rRNA Levels Research Articles

Analyses of the essential C82 subunit uncovered some differences in RNA polymerase III transcription between Trypanosoma brucei and Leishmania major.

The 17-subunit RNA polymerase III (RNAP III) synthesizes essential untranslated RNAs such as tRNAs and 5S rRNA. In yeast and vertebrates, subunit C82 forms a stable subcomplex with C34 and C31 that is necessary for promoter-specific transcription initiation. Little is known about RNAP III transcription in trypanosomatid parasites. To narrow this knowledge gap, we characterized the C82 subunit in Trypanosoma brucei and Leishmania major. Bioinformatic analyses showed that the 4 distinctive extended winged-helix (eWH) domains and the coiled-coil motif are present in C82 in these microorganisms. Nevertheless, C82 in trypanosomatids presents certain unique traits, including an exclusive loop within the eWH1 domain. We found that C82 localizes to the nucleus and binds to RNAP III-dependent genes in the insect stages of both parasites. Knock-down of C82 by RNA interference significantly reduced the levels of tRNAs and 5S rRNA and led to the death of procyclic forms of T. brucei. Tandem affinity purifications with both parasites allowed the identification of several C82-interacting partners, including C34 and some genus-specific putative regulators of transcription. However, the orthologue of C31 was not found in trypanosomatids. Interestingly, our data suggest a strong association of C82 with TFIIIC subunits in T. brucei, but not in L. major.

Evaluating the Impact of Ultrasonic Irrigation on Bacterial Levels and Activity Following Chemomechanical Procedures

IntroductionThis single-arm interventional trial aimed to investigate the efficacy of ultrasonic irrigation as a supplementary disinfection approach after chemomechanical procedures (CMP) using molecular techniques based on ribosomal RNA (rRNA) and rRNA genes (referred to as DNA). MethodsSamples were collected from 35 single-rooted teeth with radiographic evidence of apical periodontitis. Samples were taken after gaining root canal access (S1), (S2), and ultrasonic irrigation (S3). DNA-targeted quantitative polymerase chain reaction using universal primers was used to estimate total bacterial levels, while rRNA-targeted quantitative polymerase chain reaction was used to assess bacterial activity. Ratios between rRNA and DNA levels were calculated to search for active bacteria in the samples (rRNA/DNA ≥ 1). Wilcoxon matched-pairs signed-rank test was used to compare the differences in DNA levels between samples and DNA and rRNA levels within samples (P < .05). ResultsDNA-based methods revealed a significant decrease in bacterial levels from S1 to S2 and S2 to S3 (both P < .05). Notably, 11 out of 35 (31.4%) root canals did not harbor bacterial DNA after CMP, whereas ultrasonic activation increased DNA-negative samples to 17 (48.6%). However, all DNA-positive samples were also positive for rRNA, with significantly higher rRNA than DNA levels (P < .05), indicating bacterial activity at the sampling time. ConclusionsUltrasonic irrigation improved the disinfection of root canals after CMP by reducing bacterial levels. However, persisting bacteria remained active in the root canals after CMP and ultrasonic irrigation.

New ZNHIT3 Variants Disrupting snoRNP Assembly Cause Prenatal PEHO Syndrome with Isolated Hydrops.

