Disulphide Bonds Research Articles

Hemp (Cannabis sativa L.) protein concentrates from wet and dry industrial fractionation: Molecular properties, nutritional composition, and anisotropic structuring

Hemp seeds stand as a rich source of globulins and albumins with all the essential amino acids and a balanced amino acid profile. Nevertheless, the potential of dry- and wet-extracted hemp protein concentrates to create plant-based, fibrous High Moisture Meat Analogues (HMMAs) remains unknown. In this study, five distinct hemp seed protein concentrates (55.9–76.4% protein, d.b.) produced at industrial scale using dry or wet fractionation were investigated and compared for their functionality and protein molecular properties. Furthermore, non-proteinaceous components were also analysed to elaborate on the underlying mechanisms for the structuring behaviour of hemp protein concentrates during wet extrusion. Although dry fractionation resulted in lower protein concentration, hemp proteins retained their native oligomeric state and their albumin fraction, thus showing higher surface hydrophobicity and solubility, and lower gelation concentration than wet-extracted counterparts. Furthermore, hemp samples were richer in bound polyphenols (>1800 mg/100 g), presumably phenolic acids, than the control pea sample (1363 mg/100 g), which resulted in dark colours in wet-extracted samples. Selected HMMA prototypes were developed and investigated for anisotropy, viscoelasticity, and proton NMR relaxometry. Visual and instrumental anisotropy dramatically increased with the use of hemp protein concentrates, from 0.69% using pea, to 0.98% and 1.41% using dry- and wet-fractionated fractionated hemp, respectively. Nonetheless, dry-fractionated hemp HMMA showed the highest proportion of free water in the system ( T 23 proton relaxation time), and an intermediate viscoelasticity between pea and wet-fractionated hemp HMMAs. Interestingly, SDS-PAGE revealed a significant contribution of disulphide bonds on hemp protein aggregation during processing. • Wet isolation decreased hemp protein surface hydrophobicity, solubility and lightness. • Hemp protein samples resulted in meat analogues with visible fibrous-like structures. • Dry-fractionated hemp extrudates had the highest proportion of free water. • Hemp extrudates showed up to 200% higher anisotropy than the pea protein counterpart. • Disulphide bonds played a key role on hemp protein aggregation during extrusion.

A designed cyclic analogue of gomesin has potent activity against Staphylococcus aureus biofilms.

Infections caused by bacterial biofilms are very difficult to treat. The use of currently approved antibiotics even at high dosages often fails, making the treatment of these infections very challenging. Novel antimicrobial agents that use distinct mechanisms of action are urgently needed. To explore the use of [G1K,K8R]cGm, a designed cyclic analogue of the antimicrobial peptide gomesin, as an alternative approach to treat biofilm infections. We studied the activity of [G1K,K8R]cGm against biofilms of Staphylococcus aureus, a pathogen associated with several biofilm-related infections. A combination of atomic force and real-time confocal laser scanning microscopies was used to study the mechanism of action of the peptide. The peptide demonstrated potent activity against 24 h-preformed biofilms through a concentration-dependent ability to kill biofilm-embedded cells. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes across the biofilm with a half-time of 65 min. We also tested an analogue of [G1K,K8R]cGm without disulphide bonds, and a linear unfolded analogue, and found both to be inactive. The results suggest that the 3D structure of [G1K,K8R]cGm and its stabilization by disulphide bonds are essential for its antibacterial and antibiofilm activities. Moreover, our findings support the potential application of this stable cyclic antimicrobial peptide to fight bacterial biofilms.

Enhanced photo-hypoxia-activated combination therapy traced by AIE photosensitizer and targeted by hyaluronic acid: Disulphide bond interference of detoxification barrier.

