Resilience of swine nasal microbiota to influenza A virus challenge in a longitudinal study.

Influenza A virus (IAV) is an important contributing pathogen of porcine respiratory disease complex (PRDC) infections. Evidence in humans has shown that IAV can disturb the nasal microbiota and increase host susceptibility to bacterial secondary infections. Few, small-scale studies have examined the impact of IAV infection on the swine nasal microbiota. To better understand the effects of IAV infection on the nasal microbiota and its potential indirect impacts on the respiratory health of the host, a larger, longitudinal study was undertaken to characterize the diversity and community composition of the nasal microbiota of pigs challenged with an H3N2 IAV. The microbiome of challenged pigs was compared with non-challenged animals over a 6-week period using 16S rRNA gene sequencing and analysis workflows to characterize the microbiota. Minimal changes to microbial diversity and community structure were seen between the IAV infected and control animals the first 10 days post-IAV infection. However, on days 14 and 21, the microbial populations were significantly different between the two groups. Compared to the control, there were several genera showing significant increases in abundance in the IAV group during acute infection, such as Actinobacillus and Streptococcus. The results here highlight areas for future investigation, including the implications of these changes post-infection on host susceptibility to secondary bacterial respiratory infections.

Shifts in the nasal microbiota of swine in response to different dosing regimens of oxytetracycline administration.

The impacts of antibiotic treatment and dosing regimen of an antibiotic on the swine respiratory microbiota are poorly defined. To begin to address this, this study characterized the impact of oxytetracycline administration, given either parenterally or in feed, on the diversity of the nasal and tonsil microbiotas of post-weaned pigs over a two-week period. One group received a single intramuscular injection (IM) of oxytetracycline, the second was treated with oxytetracycline mixed in feed (IF), and the control group received non-medicated (NON) feed. Nasal samples were collected on days 0 (before start of treatment), 4, 7, 11, and 14. Tonsil tissue samples were collected from a subset of pigs selected for necropsy on days 4, 7, and 14. The results showed that the tonsil microbiota was stable regardless of antibiotic treatment. In contrast, the nasal bacterial diversity decreased for both oxytetracycline-treated groups compared to NON. The IF group also exhibited decreased diversity on more days than the IM group. The nasal bacterial community structures of the antibiotic treatment groups were significantly different from the NON group that persisted from day 4 until day 7 for the IM group, and up until day 11 for the IF group. This included relative increased abundances of Actinobacillus and Streptococcus, and relative decreased abundances of multiple commensal genera. The microbiota of the IF group was also more disturbed than the microbiota of the IM group, relative to NON. This study revealed that short-term exposure to broad-spectrum antibiotics like oxytetracycline can disturb the upper respiratory microbiota, and the dosing regimen has differential effects on the microbiota.

The Streptococcos suis sortases SrtB and SrtF are essential for disease in pigs.

The porcine pathogen Streptococcus suis colonizes the upper respiratory tracts of pigs, potentially causing septicaemia, meningitis and death, thus placing a severe burden on the agricultural industry worldwide. It is also a zoonotic pathogen that is known to cause systemic infections and meningitis in humans. Understanding how S. suis colonizes and interacts with its hosts is relevant for future strategies of drug and vaccine development. As with other Gram-positive bacteria, S. suis utilizes enzymes known as sortases to attach specific proteins bearing cell wall sorting signals to its surface, where they can play a role in host-pathogen interactions. The surface proteins of bacteria are often important in adhesion to and invasion of host cells. In this study, markerless in-frame deletion mutants of the housekeeping sortase srtA and the two pilus-associated sortases, srtB and srtF, were generated and their importance in S. suis infections was investigated. We found that all three of these sortases are essential to disease in pigs, concluding that their cognate-sorted proteins may also be useful in protecting pigs against infection.

Methicillin-Resistant Staphylococcus aureus Sequence Type (ST) 5 Isolates from Health Care and Agricultural Sources Adhere Equivalently to Human Keratinocytes.

