Gut-liver axis calibrates intestinal stem cell fitness.

The gut and liver are recognized to mutually communicate through the biliary tract, portal vein, and systemic circulation. However, it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy and transcriptomic and proteomic profiling, we identified pigment epithelium-derived factor (PEDF), a liver-derived soluble Wnt inhibitor, which restrains intestinal stem cell (ISC) hyperproliferation to maintain gut homeostasis by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, we found that microbial danger signals resulting from intestinal inflammation can be sensed by the liver, leading to the repression of PEDF production through peroxisome proliferator-activated receptor-α (PPARα). This repression liberates ISC proliferation to accelerate tissue repair in the gut. Additionally, treating mice with fenofibrate, a clinical PPARα agonist used for hypolipidemia, enhances colitis susceptibility due to PEDF activity. Therefore, we have identified a distinct role for PEDF in calibrating ISC expansion for intestinal homeostasis through reciprocal interactions between the gut and liver.

MYC assembles and stimulates topoisomerases 1 and 2 in a "topoisome".

High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.

Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.

Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S. cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator's key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.

Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres.

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.

Does Salmonella diarizonae 58:r:z53 Isolated from a Mallard Duck Pose a Threat to Human Health?

Salmonella diarizonae (IIIb) is frequently isolated from reptiles and less frequently from birds and mammals. However, its isolation from invasive human infections has not been widely reported. Migratory mallard ducks are excellent bioindicators of pathogen presence and pathogen antibiotic resistance (AMR). We present the first isolation from a mallard duck in central Europe of the antibiotic-resistant Salmonella enterica subsp. diarizonae with the unique antigenic pattern 58:r:z53 and report its whole-genome sequencing, serosequencing, and genotyping, which enabled the prediction of its pathogenicity and comparison with phenotypic AMR. The isolated strain was highly similar to S. diarizonae isolated from humans and food. Twenty-four AMR genes were detected, including those encoding aminoglycoside, fluoroquinolone, macrolide, carbapenem, tetracycline, cephalosporin, nitroimidazole, peptide antibiotic, and disinfecting agent/antiseptic resistance. Six Salmonella pathogenicity islands were found (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, and SPI-13). An iron transport system was detected in SPI-1 centisome C63PI. Plasmid profile analyses showed three to be present. Sequence mutations in the invA and invF genes were noted, which truncated and elongated the proteins, respectively. The strain also harbored genes encoding type-III secretion-system effector proteins and many virulence factors found in S. diarizonae associated with human infections. This study aims to elucidate the AMR and virulence genes in S. enterica subsp. diarizonae that may most seriously threaten human health.

Nonhistone Scm3 binds to AT-rich DNA to organize atypical centromeric nucleosome of budding yeast.

The molecular architecture of centromere-specific nucleosomes containing histone variant CenH3 is controversial. We have biochemically reconstituted two distinct populations of nucleosomes containing Saccharomyces cerevisiae CenH3 (Cse4). Reconstitution of octameric nucleosomes containing histones Cse4/H4/H2A/H2B is robust on noncentromere DNA, but inefficient on AT-rich centromere DNA. However, nonhistone Scm3, which is required for Cse4 deposition in vivo, facilitates in vitro reconstitution of Cse4/H4/Scm3 complexes on AT-rich centromere sequences. Scm3 has a nonspecific DNA binding domain that shows preference for AT-rich DNA and a histone chaperone domain that promotes specific loading of Cse4/H4. In live cells, Scm3-GFP is enriched at centromeres in all cell cycle phases. Chromatin immunoprecipitation confirms that Scm3 occupies centromere DNA throughout the cell cycle, even when Cse4 and H4 are temporarily dislodged in S phase. These findings suggest a model in which centromere-bound Scm3 aids recruitment of Cse4/H4 to assemble and maintain an H2A/H2B-deficient centromeric nucleosome.

Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy.

Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼ 4-µm-deep volume, with lateral and axial localization precisions of ∼ 20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.

Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

Bromodomain protein BRD4 directs mitotic cell division of mouse fibroblasts by inhibiting DNA damage.

Bromodomain protein BRD4 binds to acetylated histones to regulate transcription. BRD4 also drives cancer cell proliferation. However, the role of BRD4 in normal cell growth has remained unclear. Here, we investigated this question by using mouse embryonic fibroblasts with conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells; they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. In summary, BRD4 epigenetically marks above genes and serves as a master regulator of normal cell growth.

