Regulation of ribosomal RNA gene copy number, transcription and nucleolus organization in eukaryotes.

One of the first biological machineries to be created seems to have been the ribosome. Since then, organisms have dedicated great efforts to optimize this apparatus. The ribosomal RNA (rRNA) contained within ribosomes is crucial for protein synthesis and maintenance of cellular function in all known organisms. In eukaryotic cells, rRNA is produced from ribosomal DNA clusters of tandem rRNA genes, whose organization in the nucleolus, maintenance and transcription are strictly regulated to satisfy the substantial demand for rRNA required for ribosome biogenesis. Recent studies have elucidated mechanisms underlying the integrity of ribosomal DNA and regulation of its transcription, including epigenetic mechanisms and a unique recombination and copy-number control system to stably maintain high rRNA gene copy number. In this Review, we disucss how the crucial maintenance of rRNA gene copy number through control of gene amplification and of rRNA production by RNA polymerase I are orchestrated. We also discuss how liquid-liquid phase separation controls the architecture and function of the nucleolus and the relationship between rRNA production, cell senescence and disease.

Ferroptosis-related gene glutathione peroxidase 4 promotes reprogramming of glucose metabolism via Akt-mTOR axis in intrahepatic cholangiocarcinoma.

The role of the ferroptosis-related gene glutathione peroxidase 4 (GPX4) in oncology has been extensively investigated. However, the clinical implications of GPX4 in patients with intrahepatic cholangiocarcinoma (ICC) remain unknown. This study aimed to evaluate the prognostic impact of GPX4 and its underlying molecular mechanisms in patients with ICC. Fifty-seven patients who underwent surgical resection for ICC between 2010 and 2017 were retrospectively analyzed. Based on the immunohistochemistry, patients were divided into GPX4 high (n = 15) and low (n = 42) groups, and clinical outcomes were assessed. Furthermore, the roles of GPX4 in cell proliferation, migration and gene expression were analyzed in ICC cell lines in vitro and in vivo. The results from clinical study showed that GPX4 high group showed significant associations with high SUVmax on 18F-fluorodeoxyglucose-positron emission tomography (≥8.0, P = 0.017), multiple tumors (P = 0.004), and showed glucose transporter 1 (GLUT1) high expression with a trend toward significance (P = 0.053). Overall and recurrence-free survival in the GPX4 high expression group were significantly worse than those in the GPX4 low expression group (P = 0.038 and P < 0.001, respectively). In the experimental study, inhibition of GPX4 attenuated cell proliferation and migration in ICC cell lines. Inhibition of GPX4 also decreased the expression of glucose metabolism-related genes, such as GLUT1 or HIF1α. Mechanistically, these molecular changes are regulated in Akt-mechanistic targets of rapamycin axis. In conclusion, this study suggested the pivotal value of GPX4 serving as a prognostic marker for patients with ICC. Furthermore, GPX4 can mediate glucose metabolism of ICC.

The human ribosomal DNA array is composed of highly homogenized tandem clusters.

The structure of the human ribosomal DNA (rDNA) cluster has traditionally been hard to analyze owing to its highly repetitive nature. However, the recent development of long-read sequencing technology, such as Oxford Nanopore sequencing, has enabled us to study the large-scale structure of the genome. Using this technology, we found that human cells have a quite regular rDNA structure. Although each human rDNA copy has some variations in its noncoding region, contiguous copies of rDNA are similar, suggesting that homogenization through gene conversion frequently occurs between copies. Analysis of rDNA methylation by Nanopore sequencing further showed that all the noncoding regions are heavily methylated, whereas about half of the coding regions are clearly unmethylated. The ratio of unmethylated copies, which are speculated to be transcriptionally active, was lower in individuals with a higher rDNA copy number, suggesting that there is a mechanism that keeps the active copy number stable. In addition, the rDNA in progeroid syndrome patient cells with reduced DNA repair activity had more unstable copies compared with control normal cells, although the rate was much lower than previously reported using a fiber-FISH method. Collectively, our results clarify the view of rDNA stability and transcription regulation in human cells, indicating the presence of mechanisms for both homogenizations to ensure sequence quality and maintenance of active copies for cellular functions.