Staphylococcus aureus is part of the nasal microbiome of many humans and has become a significant public health burden due to infections with antibiotic-resistant strains, including methicillin-resistant S. aureus (MRSA) strains. Several lineages of S. aureus, including MRSA, are found in livestock species and can be acquired by humans through contact with animals. These livestock-associated MRSA (LA-MRSA) isolates raise public health concerns because of the potential for livestock to act as reservoirs for MRSA outside the hospital setting. In the United States, swine harbor a mixed population of LA-MRSA isolates, with the sequence type 398 (ST398), ST9, and ST5 lineages being detected. LA-MRSA ST5 isolates are particularly concerning to the public health community because, unlike the isolates in the ST398 and ST9 lineages, isolates in the ST5 lineage are a significant cause of human disease in both the hospital and community settings globally. The ability of swine-associated LA-MRSA ST5 isolates to adhere to human keratinocytes in vitro was investigated, and the adherence genes harbored by these isolates were evaluated and compared to those in clinical MRSA ST5 isolates from humans with no swine contact. The two subsets of isolates adhered equivalently to human keratinocytes in vitro and contained an indistinguishable complement of adherence genes that possessed a high degree of sequence identity. Collectively, our data indicate that, unlike LA-MRSA ST398 isolates, LA-MRSA ST5 isolates do not exhibit a reduced genotypic or phenotypic capacity to adhere to human keratinocytes.IMPORTANCE Our data indicate that swine-associated livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST5 isolates are as capable of adhering to human skin and have the same genetic potential to adhere as clinical MRSA ST5 isolates from humans. This suggests that humans in contact with livestock have the potential to become colonized with LA-MRSA ST5 isolates; however, the genes that contribute to the persistence of S. aureus on human skin were absent in LA-MRSA ST5 isolates. The data presented here are important evidence in evaluating the potential risks that LA-MRSA ST5 isolates pose to humans who come into contact with livestock.

Evaluation of two real-time polymerase chain reaction assays for Porcine epidemic diarrhea virus (PEDV) to assess PEDV transmission in growing pigs.

In April 2013, a Porcine epidemic diarrhea virus (PEDV) epidemic began in the United States. As part of the response, real-time reverse transcription polymerase chain reaction (RT-PCR) assays to detect PEDV were developed by several veterinary diagnostic laboratories. Our study evaluated RT-PCR PEDV assays that detect the N gene (gN) and S gene (gS) for their ability to detect PEDV infection and the transmission potential of pigs experimentally exposed to PEDV. Detection limits and quantification cycle (Cq) values of real-time RT-PCR were assayed for PEDV samples and positive controls for both gN and gS. The limit of detection for the gN assay was 10(-6) (mean Cq: 39.82 ± 0.30) and 10(-5) (mean Cq: 39.39 ± 0.72) for the gS assay with PEDV strain USA/Colorado/2013. Following recommended guidelines, rectal swabs (n = 1,064) were tested; 354 samples were positive by gN assay and 349 samples were positive by gS assay (Cq ≤ 34.99), 710 samples were negative by gN assay and 715 were negative by gS assay (Cq > 34.99) of which 355 and 344 were "undetermined" (i.e., undetected within a threshold of 40 RT-PCR cycles, by gN and gS assays, respectively). The coefficient of variation (intra-assay variation) ranged from 0.00% to 2.65% and interassay variation had an average of 2.75%. PEDV could be detected in rectal swabs from all pigs for ~2 weeks postinfection at which time the prevalence began to decrease until all pigs were RT-PCR negative by 5 weeks postinfection. Our study demonstrated that RT-PCR assays functioned well to detect PEDV and that the gN assay was slightly better.

Radical acceleration of nuclear reprogramming by chromatin remodeling with the transactivation domain of MyoD.

Induced pluripotent stem cells (iPSCs) can be created by reprogramming differentiated cells through introduction of defined genes, most commonly Oct4, Sox2, Klf4, and c-Myc (OSKM). However, this process is slow and extremely inefficient. Here, we demonstrate radical acceleration of iPSC creation with a fusion gene between Oct4 and the powerful transactivation domain (TAD) of MyoD (M(3)O). Transduction of M(3) O as well as Sox2, Klf4, and c-Myc into fibroblasts effectively remodeled patterns of DNA methylation, chromatin accessibility, histone modifications, and protein binding at pluripotency genes, raising the efficiency of making mouse and human iPSCs more than 50-fold in comparison to OSKM. These results identified that one of the most critical barriers to iPSC creation is poor chromatin accessibility and protein recruitment to pluripotency genes. The MyoD TAD has a capability of overcoming this problem. Our approach of fusing TADs to unrelated transcription factors has far-reaching implications as a powerful tool for transcriptional reprogramming beyond application to iPSC technology.

Transcriptional regulation of the Oct4 gene, a master gene for pluripotency.

