulanate-potentiated amoxycillin was encountered. The study demonstrates no difference in the clinical improvement achieved in canine acute moist dermatitis following topical or systemic therapy and that both treatment regimes represent effective treatment options for the condition.

Alkylation reactions using alpha-halolactams or lactam enolates derived from bicyclic lactam templates can proceed with high endo- or exo- diastereoselectivity respectively. In the latter case, stereochemical correction by means of enolate generation and hindered phenol quench is possible with moderate efficiency. This protocol has been applied to the synthesis of protected penmacric acid and its analogues.

The molecular mechanisms of peroxisome biogenesis have begun to emerge; in contrast, relatively little is known about how the organelle functions as cells age. In this report, we characterize age-related changes in peroxisomes of human cells. We show that aging compromises peroxisomal targeting signal 1 (PTS1) protein import, affecting in particular the critical antioxidant enzyme catalase. The number and appearance of peroxisomes are altered in these cells, and the organelles accumulate the PTS1-import receptor, Pex5p, on their membranes. Concomitantly, cells produce increasing amounts of the toxic metabolite hydrogen peroxide, and we present evidence that this increased load of reactive oxygen species may further reduce peroxisomal protein import and exacerbate the effects of aging.

Human CYP3A4 is the major cytochrome P450 isoenzyme in adult human liver and is known to metabolise many xenobiotic and endogenous compounds. There is substantial inter-individual variation in the hepatic levels of CYP3A4. Although, polymorphic mutations have been reported in the 5' regulatory region of the CYP3A4 gene, those that have been investigated so far do not appear to have any effect on gene expression. To determine whether other mutations exist in this region of the gene, we have performed a new population screen on a panel of 101 human DNA samples. A 1140 bp section of the 5' proximal regulatory region of the CYP3A4 gene, containing numerous regulatory motifs, was amplified from genomic DNA as three overlapping segments. The 300 bp distal enhancer region at -7.9kb containing additional regulatory motifs was also amplified. Mutation analysis of the resulting PCR products was carried out using non-radioactive single strand conformation polymorphism (SSCP) and confirmatory sequencing of both DNA strands in those samples showing extra SSCP bands. In addition to detection of the previously reported CYP3A4*1B allele in nine subjects, three novel alleles were found: CYP3A4*1E (having a T-->A transversion at -369 in one subject), CYP3A4*1F (having a C-->G tranversion at -747 in 17 subjects) and CYP3A4*15B containing a nine-nucleotide insertion between -845 and -844 linked to an A-->G transition at -392 and a G-->A transition in exon 6 (position 485 in the cDNA) in one subject. All the novel alleles were heterozygous. No mutations were found in the upstream distal enhancer region. Our results clearly indicate that this rapid and simple SSCP approach can reveal mutant alleles in drug metabolising enzyme genes. Detection and determination of the frequency of novel alleles in CYP3A4 will assist investigation of the relationship between genotype, xenobiotic metabolism and toxicity in the CYP3A family of isoenzymes.

Two different studies are described. The first study deals with the elimination of residues of trimethoprim (TMP), sulfatroxazole (STX) and its main metabolite N4-acetyl-sulfatroxazole (N4-acetyl-STX) in pigs. Thirty -six pigs were treated with trimethoprim/sulfatroxazole IM in the nec k at a dosage of 16 mg/kg body weight for five days. Groups of four pig s were slaughtered at different time intervals. The study showed that concentration of STX, N4-acetyl-STX and TMP in edible tissues and at the injection sites were below 0.1 ppm on day nine after the last injection. S TX was eliminated the slowest, and STX can therefore be selected as a marker for residues of the trimethoprim/sulfatroxazole formulation in the tissues. The second study deals with irritation aspects of this trimethoprim /sulfatroxazole formulation. Four pigs of 32-35 kg were treated IM w with trimethoprim/sulfatroxazole and benzylpenicillin sodium. Each pig received the same injection volume, namely four trimethoprim/sulfatroxazole injections (16 mg/kg body weight per injection site), two in the back and two in the neck muscle, and two benzylpenicillin sodium injections (20,000 I.U./kg body weight per injection site), in the back muscle. All pigs were slaughtered 14 days after treatment and the extent of the irritation was compared. There were no differences between trimethoprim/sulfatroxazole and benzylpenicillin sodium with regard to irritation at the injection site in the back muscle. The irritation in the neck site was statistically less prominent than that in the back muscle and was considered not to affect the quality of the meat.

The efficacy of 1 alpha-hydroxycholecalciferol (1 alpha-HCC) for the prevention of milk fever has been tested in a controlled field trial using a total of 601 cows on 18 farms with a history of a high incidence of milk fever. The trial protocol proposed two doses of 1 alpha-HCC with the first injection seven days before the predicted calving date and a second injection five to seven days later if the cow had not calved. Of 301 cows receiving either one or two injections of 1 alpha-HCC, 70 had milk fever, and of 300 cows that received a placebo, 102 had milk fever. The difference in incidence between the two groups was statistically significant (P = 0.005) and indicated an overall efficacy of 32 per cent for treatment with 1 alpha-HCC. An improved efficacy was demonstrated in 67 cows that received two injections of 1 alpha-HCC with the second injection administered either on the day of calving or one day previously (60 per cent efficacy). For 42 cows that received a single injection of 1 alpha-HCC between two and five days before calving an efficacy of 79 per cent was achieved.