Domains in Action: Understanding Ddi1's Diverse Functions in the Ubiquitin-Proteasome System.

The ubiquitin-proteasome system (UPS) is a pivotal cellular mechanism responsible for the selective degradation of proteins, playing an essential role in proteostasis, protein quality control, and regulating various cellular processes, with ubiquitin marking proteins for degradation through a complex, multi-stage process. The shuttle proteins family is a very unique group of proteins that plays an important role in the ubiquitin-proteasome system. Ddi1, Dsk2, and Rad23 are shuttle factors that bind ubiquitinated substrates and deliver them to the 26S proteasome. Besides mediating the delivery of ubiquitinated proteins, they are also involved in many other biological processes. Ddi1, the least-studied shuttle protein, exhibits unique physicochemical properties that allow it to play non-canonical functions in the cells. It regulates cell cycle progression and response to proteasome inhibition and defines MAT type of yeast cells. The Ddi1 contains UBL and UBA domains, which are crucial for binding to proteasome receptors and ubiquitin respectively, but also an additional domain called RVP. Additionally, much evidence has been provided to question whether Ddi1 is a classical shuttle protein. For many years, the true nature of this protein remained unclear. Here, we highlight the recent discoveries, which shed new light on the structure and biological functions of the Ddi1 protein.

Nutrient recovery from anaerobic digestate: Fertilizer informatics for circular economy.

This study explores the integration of fertilizer informatics into the circular economy, with a focus on enhancing nutrient recovery from anaerobic digestate. It utilizes advanced algorithms and data analytics to develop new nutrient management strategies essential for sustainable agriculture. This research provides a detailed assessment of current nutrient recovery technologies, evaluating their environmental impact, cost efficiency, and adaptability. Our findings highlight the importance of merging circular economy principles with fertilizer informatics, showcasing the potential for transforming waste into environmentally friendly fertilizers. This approach has significant implications for improving agricultural practices towards sustainability. The methodologies and insights presented are relevant for ongoing research in environmental stewardship and sustainable resource management. This study describes practical solutions and new perspectives, making it a valuable reference for future research.

Role of vitamin D3 in selected pulmonary diseases with particular emphasis on lung fibrosis.

INTRODUCTION AND OBJECTIVE: For many years vitamin D3 was known only as a regulator of the calcium-phosphate and water-electrolyte balances. Recent studies have paid special attention to other biological effects of calcitriol (the bioactive form of vitamin D3) with particular emphasis on its influence on immune function. Thus, any alterations, especially deficiencies, in the physiological level of calcitriol have serious health consequences. The aim of the study was to summarise the current state of knowledge concerning the role of vitamin D3 in selected pulmonary diseases. REVIEW METHODS: The review was based on data obtained from articles published in PubMed between 2000-2022. Papers were reviewed for scientific merit and relevance. BRIEF DESCRIPTION OF THE STATE OF KNOWLEDGE: In the reviewed literature, much attention was paid to clinical studies focused on the role of vitamin D3 in the pathogenesis of selected respiratory diseases. As revealed in research over the last two decades, vitamin D3 deficiency increases the risk and worsens the course of asthma, cystic fibrosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, as well as COVID-19. Surprisingly, vitamin D supplementation has not always proved to be an effective therapeutic strategy. The review also presents the unique concept of the possibility of using vitamin D3 in the prevention and treatment of pulmonary fibrosis in the course of hypersensitivity pneumonitis. SUMMARY: Due to the multiplicity and variety of factors that affect the metabolism of vitamin D3, effective counteracting, and even more eliminating the negative consequences of disorders in the level and activity of calcitriol in the respiratory tract, seems to be a breakneck action. On the other hand, only a deep understanding of the role of calcitriol in the pathogenesis of lung diseases provides the chance to develop an effective therapy.

