Cytosolic Ribosomal Protein Haploinsufficiency affects Mitochondrial Morphology and Respiration.

The interplay between ribosomal protein composition and mitochondrial function is essential for sustaining energy homeostasis. Precise stoichiometric production of ribosomal proteins is crucial to maximize protein synthesis efficiency while reducing the energy costs to the cell. However, the impact of this balance on mitochondrial ATP generation, morphology and function remains unclear. Particularly, the loss of a single copy ribosomal protein gene is observed in Mendelian disorders like Diamond Blackfan Anemia and is common in somatic tumors, yet the implications of this imbalance on mitochondrial function and energy dynamics are still unclear. In this study, we investigated the impact of haploinsufficiency for four ribosomal protein genes implicated in ribosomopathy disorders (rps-10, rpl-5, rpl-33, rps-23) in Caenorhabditis elegans and corresponding reductions in human lymphoblast cells. Our findings uncover significant, albeit variably penetrant, mitochondrial morphological differences across these mutants, alongside an upregulation of glutathione transferases, and SKN-1 dependent increase in oxidative stress resistance, indicative of increased ROS production. Specifically, loss of a single copy of rps-10 in C. elegans led to decreased mitochondrial activity, characterized by lower energy levels and reduced oxygen consumption. A similar reduction in mitochondrial activity and energy levels was observed in human leukemia cells with a 50% reduction in RPS10 transcript levels. Importantly, we also observed alterations in the translation efficiency of nuclear and mitochondrial electron transport chain components in response to reductions in ribosomal protein genes' expression in both C. elegans and human cells. This suggests a conserved mechanism whereby the synthesis of components vital for mitochondrial function are adjusted in the face of compromised ribosomal machinery. Finally, mitochondrial membrane and cytosolic ribosomal components exhibited significant covariation at the RNA and translation efficiency level in lymphoblastoid cells across a diverse group of individuals, emphasizing the interplay between the protein synthesis machinery and mitochondrial energy production. By uncovering the impact of ribosomal protein haploinsufficiency on the translation efficiency of electron transport chain components, mitochondrial physiology, and the adaptive stress responses, we provide evidence for an evolutionarily conserved strategy to safeguard cellular functionality under genetic stress.

Thrombospondin 2 is a novel biomarker of essential hypertension and associated with nocturnal Na+ excretion and insulin resistance.

BACKGROUND: Thrombospondins (TSPs) play important roles in several cardiovascular diseases. However, the association between circulating (plasma) thrombospondin 2 (TSP2) and essential hypertension remains unclear. The present study was aimed to investigate the association of circulating TSP2 with blood pressure and nocturnal urine Na+ excretion and evaluate the predictive value of circulating TSP2 in subjects with hypertension. METHODS AND RESULTS: 603 newly diagnosed essential hypertensive subjects and 508 healthy subjects were preliminarily screened, 47 healthy subjects and 40 newly diagnosed essential hypertensive subjects without any chronic diseases were recruited. The results showed that the levels of circulating TSP2 were elevated in essential hypertensive subjects. The levels of TSP2 positively associated with systolic blood pressure (SBP), diastolic blood pressure (DBP), and other clinical parameters, including homeostasis model assessment of insulin resistance (HOMA-IR), brachial-ankle pulse wave velocity, and serum triglycerides, but negatively associated with nocturnal urine Na+ concentration and excretion and high-density lipoprotein cholesterol. Results of multiple linear regressions showed that HOMA-IR and nocturnal Na+ excretion were independent factors related to circulating TSP2. Mantel-Haenszel chi-square test displayed linear relationships between TSP2 and SBP (χ2 = 35.737) and DBP (χ2 = 26.652). The area under receiver operating characteristic curve (AUROC) of hypertension prediction was 0.901. CONCLUSION: Our study suggests for the first time that the circulating levels of TSP2 may be a novel potential biomarker for essential hypertension. The association between TSP2 and blood pressure may be, at least in part, related to the regulation of renal Na+ excretion, insulin resistance, and/or endothelial function.

Ribosome biogenesis disruption mediated chromatin structure changes revealed by SRAtac, a customizable end to end analysis pipeline for ATAC-seq.