The treatment efficacy of anticancer drugs in complex physiological environments is still restricted by multi-drug resistance. To overcome this issue, a nanodrug system of HA-SS@CuS@ZIF-8@TPZ&TBMACN (HSCZTT) that breaks through the detoxification barrier for tirapazamine (TPZ) delivery was developed in this manuscript. In addition to the photothermal effect aroused by CuS in HSCZTT, which can damage tumour cells, TBMACN with photostable fluorescence in the aggregate state can also generate sufficient reactive oxygen species (ROS) to destroy tumour cells. The continuous consumption of oxygen in PDT aggravates the hypoxic environment of tumours, which further activates the TPZ released in the acidic microenvironment of the tumour to achieve apoptosis of the tumour cells. The HSCZTT can not only target the CD44 receptor overexpressed on the surface of the cancer cell, but can also effectively consume a large amount of glutathione (GSH) through the disulphide bond-modified hyaluronic acid, which serves as a targeted disulphide bond, interfering with the detoxification barrier. Our finding presents a rational strategy to overcome multidrug resistance for the improved efficacy of anticancer drugs by the targeting of Hyaluronic acid (HA), release of the drug by the acid response of ZIF-8, breakthrough of the detoxification barrier, precise positioning of the drug release and combined treatment with phototherapy and hypoxia-activated chemotherapy.

Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells.

SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main challenges is the ongoing emergence of SARS-CoV-2 variants that carry mutations within the RBD, resulting in the constant need to design and optimise the production of new recombinant protein variants. We describe here the impact of naturally occurring RBD mutations on the secretion of a recombinant Fc-tagged RBD protein expressed in HEK 293 cells. We show that mutation E484K of the B.1.351 variant interferes with the proper disulphide bond formation and folding of the recombinant protein, resulting in its retention into the endoplasmic reticulum (ER) and reduced protein secretion. Accumulation of the recombinant B.1.351 RBD-Fc fusion protein in the ER correlated with the upregulation of endogenous ER chaperones, suggestive of the unfolded protein response (UPR). Overexpression of the chaperone and protein disulphide isomerase PDIA2 further impaired protein secretion by altering disulphide bond formation and increasing ER retention. This work contributes to a better understanding of the challenges faced in producing mutant RBD proteins and can assist in the design of optimisation protocols.

Learning from hair moisture sorption and hysteresis.

The process of moisture sorption and desorption by human hair was analysed for extracting hints on the hair structure. The isotherms of moisture sorption and desorption by hair were recorded for untreated and chemically treated (permed and bleached) hair. Data of swelling were also considered. By examining the swelling and moisture sorption of keratin fibres, it is possible to conclude that hysteresis is quite improbably caused by capillary condensation. The mobility of the protein chains and the strength of the bonds binding water molecules to the active sites inside the matrix are proposed as causes instead. The concept of "breaking symmetry", derived from moisture sorption- desorption data, and the method of evaluating this parameter, is proposed as a way of characterizing the chemical treatment of hair. The results show that bleaching produces a larger breaking of symmetry than perming, and this is suggested to be due to new hydrogen bonds, created as a result of the chemical treatment, replacing the original disulphide bonds, which are of different strength compared to the bonds of untreated hair. The quantitative sorption data matched well to the model of grains of matrix enveloped in layers of water molecules at increasing relative humidity, up to 100%. The analysis suggested that, aside from the glass transition event occurring at around 60%-70% relative humidity, there is another, less examined, transition occurring at around 30% relative humidity, assigned to the opening of the hair inner structure, and accommodation of more water molecules. Both transitions are reflected by corresponding changes in the fibre mechanical behaviour. The moisture sorption-desorption by hair was shown to not only allow a quantitative differentiation among various cosmetic treatments of the hair but to also provide valuable information on the structure of the fibre.

Enhancement of Wheat Flour and Dough Properties by Non-Thermal Plasma Treatment of Wheat Flour

