The RNase PARN Controls the Levels of Specific miRNAs that Contribute to p53 Regulation.

PARN loss-of-function mutations cause a severe form of the hereditary disease dyskeratosis congenita (DC). PARN deficiency affects the stability of non-coding RNAs such as human telomerase RNA (hTR), but these effects do not explain the severe disease in patients. We demonstrate that PARN deficiency affects the levels of numerous miRNAs in human cells. PARN regulates miRNA levels by stabilizing either mature or precursor miRNAs by removing oligo(A) tails added by the poly(A) polymerase PAPD5, which if remaining recruit the exonuclease DIS3L or DIS3L2 to degrade the miRNA. PARN knockdown destabilizes multiple miRNAs that repress p53 translation, which leads to an increase in p53 accumulation in a Dicer-dependent manner, thus explaining why PARN-defective patients show p53 accumulation. This work also reveals that DIS3L and DIS3L2 are critical 3' to 5' exonucleases that regulate miRNA stability, with the addition and removal of 3' end extensions controlling miRNA levels in the cell.

Posttranscriptional modulation of TERC by PAPD5 inhibition rescues hematopoietic development in dyskeratosis congenita.

Reduced levels of TERC, the telomerase RNA component, cause dyskeratosis congenita (DC) in patients harboring mutations in TERC, PARN, NOP10, NHP2, NAF1, or DKC1. Inhibition of the noncanonical poly(A) polymerase PAPD5, or the exosome RNA degradation complex, partially restores TERC levels in immortalized DKC1 mutant cells, but it remains unknown if modulation of posttranscriptional processing of TERC could improve hematopoietic output in DC. We used human embryonic stem cells (hESCs) with a common dyskerin mutation (DKC1_A353V), which have defective telomere maintenance and reduced definitive hematopoietic potential, to understand the effects of reducing EXOSC3 activity, or silencing PAPD5-mediated oligoadenylation, on hematopoietic progenitor specification and function in DC. Reduction of EXOSC3 or PAPD5 levels in DKC1 mutant hESCs led to functional improvements in TERC levels and telomerase activity, with concomitant telomere elongation and reduced levels of DNA damage signaling. Interestingly, the silencing of PAPD5, but not EXOSC3, significantly restored definitive hematopoietic potential in DKC1 mutant cells. Mechanistically, we show that PAPD5 inhibition is sustained in differentiated CD34+ cells, with a concomitant increase in mature, functional, forms of TERC, indicating that regulation of PAPD5 is a potential strategy to reverse hematologic dysfunction in DC patients.

Quality control of assembly-defective U1 snRNAs by decapping and 5'-to-3' exonucleolytic digestion.

The accurate biogenesis of RNA-protein complexes is a key aspect of eukaryotic cells. Defects in Sm protein complex binding to snRNAs are known to reduce levels of snRNAs, suggesting an unknown quality control system for small nuclear ribonucleoprotein (snRNP) assembly. snRNA quality control may also be relevant in spinal muscular atrophy, which is caused by defects in the survival motor neuron (SMN)1 gene, an assembly factor for loading the Sm complex on snRNAs and, when severely reduced, can lead to reduced levels of snRNAs and splicing defects. To determine how assembly-defective snRNAs are degraded, we first demonstrate that yeast U1 Sm-mutant snRNAs are degraded either by Rrp6- or by Dcp2-dependent decapping/5'-to-3' decay. Knockdown of the decapping enzyme DCP2 in mammalian cells also increases the levels of assembly-defective snRNAs and suppresses some splicing defects seen in SMN-deficient cells. These results identify a conserved mechanism of snRNA quality control, and also suggest a general paradigm wherein the phenotype of an "RNP assembly disease" might be suppressed by inhibition of a competing RNA quality control mechanism.

Identification of PARN nuclease activity inhibitors by computational-based docking and high-throughput screening.

Poly(A)-specific ribonuclease (PARN) is a 3'-exoribonuclease that removes poly(A) tails from the 3' end of RNAs. PARN is known to deadenylate some ncRNAs, including hTR, Y RNAs, and some miRNAs and thereby enhance their stability by limiting the access of 3' to 5' exonucleases recruited by oligo(A) tails. Several PARN-regulated miRNAs target p53 mRNA, and PARN knockdown leads to an increase of p53 protein levels in human cells. Thus, PARN inhibitors might be used to induce p53 levels in some human tumors and act as a therapeutic strategy to treat cancers caused by repressed p53 protein. Herein, we used computational-based molecular docking and high-throughput screening (HTS) to identify small molecule inhibitors of PARN. Validation with in vitro and cell-based assays, identified 4 compounds, including 3 novel compounds and pyrimidopyrimidin-2-one GNF-7, previously shown to be a Bcr-Abl inhibitor, as PARN inhibitors. These inhibitors can be used as tool compounds and as lead compounds for the development of improved PARN inhibitors.

