OBJECTIVE

Although the beneficial health effects of regular moderate exercise are well established, there is substantial evidence that the heavy training and racing carried out by endurance athletes can cause skeletal muscle damage. This damage is repaired by satellite cells that can undergo a finite number of cell divisions. In this study, we have compared a marker of skeletal muscle regeneration of athletes with exercise-associated chronic fatigue, a condition labeled the "fatigued athlete myopathic syndrome" (FAMS), with healthy asymptomatic age- and mileage-matched control endurance athletes.

METHODS

Muscle biopsies of the vastus lateralis were obtained from 13 patients diagnosed with FAMS and from 13 healthy control subjects. DNA was extracted from the muscle samples and their telomeric restriction fragment (TRF) or telomere lengths were measured by Southern blot analysis.

RESULTS

All 13 symptomatic athletes reported a progressive decline in athletic performance, decreased ability to tolerate high mileage training, and excessive muscular fatigue during exercise. The minimum value of TRF lengths (4.0 +/- 1.8 kb) measured on the DNA from vastus lateralis biopsies from these athletes were significantly shorter than those from 13 age- and mileage-matched control athletes (5.4 +/- 0.6 kb, P < 0.05). Three of the FAMS patients had extremely short telomeres (1.0 +/- 0.3 kb). The minimum TRF lengths of the remaining 10 symptomatic athletes (4.9 +/- 0.5 kb, P < 0.05) were also significantly shorter that those of the control athletes.

CONCLUSIONS

These findings suggest that skeletal muscle from symptomatic athletes with FAMS show extensive regeneration which most probably results from more frequent bouts of satellite cell proliferation in response to recurrent training- and racing-induced muscle injury.

In chronic kidney disease (CKD), the decline in the glomerular filtration rate is associated with increased morbidity and mortality and thus poses a major challenge for healthcare systems. While the contribution of tissue-derived miRNAs and mRNAs to CKD progression has been extensively studied, little is known about the role of urinary exosomes and their association with CKD. Exosomes are small, membrane-derived endocytic vesicles that contribute to cell-to-cell communication and are present in various body fluids, such as blood or urine. Next-generation sequencing approaches have revealed that exosomes are enriched in noncoding RNAs and thus exhibit great potential for sensitive nucleic acid biomarkers in various human diseases. Therefore, in this study we aimed to identify urinary exosomal ncRNAs as novel biomarkers for diagnosis of CKD. Since up to now most approaches have focused on the class of miRNAs, we extended our analysis to several other noncoding RNA classes, such as tRNAs, tRNA fragments (tRFs), mitochondrial tRNAs, or lincRNAs. For their computational identification from RNA-seq data, we developed a novel computational pipeline, designated as ncRNASeqScan. By these analyses, in CKD patients we identified 30 differentially expressed ncRNAs, derived from urinary exosomes, as suitable biomarkers for early diagnosis. Thereby, miRNA-181a appeared as the most robust and stable potential biomarker, being significantly decreased by about 200-fold in exosomes of CKD patients compared to healthy controls. Using a cell culture system for CKD indicated that urinary exosomes might indeed originate from renal proximal tubular epithelial cells.

Posttranscriptional processing of RNA molecules is a common strategy to enlarge the structural and functional repertoire of RNomes observed in all 3 domains of life. Fragmentation of RNA molecules of basically all functional classes has been reported to yield smaller non-protein coding RNAs (ncRNAs) that typically possess different roles compared with their parental transcripts. Here we show that a valine tRNA-derived fragment (Val-tRF) that is produced under certain stress conditions in the halophilic archaeon Haloferax volcanii is capable of binding to the small ribosomal subunit. As a consequence of Val-tRF binding mRNA is displaced from the initiation complex which results in global translation attenuation in vivo and in vitro. The fact that the archaeal Val-tRF also inhibits eukaryal as well as bacterial protein biosynthesis implies a functionally conserved mode of action. While tRFs and tRNA halves have been amply identified in recent RNA-seq project, Val-tRF described herein represents one of the first functionally characterized tRNA processing products to date.

OBJECTIVE

Vitamin E is an essential fat-soluble vitamin that has been shown to induce favorable effects on animal and human immune systems. The objective of this study was to assess the effects of tocotrienol-rich fraction (TRF) supplementation on immune response following tetanus toxoid (TT) vaccine challenge in healthy female volunteers.

