High Enzyme Concentrations Research Articles

Crystal Structure of Pyrrolysyl-tRNA Synthetase from a Methanogenic Archaeon ISO4-G1 and Its Structure-Based Engineering for Highly-Productive Cell-Free Genetic Code Expansion with Non-Canonical Amino Acids.

Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including Nε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS·tRNAPyl pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion. In the present study, we determined the crystal structure of the methanogenic archaeon ISO4-G1 PylRS (ISO4-G1 PylRS) and compared it with those of structure-known PylRSs. Based on the ISO4-G1 PylRS structure, we attempted the site-specific incorporation of Nε-(p-ethynylbenzyloxycarbonyl)-L-lysine (pEtZLys) into proteins, but it was much less efficient than that of TCO*Lys with M. alvus PylRS mutants. Thus, the first-layer mutations (Y125A and M128L) of ISO4-G1 PylRS, with no additional second-layer mutations, increased the protein productivity with pEtZLys up to 57 ± 8% of that with TCO*Lys at high enzyme concentrations in the cell-free protein synthesis.

Biological Study of protease produced by clinical isolates of Staphylococcus aureus

The bacterial isolates were obtained from Al-Kindi Hospital were diagnosed by the Vitek-2 system and re confirm by 16srRNA gene as S. aurous, the results were shown 20 isolates (66.7%) out of 30 isolates were positive to protease production. All bacterial isolates (100%) were sensitive to Gentamicin and Levofloxacin. but resistant (100%) to aztreonam. The best temperature for enzyme production from bacteria was 37 °C, and the best pH for enzyme production was 7. Partial purification of the bacterial enzyme (protease) was carried out using short steps included ammonium sulfate 65% saturation, ion exchange using DEAE- cellulose column and then applied on gel filtration chromatography using Sephadex G-200 column. The enzymatic activity was determined for each purification step. The specific fold and yield of the purified enzyme were 5.91 and 38.3 % respectively. The molecular weight of the purified enzyme was 37 kDa , it was determined by SDS-PAGE. After being exposed to high concentrations of the protease enzyme (800-1000 µg/ml), the proliferation of a breast cancer cell line (MCF-7) was seen to be suppressed, however the inhibitory effect gradually diminished as the concentration of the enzyme decreased. 200–400 µg/ml is the recommended concentration.

Development of a protein concentrate for human consumption by direct enzymatic hydrolysis of antarctic krill (Euphausia superba)

Krill protein concentrates could be part of human diets as high-quality food ingredients. We have focused on food-grade enzyme hydrolysis of Antarctic krill Euphausia superba, aiming at the production of well-defined protein, phospholipid, and shell fractions. Different enzyme combinations, concentrations, and reaction times of three commercial proteases have been tested for effects on protein yield and lipid content in the soluble protein fraction. The highest protein yield (10.1 g/100 g of the initial wet weight) was obtained in the highest enzyme concentration and longest reaction time treatment, however, associated with lower cost benefits. The lipid content in the protein fractions was not statistically different among treatments, ranging within 9.2–15.8 g/100 g wet weight. The protein fractions were screened for antioxidative capacity by ORAC and minimum stable astaxanthin content. Two of the protein concentrates were fractionated by size exclusion chromatography, to define their peptide profiles. 91% of the total peptides for the single enzyme treatment and 95% after combined enzyme treatment had predominant molecular weights below 1 kDa. The detection of crustacean tropomyosin was strongly reduced after enzymatic hydrolysis in all samples. As part of food safety assessment, all hydrolysates were well tolerated by human monocytes, having no impairing effect on their viability.

