Temperature-dependent RNA editing in octopus extensively recodes the neural proteome.

In poikilotherms, temperature changes challenge the integration of physiological function. Within the complex nervous systems of the behaviorally sophisticated coleoid cephalopods, these problems are substantial. RNA editing by adenosine deamination is a well-positioned mechanism for environmental acclimation. We report that the neural proteome of Octopus bimaculoides undergoes massive reconfigurations via RNA editing following a temperature challenge. Over 13,000 codons are affected, and many alter proteins that are vital for neural processes. For two highly temperature-sensitive examples, recoding tunes protein function. For synaptotagmin, a key component of Ca2+-dependent neurotransmitter release, crystal structures and supporting experiments show that editing alters Ca2+ binding. For kinesin-1, a motor protein driving axonal transport, editing regulates transport velocity down microtubules. Seasonal sampling of wild-caught specimens indicates that temperature-dependent editing occurs in the field as well. These data show that A-to-I editing tunes neurophysiological function in response to temperature in octopus and most likely other coleoids.

Shame and Guilt in EMS: A Qualitative Analysis of Culture and Attitudes in Prehospital Emergency Care.

STUDY OBJECTIVES: The shame reaction is a highly negative emotional reaction shown to have long-term deleterious effects on the mental health of clinicians. Prior studies have focused on in-hospital personnel, but very little is known about what drives shame reactions in emergency medical services (EMS), a field with very high rates of post-traumatic stress disorder, burnout, anxiety, and depression. The objective of this study was to describe emotions, processes, and resilience associated with self-identified adverse events in the work of prehospital clinicians. METHODS: We conducted a qualitative study using a modified critical incident technique. Participants were recruited from two EMS agencies in North Carolina: one urban and one rural. They provided an open-ended, written reflection in which they were asked to self-identify particular events in their EMS careers that felt emotionally difficult. In-person or video in-depth interviews about these events were then conducted in a semi-structured fashion using an iterative interview guide. The codebook was developed through a mix of inductive and deductive analysis strategies and discussed within the research team and a content expert for validation. Interviews were transcribed and data were analyzed following a thematic content analysis approach for types of cases identified as emotionally difficult, common emotional responses and coping mechanisms, and the lingering effects of these experiences on study subjects. RESULTS: Eight interviews were conducted with EMS personnel: five from an urban agency and three from a rural agency. Participants commonly identified complex medical cases as being emotionally difficult, which led to the most robust shame reactions. Shame reactions were more common when EMS clinicians committed self-perceived errors in patient care, whereas guilt reactions were more common when patient outcomes seemed "inevitable" despite any intervention. Common themes related to coping mechanisms included both personal mechanisms, which tended to be less successful compared to interpersonal mechanisms, particularly when emotions were shared with colleagues. This reflected a perceived culture change within EMS in which sharing emotions with colleagues was seen as a departure from the "old school" where emotions tended to be kept to oneself. Feelings of inadequacy, low self-worth, and being "not good enough" were frequently identified as lingering emotions after difficult cases that were hard to move on from, corresponding to longstanding shame in these clinicians. Recovery and resilience varied but tended to be positively associated with a culture in which sharing with colleagues was encouraged, along with personal introspection on root causes for the sentinel event. CONCLUSION: EMS clinicians often identify complex patient cases as those leading to emotions such as shame and guilt, with shame reactions being more common when a perceived error was committed. Coping mechanisms were varied, but individuals often relied on their coworkers in a sharing environment to adequately process their negative feelings, which was seen as a departure from past practices in EMS personnel. Our hope is that future studies will be able to use these findings to identify targets for intervention on negative mental health outcomes in EMS personnel.

Diversity, Equity, and Inclusion in the United States Emergency Medical Services Workforce: A Scoping Review.