ZNHIT3 (zinc finger HIT type containing protein 3) is an evolutionarily conserved protein required for ribosome biogenesis by mediating the assembly of small nucleolar RNAs (snoRNAs) of class C/D into ribonucleoprotein complexes (snoRNPs). Missense mutations in the gene encoding ZNHIT3 protein have been previously reported to cause PEHO syndrome, a severe neurodevelopmental disorder typically presenting after birth. We discuss here the case of two fetuses from a single family who presented with isolated hydrops during the early second trimester of pregnancy, resulting in intrauterine demise. Autopsy revealed no associated malformation. Through whole-genome quartet analysis, we identified two novel variants within the ZNHIT3 gene, both inherited from healthy parents and occurring as compound heterozygotes in both fetuses. The c.40T>C p.Cys14Arg variant originated from the father, while the c.251_254delAAGA variant was of maternal origin. Analysis of the variants in human cell culture models reveals that both variants reduce cell growth, albeit to different extents, and impact the protein's stability and function in distinct ways. The c.251_254delAAGA results in production of a stable form of ZNHIT3 that lacks a domain required for mediating snoRNP biogenesis, whereas the c.40T>C p.Cys14Arg variation behaves similarly to the previously described PEHO-associated ZNHIT3 variants that destabilize the protein. Interestingly, both variations lead to a marked decrease in specific box C/D snoRNA levels, reduced rRNA levels and cellular translation. Analysis of rRNA methylation pattern in fetus samples reveals distinct sites of hypo 2'-O-methylation. RNA-seq analysis of undifferentiated and differentiated SHSY5Y cells transfected with the ZNHIT3 variants reveals differential expression of a set of genes, many of which are associated with developmental processes and RNA binding compared to cells expressing wild-type ZNHIT3. In summary, this work extends the phenotype of PEHO syndrome to include antenatal manifestations and describe the molecular defects induced by two novel ZNHIT3 variants.

Unveiling the consequences of early human saliva contamination on membranes for guided bone regeneration.

GBR membranes have various surface properties designed to elicit positive responses in regenerative clinical procedures; dental clinicians attempt to employ techniques to prevent the direct interaction of contaminated oral fluids with these biomaterials. However, saliva is uninterruptedly exhibited in oral surgical procedures applying GBR membranes, suggesting a persistent interaction with biomaterials and the surrounding oral tissues. This fundamental study aimed to investigate potential alterations in the physical, chemical, and key biological properties of membranes for guided bone regeneration (GBR) caused by isolated early interaction with human saliva. A reproducible step-by-step protocol for collecting and interacting human saliva with membranes was developed. Subsequently, membranes were evaluated for their physicochemical properties, protein quantification, DNA, and 16S rRNA levels viability of two different cell lines at 1 and 7 days, and ALP activity. Non-interacted membranes and pure saliva of donors were applied as controls. Qualitative morphological alterations were noticed; DNA extraction and 16S quantification revealed significantly higher values. Furthermore, the viability of HGF-1 and MC3T3-E1 cells was significantly (p < .05) reduced following saliva interaction with biodegradable membranes. Saliva contamination did not prejudice PTFE membranes significantly in any biological assay. These outcomes demonstrated a susceptible response of biodegradable membranes to isolated early human saliva interaction, suggesting impairment of structural morphology, reduced viability to HGF-1 and MC3T3-E1, and higher absorption/adherence of DNA/16S rRNA. As a result, clinical oral procedures may need corresponding refinements.

The overlooked evolutionary dynamics of 16S rRNA revises its role as the “gold standard” for bacterial species identification

The role of 16S rRNA has been and largely remains crucial for the identification of microbial organisms. Although 16S rRNA could certainly be described as one of the most studied sequences ever, the current view of it remains somewhat ambiguous. While some consider 16S rRNA to be a variable marker with resolution power down to the strain level, others consider them to be living fossils that carry information about the origin of domains of cellular life. We show that 16S rRNA is clearly an evolutionarily very rigid sequence, making it a largely unique and irreplaceable marker, but its applicability beyond the genus level is highly limited. Interestingly, it seems that the evolutionary rigidity is not driven by functional constraints of the sequence (RNA–protein interactions), but rather results from the characteristics of the host organism. Our results suggest that, at least in some lineages, Horizontal Gene Transfer (HGT) within genera plays an important role for the evolutionary non-dynamics (stasis) of 16S rRNA. Such genera exhibit an apparent lack of diversification at the 16S rRNA level in comparison to the rest of a genome. However, why it is limited specifically and solely to 16S rRNA remains enigmatic.

