Impact of pemafibrate in patients with metabolic dysfunction-associated steatotic liver disease complicated by dyslipidemia: A single-arm prospective study.

Background and Aim: This study aimed to clarify the efficacy and safety of 48-week pemafibrate treatment in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) complicated by dyslipidemia. Methods: A total of 110 patients diagnosed with MASLD complicated by dyslipidemia received pemafibrate at a dose of 0.1 mg twice daily for 48 weeks. Results: The participants were 54 males and 37 females, with a median age of 63 (52-71) years. Besides improvement in lipid profile, significant reductions from baseline to 48 weeks of treatment were found in liver-related enzymes, such as aspartate aminotransferase, alanine aminotransferase (ALT), gamma-glutamyl transpeptidase, and alkaline phosphatase (P < 0.001 for all). A significant decrease in the homeostasis model assessment-insulin resistance (HOMA-IR) was observed in patients with insulin resistance (HOMA-IR ≥ 2.5) (4.34 at baseline to 3.89 at Week 48, P < 0.05). Moreover, changes in ALT were weakly correlated with those in HOMA-IR (r = 0.34; p < 0.05). Regarding noninvasive liver fibrosis tests, platelets, Wisteria floribunda agglutinin-positive Mac-2-binding protein, type IV collagen 7s, and the non-alcoholic fatty liver disease fibrosis score significantly decreased from baseline to Week 48. Most adverse events were Grades 1-2, and no drug-related Grade 3 or higher adverse events were observed. Conclusion: This study demonstrated that 48-week pemafibrate administration improved liver-related enzymes and surrogate marker of liver fibrosis in patients with MASLD. The improvement of insulin resistance by pemafibrate may contribute to the favorable effect on MASLD complicated by dyslipidemia.

Low vitamin D levels accelerates muscle mass loss in patients with chronic liver disease.

Sarcopenia frequently and progressively occurs in patients with chronic liver disease. This study aimed to clarify the relationship between vitamin D levels and muscle mass loss. A total of 166 patients with chronic liver disease were enrolled in this study. Skeletal muscle mass index (SMI) was measured by bioelectrical impedance analysis at baseline and after 1 year. The rate of change in SMI from baseline after 1 year was calculated: ΔSMI (%) = [(1-year SMI - baseline SMI) / baseline SMI] × 100. Muscle mass loss was defined as ΔSMI ≤ -2%. The median 25-hydroxyvitamin D was 15.2 (11.2-19.3) ng/mL. The median SMI were 6.8 (5.9-7.8) kg/m2 at baseline and 6.7 (5.9-7.6) kg/m2 after 1 year. The median ΔSMI was -1.23% (-2.21% to 1.61%). Multivariate analysis identified low 25-hydroxyvitamin D as an independent factor associated with muscle mass loss. The optimal cut-off value of 25-hydroxyvitamin D to predict muscle mass loss was 12.7 ng/mL. Muscle mass loss was found in 56.4% v.s. 18.0% of patients with 25-hydroxyvitamin D < 12.7 vs. ≥ 12.7 ng/mL, respectively (p = 9.01 × 10-7); with the highest incidence in patients with non-alcoholic fatty liver disease (NAFLD). Specifically, patients with NAFLD and 25-hydroxyvitamin D < 12.7 ng/mL had a significantly higher incidence of muscle mass loss than those with ≥ 12.7 ng/mL (p = 1.23 × 10-3). Low vitamin D levels are associated with muscle mass loss after 1 year in patients with chronic liver disease, especially NAFLD.

Development of the vertebra and fin skeleton in the lamprey and its implications for the homology of vertebrate vertebrae.

