The evaluation of cystatin protein vaccines based on the stress response of ticks triggered by low-temperature and toxin stress in Haemaphysalis doenitzi.

BACKGROUND: Ticks, which are obligate blood-feeding parasites, transmit a wide range of pathogens during their hematophagic process. Certain enzymes and macromolecules play a crucial role in inhibition of several tick physiological processes, including digestion and reproduction. In the present study, genes encoding type 2 cystatin were cloned and characterized from Haemaphysalis doenitzi, and the potential role of cystatin in tick control was further assessed. RESULTS: Two cystatin genes, HDcyst-1 and HDcyst-2, were successfully cloned from the tick H. doenitzi. Their open reading frames are 390 and 426 base pairs, and the number of coding amino acids are 129 and 141, respectively. In the midgut, salivary glands, Malpighian tubules and ovaries of ticks, the relative expression of HDcyst-1 was higher in the midgut and Malpighian tubules, and HDcyst-2 was higher in the salivary glands of H. doenitzi, respectively. Lipopolysaccharide (LPS) injection and low-temperature stress elevated cystatin expression in ticks. Enzyme-linked immunosorbent assay showed that both rHDcyst-1 and rHDcyst-2 protein vaccines increased antibody levels in immunized rabbits. A vaccination trial in rabbits infected with H. doenitzi showed that both recombinant cystatin proteins significantly reduced tick engorgement weights and egg mass weight, in particular, rHDcyst-1 significantly prolonged tick engorgement time by 1 day and reduced egg hatching rates by 16.9%. In total, rHDcyst-1 and rHDcyst-2 protein vaccinations provided 64.1% and 51.8% protection to adult female ticks, respectively. CONCLUSION: This is the first report on the immunological characterization of the cystatin protein and sequencing of the cystatin gene in H. doenitzi. Cystatin proteins are promising antigens that have the potential to be used as vaccines for infestation of H. doenitzi control. © 2024 Society of Chemical Industry.

Epidemiology and risk factors for thrombosis in children and newborns: systematic evaluation and meta-analysis.

BACKGROUND: Thrombosis is a serious condition in children and neonates. However, the risk factors for thrombosis have not been conclusively determined. This study aimed to identify the risk factors for thrombosis in children and neonates in Intensive Care Unit (ICU) through a meta-analysis to better guide clinical treatment. METHODS: A systematic search of electronic databases (PubMed, Embase, Cochrane Library, WOS, CNKI, Wanfang, VIP) was conducted to retrieve studies from creation on 23 May 2022. Data on the year of publication, study design, country of origin, number of patients/controls, ethnicity, and type of thrombus were extracted. The publication bias and heterogeneity between studies were assessed, and pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using fixed or random effects models. RESULTS: A total of 18 studies met the inclusion criteria. The incidence of thrombosis in children was 2% per year (95% CI 1%-2%, P < 0.01). Infection and sepsis (OR = 1.95, P < 0.01), CVC (OR = 3.66, [95%CL 1.78-7.51], P < 0.01), mechanical ventilation (OR = 2.1, [95%CL1.47-3.01], P < 0.01), surgery (OR = 2.25, [95%CL1.2-4.22], P < 0.01), respiratory distress (OR = 1.39, [95%CL0.42-4.63], P < 0.01), ethnicities (OR = 0.88, [95%CL 0.79-0.98], P = 0.78), gestational age (OR = 1.5, [95%CL1.34-1.68], P = 0.65)were identified as risk factors for thrombosis. CONCLUSIONS: This meta-analysis suggests that CVC, Surgery, mechanical ventilation, Infection/sepsis, gestational age, Respiratory distress, and different ethnicities are risk factors for thrombosis in children and neonates in ICU. These findings may help clinicians to identify high-risk patients and develop appropriate prevention strategies. TRIAL REGISTRATION: PROSPERO (CRD 42022333449).

Rationally Designed Nonapeptides with Great Skin Photoprotective Effect through Producing Type 1 Collagen and Blocking the mTOR Pathway.

Ultraviolet (UV), as the most common environmental stress factor to human skin, causes redox imbalance and leads to photoaging and the development of cancer. In this work, we screened a nonapeptide (PWH) with good activities of antioxidant, promoting the secretion of type 1 collagen (COL-1) and repairing damaged skin from a series of rationally designed novel short peptides. PWH could alleviate UV-A-induced oxidative stress, restrain pro-inflammatory cytokine production, protect mitochondrial function, and maintain autophagy activity. We also first indicated that inhibiting the PI3K/AKT/mTOR signaling pathway and restoration of autophagy activity might delay the photoaging process in skin cells. Topical applications of PWH were further demonstrated to exhibit significant protection in full-wavelength UV-induced skin aging in mice models both in the prophylaxis and treatment way. In addition, given the good stability and without unwanted toxicity and anaphylaxis, PWH could be a promising candidate for cosmetics and pharmaceuticals.