OBJECTIVE: Emergency medical services (EMS) workforce demographics in the United States do not reflect the diversity of the population served. Despite some efforts by professional organizations to create a more representative workforce, little has changed in the last decade. This scoping review aims to summarize existing literature on the demographic composition, recruitment, retention, and workplace experience of underrepresented groups within EMS. METHODS: Peer-reviewed studies were obtained from a search of PubMed, CINAHL, Web of Science, ProQuest Thesis and Dissertations, and non-peer-reviewed ("gray") literature from 1960 to present. Abstracts and included full-text articles were screened by two independent reviewers trained on inclusion/exclusion criteria. Studies were included if they pertained to the demographics, training, hiring, retention, promotion, compensation, or workplace experience of underrepresented groups in United States EMS by race, ethnicity, sexual orientation, or gender. Studies of non-EMS fire department activities were excluded. Disputes were resolved by two authors. A single reviewer screened the gray literature. Data extraction was performed using a standardized electronic form. Results were summarized qualitatively. RESULTS: We identified 87 relevant full-text articles from the peer-reviewed literature and 250 items of gray literature. Primary themes emerging from peer-reviewed literature included workplace experience (n = 48), demographics (n = 12), workforce entry and exit (n = 8), education and testing (n = 7), compensation and benefits (n = 5), and leadership, mentorship, and promotion (n = 4). Most articles focused on sex/gender comparisons (65/87, 75%), followed by race/ethnicity comparisons (42/87, 48%). Few articles examined sexual orientation (3/87, 3%). One study focused on telecommunicators and three included EMS physicians. Most studies (n = 60, 69%) were published in the last decade. In the gray literature, media articles (216/250, 86%) demonstrated significant industry discourse surrounding these primary themes. CONCLUSIONS: Existing EMS workforce research demonstrates continued underrepresentation of women and nonwhite personnel. Additionally, these studies raise concerns for pervasive negative workplace experiences including sexual harassment and factors that negatively affect recruitment and retention, including bias in candidate testing, a gender pay gap, and unequal promotion opportunities. Additional research is needed to elucidate recruitment and retention program efficacy, the demographic composition of EMS leadership, and the prevalence of racial harassment and discrimination in this workforce.

Time- and dose-dependent volume decreases in subcortical grey matter structures of glioma patients after radio(chemo)therapy.

Background and purpose: Radiotherapy (RT) is an adjuvant treatment option for glioma patients. Side effects include tissue atrophy, which might be a contributing factor to neurocognitive decline after treatment. The goal of this study was to determine potential atrophy of the hippocampus, amygdala, thalamus, putamen, pallidum and caudate nucleus in glioma patients having undergone magnetic resonance imaging (MRI) before and after RT. Materials and methods: Subcortical volumes were measured using T1-weighted MRI from patients before RT (N = 91) and from longitudinal follow-ups acquired in three-monthly intervals (N = 349). The volumes were normalized to the baseline values, while excluding structures touching the clinical target volume (CTV) or abnormal tissue seen on FLAIR imaging. A multivariate linear effects model was used to determine if time after RT and mean RT dose delivered to the corresponding structures were significant predictors of tissue atrophy. Results: The hippocampus, amygdala, thalamus, putamen, and pallidum showed significant atrophy after RT as function of both time after RT and mean RT dose delivered to the corresponding structure. Only the caudate showed no dose or time dependant atrophy. Conversely, the hippocampus was the structure with the highest atrophy rate of 5.2 % after one year and assuming a mean dose of 30 Gy. Conclusion: The hippocampus showed the highest atrophy rates followed by the thalamus and the amygdala. The subcortical structures here found to decrease in volume indicative of radiosensitivity should be the focus of future studies investigating the relationship between neurocognitive decline and RT.

Ultra-photostable DNA FluoroCubes: Mechanism of Photostability and Compatibility with FRET and Dark Quenching.

DNA-based FluoroCubes were recently developed as a solution to photobleaching, a ubiquitous limitation of fluorescence microscopy (Niekamp; ; Stuurman; ; Vale Nature Methods, 2020). FluoroCubes, that is, compact ∼4 × 4 × 5.4 nm3 four-helix bundles coupled to ≤6 fluorescent dyes, remain fluorescent up to ∼50× longer than single dyes and emit up to ∼40× as many photons. The current work answers two important questions about the FluoroCubes. First, what is the mechanism by which photostability is enhanced? Second, are FluoroCubes compatible with Förster resonance energy transfer (FRET) and similar techniques? We use single particle photobleaching studies to show that photostability arises through interactions between the fluorophores and the four-helix DNA bundle. Supporting this, we discover that smaller ∼4 × 4 × 2.7 nm3 FluoroCubes also confer ultraphotostability. However, we find that certain dye-dye interactions negatively impact FluoroCube performance. Accordingly, 4-dye FluoroCubes lacking these interactions perform better than 6-dye FluoroCubes. We also demonstrate that FluoroCubes are compatible with FRET and dark quenching applications.