Impact of liver volumetric regeneration on survival outcomes in patients with hepatocellular carcinoma after major hepatectomy.

PURPOSE: Prognostic value of liver volumetric regeneration (LVR) in patients with hepatocellular carcinoma (HCC) who undergo major hepatectomy remains unknown. The aim of this study was to investigate the impact of LVR on long-term outcomes in these patients. METHODS: Data of 399 consecutive patients with HCC who underwent major hepatectomy between 2000 to 2018 were retrieved from a prospectively maintained institutional database. The LVR-index was defined as the relative increase in liver volume from 7 days to 3 months (RLV3m/RLV7d, where RLV3m and RLV7d is the remnant liver volume around 3 months and postoperative 7 days after surgery). The optimal cut-off value was determined using the median value of LVR-index. RESULTS: A total of 131 patients were eligible in this study. The optimal cut off value of LVR-index was 1.194. The 1-, 3-, 5- and 10-year overall survival (OS) rate of patients in the high LVR-index group were significantly better compared to those in the low LVR-index group (95.5%, 84.8%, 75.4% and 49.1% vs. 95.4%, 70.2%, 56.4%, and 19.9%, p = 0.002). Meanwhile, there was no significant difference with regards to time to recurrence between the two groups (p = 0.607). Significance of LVR-index for OS was retained after adjusting for known prognostic factors (p = 0.002). CONCLUSION: In patients with HCC undergoing major hepatectomy, LVR-index may serve as a prognostic indicator for OS.

Impact of Preoperative CEA Uptrend on Survival Outcomes in Patients with Colorectal Liver Metastasis After Hepatectomy.

BACKGROUND: Preoperative carcinoembryonic antigen (CEA) has been reported as a prognostic factor in patients with colorectal liver metastasis (CRLM) after hepatectomy. However, the impact of a preoperative "CEA uptrend" on prognosis after hepatectomy in these patients remains unknown. This study assessed the impact of CEA uptrend on prognosis in patients undergoing hepatectomy for CRLM. METHODS: Consecutive patients with CRLM who underwent hepatectomy between 2009 and 2018 were retrospectively analyzed. Patients with CRLM for whom CEA was measured both around 1 month before (CEA-1m) and within 3 days (CEA-3d) before hepatectomy were enrolled. A CEA-3d higher than both the upper limit of normal (5 ng/ml) and CEA-1m was defined as a CEA uptrend. RESULTS: Study participants comprised 212 patients with CRLM. Of these, 88 patients (41.5%) showed a CEA uptrend. CEA uptrend indicated better discriminatory ability (corrected Akaike information criteria, 733.72) and homogeneity (likelihood ratio chi-square value, 18.80) than CEA-3d or CEA-1m. Patients with CEA uptrend showed poorer overall survival than those without CEA uptrend (p < 0.001). After adjusting for known prognostic factors, the prognostic significance of CEA uptrend retained (hazard ratio 2.63, 95% confidence interval 1.63-4.26, p < 0.001). In subgroup analyses, the prognostic significance of CEA uptrend was retained irrespective of the status of RAS mutation or response to preoperative chemotherapy. CONCLUSIONS: CEA uptrend offers better prediction of survival outcomes than conventional CEA measurements in patients undergoing hepatectomy for CRLM.

Acylhydrazone Subunits as a Proton Cargo Delivery System in 7-Hydroxyquinoline.

