Patients with Marfan syndrome have a constellation of clinical features and a heterogeneous phenotype. The purpose of this study is to report a 47-year-old male patient with an unusual variant in the FBN1 gene causing Marfan syndrome. The patient with musculoskeletal, cardiovascular, and ocular findings compatible with Marfan syndrome had an unusual pathogenic mutation on the FBN1 gene. The patient was examined by at least one of the authors (NJI). The patient\'s clinical findings were compatible with Marfan syndrome. Our patient had a unique mutation in the FBN1 gene (c.8054A>G p.His2685Arg) located on exon 65. Next-generation sequencing was done using the Invitae panel. This variant was categorized as one of uncertain significance. This patient\'s variant on the FBN1 gene leading to the syndrome has scant data associated with it and this is the first time it is reported from Puerto Rico.

Earthworms are affected by physical stress, like injury, and by exposure to xenobiotics, such as the toxic metal cadmium (Cd), which enters the environment mainly through industry and agriculture. The stress response to the single and the combination of both stressors was examined in regenerative and unharmed tissue of Lumbricus terrestris to reveal if the stress response to a natural insult like injury (amputation) interferes with Cd detoxification mechanisms. We characterized the roles of metallothionein 1 (MT1) and MT2 isoforms, heat shock protein 70 as well as immune biomarkers such as the toll-like receptors (TLR) single cysteine cluster TLR and multiple cysteine cluster TLR. The role of the activated transcription factors (ATFs) ATF2, ATF7, and the cAMP responsive element binding protein as putative regulatory intersection as well as a stress-dependent change of the essential trace elements zinc and calcium was analyzed. Phosphorylated AMP activated protein kinase, the cellular energy sensor, was measured to explore the energy demand, while the energy status was determined by detecting carbohydrate and protein levels. Taken together, we were able to show that injury rather than Cd is the driving force that separates the four treatment groups - Control, Cd exposure, Injury, Cd exposure and injury. Interestingly, we found that gene expression differed regarding the tissue section that was analyzed and we hypothesize that this is due to the migration of coelomocytes, earthworm immune cells, that take over a key role in protecting the organism from a variety of environmental challenges. Surprisingly, we discovered a role for MT1 in the response to multiple stressors and an isoform-specific function for the two newly characterized TLRs. In conclusion, we gathered novel information on the relation of innate immunity, wound healing, and Cd detoxification mechanisms in earthworms.

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumours with increasing incidence, and oral leukoplakia (OLK) has a strong tendency to undergo malignant transformation. The oral microbiota may influence oral cancer progression, but the salivary bacterial composition and functional changes in OSCC and OLK have not been comprehensively elucidated. Therefore, we compared salivary bacteria in OLK and OSCC patients with healthy controls (HC).
Metagenomic sequencing was used to compare the bacterial composition and functional changes of 18 OSCC patients, 21 OLK patients and 21 HC. Spearman correlation was used to identify possible associations between functions and bacteria.
Gemella was the most differentially enriched genus in OSCC. At the species level, Streptococcus sp. NPS 308, Streptococcus agalactiae, Gemella haemolysans and Gemella morbillorum were slightly increased in OLK and OSCC. Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that OSCC was mainly associated with metabolism functions, including lipid metabolism, carbohydrate metabolism and glycan biosynthesis and metabolism. The synthesis and degradation of ketone bodies, cysteine and methionine metabolism and glycerolipid metabolism differed significantly among the three groups, and were highest in OSCC and lowest in HC. And G. haemolysans was significantly associated with these selected metabolic pathways.
Metagenomic analysis revealed significant differences in the salivary microbiota among OSCC, OLK and HC. Thus, salivary microbiota composition and functional changes may be associated with OSCC progression. Metabolism of nonessential amino acids such as cysteine and methionine in bacteria may play an important role in oral oncogenesis, and more studies of the mechanism between metabolisms of bacteria and oral oncogenesis are needed in the future.

