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Abstract Activated sludge is the centerpiece of biological wastewater treatment, as it facilitates removal of sewage-associated pollutants, fecal bacteria, and pathogens from wastewater through semi-controlled microbial ecology. It has been hypothesized that horizontal gene transfer facilitates the spread of antibiotic resistance genes within the wastewater treatment plant, in part because of the presence of residual antibiotics in sewage. However, there has been surprisingly little evidence to suggest that sewage-associated antibiotics select for resistance at wastewater treatment plants via horizontal gene transfer or otherwise. We addressed the role of sewage-associated antibiotics in promoting antibiotic resistance using lab-scale sequencing batch reactors fed field-collected wastewater, metagenomic sequencing, and our recently developed bioinformatic tool Kairos. Here, we found confirmatory evidence that fluctuating levels of antibiotics in sewage are associated with horizontal gene transfer of antibiotic resistance genes, microbial ecology, and microdiversity-level differences in resistance gene fate in activated sludge.

Background Bongkrekic acid (BA) is a mitochondrial toxin, produced by the bacteria Burkholderia gladioli pv. cocovenenans during fermentation. Outbreaks of BA poisoning have been reported in China, Indonesia, and Mozambique, but have not been previously observed in North America.Case discussion A 67-year-old male with a past medical history of hypertension presented with two days of generalized weakness, nausea, and vomiting. He had consumed ogi, a home-fermented cornmeal pudding two days earlier. The patient developed acute liver failure, encephalopathy, acute kidney injury, coagulopathy, methemoglobinemia, and metabolic acidosis. Despite intensive care, he died on hospital day 8. Workup for common causes of acute liver failure was negative. Blood testing was positive for BA, estimated at 1,000 ng/mL. This case illustrates poisoning by BA, a rare and often lethal toxin resulting from improperly fermented food.

IntroductionFluoroquinolones (FQs) are not commonly prescribed in children, yet the increasing incidence of multidrug-resistant (MDR) Enterobacterales (Ent) infections in this population often reveals FQ resistance. We sought to define the role of FQ resistance in the epidemiology of MDR Ent in children, with an overall goal to devise treatment and prevention strategies.MethodsA case–control study of children (0–18 years) at three Chicago hospitals was performed. Cases had infections by FQ-susceptible, β-lactamase-producing (bla) Ent harboring a non- or low-level expression of PMFQR genes (PMFQS Ent). Controls had FQR infections due to bla Ent with expressed PMFQR genes (PMFQR Ent). We sought bla genes by PCR or DNA (BD Max Check-Points assay®) and PMFQR genes by PCR. We performed rep-PCR, MLST, and E. coli phylogenetic grouping. Whole genome sequencing was additionally performed on PMFQS Ent positive isolates. Demographics, comorbidities, and device, antibiotic, and healthcare exposures were evaluated. Predictors of infection were assessed.ResultsOf 170 β-lactamase-producing Ent isolates, 85 (50%) were FQS; 23 (27%) had PMFQR genes (PMFQS cases). Eighty-five (50%) were FQR; 53 (62%) had PMFQR genes (PMFQR controls). The median age for children with PMFQS Ent and PMFQR Ent was 4.3 and 6.2 years, respectively (p = NS). Of 23 PMFQS Ent, 56% were Klebsiella spp., and of 53 PMFQR Ent, 76% were E. coli. The most common bla and PMFQR genes detected in PMFQS Ent were blaSHV ESBL (44%) and oqxAB (57%), and the corresponding genes detected in PMFQR Ent were blaCTX-M-1-group ESBL (79%) and aac(6’)-Ib-cr (83%). Whole genome sequencing of PMFQS Ent revealed the additional presence of mcr-9, a transferable polymyxin resistance gene, in 47% of isolates, along with multiple plasmids and mobile genetic elements propagating drug resistance. Multivariable regression analysis showed that children with PMFQS Ent infections were more likely to have hospital onset infection (OR 5.7, 95% CI 1.6–22) and isolates containing multiple bla genes (OR 3.8, 95% CI 1.1–14.5). The presence of invasive devices mediated the effects of healthcare setting in the final model. Differences in demographics, comorbidities, or antibiotic use were not found.ConclusionsParadoxically, PMFQS Ent infections were often hospital onset and PMFQR Ent infections were community onset. PMFQS Ent commonly co-harbored multiple bla and PMFQR genes, and additional silent, yet transferrable antibiotic resistance genes such as mcr-9, affecting therapeutic options and suggesting the need to address infection prevention strategies to control spread. Control of PMFQS Ent infections will require validating community and healthcare-based sources and risk factors associated with acquisition.

