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Multiple mechanisms of termination modulate the dynamics of RNAPI transcription
Full text linkTranscription elongation is stochastic, driven by a Brownian ratchet, making it subject to changes in velocity. On the rDNA, multiple polymerases are linked by "torsional entrainment" generated by DNA rotation. We report that release of entrainment by co-transcriptional 3' end cleavage, is permissive for relative movement between polymerases, promoting pausing and backtracking. Subsequent termination (polymerase release) is facilitated by the 5' exonuclease Rat1 (Xrn2) and backtracked transcript cleavage by the RNA polymerase I (RNAPI) subunit Rpa12. These activities are reproduced in vitro. Short nascent transcripts close to the transcriptional start site, combined with nascent transcript folding energy, similarly facilitate RNAPI pausing. Nascent, backtracked transcripts at pause sites are terminated by forward and reverse "torpedoes": Rat1 and the exosome cofactor Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP), respectively. Topoisomerase 2 localizes adjacent to RNAPI pause sites, potentially allowing continued elongation by downstream polymerases. Mathematical modeling supported substantial premature termination. These basic insights into transcription in vivo will be relevant to many systems
Vitamin D3 and insulin treatment affects porcine follicular fluid-derived extracellular vesicles characteristics and proteome cargo
Full text linkThe study aimed to investigate whether vitamin D 3 and insulin alone or in co-treatment can influence characteristics and protein cargo of the porcine follicular fluid (FF)-derived extracellular vesicles (EVs). To this end, medium antral follicles were cultured in vitro alone (C; control) or with 1 α ,25(OH) 2 D 3 (VD; 100 ng/mL) and insulin (I; 10 ng/mL) separately or in combination (VD+I). The properties of the FF-EVs were assessed by transmission electron microscopy, nanoparticle tracking analysis, electroforetic light scattering, and flow cytometry, while the global proteomic analysis was conducted by liquid chromatography-tandem mass spectrometry coupled with the TMT-isobaric mass tag labeling. In all groups, particles represented a typical cup- shaped morphology, ranged between 50 and 450 nm in diameter, and expressed marker proteins, such as CD63 and CD81. The I treatment decreased the concentration and average size of the FF-EVs, while VD reversed only the effect on the particle concentration. Proteomic analysis revealed 48 differentially abundant proteins (DAPs) between examined groups, whilst greater amount of DAPs was identified following VD and I treatment alone than in co-treatment. Functional analysis showed that VD alone or in combination with I decreased predominantly the abundance of ribosomal proteins. In the I group, proteins involved in oxidative stress were down- regulated. We also found that the FF-EVs are carriers of adiponectin, which was up-regulated in the VD+I group. To sum up, VD and I seem to be novel modulators of the porcine FF-EVs characteristics and protein cargo, and thereby could modify ovarian follicle function via the EV-mediated pathway
A beginners guide to Sf9 and Sf21 insect cell line culture and troubleshooting
Full text linkRecombinant proteins are not only a crucial research tool but are also widely implemented in biomedicine. There are a number of expression systems used for recombinant protein production. Among them Spodoptera frugiperda (Sf) insect cell system is a powerful tool for multiprotein expression. Most commonly used are Sf9 and Sf21 cell lines due to their cost-effectiveness and availability. While a collection of protocols describing the Sf cell lines culturing is available, we have found them incomplete and their adaptation to real laboratory conditions challenging. Here we created a user-friendly hands-on protocol suitable for beginners. Our work combines the efforts of three independent laboratories which culture Sf cells, two labs with long experience, and one which recently successfully set up this system from scratch. We propose novel tricks and tips that allow for culturing of healthy Sf cells, and high protein yield production. Besides catering for beginners, our protocol can serve as a troubleshooting guide for more experienced researchers. We believe that this work is useful for biochemistry all the way to biomedical laboratories. Starting with an exhaustive description of Sf cell lines, through baculovirus expression vector system characteristic, this publication is a protocol, troubleshooting guide and compendium in one
AlkA Glycosylase and AlkB Dioxygenase Constitute an Effective Protective System for Endogenously Arising Acrolein