Recent Advances in Multiple Strategies for the Synthesis of Terpenes by Engineered Yeast

Terpenes are an important class of natural secondary metabolites with a wide range of applications in food, pharmaceuticals, and biofuels. Currently, the traditional production methods of terpenes almost depend on plant extraction and chemical conversion. The plant extraction method consumes a lot of natural resources and makes it difficult to separate the target compound from the extractives, while the chemical conversion method has a complex synthesis route and leads to severe environmental pollution. Compared to plant extraction and chemical conversion methods, the microbial synthesis method has the advantages of preferable sustainability, low production cost and environmental friendliness, and is a potential way to achieve efficient terpenes production in the future. Yeast is a conventional platform for bio-chemical production and is also engineered to synthesize terpenes due to their abundant intracellular acetyl-CoA, high metabolic flux of the MVA pathway, high local concentrations of substrates and enzymes, and fewer by-products. At present, a variety of terpenes including α-farnesene, squalene, limonene, β-carotene have been successfully synthesized by the engineered yeast via the application of multiple strategies. This work summarized the progress of research on these strategies conducted in the synthesis of terpenes from several aspects, including the adaptive screening and expression of terpene synthases, the regulation of synthesis pathways, and the application of intracellular compartmentalized expression strategy. The perspectives and challenges were also discussed, from which it was hoped that some useful views for future research on the synthesis of terpenes in yeast would be provided.

Effects of dietary supplementation of myco-fabricated zinc oxide nanoparticles on performance, histological changes, and tissues Zn concentration in broiler chicks

A five weeks biological experiment was planned to investigate the impacts of dietary supplementation with zinc oxide nanoparticles (ZnONPs) synthesized by the endophytic fungus Alternaria tenuissima on productive performance, carcass traits, organ relative weights, serum biochemical parameters, histological alteration in some internal organs and concentration of this element in the serum, liver, thigh and breast muscle in broiler chicks. A total of 108 3-day-old commercial broiler chicks (Cobb 500) were individually weighed and equally distributed in a completely randomized design arrangement according to the dose of ZnONPs supplementation into 3 dietary experimental groups. There were 6 replications having 6 birds per replicate (n = 36/ treatment) for each treatment. The three experiential dietary treatments received corn-soybean meal-based diets enhanced with 0 (control), 40 and 60 mg/kg diet of ZnONPs respectively with feed and water were provided ad libitum consumption through 5 weeks life span. Present results indicated that after 5 weeks of feeding trial and as compared to control, the ZnONPs supplementation groups recorded higher body weight, improved feed consumption, feed conversion ratio and performance index. Serum biochemical analyses revealed that serum cholesterol, triglyceride, low density lipoprotein and uric acid decreased significantly, while high density lipoprotein and liver enzyme concentrations were increased significantly. Meanwhile, zinc accumulation in serum, liver and breast and thigh muscle were linearly increased with increasing zinc supplementation. It could be concluded that supplementation of ZnONPs to broiler diet at 40 or 60 mg/kg improved productive performance, birds' physiological status and the lower levels Zn (40 mg/kg diet) revealed promising results and can be used as an effective feed additive in broilers.

Mathematical expressions describing enzyme velocity and inhibition at high enzyme concentration.

Enzyme behaviour is characterised in the laboratory using diluted solutions of enzyme. However, in vivo processes usually occur at [S T ]≈[E T ]≈K m . Furthermore, thestudy of enzyme action involves characterisation of inhibitors and their mechanisms. However, to date, there have been no reports proposing mathematical expressions that can be used to describe enzyme activity at high enzyme concentration apart from the simplest single substrate, irreversible case. Using a continued fraction approach, equations can be easily derived for the most common cases in monosubstrate reactions, such as irreversible or reversible reactions and effector (inhibitor or activator) kinetic interactions. These expressions are an extension of the classical Michaelis-Menten equations. A first analysis using these expressions permits to deduce some differences at high versus low enzyme concentration, such as the greater effectiveness of allosteric inhibitors compared to catalytic ones. Also, they can be used to understand catalyst saturation in a reaction. Although they can be linearised, these equations also show differences that need to be taken into account. For example, the different meaning of line intersection points in Dixon plots. All in all, these expressions may be useful tools for modelling in vivo and biotechnological processes.