Mycobacterium avium and Klebsiella pneumoniae co-infection in the domestic ferret (Mustela putorius furo) - Case Report.

Introduction and Objective. Pets infected with zoonotic pathogens might become a source of infections for their owners, especially those who are immuno-compromised. The aim of this report is to describe a case of chronic, untreatable pneumonia in a domestic ferret. Materials and method. The subject was a 5-year-old female ferret suffering from recurrent pneumonia. Ante-mortally, swabs from the nasal cavity, alveolus and throat were collected from the animal. Post-mortally, lesioned organ fragments were collected. Standard microbiological testing was performed. Additionally, mycobacterial diagnosis including culture and molecular tests was performed. Results. The co-infection of Mycobacterium avium and Klebsiella pneumoniae was microbiologically confirmed. Conclusions. This case demonstrates the need to pay attention to the possibility of zoonotic pathogens in ferrets. Veterinarians diagnosing ferrets are potentially exposed to Mycobacteria spp. infections and other pathogens.

IRF4 requires ARID1A to establish plasma cell identity in multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy that exploits transcriptional networks driven by IRF4. We employ a multi-omics approach to discover IRF4 vulnerabilities, integrating functional genomics screening, spatial proteomics, and global chromatin mapping. ARID1A, a member of the SWI/SNF chromatin remodeling complex, is required for IRF4 expression and functionally associates with IRF4 protein on chromatin. Deleting Arid1a in activated murine B cells disrupts IRF4-dependent transcriptional networks and blocks plasma cell differentiation. Targeting SWI/SNF activity leads to rapid loss of IRF4-target gene expression and quenches global amplification of oncogenic gene expression by MYC, resulting in profound toxicity to MM cells. Notably, MM patients with aggressive disease bear the signature of SWI/SNF activity, and SMARCA2/4 inhibitors remain effective in immunomodulatory drug (IMiD)-resistant MM cells. Moreover, combinations of SWI/SNF and MEK inhibitors demonstrate synergistic toxicity to MM cells, providing a promising strategy for relapsed/refractory disease.

BRD4 directs mitotic cell division by inhibiting DNA damage.

BRD4 binds to acetylated histones to regulate transcription and drive cancer cell proliferation. However, the role of BRD4 in normal cell growth remains to be elucidated. Here we investigated the question by using mouse embryonic fibroblasts with conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells: they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. We suggest that BRD4 epigenetically marks those genes and serves as a master regulator of normal cell growth.

The Influence of Storage Temperature and Packaging Technology on the Durability of Ready-to-Eat Preservative-Free Meat Bars with Dried Plasma.

Food business operators must include the results of shelf life testing in their HACCP plan. Ready-to-eat preservative-free meat products enriched with blood plasma are an unfathomable area of research in food safety. We tested modified atmosphere (80% N2 and 20% CO2) and vacuum packaged RTE preservative-free baked and smoked pork bars with dried blood plasma for Aerobic Plate Count, yeast and mould, lactic acid bacteria, Staphylococcus aureus, Enterobacteriaceae, Escherichia coli, and Campylobacter spp., and the presence of Listeria monocytogenes and Salmonella spp. during storage (temperatures from 4 to 34 °C) up to 35 days after production. The obtained data on the count of individual groups of microorganisms were subjected to analysis of variance (ANOVA) and statistically tested (Student's t-test with the Bonferroni correction); for temperatures at which there were statistically significant differences and high numerical variability, the trend of changes in bacterial counts were visualised using mathematical modelling. The results show that the optimal storage conditions are refrigerated temperatures (up to 8 °C) for two weeks. At higher temperatures, food spoilage occurred due to the growth of aerobic bacteria, lactic acid bacteria, yeast, and mould. The MAP packaging method was more conducive to spoilage of the bars, especially in temperatures over 8 °C.

Ten Years of Animal Tuberculosis Monitoring in Free-Living European Bison (Bison bonasus) in Poland.