Overestimation of grey matter atrophy in glioblastoma patients following radio(chemo)therapy.

OBJECTIVE: Brain atrophy has the potential to become a biomarker for severity of radiation-induced side-effects. Particularly brain tumour patients can show great MRI signal changes over time caused by e.g. oedema, tumour progress or necrosis. The goal of this study was to investigate if such changes affect the segmentation accuracy of normal appearing brain and thus influence longitudinal volumetric measurements. MATERIALS AND METHODS: T1-weighted MR images of 52 glioblastoma patients with unilateral tumours acquired before and three months after the end of radio(chemo)therapy were analysed. GM and WM volumes in the contralateral hemisphere were compared between segmenting the whole brain (full) and the contralateral hemisphere only (cl) with SPM and FSL. Relative GM and WM volumes were compared using paired t tests and correlated with the corresponding mean dose in GM and WM, respectively. RESULTS: Mean GM atrophy was significantly higher for full segmentation compared to cl segmentation when using SPM (mean ± std: ΔVGM,full = - 3.1% ± 3.7%, ΔVGM,cl = - 1.6% ± 2.7%; p < 0.001, d = 0.62). GM atrophy was significantly correlated with the mean GM dose with the SPM cl segmentation (r = - 0.4, p = 0.004), FSL full segmentation (r = - 0.4, p = 0.004) and FSL cl segmentation (r = -0.35, p = 0.012) but not with the SPM full segmentation (r = - 0.23, p = 0.1). CONCLUSIONS: For accurate normal tissue volume measurements in brain tumour patients using SPM, abnormal tissue needs to be masked prior to segmentation, however, this is not necessary when using FSL.

Validity of near-infrared light transillumination for the assessment of proximal caries in permanent teeth.

PURPOSE: The aim of this study was to evaluate the performance of DIAGNOcam (DC) in diagnosing proximal caries and to compare its effectiveness with the International Caries Detection and Assessment System (ICDAS) and bitewing radiography (BWR). METHODS: 118 premolars extracted for orthodontic reasons were included and examined using three detection methods and validated by histological sections as the gold standard. The sensitivity, specificity and areas under the ROC curve (Az value) at the outer half enamel (D1), inner half enamel (D2) and dentine (D3) thresholds were compared between different methods. RESULTS: At all categories, the specificity of DC was almost as high as ICDAS and BWR. DC showed a significantly higher sensitivity (0.68) than both visual (0.33) and radiographic examination (0.47) at the D1 threshold. DC presented the highest Az value (area under the ROC curve) at the D1 and D2 threshold (0.81, 0.86), while BWR showed the greatest Az values at D3 (0.94). Furthermore, DC had the highest association strength with the gold standard (Spearman's ρ = 0.80). CONCLUSIONS: It can be concluded that DC could detect proximal caries effectively and showed comparable or even better performance than ICDAS and BWR.

Poly (ADP-Ribose) Polymerase 1 Regulates Cajal-Retzius Cell Development and Neural Precursor Cell Adhesion.

Poly (ADP-ribose) polymerase 1 (PARP1) is a ubiquitously expressed enzyme that regulates DNA damage repair, cell death, inflammation, and transcription. PARP1 functions by adding ADP-ribose polymers (PAR) to proteins including itself, using NAD+ as a donor. This post-translational modification known as PARylation results in changes in the activity of PARP1 and its substrate proteins and has been linked to the pathogenesis of various neurological diseases. PARP1 KO mice display schizophrenia-like behaviors, have impaired memory formation, and have defects in neuronal proliferation and survival, while mutations in genes that affect PARylation have been associated with intellectual disability, psychosis, neurodegeneration, and stroke in humans. Yet, the roles of PARP1 in brain development have not been extensively studied. We now find that loss of PARP1 leads to defects in brain development and increased neuronal density at birth. We further demonstrate that PARP1 loss increases the expression levels of genes associated with neuronal migration and adhesion in the E15.5 cerebral cortex, including Reln. This correlates with an increased number of Cajal-Retzius (CR) cells in vivo and in cultures of embryonic neural progenitor cells (NPCs) derived from the PARP1 KO cortex. Furthermore, PARP1 loss leads to increased NPC adhesion to N-cadherin, like that induced by experimental exposure to Reelin. Taken together, these results uncover a novel role for PARP1 in brain development, i.e., regulation of CR cells, neuronal density, and cell adhesion.

