Development of transcriptomics-based eukaryotes growth rate indices

Growth rate estimation is important to understand the flow of energy and nutrient elements in an ecosystem, but it has remained challenging, especially on microscopic organisms. In this study, we propose four growth rate indices that use mRNA abundance ratios between nuclear and mitochondrial genes: (1) total nuclear and mitochondrial mRNA ratio (Nuc:Mito-TmRNA), (2) nuclear and mitochondrial ribosomal protein mRNA ratio (Nuc:Mito-RPmRNA), (3) gene ontology (GO) terms and total mitochondrial mRNA ratios and (4) nuclear and mitochondrial specific gene mRNA ratio. We examine these proposed ratios using RNA-Seq datasets of Daphnia magna and Saccharomyces cerevisiae. The results showed that both Nuc:Mito-TmRNA and Nuc:Mito-RPmRNA ratio indices showed significant correlations with the growth rate for both species. A large number of GO terms mRNA ratios showed significant correlations with the growth rate of S. cerevisiae. Lastly, we identified mRNA ratios of several specific nuclear and mitochondrial gene pairs that showed significant correlations. We foresee future implications for the proposed mRNA ratios used in metatranscriptome analyses to estimate the growth rate of communities and species.

Transcriptional dynamics of methane-cycling microbiomes are linked to seasonal CH4 fluxes in two hydromorphic and organic-rich grassland soils

Soil CH4 fluxes are driven by CH4-producing and -consuming microorganisms that determine whether soils are sources or sinks of this potent greenhouse gas. Using quantitative metatranscriptomics, we linked CH4-cycling microbiomes to net surface CH4 fluxes throughout a year in two drained peatland soils differing in grassland land-use intensity and physicochemical properties. CH4 fluxes were highly dynamic; both soils were net CH4 sources in autumn and winter and sinks in spring and summer. Despite similar net CH4 emissions, methanogen and methanotroph loads, as determined by small subunit rRNA transcripts per gram soil, differed strongly between sites. In contrast, mRNA transcript abundances were similar in both soils and correlated well with CH4 fluxes. The methane monooxygenase to methanogenesis mRNA ratio was higher in spring and summer, when the soils were net CH4 sinks. CH4 uptake was linked to an increased proportion of USCα and γ and pmoA2 pmoA transcripts. We assume that methanogen transcript abundance may be useful to approximate changes in net surface CH4 emissions from drained peat soils; high methanotroph to methanogen ratios would indicate CH4 sink properties. Our study shows the strength of quantitative metatranscriptomics; mRNA transcript abundance holds promising indicator to link soil microbiome functions to ecosystem-level processes.

Antiapoptotic and antioxidative effects of cerium oxide nanoparticles on the testicular tissues of streptozotocin-induced diabetic rats: An experimental study

Background: Cerium dioxide nanoparticles (CNPs) due to the antidiabetic and antioxidant activities are proposed for the treatment of oxidative stress-associated diseases. Objective: To examine the impact of CNPs on hyperglycemia-induced apoptosis and oxidative stress in the testis of diabetic rats. Materials and Methods: Twenty-four male rats were divided into four groups (n = 6/each) as diabetic rats, CNPs group, diabetic + CNPs rats, and controls. The control group was fed only mouse food and water. Rats became diabetic through receiving streptozotocin (STZ) 60 mg/kg. CNPs were given to the rats at a dose of 30 mg/kg daily for 2 wk. Malondialdehyde and total thiol group (TTG) levels were measured using spectrofluorometer. Expression of b-cell lymphoma protein 2-associated X protein (BAX) and b-cell lymphoma protein 2 (Bcl-2) were investigated using quantitative real-time polymerase chain reaction. Western blot analysis was used to examine caspase 3 protein levels. Results: The content of malondialdehyde significantly increased in the STZ-diabetic rats, while TTG levels demonstrated a remarkable decrease. Caspase-3, BAX, and BAX/Bcl-2 mRNA ratio raised significantly in the STZ-diabetic rats. On the other hand, Bcl-2 mRNA levels reduced in the testis of diabetic rats (p = 0.006). Intervention with CNPs caused a substantial increase in the TTG levels, while the malondialdehyde contents, caspase-3, BAX levels, as well as BAX/Bcl-2 mRNA ratio were considerably decreased following CNPs treatment. Administration of CNPs increased mRNA levels of Bcl-2 (p < 0.0001). Conclusion: CNPs treatment attenuates testicular apoptosis and oxidative stress induced by diabetes. This nanoparticle might be suggested for the treatment of diabetes-associated reproductive disorders. Key words: Apoptosis, Nanoceria, Diabetes, Oxidative stress, Testis.