Full text linkAcrolein (ACR) is a ubiquitous environmental pollutant but also formed endogenously as a metabolite in oxidative stress conditions. Its adduct to adenine 1,N6-α-hydroxypropanoadenine (HPA) is a mutagenic lesion effectively repaired by the AlkB dioxygenase. Here, we provide in vivo, in vitro, and in silico evidence that it is also the substrate for the AlkA glycosylase. We studied the role of AlkA and AlkB in E. coli cells under conditions of induced adaptive response. Both alkA and alkB defective strains were not more sensitive to exogenous ACR than the wild type was. To simulate endogenously arising adducts, we used acrolein-modified plasmids, allowing monitoring of all kinds of substitutions originating from the acrolein modification of adenine. Both the AlkA and AlkB proteins were engaged in alleviating HPA-induced mutagenesis. Moreover, HPA was effectively repaired by AlkA and AlkB in vivo, even without induction of adaptive response. These findings suggest that the main contribution to acrolein mutagenicity comes from its endogenous sources, whereas AlkA and AlkB can play an additional role in controlling the level of DNA adducts of endogenous origin. Acrolein does not induce the adaptive response. HPA contains an asymmetric carbon atom in the hydroxypropano ring and exists as two stereoisomers. AlkA excises both of them in vitro. Molecular modelling demonstrated how dsDNA carrying both HPA stereoisomers could be properly bound at the AlkA catalytic centre. So, in contrast to the reaction catalyzed by AlkB, the HPA repair by AlkA is not expected to be stereoselective
Characteristics of Two Saccharomyces cerevisiae Strains and Their Extracellular Vesicles as New Candidates for Probiotics
Full text linkThere is a huge disparity between the number of bacterial and yeast probiotics in favor of the former. The latest reports indicate that extracellular vehicles (EVs) play a significant role in probiotic mechanisms. In the present work, we compared the probiotic properties of Saccharomyces cerevisiae strains (WUT3 and WUT151), which have never been previously characterized in this context, with commercial probiotic yeast—Saccharomyces cerevisiae var. boulardii CNCM-745. Notably, WUT3 and WUT151 reacted more mildly to the unfavorable simulated environment of saliva, stomach, small, and large intestines.
As a result, we confirmed that WUT3 and WUT151 were superior to S. boulardii in terms of probiotic properties. Then, we performed a complex analysis of their EVs, isolated by a multistep filtration process. The nanoparticle tracing analysis showed no significant difference in the diameter of the vesicles between the strains. MTT studies confirmed that EVs are not toxic against normal human colorectal cell lines CCD-18 Co and CCD 841 CoN. However, toxicity was observed against the HT-29 cancer line. By staining EVs with Nile Red, we successfully visualized EVs–cell interactions. Finally, we explored the profile of proteins transported with the EVs, identifying a significant overrepresentation of extracellular proteins. Based on comparison with other proteomic data, we selected marker proteins for S.cerevisiae EVs. This knowledge will be helpful for further studies on tracking the transfer of the protein cargo of yeast EVs to human cells using, for instance, specific antibodies to these marker proteins
Short coiled-coil proteins from plants and metazoans – the ‘jacks of all trades’
Full text linkThe molecular functions of short coiled-coil proteins remain poorly charac-
terized. These proteins typically act as facilitators rather than essential
components of metabolic processes, contributing to cellular homeostasis,
and are aptly described as ‘jacks of all trades but masters of none’. They
are found across diverse groups of organisms, including both plants and
animals. LSU (RESPONSE TO LOW SULFUR) are plant proteins
induced under sulfur deficiency and other environmental stresses. They par-
ticipate in metabolic pathways, including sulfate assimilation, and manage
oxidative stress by stabilizing and protecting antioxidative enzymes. In
metazoans, SCOC (SHORT COILED-COIL) proteins regulate autophagy
initiation by recruiting proteins essential for forming autophagosomes —
key vesicles involved in cellular degradation. SCOC proteins also interact
with factors critical for maintaining membrane dynamics and intracellular
transport. Despite some functional similarities, the roles of these proteins
have diverged significantly between plants and animals, reflecting
organism-specific adaptations shaped by evolutionary pressures. This diver-
gence underscores their adaptive versatility and highlights their potential as
promising targets for future biological research
Complete genome sequencing and probiotic trait analysis of Lacticaseibacillus rhamnosus LR110, a human stool isolate from the NORDBIOTIC collection