Demand to improve food quality attributes without the use of chemicals has risen exponentially in the past few years. Non-thermal plasma (NTP) (also called ‘cold plasma’) is becoming increasingly popular for this purpose due to its unique low-temperature and non-chemical nature. In the present research, the concept of in situ dielectric barrier discharge (DBD) plasma treatment inside a rotational reactor for the direct treatment of wheat flour was experimentally analyzed. The primary research goal was to determine the effects of short-period NTP treatment of DBD type on flour and dough properties. For this purpose, the influence of different operating parameters was tested, i.e., treatment time, the amount of flour placed in the reactor and the environmental (air) temperature. Changes in the structural attributes of the most commonly used flours (type 550 and 1050) and their respective doughs were studied using a set of analytical techniques. Rheological analysis demonstrated the ability of NTP to significantly intensify the visco-elastic properties of dough produced from wheat flour type 550 that was treated for less than 180 s. This indicated that plasma treatment enhanced intermolecular disulphide bonds in gluten proteins, which resulted in stronger protein–starch network formations. However, longer treatment times did not result in a significant increase in the visco-elastic properties of wheat dough. The obtained results showed a 6–7% increase in flour hydration due to NTP treatment, which also makes a contribution to hydrogen bonding due to changes in the bonded and free water phase. Experimental findings further confirmed the dependence of NTP treatment efficiency on environmental air temperature.

Extrusion of pea snack foods and control of biopolymer changes aided by rheology and simulation

Pea flour and a blend of pea starch and protein isolate with a starch-to-protein ratio close to 2/1 were processed using co-rotating twin-screw extruders. Different extruder scales operated at a moisture content of 18–35% and a screw speed of 120–700 rpm resulted in large intervals of melt temperature T (95–165 °C) and specific mechanical energy SME (150–2000 kJ/kg). With increasing T and SME, starch solubility in water increased due to starch melting and depolymerisation, and protein solubility in SDS decreased due to formation of protein aggregates linked by disulphide bonds. Extruded foods’ morphology was studied by CLSM. Protein cross-linking increased the median size of protein aggregates; starch destructuration increased the area of the starch phase to the detriment of the protein one. Melt shear viscosity was determined using a pre-shearing capillary rheometer. The melts exhibited shear-thinning behaviour according to a power-law model, with values close to those of model parameters observed for pea flour and SP 2/1 blend. The rheological model was implemented in a 1D global extrusion model to simulate pea ingredient extrusion. Satisfactory correlations between predicted extrusion variables (T, SME) and biopolymer transformation suggested that products with target structure can be obtained using simulation to tune extrusion conditions.

Refolding and characterization of a diabody against Pfs25, a vaccine candidate of Plasmodium falciparum

Pfs25, a vaccine candidate, expressed on the surface of the malarial parasite, plays an important role in the development of Plasmodium falciparum. 1269, a monoclonal antibody targeting the epidermal growth factor-like domain 1 and epidermal growth factor-like domain 3 of Pfs25, blocks the transmission of parasites in mosquitoes. In this study, we refolded 1269-Db, a dimeric antibody fragment referred as diabody, designed from 1269, with a yield of 3 mg/litre of bacterial culture. Structural integrity of the protein was validated with thermal stability, disulphide bond analysis and glutaraldehyde crosslinking experiments. To evaluate the functionality of 1269-Db, recombinant monomeric MBP-Pfs25 was produced from bacteria. Qualitative binding assays demonstrated that 1269-Db recognized the epitopes on Pfs25 in its native, but not the denatured state. An apparent KD of 2.6 nM was determined for 1269-Db with monomeric MBP-Pfs25, using isothermal titration calorimetry. 1269-Db recognized the periphery of zygotes/ookinetes, demonstrating recognition of Pfs25, expressed on the surface of the parasite. As the established refolding method resulted in a functional diabody, the optimized method pipeline for 1269-Db can potentially facilitate engineering of antibody fragments with desired properties.

Effects of innovative gelation and modified tapioca starches on the physicochemical properties of surimi gel during frozen storage

SummaryThis study presented a comparative evaluation of the effects of 5% tapioca starch, acetylated distarch phosphate and hydroxypropyl distarch phosphate (HPDSP) on the changes in surimi gel quality after the traditional gelation procedure (suwari‐cooking‐freezing) and a novel gelation procedure (suwari‐freezing‐cooking, SFC). Overall, subjected to the SFC procedure, lower hydrophobic interactions, lower total sulphydryl group content, higher disulphide bond content and a more orderly protein conformation was observed in the HPDSP‐surimi gel. These changes resulted in smaller drip loss, stabler breaking force and deformation, and less structural damage during frozen storage. Since the strongest hydrophilicity of HPDSP and the lowest gelatinisation temperature (63.15 °C), when it was reheated and then gelatinized, it interacted more closely with the protein. These findings indicated that subjected to the SFC procedure, the addition of 5% HPDSP effectively decreased the changes in the quality of the surimi gel.

