Degradation Of Ribosomal RNA Research Articles

Schlafen14 Impairs HIV-1 Expression in a Codon Usage-Dependent Manner.

Schlafen (SLFN) is a family of proteins upregulated by type I interferons with a regulatory role in translation. Intriguingly, SLFN14 associates with the ribosome and can degrade rRNA, tRNA, and mRNA in vitro, but a role in translation is still unknown. Ribosomes are important regulatory hubs during translation elongation of mRNAs rich in rare codons. Therefore, we evaluated the potential role of SLFN14 in the expression of mRNAs enriched in rare codons, using HIV-1 genes as a model. We found that, in a variety of cell types, including primary immune cells, SLFN14 regulates the expression of HIV-1 and non-viral genes based on their codon adaptation index, a measurement of the synonymous codon usage bias; consequently, SLFN14 inhibits the replication of HIV-1. The potent inhibitory effect of SLFN14 on the expression of the rare codon-rich transcript HIV-1 Gag was minimized by codon optimization. Mechanistically, we found that the endoribonuclease activity of SLFN14 is required, and that ribosomal RNA degradation is involved. Therefore, we propose that SLFN14 impairs the expression of HIV-1 transcripts rich in rare codons, in a catalytic-dependent manner.

Expand and Sensitize: Guanidine‐Functionalized Exopolysaccharide Nanoparticles Cause Bacterial Cell Expansion and Antibiotic Sensitization

AbstractConventional antibacterial agents and mechanisms are frequently observed to be ineffective due to the evolution of bacteria to the strains with stronger antibiotic resistance, and hence developing alternative antibacterial materials and mechanisms is urgently needed. Here, guanidine‐functionalized exopolysaccharide (EPS) nanoparticles (termed EPGNs) with durable antibacterial and antibiofilm activities are developed. Very interestingly, the EPGNs obtained by the reaction of EPS, epichlorohydrin, and polyhexamethylene guanidine hydrochloride exhibit an unconventional antibacterial mechanism, i.e., they can induce substantial bacterial cell expansion by upregulating the SulA and DicB proteins that are responsible for cell division inhibition, along with the increase of reactive oxygen species production, bacterial cell surface disruption, and bacterial ribosomal RNA degradation. The transcriptome analysis reveals that EPGNs can hinder cell motility, induce loss of cell integrity, decrease the resistance of bacteria to oxidative stress, and finally lead to cell death. Moreover, EPGNs can effectively accelerate the bacteria‐infected wound healing. This work provides the first example that nanomaterials can cause bacterial cell expansion by affecting intracellular structures and inhibiting cell division, and it may inspire other researchers to investigate the effect of antibacterial materials on the change of bacterial volume and design unconventional antibacterial materials/strategies.

Ribosome dysfunction underlies SLFN14-related thrombocytopenia

AbstractPathogenic missense variants in SLFN14, which encode an RNA endoribonuclease protein that regulates ribosomal RNA (rRNA) degradation, are known to cause inherited thrombocytopenia (TP) with impaired platelet aggregation and adenosine triphosphate secretion. Despite mild laboratory defects, the patients displayed an obvious bleeding phenotype. However, the function of SLFN14 in megakaryocyte (MK) and platelet biology remains unknown. This study aimed to model the disease in an immortalized MK cell line (imMKCL) and to characterize the platelet transcriptome in patients with the SLFN14 K219N variant. MK derived from heterozygous and homozygous SLFN14 K219N imMKCL and stem cells of blood from patients mainly presented with a defect in proplatelet formation and mitochondrial organization. SLFN14-defective platelets and mature MK showed signs of rRNA degradation; however, this was absent in undifferentiated imMKCL cells and granulocytes. Total platelet RNA was sequenced in 2 patients and 19 healthy controls. Differential gene expression analysis yielded 2999 and 2888 significantly (|log2 fold change| >1, false discovery rate <0.05) up- and downregulated genes, respectively. Remarkably, these downregulated genes were not enriched in any biological pathway, whereas upregulated genes were enriched in pathways involved in (mitochondrial) translation and transcription, with a significant upregulation of 134 ribosomal protein genes (RPGs). The upregulation of mitochondrial RPGs through increased mammalian target of rapamycin complex 1 (mTORC1) signaling in SLFN14 K219N MK seems to be a compensatory response to rRNA degradation. mTORC1 inhibition with rapamycin resulted in further enhanced rRNA degradation in SLFN14 K219N MK. Taken together, our study indicates dysregulation of mTORC1 coordinated ribosomal biogenesis is the disease mechanism for SLFN14-related TP.