The effect of polymorphism of PON1 gene on paraoxonase-1 levels and biochemical parameters of the blood serum in Holstein cattle

The redox state status serves a leading function in metabolism, affecting the stable course of many physiological reactions. Operation of intracellular enzymatic antioxidants is affected by both dietary and genetic control. The influence of high concentrations of the paraoxanase-1 enzyme has a positive effect, maintaining the body's resistance to oxidative stress. The aim of the research was to study the polymorphism of the promoter region of the PON1 gene (-211 A/G) in association with the biochemical parameters of the blood serum of Holstein cattle. DNA genotyping 148 Holstein cows were performed by PCR-RFLP. As a result of identification, three genotypes were established: AA – 31.8% (47 animals), GA – 49.3% (73 animals) and GG – 18.9% (28 animals), and the observed frequency of alleles A and G was – 0.564 and 0.436. An analysis of the relationship between the PON1 gene polymorphism and the level of paraoxonase-1 indicates that, depending on the genotype, the activity of these enzyme changes, as a result of which there is a variation in the biochemical parameters of the blood serum of Holstein cattle.

Differential expression of cystathionine beta synthase in adolescents with Down syndrome: impact on adiposity.

Obesity is more prevalent among people with Down Syndrome (DS) compared to general population. In this pilot study, we investigated the effect of cystathionine beta-synthase (CBS) overdosage on the regulation of transsulfuration pathway and the obesity phenotype in fifty adolescents (25 obese/overweight and 25 lean) with trisomy 21. The transcriptional levels of CBS in leukocytes and its translational levels in plasma were quantified using real time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Meanwhile, ultra performance liquid chromatography tandem mass spectrometry was used to determine the plasma concentrations of methionine, homocysteine, cystathionine and cysteine. Fasting plasma lipid profiles were assessed by colorimetric assays. The anthropometric measurements and indices of all subjects were recorded. Both DS groups had comparable levels of CBS transcripts (p = 0.2734). The plasma levels of the enzyme were significantly higher in the lean DS cases (p = 0.0174) compared to the obese/overweight participants. Total cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, methionine, homocysteine, cystathionine and cysteine showed similar plasma levels in both groups. However, the plasma cysteine levels exceeded the normal range in all DS cases. We reported a statistically significant inverse association between CBS enzyme levels and weight (r= - 0.3498, p = 0.0128), hip circumference (r= - 0.3584, p = 0.0106), body mass index (r= - 0.3719, p = 0.0078) and body adiposity index (r= - 0.3183, p = 0.0243). Our data suggests that the high concentrations of CBS enzyme together with cysteine modulate the DS obesity presumably through increased hydrogen sulfide production which has recently showed anti-adiposity effects.

Kick effect of enzymes causes filament compression

We investigate the influence of enzymes on the structure and dynamics of a filament by dissipative particle dynamics simulations. Enzyme exerts a kick force on the filament monomer. We pay particular attention to two factors: the magnitude of kick force and enzyme concentration. Large kick force as well as high enzyme concentration prefers a remarkable compression of the filament reminiscent of the effective depletion interaction owing to an effective increase in enzyme size and the reduction of solvent quality. Additionally, the kick effect gives rise to an increase of enzyme density from the center-of-mass of the filament to its periphery. Moreover, the increase of enzyme concentration and kick force also causes a decrease in relaxation time. Our finding is helpful to understand the role of catalytic force in chemo-mechano-biological function and the filament behavior under chemical reaction via kick-induced change of solvent quality.

Novel Viral DNA Polymerases From Metagenomes Suggest Genomic Sources of Strand-Displacing Biochemical Phenotypes.