Abstract Foraging behavior and interaction with prey is an integral component of the ecological niche of predators but is inherently difficult to observe for highly mobile animals in the marine environment. Billfishes have been described as energy speculators, expending a large amount of energy foraging, expecting to offset high costs with periodic high energetic gain. Surface-based group feeding of sailfish, Istiophorus platypterus, is commonly observed, yet sailfish are believed to be largely solitary roaming predators with high metabolic requirements, suggesting that individual foraging also represents a major component of predator–prey interactions. Here, we use biologging data and video to examine daily activity levels and foraging behavior, estimate metabolic costs, and document a solitary predation event for a 40 kg sailfish. We estimate a median active metabolic rate of 218.9 ± 70.5 mgO2 kg−1 h−1 which increased to 518.8 ± 586.3 mgO2 kg−1 h−1 during prey pursuit. Assuming a successful predation, we estimate a daily net energy gain of 2.4 MJ (5.1 MJ acquired, 2.7 MJ expended), supporting the energy speculator model. While group hunting may be a common activity used by sailfish to acquire energy, our calculations indicate that opportunistic individual foraging events offer a net energy return that contributes to the fitness of these highly mobile predators.

In utero drug exposure is a significant public health threat to the well-being and normal development of the neonate. Recently, testing of umbilical cord tissue (UCT) has been employed to measure illicit drug exposure, as drugs used by the mother during the third trimester may be retained in the UCT. Focus has also been given to potential adverse health effects among drug users, resulting from exposure to pharmacologically active adulterants and cutting agents in the street drug supply. The in utero effects of these substances have not been well studied in humans, nor has their presence been demonstrated as a means for assessing adverse health effects in the neonate. Here, we describe the application of a novel test method to analyze UCT for the presence of more than 20 common adulterating/cutting substances via LC/Q-TOF. In total, 300 de-identified UCT samples were analyzed–all had previously tested positive for cocaine or opiates. Generally, the positivity rates of individual compounds were similar between the Cocaine and Opiates Subgroups, apart from levamisole, xylazine, dipyrone (metabolites), and promethazine. Many of the adulterants used in the street drug supply do have legitimate medicinal/therapeutic uses, including several of the compounds most frequently detected in this study. Caffeine and lidocaine were the most frequently identified compounds both individually (>70% each) and in combination with each other. Alternatively, levamisole, an adulterant with no legitimate therapeutic use, was present in 12% of cases. Importantly, this data demonstrates that the detection of traditional drugs of abuse may serve as indicators of potential in utero exposure to toxic adulterating substances during gestation. While there is cause for concern with respect to any unintentional drug exposure, illicit drug use during pregnancy, including uncontrolled dosing, poly-adulterant consumption, and the interactions of these drug mixtures, produces a significant public health threat to the neonate which warrants further study.

Ruff et al. demonstrate that phosphorylation of the androgen receptor and 14-3-3 ζ by PIM1 kinase coordinates their interaction and chromatin occupancy at genes involved in cell migration and invasion. The authors conclude that PIM1 phosphorylation of these substrates regulates the AR transcriptome in prostate cancer cells.