Thiolated 2-Methyl-β-Cyclodextrin as a Mucoadhesive Excipient for Poorly Soluble Drugs: Synthesis and Characterization.

Thiolated cyclodextrins are structurally simple mucoadhesive macromolecules, which are able to host drugs and increase their apparent water solubility, as well as interact with the mucus layer prolonging drug residence time on the site of absorption. The aim of this study was to synthesize through green microwave-assisted process a freely soluble thiolated 2-methyl-β-cyclodextrin (MβCD-SH). Its inclusion complex properties with dexamethasone (Dex), a poor water soluble drug, and mucoadhesive characteristics were also determined. The product was deeply characterized through NMR spectroscopy (2D COSY, 2D HSQC, 1D/2D TOCSY, and 1D ROESY), showing a thiolation degree of 67%, a selective thiolation on the C6 residues and a monomeric structure. The association constant of MβCD and MβCD-SH with Dex resulted in 2514.3 ± 32.3 M−1 and 2147.0 ± 69.3 M−1, respectively, indicating that both CDs were able to host the drug. Microrheological analysis of mucin in the presence of MBCD-SH showed an increase of complex viscosity, G′ and G″, due to disulphide bond formation. The cytotoxicity screening on fibroblast BALB/3T3 clone A31 cells indicated an IC50 of 27.7 mg/mL and 30.0 mg/mL, for MβCD and MβCD-SH, respectively. Finally, MβCD-SH was able to self-assemble in water into nanometric structures, both in the presence and absence of the complexed drug.

In silico analysis of the C-terminal domain of big defensin from the Pacific oyster

Defensins are antimicrobial peptides consisting of intramolecular disulphide bonds in a complex folded arrangement of two or three antiparallel β-sheets with or without an α-helical structure. They are produced by a vast range of organisms being constitutively expressed or induced in various tissues against different stimuli like infection, injury or other inflammatory factors. Two classes of invertebrate defensin exist, namely CS-αβ and big defensin, the latter being predominantly present in molluscs. Intriguingly, an invertebrate big defensin gene has been hypothesized as the most probable ancestor of vertebrate β-defensins. Here, conserved residues were identified for both big defensin and β-defensin. In silico mutation on conserved amino acid positions of the β-defensin-like domain of big defensin from Crassostrea gigas was carried out to understand the effects of mutation on the structure and function of the protein. R64A and E71A have been identified as deleterious as well as destabilizing for the protein. Changes in amino acid network and aggregation propensity were also observed upon mutating these two charged residues. 100 ns molecular dynamics simulations of wild-type, R64A and E71A structures revealed significant conformational changes in the case of mutants. Furthermore, molecular docking highlighted the significance of R64 in ligand interaction. In conclusion, these results provide the first in-depth understanding of the structural and functional importance imparted by two conserved charged residues in the C-terminal region of big defensin. It also enhances the existing knowledge about this antimicrobial peptide for application in therapeutics and other aspects of protein engineering. Communicated by Ramaswamy H. Sarma

In silico analyses of leptin and leptin receptor of spotted snakehead Channa punctata

