Root of the Tree: The Significance, Evolution, and Origins of the Ribosome.

The ribosome is an ancient molecular fossil that provides a telescope to the origins of life. Made from RNA and protein, the ribosome translates mRNA to coded protein in all living systems. Universality, economy, centrality and antiquity are ingrained in translation. The translation machinery dominates the set of genes that are shared as orthologues across the tree of life. The lineage of the translation system defines the universal tree of life. The function of a ribosome is to build ribosomes; to accomplish this task, ribosomes make ribosomal proteins, polymerases, enzymes, and signaling proteins. Every coded protein ever produced by life on Earth has passed through the exit tunnel, which is the birth canal of biology. During the root phase of the tree of life, before the last common ancestor of life (LUCA), exit tunnel evolution is dominant and unremitting. Protein folding coevolved with evolution of the exit tunnel. The ribosome shows that protein folding initiated with intrinsic disorder, supported through a short, primitive exit tunnel. Folding progressed to thermodynamically stable ß-structures and then to kinetically trapped α-structures. The latter were enabled by a long, mature exit tunnel that partially offset the general thermodynamic tendency of all polypeptides to form ß-sheets. RNA chaperoned the evolution of protein folding from the very beginning. The universal common core of the ribosome, with a mass of nearly 2 million Daltons, was finalized by LUCA. The ribosome entered stasis after LUCA and remained in that state for billions of years. Bacterial ribosomes never left stasis. Archaeal ribosomes have remained near stasis, except for the superphylum Asgard, which has accreted rRNA post LUCA. Eukaryotic ribosomes in some lineages appear to be logarithmically accreting rRNA over the last billion years. Ribosomal expansion in Asgard and Eukarya has been incremental and iterative, without substantial remodeling of pre-existing basal structures. The ribosome preserves information on its history.

Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage.

Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and the continued use of Fe2+ by obligate anaerobes inhabiting high Fe2+ niches. Here, we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order in-line rate constant with respect to divalent cations is >200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+ and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because those ions are tightly coordinated by rRNA and deeply buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity.

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

Multiple prebiotic metals mediate translation.

Today, Mg2+ is an essential cofactor with diverse structural and functional roles in life's oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O2, high Fe2+, and high Mn2+) can revert the ribosome to a functional ancestral state. First, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) was used to compare the effect of Mg2+, Fe2+, and Mn2+ on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe2+ or Mn2+ could mediate protein production, and quantified ribosomal metal content. We found that (i) Mg2+, Fe2+, and Mn2+ had strikingly similar effects on rRNA folding; (ii) Fe2+ and Mn2+ can replace Mg2+ as the dominant divalent cation during translation of mRNA to functional protein; and (iii) Fe and Mn associate extensively with the ribosome. Given that the translation system originated and matured when Fe2+ and Mn2+ were abundant, these findings suggest that Fe2+ and Mn2+ played a role in early ribosomal evolution.

Temporal but not spatial dysmetria relates to disease severity in FA.

Friedreich's ataxia (FA) is an inherited disease that causes degeneration of the nervous system. Features of FA include proprioceptive and cerebellar deficits leading to impaired muscle coordination and, consequently, dysmetria in force and time of movement. The aim of this study is to characterize dysmetria and its association to disease severity. Also, we examine the neural mechanisms of dysmetria by quantifying the EMG burst area, duration, and time-to-peak of the agonist muscle. Twenty-seven individuals with FA and 13 healthy controls (HCs) performed the modified Functional Ataxia Rating Scale and goal-directed movements with the ankle. Dysmetria was quantified as position and time error during dorsiflexion. FA individuals exhibited greater time but not position error than HCs. Moreover, time error correlated with disease severity and was related to increased agonist EMG burst. Temporal dysmetria is associated to disease severity, likely due to altered activation of the agonist muscle.NEW & NOTEWORTHY For the first time, we quantified spatial and temporal dysmetria and its relation to disease severity in Friedreich's ataxia (FA). We found that FA individuals exhibit temporal but not spatial dysmetria relative to healthy controls. Temporal dysmetria correlated to disease severity in FA and was predicted from an altered activation of the agonist muscle. Therefore, these results provide novel evidence that FA exhibit temporal but not spatial dysmetria, which is different from previous findings on SCA6.

Improving Incidental Finding Documentation in Trauma Patients Amidst Poor Access to Follow-up Care.

