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Angiotensin-converting enzyme (ACE) inhibitors are important agents in blood pressure (BP) management. It was recently shown that the egg-protein hydrolysate NWT-03 inhibited ACE in Zucker diabetic fatty rats. We therefore designed a dose-finding study to assess the effects of 1, 2 and 5 g NWT-03 on daytime, 36-h, and night-time systolic and diastolic BP (SBP and DBP) in ninety-two generally healthy subjects with normal BP (n 29), high-normal BP (n 34) or mild hypertension (n 29). The study had a cross-over design with six treatment arms (1 g NWT-03 or placebo in period 1 and placebo or 1 g NWT-03 in period 2, 2 g NTW-03 or placebo in period 1 and placebo or 2 g NWT-03 in period 2, or 5 g NTW-03 or placebo in period 1 and placebo or 5 g NTW-03 in period 2). A comparable number of subjects from each BP class were included in each study arm. Duration of both treatments in each arm was 7 d, separated by 5-d wash-out periods. BP was measured with an ambulatory BP monitor before and after the treatments. In mild-hypertensive subjects, 2 g NWT-03 significantly decreased daytime SBP (7·9 mmHg; P=0·006), daytime DBP (4·2 mmHg; P=0·009), 36-h SBP (6·9 mmHg; P=0·015) and 36-h DBP (3·5 mmHg; P=0·035) compared with placebo subjects. In addition, in mild-hypertensive subjects, 5 g NWT-03 significantly decreased night-time SBP (14·8 mmHg; P=0·008) and night-time DBP (8·4 mmHg; P=0·020) compared with that in placebo subjects. To conclude, we found that 2 g NWT-03 lowered daytime and 36-h BP in subjects with mild hypertension, and 5 g NWT-03 lowered night-time BP in subjects with mild hypertension. As no dose–response relationship was evident, these results should be interpreted with care, and additional studies are needed.

All-aqueous emulsions exploit spontaneous liquid–liquid separation and due to their water-based nature are particular advantageous for the biocompatible storage and processing of biomacromolecules. However, the ultralow interfacial tensions characteristic of all-aqueous interfaces represent an inherent limitation to the use of thermally adsorbed particles to achieve emulsion stability. Here, we use protein nanofibrils to generate colloidosome-like two-dimensional crosslinked networks of nanostructures templated by all-aqueous emulsions, which we term fibrillosomes. We show that this approach not only allows us to operate below the thermal limit at ultra-low surface tensions but also yields structures that are stable even in the complete absence of an interface. Moreover, we show that the growth and multilayer deposition of fibrils allows us to control the thickness of the capsule shells. These results open up the possibility of stabilizing aqueous two-phase systems using natural proteins, and creating self-standing protein capsules without the requirement for three-phase emulsions or water/oil interfaces. All-aqueous emulsions are useful for delivering and processing biomolecules, but their stability is constrained by low interfacial adsorption energy. Song et al . solve this problem using protein nanofibrils that form a crosslinked network, whose stability is superior to conventional colloidal capsules.

Reversible and irreversible amyloids are two diverging cases of protein (mis)folding associated with the cross-β motif in the protein folding and aggregation energy landscape. Yet, the molecular origins responsible for the formation of reversible vs irreversible amyloids have remained unknown. Here we provide evidence at the atomic level of distinct folding motifs for irreversible and reversible amyloids derived from a single protein sequence: human lysozyme. We compare the 2.8 Å structure of irreversible amyloid fibrils determined by cryo-electron microscopy helical reconstructions with molecular insights gained by solid-state NMR spectroscopy on reversible amyloids. We observe a canonical cross-β-sheet structure in irreversible amyloids, whereas in reversible amyloids, there is a less-ordered coexistence of β-sheet and helical secondary structures that originate from a partially unfolded lysozyme, thus carrying a “memory” of the original folded protein precursor. We also report the structure of hen egg-white lysozyme irreversible amyloids at 3.2 Å resolution, revealing another canonical amyloid fold, and reaffirming that irreversible amyloids undergo a complete conversion of the native protein into the cross-β structure. By combining atomic force microscopy, cryo-electron microscopy and solid-state NMR, we show that a full unfolding of the native protein precursor is a requirement for establishing irreversible amyloid fibrils. In this paper, the authors revealed the distinct folding motifs for irreversible and reversible amyloids derived from a single protein, by combining multiple mesoscopic and molecular techniques including AFM, cryo-EM and solid-state NMR.

