Proteins are well-known to undergo a variety of post-translational modifications (PTMs). One such PTM is citrullination, an arginine modification that is catalyzed by a group of hydrolases called protein arginine deiminases (PADs). Hundreds of proteins are known to be citrullinated and hypercitrullination is associated with autoimmune diseases including rheumatoid arthritis (RA), lupus, ulcerative colitis (UC), Alzheimer's disease, multiple sclerosis (MS), and certain cancers. In this Account, we summarize our efforts to understand the structure and mechanism of the PADs and to develop small molecule chemical probes of protein citrullination. PAD activity is highly regulated by calcium. Structural studies with PAD2 revealed that calcium-binding occurs in a stepwise fashion and induces a series of dramatic conformational changes to form a catalytically competent active site. These studies also identified the presence of a calcium-switch that controls the overall calcium-dependence and a gatekeeper residue that shields the active site in the absence of calcium. Using biochemical and site-directed mutagenesis studies, we identified the key residues (two aspartates, a cysteine, and a histidine) responsible for catalysis and proposed a general mechanism of citrullination. Although all PADs follow this mechanism, substrate binding to the thiolate or thiol form of the enzyme varies for different isozymes. Substrate-specificity studies revealed that PADs 1-4 prefer peptidyl-arginine over free arginine and certain citrullination sites on a peptide substrate. Using high-throughput screening and activity-based protein profiling (ABPP), we identified several reversible (streptomycin, minocycline, and chlorotetracycline) and irreversible (streptonigrin, NSC 95397) PAD-inhibitors. Screening of a DNA-encoded library and lead-optimization led to the development of GSK199 and GSK484 as highly potent PAD4-selective inhibitors. Furthermore, use of an electrophilic, cysteine-targeted haloacetamidine warhead to mimic the guanidinium group in arginine afforded several mechanism-based pan-PAD-inhibitors including Cl-amidine and BB-Cl-amidine. These compounds are highly efficacious in various animal models, including those mimicking RA, UC, and lupus. Structure-activity relationships identified numerous covalent PAD-inhibitors with different bioavailability, in vivo stability, and isozyme-selectivity (PAD1-selective: D-Cl-amidine; PAD2-selective: compounds 16-20; PAD3-selective: Cl4-amidine; and PAD4-selective: TDFA). Finally, this Account describes the development of PAD-targeted and citrulline-specific chemical probes. While PAD-targeted probes were utilized for identifying off-targets and developing high-throughput inhibitor screening platforms, citrulline-specific probes enabled the proteomic identification of novel diagnostic biomarkers of hypercitrullination-related autoimmune diseases.
Simple Summary Modern dairy cows have elevated internal heat loads caused by high milk production, and the effects of accumulating incremental heat are exacerbated when temperature and humidity increases in the surroundings. To shed this additional heat, cows initiate a variety of adaptive mechanisms including increased respiration rate, panting, sweating, reduced milk yield, vasodilatation, and decreased reproductive performance. Hormonal changes based on reciprocal alterations to the energetic metabolism are particularly accountable for reduced efficiency of the dairy production under the heat stress. As animals experience negative energy balance; glucose, which is also a precursor of milk lactose, becomes the preferential energy fuel. In the absence of proper mitigations, heat stress possesses potential risk of economic losses to dairy sector. Besides physical measures for the timely prediction of the actual heat stress coupled with its proper amelioration, nutritional mitigation strategies should target modulating energetic metabolism and rumen environment. Abstract Higher milk yield and prolificacy of the modern dairy cattle requires high metabolism activities to support them. It causes high heat production by the body, which coupled with increasing environmental temperatures results in heat stress (HS). Production, health, and welfare of modern cattle are severely jeopardized due to their low adaptability to hot conditions. Animal activates a variety of physiological, endocrine, and behavioral mechanisms to cope with HS. Traditionally, decreased feed intake is considered as the major factor towards negative energy balance (NEBAL) leading to a decline in milk production. However, reciprocal changes related to insulin; glucose metabolism; failure of adipose mobilization; and skeletal muscle metabolism have appeared to be the major culprits behind HS specific NEBAL. There exists high insulin activity and glucose become preferential energy fuel. Physiological biochemistry of the heat stressed cows is characterized by low-fat reserves derived NEFA (non-esterified fatty acids) response, despite high energy demands. Besides these, physiological and gut-associated changes and poor feeding practices can further compromise the welfare and production of the heat-stressed cows. Better understanding of HS specific nutritional physiology and metabolic biochemistry of the dairy cattle will primarily help to devise practical interventions in this context. Proper assessment of the HS in cattle and thereby applying relevant cooling measures at dairy seems to be the basic mitigation approach. Score of the nutritional strategies be applied in the eve of HS should target supporting physiological responses of abatement and fulfilling the deficiencies possessed, such as water and minerals. Second line of abatement constitutes proper feeding, which could augment metabolic activities and synergizes energy support. The third line of supplemental supports should be directed towards modulating the metabolic (propionates, thiazolidinediones, dietary buffers, probiotics, and fermentates) and antioxidant responses (vitamins). Comprehensive understanding of the energetic metabolism dynamics under the impact of incremental heat load and complete outlook of pros and cons of the dietary ameliorating substances together with the discovery of the newer relevant supplementations constitutes the future avenues in this context.
