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Main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpromay alter structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOC) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and rate of cleavage of a viral peptide. Crystal structures of 11 Mpromutants provided structural insight into their altered functionality. Additionally, we show Mpromutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and subsequent significant decrease in cytokine secretion, providing evidence for alterations in escape of host-antiviral mechanisms. Accordingly, mutations associated with the highly virulent Delta VOC resulted in significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mprowill remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

Objectives Here we report the draft genome sequence of Staphylococcus agnetis 4244, a strain involved in bovine mastitis, and its ability to inhibit different species of antibiotic-resistant Gram-positive bacteria owing to bacteriocin production. Methods An Illumina MiSeq platform was used for genome sequencing. De novo genome assembly was done using the A5-miseq pipeline. Genome annotation was performed by the RAST server, and mining of bacteriocinogenic gene clusters was done using the BAGEL4 and antiSMASH v.5.0 platforms. Investigation of the spectrum of activity of S. agnetis 4244 was performed on BHI agar by deferred antagonism assay. Results The total scaffold size was determined to be 2 511 708 bp featuring a G+C content of 35.6%. The genome contains 2431 protein-coding sequences and 80 RNA sequences. Genome analyses revealed three prophage sequences inserted in the genome as well as several genes involved in drug resistance and two bacteriocin gene clusters (encoding a thiopeptide and a sactipeptide) encoded on the bacterial chromosome. Staphylococcus agnetis 4244 was able to inhibit all 44 strains of antibiotic-resistant Gram-positive bacteria tested in this study, including vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA) and other antibiotic-resistant staphylococcal strains. Conclusion This study emphasises the potential biotechnological application of this strain for production of bacteriocins that could be used in the food industry as biopreservatives and/or in medicine as alternative therapeutic options against VRE, MRSA, vancomycin-intermediate S. aureus and other antibiotic-resistant Gram-positive bacteria, including biofilm-forming isolates. It also provides some genetic features of the draft genome of S. agnetis 4244.

Neopetrosiamide, a 28-residue peptide from Neopetrosia sp., contains three disulfide bonds and hinders mammalian tumor cell invasion. Proper connectivity of disulfide bonds is crucial for activity. Synthetic replacement of single disulfide bridges with methylene bridges gives active analogues. Pre-stapling of one ring enhances the correct formation of the remaining disulfides by reducing isomeric possibilities and possibly initiating the correct 3D fold. Cloning and expression of neopetrosiamide in E. coli affords access to the natural linear peptide.

Three lipopeptide analogues of the lantibiotic nisin A have been synthesised on-resin using Fmoc-SPPS techniques to investigate the structure-activity relationship of the A and B ring of these types of lanthipeptides. Lanthionine and methyllanthionine macrocycles were incorporated using orthogonally protected residues for on-resin cyclisation. Unsaturated dehydroalanine and, for the first time, dehydrobutyrine were synthesised on-resin from their cysteine derivatives. However, none of the synthetic or semi-synthetic lipopeptide analogues of nisin showed inhibitory activity towards bacterial strains that are normally sensitive to nisin.

Heterologous expression in Escherichia coli is a commonly used method to produce ribosomally synthesized peptides for further study. This generally requires expression of the target protein with an affinity fusion tag, followed by isolation of the fusion protein from a cellular lysate by affinity purification, and finally by removal of the fusion tag and purification of the desired peptide. Sometimes, however, fusion proteins may be degraded during recombinant expression in E. coli. We recently reported an expression system that sandwiches the target peptide between an N-terminal small ubiquitin-like modifier (SUMO) protein and a C-terminal intein. This SUMO-peptide-intein (SPI) fusion protein protects the central peptide from degradation and can lead to improved peptide yield after purification. In this report, we detail the cloning, expression, and isolation procedures for the SPI fusion system, with comments on conditions that can be optimized for different peptides to obtain maximal yield for each construct. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cloning to construct SPI gene Basic Protocol 2: Expression of SPI fusion proteins in E. coli BL21(DE3) Support Protocol: Optimization of expression and induction conditions Basic Protocol 3: Isolation and purification of SPI fusion proteins with a chitin column Alternate Protocol: Isolation and purification of SPI fusion proteins without chitin.