The present study, in addition to molecular characterization of leptin (lepa) and its receptor (lepr) of spotted snakehead Channa punctata, is focussed on physicochemical, structural, evolutionary and selection pressure analyses which are poorly elucidated in teleosts in spite of that existence of these genes is well reported in several fish species. The putative full-length Lep and Lepr of C. punctata showed conserved structural and functional domains, especially the residues responsible for structural integrity and signal transduction. Conversely, residues predicted essential for Lep-Lepr interaction displayed divergence between teleosts and tetrapods. Impact of substitutions/deletions predicted using protein variation effect analyser tool highlighted species specificity in ligand-receptor interaction. Physicochemical properties of ligand and receptor predicted for the first time in vertebrates revealed high aliphatic and instability indices for both Lepa and Lepr, indicating thermostability of proteins but their instability under ex vivo conditions. Positive grand average of hydropathy score of Lepa suggests its hydrophobic nature conjecturing existence of leptin binding proteins in C. punctata. In addition to disulphide bonding, a novel posttranslational modification (S-126 phosphorylation) was predicted in Lepa of C. punctata. In Lepr, disulphide bond formation and N-linked glycosylation near WSXWS motif in ECD, and phosphorylation at tyrosine residues in ICD were predicted. Leptin and its receptor sequence of C. punctata cladded with its homolog from C. striata and C. argus of order Anabantiformes. Leptin system of Anabantiformes was phylogenetically closer to that of Pleuronectiformes, Scombriformes and Perciformes. Selection pressure analysis showed higher incidence of negative selection in teleostean leptin genes indicating limited adaptation in their structure and function. However, evidence of pervasive and episodic diversifying selection laid a foundation of co-evolution of Lepa and Lepr in teleosts.

Current trends in biopharmaceuticals production in Escherichia coli.

Since the manufacture of the first biotech product for a fledgling biopharmaceutical industry in 1982, Escherichia coli, has played an important role in the industrial production of recombinant proteins. It is now 40years since the introduction of Humulin® for the treatment of diabetes. E. coli remains an important production host, its use as a cell factory is well established and it has become the most popular expression platform particularly for non-glycosylated therapeutic proteins. A number of significant inherent obstacles in the use of prokaryotic expression systems to produce biologics has always restricted production. These include codon usage, the absence of post-translational modifications and proteolytic processing at the cell envelope. In this review, we reflect on the contribution that this model organism has made in the production of new biotech products for human medicine. This will include new advancements in the E. coli expression system to meet the biotechnology industry requirements, such as novel engineered strains to glycosylate heterologous proteins, add disulphide bonds and express complex proteins. The biopharmaceutical market is growing rapidly, with two production systems competing for market dominance: mammalian cells and microorganisms. In the past 10years, with increased growth of antibody-based therapies, mammalian hosts particularly CHO cells have dominated. However, with new antibody like scaffolds and mimetics emerging as future proteins of interest, E. coli has again the opportunity to be the selected as the production system of choice.

Identification of AL proteins from 10 λ-AL amyloidosis patients by mass spectrometry extracted from abdominal fat and heart tissue

Background Systemic AL amyloidosis arises from the misfolding of patient-specific immunoglobulin light chains (LCs). Potential drivers of LC amyloid formation are mutational changes and post-translational modifications (PTMs). However, little information is available on the exact primary structure of the AL proteins and their precursor LCs. Objective We analyse the exact primary structure of AL proteins extracted from 10 λ AL amyloidosis patients and their corresponding precursor LCs. Materials and Methods By cDNA sequencing of the precursor LC genes in combination with mass spectrometry of the AL proteins, the exact primary structure and PTMs were determined. This information was used to analyse their biochemical properties. Results All AL proteins comprise the VL and a small part of the CL with a common C-terminal truncation region. While all AL proteins retain the conserved native disulphide bond of the VL, we found no evidence for presence of other common PTMs. The analysis of the biochemical properties revealed that the isoelectric point of the VL is significantly increased due to introduced mutations. Conclusion Our data imply that mutational changes influence the surface charge properties of the VL and that common proteolytic processes are involved in the generation of the cleavage sites of AL proteins.

P-045 Sperm chromatin structure resilience is related to fertilization and blastocyst rates in ICSI cycles involving both oocyte and sperm donors