Chronic Prostatitis/Chronic Pelvic Pain Syndrome Leads to Impaired Semen Parameters, Increased Sperm DNA Fragmentation and Unfavorable Changes of Sperm Protamine mRNA Ratio

Background: Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a frequent disease affecting men of every age and accounting for a great number of consultations at urology departments. Previous studies suggested a negative impact of CP/CPPS on fertility. As increasing attention has been attributed to additional aspects, such as sperm DNA integrity and sperm protein alterations, besides the WHO standard semen analysis when assessing male fertility, in this prospective study, we aimed to further characterize the fertility status in CP/CPPS patients with a focus on these parameters. Methods: Sperm DNA fragmentation measured by sperm chromatin structure assay (SCSA) and protamine 1 to protamine 2 mRNA ratio assessed by RT-qPCR were analyzed along with conventional ejaculate parameters and inflammatory markers in 41 CP/CPPS patients and 22 healthy volunteers. Results: We found significant differences between the groups concerning multiple conventional ejaculate parameters. A significant increase in sperm DNA fragmentation was shown in CP/CPPS patients with association to other sperm parameters. The majority of CP/CPPS patients exhibited protamine mRNA ratios out of the range of regular fertility. Conclusions: This is a pioneering study with a strong practical orientation revealing that CP/CPPS leads to increased sperm DNA damage and changes in sperm protamine levels, emphasizing an unfavorable impact of CP/CPPS on fertility.

Protective role of ABCA1 in ischemic preconditioning is mediated by downregulation of miR-33-5p and miR-135-5p

Ischemic preconditioning (IPC) significantly reduces ischemia–reperfusion injury in the brain by inducing ischemic tolerance. Although emerging evidence suggests that microRNAs (miRNAs) contribute to the pathogenesis of brain ischemia and IPC-induced neuroprotection, the role of miRNAs and their underlying mechanisms are still unclear. IPC was induced in male C57BL/6 mice by brief bilateral common carotid artery occlusion. After 24 h, mice underwent transient middle cerebral artery occlusion followed by 3 h of reperfusion. Expression levels of messenger RNAs (mRNAs) and proteins were examined in the ipsilateral cortex, and mimics and inhibitors of selective miRNAs were transfected into Neuro-2a cells before oxygen–glucose deprivation (OGD). Post-IPC miRNA expression profiling identified neuroprotection-associated changes in miRNA expression in the ipsilateral cortex after ischemic stroke. Among them, miR-33-5p and miR-135b-5p were significantly downregulated by IPC. Inhibition of miR-33-5p and miR-135b-5p expression protected Neuro-2a cells from OGD-induced apoptosis. Inhibition of these two miRNAs significantly increased mRNA and protein levels of ATP-binding cassette subfamily A member 1 (ABCA1), and a binding assay showed that these two miRNAs showed specificity for Abca1 mRNA. Overexpression of ABCA1 decreased the Bax/Bcl2 mRNA ratio and activation of caspase-9 and caspase-3, whereas knockdown of ABCA1 expression increased the Bax/Bcl2 mRNA ratio and the percentage of Neuro-2a cells with a loss of mitochondrial membrane potential after OGD-treatment. In conclusion, ABCA1 expression is regulated by miR-33-5p and miR-135b-5p. Increased ABCA1 expression following IPC exerts a protective influence against cerebral ischemia via suppression of a mitochondria-dependent apoptosis pathway.

FLT3-ITD DNA allelic burden, but not mRNA levels, influences the biological characteristics of AML patients

FMS-like tyrosine kinase 3 gene internal tandem (FLT3-ITD) mutations represent one of the most frequent genetic lesions in acute myeloid leukemia (AML) and imparts a negative prognostic. For an optimal patient management, current clinical guidelines recommend the evaluation of the allelic ratio (AR), expressed as the DNA FLT3-ITD/WT mutational burden. We sought to evaluate the differences between the AR and FLT3-ITD/WT mRNA ratio (RR) and their respective impact on the biological characteristics of AML patients. A total of 32 DNA and mRNA samples from AML patients with FLT3-ITD were evaluated. There was a good correlation between the AR and RR (Spearman’s rho= 0.652, P <0.001). None of the biological characteristics were influenced by the RR values, whereas patients with high AR values (≥0.5) had higher WBC counts (Mann-Whitney, P= 0.01), LDH levels (Mann-Whitney, P= 0.037), and circulating blasts levels (Mann-Whitney, P= 0.023) than patients with low AR values (<0.5). Also, there was a good correlation between AR values and WBC count (Spearman’s correlation, P= 0.001), and LDH levels (Spearman’s correlation, P= 0.007). In our study population the AR, but not the RR, influenced the biological characteristic of patients suggesting a dose-independent effect of FLT3-ITD mutations.