The role of NPM1 alternative splicing in patients with chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease with heterogeneous clinical course. Recent studies revealed a link between NOTCH1 mutation and the overexpression of MYC and MYC-related genes involved in ribosome biogenesis and protein biosynthesis, such as nucleophosmin-1 (NPM1), in CLL cells. In the present study, we aim to evaluate the impact of the NOTCH1 mutation on the MYC and MYC induced NPM1 expression in CLL cells via quantification of their transcripts. METHODS: Using qRT-PCR, we analyzed the levels of MYC and three main NPM1 splice variants in 214 samples collected from CLL patients. We assessed the impact of each splice variant on CLL prognostic markers, including the IGHV, TP53, NOTCH1, SF3B1, and MYD88 mutational status, cytogenetic aberrations, and laboratory features. RESULTS: Significantly higher levels of NPM1.R1 transcripts in patients with unmutated compared to mutated IGHV status were found. The median time to first treatment (TTFT) in patients with a high level of NPM1.R1 was significantly shorter compared to the group with low NPM1.R1 levels (1.5 vs 33 months, p = 0.0002). Moreover, in Multivariate Cox Proportional Hazard Regression Model NPM1.R1 splice variant provided an independent prognostic value for TTFT. CONCLUSION: In conclusion, our study indicates the prognostic significance of the level of NPM1.R1 expression and suggests the importance of splicing alterations in the pathogenesis of CLL.

Immunomodulatory Properties of Polysaccharide-Rich Young Green Barley (Hordeum vulgare) Extract and Its Structural Characterization.

Young green barley (YGB) water extract has revealed a beneficial impact on natural killer (NK) cells' ability to recognize and eliminate human colon cancer cells, without any side effects for normal colon epithelial cells. The direct anticancer effect of the tested compounds has been also shown. The mixture of oligosaccharides found in this extract was characterized by chemical analyses and via FT-IR spectroscopy and MALDI-TOF MS techniques. The YGB preparation contained 26.9% of proteins and 64.2% of sugars, mostly glucose (54.7%) and fructose (42.7%), with a small amount of mannose (2.6%) and galactose (less than 0.5%). Mass spectrometry analysis of YGB has shown that fructose oligomers contained from 3 to 19 sugar units. The number of fructans was estimated to be about 10.2% of the dry weight basis of YGB. The presented results suggest the beneficial effect of the consumption of preparations based on young barley on the human body, in the field of colon cancer prevention.

Pro-Health and Anti-Cancer Activity of Fungal Fractions Isolated from Milk-Supplemented Cultures of Lentinus (Pleurotus) Sajor-caju.

The present study aimed to demonstrate Lentinus (formerly Pleurotus) sajor-caju (PSC) as a good source of pro-health substances. It has also shown that supplementation of its culture medium with cow milk may further improve its beneficial properties. Intracellular fractions from fungi grown on a medium supplemented with cow milk were analyzed using various biochemical methods for determination of the nutrient composition. Furthermore, anti-cancer properties of selected extracts were investigated on colorectal cancer cell lines (HT-29, LS 180, and SW948) in vitro. Biochemical analysis showed enrichment in health-enhancing compounds, such as proteins or polysaccharides (about 3.5- and 4.5-fold increase in concentration of proteins and carbohydratesin extracts of mycelia cultured on whole milk (PSC2-I), respectively), with a decrease in the level of free radicals (10-fold decrease in extract grown on milk and medium mixture (1:1) (PSC3-II)), which was related to increased catalase and superoxide dismutase activity (7.5-fold increase in catalase activity and 5-fold in SOD activity in PSC3-II compared to the control). Moreover, the viability of the cancer cells was diminished (to 60.0 ± 6.8% and 40.0 ± 8.6% of the control, on HT-29 and SW948 cells, respectively), along with pro-apoptotic (to 18.8 ± 11.8 and 14.7 ± 8.0% towards LS 180 and SW948 cells, respectively) and NO-secreting effects (about 2-fold increase) of the extracts. This study suggests that PSC has multiple nutritional and anti-cancer properties and can be used as a source of healthy biomolecules in modern medicine or functional foods.