Abstract Background The autonomous retroelement Long Interspersed Element-1 (LINE-1) mobilizes though a copy and paste mechanism using an RNA intermediate (retrotransposition). Throughout human evolution, around 500,000 LINE-1 sequences have accumulated in the genome. Most of these sequences belong to ancestral LINE-1 subfamilies, including L1PA2-L1PA7, and can no longer mobilize. Only a small fraction of LINE-1 sequences, approximately 80 to 100 copies belonging to the L1Hs subfamily, are complete and still capable of retrotransposition. While silenced in most cells, many questions remain regarding LINE-1 dysregulation in cancer cells. Results Here, we optimized CRISPR Cas9 gRNAs to specifically target the regulatory sequence of the L1Hs 5’UTR promoter. We identified three gRNAs that were more specific to L1Hs, with limited binding to older LINE-1 sequences (L1PA2-L1PA7). We also adapted the C-BERST method (dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging) to identify LINE-1 transcriptional regulators in cancer cells. Our LINE-1 C-BERST screen revealed both known and novel LINE-1 transcriptional regulators, including CTCF, YY1 and DUSP1. Conclusion Our optimization and evaluation of gRNA specificity and application of the C-BERST method creates a tool for studying the regulatory mechanisms of LINE-1 in cancer. Further, we identified the dual specificity protein phosphatase, DUSP1, as a novel regulator of LINE-1 transcription.

The nuclear factor-Kappa B (NF-κB) pathway is a crucial mediator of inflammatory signaling. Aberrant activation of NF-κB is associated with several disorders including preeclampsia (PE). Many regulators of the NF-κB pathway have been identified, including microRNAs (miRNAs). Specifically, miR-517-3p targets mRNA encoding TNFAIP3 Interacting Protein 1 (TNIP1), an inhibitor of NF-κB signaling. Activation of NF-κB increases production of the cytokine TNF superfamily member 15 (TNFSF15), leading to the upregulation of anti-angiogenic soluble vascular endothelial growth factor receptor 1 (sFlt-1). We have previously observed that Cajal bodies (CBs), subnuclear domains, are associated with the chromosome 19 miRNA gene cluster (C19MC), which encodes miR-517-3p. We have also found that coilin, the CB marker protein, is a positive regulator of miRNA biogenesis. Here we report that coilin is a regulator of miR-517-3p, sFlt-1, TNIP1, TNFSF15 and NF-κB activation, and this regulation is influenced by hypoxia. We also report that coilin and CBs are induced in the reduced uterine perfusion pressure (RUPP) rat model of PE. Collectively, the data presented here implicate coilin as a novel regulator of NF-κB activation and sFlt-1 upregulation.

To manage excessive vine vigor, Vitis vinifera L. ‘Cabernet franc’ grapevines were subjected to shoot wrap, shoot tuck, and hedge (control) techniques at one of two growth stages (shoot tips at 30 cm or at 90 cm above the top catch wire) in the Finger Lakes region of New York from 2016 to 2019. Shoot tuck and shoot wrap both reduced fruit zone lateral counts, with reductions up to 33% and 56% compared with the control, respectively. Shoot wrap reduced fruit zone lateral lengths by up to 50% and cluster compactness by up to 2.4 fewer berries per centimeter rachis. Although shoot wrap improved spray penetration to the clusters by up to 28% in one year of the study, enhanced point quadrat analysis indicated that occlusion layer number was not affected by the treatments. Shoot tip management treatments did not affect yield or fruit composition consistently. Phenological timing of shoot tip management had little impact on vine growth. Although the impacts of these modified shoot tip management practices on lateral emergence and cluster morphology were generally positive, the required hand labor to apply the treatments on a large scale may discourage the use of these management practices.

Abstract Background Long INterspersed Element-1 (LINE-1) is an autonomous retroelement able to “copy-and-paste” itself into new loci of the host genome through a process called retrotransposition. The LINE-1 bicistronic mRNA codes for two proteins, ORF1p, a nucleic acid chaperone, and ORF2p, a protein with endonuclease and reverse transcriptase activity. Both proteins bind LINE-1 mRNA in cis and are necessary for retrotransposition. While LINE-1 transcription is usually repressed in most healthy somatic cells through a plethora of mechanisms, ORF1p expression has been observed in nearly 50% of tumors, and new LINE-1 insertions have been documented in a similar fraction of tumors, including prostate cancer. Results Here, we utilized RNA ImmunoPrecipitation (RIP) and the L1EM analysis software to identify ORF1p bound RNA in prostate cancer cells. We identified LINE-1 loci that were expressed in parental androgen sensitive and androgen independent clonal derivatives. In all androgen independent cells, we found higher levels of LINE-1 RNA, as well as unique expression patterns of LINE-1 loci. Interestingly, we observed that ORF1p bound many non-LINE-1 mRNA in all prostate cancer cell lines evaluated, and polyA RNA, and RNA localized in p-bodies were especially enriched. Furthermore, the expression levels of RNAs identified in our ORF1p RIP correlated with RNAs expressed in LINE-1 positive tumors from The Cancer Genome Atlas (TCGA). Conclusion Our results show a significant remodeling of LINE-1 loci expression in androgen independent cell lines when compared to parental androgen dependent cells. Additionally, we found that ORF1p bound a significant amount of non-LINE-1 mRNA, and that the enriched ORF1p bound mRNAs are also amplified in LINE-1 expressing TCGA prostate tumors, indicating the biological relevance of our findings to prostate cancer.