Urinary podocyte mRNAs precede microalbuminuria as a progression risk marker in human type 2 diabetic nephropathy

Earlier detection of progression risk in diabetic nephropathy will allow earlier intervention to reduce progression. The hypothesis that urinary pellet podocyte mRNA is a more sensitive progression risk marker than microalbuminuria was tested. A cross sectional cohort of 165 type 2 diabetics and 41 age and sex-matched controls were enrolled. Podocyte stress (Urinary pellet podocin:nephrin mRNA ratio), podocyte detachment (Urinary pellet podocin mRNA:creatinine ratio: UPPod:CR) and a tubular marker (Urinary pellet aquaporin 2:creatinine ratio) were measured in macro-albuminuric, micro-albuminuric and norm-albuminuric groups. eGFR was reassessed after 4 years in 124 available diabetic subjects. Urinary pellet podocyte and tubular mRNA markers were increased in all diabetic groups in cross-sectional analysis. After 4 years of follow-up univariable and multivariate model analysis showed that the only urinary markers significantly related to eGFR slope were UPPod:CR (P < 0.01) and albuminuria (P < 0.01). AUC analysis using K-fold cross validation to predict eGFR loss of ≥ 3 ml/min/1.73m2/year showed that UPPod:CR and albuminuria each improved the AUC similarly such that combined with clinical variables they gave an AUC = 0.70. Podocyte markers and albuminuria had overlapping AUC contributions, as expected if podocyte depletion causes albuminuria. In the norm-albuminuria cohort (n = 75) baseline UPPod:CR was associated with development of albuminuria (P = 0.007) and, in the tertile with both normal kidney function (eGFR 84 ± 11.7 ml/min/1.73m2) and norm-albuminuria at baseline, UPPod:CR was associated with eGFR loss rate (P = 0.003). In type 2 diabetics with micro- or macro-albuminuria UPPod:CR and albuminuria were equally good at predicting eGFR loss. For norm-albuminuric type 2 diabetics UPPod:CR predicted both albuminuria and eGFR loss.

Functional conservation of mitochondrial RNA levels despite divergent mtDNA organization

Objective: Mitochondria-encoded ribosomal RNA (rRNA) genes in humans are expressed at a higher rate than protein coding genes of the mitochondria. The organization of the human mitochondrial genome (mtDNA) is amenable to differential expression of rRNAs as the rRNA encoding genes lie in tandem immediately downstream of the promoter-containing region. However, mtDNA is not organized in the same way as humans in all metazoans. In the nematode, Caenorhabditis elegans, the rRNA genes are on opposite sides of the mtDNA molecule and there are no obvious promoter sequences specific to the rRNA genes. Thus, we asked whether rRNA levels are higher relative to mRNAs in mitochondria of C. elegans as they are in humans.Results: Using droplet digital PCR, we discovered that steady-state mitochondrial rRNA transcript levels are approximately 120 times higher than the levels of mitochondrial mRNAs. These data demonstrate that despite the lack of conservation in mitochondrial genome organization, a high mitochondrial rRNA-to-mRNA ratio is a conserved feature of metazoans.

Functional Conservation of Mitochondrial RNA Levels Despite Divergent mtDNA Organization

Objective: Mitochondria-encoded ribosomal RNA (rRNA) genes in humans are expressed at a higher rate than protein coding genes of the mitochondria. The organization of the human mitochondrial genome (mtDNA) is amenable to differential expression of rRNAs as it contains a promoter specific to the transcription of the two rRNAs. However, mtDNA is not organized in the same way as humans in all metazoans. In the nematode, Caenorhabditis elegans, the rRNA genes are on opposite sides of the mtDNA molecule and there are no obvious promoter sequences specific to the rRNA genes. Thus, we asked whether rRNA levels are higher relative to mRNAs in mitochondria of C. elegans as they are in humans.Results: Using droplet digital PCR, we discovered that steady-state mitochondrial rRNA transcript levels are approximately 120 times higher than the levels of mitochondrial mRNAs. These data demonstrate that despite the lack of conservation in mitochondrial genome organization, a high mitochondrial rRNA-to-mRNA ratio is a conserved feature of metazoans.