Syntheses of stereochemically pure and selectively protected diamino diacids can be achieved by redox decarboxylation of distal N-hydroxyphthalimide esters of protected aspartic, glutamic or α-aminoadipic acids via radical addition to methylideneoxazolidinones. The products are useful for solid-supported syntheses of robust bioactive carbocyclic peptide analogs. Yields of reactive primary radical addition are superior to those of more stabilized radicals, and the reaction fails if the alkylideneoxazolidinone has a methyl substituent on its terminus (i.e., 13a/13b).

An intermediate in the synthesis of numerous antiviral protease inhibitors is the glutamine analogue, (3S)-pyrrolid-2-one-3-yl-l-alanine. Preparations of compounds based on this pharmacophore are hindered by the lack of a reliably high yielding synthesis of protected forms of this amino acid. We describe an improved scalable route with readily available reagents and facile purification. This methodology employs γ-allylation of dimethyl N-BocGlu, further Boc N-protection, OsO4-periodate oxidation, O-Me oxime formation, and RaNi-catalyzed hydrogenolysis with concomitant cyclization under basic conditions.

Recurring coronavirus outbreaks, such as the current COVID-19 pandemic, establish a necessity to develop direct-acting antivirals that can be readily administered and are active against a broad spectrum of coronaviruses. Described in this Article are novel α-acyloxymethylketone warhead peptidomimetic compounds with a six-membered lactam glutamine mimic in P1. Compounds with potent SARS-CoV-2 3CL protease and in vitro viral replication inhibition were identified with low cytotoxicity and good plasma and glutathione stability. Compounds 15e, 15h, and 15l displayed selectivity for SARS-CoV-2 3CL protease over CatB and CatS and superior in vitro SARS-CoV-2 antiviral replication inhibition compared with the reported peptidomimetic inhibitors with other warheads. The cocrystallization of 15l with SARS-CoV-2 3CL protease confirmed the formation of a covalent adduct. α-Acyloxymethylketone compounds also exhibited antiviral activity against an alphacoronavirus and non-SARS betacoronavirus strains with similar potency and a better selectivity index than remdesivir. These findings demonstrate the potential of the substituted heteroaromatic and aliphatic α-acyloxymethylketone warheads as coronavirus inhibitors, and the described results provide a basis for further optimization.

Recombinant peptide production in Escherichia coli is often accomplished through cloning and expression of a fusion protein. The fusion protein partner generally has two requirements: (a) it contains an affinity tag to assist with purification and (b) it can be cleaved off to leave only the desired peptide sequence behind. Common soluble fusion partners include small ubiquitin‐like modifier protein (SUMO), maltose‐binding protein (MBP), glutathione S‐transferase (GST), or intein proteins. However, heterologously expressed peptides can suffer from proteolytic degradation or instability. This degradation can pose a major issue for applications requiring a large amount of purified peptide, such as NMR structural assignments or biochemical assays. Improving peptide yield by testing various expression and isolation conditions requires a significant amount of effort and may not lead to improved results. Here, we cloned and expressed four different peptides as SUMO fusion proteins. These peptides (lactococcin A, leucocin A, faerocin MK, neopetrosiamide A) were truncated during expression and isolation as SUMO fusions, resulting in low yields of purified peptide. To prevent this degradation and improve yield, we designed a new expression system to create a “sandwiched” fusion protein of the form: His(6)‐SUMO‐peptide‐intein (SPI). These sandwiched peptides were more stable and protected against degradation, resulting in improved yields (up to 17‐fold) under a set of standard expression and isolation procedures. This SPI expression system uses only two commercially available vectors and standard protein purification techniques, and therefore may offer an economical and facile route to improve yields for peptides that undergo degradation.