KRAS mutation effects on the 2-[18F]FDG PET uptake of colorectal adenocarcinoma metastases in the liver.

BACKGROUND: Deriving individual tumor genomic characteristics from patient imaging analysis is desirable. We explore the predictive value of 2-[18F]FDG uptake with regard to the KRAS mutational status of colorectal adenocarcinoma liver metastases (CLM). METHODS: 2-[18F]FDG PET/CT images, surgical pathology and molecular diagnostic reports of 37 patients who underwent PET/CT-guided biopsy of CLM were reviewed under an IRB-approved retrospective research protocol. Sixty CLM in 39 interventional PET scans of the 37 patients were segmented using two different auto-segmentation tools implemented in different commercially available software packages. PET standard uptake values (SUV) were corrected for: (1) partial volume effect (PVE) using cold wall-corrected contrast recovery coefficients derived from phantom spheres with variable diameter and (2) variability of arterial tracer supply and variability of uptake time after injection until start of PET scan derived from the tumor-to-blood standard uptake ratio (SUR) approach. The correlations between the KRAS mutational status and the mean, peak and maximum SUV were investigated using Student's t test, Wilcoxon rank sum test with continuity correction, logistic regression and receiver operation characteristic (ROC) analysis. These correlation analyses were also performed for the ratios of the mean, peak and maximum tumor uptake to the mean blood activity concentration at the time of scan: SURMEAN, SURPEAK and SURMAX, respectively. RESULTS: Fifteen patients harbored KRAS missense mutations (KRAS+), while another 3 harbored KRAS gene amplification. For 31 lesions, the mutational status was derived from the PET/CT-guided biopsy. The Student's t test p values for separating KRAS mutant cases decreased after applying PVE correction to all uptake metrics of each lesion and when applying correction for uptake time variability to the SUR metrics. The observed correlations were strongest when both corrections were applied to SURMAX and when the patients harboring gene amplification were grouped with the wild type: p ≤ 0.001; ROC area under the curve = 0.77 and 0.75 for the two different segmentations, respectively, with a mean specificity of 0.69 and sensitivity of 0.85. CONCLUSION: The correlations observed after applying the described corrections show potential for assigning probabilities for the KRAS missense mutation status in CLM using 2-[18F]FDG PET images.

Observation of the pη

Data on the beam asymmetry Σ in the photoproduction of η mesons off protons are reported for tagged photon energies from 1130 to 1790 MeV (mass range from W=1748 MeV to W=2045 MeV). The data cover the full solid angle that allows for a precise moment analysis. For the first time, a strong cusp effect in a polarization observable has been observed that is an effect of a branch-point singularity at the pη^{'} threshold [E_{γ}=1447 MeV (W=1896 MeV)]. The latest BnGa partial wave analysis includes the new beam asymmetry data and yields a strong indication for the N(1895)1/2^{-} nucleon resonance, demonstrating the importance of including all singularities for a correct determination of partial waves and resonance parameters.

Maximal strength training in patients with Parkinson's disease: impact on efferent neural drive, force-generating capacity, and functional performance.