Defensing role of novel piscidins from largemouth bass (Micropterus salmoides) with evidence of bactericidal activities and inducible expressional delineation.

Micropterus salmoides is an economical important species of freshwater-cultured fish, the in-depth knowledge of its immune system is in urgent development to cope with serious infectious diseases. Piscidin is an important antimicrobial peptide (AMP) family existing in almost all teleosts. However, no piscidin has been reported in largemouth bass. In this study, three novel piscidins (MSPiscidin-1, -2, and -3) were firstly identified and characterized from the largemouth bass. The predicted mature peptides of MSPiscidin-1, -2, and -3 (consists of 24, 27, 25 amino acid residues, respectively) all adopted an amphipathic α-helical conformation representative of cationic AMPs that are important for membrane permeabilization and antibacterial activity. MSPiscidin-2 and -3 indeed displayed strong, broad-spectrum, and highly efficient antimicrobial activities in vitro against aquatic pathogens, but MSPiscidin-1 didn't show direct antimicrobial activity. MSPiscidin-2 and -3 killed bacteria mainly by inducing membrane permeabilization, in addition, they also can interact with bacterial genomic DNA, which might influence the DNA replication and transcription. Besides, MSPiscidin-2 and -3 could effectively inhibit the formation of the bacterial biofilm and eliminate the preformed biofilms. In vivo, MSPiscidin-1-3 genes showed an inducible expression pattern in the tested tissues upon Vibrio harveyi infection, which further indicated the key roles of piscidins in innate immunity in largemouth bass. Overall, this study will supplement the understanding of M. salmoides innate immune system and provide candidates for the design of novel peptide antibacterial agents used in aquaculture.

Influence of plants on microbial activity in a vertical-downflow wetland system treating waste activated sludge with high organic matter concentrations.

The rhizosphere is a key zone for pollutant removal in treatment wetlands; therefore, studies on microbial activity may provide helpful information for a better understanding of purification processes. We studied microbial activity in a vertical-downflow constructed wetland system treating waste activated sludge with high organic matter concentrations, under Mediterranean climate. The aims of the work were to study the influence of (i) the presence of plants, (ii) the plant species (Phragmites australis Cav., Typha latifolia L., Iris pseudacorus L.), and (iii) the plant growth stage (plant senescence and plant fast growing stage) on total respiration rate and phosphatase activity in the substrate (intented here as the solid support on which the plants grow). The presence of plants had a positive influence on microbial activity, since substrate respiration and both acid and alkaline phosphatase activity were always higher in planted than in unplanted mesocosms. Among the three tested species, Phragmites was the one that most stimulated both substrate respiration rate and phosphatase activity, followed by Typha and Iris. These differences of microbial activity between mesocosms were corresponding to differences of removal efficiency. Substrate respiration and phosphatase activity were of similar magnitude at the two growth stages, while the stimulating effect of plants seemed to have been delayed and microbial activity showed higher fluctuations at plant fast growing stage than at plant senescence.

Purification processes involved in sludge treatment by a vertical flow wetland system: focus on the role of the substrate and plants on N and P removal.

Vertical-flow wetland systems were tested for treatment of liquid waste activated sludge with high content of organic compounds from a soft drink factory. A mesocosm experiment was carried out on planted and unplanted systems to understand the relative importance of substrate and plants in purification processes and to compare three species: Phragmites australis Cav., Typha latifolia L., or Iris pseudacorus L. All planted mesocosms performed better than unplanted mesocosms and Phragmites showed the highest efficiencies, both in volumes and loads, closely followed by Typha. Removal efficiencies were very high in all cases, and physical filtration by the organic substrate was identified as the main processes for nutrient removal (>50%). We showed that plants played direct and indirect roles such as nutrients uptake (up to 23% of the N for Phragmites), evapotranspiration reducing outflow volumes; or improvement of filtration by the root systems and stimulation of microbial activities (respiration rate was double compared to unplanted mesocosms).

Depollution potential of three macrophytes: exudated, wall-bound and intracellular peroxidase activities plus intracellular phenol concentrations.