METHODS

In this double-blinded, placebo-controlled clinical trial, participants were randomly assigned to receive either placebo (control group) or 400 mg of TRF (study group) supplementation daily. Over the 2-month period of the study, volunteers were asked to attend three clinical sessions (that is, on days 0, 28 and 56) and blood samples were obtained from the volunteers during the follow-up. On day 28, all volunteers were also vaccinated with the TT vaccine (20 Lf) intramuscularly.

RESULTS

The results from the clinical trial showed that TRF supplementation significantly increased the total vitamin E level in the plasma of the TRF-supplemented volunteers compared with the placebo group, indicating overall compliance. Volunteers supplemented with TRF showed a significantly (P < 0.05) enhanced production of interferon-γ and interleukin (IL)-4 by the mitogen or TT-stimulated leukocytes compared with the control group. Volunteers from the TRF group produced significantly (P < 0.05) lower amounts of IL-6 compared with the placebo group. Anti-TT IgG production was also significantly (P < 0.05) augmented in the TRF-supplemented group compared with the placebo group.

CONCLUSIONS

We conclude that TRF has immunostimulatory effects and potential clinical benefits to enhance immune response to vaccines.

BACKGROUND

Rheumatoid arthritis (RA) is associated with reduced lifespan and shortened telomere length in lymphocytes, but the mechanism underlying this is unclear. Telomere loss in white blood cells (WBC) is accelerated by oxidative stress and inflammation in vitro. It was postulated that the accelerated WBC telomere shortening in RA occurs as a result of exposure to chronic inflammation.

OBJECTIVE

To measure telomere terminal restriction fragment (TRF) length in a large cohort of RA cases and healthy controls, to explore associations of TRF length with features of disease and with RA-associated HLA-DRB1 alleles.

METHODS

WBC and TRF length were measured by Southern blot in DNA from 176 hospital-based RA cases satisfying the 1987 American College of Rheumatology criteria and from 1151 controls. TRF length was compared between cases and controls, and the effects of disease duration, severity and HLA-DRB1 alleles encoding the shared epitope (SE) were assessed.

RESULTS

Age- and sex-adjusted TRF length was significantly shorter in RA cases compared with controls (p<0.001). There was no association between age- and sex-adjusted TRF length and disease duration, C reactive protein or Larsen score. The presence of one or more SE-encoding alleles was associated with reduced adjusted TRF length in RA cases (SE positive vs SE negative cases, p=0.038), but not in controls.

CONCLUSIONS

The reduced TRF length in a large group of patients with RA compared with controls has been shown. The reduction is apparently independent of disease duration and markers of disease severity, but is influenced by HLA-DRB1 genotype.

Time-restricted feeding (TRF) regimens have grown in popularity; however, very few studies have examined their weight-loss efficacy. We conducted the first human trial (Clinicaltrials.gov NCT03867773) to compare the effects of two popular forms of TRF (4 and 6 h) on body weight and cardiometabolic risk factors. Adults with obesity were randomized to 4-h TRF (eating only between 3 and 7 p.m.), 6-h TRF (eating only between 1 and 7 p.m.), or a control group (no meal timing restrictions). After 8 weeks, 4- and 6-h TRF produced comparable reductions in body weight (∼3%), insulin resistance, and oxidative stress, versus controls. Energy intake was reduced by ∼550 kcal/day in both TRF groups, without calorie counting. These findings suggest that 4- and 6-h TRF induce mild reductions in body weight over 8 weeks and show promise as interventions for weight loss. These diets may also improve some aspects of cardiometabolic health.

Keywords: blood pressure; body weight; energy intake; insulin resistance; intermittent fasting; metabolic disease; obesity; plasma lipids; time-restricted feeding.