The Predominant Prognostic Significance of NOTCH1 Mutation Defined by Emulsion PCR in Chronic Lymphocytic Leukemia.

PURPOSE: NOTCH1 mut represents a new prognostic marker in chronic lymphocytic leukaemia (CLL). The low sensitivity of the current methods may increase the risk of false-negative results, particularly in patients with low NOTCH1 mut allelic burden. This study compared two methods of the NOTCH1 mut assessment including droplet digital PCR (ddPCR) and amplification-refractory mutation system PCR (ARMS-PCR) untreated CLL patients. PATIENTS AND METHODS: This study included 319 untreated CLL patients. Two PCR-based methods; ddPCR and ARMS-PCR were performed to assess the mutational status of NOTCH1. The Mann-Whitney, Fisher's exact test, Kruskal-Wallis, Kaplan-Meier, Log rank tests and multivariate Cox proportional hazard regression model were used to analyze collected data. RESULTS: We proved that ddPCR increased the detectability of the NOTCH1 mut compared to ARMS-PCR in CLL (18.55% vs 6%). We showed a shorter time to first treatment (TTFT) in the NOTCH1 mut group of patients compared to the NOTCH1 wt defined by ddPCR (1.5 vs 33 months, p=0.01). The TTFT survival curves analysis in subgroups divided according to the mutational status of IGHV and NOTCH1 assessed by ddPCR discriminated group with the best prognosis: IGHV mut NOTCH1 wt. Multivariate analysis revealed that the mutational status of IGHV represented an independent prognostic factor for TTFT, while NOTCH1 mut determined by ddPCR constituted as a dependent prognostic factor for TTFT. CONCLUSION: The selection of the precise method of NOTCH1 mut detection as ddPCR might significantly improve prognostic stratification of CLL patient. Assessment of IGHV might be relevant to more accurate discrimination of prognostic groups of CLL patients, especially in harboring NOTCH1 mut irrespective of the quantity of allelic burden.

Synthesis and characterization of Gd2O3: Er3+, Yb3+doped with Mg2+, Li+ions-effect on the photoluminescence and biological applications.

Gd2O3:1% Er3+, 18% Yb3+,x% Mg2+(x = 0; 2.5; 4; 5; 6; 8;10; 20; 25; 50) and Gd2O3:1% Er3+, 18% Yb3+, 2,5% Mg2+,y% Li+(y = 0.5-2.5) nanoparticles were synthesized by homogenous precipitation method and calcined at 900 °C for 3 h in air atmosphere. Powder x-ray diffraction, scanning electron microscopy, cathodoluminescence, transmission electron microscopy, energy dispersive x-ray spectroscopy and photoluminescence techniques were employed to characterize the obtained nanoparticles. We observed a 8-fold increase in red luminescence for samples suspended in DMSO solution for 2.5% of Mg2+doping. The x-ray analysis shows that for the concentration of 2.5% Mg, the size of the crystallites in the NPs is the largest, which is mainly responsible for the increase in the intensity of the upconversion luminescence. But the addition of Li+ions did not improve the luminescence of the upconversion due to decreasing of crystallites size of the NPs. Synthesized nanomaterials with very effective upconverting luminescence, can act as luminescent markers inin vivoimaging. The cytotoxicity of the nanoparticles was evaluated on the 4T1 cell line for the first time.

Differential Function of a Novel Population of the CD19+CD24hiCD38hi Bregs in Psoriasis and Multiple Myeloma.

