Real-World Pitfalls of Analyzing Real-World Data: A Cautionary Note and Path Forward.

PURPOSE: Real-world data (RWD) are pervasive in oncology research and offer insights into clinical trends and patient outcomes. However, RWD have shortcomings, making them prone to pitfalls during survival analyses. The American Society of Clinical Oncology (ASCO) CancerLinQ Discovery (CLQD) multiple myeloma (MM) data set was used to demonstrate some common pitfalls when analyzing survival from RWD: using incorrect surrogate markers for missing data and/or classification errors, ignoring deaths at time zero, and failing to account for guarantee-time bias. METHODS: The ASCO CLQD MM data set (July 19, 2021, release) was used to compare overall survival (OS) in patients with a known versus presumed date of MM diagnosis, in patients with secondary AML (sAML) with early deaths (ie, 0 months) included versus dropped, and in patients with second primary malignancies (SPMs) matched versus unmatched to control for time-related confounding factors (ie, guarantee-time bias). Analyses were conducted using STATA Version 17.0 (College Station, TX). RESULTS: In the CLQD MM data set, 28% of patients were missing a diagnosis date. Attempts to use the presumed diagnosis date (ie, first bortezomib or lenalidomide administration) as a surrogate marker for missing diagnosis dates were not successful as median OS was significantly different in patients with a recorded versus presumed diagnosis date (107 v 40 months, hazard ratio [HR], 2.5; 95% CI, 2.39 to 2.64; P < .001). Dropping deaths within 1 month of sAML diagnosis resulted in an exaggerated median OS (46 v 39 months). OS in patients with MM with SPMs differed substantially before and after incorporation of matching methods to account for guarantee-time bias (HR, 0.73; 95% CI, 0.67 to 0.78; P < .001 before matching, HR, 1.30; 95% CI, 1.18 to 1.43; P < .001 after matching). CONCLUSION: To fully maximize the benefits of RWD in oncology research, clinicians must be aware of analytic methods that can overcome pitfalls in survival analyses.

Identification of a positively charged platform in Staphylococcus aureus HtsA that is essential for ferric staphyloferrin A transport.

In response to iron starvation, Staphylococcus aureus secretes both staphyloferrin A and staphyloferrin B, which are high-affinity iron-chelating molecules. The structures of both HtsA and SirA, the ferric-staphyloferrin A [Fe(III)-SA] and ferric-staphyloferrin B [Fe(III)-SB] receptors, respectively, have recently been determined. The structure of HtsA identifies a novel form of ligand entrapment composed of many positively charged residues. Through ionic interactions, the binding pocket appears highly adapted for the binding of the highly anionic siderophore SA. However, biological validation of the importance of the nine SA-interacting residues (six arginines, one tyrosine, one histidine, and one lysine) has not been previously performed. Here, we mutated each of the Fe(III)-SA-interacting residues in HtsA and found that substitutions R104A, R126A, H209A, R306A, and R306K resulted in a reduction of binding affinity of HtsA for Fe(III)-SA. While mutation of almost all proposed ligand-interacting residues decreased the ability of S. aureus cells to transport (55)Fe(III)-SA, S. aureus expressing HtsA R104A, R126A, R306A, and R306K showed the greatest transport defects and were incapable of growth in iron-restricted growth media in a SA-dependent manner. These three residues cluster together and, relative to other residues in the binding pocket, move very little between the apo and closed holo structures. Their essentiality for receptor function, together with structural information, suggests that they form a positively charged platform that is required for initial contact with the terminal carboxyl groups of the two citrates in the Fe(III)-SA complex. This is a likely mechanism by which HtsA discerns iron-bound SA from iron-free SA.

The Staphylococcus aureus siderophore receptor HtsA undergoes localized conformational changes to enclose staphyloferrin A in an arginine-rich binding pocket.