tRNA-derived small RNA fragments (tRFs) are one class of small non-coding RNAs derived from transfer RNAs (tRNAs). tRFs play important roles in cellular processes and are involved in multiple cancers. High-throughput small RNA (sRNA) sequencing experiments can detect all the cellular expressed sRNAs, including tRFs. However, distinguishing genuine tRFs from RNA fragments generated by random degradation remains a major challenge. In this study, we developed an integrated web-based computing system, tRF2Cancer, to accurately identify tRFs from sRNA deep-sequencing data and evaluate their expression in multiple cancers. The binomial test was introduced to evaluate whether reads from a small RNA-seq data set represent tRFs or degraded fragments. A classification method was then used to annotate the types of tRFs based on their sites of origin in pre-tRNA or mature tRNA. We applied the pipeline to analyze 10 991 data sets from 32 types of cancers and identified thousands of expressed tRFs. A tool called 'tRFinCancer' was developed to facilitate the users to inspect the expression of tRFs across different types of cancers. Another tool called 'tRFBrowser' shows both the sites of origin and the distribution of chemical modification sites in tRFs on their source tRNA. The tRF2Cancer web server is available at http://rna.sysu.edu.cn/tRFfinder/.

Antigen-antibody complexes efficiently inhibit the induction of antibody formation. Using Mishell-Dutton cultures, it can be demonstrated that neither T cells nor their products are required for this inhibition of IgM PFC formation. The blockade is at the level of B cells and cannot be overcome by LPS or TRF. The data demonstrate that cross-linking of antigen- and Fc-receptors by antigen-antibody complexes is a blocking signal for B cells.

Immortalization of human cells in culture is usually associated with expression of telomerase activity. In some cases, however, no telomerase activity is detectable even though comparison of the terminal restriction fragment (TRF) pattern before and after immortalization shows that lengthening of telomeres has occurred. The extreme heterogeneity in telomere length and the differences in the dynamics of telomere maintenance in telomerase-negative cell lines compared to telomerase-positive cell lines indicate that these cells have utilized one or more alternative mechanisms for lengthening of telomeres (ALT). All telomerase-negative immortalized cell lines examined to date show evidence of ALT activity, consistent with the hypothesis that telomere maintenance either by telomerase or by ALT is required for immortalization. The nature of the ALT mechanism(s) is currently unknown, but studies of telomere dynamics in an ALT cell line containing a marker just proximal to the telomeric sequences show gradual shortening of the telomere followed by rapid elongation. This is consistent with a non-reciprocal recombinational mechanism similar to that found in telomerase-defective mutant yeast strains.

Exploratory factor analyses on 569 Youth Self-Reports and 1,221 Teacher's Report Forms of clinically referred Dutch children revealed six and eight factors respectively, very similar to the eight YSR and TRF cross-informant syndromes derived by Achenbach (1991c, 1991d). Mean cross-cultural correlations were .89 for YSR syndromes and .95 for TRF syndromes. In confirmatory factor analyses of the Dutch and American YSR and TRF factor models in cross-validation samples of 570 YSRs and 1,221 TRFs, goodness-of-fit indices were only slightly better for Dutch factor models. The American cross-informant Social Problems and Attention Problems syndromes had the poorest fit. The application of the eight American cross-informant syndromes to Dutch self- and teacher reports was supported.

Normolipemic and genetically hypercholesterolemic pigs of defined lipoprotein genotype were fed a standard diet supplemented with 50 micrograms/g tocotrienol-rich fraction (TRF) isolated from palm oil. Hypercholesterolemic pigs fed the TRF supplement showed a 44% decrease in total serum cholesterol, a 60% decrease in low-density-lipoprotein (LDL)-cholesterol, and significant decreases in levels of apolipoprotein B (26%), thromboxane-B2 (41%), and platelet factor 4 (PF4; 29%). The declines in thromboxane B2 and PF4 suggest that TRF has a marked protective effect on the endothelium and platelet aggregation. The effect of the lipid-lowering diet persisted only in the hypercholesterolemic swine after 8 wk feeding of the control diet. These results support observations from previous studies on lowering plasma cholesterol in animals by tocotrienols, which are naturally occurring compounds in grain and palm oils and may have some effect on lowering plasma cholesterol in humans.