In the period 1996-2012, two outbreaks of animal tuberculosis were noted in the population of free-living European bison (Bison bonasus caucasicus) in the Bieszczady Mountains, Southern Poland. As the European bison is an endangered species and particularly susceptible to tuberculosis, not to mention a national icon, the decision was made to test all deceased bison for TB in Poland. The screened bison were obtained by elimination due to poor health or natural death. A total of 159 European bison have been examined over the last 10 years. The individuals came from four regions of Poland (Bialowieza Forest, Bieszczady Mountains, Borecka Forest, Knyszynska Forest), not only from the area where tuberculosis is still endemic. Mycobacterium bovis and Mycobacterium avium spp. hominisuis were identified in two different herds. The isolation of M. bovis from European bison was the first case described in Poland. So far, the only causative agent of tuberculosis identified in European bison in Poland, both in the wild and in captive herds, was Mycobacterium caprae. The isolated M. bovis spoligotype has not previously been registered in international spoligotype databases so far. The obtained results highlight the need to monitor TB in European bison in Poland.

A serological survey of Toxoplasma gondii in polish pigs from organic farms, other housing systems and in pigs of different age groups.

BACKGROUND: The consumption of raw or undercooked meat, especially pork, and offal containing infective tissue cysts is suspected to be a significant route of infection with Toxoplasma gondii. Although the use of "animal-friendly pig production systems" ensuring direct contact with the natural environment offers ethical benefits, it limits the ability to ensure animal health; it may also increase the probability of infections by pathogens such as T. gondii, and thus their entry into the food chain. This study determines the seroprevalence of T. gondii in pigs from different housing systems and farms with different hygiene standards in Poland, as well as among pigs of different age groups from farms with high hygiene standards. In total 760 pig serum samples were examined for the presence of specific antibodies using the PrioCHECK® Toxoplasma Ab porcine commercial ELISA test (Prionics, Switzerland). RESULTS: Test results with PP ≥ 20% were regarded as positive, as indicated by the manufacturer. Antibodies to T. gondii were found in 193 of 760 (25.4%) tested sera. Regarding different housing systems, antibodies were found in 117 pigs: of these, 52.6% (61/116) were from organic farms, 40.9% (47/115) from farms with low hygiene standards, 5.4% (9/167) from farms with high hygiene standards and 0% (0/40) from a farm with a high level of biosecurity. Regarding age groups, antibodies were found in 76 animals on farms with high hygiene standards: 11.1% (7/63) were pigs younger than 3 months, 0% (0/60) aged 3-4 months, 12.3% (7/57) aged 5-6 months (final fattening stage) and 43.7% (62/142) were sows aged 9 months and older. CONCLUSIONS: Antibodies to T. gondii were most often found in pigs from organic and low-hygiene farms, as well as in pigs aged 9 months and older. Meat derived from seropositive animals can pose a potential source of infection for humans. As maternal antibodies to T. gondii can be present in the blood of piglets aged up to 3-4 months, serological examination is unjustified in piglets up to this age.

Predicting the risk of Alaria alata infestation in wild boar on the basis of environmental factors.

Alaria alata is an emerging parasite that poses a potential risk for those consuming game, pork, snails and frogs. One paratenic host of A. alata that is known to play an important role in its spread through its feeding habitats is the wild boar. However, no statistical analysis of the influence of aquatic environments and carnivores on the occurrence of A. alata in wild boars has yet been performed. The present study combines a small-scale analysis based on hunting districts in the Mazowieckie province with a large-scale analysis based on data for all provinces in Poland. We applied various modeling approaches, including logistic regression and a generalized linear model in order to determine the presence, intensity and prevalence of A. alata. We used the Alaria mesocercariae migration technique (AMT) to estimate the risk of A. alata among wild boar in a given hunting district or province. The small-scale analysis found that mesopredators (red fox (Vulpes vulpes)) and racoon dog (Nyctereutes procyinoides) were likely to influence A. alata infestation of wild boar; however, the effect was weak, probably as a result of the large home range size of these animals. The large-scale analysis found that wetlands influence the prevalence of A. alata in wild boar, with the estimated risk increasing in the north of the country; this finding is consistent with other studies. Our findings indicate that the occurrence of A. alata in wild boar requires analysis on many levels, and environmental factors play a key role in risk assessment.

Wild Duck (Anas platyrhynchos) as a Source of Antibiotic-Resistant Salmonella enterica subsp. diarizonae O58-The First Report in Poland.

