The adaptive CRISPR-Cas immune system stores sequences from past invaders as spacers in CRISPR arrays and thereby provides direct evidence that links invaders to hosts. Mapping CRISPR spacers has ...revealed many aspects of CRISPR-Cas biology, including target requirements such as the protospacer adjacent motif (PAM). However, studies have so far been limited by a low number of mapped spacers in the database.
By using vast metagenomic sequence databases, we map approximately one-third of more than 200,000 unique CRISPR spacers from a variety of microbes and derive a catalog of more than two hundred unique PAM sequences associated with specific CRISPR-Cas subtypes. These PAMs are further used to correctly assign the orientation of CRISPR arrays, revealing conserved patterns between the last nucleotides of the CRISPR repeat and PAM. We could also deduce CRISPR-Cas subtype-specific preferences for targeting either template or coding strand of open reading frames. While some DNA-targeting systems (type I-E and type II systems) prefer the template strand and avoid mRNA, other DNA- and RNA-targeting systems (types I-A and I-B and type III systems) prefer the coding strand and mRNA. In addition, we find large-scale evidence that both CRISPR-Cas adaptation machinery and CRISPR arrays are shared between different CRISPR-Cas systems. This could lead to simultaneous DNA and RNA targeting of invaders, which may be effective at combating mobile genetic invaders.
This study has broad implications for our understanding of how CRISPR-Cas systems work in a wide range of organisms for which only the genome sequence is known.
Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design ...of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.
This study compares the effects of traditional logopedic dysphagia treatment with those of neuromuscular electrical stimulation (NMES) as adjunct to therapy on the quality of life in patients with ...Parkinson’s disease and oropharyngeal dysphagia. Eighty-eight patients were randomized over three treatment groups. Traditional logopedic dysphagia treatment and traditional logopedic dysphagia treatment combined with NMES at sensor or motor level stimulation were compared. At three times (pretreatment, post-treatment, and 3 months following treatment), two quality-of-life questionnaires (SWAL-QOL and MD Anderson Dysphagia Inventory) and a single-item Dysphagia Severity Scale were scored. The Functional Oral Intake Scale was used to assess the dietary intake. After therapy, all groups showed significant improvement on the Dysphagia Severity Scale and restricted positive effects on quality of life. Minimal group differences were found. These effects remained unchanged 3 months following treatment. No significant correlations were found between dietary intake and quality of life. Logopedic dysphagia treatment results in a restricted increased quality of life in patients with Parkinson’s disease. In this randomized controlled trial, all groups showed significant therapy effects on the Dysphagia Severity Scale and restricted improvements on the SWAL-QOL and the MDADI. However, only slight nonsignificant differences between groups were found.
This study determines the relationship between patient and investigator reported outcome measures (PROMs versus IROMs) on oropharyngeal dysphagia (OD) in Parkinson’s disease (PD). The PROMs used are ...the MD Anderson Dysphagia Inventory (MDADI) and the Dysphagia Severity Scale (DSS). The IROMs used are fiberoptic endoscopic evaluation of swallowing (FEES) and videofluoroscopy of swallowing (VFS). Ninety dysphagic PD patients were included. Multilayer perceptron (MLP) neural network analysis was used to investigate the relationship between PROMs and IROMs on OD in PD. MLP neural network analysis showed a moderate agreement between PROMs and IROMs, with an area under the curve between 0.6 and 0.7. Two-step cluster analysis revealed several clusters of patients with similar scores on FEES and/or VFS variables, but with significant different scores on MDADI and DSS variables. This study highlights that there are PD patients with similar FEES and/or VFS findings that cannot be lumped together under the same pathophysiological umbrella due to their differences in PROMs. Since the exact origin of these differences is not fully understood, it seems appropriate for the time being to take into account the different dimensions of OD during the swallowing assessment so that they can be included in a patient-tailored treatment plan.
Cancer cachexia is highly prevalent in advanced non-small cell lung cancer (NSCLC) and locally advanced head and neck squamous cell carcinoma (LAHNSCC), and compromises treatment tolerance and ...overall survival (OS). NSCLC and LAHNSCC patients share similar risk factors, and receive comparable anti-cancer treatment regimens. The aim of this study was to determine the predictive value of body composition assessed by bioelectrical impedance analysis (BIA) and handgrip strength (HGS) (baseline and early changes during therapy) on OS in NSCLC and LAHNSCC patients treated with platinum-based chemoradiotherapy (CRT) or cetuximab-based bioradiotherapy (BRT). To elucidate potential underlying determinants of early changes in body composition and HGS, specific (fat and fat free) mass loss patterns of squamous NSCLC (sNSCLC) were compared to human papilloma virus negative (HPV-) LAHNSCC patients treated with CRT.
Between 2013 and 2016, BIA and HGS were performed at baseline and after 3 weeks of CRT/BRT in LAHNSCC and NSCLC patients treated with curative intent.