Abstract B030: PARP-1 inhibition results in ovarian and endometrial cancer cell death by disrupting the nucleolar localization of the RNA helicase DDX21 which is associated with decreased survival outcomes

Abstract Cancer cells with DNA repair defects (e.g., BRCA1/2 mutant cells) are vulnerable to PARP inhibitors (PARPi) due to induction of synthetic lethality. However, recent clinical evidence has shown that PARPi can prevent the growth of some cancers irrespective of their BRCA1/2 status, suggesting alternative mechanisms of action. One such mechanism involves DDX21, an RNA helicase that localizes to the nucleoli of cells. When ADP-ribosylated by PARP-1, DDX21 promotes rDNA transcription leading to ribosome biogenesis, protein translation, and ultimately the growth of breast cancer cells. Validating this pathway in other tumor types may lead to the expanded use of PARPi as a cancer therapeutic and the potential use of DDX21 nucleolar localization as a biomarker of clinical response to PARPi. For our first aim, we hypothesized that PARPi also blocks the DDX21/PARP-1/ribosome biogenesis pathway in ovarian and endometrial cancers and can thus be used to inhibit their growth. To test this, we are determining the effect of Niraparib, an inhibitor targeting PARP-1 activity, on DDX21 subcellular localization, ribosome biogenesis, and cell growth in different ovarian (OVCAR3, OVCAR4, and HCC5012) and endometrial (KLE, Ishikawa, and HEC-1-A) cancer cell lines. We are using Western blotting to assess the levels of DDX21 and ADP-ribosylation, and immunofluorescent staining to assess the localization of DDX21 to nucleoli before and after treatment with Niraparib. We are also using qPCR to determine the levels of rRNAs and Puromycin incorporation assays to monitor ribosome translation activity before and after treatment with Niraparib. Moreover, we are using cell growth assays to determine the effect of Niraparib on the proliferation of the ovarian and endometrial cancer cell lines. Our cell-based results indicate that the nucleolar localization of DDX21 is disrupted by Niraparib treatment, leading to a pan-nuclear distribution. Furthermore, Niraparib treatment in ovarian cancer cell lines causes decreased rRNA transcription, decreased protein translation, and decreased cell proliferation. For our second aim, we are assessing possible associations among DDX21 nucleolar localization, PARP-1 protein levels, and clinical outcomes in cancer samples from patients using immunohistochemical (IHC) staining for DDX21 and PARP-1. Analysis of endometrial cancer patient samples suggests a significant association among high DDX21 nucleolar localization, high PARP-1 expression, and decreased survival. Overall, our results suggest that Niraparib works, in part, by blocking the DDX21/PARP-1/ribosome biogenesis pathway leading to endometrial and ovarian cancer cell death. The potential use of DDX21 localization as a biomarker of response to PARPi and as a prognostic factor will be further explored by conducting additional laboratory and clinical studies. Citation Format: Marwa W. Aljardali, Kevin M. Kremer, Jessica Parker, Jayanthi S. Lea, W. Lee Kraus, Cristel V. Camacho. PARP-1 inhibition results in ovarian and endometrial cancer cell death by disrupting the nucleolar localization of the RNA helicase DDX21 which is associated with decreased survival outcomes [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B030.

Evolving precision: rRNA expansion segment 7S modulates translation velocity and accuracy in eukaryal ribosomes.

Ribosome-enhancedtranslational miscoding of the genetic code causes protein dysfunction and loss of cellular fitness. During evolution, open reading frame length increased, necessitating mechanisms for enhanced translation fidelity. Indeed, eukaryal ribosomes are more accurate than bacterial counterparts, despite their virtually identical, conserved active centers. During the evolution of eukaryotic organisms ribosome expansions at the rRNA and protein level occurred, which potentially increases the options for translation regulation and cotranslational events. Here we tested the hypothesis that ribosomal RNA expansions can modulate the core function of the ribosome, faithful protein synthesis. We demonstrate that a short expansion segment present in all eukaryotes' small subunit, ES7S, is crucial for accurate protein synthesis as its presence adjusts codon-specific velocities and guarantees high levels of cognate tRNA selection. Deletion of ES7S in yeast enhances mistranslation and causes protein destabilization and aggregation, dramatically reducing cellular fitness. Removal of ES7S did not alter ribosome architecture but altered the structural dynamics of inter-subunit bridges thus affecting A-tRNA selection. Exchanging the yeast ES7S sequence with the human ES7S increases accuracy whereas shortening causes the opposite effect. Our study demonstrates that ES7S provided eukaryal ribosomes with higher accuracy without perturbing the structurally conserved decoding center.