The nucleolus is a large nuclear body that serves as the primary site for ribosome biogenesis. Recent studies have suggested that it also plays an important role in organizing chromatin architecture. However, to establish a causal relationship between nucleolar ribosome assembly and chromatin architecture, genetic tools are required to disrupt nucleolar ribosome biogenesis. In this study, we used ATAC-seq to investigate changes in chromatin accessibility upon specific depletion of two ribosome biogenesis components, RPOA-2 and GRWD-1, in the model organism Caenorhabditis elegans. To facilitate the analysis of ATAC-seq data, we introduced two tools: SRAlign, an extensible NGS data processing workflow, and SRAtac, a customizable end-to-end ATAC-seq analysis pipeline. Our results revealed highly comparable changes in chromatin accessibility following both RPOA-2 and GRWD-1 perturbations. However, we observed a weak correlation between changes in chromatin accessibility and gene expression. While our findings corroborate the idea of a feedback mechanism between ribosomal RNA synthesis, nucleolar ribosome large subunit biogenesis, and chromatin structure during the L1 stage of C. elegans development, they also prompt questions regarding the functional impact of these alterations on gene expression.

Inhibition of ribosome biogenesis in the epidermis is sufficient to trigger organism-wide growth quiescence independently of nutritional status in C. elegans.

Interorgan communication is crucial for multicellular organismal growth, development, and homeostasis. Cell nonautonomous inhibitory cues, which limit tissue-specific growth alterations, are not well characterized due to cell ablation approach limitations. In this study, we employed the auxin-inducible degradation system in C. elegans to temporally and spatially modulate ribosome biogenesis, through depletion of essential factors (RPOA-2, GRWD-1, or TSR-2). Our findings reveal that embryo-wide inhibition of ribosome biogenesis induces a reversible early larval growth quiescence, distinguished by a unique gene expression signature that is different from starvation or dauer stages. When ribosome biogenesis is inhibited in volumetrically similar tissues, including body wall muscle, epidermis, pharynx, intestine, or germ line, it results in proportionally stunted growth across the organism to different degrees. We show that specifically inhibiting ribosome biogenesis in the epidermis is sufficient to trigger an organism-wide growth quiescence. Epidermis-specific ribosome depletion leads to larval growth quiescence at the L3 stage, reduces organism-wide protein synthesis, and induced cell nonautonomous gene expression alterations. Further molecular analysis reveals overexpression of secreted proteins, suggesting an organism-wide regulatory mechanism. We find that UNC-31, a dense-core vesicle (DCV) pathway component, plays a significant role in epidermal ribosome biogenesis-mediated growth quiescence. Our tissue-specific knockdown experiments reveal that the organism-wide growth quiescence induced by epidermal-specific ribosome biogenesis inhibition is suppressed by reducing unc-31 expression in the epidermis, but not in neurons or body wall muscles. Similarly, IDA-1, a membrane-associated protein of the DCV, is overexpressed, and its knockdown in epidermis suppresses the organism-wide growth quiescence in response to epidermal ribosome biogenesis inhibition. Finally, we observe an overall increase in DCV puncta labeled by IDA-1 when epidermal ribosome biogenesis is inhibited, and these puncta are present in or near epidermal cells. In conclusion, these findings suggest a novel mechanism of nutrition-independent multicellular growth coordination initiated from the epidermis tissue upon ribosome biogenesis inhibition.

An mTOR/RNA pol I axis shapes chromatin architecture in response to fasting.

Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom. Yet, how it impacts on 3D genome organization is unknown. Here, we show that fasting induces a reversible and large-scale spatial reorganization of chromatin in C. elegans . This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, a major regulator of ribosome biogenesis. Remarkably, loss of transcription by RNA Pol I, but not RNA Pol II nor Pol III, induces a similar 3D genome reorganization in fed animals, and prevents the restoration of the fed-state architecture upon restoring nutrients to fasted animals. Our work documents the first large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients.

Dosimetric differences between intensity-modulated radiotherapy based on equivalent uniform dose and dose-volume optimization in stage III non-small cell lung cancer.