This study advances current knowledge in the field of host-microbe interactions by demonstrating that the two families of immune effectors, antimicrobial peptides and lysozymes, actively regulate the gut microbiota composition and abundance. Consequences of the loss of these antimicrobial peptides and lysozymes are exacerbated during aging, and their loss contributes to increased microbiota abundance and shifted composition in old flies. The gut microbiota affects the physiology and metabolism of animals and its alteration can lead to diseases such as gut dysplasia or metabolic disorders. Several reports have shown that the immune system plays an important role in shaping both bacterial community composition and abundance in Drosophila , and that immune deficit, especially during aging, negatively affects microbiota richness and diversity. However, there has been little study at the effector level to demonstrate how immune pathways regulate the microbiota. A key set of Drosophila immune effectors are the antimicrobial peptides (AMPs), which confer defense upon systemic infection. AMPs and lysozymes, a group of digestive enzymes with antimicrobial properties, are expressed in the gut and are good candidates for microbiota regulation. Here, we take advantage of the model organism Drosophila melanogaster to investigate the role of AMPs and lysozymes in regulation of gut microbiota structure and diversity. Using flies lacking AMPs and newly generated lysozyme mutants, we colonized gnotobiotic flies with a defined set of commensal bacteria and analyzed changes in microbiota composition and abundance in vertical transmission and aging contexts through 16S rRNA gene amplicon sequencing. Our study shows that AMPs and, to a lesser extent, lysozymes are necessary to regulate the total and relative abundance of bacteria in the gut microbiota. We also decouple the direct function of AMPs from the immune deficiency (IMD) signaling pathway that regulates AMPs but also many other processes, more narrowly defining the role of these effectors in the microbial dysbiosis observed in IMD-deficient flies upon aging. IMPORTANCE This study advances current knowledge in the field of host-microbe interactions by demonstrating that the two families of immune effectors, antimicrobial peptides and lysozymes, actively regulate the gut microbiota composition and abundance. Consequences of the loss of these antimicrobial peptides and lysozymes are exacerbated during aging, and their loss contributes to increased microbiota abundance and shifted composition in old flies. This work shows that immune effectors, typically associated with resistance to pathogenic infections, also help shape the beneficial gut community, consistent with the idea that host-symbiont interactions use the same “language” typically associated with pathogenesis.

We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions. T4 Lysozyme (T4L) is a model protein whose structure is extensively studied. Here the authors combine single-molecule and ensemble FRET measurements, FRET-positioning and screening and EPR spectroscopy to study the structural dynamics of T4L and describe its conformational landscape during the catalytic cycle by an extended Michaelis–Menten mechanism and identify an excited conformational state of the enzyme.

Niclosamide (NIC) has demonstrated promising in vitro antiviral efficacy against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Though NIC is already FDA-approved, administration of the currently available oral formulation results in systemic drug levels that are too low for the inhibition of SARS-CoV-2. We hypothesized that the co-formulation of NIC with an endogenous protein, human lysozyme (hLYS), could enable the direct aerosol delivery of the drug to the respiratory tract as an alternative to oral delivery, thereby effectively treating COVID-19 by targeting the primary site of SARS-CoV-2 acquisition and spread. To test this hypothesis, we engineered and optimized composite particles containing NIC and hLYS suitable for delivery to the upper and lower airways via dry powder inhaler, nebulizer, and nasal spray. The novel formulation demonstrates potent in vitro and in vivo activity against two coronavirus strains, MERS-CoV and SARS-CoV-2, and may offer protection against methicillin-resistance staphylococcus aureus pneumonia and inflammatory lung damage occurring secondary to SARS-CoV-2 infections. The suitability of the formulation for all stages of the disease and low-cost development approach will ensure rapid clinical development and wide-spread utilization.