The 28S rRNA RT-qPCR assay for host depletion evaluation to enhance avian virus detection in Illumina and Nanopore sequencing.

Abundant host and bacterial sequences can obscure the detection of less prevalent viruses in untargeted next-generation sequencing (NGS). Efficient removal of these non-targeted sequences is vital for accurate viral detection. This study presents a novel 28S ribosomal RNA (rRNA) RT-qPCR assay designed to assess the efficiency of avian rRNA depletion before conducting costly NGS for the detection of avian RNA viruses. The comprehensive evaluation of this 28S-test focuses on substituting DNase I with alternative DNases in our established depletion protocols and finetuning essential parameters for reliable host rRNA depletion. To validate the effectiveness of the 28S-test, we compared its performance with NGS results obtained from both Illumina and Nanopore sequencing platforms. This evaluation utilized swab samples from chickens infected with highly pathogenic avian influenza virus, subjected to established and modified depletion protocols. Both methods significantly reduced host rRNA levels, but using the alternative DNase had superior performance. Additionally, utilizing the 28S-test, we explored cost- and time-effective strategies, such as reduced probe concentrations and other alternative DNase usage, assessed the impact of filtration pre-treatment, and evaluated various experimental parameters to further optimize the depletion protocol. Our findings underscore the value of the 28S-test in optimizing depletion methods for advancing improvements in avian disease research through NGS.

Maize requires Embryo defective27 for embryogenesis and seedling development.

The essential role of plastid translation in embryogenesis has been established in many plants, but a retrograde signal triggered by defective plastid translation machinery that may leads to embryogenesis arrest remains unknown. In this study, we characterized an embryo defective27 (emb27) mutant in maize (Zea mays), and cloning indicates that Emb27 encodes the plastid ribosomal protein S13. The null mutant emb27-1 conditions an emb phenotype with arrested embryogenesis; however, the leaky mutant emb27-2 exhibits normal embryogenesis but an albino seedling-lethal phenotype. The emb27-1/emb27-2 trans-heterozygotes display varying phenotypes from emb to normal seeds but albino seedlings. Analysis of the Emb27 transcription levels in these mutants revealed that the Emb27 expression level in the embryo corresponds with the phenotypic expression of the emb27 mutants. In the W22 genetic background, an Emb27 transcription level higher than 6% of the wild-type level renders normal embryogenesis, whereas lower than that arrests embryogenesis. Mutation of Emb27 reduces the level of plastid 16S rRNA and the accumulation of the plastid-encoded proteins. As a secondary effect, splicing of several plastid introns was impaired in emb27-1 and 2 other plastid translation-defective mutants, emb15 and emb16, suggesting that plastome-encoded factors are required for the splicing of these introns, such as Maturase K (MatK). Our results indicate that EMB27 is essential for plastid protein translation, embryogenesis, and seedling development in maize and reveal an expression threshold of Emb27 for maize embryogenesis.

BRIX1 promotes ribosome synthesis and enhances glycolysis by selected translation of GLUT1 in colorectal cancer.