The aim of this study was to investigate the potential role of three macrophyte species (Iris pseudacorus, Typha latifolia and Phragmites australis) for detoxication of xenobiotics, and to study their variations with seasons or concentrations of sewage sludge from the food industry. For this purpose, some aspects of the green liver concept were explored through peroxidase measurements in three compartments in roots: intracellular, cell wall and extracellular. In addition, phenol concentrations were also measured in order to assess heavy metal detoxication potential. Enzyme activities and phenol concentrations were overall lower in winter according to the phenological stages and some sludge effects occurred. Results show that P. australis roots exuded and contained more peroxidase in all seasons: 17 U/g (1373 U/g protein), 0.8 U/g (613 U/g protein) and 4.8 U/g (1329 U/g protein) in intracellular compartments, cell wall and exudates, respectively. In contrast, the highest phenol concentration was found in I. pseudacorus roots: 3.58 mg eq. [corrected] gallic acid/g. Hence, in constructed wetlands, P. australis is suitable for organic waste water treatment, while I. pseudacorus should be used in the case of waters highly charged with heavy metals.

Feasibility of using an organic substrate in a wetland system treating sewage sludge: Impact of plant species.

A vertical-flow wetland system was tested for treatment of liquid sludge with high organic concentrations using an organic substrate (peat/crushed pine bark, 1/1) as growing medium. Mesocosms (1 m(3)) were planted with either Phragmites australis Cav., Typha latifolia L., or Iris pseudacorus L. The aim of the work was to determine the feasibility of using an organic substrate in treatment wetlands, through the study of its temporal patterns and of its impact on the water output quality. Results confirmed that the organic substrate can be used in such wetlands treating highly organic sludge, without clogging phenomena for the experimental period. The organic substrate released soluble organic matter but few mineral elements. Over the experimental period, substrate TOC concentration did not change while N concentration increased. Plants showed positive impact on substrate temporal patterns and also on the outflow water quality. Overall, Phragmites seemed to be more beneficial than Typha and Iris.

Decoding the cryptic active conformation of a protein by synthetic photoscanning: insulin inserts a detachable arm between receptor domains.

Proteins evolve in a fitness landscape encompassing a complex network of biological constraints. Because of the interrelation of folding, function, and regulation, the ground-state structure of a protein may be inactive. A model is provided by insulin, a vertebrate hormone central to the control of metabolism. Whereas native assembly mediates storage within pancreatic beta-cells, the active conformation of insulin and its mode of receptor binding remain elusive. Here, functional surfaces of insulin were probed by photocross-linking of an extensive set of azido derivatives constructed by chemical synthesis. Contacts are circumferential, suggesting that insulin is encaged within its receptor. Mapping of photoproducts to the hormone-binding domains of the insulin receptor demonstrated alternating contacts by the B-chain beta-strand (residues B24-B28). Whereas even-numbered probes (at positions B24 and B26) contact the N-terminal L1 domain of the alpha-subunit, odd-numbered probes (at positions B25 and B27) contact its C-terminal insert domain. This alternation corresponds to the canonical structure of abeta-strand (wherein successive residues project in opposite directions) and so suggests that the B-chain inserts between receptor domains. Detachment of a receptor-binding arm enables photo engagement of surfaces otherwise hidden in the free hormone. The arm and associated surfaces contain sites also required for nascent folding and self-assembly of storage hexamers. The marked compression of structural information within a short polypeptide sequence rationalizes the diversity of diabetes-associated mutations in the insulin gene. Our studies demonstrate that photoscanning mutagenesis can decode the active conformation of a protein and so illuminate cryptic constraints underlying its evolution.

The A-chain of insulin contacts the insert domain of the insulin receptor. Photo-cross-linking and mutagenesis of a diabetes-related crevice.

The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of Val(A3), which projects within a crevice between the A- and B-chains. Engineered receptor alpha-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a Pap(B25)-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and alpha-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (Gly(A3) and Ser(A3)) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of Aba(A3) and allo-Thr(A3) analogs are reduced at least 10-fold; the activity of Thr(A3)-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (Aba(A3)) or by altered polarity (allo-Thr(A3) and Thr(A3)). Our results provide evidence that Val(A3), a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the alpha-subunit in a hormone-receptor complex.

How insulin binds: the B-chain alpha-helix contacts the L1 beta-helix of the insulin receptor.