Aline Padilha de Fraga, Vinicius Proença da Silveira, Patrícia de Freitas Salla
et al.
<i>Leishmania infantum</i> is a hemopathogen of importance for the health of domestic dogs (<i>Canis lupus familiaris</i>), causing canine leishmaniasis (CanL), and it is also the etiological agent of human visceral leishmaniasis (HVL). This parasite was not reported in southern Brazil until the early 2000s, but CanL and HVL were increasingly reported in the last 15 years, mainly in cities bordering Argentina. The present study aimed to detect <i>L. infantum</i> in domestic dogs and to determine the main clinical manifestations in infected animals from Uruguaiana, a city with a high incidence of CanL. Fifty-one dogs suspected of having CanL in the urban perimeter of the city were clinically examined by veterinarians and investigated for the occurrence of <i>L. infantum</i> with two immunoassays (rapid chromatography test and ELISA) and real-time PCR (polymerase chain reaction). Clinical signs were compared in positive and negative <i>L. infantum</i> animals. A total of 31 dogs (60.8%) were infected with <i>L. infantum</i>. The main clinical manifestations associated with CanL dogs were onychogryphosis and peeling (<i>p</i> < 0.05). <i>L. infantum</i> was frequently detected in urban dogs from Uruguaiana, highlighting the concerning situation regarding health in this city. The occurrence of some clinical signs (onychogryphosis/peeling) could help to detect CanL more frequently in the canine population.
Alicja Hinz, Joanna Lewandowska-Łańcucka, Ewa Werner
et al.
Abstract Background The elastomechanical properties of nanocarriers have recently been discussed as important for the efficient delivery of various therapeutics. Some data indicate that optimal nanocarriers’ elasticity can modulate in vivo nanocarrier stability, interaction with phagocytes, and uptake by target cells. Here, we presented a study to extensively analyze the in vivo behavior of LIP-SS liposomes that were modified by forming the silicone network within the lipid bilayers to improve their elastomechanical properties. We verified liposome pharmacokinetic profiles and biodistribution, including retention in tumors on a mouse model of breast cancer, while biocompatibility was analyzed on healthy mice. Results We showed that fluorescently labeled LIP-SS and control LIP-CAT liposomes had similar pharmacokinetic profiles, biodistribution, and retention in tumors, indicating that modified elasticity did not improve nanocarrier in vivo performance. Interestingly, biocompatibility studies revealed no changes in blood morphology, liver, spleen, and kidney function but indicated prolonged activation of immune response manifesting in increased concentration of proinflammatory cytokines in sera of animals exposed to all tested liposomes. Conclusion Incorporating the silicone layer into the liposome structure did not change nanocarriers’ characteristics in vivo. Further modification of the LIP-SS surface, including decoration with hydrophilic stealth polymers, should be performed to improve their pharmacokinetics and retention in tumors significantly. Activation of the immune response by LIP-SS and LIP-CAT, resulting in elevated inflammatory cytokine production, requires detailed studies to elucidate its mechanism. Graphical Abstract
Abstract Exacerbation of scarring can originate from a minority fibroblast population that has undergone inflammatory-mediated genetic changes within the wound microenvironment. The fundamental relationship between molecular and spatial organization of the repair process at the single-cell level remains unclear. We have developed a novel, high-resolution spatial multiomics method that integrates spatial transcriptomics with scRNA-Seq; we identified new characteristic features of cell–cell communication and signaling during the repair process. Data from PU.1 -/- mice, which lack an inflammatory response, combined with scRNA-Seq and Visium transcriptomics, led to the identification of nine genes potentially involved in inflammation-related scarring, including integrin beta-like 1 (Itgbl1). Transgenic mouse experiments confirmed that Itgbl1-expressing fibroblasts are required for granulation tissue formation and drive fibrogenesis during skin repair. Additionally, we detected a minority population of Acta2 high -expressing myofibroblasts with apparent involvement in scarring, in conjunction with Itgbl1 expression. IL1β signaling inhibited Itgbl1 expression in TGFβ1-treated primary fibroblasts from humans and mice. Our novel methodology reveal molecular mechanisms underlying fibroblast–inflammatory cell interactions that initiate wound scarring.