Ribosome biogenesis protein BRX1 homolog (BRIX1) is critically required for the synthesis of the 60S ribosome subunit. However, the role and mechanism of BRIX1 in colorectal cancer (CRC) remain unclear. Kyoto Encyclopedia of Gene and Genome pathway and Gene Ontology analyses were used for bioinformatics analysis. The rRNA levels were detected in CRC tissues and cells. Nascent RNA synthesis was detected via cellular immunofluorescence. The correlation was analyzed between patient Positron Emission Tomography-Computed Tomography (PET-CT) values and their BRIX1 expression. The extracellular acidification rate (ECAR) and oxygen consumption rate were determined via live metabolic analyses. Polysome fractions were collected for BRIX1 mRNA used in translation. The orthotopic model and Cell Counting Kit-8 (CCK8) assay were used to assess BRIX1 function in CRC. BRIX1 is a core protein involved in ribosome-related pathway changes in CRC. Gene Ontology analysis showed that BRIX1 was primarily enriched in ribosome assembly and ribosome biogenesis pathways. In fresh CRC tissue, rRNA levels (5S, 5.8S, 18S and 28S) were higher in the BRIX1 high-expression group than in the BRIX1 low-expression group. Similarly, BRIX1 knockdown significantly decreased rRNA levels for 5S, 5.8S, 18S and 28S in CRC cells, whereas overexpression of BRIX1 significantly increased these levels. In addition, BRIX1 knockdown inhibited nascent RNA synthesis in CRC cells. In clinical data analysis, BRIX1 expression was related to the glucose uptake in PET-CT. BRIX1 knockdown significantly decreased the ECAR value, glucose uptake and lactic acid production in CRC cells, whereas BRIX1 overexpression significantly increased these. Furthermore, BRIX1 knockdown significantly decreased the protein expression of GLUT1, whereas BRIX1 overexpression significantly increased this; however, expression of BRIX1 mRNA was unaffected in either case. Blocking glycolysis by si-GLUT1 or galactose reversed BRIX1 promotion of glycolysis and cell proliferation in CRC cells.

Filamentous Actin in the Nucleus in Triple-Negative Breast Cancer Stem Cells: A Key to Drug-Induced Nucleolar Stress and Stemness Inhibition?

Actin, primarily a cytoplasmic cytoskeleton protein, is transported in and out of the nucleus with the help of actin-binding proteins (ABPs). Actin exists in two forms, i.e., monomeric globular (G-actin) and polymerized filamentous (F-actin). While G-actin promotes gene transcription by associating with RNA polymerases, F-actin can inhibit this effect in the nucleus. Unexpectedly, we found that lovastatin, an FDA-approved lipid-lowering drug, induces actin redistribution and its translocation into the nucleus in triple-negative breast cancer (TNBC) cancer stem cells. Lovastatin treatment also decreased levels of rRNAs and stemness markers, which are transcription products of RNA Pol I and Pol II, respectively. Bioinformatics analysis showed that actin genes were positively correlated with ABP genes involved in the translocation/polymerization and transcriptional regulation of nuclear actin in breast cancer. Similar correlations were found between actin genes and RNA Pol I genes and stemness-related genes. We propose a model to explain the roles of lovastatin in inducing nucleolar stress and inhibiting stemness in TNBC cancer stem cells. In our model, lovastatin induces translocation/accumulation of F-actin in the nucleus/nucleolus, which, in turn, induces nucleolar stress and stemness inhibition by suppressing the synthesis of rRNAs and decreasing the expression of stemness-related genes. Our model has opened up a new field of research on the roles of nuclear actin in cancer biology, offering potential therapeutic targets for the treatment of TNBC.

Dysregulated Ribosome Biogenesis Is a Targetable Vulnerability in Triple-Negative Breast Cancer: MRPS27 as a Key Mediator of the Stemness-inhibitory Effect of Lovastatin.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited effective therapeutic options readily available. We have previously demonstrated that lovastatin, an FDA-approved lipid-lowering drug, selectively inhibits the stemness properties of TNBC. However, the intracellular targets of lovastatin in TNBC remain largely unknown. Here, we unexpectedly uncovered ribosome biogenesis as the predominant pathway targeted by lovastatin in TNBC. Lovastatin induced the translocation of ribosome biogenesis-related proteins including nucleophosmin (NPM), nucleolar and coiled-body phosphoprotein 1 (NOLC1), and the ribosomal protein RPL3. Lovastatin also suppressed the transcript levels of rRNAs and increased the nuclear protein level and transcriptional activity of p53, a master mediator of nucleolar stress. A prognostic model generated from 10 ribosome biogenesis-related genes showed outstanding performance in predicting the survival of TNBC patients. Mitochondrial ribosomal protein S27 (MRPS27), the top-ranked risky model gene, was highly expressed and correlated with tumor stage and lymph node involvement in TNBC. Mechanistically, MRPS27 knockdown inhibited the stemness properties and the malignant phenotypes of TNBC. Overexpression of MRPS27 attenuated the stemness-inhibitory effect of lovastatin in TNBC cells. Our findings reveal that dysregulated ribosome biogenesis is a targetable vulnerability and targeting MRPS27 could be a novel therapeutic strategy for TNBC patients.