RNA disruption is a widespread phenomenon associated with stress-induced cell death in tumour cells

We have previously shown that neoadjuvant chemotherapy can induce the degradation of tumour ribosomal RNA (rRNA) in patients with advanced breast cancer, a phenomenon we termed “RNA disruption”. Extensive tumour RNA disruption during chemotherapy was associated with a post-treatment pathological complete response and improved disease-free survival. The RNA disruption assay (RDA), which quantifies this phenomenon, is now being evaluated for its clinical utility in a large multinational clinical trial. However, it remains unclear if RNA disruption (i) is manifested across many tumour and non-tumour cell types, (ii) can occur in response to cell stress, and (iii) is associated with tumour cell death. In this study, we show that RNA disruption is induced by several mechanistically distinct chemotherapy agents and report that this phenomenon is observed in response to oxidative stress, endoplasmic reticulum (ER) stress, protein translation inhibition and nutrient/growth factor limitation. We further show that RNA disruption is dose- and time-dependent, and occurs in both tumourigenic and non-tumourigenic cell types. Northern blotting experiments suggest that the rRNA fragments generated during RNA disruption stem (at least in part) from the 28S rRNA. Moreover, we demonstrate that RNA disruption is reproducibly associated with three robust biomarkers of cell death: strongly reduced cell numbers, lost cell replicative capacity, and the generation of cells with a subG1 DNA content. Thus, our findings indicate that RNA disruption is a widespread phenomenon exhibited in mammalian cells under stress, and that high RNA disruption is associated with the onset of cell death.

Escherichia coli protein synthesis is limited by mRNA availability rather than ribosomal capacity during phosphate starvation.

Protein synthesis is the most energetically costly process in the cell. Consequently, it is a tightly regulated process, and regulation of the resources allocated to the protein synthesis machinery is at the heart of bacterial growth optimization theory. However, the molecular mechanisms that result in dynamic downregulation of protein synthesis in response to nutrient starvation are not well described. Here, we first quantify the Escherichia coli response to phosphate starvation at the level of accumulation rates for protein, RNA and DNA. Escherichia coli maintains a low level of protein synthesis for hours after the removal of phosphate while the RNA contents decrease, primarily as a consequence of ribosomal RNA degradation combined with a reduced RNA synthesis rate. To understand the molecular basis for the low protein synthesis rate of phosphate-starved cells, template mRNA for translation was overproduced in the form of a highly induced long-lived mRNA. Remarkably, starved cells increased the rate of protein synthesis and reduced the rate of ribosomal RNA degradation upon mRNA induction. These observations suggest that protein synthesis in phosphate-starved cells is primarily limited by the availability of template, and does not operate at the maximum capacity of the ribosomes. We suggest that mRNA limitation is an adaptive response to phosphate starvation that prevents the deleterious consequences of overcommitting resources to protein synthesis. Moreover, our results support the model that degradation of ribosomal RNA occurs as a consequence of the availability of idle ribosomal subunits.

Evaluation of the Collateral Activity Of CRISPR/Cas13a-Ribonuclease on the Agilent 2100 Bioanalyzer

The paper describes an original technique for detecting the collateral activity of CRISPR/Cas 13a ribonuclease based on the assessment of ribosomal RNA degradation. The Agilent 2100 bioanalyzer is used as an analyzing device. This approach is an alternative to existing detection methods and has a number of advantages over them in the case when a quantitative assessment of activity is not required. On the example of the test sample, the optimal concentrations and ratios of the components of the reaction mixture, which are necessary to obtain the most indicative result, were determined. The proposed technique can be used for qualitative assessment of the activity of recombinant ribonuclease Cas13a preparations obtained under different conditions of heterologous protein expression and purification, as well as for testing guide RNAs.