Microbial community profiling of samples differing in a specific ecological function, i.e., soilborne plant disease suppression, can be used to mark, recover, and ultimately identify the bacteria responsible for that specific function. Previously, several terminal restriction fragments (TRF) of 16S rRNA genes were statistically associated with damping-off disease suppression. This work presents the development of sequence-based TRF length polymorphism (T-RFLP)-derived molecular markers to direct the identification and isolation of novel bacteria involved in damping-off pathogen suppression. Multiple sequences matching TRF M139 and M141 were cloned and displayed identity to multiple database entries in the genera incertae sedis of the Burkholderiales. Sequences matching TRF M148, in contrast, displayed greater sequence diversity. A sequence-directed culturing strategy was developed using M139- and M141-derived markers and media reported to be selective for the genera identified within this group. Using this approach, we isolated and identified novel Mitsuaria and Burkholderia species with high levels of sequence similarity to the targeted M139 and M141 TRF, respectively. As predicted, these Mitsuaria and Burkholderia isolates displayed the targeted function by reducing fungal and oomycete plant pathogen growth in vitro and reducing disease severity in infected tomato and soybean seedlings. This work represents the first successful example of the use of T-RFLP-derived markers to direct the isolation of microbes with pathogen-suppressing activities, and it establishes the power of low-cost molecular screening to identify and direct the recovery of functionally important microbes, such as these novel biocontrol strains.

BACKGROUND

Vitamin E supplements containing tocotrienols are now being recommended for optimum health but its effects are scarcely known. The objective was to determine the effects of Tocotrienol Rich Fraction (TRF) supplementation on lipid profile and oxidative status in healthy older individuals at a dose of 160 mg/day for 6 months.

METHODS

Sixty-two subjects were recruited from two age groups: 35-49 years (n = 31) and above 50 years (n = 31), and randomly assigned to receive either TRF or placebo capsules for six months. Blood samples were obtained at 0, 3rd and 6th months.

RESULTS

HDL-cholesterol in the TRF-supplemented group was elevated after 6 months (p < 0.01). Protein carbonyl contents were markedly decreased (p < 0.001), whereas AGE levels were lowered in the>> 50 year-old group (p < 0.05). Plasma levels of total vitamin E particularly tocopherols were significantly increased in the TRF-supplemented group after 3 months (p < 0.01). Plasma total tocotrienols were only increased in the>> 50 year-old group after receiving 6 months of TRF supplementation. Changes in enzyme activities were only observed in the>> 50 year-old group. SOD activity was decreased after 3 (p < 0.05) and 6 (p < 0.05) months of TRF supplementation whereas CAT activity was decreased after 3 (p < 0.01) and 6 (p < 0.05) months in the placebo group. GPx activity was increased at 6 months for both treatment and placebo groups (p < 0.05).

CONCLUSIONS

The observed improvement of plasma cholesterol, AGE and antioxidant vitamin levels as well as the reduced protein damage may indicate a restoration of redox balance after TRF supplementation, particularly in individuals over 50 years of age.

BACKGROUND

Telomere shortening at every replication cycle is postulated to limit the life span of human somatic cells. In contrast, activation of telomerase is proposed to be an essential step for cancer cell immortalization. Head and neck cancer is the most common malignancy in the Indian population compared with Western countries. However, there are very few reports on telomerase activity and telomere length in head and neck cancer.

METHODS

Telomerase activation and telomere length alterations were studied in tumor and adjacent normal tissues in 110 patients with head and neck cancer and 40 patients with precancerous/benign conditions. Telomerase activity and telomere lengths were determined by Telomeric Repeat Amplification Protocol (TRAP assay) and Southern blot analysis, respectively.

RESULTS

Telomerase activation was observed in 78.2% of the malignant tissues, 85% of the precancerous tissues, and 53.1% of the adjacent normal tissues. Peak terminal restriction fragment length (TRF) was observed to be significantly lower in malignant tissues compared with the adjacent normal tissues. No significant correlation could be observed between telomerase activation and clinicopathologic characteristics of the patients. Two-year disease-free survival analysis showed that patients showing telomerase activation in the adjacent normal tissues and patients showing higher telomere length in malignant tissues had poor disease-free survival.

CONCLUSIONS

Our results demonstrate the significant clinical usefulness of telomerase activation and telomere length for head and neck cancer patients. These markers may be helpful in predicting the clinical course of the disease and thus in identifying the patients in need of a close follow-up and vigorous adjuvant treatment.