The mountain pine beetle, Dendroctonus ponderosae, has infested over ~16 Mha of pine forests in British Columbia killing >50% of mature lodgepole pine, Pinus contorta, trees in affected stands. At present, it is functionally an invasive species in Alberta, killing and reproducing in evolutionarily naive populations of lodgepole pine (P. contorta), novel jack pine (P. banksiana), and their hybrids. The entomopathogenic fungus Beauveria bassiana has shown some potential as a biocontrol agent of several bark beetle species. In this study, nine isolates of B. bassiana were examined for insect virulence characteristics, including conidiation rate, pigmentation, and infection rate in laboratory-reared D. ponderosae, to assess for their potential as biocontrol agents. The strains were categorized into three phenotypic groups based on pigmentation, conidial density, and myceliation rate. Virulence screening utilizing insect-based agar medium (D. ponderosae and European honeybee Apis mellifera carcasses) revealed no difference in selection of fungal growth. However, infection studies on D. ponderosae and A. mellifera showed contrasting results. In vivo A. mellifera infection model revealed ~5% mortality, representing the natural death rate of the hive population, whereas laboratory-reared D. ponderosae showed 100% mortality and mycosis. The LT50 (median lethal time 50) ranges from 2 to 5 ± 0.33 days, and LT100 ranges from 4 to 6 ± 0.5 days. We discuss the selective advantages of the three phenotypic groups in terms of virulence, pigmentation, conidial abundance, and tolerance to abiotic factors like UV and host tree monoterpenes. These results can further provide insights into the development of several phenotypically diverse B. bassiana strains in controlling the spread of the invasive D. ponderosae in Western Canada. • Three B. bassiana morphotype groups have been demonstrated to kill D. ponderosae. • A range of effective lethal times (LT50 and LT100) was established against D. ponderosae. • Variable tolerance to UV light and pine monoterpenes were observed in B. bassiana.

High blood pressure and consequential cardiovascular diseases are among the top causes of death worldwide. The apelinergic (APJ) system has emerged as a promising target for the treatment of cardiovascular issues, especially prevention of ischemia reperfusion (IR) injury after a heart attack or stroke. However, rapid degradation of the endogenous apelin peptides in vivo limits their use as therapeutic agents. Here, we study the effects of simple homologue substitutions, i.e. incorporation of non-canonical amino acids l-cyclohexylalanine (l-Cha) and l-homoarginine (l-hArg), on the proteolytic stability of pyr-1-apelin-13 and apelin-17 analogues. The modified 13-mers display up to 40 times longer plasma half-life than native apelin-13 and in preliminary in vivo assay show moderate blood pressure-lowering effects. The corresponding apelin-17 analogues show pronounced blood pressure-lowering effects and up to a 340-fold increase in plasma half-life compared to the native apelin-17 isoforms, suggesting their potential use in the design of metabolically stable apelin analogues to prevent IR injury.

Tragically, the death toll from the COVID-19 pandemic continues to rise, and with variants being observed around the globe new therapeutics, particularly direct-acting antivirals that are easily administered, are desperately needed. Studies targeting the SARS-CoV-2 3CL protease, which is critical for viral replication, with different peptidomimetics and warheads is an active area of research for development of potential drugs. To date, however, only a few publications have evaluated the nitrile warhead as a viral 3CL protease inhibitor, with only modest activity reported. This article describes our investigation of P3 4-methoxyindole peptidomimetic analogs with select P1 and P2 groups with a nitrile warhead that are potent inhibitors of SARS-CoV-2 3CL protease and demonstrate in vitro SARS-CoV-2 antiviral activity. A selectivity for SARS-CoV-2 3CL protease over human cathepsins B, S and L was also observed with the nitrile warhead, which was superior to that with the aldehyde warhead. A co-crystal structure with SARS-CoV-2 3CL protease and a reversibility study indicate that a reversible, thioimidate adduct is formed when the catalytic sulfur forms a covalent bond with the carbon of the nitrile. This effort also identified efflux as a property limiting antiviral activity of these compounds, and together with the positive attributes described these results provide insight for further drug development of novel nitrile peptidomimetics targeting SARS-CoV-2 3CL protease.

Apelin is an important contributor to the renin-angiotensin axis, regulating cardiovascular, metabolic, and neurological functions. Apelin-17 has especially potent cardio-physiological effects but is rapidly degraded in human blood (t0.5 ∼ 4 min). Angiotensin-converting enzyme 2 (ACE-2), neprilysin (NEP), and plasma kallikrein (KLKB1) cleave and inactivate it, with the latter cutting within the arginine-arginine site. Here, we show that analogues with an N-terminal polyethylene glycol (PEG) extension as well as peptide bond isosteres resist KLKB1 cleavage but that only the PEG-extended analogues significantly improve physiologically activity. The PEGylated analogues feature comparatively high log D7.4 values and high plasma protein binding, adding to their stability. An alanine scan of apelin-17 reveals that the integrity and conformational flexibility of the KFRR motif are necessary for cardio-physiological activity. An optimized Cbz-PEG6 analogue is presented that is stable in blood (t0.5 ∼ 18 h), has significant blood-pressure lowering effect, and shows fast recovery of heart function in Langendorff assay.