Ribosomal RNA degradation induced by the bacterial RNA polymerase inhibitor rifampicin.

Rifampicin, a broad-spectrum antibiotic, inhibits bacterial RNA polymerase. Here we show that rifampicin treatment of Escherichia coli results in a 50% decrease in cell size due to a terminal cell division. This decrease is a consequence of inhibition of transcription as evidenced by an isogenic rifampicin-resistant strain. There is also a 50% decrease in total RNA due mostly to a 90% decrease in 23S and 16S rRNA levels. Control experiments showed this decrease is not an artifact of our RNA purification protocol and therefore due to degradation in vivo. Since chromosome replication continues after rifampicin treatment, ribonucleotides from rRNA degradation could be recycled for DNA synthesis. Rifampicin-induced rRNA degradation occurs under different growth conditions and in different strain backgrounds. However, rRNA degradation is never complete, thus permitting the reinitiation of growth after removal of rifampicin. The orderly shutdown of growth under conditions where the induction of stress genes is blocked by rifampicin is noteworthy. Inhibition of protein synthesis by chloramphenicol resulted in a partial decrease in 23S and 16S rRNA levels whereas kasugamycin treatment had no effect. Analysis of temperature-sensitive mutant strains implicate RNase E, PNPase, and RNase R in rifampicin-induced rRNA degradation. We cannot distinguish between a direct role for RNase E in rRNA degradation versus an indirect role involving a slowdown of mRNA degradation. Since mRNA and rRNA appear to be degraded by the same ribonucleases, competition by rRNA is likely to result in slower mRNA degradation rates in the presence of rifampicin than under normal growth conditions.

Quantification of immune cell-mediated destruction of tumor cells in vitro using the RNA disruption assay.

e15017 Background: Immune checkpoint inhibitors (ICIs) are increasingly being used in the treatment of human cancers, often in combination with cytotoxic chemotherapy drugs. However, current ICIs are costly and are only effective against a fraction of patient tumours. Thus, a tool that could reliably quantify tumour response to ICIs and predict outcome post-treatment would be highly valuable. Patients with non-responding tumours could be spared the costs and toxic side-effects of the ineffective drugs and moved promptly to alternate treatments. We have observed that a variety of structurally and mechanistically distinct chemotherapy agents induce the degradation of ribosomal RNA (rRNA) into a large number of high molecular weight fragments (between the 28S and 18S rRNAs)---a phenomenon we have termed “RNA disruption”. High tumour RNA disruption during treatment, as quantified using the RNA disruption assay (RDA), has been shown to predict pathologic complete response and improved disease-free survival in breast cancer patients. Methods: To assess whether RDA could be used to monitor immune cell killing of tumour cells, we collected peripheral blood mononuclear cells (PBMCs) from consenting healthy human donors and enriched the cell population for natural killer (NK) cells to 90% using a negative selection approach. The cells, with or without IL-2 pre-treatment, were incubated with K562 cells, after which K562 cell killing was quantified over time using a standard immunocytotoxicity assay and K562 cell RNA disruption was measured using RDA. Results: Freshly isolated human NK cells when incubated with K562 chronic myeloid leukemia cells induced cell destruction and RNA disruption in K562 cells in a dose-dependent manner. Pre-incubation with IL-2 augmented both NK cell-mediated RNA disruption and NK-mediated cytotoxicity in K562 cells. Interestingly, the pattern of K562 RNA disruption fragments generated by NK cells was similar to that generated by chemotherapy drugs. Conclusions: RDA was successfully used to quantify immune cell-mediated destruction of tumour cells, raising the prospect of its possible use to monitor tumour destruction by immune cells in vivo and to predict response to ICIs (alone or in concert with cytotoxic chemotherapy drugs).