Outcome of metal vs plastic stents for biliary obstruction in patients with pancreatic carcinoma undergoing neoadjuvant chemoradiotherapy: A systematic review and meta-analysis.

BACKGROUND: Preoperative biliary drainage (PBT) may be warranted in patients with borderline resectable or locally advanced pancreatic carcinoma before neoadjuvant therapy (NAT) to relieve obstructive jaundice. However, it is unclear if the use of self-expanding metal stents (SEMS) has any benefit over plastic stents in this setting. METHODS: We searched electronic databases from inception to February 11, 2022 to identify studies comparing SEMS and plastic stents for PBT in patients with pancreatic carcinoma undergoing NAT. Random effect models were used to determine pooled rates of recurrent biliary obstruction (RBO) and/or need for reintervention, stent-related complications and surgical outcome. RESULTS: A total of 10 studies (474 patients; metal group-37.1%) were included. Pooled risk ratio of RBO and/or need for reintervention was lower in the metal group (RR, 0.23 [95% CI: 0.11-0.45, I2  = 60%]). Pooled risks of stent occlusion (RR, 0.43 [95% CI: 0.24-0.80, I2  = 45%]) and stent-related cholangitis (RR, 0.37 [95% CI: 0.17-0.78, I2  = 1%]) were lower in the metal group. However, risks of stent-related cholecystitis (RR, 1.51 [95% CI: 0.36-6.41, I2  = 0%]) and pancreatitis (RR, 1.52 [95% CI: 0.07-31.84, I2  = 66%]) were higher in the metal group. The metal group was also associated with a reduced risk of delay in NAT (RR, 0.38 [95% CI: 0.18-0.80, I2  = 14%]). Pooled risk ratio of R0 resection and postoperative complications was equal amongst both groups. CONCLUSION: Metal stents are associated with reduced risk of RBO and/or need for reintervention, reduced risk of stent occlusion and cholangitis as compared to plastic stents in patients with pancreatic carcinoma undergoing NAT.

USB1 is a miRNA deadenylase that regulates hematopoietic development.

Mutations in the 3' to 5' RNA exonuclease USB1 cause hematopoietic failure in poikiloderma with neutropenia (PN). Although USB1 is known to regulate U6 small nuclear RNA maturation, the molecular mechanism underlying PN remains undetermined, as pre-mRNA splicing is unaffected in patients. We generated human embryonic stem cells harboring the PN-associated mutation c.531_delA in USB1 and show that this mutation impairs human hematopoiesis. Dysregulated microRNA (miRNA) levels in USB1 mutants during blood development contribute to hematopoietic failure, because of a failure to remove 3'-end adenylated tails added by PAPD5/7. Modulation of miRNA 3'-end adenylation through genetic or chemical inhibition of PAPD5/7 rescues hematopoiesis in USB1 mutants. This work shows that USB1 acts as a miRNA deadenylase and suggests PAPD5/7 inhibition as a potential therapy for PN.

Solar array placement, electricity generation, and cropland displacement across California's Central Valley.

Understanding agriculturally co-located solar photovoltaic (PV) installation capacity, practices, and preferences is imperative to foster a future where solar power and agriculture co-exist with limited impacts on food production. Crops and PV panels are often co-located as they have similar ideal conditions for maximum yield. The recent boom in solar photovoltaics is displacing a significant amount of cropland. The literature on agriculturally co-located PV array installations lacks important spatiotemporal details that could help inform future array installations and improve associated policies and incentive programs. This study used imagery from the National Agriculture Imagery Program for object-based analysis (within eCognition Developer), and from Landsat 5 TM, 7 ETM+ and 8 OLI for temporal analysis (using LandTrendr) to identify and characterize non-residential ground-mounted PV arrays in California's Central Valley installed between 2008 and 2018. This dataset includes over 210,000 individually identified panels grouped by mount and installation year into 1006 PV arrays (69% are agriculturally co-located). The most common type of mounting system is fixed-axis, and individual co-located systems tend to be small (0.34 MW). There were fewer single-axis tracking arrays, although the average capacity per system is nearly four times higher (1.20 MW). In total, the mapped arrays accounted for 3.6 GW of capacity and generated a cumulative of 32,700 GWh within the Central Valley during the study period. For the 694 identified agriculturally co-located arrays (2.1 GW), significantly sub-optimal installation practices were observed in the spacing and spatial field placement of the arrays. In terms of crop conversion preferences, commodity crops (pastureland) dominated the total cumulative area converted although specialty crops (orchards) also contributed to a large number of solar installations on cropland. These results provide important details of current PV placement practices; understanding these can help to inform future practices and guide future regulations that might promote solar installations while preserving agricultural production.