The reimagined concept of long-range tautomeric proton transfer using crane subunits is shown by designing and synthesising two new acylhydrazones containing a 7-hydroxyquinoline (7-OHQ) platform. The acylhydrazone subunits attached to the 7-OHQ at the 8th position act as crane arms for delivering proton cargo to the quinoline nitrogen. Light-induced tautomerization to their keto forms leads to Z/E isomerization of the C=C axle bond, followed by proton delivery to the quinoline nitrogen by the formation of covalent or hydrogen bonds. The axle's being either an imine or ketimine bond is the structural difference between the studied compounds. The -CH3 group in the latter provides steric strain, resulting in different proton transport pathways. Both compounds show long thermal stability in the switched state, which creates a tuneable action of bidirectional proton cargo transport by using different wavelengths of irradiation. Upon the addition of acid, the quinoline nitrogen is protonated; this results in E/Z configuration switching of the acylhydrazone subunits. This was proven by single-crystal X-ray structure analysis and NMR spectroscopy.

Preoperative CT volumetry of estimated residual kidney for prediction of postoperative chronic kidney disease in patients with renal cell carcinoma.

BACKGROUND: Surgical treatments for renal cell carcinoma reduces kidney volume to some degree and may derive postsurgical chronic kidney disease. We made a new marker for postoperative renal function using CT volumetry. To determine the impact of various parameters including this marker, we observed pre- and postsurgical renal function of experienced cases. METHODS: From 2004 to 2014, we underwent total or partial nephrectomy for 181 patients with renal carcinoma in a single institution. Of the total, 138 cases with presurgical CT volumetry were included in this study. We evaluated parameters for assessments of peri- and postoperative renal function including age, gender, serum creatinine, eGFR, performed surgery, pathology, estimated residual kidney volume and associated disease. Presence or absence of acute kidney injury (AKI) and chronic kidney disease (CKD) were also evaluated before, immediately after and 5 years after surgery. RESULTS: Multiple logistic regression analysis identified AKI, preoperative eGFR and estimated residual kidney volume as significant prognostic factors for the postoperative CKD. Moreover, cases with triple positive of these factors suffer postoperative CKD more significantly than those with one or two positives. CONCLUSION: Using these predictive factors, we may determine patients with high risk for CKD who require an early intervention of renal protective treatment.

Laparoscopic sacrocolpopexy for pelvic organ prolapse: Comparison of standard versus tacker combination method.

OBJECTIVE: To compare the surgical outcomes of laparoscopic sacrocolpopexy for pelvic organ prolapse between a group in which only sutures were used (standard method), and a group in which a combination of tackers and sutures were used (tacker combination method). METHODS: A total of 77 patients who underwent laparoscopic sacrocolpopexys from June 2016 to October 2019 were divided into a suture group (36 patients) and a suture + tacker group (41 patients). We retrospectively compared operation time, amount of blood loss, postoperative length of hospital stay, incidence of perioperative complications and anatomical cure rate 1 year after surgery. Lower urinary tract symptoms were evaluated using symptom questionnaires and objective parameters. RESULTS: Operation time in the suture + tacker group was shorter (104.9 ± 27.0 vs 147.5 ± 33.7 min; P < 0.0001). The incidence of perioperative complications in the suture group and the suture + tacker group was 2.8% and 2.4%, respectively (P = 0.9409). Anatomical cure rates at 1 year after surgery were 94.4% and 100%, respectively (P = 0.2153). Both groups showed significant improvement after 1 year for International Prostate Symptom Score total and quality of life score, Overactive Bladder Symptom Score total score, voided volume, maximum urinary flow rate and post-void residual. [Corrections added on 7 September 2021 after first online publication: the first two P-values have been updated.] CONCLUSIONS: The combined use of sutures and tackers in laparoscopic sacrocolpopexy simplifies the procedure and translates into shorter operation time. Surgical outcomes at 1 year and improvement of lower urinary tract symptoms are similar regardless of the technique.

The Combination of Cholecystokinin and Stress Amplifies an Inhibition of Appetite, Gastric Emptying, and an Increase in c-Fos Expression in Neurons of the Hypothalamus and the Medulla Oblongata.