The strengths and limitations of the major methods developed to measure telomere lengths (TLs) in cells and tissues are presented in this review. These include Q-PCR (Quantitative Polymerase Chain Reaction), TRF (Terminal Restriction Fragment) analysis, a variety of Q-FISH (Quantitative Fluorescence In Situ Hybridization) methods, STELA (Single TElomere Length Analysis) and TeSLA (Telomere Shortest Length Assay). For each method, we will cover information about validation studies, including reproducibility in independent laboratories, accuracy, reliability and sensitivity for measuring not only the average but also the shortest telomeres. There is substantial evidence that it is the shortest telomeres that trigger DNA damage responses leading to replicative senescence in mammals. However, the most commonly used TL measurement methods generally provide information on average or relative TL, but it is the shortest telomeres that leads to telomere dysfunction (identified by TIF, Telomere dysfunction Induced Foci) and limit cell proliferation in the absence of a telomere maintenance mechanism, such as telomerase. As the length of the shortest telomeres is a key biomarker determining cell fate and the onset of senescence, a new technique (TeSLA) that provides quantitative information about all the shortest telomeres will be highlighted.This article is part of the theme issue 'Understanding diversity in telomere dynamics'.

Psychosocial adjustment and quality of life has been reported good in children after a successful renal transplantation (Tx). There are, however, few reports of using standardized methods in evaluating these issues, particularly in small children. We investigated the psychosocial adjustment in 32 children at school age (mean 9.6 +/- 1.6), who had received a renal Tx under the age of 5 yr, using the Achenbach Child Behavior Checklist with data collected from both parents (CBCL) and teachers (CBCL-TRF). Health-related quality of life (HRQOL) was assessed by interviewing the children using a 17-dimensional (17D) health-related measure and compared to HRQOL of 244 normal school children. The effect of additional diseases and comorbidity on psychosocial adjustment and HRQOL was assessed. The total scores on the CBCL did not differ from normative samples of healthy children. However, somatic complaints and social problems were reported more frequently in boys, and attention problems in both boys and girls. Patients with pathological scores had significantly more comorbidity (p = 0.03) and were more often attending a special school (p = 0.007) than patients with normal scores. The global 17D HRQOL index was significantly lower than measured in healthy controls (94 +/- 5 for controls and 85 +/- 7 for patients, p < 0.0001). It is of crucial importance to further minimize the risk factors leading to comorbidity in children after Tx. HRQOL assessment by the children themselves can be used to direct interventions and support the children's psychosocial adjustment.

Measuring the DNA telomere length of skeletal muscle in experienced endurance runners may contribute to our understanding of the effects of chronic exposure to endurance exercise on skeletal muscle. This study compared the minimum terminal restriction fragment (TRF) length in the vastus lateralis muscle of 18 experienced endurance runners (mean age: 42 +/- 7 years) to those of 19 sedentary individuals (mean age: 39 +/- 10 years). The runners had covered almost 50,000 km in training and racing over 15 years. Minimum TRF lengths measured in the muscle of both groups were similar (P = 0.805) and within the normal range. Minimum TRF length in the runners, however, was inversely related to their years spent running (r = -0.63, P = 0.007) and hours spent training (r = -0.52, P = 0.035). Therefore, since exposure to endurance running may influence minimum TRF length, and by implication, the proliferative potential of the satellite cells, chronic endurance running may be seen as a stressor to skeletal muscle.

The effects of diets supplemented with either chickpea or its main oligosaccharide raffinose on the composition of the faecal microbial community were examined in 12 healthy adults (18-65 years) in a randomised crossover intervention study. Subjects consumed their usual diet supplemented with soups and desserts that were unfortified, or fortified with either 200 g/d of canned chickpeas or 5 g/d of raffinose for 3 week periods. Changes in faecal bacterial populations of subjects were examined using 16S rRNA-based terminal restriction fragment length polymorphisms (T-RFLP) and clone libraries generated from the diet pools. Classification of the clone libraries and T-RFLP analysis revealed that Faecalibacterium prausnitzii, reported to be an efficient butyrate producer and a highly metabolically active bacterium in the human intestinal microbiota, was more abundant in the raffinose diet and the chickpea diet compared to the control diet. However, no significant difference was observed in the faecal total short chain fatty acid concentration or in the levels of the components (butyrate, acetate and propionate) with the chickpea diet or the raffinose diet compared to the control diet. Bifidobacterium species were detected by T-RFLP in all three diet groups and quantitative real-time PCR (qPCR) analysis showed a marginal increase in 16S rRNA gene copies of Bifidobacterium with the raffinose diet compared to control (P>0.05). The number of individuals showing TRFs for the Clostridium histolyticum - Clostridum lituseburense groups, which include pathogenic bacteria species and putrefactive bacteria, were lower in the chickpea diet compared to the other two treatments. Diet appeared to affect colonisation by a high ammonia-producing bacterial isolate which was detected in 83%, 92% and 42% of individuals in the control, raffinose and chickpea groups, respectively. Our results indicate that chickpea and raffinose have the potential to modulate the intestinal microbial composition to promote intestinal health in humans.

