Misinformation and Impact on Interprofessional Healthcare Students' Knowledge, Attitudes, Perceptions, and Concerns of COVID-19.

BACKGROUND: The COVID-19 pandemic has highlighted the importance of information sources (e.g., Internet, social media) and their role in spreading misinformation. PURPOSE: To describe the information sources and frequency of use by health professional students and to compare users of dependable and nontrustworthy news sources on stressors, stress relievers, safety, and preventative activities, worries, and attitudes toward COVID-19. METHODS: 123 students from nursing (38%), medicine (33%), and health professions (28%) completed online surveys on disaster preparedness training, knowledge of the COVID-19 virus, and safety and prevention practices. Students were mostly female (81%), white (59%), and aged 21-30 yrs (72%). RESULTS: Students who relied on credible news sources scored higher on knowledge of the COVID-19 condition and reported less stress than their counterparts. CONCLUSION: The findings emphasize the importance of students avoiding untrustworthy news sources. Informed students are less stressed and can help initiate necessary safety measures in the areas they serve.

Preparing BSN students for a new workplace: Experiences in a COVID-19 designated unit as an RN extender.

Academic nursing programs were impacted by the onset of the pandemic with the challenges of social distancing, detecting, and controlling the spread of the virus. Many traditionally campus-based, face-to-face programs transitioned quickly to include virtual activities for classes, laboratories, and clinical experiences for students. Initially direct patient care clinical activities came to a halt due to concerns for student safety and overwhelming numbers of COVID-19 cases stressing health system resources. However, as COVID-19 cases began to rise and hospitals struggled with staffing shortages, it became necessary to explore how nursing students could contribute to relieving the staffing burden while obtaining critical education as RN Extenders. Students that participated in the RN Extender program were seniors entering the nursing workforce within the next two months. Significant student screening and preparation was provided to support students serving as RN Extenders in COVID-19 units. The prior working relationships and personal contact between academic and practice leaders, a sense of equal participation, clear and realistic expectations of the benefits and responsibilities of each group were crucial. Building ongoing and frequent opportunities for formal and informal communication among all stakeholders was vital and the major contributor to the success of this program.

Dynamic sumoylation of promoter-bound general transcription factors facilitates transcription by RNA polymerase II.

Transcription-related proteins are frequently identified as targets of sumoylation, including multiple subunits of the RNA polymerase II (RNAPII) general transcription factors (GTFs). However, it is not known how sumoylation affects GTFs or whether they are sumoylated when they assemble at promoters to facilitate RNAPII recruitment and transcription initiation. To explore how sumoylation can regulate transcription genome-wide, we performed SUMO ChIP-seq in yeast and found, in agreement with others, that most chromatin-associated sumoylated proteins are detected at genes encoding tRNAs and ribosomal proteins (RPGs). However, we also detected 147 robust SUMO peaks at promoters of non-ribosomal protein-coding genes (non-RPGs), indicating that sumoylation also regulates this gene class. Importantly, SUMO peaks at non-RPGs align specifically with binding sites of GTFs, but not other promoter-associated proteins, indicating that it is GTFs specifically that are sumoylated there. Predominantly, non-RPGs with SUMO peaks are among the most highly transcribed, have high levels of TFIIF, and show reduced RNAPII levels when cellular sumoylation is impaired, linking sumoylation with elevated transcription. However, detection of promoter-associated SUMO by ChIP might be limited to sites with high levels of substrate GTFs, and promoter-associated sumoylation at non-RPGs may actually be far more widespread than we detected. Among GTFs, we found that TFIIF is a major target of sumoylation, specifically at lysines 60/61 of its Tfg1 subunit, and elevating Tfg1 sumoylation resulted in decreased interaction of TFIIF with RNAPII. Interestingly, both reducing promoter-associated sumoylation, in a sumoylation-deficient Tfg1-K60/61R mutant strain, and elevating promoter-associated SUMO levels, by constitutively tethering SUMO to Tfg1, resulted in reduced RNAPII occupancy at non-RPGs. This implies that dynamic GTF sumoylation at non-RPG promoters, not simply the presence or absence of SUMO, is important for maintaining elevated transcription. Together, our findings reveal a novel mechanism of regulating the basal transcription machinery through sumoylation of promoter-bound GTFs.