Through a chemical genetics screen and phospho-proteomics, Ledet and Ruff et al identify substrates of the oncogenic kinase PIM1 in prostate cancer cells. They find that NDRG1 protein is destabilized by PIM1-mediated phosphorylation to reduce its metastasis suppressor function. These findings shed light on the role of PIM1 in prostate cancer.

Abstract Background We previously examined expression of Long Interspersed Element-1 (LINE-1) in a variety of prostate cancer cells including hormone-dependent LNCaP cells. These studies demonstrated expression and sub-cellular localization of LINE-1 proteins, ORF1p, with antibody 4H1, and ORF2p, with antibody chA1-L1. Results Here we conduct immunoprecipitation/mass spectrometry analysis using chA1-L1 antibody against ORF2p in LNCaP cells. Our results indicate that antigens detected by the antibody include the transcriptional regulator BCLAF1. We show that chA1-L1 recognizes BCLAF1 using siRNA knockdown and overexpression of a tagged BCLAF1. We also show that chA1-L1 antibody recognizes ORF2p in HEK293 cells overexpressing LINE-1. Further, analysis of ORF2p (chA1-L1) and BCLAF1 foci using immunofluorescence in LNCaP cells showed significant colocalization. Conclusions Overall, our findings indicate that chA1-L1 antibody recognizes both BCLAF1 and ORF2p but the majority of antigen recognized in LNCaP cells is BCLAF1.

Many terrestrial and aquatic taxa are known to form periodic aggregations, whether across life history or solely during specific life stages, that are generally governed by the availability and distribution of resources. Associations between individuals during such aggregation events are considered random and not driven by social attraction or underlying community structure. White sharks (Carcharodon carcharias) have been described as a species that exhibits resource-driven aggregative behaviors across ontogenetic stages and juvenile white sharks are known to form aggregations at specific nursery sites where individuals may remain for extended periods of time in the presence of other individuals. We hypothesized juvenile white sharks form distinct communities during these critical early phases of ontogeny and discuss how a tendency to co-occur across life stages may be seeded by the formation of these communities in early ontogeny. We present results from a series of social network analyses of 86 juvenile white sharks derived from 6 years of passive acoustic telemetry data in southern California, demonstrating the likelihood of association of tagged juvenile white sharks is greater when sharks are of similar size-classes. Individuals in observed networks exhibited behaviors that best approximated fission-fusion dynamics with spatiotemporally unstable group membership. These results provide evidence of possible non-resource driven co-occurrence and community structure in juvenile white sharks during early life stages.