The EAT-Lancet commission recently suggested that transformation to healthy diets by 2050 will require a reduction of at least 50% in consumption of foods such as red meat and sugar, and a doubling in the global consumption of fruits, vegetables, nuts, and legumes. A diet rich in plant-based foods and with fewer animal source foods confers both improved health and environmental benefits. Notably, the risk of vitamin B12 deficiency increases when consuming a diet low in animal products. Humans are dependent on animal foods such as dairy products, meat, fish and eggs. Vitamin B12 deficiency is common worldwide, especially in populations with low consumption of animal foods because of low socioeconomic status, ethical reasons, or because of their lifestyle (i.e., vegans). According to the European Food Safety Authoroty, the recommended adequate intake of vitamin B12 is 4.0 μg/d for adults, and vitamin B12 requirements are higher during pregnancy and lactation. Infants and children from deficient mothers and elderly people are at risk for vitamin B12 deficiency. Diagnosis of vitamin B12 deficiency is hampered by low specificity of available biomarkers, and there is no consensus yet regarding the optimal definition of low vitamin B12 status. In general, a combination of at least two biomarkers is recommended. Therefore, this review presents an overview of vitamin B12 biochemistry and its biomarkers. We further summarize current recommendations of vitamin B12 intake, and evidence on the associations of vitamin B12 intake from different nutrient-dense animal foods with vitamin B12 status markers. Finally, potential consequences of low vitamin B12 status on different health outcomes for pregnant women, infants and elderly are presented.
A. Francioso, A. Baseggio Conrado, L. Mosca
et al.
Sulfur contributes significantly to nature chemical diversity and thanks to its particular features allows fundamental biological reactions that no other element allows. Sulfur natural compounds are utilized by all living beings and depending on the function are distributed in the different kingdoms. It is no coincidence that marine organisms are one of the most important sources of sulfur natural products since most of the inorganic sulfur is metabolized in ocean environments where this element is abundant. Terrestrial organisms such as plants and microorganisms are also able to incorporate sulfur in organic molecules to produce primary metabolites (e.g., methionine, cysteine) and more complex unique chemical structures with diverse biological roles. Animals are not able to fix inorganic sulfur into biomolecules and are completely dependent on preformed organic sulfurous compounds to satisfy their sulfur needs. However, some higher species such as humans are able to build new sulfur-containing chemical entities starting especially from plants' organosulfur precursors. Sulfur metabolism in humans is very complicated and plays a central role in redox biochemistry. The chemical properties, the large number of oxidation states, and the versatile reactivity of the oxygen family chalcogens make sulfur ideal for redox biological reactions and electron transfer processes. This review will explore sulfur metabolism related to redox biochemistry and will describe the various classes of sulfur-containing compounds spread all over the natural kingdoms. We will describe the chemistry and the biochemistry of well-known metabolites and also of the unknown and poorly studied sulfur natural products which are still in search for a biological role.
Sympathetic neurons are affected in familial dysautonomia, a rare disease associated with a mutation in ELP1, but the mechanisms are not fully understood. Here the authors show, using neurons derived from participants with familial dysauotnomia, that spontaneous sympathetic neuron hyperactivity is observed and is associated with norepinephrine transporter deficits.