Chemical inhibition of PAPD5/7 rescues telomerase function and hematopoiesis in dyskeratosis congenita.

Dyskeratosis congenita (DC) is a pediatric bone marrow failure syndrome caused by germline mutations in telomere biology genes. Mutations in DKC1 (the most commonly mutated gene in DC), the 3' region of TERC, and poly(A)-specific ribonuclease (PARN) cause reduced levels of the telomerase RNA component (TERC) by reducing its stability and accelerating TERC degradation. We have previously shown that depleting wild-type DKC1 levels by RNA interference or expression of the disease-associated A353V mutation in the DKC1 gene leads to decay of TERC, modulated by 3'-end oligoadenylation by noncanonical poly(A) polymerase 5 (PAPD5) followed by 3' to 5' degradation by EXOSC10. Furthermore, the constitutive genetic silencing of PAPD5 is sufficient to rescue TERC levels, restore telomerase function, and elongate telomeres in DKC1_A353V mutant human embryonic stem cells (hESCs). Here, we tested a novel PAPD5/7 inhibitor (RG7834), which was originally discovered in screens against hepatitis B viral loads in hepatic cells. We found that treatment with RG7834 rescues TERC levels, restores correct telomerase localization in DKC1 and PARN-depleted cells, and is sufficient to elongate telomeres in DKC1_A353V hESCs. Finally, treatment with RG7834 significantly improved definitive hematopoietic potential from DKC1_A353V hESCs, indicating that the chemical inhibition of PAPD5 is a potential therapy for patients with DC and reduced TERC levels.

Evaluating the cost and carbon footprint of second-life electric vehicle batteries in residential and utility-level applications.

The volume of end-of-life automotive batteries is increasing rapidly as a result of growing electric vehicle adoption. Most automotive lithium-ion batteries (LIBs) are recycled but could be repurposed as second-life batteries (SLBs) since they have 70-80% residual capacity, which can be adequate for stationary applications. SLBs have been proposed as potential, inexpensive, low-carbon energy storage for residential and utility-level applications, with or without photovoltaics (PV). However, it is unknown whether SLBs will be better than new batteries and whether SLBs will provide similar cost and carbon emission reduction for the different stationary applications in all locations. This work compared the levelized cost of electricity and life-cycle carbon emissions associated with using SLBs and new LIBs in the US for three energy storage applications: (1) residential energy storage with rooftop PV, (2) utility-level PV firming, and (3) utility-level peak-shaving, leading to a total of 41 scenarios. SLBs reduced the levelized cost of electricity by 12-57% and carbon emissions by 7-31% compared to new LIBs in the considered applications, with higher reductions for utility-level applications. SLBs still provided benefits at the residential level when compared to rooftop PV alone by reducing the levelized cost by 15-25% and carbon emissions by 22-51%, making SLBs attractive to residential consumers as well. SLBs offer an opportunity to utilize an end-of-life product for energy storage applications, provided the uncertainty in SLB quality and availability is addressed.

Utility of tissue Xpert-Mtb/Rif for the diagnosis of intestinal tuberculosis in patients with ileocolonic ulcers.

INTRODUCTION: Data on the use of Xpert Mtb/Rif for the diagnosis of intestinal tuberculosis is sparse. We report on the utility of Xpert Mtb/Rif testing for diagnosis of intestinal tuberculosis (ITB) in patients with ileocecal ulcers. METHODOLOGY: We performed a retrospective analysis of patients with ileocecal ulcers and suspected to have ITB and in whom testing of intestinal tissue for Xpert Mtb/Rif was performed. The patients were divided into two groups: those with a final diagnosis of intestinal tuberculosis and those with other diagnosis. These patients were compared for clinical features and presentation. The sensitivity, specificity, positive predictive value, and negative predictive value of Xpert Mtb/Rif for the diagnosis of ITB were calculated. RESULTS: Of the 40 patients studied, 23 were women and the mean age was 32.92 ±â€„12.78 years. Abdominal pain was present in 33 (88.5%) patients and diarrhea in 12 (30%). A total of 25 patients had underlying ITB whereas 15 patients had other diagnoses (Crohn's disease, amebiasis, nonspecific ileitis, etc.). The sensitivity, specificity, negative predictive value, positive predictive value, and accuracy of GeneXpert-Mtb/Rif was 32% (CI: 14.95-53.50%), 100% (78.2-100), 46.88% (40.27-53.59%), 100 & 57.50 (40.89-72.89%) respectively. CONCLUSION: A positive GeneXpert-Mtb/Rif helps in the diagnosis of ITB, but the sensitivity is low.