Cryptic peptides (cryptides) are biologically active peptides formed after proteolysis of native precursors present in animal venoms, for example. Proteolysis is an overlooked post-translational modification that increases venom complexity. The tripeptide KPP (Lys-Pro-Pro) is a peptide encrypted in the C-terminus of Ts14-a 25-mer peptide from the venom of the Tityus serrulatus scorpion that has a positive impact on the cardiovascular system, inducing vasodilation and reducing arterial blood pressure of hypertensive rats among other beneficial effects. A previous study reported that KPP and its native peptide Ts14 act via activation of the bradykinin receptor B2 (B2R). However, the cellular events underlying the activation of B2R by KPP are unknown. To study the cell signaling triggered by the Ts14 cryptide KPP, we incubated cardiac myocytes isolated from C57BL/6 mice with KPP (10 -7 mol·L -1) for 0, 5, or 30 min and explored the proteome and phosphoproteome. Our results showed that KPP regulated cardiomyocyte proteins associated with, but not limited to, apoptosis, muscle contraction, protein turnover, and the respiratory chain. We also reported that KPP led to AKT phosphorylation, activating AKT and its downstream target nitric oxide synthase. We also observed that KPP led to dephosphorylation of phospholamban (PLN) at its activation sites (S16 and T17), leading to reduced contractility of treated cardiomyocytes. Some cellular targets reported here for KPP (e.g., AKT, PLN, and ERK) have already been reported to protect the cardiac tissue from hypoxia-induced injury. Hence, this study suggests potential beneficial effects of this scorpion cryptide that needs to be further investigated, for example, as a drug lead for cardiac infarction.

Opines are widely distributed natural products formed by the reductive condensation of amino acids with α-keto acids or carbonyls of carbohydrates. They have important biological roles in bacteria, higher plants, fungi, invertebrates and mammals, including humans. An unusual cyclic opine of undefined stereochemistry, cycloalanopine, was previously isolated from Lactobacillus rhamnosus LS8 and reported to have antimicrobial activity against both Gram-negative and Gram-positive bacteria. In this work, we report a three-step strategy to synthetically access pure isomers of this cyclic compound and analogs thereof. In the key step, acyclic bis-hydrazides can be oxidized with (diacetoxyiodo) benzene to corresponding cyclic N,N′-diacylhydrazides. The three cycloalanopine isomers, along with several analogs, were synthesized and tested against a panel of Gram-positive and Gram-negative bacteria. We identified the active isomer as the meso compound: (4R,6S)-4,6-dimethyl-1,2,5-triazepan-3,7-dione. Additionally, a glycine derivative, (R)-4-methyl-1,2,5-triazepan-3,7-dione, was ascertained to be more potent. This compound was active against both Gram-positive and Gram-negative organisms with the strongest potency against Escherichia coli and Acinetobacter baumannii, an opportunistic pathogen found in hospital-derived infections.

Sustained, indolent immune injury of the vasculature of a heart transplant limits long-term graft and recipient survival. This injury is mitigated by a poorly characterized, maladaptive repair response. Vascular endothelial cells respond to proangiogenic cues in the embryo by differentiation to specialized phenotypes, associated with expression of apelin. In the adult, the role of developmental proangiogenic cues in repair of the established vasculature is largely unknown. We found that human and minor histocompatibility-mismatched donor mouse heart allografts with alloimmune-mediated vasculopathy upregulated expression of apelin in arteries and myocardial microvessels. In vivo, loss of donor heart expression of apelin facilitated graft immune cell infiltration, blunted vascular repair, and worsened occlusive vasculopathy in mice. In vitro, an apelin receptor agonist analog elicited endothelial nitric oxide synthase activation to promote endothelial monolayer wound repair and reduce immune cell adhesion. Thus, apelin acted as an autocrine growth cue to sustain vascular repair and mitigate the effects of immune injury. Treatment with an apelin receptor agonist after vasculopathy was established markedly reduced progression of arterial occlusion in mice. Together, these initial data identify proangiogenic apelin as a key mediator of coronary vascular repair and a pharmacotherapeutic target for immune-mediated injury of the coronary vasculature.