BACKGROUND: Incidental findings (IFs) are common among injured patients and create a complex problem with no standardized solution. MATERIALS AND METHODS: This is a retrospective review of adult trauma patients admitted to a level I trauma center from January to May 2017. IFs from abdominal, chest, and neck imaging were categorized based on previously published guidelines focused on clinically significant IFs. Patient demographics related to access to care were collected. Outcome measures included documentation and patient notification of IFs. A univariate analysis was performed to identify characteristics that were associated with these outcomes. RESULTS: Of 1671 patients, 682 met inclusion criteria, and 418 (61.3%) had any IF based on the a priori categorization scheme. In total, 67 (9.8%) were homeless, 58 (8.5%) had no health insurance, and 115 (16.9%) had no established primary care provider prior to admission. Documentation of IFs was included in discharge summaries and instructions 76.5% and 40.2% of the time, respectively. Physicians were statistically more likely to appropriately document IFs when radiologists provided specific recommendations. Transfer to another hospital service prior to discharge and discharge to another acute care facility were associated with reduced rates of successful documentation. No factors significantly affected documentation of patient notification. CONCLUSIONS: Trauma patients are at risk for poor access to follow-up care of IFs. Expanding IF-specific guidelines, collaborating with radiologists to facilitate their inclusion in reports, and ensuring that IFs are part of patient hand-offs could provide systematic methods of improving their documentation.

Older Adults With Isolated Rib Fractures Do Not Require Routine Intensive Care Unit Admission.

BACKGROUND: Older adults with isolated rib fractures are often admitted to an intensive care unit (ICU) because of presumedly increased morbidity and mortality. However, evidence-based guidelines are limited. We sought to identify characteristics of these patients that predict the need for ICU care. MATERIALS AND METHODS: We analyzed patients ≥50 y old at our center during 2013-2017 whose only indication for ICU admission, if any, was isolated rib fractures. The primary outcome was any critical care intervention (e.g., intubation) or adverse event (e.g., hypoxemia) (CCIE) based on accepted critical care guidelines. We used stepwise logistic regression to identify characteristics that predict CCIEs. RESULTS: Among 401 patients, 251 (63%) were admitted to an ICU. Eighty-three patients (33%) admitted to an ICU and 7 (5%) admitted to the ward experienced a CCIE. The most common CCIEs were hypotension (10%), frequent respiratory therapy (9%), and oxygen desaturation (8%). Predictors of CCIEs included incentive spirometry <1 L (OR 4.72, 95% CI 2.14-10.45); use of a walker (OR 2.86, 95% CI 1.29-6.34); increased chest Abbreviated Injury Scale score (AIS 3 OR 5.83, 95% CI 2.34-14.50); age ≥72 y (OR 2.68, 95% CI 1.48-4.86); and active smoking (OR 2.11, 95% CI 1.06-4.20). CONCLUSIONS: Routine ICU admission is not necessary for most older adults with isolated rib fractures. The predictors we identified warrant prospective evaluation for development of a clinical decision rule to preclude unnecessary ICU admissions.

A Rare Case of an Intracardiac Myoepithelial Carcinoma in an Infant.

Myoepithelial carcinomas (MC) represent aggressive tumors that occur in a myriad of ages and anatomic locations. The rarity and histologic similarity with other tumors make them difficult to diagnosis. We report an extremely rare case of a right ventricular outflow tract mass identified to be an intracardiac MC in a 4-month-old male infant. Pathology revealed an EWS-KLF15 translocation. Treatment included gross total resection and intensive chemotherapy. Recurrent cardiac mass with brain metastasis was seen 16 months after primary diagnosis. We describe the rarity of intracardiac MC in pediatric patients and the challenges encountered in the multimodal management of this patient.

Functional RNAs: combined assembly and packaging in VLPs.

We describe here a one pot RNA production, packaging and delivery system based on bacteriophage Qß. We demonstrate a method for production of a novel RNAi scaffold, packaged within Qß virus-like particles (VLPs). The RNAi scaffold is a general utility chimera that contains a functional RNA duplex with paired silencing and carrier sequences stabilized by a miR-30 stem-loop. The Qß hairpin on the 5΄ end confers affinity for the Qß coat protein (CP). Silencing sequences can include mature miRNAs and siRNAs, and can target essentially any desired mRNA. The VLP-RNAi assembles upon co-expression of CP and the RNAi scaffold in E. coli. The annealing of the scaffold to form functional RNAs is intramolecular and is therefore robust and concentration independent. We demonstrate dose- and time-dependent inhibition of GFP expression in human cells with VLP-RNAi. In addition, we target the 3΄UTR of oncogenic Ras mRNA and suppress Pan-Ras expression, which attenuates cell proliferation and promotes mortality of brain tumor cells. This combination of RNAi scaffold design with Qß VLP packaging is demonstrated to be target-specific and efficient.

Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event.