Parkinson's disease (PD) is characterized by progressive neurological deterioration, typically accompanied by reductions in skeletal muscle force-generating capacity (FGC) and functional performance. Physical activity has the potential to counteract this debilitating outcome, however, it is elusive if high-intensity strength training included in conventional treatment may improve results. Therefore, we randomly assigned 22 PD patients (74 ± 9 yr) to conventional rehabilitation with or without maximal strength training (MST) performed as leg press and chest press at ~90% of one repetition maximum (1RM), five times per week for 4 wk. FGC, physical performance, and efferent neural drive assessed as evoked potentials (V-wave normalized to M-wave in m. soleus) were measured following training. Results revealed that only MST improved 1RM leg press (101 ± 23 to 118 ± 18 kg) and chest press (36 ± 15 to 41 ± 15 kg), plantar flexion maximal voluntary contraction (235 ± 125 to 293 ± 158 N·m), and rate of force development (373 ± 345 to 495 ± 446 N·m·s-1; all P < 0.05; different from controls P < 0.05). FGC improvements were accompanied by an increased efferent neural drive to maximally contracting musculature (V-to-M ratio: 0.17 ± 0.12 to 0.24 ± 0.15; P < 0.05; different from controls P < 0.05), improved physical performance (stair climbing: 21.0 ± 9.2 to 14.4 ± 5.2 s; timed up and go: 7.8 ± 3.3 to 6.2 ± 2.5 s; both P < 0.05), and self-perceived improvement in health (3.1 ± 0.5 to 2.6 ± 0.9) and social activities functioning (2.2 ± 1.0 to 1.5 ± 1.1; both P < 0.05). No changes were observed in the control group. In conclusion, this study shows that MST improves FGC, neuromuscular function, and functional performance and advocates that high-intensity strength training should be implemented as an adjunct therapy in the treatment of PD patients.NEW & NOTEWORTHY This randomized, controlled trial documents that supervised high-intensity strength training improves efferent neural drive, maximal muscle strength, rate of force development, and functional performance in patients with Parkinson's disease (PD). In contrast, no differences were observed in these outcome variables in patients receiving conventional treatment consisting of recreational physical activity with low-to-medium intensity. Consequently, this study advocates that high-intensity strength training should be implemented in the clinical treatment of PD patients.

The anti-epileptic drug valproic acid causes malformations in the developing craniofacial skeleton of zebrafish larvae.

Valproic acid (VPA) is an anti-epileptic drug known to cause congenital craniofacial abnormalities, including orofacial clefts (OFC). The exact mechanisms by which VPA leads to craniofacial skeletal malformations are poorly understood. In this study, we investigated the effects of VPA on cartilage and bone formation in the zebrafish larval head during 1-13 hpf (early) and 25-37 hpf (late) development in which cranial neural crest cells (CNCCs) arise and then proliferate and differentiate, respectively. Double-staining for cartilage and bone at 5 dpf revealed that VPA reduced cartilage and bone formation in a dose-dependent manner after both early or late exposure. Several different CNCC-derived cartilage and bone elements were affected in both groups. In the early group (100 µM VPA), the posterior head length and the ethmoid plate were reduced in length (both p < 0.01), while mineralization of 4 out of 9 bone elements was often lacking (all p < 0.01). In the late group (100 µM VPA), also the posterior head length was reduced as well as the length of the ceratohyals (both p < 0.01). Similar to early exposure, mineralization of 3 out of 9 bone elements was often lacking (all p < 0.01). These results indicate that both CNCC formation (early) and differentiation (late) are hampered by VPA treatment, of which the consequences for bone and cartilage formation are persistent at 5 dpf. Indeed, we also found that the expression of several genes related to cartilage and bone was upregulated at 5 dpf. These data indicate a compensatory reaction to the lack of cartilage and bone. Altogether, VPA seems to induce craniofacial malformations via disturbed CNCC function leading to defects in cartilage and bone formation.

Orofacial Muscles: Embryonic Development and Regeneration after Injury.

Orofacial congenital defects such as cleft lip and/or palate are associated with impaired muscle regeneration and fibrosis after surgery. Also, other orofacial reconstructions or trauma may end up in defective muscle regeneration and fibrosis. The aim of this review is to discuss current knowledge on the development and regeneration of orofacial muscles in comparison to trunk and limb muscles. The orofacial muscles include the tongue muscles and the branchiomeric muscles in the lower face. Their main functions are chewing, swallowing, and speech. All orofacial muscles originate from the mesoderm of the pharyngeal arches under the control of cranial neural crest cells. Research in vertebrate models indicates that the molecular regulation of orofacial muscle development is different from that of trunk and limb muscles. In addition, the regenerative ability of orofacial muscles is lower, and they develop more fibrosis than other skeletal muscles. Therefore, specific approaches need to be developed to stimulate orofacial muscle regeneration. Regeneration may be stimulated by growth factors such fibroblast growth factors and hepatocyte growth factor, while fibrosis may be reduced by targeting the transforming growth factor ß1 (TGFß1)/myofibroblast axis. New approaches that combine these 2 aspects will improve the surgical treatment of orofacial muscle defects.

Fgf8a mutation affects craniofacial development and skeletal gene expression in zebrafish larvae.