Psoriasis (Ps), an autoimmune disease, and multiple myeloma (MM), a blood neoplasm, are characterized by immune dysregulation resulting from the imbalance between the effector and regulatory cells, including B regulatory (Breg) lymphocytes. Peripheral blood samples from 80 Ps patients, 17 relapsed/refractory MM patients before and after daratumumab (anti-CD38 monoclonal antibody) treatment, 23 healthy volunteers (HVs), and bone marrow samples from 59 MM patients were used in the study. Bregs were determined by flow cytometry using CD19, CD24, and CD38. Intracellular production of interleukin-10 (IL-10) was assessed by flow cytometry after CD40L, LPS, and CpG stimulation. IL-10 serum or plasma concentrations were tested using ELISA method. The percentage of CD19+CD24hiCD38hi Bregs was not different whereas the production of IL-10 in Bregs was significantly higher in Ps patients in comparison with HVs. The percentage of CD19+CD24hiCD38hi Bregs in MM patients was significantly higher than in HVs (p < 0.0001). The percentage of CD19+CD24hiCD38hi Bregs was significantly higher in MM patients with the ISS stage I (p = 0.0233) while IL-10 production in Bregs was significantly higher in ISS stage III (p = 0.0165). IL-10 serum or plasma concentration was significantly higher in Ps and MM patients when compared to HVs (p < 0.0001). Following the treatment with daratumumab the percentages of CD19+CD24hiCD38hi Bregs significantly decreased (p < 0.0003). Here, in the two opposite immune conditions, despite the differences in percentages of Bregs in Ps and MM we have identified some similarities in the IL-10 producing Bregs. Effective treatment of daratumumab besides the anti-myeloma effect was accompanied by the eradication of Bregs.

Cofilin-1 Maintains Prosurvival Signaling in Chronic Lymphocytic Leukemia Cells.

BACKGROUND/AIM: Despite numerous studies, the etiology of chronic lymphocytic leukemia (CLL) remains unknown. A hypothesis of autoantigen stimulation in leukemic clone selection might explain 'stereotypy' of B-cell receptors. In healthy cells, cofilin-1 (CFL1) has multiple functions. Its role was described in several malignancies. The aim of this study was characterization of the role of CFL1 in CLL. Materialas and Methods: Cells from peripheral blood of 180 patients and 42 healthy volunteers (HVs) were isolated. Gene expression was assessed with reverse transcription polymerase chain reaction (RT-qPCR); western blot was performed for determination of protein level and activity. After silencing of CFL1 gene, cell ability for migration and chemotaxis was investigated with Transwell method. Post-silencing, apoptosis and cell cycle was determined by flow cytometry. RESULTS: In RT-qPCR, we observed significantly higher expression of CFL1. Higher activity of protein in CLL cells when compared to HVs was detected. Knock-down of CFL1 led to decreased chemotaxis and migration of CLL cells versus cells from HVs. Apoptosis was increased amongst cells with silenced CFL1 and correlated with higher proportion of cells in the G2/M phase. CONCLUSION: Significantly higher expression of CFL1 mRNA in CLL and higher protein activity might indicate high utilization of CFL1 in malignant cells, maintaining their viability, as its inhibition affected viability, cell-cycle progression and motility of leukemia cells.

Structural, optical and magnetic properties of Y3-0.02-xEr0.02Yb x Al5O12 (0 < x < 0.20) nanocrystals: effect of Yb content.