Functional characterization of Nosema bombycis (microsporidia) trehalase 3.

Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription-quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell's energy resources.

Recent insights into the morphology, molecular characterization and tissue localization of the caprine Sarcocystis species infecting domestic goats (Capra hiricus): Sarcocystis moulei, Sarcocystis capracanis, and Sarcocystis hircicanis

Ninety-seven (64.67%) out of 150 domestic goats (Capra hiricus) carcasses were found to be infected by Sarcocystis moulei, Sarcocystis capracanis, and Sarcocystis hircicanis sarcocysts. Sarcocystis moulei macrosarcocysts were detected in the cardiac, esophageal, skeletal, lingual, and diaphragmatic muscles of seven goats (4.67%) out of the 150 examined animals, whereas the microscopic Sarcocystis species were found in (90/150 = 60%). Two morphotypes of S. moulei were observed. Morphotype (I) macrosarcocysts were large-sized oval, ovoid, spherical, and measured 2–7 mm in length x 2–6 mm in width. Sarcocystis moulei morphotype (II) macrosarcocysts were spindle-shaped, spheroid, sometimes elongated, and measured 1.8–6 x 0.5–2 mm. By TEM, all S. moulei morphotypes were ultrastructurally the same and had a sarcocyst wall that was characterized by highly branched or cauliflower-like villar protrusions (VP) with dumbbell-like structures. The VP interior was packed with well-developed microtubules in longitudinal and cross arrangements. Sarcocystis moulei cyst wall was 3–6 μm thick. Sarcocystis capracanis microsarcocysts detected herein had a cyst wall that ranged from 4–8 μm in thickness. The VP was upright finger-like or cylindrical. The PVM had electron-dense corrugations in the region of the VP. Few amounts of microfilaments were detected inside the cores of VP. Sarcocystis hircicanis had a thinner cyst wall (~1–3 μm) with hairy long VP that ranged from 1 to 7.5 μm in length. Microtubules were missing inside the cores of the VP. The three caprine Sarcocystis species were molecularly characterized on the level of the 18S rRNA, 28S rRNA, and Cox1 genes.

Collaborative cytometric inter-laboratory ring test for probiotics quantification.

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. From this definition, accurate enumeration of probiotic products is a necessity. Nonetheless, this definition does not specify the methods for assessing such viability. Colony forming units is the de facto gold standard for enumerating viable in probiotic products. The notion of microbial viability has been anchored in the concept of cultivability, which refers to a cell's capacity to replicate and form colonies on agar media. However, there is a growing consensus that the term "viability" should not be exclusively tied to the ability to cultivate cells. For example, bacterial cells can exist in a Viable But Non-Culturable (VBNC) state, characterized by the maintenance of characteristics such as membrane integrity, enzymatic activity, pH gradients, and elevated levels of rRNA, despite losing the ability to form colonies. Herein we present the results of a collaborative inter-laboratory ring test for cytometric bacterial quantification. Specifically, membrane integrity fluorescence flow cytometry (FFC) method and the newer impedance flow cytometry (IFC) method have been used. Both methods interrogate single cells in solution for the presence of intact membranes. FFC exploits fluorochromes that reflect the presence or absence of an intact membrane. IFC probes membrane integrity in a label-free approach by detecting membrane-induced hindrances to the propagation of electricity. A performance ring-test and comparison design on the FFC method showed that the method is robust against the exchange of equipment, procedures, materials, and operators. After initial method optimization with assessments of rehydration medium, wake-up duration, and phase shift gating on the individual strains, the IFC method showed good agreement with the FFC results. Specifically, we tested 6 distinct species of probiotic bacteria (3 Lactobacillus and 3 Bifidobacterium strains) finding good agreement between FFC and IFC results in terms of total and live cells. Together, these results demonstrate that flow cytometry is a reliable, precise, and user-friendly culture-independent method for bacterial enumeration.

Daxx knockdown promoted rDNA transcription without impairing H3.3 expression in mouse preimplantation embryos.