Two intermittent fasting variants, intermittent energy restriction (IER) and time-restricted feeding (TRF), have received considerable interest as strategies for weight-management and/or improving metabolic health. With these strategies, the pattern of energy restriction and/or timing of food intake are altered so that individuals undergo frequently repeated periods of fasting. This review provides a commentary on the rodent and human literature, specifically focusing on the effects of IER and TRF on glucose and lipid metabolism. For IER, there is a growing evidence demonstrating its benefits on glucose and lipid homeostasis in the short-to-medium term; however, more long-term safety studies are required. Whilst the metabolic benefits of TRF appear quite profound in rodents, findings from the few human studies have been mixed. There is some suggestion that the metabolic changes elicited by these approaches can occur in the absence of energy restriction, and in the context of IER, may be distinct from those observed following similar weight-loss achieved via modest continuous energy restriction. Mechanistically, the frequently repeated prolonged fasting intervals may favour preferential reduction of ectopic fat, beneficially modulate aspects of adipose tissue physiology/morphology, and may also impinge on circadian clock regulation. However, mechanistic evidence is largely limited to findings from rodent studies, thus necessitating focused human studies, which also incorporate more dynamic assessments of glucose and lipid metabolism. Ultimately, much remains to be learned about intermittent fasting (in its various forms); however, the findings to date serve to highlight promising avenues for future research.

A new approach to characterize growing microorganisms in environmental samples based on labeling microbial DNA with H(2)(18)O is described. To test if sufficient amounts of (18)O could be incorporated into DNA to use water as a labeling substrate for stable isotope probing, Escherichia coli DNA was labeled by cultivating bacteria in Luria broth with H(2)(18)O and labeled DNA was separated from [(16)O]DNA on a cesium chloride gradient. Soil samples were incubated with H(2)(18)O for 6, 14, or 21 days, and isopycnic centrifugation of the soil DNA showed the formation of two bands after 6 days and three bands after 14 or 21 days, indicating that (18)O can be used in the stable isotope probing of soil samples. DNA extracted from soil incubated for 21 days with H(2)(18)O was fractionated after isopycnic centrifugation and DNA from 17 subsamples was used in terminal restriction fragment length polymorphism (TRFLP) analysis of bacterial 16S rRNA genes. The TRFLP patterns clustered into three groups that corresponded to the three DNA bands. The fraction of total fluorescence contributed by individual terminal restriction fragments (TRF) to a TRFLP pattern varied across the 17 subsamples so that a TRF was more prominent in only one of the three bands. Labeling soil DNA with H(2)(18)O allows the identification of newly grown cells. In addition, cells that survive but do not divide during an incubation period can also be characterized with this new technique because their DNA remains without the label.

BACKGROUND

The detection of tocotrienols in human plasma has proven elusive, and it is hypothesized that they are rapidly assimilated and redistributed in various mammalian tissues.

OBJECTIVE

The primary study objective was to evaluate the postprandial fate of tocotrienols and alpha-tocopherol in human plasma and lipoproteins.

METHODS

Seven healthy volunteers (4 males, 3 females) were administered a single dose of vitamin E [1011 mg palm tocotrienol-rich fraction (TRF) or 1074 mg alpha-tocopherol] after a 7-d conditioning period with a tocotrienol-free diet. Blood was sampled at baseline (fasted) and 2, 4, 5, 6, 8, and 24 h after supplementation. Concentrations of tocopherol and tocotrienol isomers in plasma, triacylglycerol-rich particles (TRPs), LDLs, and HDLs were measured at each interval.