Abstract Introduction The pandemic of extended-spectrum beta-lactamase-(ESBL)-producing Enterobacteriaceae (Ent) is strongly linked to the dissemination of CTX-M-type-ESBL-Ent. We sought to define the epidemiology of infections in children due to an emerging resistance type, CTX-M-9-group-producing-Ent (CTX-M-9-grp-Ent). Methods A retrospective matched case-control analysis of children with CTX-M-9-grp-Ent infections who received medical care at three Chicago area hospitals was performed. Cases were defined as children possessing extended-spectrum cephalosporin-resistant (ESC-R) infections due to bla CTX-M-9. PCR and DNA analysis assessed beta-lactamase (bla) genes, multi-locus sequence types (MLST) and phylogenetic grouping of E. coli. Controls were children with ESC-susceptible (ESC-S)-Ent infections matched one case to three controls by age, source, and hospital. The clinical-epidemiologic predictors of CTX-M-9-grp-Ent infection were assessed. Results Of 356 ESC-R-Ent isolates from children (median age 4.1 years), the CTX-M-9-group was the solely detected bla gene in 44 (12.4%). The predominant species was E. coli (91%) of virulent phylogroups D (60%) and B2 (40%). MLST revealed multiple strain types. On multivariable analysis, CTX-M-9-grp-Ent occurred more often in E. coli than other Ent genera (OR 7.4, 95% CI 2.4, 27.2), children of non-Black-White-Hispanic race (OR 7.4, 95% CI 2.4, 28.2), and outpatients (OR 4.5, 95% CI 1.7, 12.3), which was a very unexpected finding for infections due to antibiotic-resistant bacteria. Residents of South Chicago had a 6.7 times higher odds of having CTX-M-9-grp-Ent infections than those in the reference region (West), while residence in Northwestern Chicago was associated with an 81% decreased odds of infection. Other demographic, comorbidity, invasive-device, and antibiotic use differences were not found. Conclusion CTX-M-9-grp-Ent infection may be associated with patient residence and is occurring in children without traditional in-patient exposure risk factors. This suggests that among children, the community environment may be a key contributor in the spread of these resistant pathogens.

In today's environment in the field of forensic science where continual advancements in technology and analytical approaches are the norm, the need for forensic practitioners with more specialized and subject-specific knowledge is critical. An updated survey targeting the preferred educational requirements by senior practitioners, crime laboratory directors and managers for entry level applicants was conducted. Results underscored a preference for specialized coursework within specific disciplines in preparing the next generation of forensic scientists while maintaining a strong foundation in the natural sciences at the undergraduate level. Practitioners, regardless of discipline, are seeking applicants with exposure to advanced curriculum content in addition to refined professional skills and critical thinking capabilities. The results of this survey reflect a transition in the needs of crime laboratory employers from a general, broad based criminalistics curriculum as described under current accreditation guidelines, to a focused subject matter rich curriculum with additional management and professional content. Keywords: Forensic science, Undergraduate education, Graduate education, Laboratory survey, Educational requirements

Small molecules and peptide inhibitors have their benefits and faults when it comes to inhibiting protein-protein interactions. Here, the authors designed a peptoid-peptide hybrid that inhibited β-catenin/TCF interactions, leading to inhibition of Wnt signalling in models of prostate cancer.

Young-of-the-year (YOY) and juvenile-stage white sharks may use southern California nearshore beach habitats more extensively than previously known, within meters of some of the most heavily used beaches in the world. Such knowledge forms a critical component of species management and conservation plans, in addition to public safety and risk mitigation planning. We used data derived from a combination of satellite tag locations (13 animals over 3 years) and passive acoustic monitoring (34 animals over 8 years) to examine the occurrence, relative abundance, and residency patterns of YOY white sharks in southern California waters. Our results suggest that southern California contains spatiotemporally dynamic centers of primary nursery habitat. Tagged YOY white sharks formed loose aggregations at “hotspot” locations that were interannually variable, where individuals exhibited temporal fidelity, higher levels of residency, and spatially restricted movements, with multiple YOY individuals simultaneously displaying this behavior. While models of biotic and abiotic variables suggested relative abundance of tagged sharks may be predicted by sea surface temperature, salinity and productivity (chlorophyll-A), these predictors were not consistent across all years of the study. Thus, novel approaches that incorporate technologies to derive high resolution environmental data, paired with more comprehensive telemetry datasets are therefore required to better understand the extrinsic factors that drive habitat selection and residency patterns in juvenile white sharks.