Sivabalan Sivasamy, Antony Diwakar Chandran, Prakash Shyam Karuppiah
et al.
In Allium cepa (L.) cultivation, salinity stress is the most important environmental factor worldwide that affects plant growth, protection as well as yield stability. The present study was carried out to examine the alterations in soil dynamics, antioxidants, biochemical and secondary metabolic profile of A. cepa under alkaline stress. Various parameters of A. cepa were studied under salinity stress by exposing them to different concentrations of sodium carbonate (SC) and sodium bicarbonate (SBC) salts (0, 10 and 100 mM). Treatment of both SC and SBC in A. cepa increases superoxide dismutase, glutathione peroxidase and catalase concentrations. These were attributed to increase in peroxide, lipid peroxides as well as carbonyl groups. The salinity stress was driven by the alterations in the soil dynamics. Of note, the salt stress induces secondary metabolites changes in A. cepa. The compounds like phenol 2 4-bis (1 1-dimethylethyl), hexadecane, 7,9-di‑tert‑butyl‑1-oxaspiro(4,5) deca-6,9-diene-2,8‑dione, 1,2-benzenedicarboxylic acid, butyl octyl ester, 1,2-benzenedicarboxylic acid, diisooctyl ester are elevated. In addition, the level of total proteins is reduced in all the groups except 10 mM and Proline amino acid in the shoot is elevated. Reversing all the parameters tested to achieve control levels in A. cepa indicates that significant cellular mechanism appeared in stress condition that brings back the altered parameters to normal levels. The biochemical and secondary metabolite profile analysis of the petroleum ether extracts of A. cepa under various alkaline stress conditions reveals that the composition of secondary metabolites and their concentration in A. cepa varies depending on the extent of alkaline stress.
Ross W. Cheloha, Thibault J. Harmand, Charlotte Wijne
et al.
Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain–only antibodies, discovered in camelids, have been truncated to yield single-domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. Whereas they are known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.
Lead (Pb) is a harmful metal element for aquatic animals. The aim of this study was to determine waterborne Pb exposure on oxidative stress, serum biochemistry and heat shock proteins (HSPs) genes expression in Channa argus. Fish were randomly divided into four groups and the Pb concentrations were 0, 50, 200, and 800 μg/L, respectively. The results showed that the accumulation of Pb was detected in the gill, intestine, liver and muscle following exposure to Pb. Pb accumulation content in tissues was gill > intestinal > liver > muscle. With the increased of Pb exposure concentrations, the levels of catalase (CAT), glutathione peroxidase (GPx), lysozyme (LZM) and immunoglobulin M (IgM) significantly decreased. Serum biochemistry, oxidative stress parameters and HSPs gene expression were all enhanced with the increase following Pb expose concentration. Our results suggest that waterborne Pb exposure can induce Pb accumulation, oxidative stress and immune response in C. argus.
Body size is an important indicator of growth and health in sheep. In the present study, we performed Genome-Wide Association Studies (GWAS) to detect significant single-nucleotide polymorphisms (SNPs) associated with Hu sheep’s body size. After genotyping parental (G1) and offspring (G2) generation of the nucleus herd for meat production of Hu sheep and conducting GWAS on the body height, chest circumference, body length, tail length, and tail width of the two groups, 5 SNPs associated with body height and 4 SNPs correlated with chest circumference were identified at the chromosomal significance level. No SNPs were significantly correlated to body length, tail length, and width. Four out of the 9 SNPs were found to be located within the 4 genes. KITLG and CADM2 are considered as candidate functional genes related to body height; MCTP1 and COL4A6 are candidate functional genes related to chest circumference. The 9 SNPs found in GWAS were verified using the G3 generation of the nucleus herd for meat production. Nine products were amplified around the 9 sites, and 29 SNPs were found; 3 mutation sites, G > C mutation at 134 bp downstream of s554331, T > G mutation at 19 bp upstream of s26859.1, and A > G mutation at 81 bp downstream of s26859.1, were significantly correlated to the body height. Dual-luciferase reporter gene experiments showed that the 3 SNPs could significantly impact dual-luciferase and gene transcription activity.