Cholecystokinin (CCK) had been the first gastrointestinal hormone known to exert anorexic effects. CCK had been inferred to contribute to the onset of functional dyspepsia (FD) symptoms. To understand the pathophysiology of FD, the roles of stress have to be clarified. In this study, we aimed to clarify the influence of stress on the action of cholecystokinin (CCK) on appetite and gastric emptying. Using rats, stress was simulated by giving restraint stress or intraperitoneal injection of the stress-related peptide hormone urocortin 1 (UCN1). The effects of CCK and restraint stress, alone or in combination, on food intake and gastric motility were examined, and c-Fos expression in the neurons of appetite control network in the central nervous system was assessed by immunohistochemical staining. CCK inhibited food intake and gastric emptying in a dose-dependent manner. Food intake for 1 h was significantly lower with UCN1 (2 nmol/kg) than with the saline control. Restraint stress amplified the suppressive effects of CCK on food intake for 1 h and on gastric emptying. With regard to brain function, the CCK induced c-Fos expression in the neurons of the nucleus tractus solitarius and paraventricular nucleus of the hypothalamus was markedly and significantly amplified by the addition of restraint stress with CCK. The results suggested that stress might amplify the anorexic effects of CCK through activation of the nuclei that comprise the brain neuronal network for satiation; this might play a role in the pathogenesis of the postprandial distress syndromes of functional dyspepsia.

Important cardiac transcription factor genes are accompanied by bidirectional long non-coding RNAs.

BACKGROUND: Heart development is a relatively fragile process in which many transcription factor genes show dose-sensitive characteristics such as haploinsufficiency and lower penetrance. Despite efforts to unravel the genetic mechanism for overcoming the fragility under normal conditions, our understanding still remains in its infancy. Recent studies on the regulatory mechanisms governing gene expression in mammals have revealed that long non-coding RNAs (lncRNAs) are important modulators at the transcriptional and translational levels. Based on the hypothesis that lncRNAs also play important roles in mouse heart development, we attempted to comprehensively identify lncRNAs by comparing the embryonic and adult mouse heart and brain. RESULTS: We have identified spliced lncRNAs that are expressed during development and found that lncRNAs that are expressed in the heart but not in the brain are located close to genes that are important for heart development. Furthermore, we found that many important cardiac transcription factor genes are located in close proximity to lncRNAs. Importantly, many of the lncRNAs are divergently transcribed from the promoter of these genes. Since the lncRNA divergently transcribed from Tbx5 is highly evolutionarily conserved, we focused on and analyzed the transcript. We found that this lncRNA exhibits a different expression pattern than that of Tbx5, and knockdown of this lncRNA leads to embryonic lethality. CONCLUSION: These results suggest that spliced lncRNAs, particularly bidirectional lncRNAs, are essential regulators of mouse heart development, potentially through the regulation of neighboring transcription factor genes.

Case of mumps orchitis after vaccination.

We present a case of mumps orchitis after vaccination. A 35-year-old man visited the emergency unit of our hospital for fever and right testicular pain on 7 January 2012. Two weeks before the visit, he underwent vaccination for mumps. After 7 days of hospitalization, the patient recovered from fever and testicular pain. Scrotal magnetic resonance imaging suggested right orchitis, and an increased level of serum mumps immunoglobulin M and immunoglobulin G indicated an initial infection of mumps virus. The peak value of immunoglobulin G showed 106 enzyme immunoassay unit 5 weeks after vaccination, which was 10-fold higher than the ordinary rise of a vaccinated individual. This fact leads to two possibilities. One is this happened as an adverse event of vaccination, and the other is that he actually had a wild-type infection at the same time as vaccination. There are some reports regarding adverse events of mumps vaccine; however, they might include wild-type infection.

Replication fork blocking deficiency leads to a reduction of rDNA copy number in budding yeast.

The ribosomal RNA genes are encoded as hundreds of tandem repeats, known as the rDNA, in eukaryotes. Maintaining these copies seems to be necessary, but copy number changes in an active manner have been reported in only frogs, flies, Neurospora, and yeast. In the best-studied system, yeast, a protein (Fob1) binds to the rDNA and unidirectionally blocks the replication fork. This block stimulates rDNA double-strand breaks (DSBs) leading to recombination and copy number change. To date, copy number maintenance and concerted evolution mediated by rDNA repeat turnover were the proposed benefits of Fob1-dependent replication fork arrest. In this study, we tested whether Fob1 provides these benefits and found that rDNA copy number decreases when FOB1 is deleted, suggesting that Fob1 is important for recovery from low copy number. We suppose that replication fork stalling at rDNA is necessary for recovering from rDNA copy number loss in other species as well.