Infectious diseases outbreaks are a cause of significant morbidity and mortality among hospitalized patients. Infants admitted to the neonatal intensive care unit (NICU) are particularly vulnerable to infectious complications during hospitalization. Thus, rapid recognition of and response to outbreaks in the NICU is essential. At Rush University Medical Center, whole-genome sequencing (WGS) has been utilized since early 2016 as an adjunctive method for outbreak investigations. The use of WGS and potential lessons learned are illustrated for 3 different NICU outbreak investigations involving methicillin-resistant Staphylococcus aureus (MRSA), group B Streptococcus (GBS), and Serratia marcescens. WGS has contributed to the understanding of the epidemiology of outbreaks in our NICU, and it has also provided further insight in settings of unusual diseases or when lower-resolution typing methods have been inadequate. WGS has emerged as the new gold standard for evaluating strain relatedness. As barriers to implementation are overcome, WGS has the potential to transform outbreak investigation in healthcare settings.

There has been an alarming increase of novel synthetic opioids (NSOs) in the illegal drug market since 2016. These substances are still detected in forensic case investigations in biological samples as well as in seized materials. Although NSOs are steadily emerging, other potent “old” synthetic opioids are still circulating and involved in fatal overdose cases. This is the case of the potent designer drugs carfentanil and 3-methylfentanyl. These drugs were associated with several deaths since at least 20 years ago, and they are still a public health threat.In this study, we presented postmortem (PM) forensic cases that were positive for carfentanil or 3-methylfentanyl over a four-year period 2017–2020. Quantitative confirmation was via a quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method that targets 21 designer opioids, able to differentiate isomers such as cis- and trans-3-methylfentanyl, with a limit of quantification of 0.05 ng/mL.A total of 1136 and 153 PM cases were received in the laboratory from different regions of the United States positive for carfentanil and 3-methylfentanyl, respectively. PM peripheral blood ranged from 0.050‒32 ng/mL for carfentanil, and 0.057‒7.2 and 0.057‒2.2 ng/mL for cis- and trans-3-methylfentanil, respectively. During this period, fentanyl was the most common concomitant drug detected within these cases. Heroin and cocaine were also highly prevalent in those cases positive for carfentanil. Other NSOs such as 2-furanylfentanyl, cyclopropylfentanyl and para-fluorobutyrylfentanyl were also significant findings in positive carfentanil cases. For 3-methylfentanyl, cocaine was the second most prevalent co-occurrence drug, while heroin decreased over the years in those cases where 3-methylfentanyl was confirmed.

The Cajal body (CB) is a subnuclear domain that participates in the biogenesis of many different types of ribonucleoproteins (RNPs), including small nuclear RNPs (snRNPs), small Cajal body-specific RNPs (scaRNPs) and telomerase. Most scaRNAs, the RNA component of scaRNPs, accumulate in CBs. However, there are three scaRNAs (scaRNA 2, 9, and 17) that are known to be processed into small, nucleolar-enriched fragments. Evidence suggests that these fragments are packaged into a new class of RNPs, called regulatory RNPs (regRNPs), and may modify small nucleolar RNP (snoRNP) activity, thus playing a role in rRNA modification. However, the mechanism by which these fragments are produced is unknown. Previous work has reported the involvement of Drosha and DGCR8 in the cleavage of primary-scaRNA9. Here, we expand on that knowledge by identifying sequence elements necessary for the efficient production of these RNA fragments and demonstrate that primary scaRNA 2 and 17 are also processed by the Drosha-DGCR8 complex. Collectively, our work establishes new factors in the scaRNP biogenesis pathway and adds to the ever-expanding list of noncanonical functions for the microprocessor complex.

Upper trophic level predators dramatically impacted by fisheries include the large-bodied hammerhead sharks, which have become species of conservation concern worldwide. Implementing spatial management for conservation of hammerhead populations requires knowledge of temporal distribution patterns and habitat use, identification of essential habitat for protection, and quantification of interactions with human activities. There is little such information for the smooth hammerhead shark, Sphyrna zygaena. We used fin-mounted satellite tags to examine the movements and habitat use of juvenile smooth hammerheads, a demographic segment particularly threatened by exploitation. Six sharks were tagged off the US mid-Atlantic and tracked for 49–441 days (mean 187 ± 136 days). Sharks consistently showed area-restricted movements within a summer core area in waters of the New York Bight and a winter core area off Cape Hatteras, North Carolina, with directed movements between them in autumn. There was high overlap of shark winter core area use and the Mid-Atlantic Shark Area (MASA) – a 7 month per year, bottom-longline fishery closure – indicating that this area closure offers seasonal reduction in fishing pressure for this species. Based on timing of shark movements and the MASA closure, protection for juvenile smooth hammerheads may be increased by beginning the closure period 1 month earlier than currently scheduled. Generalized additive mixed models revealed that area-restricted movements of sharks in their summer and winter core areas coincided with high primary productivity, and elevated sea surface temperature. Consistency in use of summer and winter core areas suggests that the coastal waters of the New York Bight and Cape Hatteras, North Carolina could be considered for Essential Fish Habitat designation for this species. This study reveals the first high resolution movements and habitat use for smooth hammerheads in the western North Atlantic to inform management planning for this population.