Queuine, a bacterial-derived hypermodified nucleobase, shows protection in in vitro models of neurodegeneration.

Growing evidence suggests that human gut bacteria, which comprise the microbiome, are linked to several neurodegenerative disorders. An imbalance in the bacterial population in the gut of Parkinson's disease (PD) and Alzheimer's disease (AD) patients has been detected in several studies. This dysbiosis very likely decreases or increases microbiome-derived molecules that are protective or detrimental, respectively, to the human body and those changes are communicated to the brain through the so-called 'gut-brain-axis'. The microbiome-derived molecule queuine is a hypermodified nucleobase enriched in the brain and is exclusively produced by bacteria and salvaged by humans through their gut epithelium. Queuine replaces guanine at the wobble position (position 34) of tRNAs with GUN anticodons and promotes efficient cytoplasmic and mitochondrial mRNA translation. Queuine depletion leads to protein misfolding and activation of the endoplasmic reticulum stress and unfolded protein response pathways in mice and human cells. Protein aggregation and mitochondrial impairment are often associated with neural dysfunction and neurodegeneration. To elucidate whether queuine could facilitate protein folding and prevent aggregation and mitochondrial defects that lead to proteinopathy, we tested the effect of chemically synthesized queuine, STL-101, in several in vitro models of neurodegeneration. After neurons were pretreated with STL-101 we observed a significant decrease in hyperphosphorylated alpha-synuclein, a marker of alpha-synuclein aggregation in a PD model of synucleinopathy, as well as a decrease in tau hyperphosphorylation in an acute and a chronic model of AD. Additionally, an associated increase in neuronal survival was found in cells pretreated with STL-101 in both AD models as well as in a neurotoxic model of PD. Measurement of queuine in the plasma of 180 neurologically healthy individuals suggests that healthy humans maintain protective levels of queuine. Our work has identified a new role for queuine in neuroprotection uncovering a therapeutic potential for STL-101 in neurological disorders.

A brief educational intervention can improve nursing students' knowledge of the human papillomavirus vaccine and readiness to counsel.

Provider recommendation is a primary reason for patient uptake of the human papillomavirus (HPV) vaccine. Most provider-focused educational interventions are focused on physicians, even though nurses are also important sources of vaccine-related information for their patients. This study examined whether a HPV educational intervention could improve nursing students' HPV knowledge, beliefs, and comfort with counseling. The same lecture on HPV and HPV vaccination was given to both medical and nursing students. To determine the effects of the lecture, students were asked to complete identical pre- and post-lecture tests with questions on demographics, knowledge, attitudes, and comfort with counseling on the HPV vaccine. Pre- and post-lecture test scores were compared between nursing and medical students to assess whether there were differences in pre-lecture test scores and/or changes in post-lecture test scores. On the pre-lecture tests, fewer nursing students responded correctly to knowledge questions, indicated positive attitudes or comfort with counseling about the HPV vaccine compared to medical students. However, similar frequencies of nursing and medical students responded correctly to knowledge questions and indicated a positive attitude, as well as a high comfort level with counseling on the post-lecture tests. Study results show that integrating lectures in a nursing program curriculum could be a feasible way to increase students' HPV knowledge. Having health-care providers with similar levels of knowledge, attitudes, and comfort with counseling on HPV vaccination is ideal, as all share the responsibility of recommending the vaccine to patients.

SETX (senataxin), the helicase mutated in AOA2 and ALS4, functions in autophagy regulation.