Rikkunshito improves anorexia through ghrelin- and orexin-dependent activation of the brain hypothalamus and mesolimbic dopaminergic pathway in rats.

BACKGROUND: Rikkunshito (RKT), a traditional Japanese medicine, can relieve epigastric discomfort and anorexia in patients with functional dyspepsia. RKT enhances the orexigenic hormone, ghrelin. Ghrelin regulates food motivation by stimulating the appetite control center in the hypothalamus and the brain mesolimbic dopaminergic pathway (MDPW). However, the effect of RKT on MDPW remains unclear. Here, we aimed to investigate the central neural mechanisms underlying the orexigenic effects of RKT, focusing on the MDPW. METHODS: We examined the effects of RKT on food intake and neuronal c-Fos expression in restraint stress- and cholecystokinin octapeptide-induced anorexia in male rats. KEY RESULTS: RKT treatment significantly restored stress- and cholecystokinin octapeptide-induced decreased food intake. RKT increased c-Fos expression in the ventral tegmental area (VTA), especially in tyrosine hydroxylase-immunoreactive neurons, and nucleus accumbens (NAc). The effects of RKT were suppressed by the ghrelin receptor antagonist [D-Lys3]-GHRP-6. RKT increased the number of c-Fos/orexin-double-positive neurons in the lateral hypothalamus (LH), which project to the VTA. The orexin receptor antagonist, SB334867, suppressed RKT-induced increase in food intake and c-Fos expression in the LH, VTA, and NAc. RKT increased c-Fos expression in the arcuate nucleus and nucleus of the solitary tract of the medulla, which was inhibited by [D-Lys3]-GHRP-6. CONCLUSIONS & INFERENCES: RKT may restore appetite in subjects with anorexia through ghrelin- and orexin-dependent activation of neurons regulating the brain appetite control network, including the hypothalamus and MDPW.

Comparative analysis and classification of highly divergent mouse rDNA units based on their intergenic spacer (IGS) variability.

Ribosomal DNA (rDNA) repeat units are organized into tandem clusters in eukaryotic cells. In mice, these clusters are located on at least eight chromosomes and show extensive variation in the number of repeats between mouse genomes. To analyze intra- and inter-genomic variation of mouse rDNA repeats, we selectively isolated 25 individual rDNA units using Transformation-Associated Recombination (TAR) cloning. Long-read sequencing and subsequent comparative sequence analysis revealed that each full-length unit comprises an intergenic spacer (IGS) and a ∼13.4 kb long transcribed region encoding the three rRNAs, but with substantial variability in rDNA unit size, ranging from ∼35 to ∼46 kb. Within the transcribed regions of rDNA units, we found 209 variants, 70 of which are in external transcribed spacers (ETSs); but the rDNA size differences are driven primarily by IGS size heterogeneity, due to indels containing repetitive elements and some functional signals such as enhancers. Further evolutionary analysis categorized rDNA units into distinct clusters with characteristic IGS lengths; numbers of enhancers; and presence/absence of two common SNPs in promoter regions, one of which is located within promoter (p)RNA and may influence pRNA folding stability. These characteristic features of IGSs also correlated significantly with 5'ETS variant patterns described previously and associated with differential expression of rDNA units. Our results suggest that variant rDNA units are differentially regulated and open a route to investigate the role of rDNA variation on nucleolar formation and possible associations with pathology.

Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc.