Abstract Study question Does the structural maintenance of sperm chromatin have an implication in assisted reproduction outcomes? Summary answer Sperm deprotamination is related to fertilizing capacity. Also, the time needed for the reduction of protamine disulphide bridges is a parameter underlying early embryo development. What is known already How male factor affects embryo development has been a controversial topic for decades, with opposite results. A meta-analysis recently showed that whereas sperm genotoxic damage has an impact on IVF success, its repercussion on ICSI remains unclear. Revealing the factors that may predict embryo development is much warranted to understand the sperm contribution in the ICSI era. Sperm DNA is preferentially protamine-condensed and necessarily needs to be switched to histone-condensed chromatin upon fertilization. This study aims to determine the degree of protamination and the disulphide bond integrity to provide new insights into the influence of chromatin resilience on embryo development. Study design, size, duration The study setting included a public university and a private sperm bank. The design was a blinded retrospective study, including samples from 51 sperm donors that were used for a total of 55 cycles conducted with donated oocytes in the fertility clinic. Sperm quality parameters, including those focused on chromatin structure, were evaluated after 0 min and 240 min post-thawing and compared to embryo outcomes. Participants/materials, setting, methods Sperm samples from donors selected by the germplasm bank were collected and used to fertilize oocytes in double-donation cycles (oocyte donation) with standard intracytoplasmic sperm injection. Cryopreserved samples were used to assess sperm viability (SYBR14/PI), incidence of thiol reduction (Dibromobimane, DBB) and chromatin deprotamination (Chromomycin A3, CMA3) through flow cytometry, at 0 min and 240 min after thawing. Positive controls after incubation with Triton-X100, DTT and DTT+NaCl were used to establish the flow cytometry thresholds. Main results and the role of chance In fresh semen samples, sperm concentration was 34.12±18.13 sperm/mL and progressive motility was 37.49±13.22% (mean±standard deviation). At 0 min and 240 min post-thawing, sperm viability was 24.80±12.12% and 18.65±10.37%, respectively, and the death rate was -1.54±1.24%/hour. Percentages of sperm with reduced thiols were 17.37±11.26% and 11.53±9.16% after 0 min and 240 min post-thawing, the reduction rate being -1.46±2.31%. Percentages of sperm with chromatin deprotamination were 21.12±9.03% and 35.34±14.10% after 0 min and 240 min post-thawing, and the deprotamination rate was 14.22±11.90%/hour. ICSI was conducted with an average of 11.86±7.89 oocytes per sperm sample, of which 8.78±5.95 were fertilized; the mean fertilization rate was 75.77±17.61%. For each sperm sample, 5.98±4.93 blastocysts at day 5 were obtained, leading to an average blastocyst rate of 65.17±22.22%. Interestingly, the deprotamination status at 0 min post-thaw was correlated to fertilization rates (Rs= -0.388; P = 0.005) and the decreasing rate of sperm with reduced thiols was correlated to blastocyst rate (Rs= -0.351; P = 0.012). Reduced thiols and deprotamination were also correlated with each other (Rs= -0.320; P = 0.022). Limitations, reasons for caution The main limitation of the present study was the sample size. More donors are thus needed in order for the promising results found in this study to be confirmed. Wider implications of the findings The research conducted herein has evidenced that the resilience of the sperm chromatin structure is related to embryo development outcomes from ICSI cycles conducted with oocyte donors. Trial registration number N/A

Cysteine Oxidation Promotes Dimerization/Oligomerization of Circadian Protein Period 2.

The molecular circadian clock is based on a transcriptional/translational feedback loop in which the stability and half-life of circadian proteins is of importance. Cysteine residues of proteins are subject to several redox reactions leading to S-thiolation and disulfide bond formation, altering protein stability and function. In this work, the ability of the circadian protein period 2 (PER2) to undergo oxidation of cysteine thiols was investigated in HEK-293T cells. PER2 includes accessible cysteines susceptible to oxidation by nitroso cysteine (CysNO), altering its stability by decreasing its monomer form and subsequently increasing PER2 homodimers and multimers. These changes were reversed by treatment with 2-mercaptoethanol and partially mimicked by hydrogen peroxide. These results suggest that cysteine oxidation can prompt PER2 homodimer and multimer formation in vitro, likely by S-nitrosation and disulphide bond formation. These kinds of post-translational modifications of PER2 could be part of the redox regulation of the molecular circadian clock.