The paramagnetic Y3-0.02-x Er0.02Yb x Al5O12 (x = 0.02, 0.06, 0.10, 0.12, 0.18, 0.20) nanocrystals (NCs) were synthesized by the microwave-induced solution combustion method. The XRD, TEM and SEM techniques were applied to determine the NCs' structures and sizes. The XRD patterns confirmed that the NCs have for the most part a regular structure of the Y3Al5O12 (YAG) phase. The changes of the distance between donor Yb3+ (sensitizer) and acceptor Er3+ (activator) were realized by changing the donor's concentration with a constant amount of acceptor. Under 980 nm excitation, at room temperature, the NCs exhibited strong red emission near 660 and 675 nm, and green upconversion emission at 550 nm, corresponding to the intra 4f transitions of Er3+ (4F9/2, 2H11/2, 4S3/2) â†’ Er3+ (4I15/2). The strongest emission was observed in a sample containing 18% Yb3+ ions. The red and green emission intensities are respectively about 5 and 12 times higher as compared to NCs doped with 2% of Yb3+. In order to prove that the main factor responsible for the increase of the upconversion luminescence efficiency is reduction of the distance between Yb3+ and Er3+, we examined, for the first time the influence of hydrostatic pressure on luminescence and luminescence decay time of the radiative transitions inside donor ion. The decrease of both luminescence intensity and luminescence decay times, with increasing hydrostatic pressure was observed. After applying hydrostatic pressure to samples with e.g. 2% and 6% Yb3+, the distance between the donor and acceptor decreases. However, for higher concentrations of the donor, this distance is smaller, and this leads to the effective energy transfer to Er3+ ions. With increasing pressure, the maximum intensity of near infrared emission is observed at 1029, 1038 and 1047 nm, what corresponds to 2F5/2 â†’ 2F7/2 transition of Yb3+.

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit.

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (UbPT), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of UbPT, and we expect that its future uses will help define and investigate the ubiquitin interactome.

[The involvement of matrix metalloproteinases in the development and progression of neoplasm diseases]. / Udzial metaloproteinaz macierzy zewnatrzkomórkowej w rozwoju i progresji chorób nowotworowych.

Neoplasm diseases are one of the main causes of death in Poland and worldwide. Forming and progression of tumour are regulated by the number of factors, among which one of the most important are matrix metalloproteinases (MMPs), zinc-dependant proteases, responsible for remodeling of extracellular matrix (ECM). They may induce cancer progression directly by modifying the ECM, enabling cancer growth and migrating of cells released from tumour, as well as invading adjacent tissue and blood or lymphatic vessels. MMPs may also induce carcinogenesis in indirect way by modifying tumour microenvironment and secreting factors promoting or inhibiting particular processes. There is number of factors secreted by cancer cells, stromal components and ECM elements regulating activation and functionality of matrix metalloproteinases. Understanding the mechanisms and pathways underlying regulation and activation of MMPs is crucial for comprehension of carcinogenesis and metastasis, and may contribute to developing of new therapeutic strategies.

Characterizing polyubiquitinated forms of the neurodegenerative ubiquitin mutant UBB+1.

The ubiquitin mutant UBB+1 has been identified as a hallmark of neurodegenerative diseases. In this study, we characterize polyubiquitinated forms of UBB+1 in vitro and from patient samples. The ability of UBB+1 to be readily ubiquitinated by several E2 enzymes provided a mechanism for the controlled synthesis and purification of defined conjugates. This allowed us to utilize polyUb-UBB+1 conjugates for biochemical assays, as well as solution NMR. Coupled with our immunoassay for detection of ubiquitinated forms of UBB+1 in patient blood samples, we gain a clearer picture of the molecular mechanisms underlying neurodegenerative diseases.

[Postpartum levator ani muscle injuries. Diagnosis and treatment]. / Poporodowe uszkodzenia dzwigacza odbytu. Diagnostyka i terapia.

Levator ani muscle (LAM) injuries are much more frequent than trauma to sphincter ani muscles, but so far they have been omitted in obstetric handbooks. Levator ani avulsion is observed only after vaginal delivery. Forceps delivery second stage of labor ≥ 110 min., fetal head circumference ≥ 35 cm, episiotomy and coincidence of anal sphincter trauma are risk factors for levator ani avulsion. The most vital issue in that type of trauma is pelvic organ prolapse and 2-4-fold higher risk of recurrence after prolapse surgery. The current level of evidence does not allow to conclusively determine the of role of levator avulsion in urinary incontinence. Levator injuries are occult, what constitutes the main diagnostic problem. Until recently magnetic resonance imaging has been the only diagnostic method until the development of 3-dimensional ultrasound. Nowadays, 3-D ultrasound is an essential technique in static and functional diagnosis of the levator ani. There are no effective methods of levator trauma prevention. Except the risk factors reduction, there are some pilot data about positive role of antepartal perineal muscle training. Physiotherapy plays the main role in reducing the effects of levator trauma. Mesh techniques are the most effective operative methods in coincident pelvic organ prolapse with levator avulsion, but there is still a 2-fold higher risk of recurrence.