Craniofacial development is tightly regulated and therefore highly vulnerable to disturbance by genetic and environmental factors. Fibroblast growth factors (FGFs) direct migration, proliferation and survival of cranial neural crest cells (CNCCs) forming the human face. In this study, we analyzed bone and cartilage formation in the head of five dpf fgf8ati282 zebrafish larvae and assessed gene expression levels for 11 genes involved in these processes. In addition, in situ hybridization was performed on 8 and 24 hours post fertilization (hpf) larvae (fgf8a, dlx2a, runx2a, col2a1a). A significant size reduction of eight out of nine craniofacial cartilage structures was found in homozygous mutant (6-36%, P<0.01) and heterozygous (7-24%, P<0.01) larvae. Also, nine mineralized structures were not observed in all or part of the homozygous (0-71%, P<0.0001) and heterozygous (33-100%, P<0.0001) larvae. In homozygote mutants, runx2a and sp7 expression was upregulated compared to wild type, presumably to compensate for the reduced bone formation. Decreased col9a1b expression may compromise cartilage formation. Upregulated dlx2a in homozygotes indicates impaired CNCC function. Dlx2a expression was reduced in the first and second stream of CNCCs in homozygous mutants at 24 hpf, as shown by in situ hybridization. This indicates an impairment of CNCC migration and survival by fgf8 mutation.

Ionophore-Based Biphasic Chemical Sensing in Droplet Microfluidics.

Droplet microfluidics is an enabling platform for high-throughput screens, single-cell studies, low-volume chemical diagnostics, and microscale material syntheses. Analytical methods for real-time and in situ detection of chemicals in the droplets will benefit these applications, but they remain limited. Reported herein is a novel heterogeneous chemical sensing strategy based on functionalization of the oil phase with rationally combined sensing reagents. Sub-nanoliter oil segments containing pH-sensitive fluorophores, ionophores, and ion-exchangers enable highly selective and rapid fluorescence detection of physiologically important electrolytes (K+ , Na+ , and Cl- ) and polyions (protamine) in sub-nanoliter aqueous droplets. Electrolyte analysis in whole blood is demonstrated without suffering from optical interference from the sample matrix. Moreover, an oil phase doped with an aza-BODIPY dye allows indication of H2 O2 in the aqueous droplets, exemplifying sensing of targets beyond ionic species.

Aberrant resting-state oscillatory brain activity in Parkinson's disease patients with visual hallucinations: An MEG source-space study.

To gain insight into possible underlying mechanism(s) of visual hallucinations (VH) in Parkinson's disease (PD), we explored changes in local oscillatory activity in different frequency bands with source-space magnetoencephalography (MEG). Eyes-closed resting-state MEG recordings were obtained from 20 PD patients with hallucinations (Hall+) and 20 PD patients without hallucinations (Hall-), matched for age, gender and disease severity. The Hall+ group was subdivided into 10 patients with VH only (unimodal Hall+) and 10 patients with multimodal hallucinations (multimodal Hall+). Subsequently, neuronal activity at source-level was reconstructed using an atlas-based beamforming approach resulting in source-space time series for 78 cortical and 12 subcortical regions of interest in the automated anatomical labeling (AAL) atlas. Peak frequency (PF) and relative power in six frequency bands (delta, theta, alpha1, alpha2, beta and gamma) were compared between Hall+ and Hall-, unimodal Hall+ and Hall-, multimodal Hall+ and Hall-, and unimodal Hall+ and multimodal Hall+ patients. PF and relative power per frequency band did not differ between Hall+ and Hall-, and multimodal Hall+ and Hall- patients. Compared to the Hall- group, unimodal Hall+ patients showed significantly higher relative power in the theta band (p = 0.005), and significantly lower relative power in the beta (p = 0.029) and gamma (p = 0.007) band, and lower PF (p = 0.011). Compared to the unimodal Hall+, multimodal Hall+ showed significantly higher PF (p = 0.007). In conclusion, a subset of PD patients with only VH showed slowing of MEG-based resting-state brain activity with an increase in theta activity, and a concomitant decrease in beta and gamma activity, which could indicate central cholinergic dysfunction as underlying mechanism of VH in PD. This signature was absent in PD patients with multimodal hallucinations.