BACKGROUND: Although vertebrae are the defining character of vertebrates, they are found only in rudimentary form in extant agnathans. In addition, the vertebrae of agnathans possess several unique features, such as elastin-like molecules as the main matrix component and late (post-metamorphosis) differentiation of lamprey vertebrae. In this study, by tracing the developmental process of vertebrae in lamprey, we examined the homology of vertebrae between lampreys and gnathostomes. RESULTS: We found that the lamprey somite is first subdivided mediolaterally, with myotome cells differentiating medially and non-myotome cells emerging laterally. Subsequently, collagen-positive non-myotome cells surround the myotome. This pattern of somitogenesis is rather similar to that in amphioxi and sheds doubt on the presence of a sclerotome, in terms of mesenchyme cells induced by a signal from the notochord, in lamprey. Further tracing of non-myotome cell development revealed that fin cartilage develops in ammocoete larvae approximately 35 mm in body length. The development of the fin cartilage occurs much earlier than that of the vertebra whose development proceeds during metamorphosis. CONCLUSION: We propose that the homology of vertebrae between agnathans and gnathostomes should be discussed carefully, because the developmental process of the lamprey vertebra is different from that of gnathostomes.

A novel formula used for predicting hepatocellular carcinoma after the achievement of sustained virologic response by direct-acting antivirals in patients with chronic hepatitis C.

Although eliminating HCV can prevent hepatocellular carcinoma (HCC), some patients develop HCC even after obtaining sustained virologic response (SVR). Previously, we developed a new formula to predict advanced liver fibrosis. This study aimed to clarify the usefulness of this formula for predicting HCC after achieving SVR. Among 351 consecutive patients who had been treated with direct-acting antivirals, 299 were included in this study. New formula scores were used as a marker for predicting liver fibrosis and as a predictive model for HCC incidence. The participants were 172 men and 127 women with a median age of 68 years. The median new formula score was -1.291. The cumulative HCC incidence rates were 4.3%, 9.7%, and 12.5% at 1, 3, and 5 years, respectively. The cumulative incidence of HCC was significantly higher in patients with a history of HCC than in those without treatment history of HCC (P = 2.52×10-26). Multivariate analysis revealed that male (HR = 6.584, 95% CI = 1.291-33.573, P = 0.023) and new formula score (HR = 1.741, 95% CI = 1.041-2.911, P = 0.035) were independent factors associated with the development of HCC in patients without a treatment history of HCC. The optimal cutoff value for predicting the development of HCC was -0.214. The cumulative incidence rates of HCC in patients with new formula scores ≥-0.214 were 5.4%, 15.3%, and 15.3% at 1, 3, and 5 years, respectively, whereas the incidence rates of HCC in patients with new formula scores <-0.214 were 0.0%, 0.6%, and 4.8%, respectively (P = 2.12×10-4). In conclusion, this study demonstrated the usefulness of new formula scores as a predictor of HCC after achieving SVR, especially in patients without past treatment history of treatment for HCC.

Sensing intracellular signatures with synthetic mRNAs.

The bottom-up assembly of biological components in synthetic biology has contributed to a better understanding of natural phenomena and the development of new technologies for practical applications. Over the past few decades, basic RNA research has unveiled the regulatory roles of RNAs underlying gene regulatory networks; while advances in RNA biology, in turn, have highlighted the potential of a wide variety of RNA elements as building blocks to construct artificial systems. In particular, synthetic mRNA-based translational regulators, which respond to signals in cells and regulate the production of encoded output proteins, are gaining attention with the recent rise of mRNA therapeutics. In this Review, we discuss recent progress in RNA synthetic biology, mainly focusing on emerging technologies for sensing intracellular protein and RNA molecules and controlling translation.

Programmable mammalian translational modulators by CRISPR-associated proteins.

Translational modulation based on RNA-binding proteins can be used to construct artificial gene circuits, but RNA-binding proteins capable of regulating translation efficiently and orthogonally remain scarce. Here we report CARTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Gene control) to repurpose Cas proteins as translational modulators in mammalian cells. We demonstrate that a set of Cas proteins efficiently and orthogonally repress or activate the translation of designed mRNAs that contain a Cas-binding RNA motif in the 5'-UTR. By linking multiple Cas-mediated translational modulators, we designed and built artificial circuits like logic gates, cascades, and half-subtractor circuits. Moreover, we show that various CRISPR-related technologies like anti-CRISPR and split-Cas9 platforms could be similarly repurposed to control translation. Coupling Cas-mediated translational and transcriptional regulation enhanced the complexity of synthetic circuits built by only introducing a few additional elements. Collectively, CARTRIDGE has enormous potential as a versatile molecular toolkit for mammalian synthetic biology.