SETX (senataxin) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response through resolution of R-loop structures. Mutations in SETX result in either of two distinct neurodegenerative disorders. SETX dominant mutations result in a juvenile form of amyotrophic lateral sclerosis (ALS) called ALS4, whereas recessive mutations are responsible for ataxia called ataxia with oculomotor apraxia type 2 (AOA2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed the effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX-depleted cells. Importantly, we discovered a strong connection between SETX and the macroautophagy/autophagy pathway, reflecting a direct effect on transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2.Abbreviations: 3'READS: 3' region extraction and deep sequencing; ACTB: actin beta; ALS4: amyotrophic lateral sclerosis type 4; AOA2: ataxia with oculomotor apraxia type 2; APA: alternative polyadenylation; AS: alternative splicing; ATG7: autophagy-related 7; ATP6V0D2: ATPase H+ transporting V0 subunit D2; BAF: bafilomycin A1; BECN1: beclin 1; ChIP: chromatin IP; Chloro: chloroquine; CPT: camptothecin; DDR: DNA damage response; DNMT1: DNA methyltransferase 1; DRIP: DNA/RNA IP; DSBs: double strand breaks; EBs: embryoid bodies; FTD: frontotemporal dementia; GABARAP: GABA type A receptor-associated protein; GO: gene ontology; HR: homologous recombination; HTT: huntingtin; IF: immunofluorescence; IP: immunoprecipitation; iPSCs: induced pluripotent stem cells; KD: knockdown; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; PASS: PolyA Site Supporting; PFA: paraformaldehyde; RNAPII: RNA polymerase II; SCA: spinocerebellar ataxia; SETX: senataxin; SMA: spinal muscular atrophy; SMN1: survival of motor neuron 1, telomeric; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TSS: transcription start site; TTS: transcription termination site; ULK1: unc-51 like autophagy activating kinase 1; WB: western blot; WIPI2: WD repeat domain, phosphoinositide interacting 2; XRN2: 5'-3' exoribonuclease 2.

The C9ORF72 Gene, Implicated in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia, Encodes a Protein That Functions in Control of Endothelin and Glutamate Signaling.

A GGGGCC repeat expansion in the C9ORF72 (C9) gene is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Several mechanisms have been proposed to account for its toxicity, including the possibility that reduced C9 protein levels contribute to disease. To investigate this possibility, we examined the effects of reduced C9 levels in several cell systems. We first showed that C9 knockdown (KD) in U87 glioblastoma cells results in striking morphological changes, including vacuolization and alterations in cell size. Unexpectedly, RNA analysis revealed changes in expression of many genes, including genes involved in endothelin (EDN) signaling and immune system pathways and multiple glutamate cycling genes (e.g., EAAT2), which were verified in several cell models, including astrocytes and brain samples from C9-positive patients. Consistent with deregulation of the glutamate cycling genes, elevated intracellular glutamate was detected in both KD cells and patient astrocytes. Importantly, levels of mRNAs encoding EDN1 and its receptors, known to be elevated in ALS, were sharply increased by C9 KD, likely resulting from an observed activation of NF-κB signaling and/or a possible role of a C9 isoform in gene control.

NRDE-2, the human homolog of fission yeast Nrl1, prevents DNA damage accumulation in human cells.

The RNA helicase Mtr4 is a versatile protein that is a crucial component of several distinct RNA surveillance complexes. Here we describe a novel complex that contains Mtr4, but has a role distinct from any of those previously described. We found that Mtr4 association with the human homolog of fission yeast Nrl1, NRDE-2, defines a novel function for Mtr4 in the DNA damage response pathway. We provide biochemical evidence that Mtr4 and NRDE-2 are part of the same complex and show that both proteins play a role in the DNA damage response by maintaining low DNA double-strand break levels. Importantly, the DNA damage response function of the Mtr4/NRDE-2 complex does not depend on the formation of R loops. We show however that NRDE-2 and Mtr4 can affect R-loop signals at a subset of distinct genes, possibly regulating their expression. Our work not only expands the wide range of Mtr4 functions, but also elucidates an important role of the less characterized human NRDE-2 protein.

An Mtr4/ZFC3H1 complex facilitates turnover of unstable nuclear RNAs to prevent their cytoplasmic transport and global translational repression.

Many long noncoding RNAs (lncRNAs) are unstable and rapidly degraded in the nucleus by the nuclear exosome. An exosome adaptor complex called NEXT (nuclear exosome targeting) functions to facilitate turnover of some of these lncRNAs. Here we show that knockdown of one NEXT subunit, Mtr4, but neither of the other two subunits, resulted in accumulation of two types of lncRNAs: prematurely terminated RNAs (ptRNAs) and upstream antisense RNAs (uaRNAs). This suggested a NEXT-independent Mtr4 function, and, consistent with this, we isolated a distinct complex containing Mtr4 and the zinc finger protein ZFC3H1. Strikingly, knockdown of either protein not only increased pt/uaRNA levels but also led to their accumulation in the cytoplasm. Furthermore, all pt/uaRNAs examined associated with active ribosomes, but, paradoxically, this correlated with a global reduction in heavy polysomes and overall repression of translation. Our findings highlight a critical role for Mtr4/ZFC3H1 in nuclear surveillance of naturally unstable lncRNAs to prevent their accumulation, transport to the cytoplasm, and resultant disruption of protein synthesis.