Viruses are the most abundant and diverse biological entities on the planet and constitute a significant proportion of Earth’s genetic diversity. Most of this diversity is not represented by isolated viral-host systems and has only been observed through sequencing of viral metagenomes (viromes) from environmental samples. Viromes provide snapshots of viral genetic potential, and a wealth of information on viral community ecology. These data also provide opportunities for exploring the biochemistry of novel viral enzymes. The in vitro biochemical characteristics of novel viral DNA polymerases were explored, testing hypothesized differences in polymerase biochemistry according to protein sequence phylogeny. Forty-eight viral DNA Polymerase I (PolA) proteins from estuarine viromes, hot spring metagenomes, and reference viruses, encompassing a broad representation of currently known diversity, were synthesized, expressed, and purified. Novel functionality was shown in multiple PolAs. Intriguingly, some of the estuarine viral polymerases demonstrated moderate to strong innate DNA strand displacement activity at high enzyme concentration. Strand-displacing polymerases have important technological applications where isothermal reactions are desirable. Bioinformatic investigation of genes neighboring these strand displacing polymerases found associations with SNF2 helicase-associated proteins. The specific function of SNF2 family enzymes is unknown for prokaryotes and viruses. In eukaryotes, SNF2 enzymes have chromatin remodeling functions but do not separate nucleic acid strands. This suggests the strand separation function may be fulfilled by the DNA polymerase for viruses carrying SNF2 helicase-associated proteins. Biochemical data elucidated from this study expands understanding of the biology and ecological behavior of unknown viruses. Moreover, given the numerous biotechnological applications of viral DNA polymerases, novel viral polymerases discovered within viromes may be a rich source of biological material for further in vitro DNA amplification advancements.

Enzyme Nanoreactor for In Vivo Detoxification of Organophosphates.

A nanoreactor containing an evolved mutant of Saccharolobus solfataricus phosphotriesterase (L72C/Y97F/Y99F/W263V/I280T) as a catalytic bioscavenger was made for detoxification of organophosphates. This nanoreactor intended for treatment of organophosphate poisoning was studied against paraoxon (POX). Nanoreactors were low polydispersity polymersomes containing a high concentration of enzyme (20 μM). The polyethylene glycol-polypropylene sulfide membrane allowed for penetration of POX and exit of hydrolysis products. In vitro simulations under second order conditions showed that 1 μM enzyme inactivates 5 μM POX in less than 10 s. LD50-shift experiments of POX-challenged mice through intraperitoneal (i.p.) and subcutaneous (s.c.) injections showed that intravenous administration of nanoreactors (1.6 nmol enzyme) protected against 7 × LD50 i.p. in prophylaxis and 3.3 × LD50 i.p. in post-exposure treatment. For mice s.c.-challenged, LD50 shifts were more pronounced: 16.6 × LD50 in prophylaxis and 9.8 × LD50 in post-exposure treatment. Rotarod tests showed that transitory impaired neuromuscular functions of challenged mice were restored the day of experiments. No deterioration was observed in the following days and weeks. The high therapeutic index provided by prophylactic administration of enzyme nanoreactors suggests that no other drugs are needed for protection against acute POX toxicity. For post-exposure treatment, co-administration of classical drugs would certainly have beneficial effects against transient incapacitation.

Kinetic Processes in Enzymatic Nanoreactors for In Vivo Detoxification.

Enzymatic nanoreactors are enzyme-encapsulated nanobodies that are capable of performing biosynthetic or catabolic reactions. For this paper, we focused on therapeutic enzyme nanoreactors for the neutralization of toxicants, paying special attention to the inactivation of organophosphorus compounds (OP). Therapeutic enzymes that are capable of detoxifying OPs are known as bioscavengers. The encapsulation of injectable bioscavengers by nanoparticles was first used to prevent fast clearance and the immune response to heterologous enzymes. The aim of enzyme nanoreactors is also to provide a high concentration of the reactive enzyme in stable nanocontainers. Under these conditions, the detoxification reaction takes place inside the compartment, where the enzyme concentration is much higher than in the toxicant diffusing across the nanoreactor membrane. Thus, the determination of the concentration of the encapsulated enzyme is an important issue in nanoreactor biotechnology. The implications of second-order reaction conditions, the nanoreactor’s permeability in terms of substrates, and the reaction products and their possible osmotic, viscosity, and crowding effects are also examined.