Experimental inoculation of a tissue homogenate containing porcine circovirus type 3 obtained after two in vivo passages in NIBS miniature pigs.

Porcine circovirus type 3 (PCV3) is a novel porcine circovirus that has been detected in pigs showing various clinical and pathological conditions, as well as in many asymptomatic pigs. The pathogenesis of PCV3 infection in pigs remains unclear. To evaluate the in vivo growth and pathogenicity of PCV3, we performed two experiments on PCV3 infection in laboratory-grade miniature pigs with strictly controlled genetic backgrounds and microbiological status. A PCV3 passage experiment confirmed PCV3 genome detection in the sera and multiple organs via in vivo serial passage generations. PCV3 was successively passaged in miniature pigs by inoculating tissue homogenates from infected pigs supporting Koch's principles. In the PCV3 infection experiment, viremia was observed in all the inoculated pigs, and transient neurological signs were observed in one of the three pigs. Histopathologically, all three pigs in the PCV3 inoculation group exhibited lung disorders such as interstitial pneumonia and lymphoplasmacytic perivasculitis. In addition, one pig with neurological signs in the PCV3 inoculation group showed focal thrombosis in the meninges of the cerebellum. Vascular lesions in both the lungs and brain suggest that PCV3 may cause injury to vascular tissues. In situ hybridization (ISH)-RNA analysis demonstrated that the PCV3 genome was localized in the lymph nodes of pigs inoculated with PCV3. The PCV3 in vivo passage system in NIBS miniature pigs will help investigate the pathogenicity of PCV3.

Pseudo-hyperacute T Waves in a Giant Hiatal Hernia.

An 89-year-old woman with a giant hiatal hernia complained of persistent chest pain. An electrocardiogram (ECG) showed hyperacute T waves, suggesting the early phase of ST-elevation myocardial infarction. After endoscopic drainage for hiatal hernia, the chest pain disappeared, and the ECG abnormalities resolved. The present case illustrates that compression of the heart by a giant hiatal hernia can induce T wave elevation mimicking acute coronary syndrome.

Review of the intertidal rove beetle tribe Liparocephalini Fenyes (Coleoptera, Staphylinidae, Aleocharinae) from Japan.

Japanese fauna of the intertidal rove beetle of tribe Liparocephalini Fenyes, 1918 is reviewed and six genera and 26 species are recognized. A new genus, Rotundicephala Tasaku, Ono & Maruyama, gen. n., is described to include R. pacifica (Sawada, 1971) comb. n. (type species; transferred from Diaulota), R. koreana (Yoo & Ahn, 2021), comb. n. (transferred from Diaulota) and R. koheii Tasaku, Ono & Maruyama, sp. n. Four new species are described: Diaulota decipiens Tasaku, Ono & Maruyama, sp. n., which has been confused with D. aokii, D. orientalis Tasaku, Ono & Maruyama, sp. n., R. koheii Tasaku, Ono & Maruyama, sp. n., and Paramblopusa sumikawai Tasaku, Ono & Maruyama, sp. n. Three species, Amblopusa brevipes Casey, 1893, D. submarina Ahn, 2023 and R. koreana, are reported from Japan for the first time. We provide keys to genera and species, illustrations of mouth parts and diagnostic characteristics of each genus, and diagnostic characteristics and illustrations of the genitalia of almost all species. Biogeographical patterns of Diaulota and Rotundicephala gen. n., and the tribal range and monophyly of Liparocephalini are discussed. Three genus-group: the Liparocephalus genus-group, the Amblopusa genus-group, and the Paramblopusa genus-group are recognized in the tribe, but Baeostethus Broun, 1909 known from New Zealand is regarded as incertae sedis. The genera Ashella Klimaszewski, 2020 and Ianmoorea Ahn, 2006 are excluded from Liparocephalini.

Pseudoxanthoma elasticum resulting in acute coronary syndrome.

Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder that causes elastic tissue degeneration in the skin, eyes, and cardiovascular system. Gastrointestinal bleeding and fundus hemorrhage are serious complications associated with PXE prognosis as well as cardiovascular involvement. This is a rare case of acute coronary syndrome in a PXE patient with high bleeding risk. Learning objective: Pseudoxanthoma elasticum (PXE) resulting in acute coronary syndrome (ACS) is rare. Given PXE patients are generally at very high bleeding risk, antithrombotic therapy as secondary prevention after ACS onset should be taken into full consideration.

Black pleural effusion caused by a pancreaticopleural fistula associated with autoimmune pancreatitis: A case report.

RATIONALE: Black pleural effusion is a rare medical condition and a diagnostic marker. Pancreaticopleural fistula is one of the causes of black pleural effusion. Thus far, black pleural effusions caused by pancreaticopleural fistulae have mostly been reported in patients with alcohol-induced chronic pancreatitis. In this report, we present a case of black pleural effusion caused by a pancreaticopleural fistula associated with autoimmune pancreatitis. PATIENT CONCERNS AND DIAGNOSIS: A 59-year-old female without a history of alcohol drinking presented to our hospital with a chief complaint of dyspnea, as well as chest and back discomfort. She had left pleural effusion, and thoracentesis showed black pleural effusion. Computed tomography revealed the presence of encapsulated fluid from the pancreatic tail to the left pleural cavity, which was diagnosed as a pancreaticopleural fistula. It also showed diffuse pancreatic swelling. Serum testing showed a high IgG4 level (363 mg/dL). These findings led to the diagnosis of autoimmune pancreatitis. INTERVENTIONS AND OUTCOME: The patient underwent endoscopic pancreatic sphincterotomy and pancreatic duct stent placement and received treatment with steroids. After treatment, there was no further accumulation of pleural effusion observed. CONCLUSION: This is the first report of black pleural effusion due to a pancreaticopleural fistula associated with autoimmune pancreatitis. The characteristic appearance of black pleural effusion may assist diagnosis. We report this case to emphasize that autoimmune pancreatitis can be a cause of black pleural effusion.

Antifibrotic effect and long-term outcome of SGLT2 inhibitors in patients with NAFLD complicated by diabetes mellitus.

The aim of this retrospective multicenter study was to clarify the antifibrotic effect and long-term outcome of sodium glucose cotransporter 2 inhibitors (SGLT2-Is) in patients with nonalcoholic fatty liver disease (NAFLD) complicated by type 2 diabetes mellitus (T2DM). Of the 1262 consecutive patients with T2DM who recently received SGLT2-Is, 202 patients with NAFLD had been receiving SGLT2-Is for more than 48 weeks and were subjected to this analysis. Furthermore, 109 patients who had been on SGLT2-I therapy for more than 3 years at the time of analysis were assessed for the long-term effects of SGLT2-Is. Significant decreases in body weight, liver transaminases, plasma glucose, hemoglobin A1c, and Fibrosis-4 (FIB-4) index were found at week 48. Overall, the median value of FIB-4 index decreased from 1.42 at baseline to 1.25 at week 48 (p < 0.001). In the low-risk group (FIB-4 index < 1.3), there was no significant change in the FIB-4 index. In the intermediate-risk (≥1.3 and <2.67) and high-risk (≥2.67) groups, the median levels significantly decreased from 1.77 and 3.33 at baseline to 1.58 and 2.75 at week 48, respectively (p < 0.001 for both). Improvements in body weight, glucose control, liver transaminases, and FIB-4 index were found at 3 years of SGLT2-I treatment. In the intermediate-risk and high-risk groups (≥1.3 FIB-4 index), the FIB-4 index maintained a significant reduction from baseline throughout the 3 years of treatment. Conclusion: This study showed that SGLT2-Is offered a favorable effect on improvement in FIB-4 index as a surrogate marker of liver fibrosis in patient with NAFLD complicated by T2DM, especially those with intermediate and high risks of advanced fibrosis, and this antifibrotic effect is sustained for the long term.