Life originated in an anoxic, Fe2+-rich environment. We hypothesize that on early Earth, Fe2+ was a ubiquitous cofactor for nucleic acids, with roles in RNA folding and catalysis as well as in processing of nucleic acids by protein enzymes. In this model, Mg2+ replaced Fe2+ as the primary cofactor for nucleic acids in parallel with known metal substitutions of metalloproteins, driven by the Great Oxidation Event. To test predictions of this model, we assay the ability of nucleic acid processing enzymes, including a DNA polymerase, an RNA polymerase and a DNA ligase, to use Fe2+ in place of Mg2+ as a cofactor during catalysis. Results show that Fe2+ can indeed substitute for Mg2+ in catalytic function of these enzymes. Additionally, we use calculations to unravel differences in energetics, structures and reactivities of relevant Mg2+ and Fe2+ complexes. Computation explains why Fe2+ can be a more potent cofactor than Mg2+ in a variety of folding and catalytic functions. We propose that the rise of O2 on Earth drove a Fe2+ to Mg2+ substitution in proteins and nucleic acids, a hypothesis consistent with a general model in which some modern biochemical systems retain latent abilities to revert to primordial Fe2+-based states when exposed to pre-GOE conditions.

A genome-wide association study of congenital cardiovascular left-sided lesions shows association with a locus on chromosome 20.

Congenital heart defects involving left-sided lesions (LSLs) are relatively common birth defects with substantial morbidity and mortality. Previous studies have suggested a high heritability with a complex genetic architecture, such that only a few LSL loci have been identified. We performed a genome-wide case-control association study to address the role of common variants using a discovery cohort of 778 cases and 2756 controls. We identified a genome-wide significant association mapping to a 200 kb region on chromosome 20q11 [P= 1.72 × 10-8 for rs3746446; imputed Single Nucleotide Polymorphism (SNP) rs6088703 P= 3.01 × 10-9, odds ratio (OR)= 1.6 for both]. This result was supported by transmission disequilibrium analyses using a subset of 541 case families (lowest P in region= 4.51 × 10-5, OR= 1.5). Replication in a cohort of 367 LSL cases and 5159 controls showed nominal association (P= 0.03 for rs3746446) resulting in P= 9.49 × 10-9 for rs3746446 upon meta-analysis of the combined cohorts. In addition, a group of seven SNPs on chromosome 1q21.3 met threshold for suggestive association (lowest P= 9.35 × 10-7 for rs12045807). Both regions include genes involved in cardiac development-MYH7B/miR499A on chromosome 20 and CTSK, CTSS and ARNT on chromosome 1. Genome-wide heritability analysis using case-control genotyped SNPs suggested that the mean heritability of LSLs attributable to common variants is moderately high ([Formula: see text] range= 0.26-0.34) and consistent with previous assertions. These results provide evidence for the role of common variation in LSLs, proffer new genes as potential biological candidates, and give further insight to the complex genetic architecture of congenital heart disease.

Folding, Assembly, and Persistence: The Essential Nature and Origins of Biopolymers.

Life as we know it requires three basic types of polymers: polypeptide, polynucleotide, and polysaccharide. Here we evaluate both universal and idiosyncratic characteristics of these biopolymers. We incorporate this information into a model that explains much about their origins, selection, and early evolution. We observe that all three biopolymer types are pre-organized, conditionally self-complementary, chemically unstable in aqueous media yet persistent because of kinetic trapping, with chiral monomers and directional chains. All three biopolymers are synthesized by dehydration reactions that are catalyzed by molecular motors driven by hydrolysis of phosphorylated nucleosides. All three biopolymers can access specific states that protect against hydrolysis. These protected states are folded, using self-complementary interactions among recurrent folding elements within a given biopolymer, or assembled, in associations between the same or different biopolymer types. Self-association in a hydrolytic environment achieves self-preservation. Heterogeneous association achieves partner-preservation. These universal properties support a model in which life's polymers emerged simultaneously and co-evolved in a common hydrolytic milieu where molecular persistence depended on folding and assembly. We believe that an understanding of the structure, function, and origins of any given type of biopolymer requires the context of other biopolymers.

Assessment of large copy number variants in patients with apparently isolated congenital left-sided cardiac lesions reveals clinically relevant genomic events.