The Ins and Outs of Autophagic Ribosome Turnover.

Ribosomes are essential for protein synthesis in all organisms and their biogenesis and number are tightly controlled to maintain homeostasis in changing environmental conditions. While ribosome assembly and quality control mechanisms have been extensively studied, our understanding of ribosome degradation is limited. In yeast or animal cells, ribosomes are degraded after transfer into the vacuole or lysosome by ribophagy or nonselective autophagy, and ribosomal RNA can also be transferred directly across the lysosomal membrane by RNautophagy. In plants, ribosomal RNA is degraded by the vacuolar T2 ribonuclease RNS2 after transport by autophagy-related mechanisms, although it is unknown if a selective ribophagy pathway exists in plants. In this review, we describe mechanisms of turnover of ribosomal components in animals and yeast, and, then, discuss potential pathways for degradation of ribosomal RNA and protein within the vacuole in plants.

The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA.

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.

Autophagy-dependent ribosomal RNA degradation is essential for maintaining nucleotide homeostasis during C. elegans development.

Ribosome degradation through the autophagy-lysosome pathway is crucial for cell survival during nutrient starvation, but whether it occurs under normal growth conditions and contributes to animal physiology remains unaddressed. In this study, we identified RNST-2, a C. elegans T2 family endoribonuclease, as the key enzyme that degrades ribosomal RNA in lysosomes. We found that loss of rnst-2 causes accumulation of rRNA and ribosomal proteins in enlarged lysosomes and both phenotypes are suppressed by blocking autophagy, which indicates that RNST-2 mediates autophagic degradation of ribosomal RNA in lysosomes. rnst-2(lf) mutants are defective in embryonic and larval development and are short-lived. Remarkably, simultaneous loss of RNST-2 and de novo synthesis of pyrimidine nucleotides leads to complete embryonic lethality, which is suppressed by supplements of uridine or cytidine. Our study reveals an essential role of autophagy-dependent degradation of ribosomal RNA in maintaining nucleotide homeostasis during animal development.

Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq.

Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types (INTACT). As nuclear RNA is low in polyadenylated transcripts and conventional pulldown methods would not capture non-polyadenylated pre-mRNA, we also present our method to remove ribosomal RNA from the total nuclear RNA in preparation for nuclear RNA-Seq.

Acrolein toxicity at advanced age: present and future.

It is thought that tissue damage at advanced age is mainly caused by ROS (reactive oxygen species, O2-, H2O2, and ·OH). However, it was found that acrolein (CH2=CH-CHO) is more toxic than ROS, and is mainly produced from spermine (SPM), one of the polyamines, rather than from unsaturated fatty acids. Significant amounts of SPM are present normally as SPM-ribosome complexes, and contribute to protein synthesis. However, SPM was released from ribosomes due to the degradation of ribosomal RNA by ·OH or the binding of Ca2+ to ribosomes, and acrolein was produced from free SPM by polyamine oxidases, particularly by SPM oxidase. Acrolein inactivated several proteins such as GAPDH (glycelaldehyde-3-phosphate dehydrogenase), and also stimulated MMP-9 (matrix metalloproteinase-9) activity. Acrolein-conjugated GAPDH translocated to nucleus, and caused apoptosis like nitrosylated GAPDH. Through acrolein conjugation with several proteins, acrolein causes tissue damage during brain stroke, dementia, renal failure, and primary Sjögren's syndrome. Thus, development of acrolein scavengers with less side effects is very important to maintain QOL (quality of life) of elderly people.

Nuclear Transcriptomes at High Resolution Using Retooled INTACT.