During fertilization, DAXX (death domain-associated protein) mediates histone variant H3.3 incorporation into heterochromatin, which plays an important role in the maintenance of genomic integrity. rDNA, the ribosomal gene, is included in the first wave of gene activation after fertilization. Our and other studies indicated that loss of Daxx disturbs rDNA heterochromatinization and promotes rDNA transcription without change in protein expression of H3.3. However, maternal and zygotic deletion of Daxx impairs blastocyst development. Whether Daxx knockdown affects H3.3 expression and improves the rDNA transcription in preimplantation development has not been reported. In the present study, we injected HA-labelled H3.3 (H3.3-HA) into oocytes during ICSI procedure, and detected H3.3 and DAXX by immunofluorescent staining. Then, we knockdowned Daxx and detected the gene expression levels of Daxx, H3.3, 18s and 47s rRNA. We also performed immunofluorescent staining of B23, γH2A and EdU incorporation to demonstrate nuclear structure, DNA damage and replication. We found injection of H3.3-HA did not impair preimplantation development. Daxx siRNA did not change expression of H3.3 mRNA, and the development of two-cell embryos and blastocysts, but the overall replication and expression levels of rRNA were increased compared with that in the control group. Finally, knockdown of DAXX did not aggravate the DNA damage but loosened the nucleolus. We concluded that Daxx knockdown promoted DNA replication and rDNA transcription, but did not affect H3.3 expression and subsequent preimplantation development.

Effects of In Utero EtOH Exposure on 18S Ribosomal RNA Processing: Contribution to Fetal Alcohol Spectrum Disorder.

Fetal alcohol spectrum disorders (FASD) are leading causes of neurodevelopmental disability. The mechanisms by which alcohol (EtOH) disrupts fetal brain development are incompletely understood, as are the genetic factors that modify individual vulnerability. Because the phenotype abnormalities of FASD are so varied and widespread, we investigated whether fetal exposure to EtOH disrupts ribosome biogenesis and the processing of pre-ribosomal RNAs and ribosome assembly, by determining the effect of exposure to EtOH on the developmental expression of 18S rRNA and its cleaved forms, members of a novel class of short non-coding RNAs (srRNAs). In vitro neuronal cultures and fetal brains (11-22 weeks) were collected according to an IRB-approved protocol. Twenty EtOH-exposed brains from the first and second trimester were compared with ten unexposed controls matched for gestational age and fetal gender. Twenty fetal-brain-derived exosomes (FB-Es) were isolated from matching maternal blood. RNA was isolated using Qiagen RNA isolation kits. Fetal brain srRNA expression was quantified by ddPCR. srRNAs were expressed in the human brain and FB-Es during fetal development. EtOH exposure slightly decreased srRNA expression (1.1-fold; p = 0.03). Addition of srRNAs to in vitro neuronal cultures inhibited EtOH-induced caspase-3 activation (1.6-fold, p = 0.002) and increased cell survival (4.7%, p = 0.034). The addition of exogenous srRNAs reversed the EtOH-mediated downregulation of srRNAs (2-fold, p = 0.002). EtOH exposure suppressed expression of srRNAs in the developing brain, increased activity of caspase-3, and inhibited neuronal survival. Exogenous srRNAs reversed this effect, possibly by stabilizing endogenous srRNAs, or by increasing the association of cellular proteins with srRNAs, modifying gene transcription. Finally, the reduction in 18S rRNA levels correlated closely with the reduction in fetal eye diameter, an anatomical hallmark of FASD. The findings suggest a potential mechanism for EtOH-mediated neurotoxicity via alterations in 18S rRNA processing and the use of FB-Es for early diagnosis of FASD. Ribosome biogenesis may be a novel target to ameliorate FASD in utero or after birth. These findings are consistent with observations that gene-environment interactions contribute to FASD vulnerability.

The induction of p53 correlates with defects in the production, but not the levels, of the small ribosomal subunit and stalled large ribosomal subunit biogenesis.