Binding of insulin to the insulin receptor plays a central role in the hormonal control of metabolism. Here, we investigate possible contact sites between the receptor and the conserved non-polar surface of the B-chain. Evidence is presented that two contiguous sites in an alpha-helix, Val(B12) and Tyr(B16), contact the receptor. Chemical synthesis is exploited to obtain non-standard substitutions in an engineered monomer (DKP-insulin). Substitution of Tyr(B16) by an isosteric photo-activatable derivative (para-azido-phenylalanine) enables efficient cross-linking to the receptor. Such cross-linking is specific and maps to the L1 beta-helix of the alpha-subunit. Because substitution of Val(B12) by larger side-chains markedly impairs receptor binding, cross-linking studies at B12 were not undertaken. Structure-function relationships are instead probed by side-chains of similar or smaller volume: respective substitution of Val(B12) by alanine, threonine, and alpha-aminobutyric acid leads to activities of 1(+/-0.1)%, 13(+/-6)%, and 14(+/-5)% (relative to DKP-insulin) without disproportionate changes in negative cooperativity. NMR structures are essentially identical with native insulin. The absence of transmitted structural changes suggests that the low activities of B12 analogues reflect local perturbation of a "high-affinity" hormone-receptor contact. By contrast, because position B16 tolerates alanine substitution (relative activity 34(+/-10)%), the contribution of this neighboring interaction is smaller. Together, our results support a model in which the B-chain alpha-helix, functioning as an essential recognition element, docks against the L1 beta-helix of the insulin receptor.

Diabetes-associated mutations in insulin identify invariant receptor contacts.

Mutations in human insulin cause an autosomal-dominant syndrome of diabetes and fasting hyperinsulinemia. We demonstrate by residue-specific photo cross-linking that diabetes-associated mutations occur at receptor-binding sites. The studies use para-azido-phenylalanine, introduced at five sites by total protein synthesis. Because two such sites (Val(A3) and Phe(B24)) are largely buried in crystal structures of the free hormone, their participation in receptor binding is likely to require a conformational change to expose a hidden functional surface. Our results demonstrate that this surface spans both chains of the insulin molecule and includes sites of rare human mutations that cause diabetes.

Mechanism of insulin chain combination. Asymmetric roles of A-chain alpha-helices in disulfide pairing.

The A and B chains of insulin combine to form native disulfide bridges without detectable isomers. The fidelity of chain combination thus recapitulates the folding of proinsulin, a precursor protein in which the two chains are tethered by a disordered connecting peptide. We have recently shown that chain combination is blocked by seemingly conservative substitutions in the C-terminal alpha-helix of the A chain. Such analogs, once formed, nevertheless retain high biological activity. By contrast, we demonstrate here that chain combination is robust to non-conservative substitutions in the N-terminal alpha-helix. Introduction of multiple glycine substitutions into the N-terminal segment of the A chain (residues A1-A5) yields analogs that are less stable than native insulin and essentially without biological activity. (1)H NMR studies of a representative analog lacking invariant side chains Ile(A2) and Val(A3) (A chain sequence GGGEQCCTSICSLYQLENYCN; substitutions are italicized and cysteines are underlined) demonstrate local unfolding of the A1-A5 segment in an otherwise native-like structure. That this and related partial folds retain efficient disulfide pairing suggests that the native N-terminal alpha-helix does not participate in the transition state of the reaction. Implications for the hierarchical folding mechanisms of proinsulin and insulin-like growth factors are discussed.

Protein structure and the spandrels of San Marco: insulin's receptor-binding surface is buttressed by an invariant leucine essential for its stability.

Insulin provides a model of induced fit in macromolecular recognition: the hormone's conserved core is proposed to contribute to a novel receptor-binding surface. The core's evolutionary invariance, unusual among globular proteins, presumably reflects intertwined constraints of structure and function. To probe the architectural basis of such invariance, we have investigated hydrophobic substitutions of a key internal side chain (Leu(A16)). Although the variants exhibit perturbed structure and stability, moderate receptor-binding activities are retained. These observations suggest that the A16 side chain provides an essential structural buttress but unlike neighboring core side chains, does not itself contact the receptor. Among invertebrate insulin-like proteins, Leu(A16) and other putative core residues are not conserved, suggesting that the vertebrate packing scheme is not a general requirement of an insulin-like fold. We propose that conservation of Leu(A16) among vertebrate insulins and insulin-like growth factors is a side consequence of induced fit: alternative packing schemes are disallowed by lack of surrounding covariation within the hormone's hidden receptor-binding surface. An analogy is suggested between Leu(A16) and the spandrels of San Marco, tapering triangular spaces at the intersection of the dome's arches. This celebrated metaphor of Gould and Lewontin emphasizes the role of interlocking constraints in the evolution of biological structures.