OBJECTIVE: To determine the dosimetric differences between biological and physical functions of equivalent uniform dose (EUD) and dose volume (DV) therapy in patients with phase III non-small cell lung cancer. METHODS: Four different radiotherapy plans (DV+DV, DV-EUD+DV, EUD+EUD and EUD-DV+EUD) were developed for 15 patients with stage III NSCLC. To study physical function (DV+DV) the target area was optimized by introducing the conditions of biological function optimization, while the organs at risk were optimized by means of physical function (DV-EUD+DV). Biological function optimization (EUD+EUD) was performed for the target area by applying conditions of physical function optimization while biological function optimization (EUD-DV+DV) was conducted for the organs at risk to compare dosimetric parameters among the four groups of treatment plans. RESULTS: PTV: D2%, D98%, D50%, V105% and Dmax of both the DV-EUD+DV group and EDU-DV+EUD group were the minimum (P<0.05). The minimum and average dose of the EUD+EUD group showed an increasing trend and high-dose area became observable. For homogeneity index (HI), DV-EUD+DV group and EUD-DV+EUD results were compared with the other groups (P<0.05), no significant difference was observed statistically between the DV-EUD+DV group and EUD DV+EUD (P=0.659). With regard to conformability index (CI), the results of the four groups showed no significant difference (P>0.05). For the organs at risk, the mean dose of lung tissue (MLD), V5, V10, V20, V30, heart V30, V40, and Dmean also revealed no significant difference (P>0.05). For the spinal cord, the D1 % of the EUD+EUD group and EUD-DV+EUD groups were significantly different (P<0.05) than the other groups. While no significant difference (P=0.32) was found between the EUD+EUD and EUD-DV+EUD groups. When comparing the number of machine unions (MU) no significant difference was revealed (P>0.05) among the results of the 4 groups. CONCLUSION: The methods featuring optimization of physical and biological functions are effective in improving the uniformity of target area to have better outcome of the treatment. Biological function optimization or the combination of biological and physical function optimization is conducive to significantly reduce the required dose for the spinal cord.

Methylphenidate and atomoxetine treatment negatively affect physical growth indexes of school-age children and adolescents with attention-deficit/hyperactivity disorder.

AIM: To determine the effects of drug therapy on the physical growth of school-age children and adolescents with attention-deficit/hyperactivity disorder (ADHD). METHOD: The medical records of 86 participants (average age: 8.9 ± 2.2 years) with ADHD prescribed methylphenidate (MPH) or atomoxetine (ATX) for ≥24 weeks from the Children's Hospital of Chongqing Medical University were analysed. RESULTS: The Z-scores of height, weight and body mass index (BMI) of children with ADHD decreased significantly over the first six months of MPH treatment (P < 0.001). The slopes of the fitting lines after the first six months of MPH (-0.18, -0.58 and -0.69, respectively) returned over the entire treatment (the slopes changed to -0.027, -0.26 and -0.20, respectively). For ATX, the Z-scores of height of children decreased significantly over the first six months (P < 0.001), but the Z-scores of weight and BMI did not (P > 0.05). The slopes of the fitting lines after the first six months of ATX (-0.058, -0.032 and 0.0094, respectively) changed over the entire treatment (slopes were 0.16, 0.52 and 0.26, respectively). Children taking MPH were more likely to report decreased appetite (P < 0.05). The weight and BMI of the children receiving MPH were significantly correlated with decreased appetite (P < 0.01). CONCLUSION: The physical growth indexes (PGIs) of school-age children and adolescents with ADHD were negatively affected while taking MPH, and these effects were gradually mitigated with continued treatment. ATX hardly had negative effects on weight and BMI. Neither MPH nor ATX had a significant negative effect on the height of children in long-term ADHD treatment. It is necessary for clinicians to consider children's diet during treatment.

Cytoplasmic HYL1 modulates miRNA-mediated translational repression.

MicroRNAs (miRNAs) control various biological processes by repressing target mRNAs. In plants, miRNAs mediate target gene repression via both mRNA cleavage and translational repression. However, the mechanism underlying this translational repression is poorly understood. Here, we found that Arabidopsis thaliana HYPONASTIC LEAVES1 (HYL1), a core component of the miRNA processing machinery, regulates miRNA-mediated mRNA translation but not miRNA biogenesis when it localized in the cytoplasm. Cytoplasmic HYL1 localizes to the endoplasmic reticulum and associates with ARGONAUTE1 (AGO1) and ALTERED MERISTEM PROGRAM1. In the cytoplasm, HYL1 monitors the distribution of AGO1 onto polysomes, binds to the mRNAs of target genes, represses their translation, and partially rescues the phenotype of the hyl1 null mutant. This study uncovered another function of HYL1 and provides insight into the mechanism of plant gene regulation.