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS–protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS–protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective.

Peptidoglycan is the major structural constituent of the bacterial cell wall, forming a meshwork outside the cytoplasmic membrane that maintains cell shape and prevents lysis. In Gram-negative bacteria, peptidoglycan is located in the periplasm, where it is protected from exogenous lytic enzymes by the outer membrane. Here we show that the type VI secretion system of Pseudomonas aeruginosa breaches this barrier to deliver two effector proteins, Tse1 and Tse3, to the periplasm of recipient cells. In this compartment, the effectors hydrolyse peptidoglycan, thereby providing a fitness advantage for P. aeruginosa cells in competition with other bacteria. To protect itself from lysis by Tse1 and Tse3, P. aeruginosa uses specific periplasmically localized immunity proteins. The requirement for these immunity proteins depends on intercellular self-intoxication through an active type VI secretion system, indicating a mechanism for export whereby effectors do not access donor cell periplasm in transit. Duelling bacterial pathogens The type VI secretion system (T6SS) is a protein-export machine that is present in about one-quarter of all sequenced bacteria. Bacteria can use this system to deliver toxic effector proteins in a contact-dependent manner to other bacterial cells. However, what these proteins do once their destination is reached has remained largely unknown. It is now shown that the opportunistic human pathogen Pseudomonas aeruginosa uses its T6SS to kill competing Gram-negative bacteria by injecting them with two peptidoglycan-degradative enzymes, the effector proteins Tse1 and Tse3. P. aeruginosa protects itself from these effectors by expressing immunity proteins that bind the toxins.

Lysozymes are among the best-characterized enzymes, acting upon the cell wall substrate peptidoglycan. Here, examining the invasive bacterial periplasmic predator Bdellovibrio bacteriovorus , we report a diversified lysozyme, DslA, which acts, unusually, upon (GlcNAc-) deacetylated peptidoglycan. B. bacteriovorus are known to deacetylate the peptidoglycan of the prey bacterium, generating an important chemical difference between prey and self walls and implying usage of a putative deacetyl-specific “exit enzyme”. DslA performs this role, and ΔDslA strains exhibit a delay in leaving from prey. The structure of DslA reveals a modified lysozyme superfamily fold, with several adaptations. Biochemical assays confirm DslA specificity for deacetylated cell wall, and usage of two glutamate residues for catalysis. Exogenous DslA, added ex vivo, is able to prematurely liberate B. bacteriovorus from prey, part-way through the predatory lifecycle. We define a mechanism for specificity that invokes steric selection, and use the resultant motif to identify wider DslA homologues. The bacterial periplasmic predator Bdellovibrio bacteriovorus deacetylates the peptidoglycan of the prey bacterium early upon invasion. Here, the authors identify and characterize a Bdellovibrio lysozyme that acts specifically on deacetylated peptidoglycan and is important for periplasmic exit.

The scientific community has definitely demonstrated the importance of the use of mouthwash in daily oral hygiene. In our pilot study, we tested the effectiveness of a novel mouth rinse containing sea salt, xylitol, and lysozyme. Streptococcus mutans (S. mutans) growth, and plaque index in adolescent patients aged 14–17 years, were observed. The bacterial load was investigated by in vitro microbiological analysis; the plaque index was assessed through the O’Leary’s Plaque Control Record (PCR). The study has shown that the use of a sea salt-based mouthwash in daily oral hygiene reduces the bacterial levels of S. mutans (p < 0.01) linked to the combined action of xylitol and lysozyme, together with the action of sea salt. Our preliminary data confirm and improve the main results reported in the scientific literature on the importance of the use of xylitol, lysozyme, and sea salt in oral health.