Two hundred thirty-three patients were included for baseline measurements. Fat free mass index (FFMI) and HGS<10th percentile of reference values at baseline were both prognostic for poor OS in NSCLC and LAHNSCC HR 1.64 95%CI 1.13-2.39,
= 0.01 and HR 2.30 95%CI 1.33-3.97,
= 0.003, respectively, independent of Charlson Comorbidity Index, cancer site, and gross tumor volume. Early fat mass (FM) loss during CRT was predictive for poor OS in sNSCLC (
= 64) HR 3.80 95%CI 1.79-8.06
≤ 0.001 but not in HPV- LAHNSCC (
= 61). In patients with significant weight loss (>2%) in the first 3 weeks of CRT (sNSCLC
= 24, HPV- LAHNSCC
= 23), the FM change was -1.4 ± 14.5% and -8.7 ± 9.0% in sNSCLC and HPV- LAHNSCC patients, respectively (
< 0.05). Fat fee mass change was -5.6 ± 6.3% and -4.0 ± 4.3% for sNSCLC and HPV- LAHNSCC, respectively (
= 0.31).
FFMI and HGS<10th percentile at baseline are independent prognostic factors for poor OS in NSCLC and LAHNSCC patients treated with CRT/BRT. The specific composition of mass loss during first 3 weeks of CRT significantly differs between sNSCLC and HPV- LAHNSCC patients. Early FM loss was prognostic in sNSCLC only.
Patients who receive chemoradiotherapy or bioradiotherapy (CRT/BRT) for locally advanced head and neck squamous cell carcinoma (LAHNSCC) often experience high toxicity rates interfering with oral ...intake, causing tube feeding (TF) dependency. International guidelines recommend gastrostomy insertion when the expected use of TF exceeds 4 weeks. We aimed to develop and externally validate a prediction model to identify patients who need TF ≥ 4 weeks and would benefit from prophylactic gastrostomy insertion.
A retrospective multicenter cohort study was performed in four tertiary head and neck cancer centers in the Netherlands. The prediction model was developed using data from University Medical Center Utrecht and the Netherlands Cancer Institute and externally validated using data from Maastricht University Medical Center and Radboud University Medical Center. The primary endpoint was TF dependency ≥4 weeks initiated during CRT/BRT or within 30 days after CRT/BRT completion. Potential predictors were extracted from electronic health records and radiotherapy dose–volume parameters were calculated.
The developmental and validation cohort included 409 and 334 patients respectively. Multivariable analysis showed predictive value for pretreatment weight change, texture modified diet at baseline, ECOG performance status, tumor site, N classification, mean radiation dose to the contralateral parotid gland and oral cavity. The area under the receiver operating characteristics curve for this model was 0.73 and after external validation 0.62. Positive and negative predictive value for a risk of 90% or higher for TF dependency ≥4 weeks were 81.8% and 42.3% respectively.
We developed and externally validated a prediction model to estimate TF-dependency ≥4 weeks in LAHNSCC patients treated with CRT/BRT. This model can be used to guide personalized decision-making on prophylactic gastrostomy insertion in clinical practice.
The aim of this work was to translate the 44-item SWAL-QoL into Dutch (SWAL-QoL-NL) and compare the validity of this questionnaire against Euroqol in a Dutch population with dysphagia. SWAL-QoL was ...translated according to international guidelines. SWAL-QoL-NL and Euroqol were completed by 152 patients in seven diagnosis groups. Internal consistency and correlations were calculated. Scores for nine subscales (General burden, Food selection, Eating duration, Fear of eating, Sleep, Fatigue, Mental health, Social functioning and a symptom score) ranged between 0.80 and 0.92 (Cronbach’s α). Two subscales (Eating desire and Communication scored 0.67 and 0.60, respectively, and were removed from the questionnaire. The 14-item battery on clinical symptoms showed an internal consistency of 0.80, allowing the use of a sum score on group level in clinical research. Correlation of SWAL-QoL-NL subscales with the Euroqol was negligible to low (Pearson’s correlations range = 0.09–0.36). The 39-item SWAL-QoL-NL proved to be a reliable tool to examine the impact of dysphagia on quality of life in a Dutch population. Internal consistency allows the use of nine subscales of SWAL-QoL-NL for comparisons on a group level (0.80 < α < 0.92) only. Also a Symptom score can be derived from the raw data.
The green micro-algae Chlamydomonas reinhardtii and Dunaliella tertiolecta were cultivated under medium-duration square-wave light/dark cycles with a cycle time of 15 s. These cycles were used to ...simulate the light regime experienced by micro-algae in externally-illuminated (sunlight) air-lift loop bioreactors with internal draft tube. Biomass yield in relation to light energy was determined as g protein per mol of photons (400-700 nm). Between 600 and 1200 mu mol m super(-2) s super(-1) the yield at a 10/5 s light/dark cycle was equal to the yield at continuous illumination. Consequently, provided that the liquid circulation time is 15 s, a considerable dark zone seems to be allowed in the interior of air-lift loop photobioreactors (33% v/v) without loss of light utilization efficiency. However, at a 5/10 s light/dark cycle, corresponding to a 67% v/v dark zone, biomass yield decreased. Furthermore, both algae, C. reinhardtii and D. tertiolecta, responded similarly to these cycles with respect to biomass yield. This was interesting because they were reported to exhibit a different photoacclimation strategy. Finally, it was demonstrated that D. tertiolecta was much more efficient at low (average) photon flux densities (57-370 mu mol m super(-2) s super(-1)) than at high PFDs (> 600 mu mol m super(-2) s super(-1)) and it was shown that D. tertiolecta was cultivated at a sub-optimal temperature (20 degree C).
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