Ribosome biogenesis is one of the biggest consumers of cellular energy. More than 20 genetic diseases (ribosomopathies) and multiple cancers arise from defects in the production of the 40S (SSU) and 60S (LSU) ribosomal subunits. Defects in the production of either the SSU or LSU result in p53 induction through the accumulation of the 5S RNP, an LSU assembly intermediate. While the mechanism is understood for the LSU, it is still unclear how SSU production defects induce p53 through the 5S RNP since the production of the two subunits is believed to be uncoupled. Here, we examined the response to SSU production defects to understand how this leads to the activation of p53 via the 5S RNP. We found that p53 activation occurs rapidly after SSU production is blocked, prior to changes in mature ribosomal RNA (rRNA) levels but correlated with early, middle and late SSU pre-rRNA processing defects. Furthermore, both nucleolar/nuclear LSU maturation, in particular late stages in 5.8S rRNA processing, and pre-LSU export were affected by SSU production defects. We have therefore uncovered a novel connection between the SSU and LSU production pathways in human cells, which explains how p53 is induced in response to SSU production defects.

Selection and Evaluation of Reference Genes for RT-qPCR Analysis in Amorphophallus Konjac Based on Transcriptome Data.

Combined with the Konjac transcriptome database of our laboratory and internal reference genes commonly used in plants, the eight candidate internal reference genes were screened and detected. They are the 25S ribosomal RNA gene (25S rRNA), 18S ribosomal RNA gene (18S rRNA), actin gene (ACT), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), ubiquitin gene (UBQ), β-tubulin gene (β-TUB), eukaryotic elongation factor 1-αgene(eEF-1α), and eukaryotic translation initiation factor 4α-1 gene (eIF-4α). The results of GeNorm, Normfinder, and BestKeeper were analyzed comprehensively. The data showed that the expression levels of 25S rRNA, 18S rRNA, and ACT at the reproductive periods, eEF-1α and eIF-4α at the nutritional periods, and eEF-1α, UBQ, and ACT at different leaf developmental periods were stable. These identified and stable internal reference genes will provide the basis for the subsequent molecular biology-related studies of Konjac.

Mt-LAF3 is a pseudouridine synthase ortholog required for mitochondrial rRNA and mRNA gene expression in Trypanosoma brucei

Trypanosoma brucei and related kinetoplastid parasites possess unique RNA processing pathways, including in their mitochondria, that regulate metabolism and development. Altering RNA composition or conformation through nucleotide modifications is one such pathway, and modifications including pseudouridine regulate RNA fate and function in many organisms. We surveyed pseudouridine synthase (PUS) orthologs in trypanosomatids, with a particular interest in mitochondrial enzymes due to their potential importance for mitochondrial function and metabolism. Trypanosoma brucei mitochondrial (mt)-LAF3 is an ortholog of human and yeast mitochondrial PUS enzymes, and a mitoribosome assembly factor, but structural studies differ in their conclusion as to whether it has PUS catalytic activity. Here, we generated T. brucei cells that are conditionally null (CN) for mt-LAF3 expression and showed that mt-LAF3 loss is lethal and disrupts mitochondrial membrane potential (ΔΨm). Addition of a mutant gamma ATP synthase allele to the CN cells permitted ΔΨm maintenance and cell survival, allowing us to assess primary effects on mitochondrial RNAs. As expected, these studies showed that loss of mt-LAF3 dramatically decreases levels of mitochondrial 12S and 9S rRNAs. Notably, we also observed decreases in mitochondrial mRNA levels, including differential effects on edited vs. pre-edited mRNAs, indicating that mt-LAF3 is required for mitochondrial rRNA and mRNA processing, including of edited transcripts. To assess the importance of PUS catalytic activity in mt-LAF3 we mutated a conserved aspartate that is necessary for catalysis in other PUS enzymes and showed it is not essential for cell growth, or maintenance of ΔΨm and mitochondrial RNA levels. Together, these results indicate that mt-LAF3 is required for normal expression of mitochondrial mRNAs in addition to rRNAs, but that PUS catalytic activity is not required for these functions. Instead, our work, combined with previous structural studies, suggests that T. brucei mt-LAF3 acts as a mitochondrial RNA-stabilizing scaffold.