Recurring coronavirus outbreaks, such as the current COVID-19 pandemic, establish a necessity to develop direct-acting antivirals that can be readily administered and are active against a broad spectrum of coronaviruses. Described in this Article are novel α-acyloxymethylketone warhead peptidomimetic compounds with a six-membered lactam glutamine mimic in P1. Compounds with potent SARS-CoV-2 3CL protease and in vitro viral replication inhibition were identified with low cytotoxicity and good plasma and glutathione stability. Compounds 15e, 15h, and 15l displayed selectivity for SARS-CoV-2 3CL protease over CatB and CatS and superior in vitro SARS-CoV-2 antiviral replication inhibition compared with the reported peptidomimetic inhibitors with other warheads. The cocrystallization of 15l with SARS-CoV-2 3CL protease confirmed the formation of a covalent adduct. α-Acyloxymethylketone compounds also exhibited antiviral activity against an alphacoronavirus and non-SARS betacoronavirus strains with similar potency and a better selectivity index than remdesivir. These findings demonstrate the potential of the substituted heteroaromatic and aliphatic α-acyloxymethylketone warheads as coronavirus inhibitors, and the described results provide a basis for further optimization.

Significance Vascular diseases remain a major health burden, and AAAs lack effective medical therapy. We demonstrate a seminal role for APLN in AAA pathogenesis based on loss-of-function and gain-of-function approaches and included human vascular SMCs and AA tissue obtained from patients. We identified NEP as a dominant inactivating enzyme for native APLN-17. This allowed us to design and synthesize a stable and bioactive APLN analog resistant to NEP degradation that showed profound therapeutic effects against AAA. Our study clearly defines the APLN pathway as a central node in the pathogenesis of AAA and elucidate a therapeutic strategy of enhancing the APLN pathway by using a stable APLN analog to treat AAA., Abdominal aortic aneurysm (AAA) remains the second most frequent vascular disease with high mortality but has no approved medical therapy. We investigated the direct role of apelin (APLN) in AAA and identified a unique approach to enhance APLN action as a therapeutic intervention for this disease. Loss of APLN potentiated angiotensin II (Ang II)-induced AAA formation, aortic rupture, and reduced survival. Formation of AAA was driven by increased smooth muscle cell (SMC) apoptosis and oxidative stress in Apln−/y aorta and in APLN-deficient cultured murine and human aortic SMCs. Ang II-induced myogenic response and hypertension were greater in Apln−/y mice, however, an equivalent hypertension induced by phenylephrine, an α-adrenergic agonist, did not cause AAA or rupture in Apln−/y mice. We further identified Ang converting enzyme 2 (ACE2), the major negative regulator of the renin-Ang system (RAS), as an important target of APLN action in the vasculature. Using a combination of genetic, pharmacological, and modeling approaches, we identified neutral endopeptidase (NEP) that is up-regulated in human AAA tissue as a major enzyme that metabolizes and inactivates APLN-17 peptide. We designed and synthesized a potent APLN-17 analog, APLN-NMeLeu9-A2, that is resistant to NEP cleavage. This stable APLN analog ameliorated Ang II-mediated adverse aortic remodeling and AAA formation in an established model of AAA, high-fat diet (HFD) in Ldlr−/− mice. Our findings define a critical role of APLN in AAA formation through induction of ACE2 and protection of vascular SMCs, whereas stable APLN analogs provide an effective therapy for vascular diseases.

The main protease (Mpro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drug's properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar Ki values with the Mpro from both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 Mpro. The comparison of a new X-ray crystal structure of Mpro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 Mpro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the Mpro, which facilitates coordination of the drug's P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials.

The main protease (Mpro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drug’s properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar Kivalues with the Mprofrom both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 Mpro. The comparison of a new X-ray crystal structure of Mprofrom SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 Mpro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the Mpro, which facilitates coordination of the drug’s P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials.

The main viral protease (3CLpro) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CLpro by TAILS substrate-targeted N-terminomics. We identify >100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CLpro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CLpro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike healthy lung cells. The 3CLpro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design., Graphical Abstract, Pablos et al report diverse SARS-CoV-2 3CLpro host substrates and interactors, providing insights into pathological mechanisms. In addition to blocking viral polyprotein processing, 3CLpro inhibitor-drugs should restore multiple antiviral defenses and intracellular sensing of CoV-2 Spike protein by galectin-8, which triggers protective xenophagy in infection.