RESULTS

After intervention with TRF, plasma tocotrienols peaked at 4 h (4.79 +/- 1.2 microg/mL), whereas alpha-tocopherol peaked at 6 h (13.46 +/- 1.68 microg/mL). Although tocotrienols were similarly detected in TRPs, LDLs, and HDLs, tocotrienol concentrations were significantly lower than alpha-tocopherol concentrations. In comparison, plasma alpha-tocopherol peaked at 8 h (24.3 +/- 5.22 microg/mL) during the alpha-tocopherol treatment and emerged as the major vitamin E isomer detected in plasma and lipoproteins during both the TRF and the alpha-tocopherol treatments.

CONCLUSIONS

Tocotrienols are detected in postprandial plasma, albeit in significantly lower concentrations than is alpha-tocopherol. This finding confirms previous observations that, in the fasted state, tocotrienols are not detected in plasma. Tocotrienol transport in lipoproteins appears to follow complex biochemically mediated pathways within the lipoprotein cascade.

Rhizobial infection and root nodule formation in legumes require recognition of signal molecules produced by the bacteria and their hosts. Here, we show that rhizobial transfer RNA (tRNA)-derived small RNA fragments (tRFs) are signal molecules that modulate host nodulation. Three families of rhizobial tRFs were confirmed to regulate host genes associated with nodule initiation and development through hijacking the host RNA-interference machinery that involves ARGONAUTE 1. Silencing individual tRFs with the use of short tandem target mimics or by overexpressing their targets represses root hair curling and nodule formation, whereas repressing these targets with artificial microRNAs identical to the respective tRFs or mutating these targets with CRISPR-Cas9 promotes nodulation. Our findings thus uncover a bacterial small RNA-mediated mechanism for prokaryote-eukaryote interaction and may pave the way for enhancing nodulation efficiency in legumes.

Although the serum-abundant metal-binding protein transferrin (encoded by the Trf gene) is synthesized primarily in the liver, its function in the liver is largely unknown. Here, we generated hepatocyte-specific Trf knockout mice (Trf-LKO), which are viable and fertile but have impaired erythropoiesis and altered iron metabolism. Moreover, feeding Trf-LKO mice a high-iron diet increased their susceptibility to develop ferroptosis-induced liver fibrosis. Importantly, we found that treating Trf-LKO mice with the ferroptosis inhibitor ferrostatin-1 potently rescued liver fibrosis induced by either high dietary iron or carbon tetrachloride (CCl4) injections. In addition, deleting hepatic Slc39a14 expression in Trf-LKO mice significantly reduced hepatic iron accumulation, thereby reducing ferroptosis-mediated liver fibrosis induced by either high dietary iron diet or CCl4 injections. Finally, we found that patients with liver cirrhosis have significantly lower levels of serum transferrin and hepatic transferrin, as well as higher levels of hepatic iron and lipid peroxidation compared to healthy controls. Taken together, these data indicate that hepatic transferrin plays a protective role in maintaining liver function, providing a possible therapeutic target for preventing ferroptosis-induced liver fibrosis.

Erythropoietin (Epo) is the central regulator of red blood cell production and acts primarily by inducing proliferation and differentiation of erythroid progenitor cells. Because a sufficient supply of iron is a prerequisite for erythroid proliferation and hemoglobin synthesis, we have investigated whether Epo can regulate cellular iron metabolism. We present here a novel biologic function of Epo, namely as a potential modulator of cellular iron homeostasis. We show that, in human (K562) and murine erythroleukemic cells (MEL), Epo enhances the binding affinity of iron-regulatory protein (IRP)-1, the central regulator of cellular iron metabolism, to specific RNA stem-loop structures, known as iron-responsive elements (IREs). Activation of IRP-1 by Epo is associated with a marked increase in transferrin receptor (trf-rec) mRNA levels in K562 and MEL, enhanced cell surface expression of trf-recs, and increased uptake of iron into cells. These findings are in agreement with the well-established mechanism whereby high-affinity binding of IRPs to IREs stabilizes trf-rec mRNA by protecting it from degradation by a specific RNase. The effects of Epo on IRE-binding of IRPs were not observed in human myelomonocytic cells (THP-1), which indicates that this response to Epo is not a general mechanism observed in all cells but is likely to be erythroid-specific. Our results provide evidence for a direct functional connection between Epo biology and iron metabolism by which Epo increases iron uptake into erythroid progenitor cells via posttranscriptional induction of trf-rec expression. Our data suggest that sequential administration of Epo and iron might improve the response to Epo therapy in some anemias.