Comparative study of the phenotype and virulence of recent serovar 1a, 1b, and 2a isolates of Erysipelothrix rhusiopathiae in Japan.

Erysipelothrix rhusiopathiae causes swine erysipelas (SE) and is classified -into 16 serovars based on cell surface antigens. Our previous study suggested that recent SE outbreaks were mostly caused by serovar 1a of E. rhusiopathiae with the surface protective antigen (Spa)A protein characterized by methionine and isoleucine at positions 203 and 257 (M203/I257 SpaA). In this study, four recent E. rhusiopathiae isolates comprising two serovar 1a with M203/I257 SpaA strains (2012 Miyazaki and 2012 Chiba), one serovar 1b strain (2015 Miyazaki), and one serovar 2a strain (2012 Nagano) were compared with each other and with the serovar 1a Fujisawa reference strain regarding in vitro phenotypes and in vivo virulence in mice and pigs. The serovar 1b and 2a strains, which are the less prevalent strains in the field in Japan, showed lower growth in liquid culture and lower virulence in animals than the serovar 1a variants. Adhesion of the serovar 2a strain to porcine endothelial cells was weaker than that of the serovar 1a and 1b strains. Several advantages of serovar 1a strains were found, but no plausible cause of the M203/I257 SpaA type variants to be selected for the most prevalent strains among serovar 1a strains was identified in this study.

Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex.

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3' untranslated region (3'-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3'-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3'-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node.

Effect of Vitamin D Supplementation on Skeletal Muscle Volume and Strength in Patients with Decompensated Liver Cirrhosis Undergoing Branched Chain Amino Acids Supplementation: A Prospective, Randomized, Controlled Pilot Trial.

BACKGROUND: Sarcopenia worsens patient prognoses in chronic liver disease. This study aimed to elucidate the effects of vitamin D supplementation on skeletal muscle volume and strength in patients with decompensated cirrhosis. METHODS: Thirty-three patients were entered into the study based on the criteria and then randomly assigned to two groups: Group A (n = 17), the control group, and Group B (n = 16), those who received oral native vitamin D3 at a dose of 2000 IU once a day for 12 months. RESULTS: SMI values in Group B were significantly increased at 12 months (7.64 × 10-3). The extent of changes in the SMI and grip strength in Group B were significantly greater than that in Group A at 12 months (p = 2.57 × 10-3 and 9.07 × 10-3). The median change rates in the SMI were +5.8% and the prevalence of sarcopenia was significantly decreased from 80.0% (12/15) to 33.3% (5/15; p = 2.53 × 10-2) in Group B. CONCLUSIONS: Vitamin D supplementation might be an effective and safe treatment option for patients with decompensated cirrhosis to increase or restore the skeletal muscle volume and strength or prevent the muscle volume and strength losses.

Effect of sodium-glucose cotransporter 2 inhibitor in patients with non-alcoholic fatty liver disease and type 2 diabetes mellitus: a propensity score-matched analysis of real-world data.

BACKGROUND: Although sodium-glucose cotransporter 2 inhibitors (SGLT2-Is) improve not only glycemic control but also liver inflammation and fatty changes in patients with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM), its sustainability and effect on liver fibrosis have remained unclear. The current study aimed to clarify the effects of 48-week SGLT2-I therapy on liver inflammation, fatty changes, and fibrosis in NAFLD patients with T2DM. METHODS: This study evaluated the effects of SGLT2-I on NAFLD, including liver fibrosis assessed via transient elastography, in 56 patients with NAFLD who received SGLT2-I for 48 weeks. Moreover, changes in each clinical parameter between patients receiving SGLT2-I (the SGLT2-I group) and those receiving other oral hypoglycemic agents (OHAs) (the non-SGLT2-I group) were compared, using 1:1 propensity score matching to adjust for baseline factors. RESULTS: The SGLT2-I group exhibited a significant decrease in controlled attenuation parameter (312 dB/m at baseline to 280 dB/m at week 48) and liver stiffness measurement (9.1-6.7 kPa) (p < 0.001 for both). After propensity score matching (44 patients each in the SGLT2-I and non-SGLT2-I groups), no significant difference in HbA1c decrease was observed between the two groups. However, compared with the non-SGLT2-I group, the SGLT2-I group showed a significant decrease in body weight (p < 0.001), alanine aminotransferase (p = 0.02), uric acid (p < 0.001), and Fibrosis-4 (FIB-4) index (p = 0.01) at week 48. The improvement in FIB-4 index, defined as a ⩾10% decline from baseline at week 48, was 56.8% (25/44) in the SGLT2-I group and 20.5% (9/44) in the non-SGLT2-I group (p < 0.001). CONCLUSION: SGLT2-Is improved not only glycemic control but also liver fatty infiltration and fibrosis in patients with NAFLD and T2DM, suggesting their possible superiority to other OHAs concerning these effects.