Modeling and Experimental Validation of Continuous Biocatalytic Oxidation in Two Continuous Stirred Tank Reactors in Series

The rate of oxygen transfer from the gas phase to the liquid phase is a critical process parameter for biocatalytic oxidations due to the poor water solubility of molecular oxygen and the low oxygen affinity of many of the relevant enzymes, such as oxidases. In gas–liquid systems, mechanical mixing can be used to increase the interfacial area available for mass transfer and thereby increase the volumetric mass transfer coefficient (kLa). As such, the operation of these reactions in a continuous stirred tank reactor (CSTR) may allow for better performance in a readily scalable way. Even so, achieving a high substrate conversion in a single reactor would require operation at high pressure, to improve the solubility of oxygen, as well as a high enzyme concentration. An alternative and more cost-effective means of improving the substrate conversion might be to operate a series of multiple CSTRs. As such, the oxidation of glucose to gluconic acid by glucose oxidase, coupled with catalase, was modeled in a series of identical well-mixed reactors. It was found that achieving full conversion would require an impractical number of reactors at atmospheric pressure. However, the overall conversion of the reaction could be doubled by simply using two CSTRs in series. Subsequently, experiments were carried out to validate this, and the results showed that the overall conversion was in fact tripled. This likely resulted from a higher kLa in the second reactor, which was potentially caused by the change in the media composition from the first reactor.

Reduction of Promiscuous Peptides-Enzyme Inhibition and Aggregation by Negatively Charged Biopolymers.

In this work, peptides selected from a microarray were found to inhibit β-gal with promiscuous mechanisms. Peptides inhibited the enzyme in a noncompetitive kinetics, and the inhibition of enzyme activities was reduced under high enzyme concentrations and the addition of detergent. Dynamic light scattering and atomic force microscope revealed that peptide/enzyme aggregation was related to inhibited enzyme activities. Positively charged residues of arginine and lysine were critical for the enzyme inhibition. The preincubation of peptide inhibitors with negatively charged biopolymers of polyphosphates, ssDNA, and low pI peptides could increase the residual activity of peptide-inhibited enzyme, possibly due to the disruption of the electrostatic interaction between positively charged peptide residues and the β-gal surface. Further, negative biopolymers were able to recover the activity of the aggregated peptide/β-gal complex. Negatively charged biopolymers could be used in high-throughput screening assays to reduce peptides/protein aggregation and thereby minimize promiscuous inhibitions.

Toxicokinetics of Arenobufagin and its Cardiotoxicity Mechanism Exploration Based on Lipidomics and Proteomics Approaches in Rats.

Arenobufagin (ArBu), one of the main active bufadienolides of toad venom with cardiotonic effect, analgesic effect, and outstanding anti-tumor potentiality, is also a potential cardiotoxic component. In the present study, the cardiac effect of ArBu and its underlying mechanism were explored by integrating data such as heart rates, toxicokinetics, myocardial enzyme and brain natriuretic peptide (BNP) activity, pathological sections, lipidomics and proteomics. Under different doses, the cardiac effects turned out to be different. The oral dose of 60 mg/kg of ArBu sped up the heart rate. However, 120 mg/kg ArBu mainly reduced the heart rate. Over time, they all returned to normal, consisting of the trend of ArBu concentration-time curve. High concentrations of myocardial enzymes and BNP indicated that ArBu inhibited or impaired the cardiac function of rats. Pathological sections of hearts also showed that ArBu caused myocardial fiber disorder and rupture, in which the high-dose group was more serious. At the same time, serum and heart tissue lipidomics were used to explore the changes in body lipid metabolism under different doses. The data indicated a larger difference in the high-dose ArBu group. There were likewise many significant differences in the proteomics of the heart. Furthermore, a multi-layered network was used to integrate the above information to explore the potential mechanism. Finally, 4 proteins that were shown to be significantly and differentially expressed were validated by targeted proteomics using parallel reaction monitoring (PRM) analysis. Our findings indicated that ArBu behaved as a bidirectional regulation of the heart. The potential mechanism of cardiac action was revealed with the increased dose, which provided a useful reference for the safety of clinical application of ArBu.