Oct4 is one of the most important transcription factors required to maintain an undifferentiated state (self-renewal) and pluripotency of human and mouse embryonic stem (ES) cells as well as early embryonic cells. In addition, Oct4 is the only known transcription factor that has to be exogenously introduced into differentiated cells to make induced pluripotent stem (iPS) cells. Therefore, it is of great importance to understand how Oct4 transcription is regulated in ES cells and embryos and how it becomes activated during iPS cell formation. In this article, we will review the regulation of the mouse Oct4 gene from the viewpoint of DNA methylation, binding of orphan nuclear receptors, histone modifications and synergistic effects with other pluripotency factors. We will also raise several key questions that need to be addressed in future work to improve our understanding of Oct4 gene regulation and its essential role in self-renewal and pluripotency.

Post-mitotic role of nucleostemin as a promoter of skeletal muscle cell differentiation.

Nucleostemin (NS) is a nucleolar protein abundantly expressed in a variety of proliferating cells and undifferentiated cells. Its known functions include cell cycle regulation and the control of pre-rRNA processing. It also has been proposed that NS has an additional role in undifferentiated cells due to its downregulation during stem cell differentiation and its upregulation during tissue regeneration. Here, however, we demonstrate that skeletal muscle cell differentiation has a unique expression profile of NS in that it is continuously expressed during differentiation. NS was expressed at similar levels in non-proliferating muscle stem cells (satellite cells), rapidly proliferating precursor cells (myoblasts) and post-mitotic terminally differentiated cells (myotubes and myofibers). The sustained expression of NS during terminal differentiation is necessary to support increased protein synthesis during this process. Downregulation of NS inhibited differentiation of myoblasts to myotubes, accompanied by striking downregulation of key myogenic transcription factors, such as myogenin and MyoD. In contrast, upregulation of NS inhibited proliferation and promoted muscle differentiation in a p53-dependent manner. Our findings provide evidence that NS has an unexpected role in post-mitotic terminal differentiation. Importantly, these findings also indicate that, contrary to suggestions in the literature, the expression of NS cannot always be used as a reliable indicator for undifferentiated cells or proliferating cells.

Novel role of nucleostemin in the maintenance of nucleolar architecture and integrity of small nucleolar ribonucleoproteins and the telomerase complex.

Nucleostemin (NS) is a nucleolar protein involved in the regulation of cell proliferation. Both overexpression and knockdown of NS increase the activity of the tumor suppressor protein p53, resulting in cell cycle arrest. In addition, NS regulates processing of pre-rRNA and consequently the level of total protein synthesis. Here, we describe a previously uncharacterized function of NS in the maintenance of the tripartite nucleolar structure as well as the integrity of small nucleolar ribonucleoproteins (snoRNPs). NS is also necessary to maintain the telomerase complex which shares common protein subunits with the H/ACA box snoRNPs. First, immunofluorescence microscopy and electron microscopy demonstrated that knockdown of NS disorganized the nucleolar architecture, in particular, the dense fibrillar component where snoRNPs are localized. Second, gel filtration chromatography and immunoprecipitation indicated that NS depletion leads to dissociation of the components of snoRNPs and the telomerase complex. Third, NS depletion reduced both telomerase activity and the cellular level of pseudouridine, an H/ACA snoRNP-mediated modification of rRNA and other RNAs that are important for their folding and stability. These morphological, biochemical and functional studies demonstrate that NS plays an important role to maintain nucleolar structure and function on a more fundamental level than previously thought.

Critical role of nucleostemin in pre-rRNA processing.

Nucleostemin is a nucleolar protein widely expressed in proliferating cells. Nucleostemin is involved in the regulation of cell proliferation, and both depletion and overexpression of nucleostemin induce cell cycle arrest through the p53 signaling pathway. Although the presence of p53-independent functions of nucleostemin has been previously suggested, the identities of these additional functions remained to be investigated. Here, we show that nucleostemin has a novel role as an integrated component of ribosome biogenesis, particularly pre-rRNA processing. Nucleostemin forms a large protein complex (>700 kDa) that co-fractionates with the pre-60 S ribosomal subunit in a sucrose gradient. This complex contains proteins related to pre-rRNA processing, such as Pes1, DDX21, and EBP2, in addition to several ribosomal proteins. We show that the nucleolar retention of DDX21 and EBP2 is dependent on the presence of nucleostemin in the nucleolus. Furthermore, the knockdown of nucleostemin delays the processing of 32 S pre-rRNA into 28 S rRNA. This is accompanied by a substantial decrease of protein synthesis as well as the levels of rRNAs and some mRNAs. In addition, overexpressed nucleostemin significantly promotes the processing of 32 S pre-rRNA. Collectively, these biochemical and functional studies demonstrate a novel role of nucleostemin in ribosome biogenesis. This is a key aspect of the role of nucleostemin in regulating cell proliferation.