In-vitro protein refolding is one of the key rate-limiting unit operations in manufacturing of fusion proteins such as peptibodies expressed using E. coli. Dilution-assisted refolding is the most commonly used industrial practice to achieve the soluble, native functional form of the recombinant protein from the inclusion bodies. This study is focused on developing a chromatography-assisted in-vitro refolding platform to produce the biologically active, native form of recombinant peptibody. Recombinant Romiplostim was selected as a model protein for the study. A plug flow tubular reactor was connected in series with capture step affinity chromatography to achieve simultaneous in-vitro refolding and capture step purification of recombinant Romiplostim. Effect of various critical process parameters like fold dilution, temperature, residence time, and Cysteine: DTT ratio was studied using a central composite based design of experiment strategy to achieve a maximum refolding yield of selected peptibody. Under optimum refolding conditions, the maximum refolding yield of 57.0 ± 1.5 % and a purity of over 79.73 ± 3.4 % were achieved at 25-fold dilution, 15 °C temperature, 6 h residence time with 6 mM and 10 mM of cysteine and DTT, respectively. The formation of native peptibody structure was examined using various orthogonal analytical tools to study the protein\'s primary, secondary, and tertiary structure. The amino acid sequence for the disulfide-linked peptide was mapped using collision-induced dissociation (CID) to confirm the formation of interchain disulfide bonds between Cys7-Cys7 and Cys10-Cys10 similarly for intra-chain disulfide bonds between Cys42-Cys102, and Cys148-Cys206. The developed protocol here is a valuable tool to identify high-yield scalable refolding conditions for multi-domain proteins involving inter-domain disulfide bonds.

Considering the biological and ecological importance of Cu-thiol interactions and the discrepancies in previous research, this study focuses on Cu interactions with biologically and ecologically relevant thiols: glutathione (GSH), L-cysteine (L-cys), 3-mercaptopropionic acid (MPA), and thioacetic acid (TAA) in aqueous solution. The addition of Cu(II) to a thiol-containing solution led to a rapid reduction of Cu(II) and the formation of a Cu(I)-thiol complex. The mechanism of Cu(II) reduction and Cu(I) complex formation as well as the kinetics of Cu(I) oxidation strongly depend on the structural properties of the individual thiols investigated. The reducing power of the investigated thiols can be summarized as follows: L-cys ≅ GSH > MPA > TAA. The reaction order, with respect to Cu(I) oxidation, also changes over the time of the reaction course. The deviation of the reaction kinetics from the first order with respect to Cu(I) in the later stages of the reaction course can be attributed to a Fenton-like reaction occurring under low thiol concentration conditions. At high Cu:thiol ratios, in the case of GSH, L-cys, and MPA, the early stage of the reaction course is characterized by high Cu(I) stability, most likely as a result of Cu(I) complexation by the thiols present in excess in the reaction mixture.