Abstract 17237: A Pair Of Diaphragm-Crossed Organs: A Pancreatico-Pericardial Fistula

Introduction: Pseudocysts are a known complication of chronic pancreatitis, occurring in 30-40% of patients. They form when disruption of the pancreatic duct system creates fluid-filled pockets with high concentrations of digestive enzymes. Leakage of these enzymes can create pancreatic fistulas. Here, we present a case of an unusual fistula that occurred in an immunocompromised individual. Case Description: A 34-year-old man with a history of Acquired Immunodeficiency Syndrome, alcohol use disorder, and chronic pancreatitis who had recently recovered from acute pancreatitis complicated by pericardial effusion was readmitted with worsening pleuritic chest pain and shortness of breath. He was hemodynamically stable with a normal cardiopulmonary exam without jugular venous distention or pulsus paradoxus. Labs were at his baseline other than an elevated d-dimer level. Computed tomography scan revealed a large, loculated pericardial effusion with a fistula extending through the diaphragm to a large peripancreatic pseudocyst. Gastroenterology performed pancreatic ductal stenting via endoscopic retrograde cholangiopancreatography and Interventional Radiology placed a percutaneous drainage catheter to facilitate resolution of the pseudocyst. Discussion: A pancreatico-pericardial fistula is a very rare complication of chronic pancreatitis. A 2016 review detailed only fifteen cases over a 40-year span with surgical management providing the best outcomes. Given this patient’s immunocompromise and malnutrition surgery was not offered, but endoscopic stenting and percutaneous drainage allowed for successful resolution of his fistula.

Anaerobic granulation of single culture Clostridium beijerinckii

This study utilizes Clostridium beijerinckii P260, an anaerobic bacterium capable of acetone-butanol-ethanol (ABE) production, as model bacteria to test the hypothesis that single culture anaerobic granulation can be achieved in accordance with the biogranulation theory developed from the mixed culture studies. After four months of cultivation, C. beijerinckii granules formed under the selection pressure of a selective settling velocity at 9.0 m h−1 in a sequential batch reactor (SBR). These C. beijerinckii granules possess excellent settleability and exceptionally high cell and enzyme concentrations, which demonstrate promising potential in the application for high-rate fermentation and separation of value-added products. However, C. beijerinckii granules only accounted for 25% of the cell mass in the SBR, leaving the rest 75% still in the form of planktonic cells, which indicates an incomplete anaerobic granulation. The lack of ideal feast and famine conditions in the cyclic SBR operation was regarded as the reason. Future research should focus on the optimization of the bioreactor operation to create optimal conditions for inducing the phenotype switch from planktonic to granulated growth mode.

Quality of Death in Fighting Bulls during Bullfights: Neurobiology and Physiological Responses.