Congenital left-sided cardiac lesions (LSLs) are a significant contributor to the mortality and morbidity of congenital heart disease (CHD). Structural copy number variants (CNVs) have been implicated in LSL without extra-cardiac features; however, non-penetrance and variable expressivity have created uncertainty over the use of CNV analyses in such patients. High-density SNP microarray genotyping data were used to infer large, likely-pathogenic, autosomal CNVs in a cohort of 1,139 probands with LSL and their families. CNVs were molecularly confirmed and the medical records of individual carriers reviewed. The gene content of novel CNVs was then compared with public CNV data from CHD patients. Large CNVs (>1 MB) were observed in 33 probands (∼3%). Six of these were de novo and 14 were not observed in the only available parent sample. Associated cardiac phenotypes spanned a broad spectrum without clear predilection. Candidate CNVs were largely non-recurrent, associated with heterozygous loss of copy number, and overlapped known CHD genomic regions. Novel CNV regions were enriched for cardiac development genes, including seven that have not been previously associated with human CHD. CNV analysis can be a clinically useful and molecularly informative tool in LSLs without obvious extra-cardiac defects, and may identify a clinically relevant genomic disorder in a small but important proportion of these individuals.

A group-mediated physical activity intervention in older knee osteoarthritis patients: effects on social cognitive outcomes.

The objective of the present study was to compare a group-mediated cognitive behavioral (GMCB) physical activity intervention with traditional exercise therapy (TRAD) upon select social cognitive outcomes in sedentary knee osteoarthritis (knee OA) patients. A total of 80 patients (mean age = 63.5 years; 84% women) were recruited using clinic and community-based strategies to a 12-month, single-blind, two-arm, randomized controlled trial. Mobility-related self-efficacy, self-regulatory self-efficacy (SRSE), and satisfaction with physical function (SPF) were assessed at baseline, 3, and 12 months. Results of intent-to-treat 2 (Treatment: GMCB and TRAD) × 2 (Time: 3 and 12 month) analyses of covariance yielded significantly greater increases in SRSE and SPF (P < 0.01) relative to TRAD. Partial correlations revealed that changes in SRSE and SPF were significantly related (P < 0.05) to improvements in physical activity and mobility at 3 and 12-months. The GMCB intervention yielded more favorable effects on important social cognitive outcomes than TRAD; these effects were related to improvements in physical activity and mobility.

Abnormal Longitudinal Growth of the Aorta in Children with Familial Bicuspid Aortic Valve.

Bicuspid aortic valve (BAV) is the most common type of congenital heart defect (CHD) and is associated with clinically significant cardiovascular complications including valve calcification and ascending aortopathy (AscAo), predominantly occurring in adulthood. While a limited number of genetic etiologies for BAV have been defined, family members of affected individuals display BAV along with other left-sided CHD. This has led to guidelines from the American Heart Association and American College of Cardiology that recommend echocardiographic screening of first-degree relatives of affected adults. While potentially beneficial in adults, the yield of such screening in children is unknown. The purpose of this study was to investigate a cohort of children with familial BAV to determine the frequency of development of AscAo, and to identify risk factors that contribute to abnormal aortic growth. Echocardiograms over a 10-year follow-up period were reviewed on 26 patients with familial BAV [22 male, 4 female; 22 with isolated BAV, 6 with BAV and aortic coarctation (CoA)]. All had a family history of CHD and were recruited from 2005 to 2010 as part of a genetics research study. Four aortic segments (annulus, root, sinotubular junction, ascending aorta) on parasternal long-axis echocardiographic images were measured by a single observer. The mean age at first echocardiogram was 7.1 ± 5.5 and that was 13.8 ± 6.2 years at the last echocardiogram. Only patients with > 2 echocardiograms in the 10-year period were included. Z score measurements of the aorta were plotted over time and based on these the cohort was divided into two groups: Group 1 (abnormal)-Z score for any segment > 2 or a change in Z score > 2 over follow-up; Group 2 (normal)-Z score < 2 throughout follow-up and change in Z score < 2. Nineteen out of 26 children displayed abnormal aortic growth or dilation of the aorta. BAV with right/left cusp fusion was more frequent in Group 1 (15/18) versus Group 2 (3/7) (p < 0.05). There were no significant differences in gender, aortic valve dysfunction, presence of CoA, family history, cardiac function, presence of left ventricular hypertrophy, or medication use between the 2 groups. In our longitudinal study of children with familial BAV, the majority display evidence of abnormal growth of the ascending aorta during the follow-up period consistent with AscAo and support the extension of current adult guidelines to the pediatric population. While we find that right/left cusp fusion is a risk factor for abnormal aortic growth, additional studies are needed to identify other factors to better select children who require serial screening.

The ribosome challenge to the RNA world.