How morphogen gradients control patterning and growth in developing tissues remains largely unknown due to lack of tools manipulating morphogen gradients. Here, we generate two membrane-tethered protein binders that manipulate different aspects of Decapentaplegic (Dpp), a morphogen required for overall patterning and growth of the Drosophila wing. One is "HA trap" based on a single-chain variable fragment (scFv) against the HA tag that traps HA-Dpp to mainly block its dispersal, the other is "Dpp trap" based on a Designed Ankyrin Repeat Protein (DARPin) against Dpp that traps Dpp to block both its dispersal and signaling. Using these tools, we found that, while posterior patterning and growth require Dpp dispersal, anterior patterning and growth largely proceed without Dpp dispersal. We show that dpp transcriptional refinement from an initially uniform to a localized expression and persistent signaling in transient dpp source cells render the anterior compartment robust against the absence of Dpp dispersal. Furthermore, despite a critical requirement of dpp for the overall wing growth, neither Dpp dispersal nor direct signaling is critical for lateral wing growth after wing pouch specification. These results challenge the long-standing dogma that Dpp dispersal is strictly required to control and coordinate overall wing patterning and growth.

Age-Dependent Ribosomal DNA Variations in Mice.

The rRNA gene, which consists of tandem repetitive arrays (ribosomal DNA [rDNA] repeat), is one of the most unstable regions in the genome. The rDNA repeat in the budding yeast Saccharomyces cerevisiae is known to become unstable as the cell ages. However, it is unclear how the rDNA repeat changes in aging mammalian cells. Using quantitative single-cell analyses, we identified age-dependent alterations in rDNA copy number and levels of methylation in mice. The degree of methylation and copy number of rDNA from bone marrow cells of 2-year-old mice were increased by comparison to levels in 4-week-old mice in two mouse strains, BALB/cA and C57BL/6. Moreover, the level of pre-rRNA transcripts was reduced in older BALB/cA mice. We also identified many sequence variations in the rDNA. Among them, three mutations were unique to old mice, and two of them were found in the conserved region in budding yeast. We established yeast strains with the old-mouse-specific mutations and found that they shortened the life span of the cells. Our findings suggest that rDNA is also fragile in mammalian cells and that alterations within this region have a profound effect on cellular function.

Successful lymphatic embolization using N-butyl-2-cyanoacrylate for postoperative lymphorrhea in a patient with renal pelvic cancer.

We present a successful case of percutaneous lymphatic embolization using N-butyl-2-cyanoacrylate (NBCA) for postoperative lymphorrhea in a patient with urothelial carcinoma of renal pelvis. A 75-year-old man with urothelial carcinoma of left renal pelvis with para-aortic lymph nodes metastases. The patient presented severe lymphorrhea after neo-adjuvant chemotherapy followed by laparoscopic total left nephroureterectomy and lymph nodes dissection. Since conservative treatments were ineffective, percutaneous lymphatic embolization using NBCA resulted in healing of the lymphorrhea without recurrence. Percutaneous lymphatic embolization using NBCA followed by intranodal lymphography is a powerful treatment option for intractable postoperative lymphorrhea after lymph nodes dissection.

Shotgun proteomics deciphered age/division of labor-related functional specification of three honeybee (Apis mellifera L.) exocrine glands.

The honeybee (Apis mellifera L.) uses various chemical signals produced by the worker exocrine glands to maintain the functioning of its colony. The roles of worker postcerebral glands (PcGs), thoracic glands (TGs), and mandibular glands (MGs) and the functional changes they undergo according to the division of labor from nursing to foraging are not as well studied. To comprehensively characterize the molecular roles of these glands in workers and their changes according to the division of labor of workers, we analyzed the proteomes of PcGs, TGs, and MGs from nurse bees and foragers using shotgun proteomics technology. We identified approximately 2000 proteins from each of the nurse bee or forager glands and highlighted the features of these glands at the molecular level by semiquantitative enrichment analyses of frequently detected, gland-selective, and labor-selective proteins. First, we found the high potential to produce lipids in PcGs and MGs, suggesting their relation to pheromone production. Second, we also found the proton pumps abundant in TGs and propose some transporters possibly related to the saliva production. Finally, our data unveiled candidate enzymes involved in labor-dependent acid production in MGs.