The mountain pine beetle, Dendroctonus ponderosae, has infested over ~16 Mha of pine forests in British Columbia killing50% of mature lodgepole pine, Pinus contorta, trees in affected stands. At present, it is functionally an invasive species in Alberta, killing and reproducing in evolutionarily naïve populations of lodgepole pine (P. contorta), novel jack pine (P. banksiana), and their hybrids. The entomopathogenic fungus Beauveria bassiana has shown some potential as a biocontrol agent of several bark beetle species. In this study, nine isolates of B. bassiana were examined for insect virulence characteristics, including conidiation rate, pigmentation, and infection rate in laboratory-reared D. ponderosae, to assess for their potential as biocontrol agents. The strains were categorized into three phenotypic groups based on pigmentation, conidial density, and myceliation rate. Virulence screening utilizing insect-based agar medium (D. ponderosae and European honeybee Apis mellifera carcasses) revealed no difference in selection of fungal growth. However, infection studies on D. ponderosae and A. mellifera showed contrasting results. In vivo A. mellifera infection model revealed ~5% mortality, representing the natural death rate of the hive population, whereas laboratory-reared D. ponderosae showed 100% mortality and mycosis. The LT

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The apelin/apelin receptor (ApelinR) signal transduction pathway exerts essential biological roles, particularly in the cardiovascular system. Disturbances in the apelin/ApelinR axis are linked to vascular, heart, kidney, and metabolic disorders. Therefore, the apelinergic system has surfaced as a critical therapeutic strategy for cardiovascular diseases (including pulmonary arterial hypertension), kidney disease, insulin resistance, hyponatremia, preeclampsia, and erectile dysfunction. However, apelin peptides are susceptible to rapid degradation through endogenous peptidases, limiting their use as therapeutic tools and translational potential. These proteases include angiotensin converting enzyme 2, neutral endopeptidase, and kallikrein thereby linking the apelin pathway with other peptide systems. In this context, apelin analogs with enhanced proteolytic stability and synthetic ApelinR agonists emerged as promising pharmacological alternatives. In this review, we focus on discussing the putative roles of the apelin pathway in various physiological systems from function to dysfunction, and emphasizing the therapeutic potential of newly generated metabolically stable apelin analogs and non-peptide ApelinR agonists.

The main protease, Mpro (or 3CLpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19., Coronavirus main protease is essential for viral polyprotein processing and replication. Here Vuong et al. report efficient inhibition of SARS-CoV-2 replication by the dipeptide-based protease inhibitor GC376 and its parent GC373, which were originally used to treat feline coronavirus infection.

Bacillus licheniformis SMIA-2, a thermophilic and thermostable enzyme-producing bacterium, is found to be active against several strains of Staphylococcus aureus and several Bacillus species. Here, we report the 4.30-Mbp draft genome and bioinformatic predictions supporting gene inventories for amylase, protease, cellulase, xylanase, and antimicrobial compound biosynthesis.

Spin-labeled amino acids (SLAAs) are often used to determine intermolecular distances and conformations in proteins via double electron-electron resonance. Currently available SLAAs can be difficult to incorporate selectively and have little resemblance to natural side chains in proteins. Enantioselective synthesis of three spin-labeled l-amino acids is described, starting from readily available 2,2,6,6-tetramethyl-4-piperidinone. These SLAAs better replicate canonical residues in proteins and aim for biological incorporation via genetic incorporation or solid-phase peptide synthesis.

Bacteria use various strategies to compete in an ecological niche, including the production of bacteriocins. Bacteriocins are ribosomally synthesized antibacterial peptides, and it has been postulated that the majority of Gram-positive bacteria produce one or more of these natural products. Bacteriocins can be used in food preservation and are also considered as potential alternatives to antibiotics. The majority of bacteriocins from Gram-positive bacteria had been traditionally divided into two major classes, namely lantibiotics, which are post-translationally modified bacteriocins, and unmodified bacteriocins. The last decade has seen an expanding number of ribosomally synthesized and post-translationally modified peptides (RiPPs) in Gram-positive bacteria that have antibacterial activity. These include linear azol(in)e-containing peptides, thiopeptides, bottromycins, glycocins, lasso peptides and lipolanthines. In addition, the three-dimensional (3D) structures of a number of modified and unmodified bacteriocins have been elucidated in recent years. This review gives an overview on the structural variety of bacteriocins from Gram-positive bacteria. It will focus on the chemical and 3D structures of these peptides, and their interactions with receptors and membranes, structure-function relationships and possible modes of action.