Isolated nuclei provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here, we describe transfer of the isolation of nuclei from tagged specific cell types (INTACT) to the monocot rice (Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear-envelope-targeting domain of the nuclear tagging fusion (NTF) protein with an outer nuclear-envelope-anchored domain. This modified NTF was combined with codon-optimized Escherichia coli BirA in a single T-DNA construct. We also developed inexpensive methods for INTACT, T-DNA insertion mapping, and profiling of the complete nuclear transcriptome, including a ribosomal RNA degradation procedure that minimizes pre-ribosomal RNA (pre-rRNA) transcripts. A high-resolution comparison of nuclear and steady-state poly(A)+ transcript populations of seedling root tips confirmed the capture of pre-messenger RNA (pre-mRNA) and exposed distinctions in diversity and abundance of the nuclear and total transcriptomes. This retooled INTACT can enable high-resolution monitoring of the nuclear transcriptome and chromatin in specific cell types of rice and other species.

Elucidation of pathways of ribosomal RNA degradation: an essential role for RNase E.

Although normally stable in growing cells, ribosomal RNAs are degraded under conditions of stress, such as starvation, and in response to misassembled or otherwise defective ribosomes in a process termed RNA quality control. Previously, our laboratory found that large fragments of 16S and 23S rRNA accumulate in strains lacking the processive exoribonucleases RNase II, RNase R, and PNPase, implicating these enzymes in the later steps of rRNA breakdown. Here, we define the pathways of rRNA degradation in the quality control process and during starvation, and show that the essential endoribonuclease, RNase E, is required to make the initial cleavages in both degradative processes. We also present evidence that explains why the exoribonuclease, RNase PH, is required to initiate the degradation of rRNA during starvation. The data presented here provide the first detailed description of rRNA degradation in bacterial cells.

RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines.

BackgroundCellular stressors and apoptosis-inducing agents have been shown to induce ribosomal RNA (rRNA) degradation in eukaryotic cells. Recently, RNA degradation in vivo was observed in patients with locally advanced breast cancer, where mid-treatment tumor RNA degradation was associated with complete tumor destruction and enhanced patient survival. However, it is not clear how widespread chemotherapy induced “RNA disruption” is, the extent to which it is associated with drug response or what the underlying mechanisms are.MethodsOvarian (A2780, CaOV3) and breast (MDA-MB-231, MCF-7, BT474, SKBR3) cancer cell lines were treated with several cytotoxic chemotherapy drugs and total RNA was isolated. RNA was also prepared from docetaxel resistant A2780DXL and carboplatin resistant A2780CBN cells following drug exposure. Disruption of RNA was analyzed by capillary electrophoresis. Northern blotting was performed using probes complementary to the 28S and 18S rRNA to determine the origins of degradation bands. Apoptosis activation was assessed by flow cytometric monitoring of annexin-V and propidium iodide (PI) binding to cells and by measuring caspase-3 activation. The link between apoptosis and RNA degradation (disruption) was investigated using a caspase-3 inhibitor.ResultsAll chemotherapy drugs tested were capable of inducing similar RNA disruption patterns. Docetaxel treatment of the resistant A2780DXL cells and carboplatin treatment of the A2780CBN cells did not result in RNA disruption. Northern blotting indicated that two RNA disruption bands were derived from the 3’-end of the 28S rRNA. Annexin-V and PI staining of docetaxel treated cells, along with assessment of caspase-3 activation, showed concurrent initiation of apoptosis and RNA disruption, while inhibition of caspase-3 activity significantly reduced RNA disruption.ConclusionsSupporting the in vivo evidence, our results demonstrate that RNA disruption is induced by multiple chemotherapy agents in cell lines from different tissues and is associated with drug response. Although present, the link between apoptosis and RNA disruption is not completely understood. Evaluation of RNA disruption is thus proposed as a novel and effective biomarker to assess response to chemotherapy drugs in vitro and in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2197-1) contains supplementary material, which is available to authorized users.