Broccoli serves as a functional food because it can accumulate selenium (Se), well-known bioactive amino-acid-derived secondary metabolites, and polyphenols. The chemical and physical properties of Se are very similar to those of sulfur (S), and competition between sulfate and selenate for uptake and assimilation has been demonstrated. Towards an efficient agronomic fortification of broccoli florets, the working questions were whether we could overcome this competition by exogenously applying the S-containing amino acids cysteine (Cys) or/and methionine (Met), or/and the precursors of Glucosinolate (GSL) types along with Se application. Broccoli plants were cultivated in a greenhouse and at the beginning of floret growth, we exogenously applied sodium selenate in the concentration gradient of 0, 0.2, 1.5, and 3.0 mM to study the impact of increased Se concentration on the organic S (Sorg) content of the floret. The Se concentration of 0.2 mM (Se0.2) was coupled with the application of Cys, Met, their combination, or a mixture of phenylalanine, tryptophane, and Met. The application took place through fertigation or foliar application (FA) by adding isodecyl alcohol ethoxylate (IAE) or a silicon ethoxylate (SiE) surfactant. Fresh biomass, dry mass, and Se accumulation in florets were evaluated, along with their contents of Sorg, chlorophylls (Chl), carotenoids (Car), glucoraphanin (GlRa), glucobrassicin (GlBra), glucoiberin (GlIb), and polyphenols (PPs), for the biofortification efficiency of the three application modes. From the studied selenium concentration gradient, the foliar application of 0.2 mM Se using silicon ethoxylate (SiE) as a surfactant provided the lowest commercially acceptable Se content in florets (239 μg or 0.3 μmol g-1 DM); it reduced Sorg (-45%), GlIb (-31%), and GlBr (-27%); and it increased Car (21%) and GlRa (27%). Coupled with amino acids, 0.2 mM Se provided commercially acceptable Se contents per floret only via foliar application. From the studied combinations, that of Met,Se0.2/FA,IAE provided the lowest Se content per floret (183 μg or 0.2 μmol g-1 DM) and increased Sorg (35%), Car (45%), and total Chl (27%), with no effect on PPs or GSLs. Cys,Met,Se0.2/FA,IAE and amino acid mix,Se0.2/FA,IAE increased Sorg content, too, by 36% and 16%, respectively. Thus, the foliar application with the IAE surfactant was able to increase Sorg, and methionine was the amino acid in common in these treatments, with varying positive effects on carotenoids and chlorophylls. Only the Cys,Met,Se0.2 combination presented positive effects on GSLs, especially GlRa, but it reduced the fresh mass of the floret. The foliar application with SiE as a surfactant failed to positively affect the organic S content. However, in all studied combinations of Se 0.2 mM with amino acids, the Se content per floret was commercially acceptable, the yield was not affected, the content of GSLs was increased (especially that of GlRa and GlIb), and PPs were not affected. The content of GlBr decreased except for the treatment with methionine (Met,Se0.2/FA,SiE) where GlBr remained unaffected. Hence, the combination of Se with the used amino acids and surfactants can provide enhanced biofortification efficiency in broccoli by providing florets as functional foods with enhanced functional properties.

The pyruvate oxidases from Escherichia coli (EcPOX) and Lactobacillus plantarum (LpPOX) are both thiamin-dependent flavoenzymes. Their sequence and structure are closely related, and they catalyse similar reactions-but they differ in their activity pattern: LpPOX is always highly active, EcPOX only when activated by lipids or limited proteolysis, both involving the protein\'s C-terminal 23 residues (the \'α-peptide\'). Here, we relate the redox-induced infrared (IR) difference spectrum of EcPOX to its unusual activation mechanism. The IR difference spectrum of EcPOX is marked by contributions from the protein backbone, reflecting major conformational changes. A rare sulfhydryl (-SH) difference signal indicates changes in the vicinity of cysteines. We could pin the Cys-SH difference signal to Cys88 and Cys494, both being remote from the moving α-peptide and the redox-active flavin cofactor. Yet, when the α-peptide is proteolytically removed, the Cys-SH difference signal disappears, together with several difference signals in the amide range. The remaining IR signature of the permanently activated EcPOXΔ23 is strikingly similar to the simpler signature of LpPOX. The loss of the α-peptide \'transforms\' the catalytically complex EcPOX into the catalytically \'simpler\' LpPOX.

Marfan syndrome, an autosomal dominant disorder of connective tissue, is primarily caused by mutations in the fibrillin-1 (FBN1) gene, which encodes the protein fibrillin-1. The protein is composed of epidermal growth factor-like (EGF-like) domains, transforming growth factor beta-binding protein-like (TB) domains, and hybrid (Hyb) domains and is an important component of elastin-related microfibrils in elastic fiber tissue. In this study, we report a cysteine to tyrosine substitution in two different domains of fibrillin-1, both of which cause Marfan syndrome with ocular abnormalities, in two families. Using protease degradation and liquid chromatography-tandem mass spectrometry analyses, we explored the different effects of substitution of cysteine by tyrosine in an EGF-like and a calcium-binding (cb) EGF-like domain on protein stability. The results showed that cysteine mutations in the EGF domain are more likely to result in altered proteolytic sensitivity and thermostability than those in the cbEGF domain. Furthermore, cysteine mutations can lead to new enzymatic sites exposure or hidden canonical cleavage sites. These results indicate the differential clinical phenotypes and molecular pathogenesis of Marfan syndrome caused by cysteine mutations in different fibrillin-1 domains. These results strongly suggest that failure to form disulfide bonds and abnormal proteolysis of fibrillin-1 caused by cysteine mutations may be an important factor underlying the pathogenesis of diseases caused by fibrillin-1 mutations, such as Marfan syndrome.