To improve sustainability in the aquaculture industry plant meals are increasingly used to replace fish meal in fish feed. Solvent-extracted soybean meal (SBM) is an attractive protein source for fish feed because of its high protein content, favorable amino acid profile, and low cost. In Atlantic salmon (Salmo salar), SBM at low levels causes soybean meal-induced enteritis (SBMIE). Few studies have been done with SBM in Pacific salmon, and none of those have included intestinal inflammation analysis. To gain more insight into salmonid responses to SBM, we assessed and compared the effects of SBM on intestinal morphology, inflammation and microbiome composition of Chinook salmon (Oncorhynchus tshawytscha), pink salmon (O. gorbuscha) and Atlantic salmon (Salmo salar). Atlantic, Chinook and pink salmon were fed for three weeks on a diet with 20% inclusion of SBM, or a control diet with fish meal. After one week on the SBM diet, Atlantic and Chinook salmon showed increased submucosa thickness in the distal intestine compared to the fish fed on the fishmeal diet. Intestinal inflammation in these species increased over time, with a further increase in submucosa thickness coincident with an infiltration of eosinophilic granular and mononuclear leucocytes. After 3 weeks on the SBM diet, intestinal inflammation was most severe in Chinook salmon. In contrast, pink salmon only showed a slight increase in submucosa thickness after three weeks on the SBM diet, and no significant increase in inflammatory cell infiltrate. Sequence-based analysis of the intestinal microbiome showed a significant difference in overall microbiome composition between species, but did not show an effect of the SBM diet on microbiome diversity or composition in any of the three salmon species. In conclusion, SBM-fed Chinook salmon were more susceptible to SBMIE than Atlantic salmon whereas pink salmon were not susceptible to SBMIE at the levels of SBM tested.

Hyoscyamine-6β-hydroxylase (H6H, EC 1.14.11.11) is a plant enzyme that catalyses the last two steps in the biosynthesis of the anticholinergic drug scopolamine, i.e. the hydroxylation of hyoscyamine to 6β-hydroxyhyoscyamine (anisodamine) and subsequent oxidative ring-closure to the 6,7-β-epoxide. A H6H gene homologue was isolated from the plant Brugmansia sanguinea (BsH6H) and recombinantly cloned into Escherichia coli, expressed and purified using an effective SUMO-fusion procedure. Enzymatic activity is approximately 40-fold higher for the first reaction step and the substrate affinity is comparable to other characterized H6H homologues (Km ∼ 60 μM). Truncation of an H6H enzyme flexible N-terminal region yields an active and stable yet more compact enzyme version.

Lacticin 3147 is a two peptide lantibiotc (LtnA1 and LtnA2) that displays nanomolar activity against many Gram-positive bacteria. Lacticin 3147 may exert its antimicrobial effect by several mechanisms. Isothermal titration calorimetry experiments show that only LtnA1 binds to the peptidoglycan precursor lipid II, which could inhibit peptidoglycan biosynthesis. An experimentally supported model of the resulting complex suggests that the key binding partners are the C-terminus of LtnA1 and pyrophosphate of lipid II. A combination of in vivo and in vitro assays indicates that LtnA1 and LtnA2 can induce rapid membrane lysis without the need for lipid II binding. However, the presence of lipid II substantially increases the activity of lacticin 3147. Furthermore, studies with synthetic LtnA2 analogues containing either desmethyl- or oxa-lanthionine rings confirm that the precise geometry of these rings is essential for this synergistic activity.

Current chemical therapies for Chagas Disease (CD) lack ability to clear Trypanosoma cruzi (Tc) parasites and cause severe side effects, making search for new strategies extremely necessary. We evaluated the action of Tityus serrulatus venom (TsV) components during Tc infection. TsV treatment increased nitric oxide and pro-inflammatory cytokine production by Tc-infected macrophages (MO), decreased intracellular parasite replication and trypomastigotes release, also triggering ERK1/2, JNK1/2 and p38 activation. Ts7 demonstrated the highest anti-Tc activity, inducing high levels of TNF and IL-6 in infected MO. TsV/Ts7 presented synergistic effect on p38 activation when incubated with Tc antigen. KPP-treatment of MO also decreased trypomastigotes releasing, partially due to p38 activation. TsV/Ts7-pre-incubation of Tc demonstrated a direct effect on parasite decreasing MO-trypomastigotes releasing. In vivo KPP-treatment of Tc-infected mice resulted in decreased parasitemia. Summarizing, this study opens perspectives for new bioactive molecules as CD-therapeutic treatment, demonstrating the TsV/Ts7/KPP-trypanocidal and immunomodulatory activity during Tc infection.