Roles of Sumoylation in mRNA Processing and Metabolism.

SUMO has gained prominence as a regulator in a number of cellular processes. The roles of sumoylation in RNA metabolism, however, while considerable, remain less well understood. In this chapter we have assembled data from proteomic analyses, localization studies and key functional studies to extend SUMO's role to the area of mRNA processing and metabolism. Proteomic analyses have identified multiple putative sumoylation targets in complexes functioning in almost all aspects of mRNA metabolism, including capping, splicing and polyadenylation of mRNA precursors. Possible regulatory roles for SUMO have emerged in pre-mRNA 3' processing, where SUMO influences the functions of polyadenylation factors and activity of the entire complex. SUMO is also involved in regulating RNA editing and RNA binding by hnRNP proteins, and recent reports have suggested the involvement of the SUMO pathway in mRNA export. Together, these reports suggest that SUMO is involved in regulation of many aspects of mRNA metabolism and hold the promise for exciting future studies.

R Loops and Links to Human Disease.

Aberrant R-loop structures are increasingly being realized as an important contributor to human disease. R loops, which are mainly co-transcriptional, abundant RNA/DNA hybrids, form naturally and can indeed be beneficial for transcription regulation at certain loci. However, their unwanted persistence elsewhere or in particular situations can lead to DNA double-strand breaks, chromosome rearrangements, and hypermutation, which are all sources of genomic instability. Mutations in genes involved in R-loop resolution or mutations leading to R-loop formation at specific genes affect the normal physiology of the cell. We discuss here the examples of diseases for which a link with R loops has been described, as well as how disease-causing mutations might participate in the development and/or progression of diseases that include repeat-associated conditions, other neurological disorders, and cancers.

XRN2 Links Transcription Termination to DNA Damage and Replication Stress.

XRN2 is a 5'-3' exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability.

Probing the effect of force on HIV-1 receptor CD4.

Cell-surface proteins are central for the interaction of cells with their surroundings and are also associated with numerous diseases. These molecules are exposed to mechanical forces, but the exact relation between force and the functions and pathologies associated with cell-surface proteins is unclear. An important cell-surface protein is CD4, the primary receptor of HIV-1. Here we show that mechanical force activates conformational and chemical changes on CD4 that may be important during viral attachment. We have used single-molecule force spectroscopy and analysis on HIV-1 infectivity to demonstrate that the mechanical extension of CD4 occurs in a time-dependent manner and correlates with HIV-1 infectivity. We show that Ibalizumab, a monoclonal antibody that blocks HIV-1, prevents the mechanical extension of CD4 domains 1 and 2. Furthermore, we demonstrate that thiol/disulfide exchange in CD4 requires force for exposure of cryptic disulfide bonds. This mechanical perspective provides unprecedented information that can change our understanding on how viruses interact with their hosts.

SETX sumoylation: A link between DNA damage and RNA surveillance disrupted in AOA2.

Senataxin (SETX) is a putative RNA:DNA helicase that is mutated in two distinct juvenile neurological disorders, AOA2 and ALS4. SETX is involved in the response to oxidative stress and is suggested to resolve R loops formed at transcription termination sites or at sites of collisions between the transcription and replication machineries. R loops are hybrids between RNA and DNA that are believed to lead to DNA damage and genomic instability. We discovered that Rrp45, a core component of the exosome, is a SETX-interacting protein and that the interaction depends on modification of SETX by sumoylation. Importantly, we showed that AOA2 but not ALS4 mutations prevented both SETX sumoylation and the Rrp45 interaction. We also found that upon replication stress induction, SETX and Rrp45 co-localize in nuclear foci that constitute sites of R-loop formation generated by transcription and replication machinery collisions. We suggest that SETX links transcription, DNA damage and RNA surveillance, and discuss here how this link can be relevant to AOA2 disease.

Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.

A SUMO-dependent interaction between Senataxin and the exosome, disrupted in the neurodegenerative disease AOA2, targets the exosome to sites of transcription-induced DNA damage.

Senataxin (SETX) is an RNA/DNA helicase implicated in transcription termination and the DNA damage response and is mutated in two distinct neurological disorders: AOA2 (ataxia oculomotor apraxia 2) and ALS4 (amyotrophic lateral sclerosis 4). Here we provide evidence that Rrp45, a subunit of the exosome, associates with SETX in a manner dependent on SETX sumoylation. We show that the interaction and SETX sumoylation are disrupted by SETX mutations associated with AOA2 but not ALS4. Furthermore, Rrp45 colocalizes with SETX in distinct foci upon induction of transcription-related DNA damage. Our results thus provide evidence for a SUMO-dependent interaction between SETX and the exosome, disrupted in AOA2, that targets the exosome to sites of DNA damage.

Variability of 13C-labeling in plant leaves.

RATIONALE: Plant tissues artificially labeled with (13)C are increasingly used in environmental studies to unravel biogeochemical and ecophysiological processes. However, the variability of (13)C-content in labeled tissues has never been carefully investigated. Hence, this study aimed at documenting the variability of (13)C-content in artificially labeled leaves. METHODS: European beech and Italian ryegrass were subjected to long-term (13)C-labeling in a controlled-environment growth chamber. The (13)C-content of the leaves obtained after several months labeling was determined by isotope ratio mass spectrometry. RESULTS: The (13)C-content of the labeled leaves exhibited inter- and intra-leaf variability much higher than those naturally occurring in unlabeled plants, which do not exceed a few per mil. This variability was correlated with labeling intensity: the isotope composition of leaves varied in ranges of ca 60‰ and 90‰ for experiments that led to average leaf (13)C-content of ca +15‰ and +450‰, respectively. CONCLUSIONS: The reported variability of isotope composition in (13)C-enriched leaves is critical, and should be taken into account in subsequent experimental investigations of environmental processes using (13)C-labeled plant tissues.

Reducing and correcting for contamination of ecosystem water stable isotopes measured by isotope ratio infrared spectroscopy.

Concern exists about the suitability of laser spectroscopic instruments for the measurement of the (18)O/(16)O and (2)H/(1)H values of liquid samples other than pure water. It is possible to derive erroneous isotope values due to optical interference by certain organic compounds, including some commonly present in ecosystem-derived samples such as leaf or soil waters. Here we investigated the reliability of wavelength-scanned cavity ring-down spectroscopy (CRDS) (18)O/(16)O and (2)H/(1)H measurements from a range of ecosystem-derived waters, through comparison with isotope ratio mass spectrometry (IRMS). We tested the residual of the spectral fit S(r) calculated by the CRDS instrument as a means to quantify the difference between the CRDS and IRMS δ-values. There was very good overall agreement between the CRDS and IRMS values for both isotopes, but differences of up to 2.3‰ (δ(18)O values) and 23‰ (δ(2)H values) were observed in leaf water extracts from Citrus limon and Alnus cordata. The S(r) statistic successfully detected contaminated samples. Treatment of Citrus leaf water with activated charcoal reduced, but did not eliminate, δ(2)H(CRDS) - δ(2)H(IRMS) linearly for the tested range of 0-20% charcoal. The effect of distillation temperature on the degree of contamination was large, particularly for δ(2)H values but variable, resulting in positive, negative or no correlation with distillation temperature. S(r) and δ(CRDS) - δ(IRMS) were highly correlated, in particular for δ(2)H values, across the range of samples that we tested, indicating the potential to use this relationship to correct the δ-values of contaminated plant water extracts. We also examined the sensitivity of the CRDS system to changes in the temperature of its operating environment. We found that temperature changes ≥4 °C for δ(18)O values and ≥10 °C for δ(2)H values resulted in errors larger than the CRDS precision for the respective isotopes and advise the use of such instruments only in sufficiently temperature-stabilised environments.