Prevalence and grade of RLS in migraine: A prospective study of 251 migraineurs by synchronous test of c-TTE and c-TCD.

BACKGROUND: Right-to left shunt (RLS) is regarded as a risk factor resulting in migraine, but the relevance between the RLS and migraine remains controversial. This paper aims at investigating the prevalence and RLS grade of patent foramen ovale (PFO) in cases of migraine (including migraine with and without aura) and evaluate the relationship between PFO and migraine. METHODS: Synchronous test of contrast transthoracic echocardiography and contrast transcranial Doppler ultrasonography was performed in 251 cases of migraine, which contains 62 cases of migraine with aura (MA) and 189 cases without aura (MO) and 275 healthy adults. Among these cases, 25 cases with migraine and 14 healthy adults were evaluated through transesophageal echocardiography. RESULTS: (1). The prevalence of permanent RLS, total RLS, and large RLS in migraine was 11.16%, 39.04%, and 17.13%, respectively, which was significantly higher than that of the controls (P = .042, <.001, and.001, respectively). (2). Permanent RLS was detected as 7.93% of the cases in MO, 20.96% in MA, and 6.18% in controls. Total RLS was detected as 35.98% of the cases in MO, 48.38% in MA, and 23.64% in controls. Large RLS was detected as 13.76% of the cases in MO, 27.41% in MA, and 7.27% in controls. Compared with controls, the positive rate of total RLS and large RLS in MO increased (P = .004 and.022, respectively), the that of permanent RLS, total RLS, and large RLS in MA also increased (P < .001 for each of the comparisons). The positive rate of permanent RLS and large RLS in MA was remarkably higher than that in MO (P = .005 and.013, respectively). (3) The presence of large-size PFO (≥2.0 mm) of migraine showed higher than that of the controls (P = .048). CONCLUSIONS: PFO is associated with the migraine (especially with aura), when it is permanent RLS, large RLS, and large-size PFO (≥2.0 mm).

Preparation of a Broad-Spectrum Heterocyclic Aromatic Amines (HAAs) Antibody and Its Application in Detection of Eight HAAs in Heat Processed Meat.

Heterocyclic aromatic amines (HAAs) are potential human mutagens and carcinogens mainly generated in heat-treated meat. In this work, a broad-spectrum HAAs antibody was prepared and used to develop an indirect competitive ELISA (ic-ELISA) for simultaneous determination of eight HAAs, including 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f] quinoline (MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx), 2-amino-3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline (4,7,8-TriMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in grilled and fried meat samples. The limit of detection (LOD, calculated as IC10) and 50% inhibition concentration (IC50) of ic-ELISA were 5.29 µg/L and 99.08 µg/L, respectively. The detection results of this ic-ELISA were in good agreement with the detection results of UPLC-MS/MS in real samples, which indicated that this ic-ELISA can be applied to detect the total content of eight HAAs in heat processed meat. Use of a broad-spectrum antibody is an efficient strategy in developing immunoassay for simultaneous measuring food risk factors with similar structure.

MicroRNA166 Monitors SPOROCYTELESS/NOZZLE for Building of the Anther Internal Boundary.

The internal boundary between inner and outer microsporangia within anthers is essential for male fertility of vascular plants. Dehiscence zones embedded in the boundary release pollen for fertilization. However, the molecular mechanism underlying boundary formation in anthers remains poorly understood. Here, we report that microRNA166 (miR166) and its target PHABULOSA (PHB) regulate SPOROCYTELESS/NOZZLE (SPL/NZZ), which controls microsporogenesis. In developing anthers of Arabidopsis (Arabidopsis thaliana), the expression domains of miR165/6 and SPL/NZZ are overlapped and rearranged synchronously. Dominant mutation of PHB suppresses SPL/NZZ expression on the adaxial sides of stamens, resulting in a thickened boundary, whereas activation of MIR166g up-regulates SPL/NZZ expression, leading to ectopic microsporogenesis in the boundary. PHB limits the expression domains of SPL/NZZ to facilitate construction of the boundary, while miR166 preserves the expression domains of SPL/NZZ by inhibiting PHB to allow the inner microsporangia to take shape. Subsequently, PHB activates the key stem cell maintainer WUSCHEL in anthers to restrict the stomium cells to the boundary so that dehiscence zones develop and release pollen properly. These findings link adaxial/abaxial polarity to microsporogenesis in building of the internal boundary of anthers and thus advance the concepts underlying the establishment of the internal structure of male organs.