Analysis of protein denaturation, and chemical visualisation, of frozen grass carp surimi containing soluble soybean polysaccharides

SummaryThe mechanism of protein denaturation of frozen surimi enriched with soluble soybean polysaccharides (SSPS) was investigated. Near‐infrared (NIR) and hyperspectral imaging (HSI) technology were used to predict protein denaturation. The fresh grass carp surimi was divided into four groups with SSPS additions of 0%, 1%, 3% and 5%, respectively, which were frozen and stored at −18 °C. Samples were examined after 0, 1, 2, 4 and 8 weeks, for salt‐soluble protein content, total SH, NIR hyperspectral image and Raman spectrum features. The results showed that addition of SSPS decelerated protein degradation. After 8 weeks of storage, the 5% SSPS addition maintained the highest salt‐soluble protein content, while for total SH, 3% and 5% SSPS addition had similar effect. Raman spectra illustrated that SSPS had the ability of maintaining an α‐helix content, as well as decreasing the exposure of polar groups, and reducing the oxidation of SH group into disulphide bonds. NIR spectra showed that the overall reflectance of frozen surimi increased with the increase in SSPS. Two partial least squares regression (PLSR) models were established after pretreatment of HSI spectral statistics and selection of characteristic wavelengths. The distribution maps were finally generated based on the simplified PLSR models.

Redox-responsive nano-micelles containing trisulfide bonds to enhance photodynamic efficacy of zinc naphthalocyanine

A novel amphiphilic polymer with the trisulfide bond was developed. The nano-micelles could be formed through the self-assembling of this polymer with hydrophobic zinc naphthalocyanine, a NIR-absorbing photosensitizer. The micelles showed unique rods, high drug loading, high stability, high sensitivity to GSH, and excellent biosafety. The singlet oxygen production quantum yield of micelles was obviously higher than that of free photosensitizers and micelles containing disulphide bonds in the simulated tumor environment. The phototoxicity of micelles in cancer cells was significant. The redox response drug-loaded micelle was promising to enhance the photodynamic efficacy of zinc naphthalocyanine in cancer cells.

A β-defensin isoform from the Flathead grey mullet, Mugil cephalus (Linnaeus 1758): Structural and functional characterisation in silico

Defensins are an important class of AMPs, rich in cysteine with antibacterial, antifungal and antiviral functions. The present study identified and functionally characterised a β-defensin gene from Grey mullet, Mugil cephalus, termed as Mc-Def. A 192 base pair complementary DNA with an open reading frame encoding 63 amino acids comprising 20 amino acid signal and 43 amino acid mature peptide region was obtained. Mc-Def has a net charge of 3.9 and a molecular weight of 7.27 kDa with an isoelectric point of 8.88. It possessed six cysteines forming 3 disulphide bonds between C1–C5, C2–C4 and C3–C6. Three dimensional structure identified a short N-terminal α helix followed by β sheet strands stabilized by disulfide bonds. The mRNA structure was predicted by Biomodel server with significant MEF score. Multiple sequence alignment and phylogeny revealed the sequence similarity of Mc-Def with fish β-defensin 2 type of AMPs. Molecular diversity and evolution of β-defensin provided immunity to host system against various infections. In silico analysis identified its potential as antibacterial, antifungal, anticancer, antioxidant and non-hemolytic peptide activity. These intriguing results firmly suggest that the Mc-Def can be used as a potent drug molecule in fish health management.

Trichothiodystrophy hair shafts display distinct ultrastructural features.

Hair shafts from three trichothiodystrophy (TTD) patients with mutations in the ERCC2 (XPD) gene were examined by transmission electron microscopy. TTD is a rare, recessive disorder with mutations in several genes in the DNA repair/transcription pathway, including ERCC2. Unlike previous studies, the hair shafts were examined after relaxation of their structure by partial disulphide bond reduction in the presence of sodium dodecyl sulphate, permitting improved visualization. Compared with hair shafts of normal phenotype, TTD cuticle cells displayed aberrant marginal bands and exocuticle layers. Clusters of cells stained differently (light versus dark) in the cortex of aberrant shafts, and the keratin macrofibrils appeared much shorter in the cytoplasm. Considerable heterogeneity in these properties was evident among samples and even along the length of single hair shafts. The results are consistent with not only a paucity of high sulphur components, such as keratin-associated proteins, but also a profound imbalance in protein content and organization.