Viperin interacts with the kinase IRAK1 and the E3 ubiquitin ligase TRAF6, coupling innate immune signaling to antiviral ribonucleotide synthesis.

Virus-inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) is a radical SAM enzyme that plays a multifaceted role in the cellular antiviral response. Viperin has recently been shown to catalyze the SAM-dependent formation of 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), which inhibits some viral RNA polymerases. Viperin is also implicated in regulating Lys-63-linked polyubiquitination of interleukin-1 receptor-associated kinase-1 (IRAK1) by the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) as part of the Toll-like receptor-7 and -9 (TLR7/9) innate immune signaling pathways. In these pathways, the poly-ubiquitination of IRAK1 by TRAF6 is necessary to activate IRAK1, which then phosphorylates downstream targets and ultimately leads to the production of type I interferons. That viperin is a component of these pathways suggested that its enzymatic activity might be regulated by interactions with partner proteins. To test this idea, we have reconstituted the interactions between viperin, IRAK1, and TRAF6 by transiently expressing these enzymes in HEK 293T cells. We show that IRAK1 and TRAF6 increase viperin activity ∼10-fold to efficiently catalyze the radical-mediated dehydration of CTP to ddhCTP. Furthermore, we found that TRAF6-mediated ubiquitination of IRAK1 requires the association of viperin with both IRAK1 and TRAF6. Ubiquitination appears to depend on structural changes in viperin induced by SAM binding, but, significantly, does not require catalytically active viperin. We conclude that the synergistic activation of viperin and IRAK1 provides a mechanism that couples innate immune signaling with the production of the antiviral nucleotide ddhCTP.

Product and Microbial Population Kinetics During Balsamic-Styled Vinegar Production.

Balsamic-styled vinegar is a nutraceutical product obtained from a two-stage fermentation process of grape must. However, little is known about how fermentation conditions affect growth kinetics, bio-product development, population dynamics and the final product quality. As a result, the current study investigated the effect of fermentation temperature and inoculation strategy on the fermentation dynamics of Balsamic-styled vinegar production. A microbial consortium of non-Saccharomyces yeasts (n = 13) and acetic acid bacteria (n = 5) was tested at various fermentation temperatures (22 °C, 28 °C and a fluctuating temperature regimen). Different inoculation strategies (co-inoculation and sequential inoculation) were investigated, and population dynamics of the product selected due to a rapid fermentation period were confirmed using a 16S and 18S gene sequencing. A higher fermentation temperature (28 °C) and co-inoculation strategy resulted in a shorter fermentation cycle, whilst the desired acetic acid concentration of 60 g/L was achieved within 38 days. 16S and 18S gene sequencing showed that 50.84% of Acetobacter species were abundant at the end of the fermentation cycle, while 40.18% bacteria were unculturable. The study provides a better understanding of how fermentation temperature and inoculation strategy affect the fermentation period, population dynamics and the growth kinetics of the microbial consortium during the production of Balsamic-styled vinegar. PRACTICAL APPLICATION: Lower quality South African wine grapes could be channelled to an alternative high-priced product (Balsamic-styled vinegar), with low technological input requirements. Thus, making it easier to incorporate a low capital start-up business while empowering small business entrepreneurs and boosting the economy.

Super-resolution imaging identifies PARP1 and the Ku complex acting as DNA double-strand break sensors.

DNA double-strand breaks (DSBs) are fatal DNA lesions and activate a rapid DNA damage response. However, the earliest stage of DSB sensing remains elusive. Here, we report that PARP1 and the Ku70/80 complex localize to DNA lesions considerably earlier than other DSB sensors. Using super-resolved fluorescent particle tracking, we further examine the relocation kinetics of PARP1 and the Ku70/80 complex to a single DSB, and find that PARP1 and the Ku70/80 complex are recruited to the DSB almost at the same time. Notably, only the Ku70/80 complex occupies the DSB exclusively in the G1 phase; whereas PARP1 competes with the Ku70/80 complex at the DSB in the S/G2 phase. Moreover, in the S/G2 phase, PARP1 removes the Ku70/80 complex through its enzymatic activity, which is further confirmed by in vitro DSB-binding assays. Taken together, our results reveal PARP1 and the Ku70/80 complex as critical DSB sensors, and suggest that PARP1 may function as an important regulator of the Ku70/80 complex at the DSBs in the S/G2 phase.