Simple SummaryFighting bulls that participate in bullfighting face energy and metabolic demands due to the high intensity and duration of the exercise performed. Under these conditions, specific corporal mechanisms, such as the acid–base balance, are affected, causing metabolic acidosis. However, fighting bulls also undergo muscular injuries, physiological changes, and high enzyme concentrations that reflect the stress to which they are subjected, and in some bulls, bullfights can trigger electrolytic imbalances that include hypercalcaemia, hypermagnesaemia, and hyperphosphataemia, exacerbated by muscular necrosis and myoglobinuria.During bullfights, bulls undergo physiometabolic responses such as glycolysis, anaerobic reactions, cellular oedema, splenic contraction, and hypovolemic shock. The objective of this review article is to present the current knowledge on the factors that cause stress in fighting bulls during bullfights, including their dying process, by discussing the neurobiology and their physiological responses. The literature shows that biochemical imbalances occur during bullfights, including hypercalcaemia, hypermagnesaemia, hyperphosphataemia, hyperlactataemia, and hyperglycaemia, associated with increased endogenous cortisol and catecholamine levels. Creatine kinase, citrate synthase, and lactate dehydrogenase levels also increase, coupled with decreases in pH, blood bicarbonate levels, excess base, partial oxygen pressure, and oxygen saturation. The intense exercise also causes a marked decrease of glycogen in type I and II muscle fibres that can produce myoglobinuria and muscular necrosis. Other observations suggest the presence of osteochondrosis. The existing information allows us to conclude that during bullfights, bulls face energy and metabolic demands due to the high intensity and duration of the exercise performed, together with muscular injuries, physiological changes, and high enzyme concentrations. In addition, the final stage of the bullfight causes a slow dying process for an animal that is sentient and conscious of its surroundings.

Nuclear speckles: dynamic hubs of gene expression regulation.

Complex, multistep biochemical reactions that routinely take place in our cells require high concentrations of enzymes, substrates, and other structural components to proceed efficiently and typically require chemical environments that can inhibit other reactions in their immediate vicinity. Eukaryotic cells solve these problems by restricting such reactions into diffusion-restricted compartments within the cell called organelles that can be separated from their environment by a lipid membrane, or into membrane-less compartments that form through liquid-liquid phase separation (LLPS). One of the most easily noticeable and the earliest discovered organelle is the nucleus, which harbors the genetic material in cells where transcription by RNA polymerases produces most of the messenger RNAs and a plethora of noncoding RNAs, which in turn are required for translation of mRNAs in the cytoplasm. The interior of the nucleus is not a uniform soup of biomolecules and rather consists of a variety of membrane-less bodies, such as the nucleolus, nuclear speckles (NS), paraspeckles, Cajal bodies, histone locus bodies, and more. In this review, we will focus on NS with an emphasis on recent developments including our own findings about the formation of NS by two large IDR-rich proteins SON and SRRM2.

Urinary Carnosinase-1 Excretion is Associated with Urinary Carnosine Depletion and Risk of Graft Failure in Kidney Transplant Recipients: Results of the TransplantLines Cohort Study

Carnosine affords protection against oxidative and carbonyl stress, yet high concentrations of the carnosinase-1 enzyme may limit this. We recently reported that high urinary carnosinase-1 is associated with kidney function decline and albuminuria in patients with chronic kidney disease. We prospectively investigated whether urinary carnosinase-1 is associated with a high risk for development of late graft failure in kidney transplant recipients (KTRs). Carnosine and carnosinase-1 were measured in 24 h urine in a longitudinal cohort of 703 stable KTRs and 257 healthy controls. Cox regression was used to analyze the prospective data. Urinary carnosine excretions were significantly decreased in KTRs (26.5 [IQR 21.4–33.3] µmol/24 h versus 34.8 [IQR 25.6–46.8] µmol/24 h; p < 0.001). In KTRs, high urinary carnosinase-1 concentrations were associated with increased risk of undetectable urinary carnosine (OR 1.24, 95%CI [1.06–1.45]; p = 0.007). During median follow-up for 5.3 [4.5–6.0] years, 84 (12%) KTRs developed graft failure. In Cox regression analyses, high urinary carnosinase-1 excretions were associated with increased risk of graft failure (HR 1.73, 95%CI [1.44–2.08]; p < 0.001) independent of potential confounders. Since urinary carnosine is depleted and urinary carnosinase-1 imparts a higher risk for graft failure in KTRs, future studies determining the potential of carnosine supplementation in these patients are warranted.