The "One Health" approach increasingly demonstrates the global spread of pathogenic microorganisms and their antimicrobial resistance in the environment, both in animals and humans. Salmonella enterica subsp. diarizonae is nowadays very often isolated from cold-blooded reptiles to a lesser extent from sheep, but unfortunately more and more often from humans. However, there are a few studies describing the isolation of Salmonella enterica subsp. diarizonae from migratory wild birds. The mallard duck (Anas platyrhynchos), a wild animal that traverses the continent of Eurasia, can be an excellent indicator of the spread of intestinal microbes as well as their resistance to antibiotics. This is the first report of the Salmonella enterica subsp. diarizonae detection in Poland in a migrating mallard duck. This research presented the identification difficulties associated with the isolation of Salmonella enterica subsp. diarizonae using three different biochemical tests and advanced serology tests. At the same time, we detected very high antimicrobial resistance in the isolated strain. By using the minimum inhibitory concentration (MIC) method, it was found that the isolated strain of S. enterica subsp. diarizonae has high antibiotic resistance against 14 of the 33 tested antimicrobials agents. The resistance genes that have been identified in S. enterica subsp. diarizonae include aadA, strA/strB, and blaTEM.

Kinetic principles underlying pioneer function of GAGA transcription factor in live cells.

How pioneer factors interface with chromatin to promote accessibility for transcription control is poorly understood in vivo. Here, we directly visualize chromatin association by the prototypical GAGA pioneer factor (GAF) in live Drosophila hemocytes. Single-particle tracking reveals that most GAF is chromatin bound, with a stable-binding fraction showing nucleosome-like confinement residing on chromatin for more than 2 min, far longer than the dynamic range of most transcription factors. These kinetic properties require the full complement of GAF's DNA-binding, multimerization and intrinsically disordered domains, and are autonomous from recruited chromatin remodelers NURF and PBAP, whose activities primarily benefit GAF's neighbors such as Heat Shock Factor. Evaluation of GAF kinetics together with its endogenous abundance indicates that, despite on-off dynamics, GAF constitutively and fully occupies major chromatin targets, thereby providing a temporal mechanism that sustains open chromatin for transcriptional responses to homeostatic, environmental and developmental signals.

Intra-Palpebral Tuberculin Skin Test and Interferon Gamma Release Assay in Diagnosing Tuberculosis Due to Mycobacterium caprae in European Bison (Bison bonasus).

Despite the threat posed by tuberculosis (TB) to the protected European bison (Bison bonasus), no validated TB tests exist for this species. This pilot study evaluates two tests based on detecting cellular immunity for this purpose: interferon gamma release assay (IGRA) and tuberculin skin test (TST). Ten animals were subjected to ante-mortem and post-mortem examinations. IGRA was performed using a commercial test, and the comparative TST was performed in the eyelids. The lesions were assessed post-mortem and material was collected for mycobacterial culture. The isolated strains were subjected to genotyping. At post-mortem examination, five out of ten individuals demonstrated both tuberculous lesions and positive culture results (Mycobacterium caprae). Compared to the palpebral TST, the findings of the IGRA are easier to interpret when diagnosing tuberculosis in European bison.

Oncogenic RAS commandeers amino acid sensing machinery to aberrantly activate mTORC1 in multiple myeloma.

Oncogenic RAS mutations are common in multiple myeloma (MM), an incurable malignancy of plasma cells. However, the mechanisms of pathogenic RAS signaling in this disease remain enigmatic and difficult to inhibit therapeutically. We employ an unbiased proteogenomic approach to dissect RAS signaling in MM. We discover that mutant isoforms of RAS organize a signaling complex with the amino acid transporter, SLC3A2, and MTOR on endolysosomes, which directly activates mTORC1 by co-opting amino acid sensing pathways. MM tumors with high expression of mTORC1-dependent genes are more aggressive and enriched in RAS mutations, and we detect interactions between RAS and MTOR in MM patient tumors harboring mutant RAS isoforms. Inhibition of RAS-dependent mTORC1 activity synergizes with MEK and ERK inhibitors to quench pathogenic RAS signaling in MM cells. This study redefines the RAS pathway in MM and provides a mechanistic and rational basis to target this mode of RAS signaling.