[Osteoarthritis: etiology, risk factors, molecular mechanisms]. / Osteoartroza: etiologia, czynniki ryzyka, mechanizmy molekularne.

Osteoarthritis is an incurable joint disease manifesting itself with gradually progressing degenerative changes, leading to premature motor disability. These changes mainly occur owing to an imbalance between the processes of degeneration and regeneration of articular cartilage structures. Until now many risk factors favoring the development of degenerative joint disease have been identified. These include age, weight, previously sustained traumas to joints, sports, sex and genetic predisposition. The latest scientific reports confirm that the pathogenesis of changes in osteoarthritic joints is complex and occurs on many levels. Enzymes belonging to the metalloproteinases family are mainly responsible for the degeneration of articular cartilage. Their activity is regulated by numerous pro-inflammatory cytokines, transcription factors and miRNA. A thorough analysis of all processes occurring in the afflicted joints needs to be carried out before effective therapeutic strategies can be developed.

Do gene expression changes in articular cartilage proteases of the synovial membrane correlate with expression changes of the same genes in systemic blood cells?

PURPOSE: The aim of our study was to find whether an injury of the knee joint tissues increases gene expression of selected hyaline cartilage degenerating enzymes such as matrix metaloproteinases (MMP) and aggreacaneses (Agg). METHODS: A total of 138 patients (81 female, 57 male) were admitted for knee arthroscopy with a mean age of 38.8 years. Full blood samples were collected preoperatively and synovium samples intraoperatively. Joint tissue lesions such as menisci, anterior cruciate ligament (ACL) and hyaline cartilage were estimated. Real time PCR with spectrophotometric analysis was performed. RESULTS: An ACL lesion was found in 56 patients, medial menisci (MM) in 65, and lateral menisci (LM) in five. Chondral lesions were estimated according to Outerbridge's grading system. In laboratory tests correlation between ACL tear and gene expression was seen except TIMP1 in serum (p < 0.05). In MM lesions MMP9, Agg2 elevation in serum was observed. LM lesions erased MMP13, MMP14 in serum and MMP8 in synovium. Chondral lesions revealed that many genes had higher expression in patients without hyaline degeneration. All of the gene expressions correlated between serum and synovium. CONCLUSION: An ACL lesion provokes elevation in expression of proteases genes, while the influence of other lesions remains elusive. Gene expression in synovium correlates with peripheral blood.

The mechanism of copper uptake by tyrosinase from Bacillus megaterium.

Tyrosinase belongs to the type 3 copper enzyme family, containing a dinuclear copper center, CuA and CuB. It is mainly responsible for melanin production in a wide range of organisms. Although copper ions are essential for the activity of tyrosinase, the mechanism of copper uptake is still unclear. We have recently determined the crystal structure of tyrosinase from Bacillus megaterium (TyrBm) and revealed that this enzyme has tighter binding of CuA in comparison with CuB. Investigating copper accumulation in TyrBm, we found that the presence of copper has a more significant effect on the diphenolase activity. By decreasing the concentration of copper, we increased the diphenolase to monophenolase activity ratio twofold. Using a rational design approach, we identified five variants having an impact on copper uptake. We have found that a major role of the highly conserved Asn205 residue is to stabilize the orientation of the His204 imidazole ring in the binding site, thereby promoting the correct coordination of CuB. Further investigation of these variants revealed that Phe197, Met61, and Met184, which are located at the entrance to the binding site, not only play a role in copper uptake, but are also important for enhancing the diphenolase activity. We propose a mechanism of copper accumulation by the enzyme as well as an approach to changing the selectivity of TyrBm towards L-dopa production.