CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells.

5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC). 5hmC and TET proteins have been implicated in stem cell biology and cancer, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the 'poised' chromatin signature found at developmentally-regulated genes in ES cells.

Glial-derived mitochondrial signals impact neuronal proteostasis and aging.

The nervous system plays a critical role in maintaining whole-organism homeostasis; neurons experiencing mitochondrial stress can coordinate the induction of protective cellular pathways, such as the mitochondrial unfolded protein response (UPRMT), between tissues. However, these studies largely ignored non-neuronal cells of the nervous system. Here, we found that UPRMT activation in four, astrocyte-like glial cells in the nematode, C. elegans, can promote protein homeostasis by alleviating protein aggregation in neurons. Surprisingly, we find that glial cells utilize small clear vesicles (SCVs) to signal to neurons, which then relay the signal to the periphery using dense-core vesicles (DCVs). This work underlines the importance of glia in establishing and regulating protein homeostasis within the nervous system, which can then impact neuron-mediated effects in organismal homeostasis and longevity.

Glial-derived mitochondrial signals affect neuronal proteostasis and aging.

The nervous system plays a critical role in maintaining whole-organism homeostasis; neurons experiencing mitochondrial stress can coordinate the induction of protective cellular pathways, such as the mitochondrial unfolded protein response (UPRMT), between tissues. However, these studies largely ignored nonneuronal cells of the nervous system. Here, we found that UPRMT activation in four astrocyte-like glial cells in the nematode, Caenorhabditis elegans, can promote protein homeostasis by alleviating protein aggregation in neurons. Unexpectedly, we find that glial cells use small clear vesicles (SCVs) to signal to neurons, which then relay the signal to the periphery using dense-core vesicles (DCVs). This work underlines the importance of glia in establishing and regulating protein homeostasis within the nervous system, which can then affect neuron-mediated effects in organismal homeostasis and longevity.

Autophagy-mediated longevity is modulated by lipoprotein biogenesis.

Autophagy-dependent longevity models in C. elegans display altered lipid storage profiles, but the contribution of lipid distribution to life-span extension is not fully understood. Here we report that lipoprotein production, autophagy and lysosomal lipolysis are linked to modulate life span in a conserved fashion. We find that overexpression of the yolk lipoprotein VIT/vitellogenin reduces the life span of long-lived animals by impairing the induction of autophagy-related and lysosomal genes necessary for longevity. Accordingly, reducing vitellogenesis increases life span via induction of autophagy and lysosomal lipolysis. Life-span extension due to reduced vitellogenesis or enhanced lysosomal lipolysis requires nuclear hormone receptors (NHRs) NHR-49 and NHR-80, highlighting novel roles for these NHRs in lysosomal lipid signaling. In dietary-restricted worms and mice, expression of VIT and hepatic APOB (apolipoprotein B), respectively, are significantly reduced, suggesting a conserved longevity mechanism. Altogether, our study demonstrates that lipoprotein biogenesis is an important mechanism that modulates aging by impairing autophagy and lysosomal lipolysis.

The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine.

5-Hydroxymethylcytosine (5hmC) is a newly discovered DNA base present at detectable levels in most mammalian cell types and tissues. It is generated by Tet-enzyme-mediated oxidation of 5-methylcytosine (5mC). 5hmC is important both because of its potential role in regulating gene expression and because it may be an intermediate in DNA demethylation. Here we describe a technique termed GLIB (glucosylation, periodate oxidation and biotinylation), which combines several enzymatic and chemical modification steps to attach biotin to 5hmC. Biotin-containing genomic DNA fragments are then enriched using streptavidin beads, eluted and sequenced. GLIB is capable of quantitatively tagging and precipitating fragments containing a single 5hmC molecule. Sample preparation and GLIB can be conducted in 2-3 d.

Race, treatment preferences, and hospice enrollment: eligibility criteria may exclude patients with the greatest needs for care.

BACKGROUND: The requirement that patients give up curative treatment makes hospice enrollment unappealing for some patients and may particularly limit use among African-American patients. The current study was conducted to determine whether African-American patients with cancer are more likely than white patients to have preferences for cancer treatment that exclude them from hospice and whether they are less likely to want specific hospice services. METHODS: Two hundred eighty-three patients who were receiving treatment for cancer at 6 oncology clinics within the University of Pennsylvania Cancer Network completed conjoint interviews measuring their perceived need for 5 hospice services and their preferences for continuing cancer treatment. Patients were followed for 6 months or until death. RESULTS: African-American patients had stronger preferences for continuing their cancer treatments on a 7-point scale even after adjusting for age, sex, finances, education, Eastern Cooperative Oncology Group performance status, quality of life, and physical and psychologic symptom burden (adjusted mean score, 4.75 vs 3.96; beta coefficient, 0.82; 95% confidence interval, 0.22-1.41 [P = .007]). African-American patients also had greater perceived needs for hospice services after adjusting for these characteristics (adjusted mean score, 2.31 vs 1.83; beta coefficient, 0.51; 95% confidence interval, 0.11-0.92 [P = .01]). However, this effect disappeared after adjusting for household finances. CONCLUSIONS: Hospice eligibility criteria may exclude African-American patients disproportionately despite greater perceived needs for hospice services in this population. The mechanisms driving this health disparity likely include both cultural differences and economic characteristics, and consideration should be given to redesigning hospice eligibility criteria.

Do phase 1 patients have greater needs for palliative care compared with other cancer patients?

BACKGROUND: Phase 1 oncology trial participants often are excluded from hospice. However, it is not known whether they would benefit from hospice services. The objectives of the current study were to define the palliative care needs of these patients and to determine whether their needs are greater than those of other cancer patients. METHODS: Two hundred ninety-seven patients who were undergoing cancer therapy and 69 patients who were enrolled in phase 1 trials at 7 oncology clinics in an urban cancer network were recruited and consented to participate in interviews. Interviewers assessed the prevalence and severity of 10 symptoms using the Global Distress Index of the Memorial Symptom Assessment Scale and patients' perceived need for 4 services typically provided through hospice: a chaplain, counselor, home health aide, and visiting nurse. RESULTS: Patients in the 2 groups had a similar symptom burden. However, after adjusting for Eastern Cooperative Oncology Group performance status scores, phase 1 patients were more likely to have 5 of the 10 symptoms and reported greater severity for 6 of the 10 symptoms. Compared with other patients, phase 1 patients were less likely to say they needed a home health aide (4 of 69 patients [6%] vs 198 of 297 patients [67%]), a chaplain (7 of 69 patients [10%] vs 134 of 297 patients [45%]), or a counselor (11 of 69 patients [16%] vs 160 of 297 patients [54%]; chi-square test: P<.001 for all). They were equally likely to say they needed a visiting nurse (30 of 69 patients [44%] vs 142 of 297 patients [48%]; chi-square test: P=.516). CONCLUSIONS: Compared with other patients who had cancer, patients who were participating in phase 1 trials were less likely to want several home care services, although they experienced a greater symptom burden. Further research will be needed to define the palliative care needs of this population.