Fe2+ Ions Alleviate the Symptom of Citrus Greening Disease.

Citrus greening (CG) is among the most devastating citrus diseases worldwide. CG-infected trees exhibit interveinal chlorotic leaves due to iron (Fe) deficiency derived from CG; thus, Fe content is lower in infected leaves than in healthy leaves. In this study, we demonstrated that the foliar application of Fe2+ relieves the symptom of CG infection in citrus trees. We applied Fe2+ and citrate to the leaves of infected rough lemon plants. Following this treatment, a reduction in the number of yellow symptomatic leaves was observed, and their growth was restored. Using chlorophyll content as an index, we screened for effective Fe complexes and found that a high ratio of citrate to Fe2+ in the applied solution led to effects against CG in Shikuwasa trees. A high proportion of Fe2+ to total Fe was another key factor explaining the effectiveness of the solution in CG infection, indicating the importance of Fe2+ absorption into plant cells. We confirmed the proportion of Fe2+ to total Fe through the high correlation of reflectometry data via a triazine reaction and X-ray absorption fine structure analysis. These results demonstrate that the foliar application of a high-Fe2+ citrate solution can restore the growth of CG diseased trees.

A new species and new record of the intertidal rove beetle genus Bryothinusa Casey (Staphylinidae: Aleocharinae: Myllaenini) from Japan.

A new species of the intertidal rove beetle genus Bryothinusa, B. japonica sp. nov. is described, and B. koreana Ahn Jeon, 2004 is recorded from Japan for the first time. These species are illustrated and compared with similar species. A key to the Japanese Bryothinusa species and a checklist of the known Bryothinusa species are provided.

Direct RNA sequencing approach to compare non-model mitochondrial transcriptomes: An application to a cephalopod host and its mesozoan parasite.

To identify non-protein coding as well as truncated or premature RNA sequences expressed and obtain more complete transcriptome information, we combined the MinION direct RNA-sequencing of a conventional poly(A) RNA purification method with poly(A)-tagging of the non-coding RNA (ncRNA) fraction. This approach was applied to transcriptome sequencing of the dicyemid mesozoan, Dicyema misakiense, which has minicircular mitochondrial DNA molecules where each molecule encodes a single gene, as well as the host. Using informatics analysis, we distinguished dicyemid RNAs from those of the host squid. The poly(A) RNAs were assigned to host mitochondrial genes, host nuclear protein-coding genes, Dicyema nuclear protein-coding genes, and Dicyema mitochondrial genes in the decreasing order. Our poly(A)-tailing method recovered significantly more ncRNAs from the host compared with the sequencing of poly(A) RNAs. Furthermore, our method captured various lengths of squid mitochondrial DNA (mtDNA) transcripts at different steps of maturation including a read of 3,500 bp, which covers 21% of the squid mitochondrial genome, possibly a premature host RNA product. In contrast, shorter and less abundant reads were recovered from the dicyemid mitochondrial RNAs (mtRNAs). Even the longest read was 307 bp covering only a part of a minicircle. This study revealed significantly different modes of the mitochondrial transcription between a mesozoan and the host. Our approach to perform direct RNA-sequencing combined with the poly(A)-tailing reaction can be an effective method to fully capture non-poly(A) transcripts in a wide range of organisms.