Homodimerization of HYL1 ensures the correct selection of cleavage sites in primary miRNA.

MicroRNA (miRNA) plays an important role in the control of gene expression. HYPONASTIC LEAVES1 (HYL1) is a double-stranded RNA-binding protein that forms a complex with DICER-LIKE1 (DCL1) and SERRATE (SE) to process primary miRNA (pri-miRNA) into mature miRNA. Although HYL1 has been shown to partner with DCL1 to enhance miRNA accuracy, the mechanism by which HYL1 selects the DCL1-targeted cleavage sites in pri-miRNA has remained unknown. By mutagenesis of HYL1 and analysis of in vivo pri-miRNA processing, we investigated the role of HYL1 in pri-miRNA cleavage. HYL1 forms homodimers in which the residues Gly147 and Leu165 in the dsRBD2 domain are shown to be critical. Disruption of HYL1 homodimerization causes incorrect cleavage at sites in pri-miRNA without interrupting the interaction of HYL1 with DCL1 and accumulation of pri-miRNAs in HYL1/pri-miRNA complexes, leading to a reduction in the efficiency and accuracy of miRNAs that results in strong mutant phenotypes of the plants. HYL1 homodimers may function as a molecular anchor for DCL1 to cleave at a distance from the ssRNA-dsRNA junction in pri-miRNA. These results suggest that HYL1 ensures the correct selection of pri-miRNA cleavage sites through homodimerization and thus contributes to gene silencing and plant development.

[Effect of tetrandrine, toremifene and their combination on the reversion of multidrug resistance of K562/A02 cell line].

This study was aimed to investigate the reversible effect of tetrandrine, toremifene and their combination on multidrug resistance of K562/A02 cell line. The IC(50) (the concentration causing 50% inhibition of cell growth) of adriamycin (ADR) were assayed by MTT method, the expression of MDR1 mRNA was measured by RT-PCR, the concentration of p-glycoprotein (P-gp) and intracellular ADR were detected by flow cytometry. The results showed that the IC(50) of ADR on K562/A02 and K562 cells were 57.43 and 1.16 mg/L, respectively. The IC(50) of ADR on K562/A02 cells after treatment with tetrandrine, toremifene and both combination were 14.12, 20.74 and 9.14 mg/L respectively, but both drugs did not influence the IC(50) of ADR on K562 cells. Pretreating K562/A02 cells with toremifene (2.5 micromol/L), tetrandrine (1 micromol/L) or both for 72 hours partially restored the sensitivity of K562/A02 cells to ADR. Tetrandrine and toremifene (alone or combination) elevated the ADR concentration in K562/A02, down regulated the expressions of P-gp and MDR1 mRNA. It is concluded that multidrug resistance of K562/A02 cells can be partially reversed by tetrandrine or toremifene, the combination of both drugs shows a higher synergistic reversal effect.

PEG prodrugs of 6-mercaptopurine for parenteral administration using benzyl elimination of thiols.

6-Mercaptopurine (6-MP) is an orally administered, water-insoluble purine analog that is effective against acute lymphatic leukemia. Oral absorption of 6-MP, however, is quite erratic, with only 16-50% of the administered dose reaching the blood. In this report, water-soluble parenterally administered poly(ethylene glycol) (PEG) prodrugs of 6-MP were synthesized using several chemical approaches that enabled the protection of the thiol group through a modification of the benzyl elimination (BE) system. In our earlier work on antimetabolites, it was found that branching of the PEG allowed greater loading of the active drug. This approach was also utilized within this work to give multiloaded systems. The resulting conjugates were stable in pH 7.4 PBS buffer as well as in rat plasma for extended periods. However, these conjugates did act as prodrugs in vivo and a number of PEG-6-MP constructs had significant (P < 0.05) activity in murine leukemia, as well as certain solid tumors, compared with unconjugated 6-MP in a solubilizing vehicle. The fact that some PEG-6-MP conjugates were stable during in vitro plasma dissociation assays, but demonstrated in vivo anticancer activity, suggests extravascular cleavage of the linking group. This work demonstrates that PEG conjugation is an effective means of solubilizing 6-MP for parenteral administration.