Full text linkWe present the complete genome of Lacticaseibacillus rhamnosus strain LR110, a human stool isolate from the NORDBIOTIC strain collection. The genome consists of a 2,867,184-bp chromosome with 46.8% GC content. Genomic analysis revealed genes related to thiamine salvage, lactose metabolism, and putrescine biosynthesis, providing insight into the strain’s potential probiotic properties
Exon 4 and intron 4 TP53 are both methylated in advanced‑stage ovarian carcinomas
Full text linkAlthough intragenic CpG dinucleotides are
conserved during evolution, they are also sensitive to
methylation‑dependent mechanisms. Methylation status of
the TP53 introns 1, 3 and 4 have been analysed in stage III
ovarian carcinoma (OC). In the present study, the methylation of exon 4 (10 CpG pairs) was analysed in advanced‑stage OC to investigate TP53 methylation and compare exon and intron 4 methylation patterns. A total of 80 samples from patients with advanced‑stage OC and metastatic lesions were examined, along with 80 samples derived from healthy patients who had never been diagnosed with cancer. Methylation analysis of the human A2780 ovarian cancer cell line was also performed. Exon and intron 4 were methylated in OC, corresponding metastases and paired healthy tissue. The DNA from the human A2780 ovarian cancer cell line and the normal samples from healthy subjects was also methylated. The data indicate the existence of an intragenic mechanism of regulation of TP53 activity that involves demethylation/methylation processes. This mechanism provides the ability to alter the response from cell cycle arrest to apoptosis by manipulating only the expression of long or short p53 isoforms
A Comparison of the Response of the Human Intestinal Microbiota to Probiotic and Nutritional Interventions In Vitro and In Vivo—A Case Study
Full text linkBackground/Objectives: With increasing knowledge of the role of the microbiota in health and disease, the need for the reliable simulation of its behavior in response to various factors, such as diet and probiotic administration in in vitro conditions, has emerged. Although many studies utilize developed systems, data on how accurately these systems represent individual microbiota responses are scarce. Methods: In the present study, the Simulator of Human Intestinal Microbial Ecosystem (SHIME®) was exposed to experimental conditions mimicking the application of probiotics and dietary changes in the study participant. Next-generation 16S rRNA sequencing was used to reveal the structure of the microbial communities in the analyzed samples. Results: Analysis of 17 samples revealed that predominantly diet and, to a lesser extent, probiotics had a divergent effect on the microbiota’s fluctuations dependent on the culture environment. Despite this, results from both in vitro and in vivo conditions aligned well with previously published data on the expected impact of dietary changes on the intestinal microbial community. Conclusions: The anecdotal evidence presented in this study suggested that current in vitro technology enables the reproduction of some of the microbiota responses that are well known from in vivo research. However, further work is required to enable simulations of an individual microbiota
Severity of atopic dermatitis is associated with gut-derived metabolites and leaky gut-related biomarkers
Full text linkDysbiosis of the gut microbiota may contribute to metabolic dysregulation, intestinal barrier disruption, and inflammatory disorders. The objective was to identify gut-derived metabolites and leaky gut biomarkers linked to atopic dermatitis. Fifty adult patients with atopic dermatitis and 25 controls were studied. Blood levels of 30 biomarkers were analyzed using liquid chromatography-mass spectrometry, ELISA, and Luminex assays, and correlated with clinical outcomes (EASI, SCORAD, extent of skin lesions). We discovered higher concentrations of caproic acid, glycerophosphocholine, Reg3A, I-FABP, IL-10, and IL-22 in patients with atopic dermatitis compared to controls, while the concentration of trimethylamine was lower. Disease severity was associated with lower caproic acid and isocaproic acid levels. Indoxyl and leaky gut biomarkers (LBP, Reg3A, IL-10, IL-22) correlated with higher disease activity. Leaky gut-related biomarkers were positively associated with C6 short-chain fatty acids and negatively with indoxyl. These findings highlight potential biomarkers of the gut-skin axis that could aid in predicting the onset and evolution of atopic dermatitis. Given that short-chain fatty acids and indoxyl are fermentation products of fiber and protein, respectively, our results suggest that a fiber-rich diet and moderation of protein intake could be beneficial not only for metabolic health but also in managing atopic dermatitis