Abstract Background Long Interspersed Nuclear Element-1 (LINE-1) is an autonomous retrotransposon that generates new genomic insertions through the retrotransposition of a RNA intermediate. Expression of LINE-1 is tightly repressed in most somatic tissues to prevent DNA damage and ensure genomic integrity. However, the reactivation of LINE-1 has been documented in cancer and the role of LINE-1 protein expression and retrotransposition has become of interest in the development, progression, and adaptation of many epithelial neoplasms, including prostate cancer. Results Here, we examined endogenous LINE-1 protein expression and localization in a panel of prostate cancer cells and observed a diverse range of LINE-1 expression patterns between cell lines. Subcellular localization of LINE-1 proteins, ORF1p and ORF2p, revealed distinct expression patterns. ORF1p, a nucleic acid chaperone that binds LINE-1 mRNA, was predominantly expressed in the cytoplasm, with minor localization in the nucleus. ORF2p, containing endonuclease and reverse transcriptase domains, exhibited punctate foci in the nucleus and also displayed co-localization with PCNA and γH2AX. Using a retrotransposition reporter assay, we found variations in LINE-1 retrotransposition between cell lines. Conclusions Overall, our findings reveal new insight into the expression and retrotransposition of LINE-1 in prostate cancer. The prostate cancer cells we investigated provide a unique model for investigating endogenous LINE-1 activity and provide a functional model for studying LINE-1 mechanisms in prostate cancer.

Molecular typing using repetitive sequenced-based PCR (rep-PCR) and hsp60 sequencing were applied to a collection of diverse Enterobacter cloacae complex isolates. To determine the most practical method for reference laboratories, we analyzed 71 E. cloacae complex isolates from sporadic and outbreak occurrences originating from 4 geographic areas. While rep-PCR was more discriminating, hsp60 sequencing provided a broader and a more objective geographical tracking method similar to multilocus sequence typing (MLST). In addition, we suggest that MLST may have higher discriminative power compared to hsp60 sequencing, although rep-PCR remains the most discriminative method for local outbreak investigations. In addition, rep-PCR can be an effective and inexpensive method for local outbreak investigation.

The most common types of modification in human rRNA are pseudouridylation and 2′-O ribose methylation. These modifications are performed by small nucleolar ribonucleoproteins (snoRNPs) which contain a guide RNA (snoRNA) that base pairs at specific sites within the rRNA to direct the modification. rRNA modifications can vary, generating ribosome heterogeneity. One possible method that can be used to regulate rRNA modifications is by controlling snoRNP activity. RNA fragments derived from some small Cajal body-specific RNAs (scaRNA 2, 9 and 17) may influence snoRNP activity. Most scaRNAs accumulate in the Cajal body – a subnuclear domain – where they participate in the biogenesis of small nuclear RNPs, but scaRNA 2, 9 and 17 generate nucleolus-enriched fragments of unclear function, and we hypothesize that these fragments form regulatory RNPs that impact snoRNP activity and modulate rRNA modifications. Our previous work has shown that SMN, Drosha and various stresses, including etoposide treatment, may alter regulatory RNP formation. Here we demonstrate that etoposide treatment decreases the phosphorylation of SMN, reduces Drosha levels and increases the 2′-O-methylation of two sites within 28S rRNA. These findings further support a role for SMN and Drosha in regulating rRNA modification, possibly by affecting snoRNP or regulatory RNP activity.