BACKGROUND: Standard treatment of epileptic seizures involves the use of antiepileptic drugs (AEDs). Both AEDs themselves and treatment duration may influence the levels of biochemical parameters, e.g., lipids or homocysteine (HCys), that may increase the risk of cardiovascular diseases. The aim of the present study was to compare the levels of lipid parameters, as well as the concentrations of selected aminothiols (i.e., HCys, cysteine, and glutathione) between epileptic children treated with multiple AEDs and children without epilepsy.
METHODS: In the study, 21 children with epilepsy treated with two or more AEDs for at least 6 months (8 girls and 13 boys, mean age 7.03 ± 4.51) and 23 children without epilepsy (7 girls and 16 boys, mean age 7.54 ± 3.90) were prospectively analyzed. Lipid parameters, i.e., total cholesterol (TC), triglycerides (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL), and levels of selected aminothiols were determined in the blood serum.
RESULTS: No differences in the mean levels of lipid parameters and in the mean values of lipid ratios (TC/HDL, TG/HDL, LDL/HDL) were observed between the total groups as well as in the sex subgroups. HCys and cysteine levels did not differ between the patients and controls. We observed significantly lower levels of glutathione in children with epilepsy than in children without epilepsy (1.49 ± 0.35 µmol/L vs. 2.39 ± 1.17 µmol/L, respectively) (p < 0.001). Glutathione level was also lower in boys with epilepsy than in boys without epilepsy (p = 0.007). Similarly, epileptic girls had statistically decreased levels of glutathione when compared to girls without epilepsy (p = 0.006).
CONCLUSIONS: A lower level of glutathione is observed in pediatric patients with epilepsy treated with two or more AEDs for at least 6 months. This indicates the oxidative stress of the patients treated with AEDs, which in turn may affect their well-being, and in the case of chronic occurrence resulting from long-term treatment, also on the function of the liver and the condition of the cardiovascular system.

Oral halitosis is characterized by a foul, unpleasant breath that emanates from the oral cavity due to local or systemic conditions. Approximately 90% of offensive odors are caused by volatile sulfur compounds (VSCs). L-cysteine, used as a test solution to control bad breath, induces the formation of VSCs and serves as a preliminary rinse. The study aim was to investigate the effectiveness of L-cysteine solution in differentiating the origin of oral halitosis using a gas chromatography apparatus. Methods: In total, 37 patients with an average age of 49.56 years were evaluated and divided into two groups: halimetry before the use of L-cysteine (n= 37) and halimetry after the use of L-cysteine (n= 37). Patients over 18 years of age, without severe systemic health impairment or infectious/contagious diseases, and who did not use medicines that influenced their breath were included. Halimetry was performed using the OralCroma™ device. In the halimetry before the use of L-cysteine group, 5.40%, 5.40%, and 64.86% of the patients had high levels of sulfide, methyl mercaptan, and dimethyl sulfide, respectively. After the use of L-cysteine, 48.64%, 8.10%, and 37.84% of the patients had high levels of sulfide, methyl mercaptan, and dimethyl sulfide. In this study, L-cysteine proved to be important for the assessment of oral halitosis and effective in differentiating the origin of oral halitosis; therefore, this compound could be used for the differential diagnosis of oral halitosis origin using the OralChroma™ device.