PARN Modulates Y RNA Stability and Its 3'-End Formation.

Loss-of-function mutations in 3'-to-5' exoribonucleases have been implicated in hereditary human diseases. For example, PARN mutations cause a severe form of dyskeratosis congenita (DC), wherein PARN deficiency leads to human telomerase RNA instability. Since the DC phenotype in PARN patients is even more severe than that of loss-of-function alleles in telomerase components, we hypothesized that PARN would also be required for the stability of other RNAs. Here, we show that PARN depletion reduces the levels of abundant human Y RNAs, which might contribute to the severe phenotype of DC observed in patients. Depletion of PAPD5 or the cytoplasmic exonuclease DIS3L rescues the effect of PARN depletion on Y RNA levels, suggesting that PARN stabilizes Y RNAs by removing oligoadenylated tails added by PAPD5, which would otherwise recruit DIS3L for Y RNA degradation. Through deep sequencing of 3' ends, we provide evidence that PARN can also deadenylate the U6 and RMRP RNAs without affecting their levels. Moreover, we observed widespread posttranscriptional oligoadenylation, uridylation, and guanylation of U6 and Y RNA 3' ends, suggesting that in mammalian cells, the formation of a 3' end for noncoding RNAs can be a complex process governed by the activities of various 3'-end polymerases and exonucleases.

Hypo- and Hyper-Assembly Diseases of RNA-Protein Complexes.

A key aspect of cellular function is the proper assembly and utilization of ribonucleoproteins (RNPs). Recent studies have shown that hyper- or hypo-assembly of various RNPs can lead to human diseases. Defects in the formation of RNPs lead to 'RNP hypo-assembly diseases', which can be caused by RNA degradation outcompeting RNP assembly. By contrast, excess RNP assembly, either in higher order RNP granules, or due to the expression of repeat-containing RNAs, can lead to 'RNP hyper-assembly diseases'. Here, we discuss the most recent advances in understanding the cause of disease onset, as well as potential therapies from the aspect of modulating RNP assembly in the cell, which presents a novel route to the treatment of these diseases.

Inhibition of telomerase RNA decay rescues telomerase deficiency caused by dyskerin or PARN defects.

Mutations in the human telomerase RNA component (hTR), the telomerase ribonucleoprotein component dyskerin (DKC1) and the poly(A) RNase (PARN) can lead to reduced levels of hTR and to dyskeratosis congenita (DC). However, the enzymes and mechanisms responsible for hTR degradation are unknown. We demonstrate that defects in dyskerin binding lead to hTR degradation by PAPD5-mediated oligoadenylation, which promotes 3'-to-5' degradation by EXOSC10, as well as decapping and 5'-to-3' decay by the cytoplasmic DCP2 and XRN1 enzymes. PARN increased hTR levels by deadenylating hTR, thereby limiting its degradation by EXOSC10. Telomerase activity and proper hTR localization in dyskerin- or PARN-deficient cells were rescued by knockdown of DCP2 and/or EXOSC10. Prevention of hTR RNA decay also led to a rescue of localization of DC-associated hTR mutants. These results suggest that inhibition of RNA decay pathways might be a useful therapy for some telomere pathologies.

Exploring chemical modifications for siRNA therapeutics: a structural and functional outlook.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism induced by small interfering RNAs (siRNAs) and micro-RNAs (miRNAs), and has proved to be one of the most important scientific discoveries made in the last century. The robustness of RNAi has opened up new avenues in the development of siRNAs as therapeutic agents against various diseases including cancer and HIV. However, there had remained a lack of a clear mechanistic understanding of messenger RNA (mRNA) cleavage mediated by Argonaute2 of the RNA-induced silencing complex (RISC), due to inadequate structural data. The X-ray crystal structures of the Argonaute (Ago)-DNA-RNA complexes reported recently have proven to be a breakthrough in this field, and the structural details can provide guidelines for the design of the next generation of siRNA therapeutics. To harness siRNAs as therapeutic agents, the prudent use of various chemical modifications is warranted to enhance nuclease resistance, prevent immune activation, decrease off-target effects, and to improve pharmacokinetic and pharmacodynamic properties. The focus of this review is to interpret the tolerance of various chemical modifications employed in siRNAs toward RNAi by taking into account the crystal structures and biochemical studies of Ago-RNA complexes. Moreover, the challenges and recent progress in imparting druglike properties to siRNAs along with their delivery strategies are discussed.