ID: 203: Middle East respiratory syndrome coronavirus accessory protein NS4b is a 2H-Phosphoesterase that degrades 2′,5′-oligoadenylate activators of RNase L

Efficient and productive virus infection often requires viral countermeasures that block innate immunity. The interferon-inducible 2′,5′-oligoadenylate (2-5A) synthetases (OAS) and ribonuclease L (RNase L) are components of a potent host antiviral pathway. We previously showed that murine coronavirus (MHV) accessory protein ns2, group A rotavirus (RVA) VP3 carboxy-terminal domain (VP3-CTD), and mammalian AKAP7 are members of 2H phosphoesterase superfamily with 2′-phosphodiesterase (2′-PDE) activity that potently cleaves 2-5A thereby preventing activation of RNase L (Zhao, Jha et al., PMID: 22704621; Zhang, Jha et al., PMID: 23878220 and Gusho, Zhang, Jha et al., PMID: 24987090). Here, we will demonstrate that Middle East respiratory syndrome (MERS)-CoV gene NS4b encodes a homologous and similar PDE that cleaves 2-5A in vitro (km/Kcat = 12.1 M−1 s−1), inhibits 2-5A accumulation in cell culture and prevents ribosomal (r) RNA degradation in murine bone marrow macrophages (BMM), a hallmark of RNase L antagonism, and rescues an MHV mutant virus with a catalytically inactive NS2a protein unable to antagonize RNase L in vivo. Interestingly, NS4b has a nuclear localization signal however there is a mixed nuclear/cytoplasmic localization when overexpressed in human airway cell line A549 and in BMM when expressed from chimeric MHV. Viral evasion of OAS/RNase L pathway is a critical hepatovirulence determinant for lineage A Betacoronavirus mouse hepatitis virus (MHV). Taken together, our data suggest that RNase L antagonism may be a critical component of MERS-CoV pathogenesis. Additionally, this is the first evidence of RNase L antagonism by a lineage C Betacoronavirus.

E. coli 16S Ribosomal RNA Degradation Pathways during Starvation and by Quality Control

Ribosomes are generally stable in growing E. coli cells. However, under conditions of starvation leading to slow cell growth, the need for functional ribosomes decreases and they may become substrates for degradation. In addition, assembling ribosomes that are somehow defective also are eliminated by a quality control process in exponentially growing cells. We examined the pathways of rRNA degradation during starvation and in quality control. During starvation, RNase PH initiates removal of nucleotides from the 3′ end of 16S rRNA until nt 1403. This process leads to an endonucleolytic cleavage by RNase E at position 919. The resulting fragments are further degraded to mononucleotides by RNase R and RNase II. Our findings further suggest that although RNase E makes the internal cut in the quality control process as well, the cutting site is different; moreover, RNase PH plays no role in 16S rRNA degradation in the quality control process demonstrating that the two degradation pathways differ. The pathways also differ in the exoribonucleases which complete the degradation as RNase R and PNPase are required to degrade the intermediate fragments produced in quality control. These differences can be explained as the starting substrates in the quality control pathway are defective ribosomes, whereas in starvation, they are fully assembled, functional particles. These studies provide a detailed description of the pathways of degradation of rRNA during starvation and during steady-state growth due to quality control.

Ribonucleic acids of wheat grain and its endosperm during development and ripening

The work present quantitative changes and synthesis od: RNA fractions in wheat grain and endosperm during development and ripening. It was found that share of rRNA in total RNA decreases during development both, in the endosperm and in grain. Rapid drop of rRNA content in total RNA takes place in the endosperm since 31st day after blooming, and in grain - since 45th day after blooming. Absolute amount of rRNA, as calculated per 100 grains or endosperms, increases in the first half of grain formation period, and then decreases till the end of development. As a result of degradation of ribosomal RNA in endosperm, low-molecule fraction – 4 S, significantly increased at final stage of development. Decrease of rRNA fraction at final stage of development is not accompanied by an increase of absolute amount of 4 S RNA fraction. Incorporation of 3H-,uriidine into grain and endosperm RNA is highest at the begining of development. Further on it gradually decreases till the end of development. Synthesis of RNA in grain takes place throughout the whole development. Contrary to this, no incorporation of precursor into rRNA was noted during waxy and full ripeness of wheat grain.

What caused hemolytic anemia and colicky abdominal pain? Lead!

What caused hemolytic anemia and colicky abdominal pain? Lead!