An RNA World that predated the modern world of polypeptide and polynucleotide is one of the most widely accepted models in origin of life research. In this model, the translation system shepherded the RNA World into the extant biology of DNA, RNA, and protein. Here, we examine the RNA World Hypothesis in the context of increasingly detailed information available about the origins, evolution, functions, and mechanisms of the translation system. We conclude that the translation system presents critical challenges to RNA World Hypotheses. Firstly, a timeline of the RNA World is problematic when the ribosome is incorporated. The mechanism of peptidyl transfer of the ribosome appears distinct from evolved enzymes, signaling origins in a chemical rather than biological milieu. Secondly, we have no evidence that the basic biochemical toolset of life is subject to substantive change by Darwinian evolution, as required for the transition from the RNA world to extant biology. Thirdly, we do not see specific evidence for biological takeover of ribozyme function by protein enzymes. Finally, we can find no basis for preservation of the ribosome as ribozyme or the universality of translation, if it were the case that other information transducing ribozymes, such as ribozyme polymerases, were replaced by protein analogs and erased from the phylogenetic record. We suggest that an updated model of the RNA World should address the current state of knowledge of the translation system.

Molecular paleontology: a biochemical model of the ancestral ribosome.

Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.

Antiapicoplast and gametocytocidal screening to identify the mechanisms of action of compounds within the malaria box.

Malaria remains a significant infectious disease that causes millions of clinical cases and >800,000 deaths per year. The Malaria Box is a collection of 400 commercially available chemical entities that have antimalarial activity. The collection contains 200 drug-like compounds, based on their oral absorption and the presence of known toxicophores, and 200 probe-like compounds, which are intended to represent a broad structural diversity. These compounds have confirmed activities against the asexual intraerythrocytic stages of Plasmodium falciparum and low cytotoxicities, but their mechanisms of action and their activities in other stages of the parasite's life cycle remain to be determined. The apicoplast is considered to be a promising source of malaria-specific targets, and its main function during intraerythrocytic stages is to provide the isoprenoid precursor isopentenyl diphosphate, which can be used for phenotype-based screens to identify compounds targeting this organelle. We screened 400 compounds from the Malaria Box using apicoplast-targeting phenotypic assays to identify their potential mechanisms of action. We identified one compound that specifically targeted the apicoplast. Further analyses indicated that the molecular target of this compound may differ from those of the current antiapicoplast drugs, such as fosmidomycin. Moreover, in our efforts to elucidate the mechanisms of action of compounds from the Malaria Box, we evaluated their activities against other stages of the life cycle of the parasite. Gametocytes are the transmission stage of the malaria parasite and are recognized as a priority target in efforts to eradicate malaria. We identified 12 compounds that were active against gametocytes with 50% inhibitory concentration values of <1 µM.

Domain III of the T. thermophilus 23S rRNA folds independently to a near-native state.

The three-dimensional structure of the ribosomal large subunit (LSU) reveals a single morphological element, although the 23S rRNA is contained in six secondary structure domains. Based upon maps of inter- and intra-domain interactions and proposed evolutionary pathways of development, we hypothesize that Domain III is a truly independent structural domain of the LSU. Domain III is primarily stabilized by intra-domain interactions, negligibly perturbed by inter-domain interactions, and is not penetrated by ribosomal proteins or other rRNA. We have probed the structure of Domain III rRNA alone and when contained within the intact 23S rRNA using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension), in the absence and presence of magnesium. The combined results support the hypothesis that Domain III alone folds to a near-native state with secondary structure, intra-domain tertiary interactions, and inter-domain interactions that are independent of whether or not it is embedded in the intact 23S rRNA or within the LSU. The data presented support previous suggestions that Domain III was added relatively late in ribosomal evolution.

Isolation of antiplasmodial anthraquinones from Kniphofia ensifolia, and synthesis and structure-activity relationships of related compounds.

Bioassay-guided separation of the South African plant Kniphofia ensifolia for antiplasmodial activity led to the isolation of two new anthraquinones, named kniphofiones A and B (3 and 4), together with three known bioactive anthraquinone monomers (1, 2 and 5), and four known bisanthraquinones (6-9). The structures of the two new compounds were elucidated based on analyses of their 1D and 2D NMR spectra and mass spectrometric data. The dimeric compounds 6 and 7 displayed the strongest antiplasmodial activity among all the isolated compounds, with IC50 values of 0.4 ± 0.1 and 0.2 ± 0.1 µM, respectively. The two new compounds displayed modest activities, with IC50 values of 26 ± 4 and 9 ± 1 µM, respectively. Due to the synthetic accessibility of the new compounds and the increased activity shown by the dimeric compounds, a structure-activity relationship study was conducted. As a result, one analogue of kniphofione B (4), the caffeic acid derivative of aloe-emodin, was found to have the highest activity among all the aloe-emodin derivatives, with an IC50 value of 1.3 ± 0.2 µM.