Staphylococcus aureus uses several efficient iron acquisition strategies to overcome iron limitation. Recently, the genetic locus encoding biosynthetic enzymes for the iron chelating molecule, staphyloferrin A (SA), was determined. S. aureus synthesizes and secretes SA into its environment to scavenge iron. The membrane-anchored ATP binding cassette-binding protein, HtsA, receives the ferric-chelate for import into the cell. Recently, we determined the apoHtsA crystal structure, the first siderophore receptor from gram-positive bacteria to be structurally characterized. Herein we present the x-ray crystal structure of the HtsA-ferric-SA complex. HtsA adopts a class III binding protein fold composed of separate N- and C-terminal domains bridged by a single alpha-helix. Recombinant HtsA can efficiently sequester ferric-SA from S. aureus culture supernatants where it is bound within the pocket formed between distinct N- and C-terminal domains. A basic patch composed mainly of six Arg residues contact the negatively charged siderophore, securing it within the pocket. The x-ray crystal structures from two different ligand-bound crystal forms were determined. The structures represent the first structural characterization of an endogenous alpha-hydroxycarboxylate-type siderophore-receptor complex. One structure is in an open form similar to apoHtsA, whereas the other is in a more closed conformation. The conformational change is highlighted by isolated movement of three loops within the C-terminal domain, a domain movement unique to known class III binding protein structures.

Tall fescue genomic SSR markers: development and transferability across multiple grass species.

Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies. Currently, a very limited number of SSR markers are available for tall fescue (Festuca arundinacea Schreb.) and other forage grass species. A tall fescue genomic library enriched in (GA/CT)( n ) repeats was used to develop primer pairs (PPs) flanking SSRs and assess PP functionality across different forage, cereal, and turf grass species. A total of 511 PPs were developed and assessed for their utility in six different grass species. The parents and a subset of a tall fescue mapping population were used to select PPs for mapping in tall fescue. Survey results revealed that 48% (in rice) to 66% (in tall fescue) of the PPs produced clean SSR-type amplification products in different grass species. Polymorphism rates were higher in tall fescue (68%) compared to other species (46% ryegrass, 39% wheat, and 34% rice). A set of 194 SSR loci (38%) were identified which amplified across all six species. Loci segregating in the tall fescue mapping population were grouped as loci segregating from the female parent (HD28-56, 37%), the male parent (R43-64, 37%), and both parents (26%). Three percent of the loci that were polymorphic between parents were monomorphic in the pseudo F1 mapping population and the remaining loci segregated. Sequencing of amplified products obtained from PP NFFAG428 revealed a very high level of sequence similarity among the grass species under study. Our results are the first report of genomic SSR marker development from tall fescue and they demonstrate the usefulness of these SSRs for genetic linkage mapping in tall fescue and cross-species amplification.

Molecular and biochemical analysis of two cDNA clones encoding dihydroflavonol-4-reductase from Medicago truncatula.

Dihydroflavonol-4-reductase (DFR; EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Two DFR cDNA clones (MtDFR1 and MtDFR2) were isolated from the model legume Medicago truncatula cv Jemalong. Both clones were functionally expressed in Escherichia coli, confirming that both encode active DFR proteins that readily reduce taxifolin (dihydroquercetin) to leucocyanidin. M. truncatula leaf anthocyanins were shown to be cyanidin-glucoside derivatives, and the seed coat proanthocyanidins are known catechin and epicatechin derivatives, all biosynthesized from leucocyanidin. Despite high amino acid similarity (79% identical), the recombinant DFR proteins exhibited differing pH and temperature profiles and differing relative substrate preferences. Although no pelargonidin derivatives were identified in M. truncatula, MtDFR1 readily reduced dihydrokaempferol, consistent with the presence of an asparagine residue at a location known to determine substrate specificity in other DFRs, whereas MtDFR2 contained an aspartate residue at the same site and was only marginally active on dihydrokaempferol. Both recombinant DFR proteins very efficiently reduced 5-deoxydihydroflavonol substrates fustin and dihydrorobinetin, substances not previously reported as constituents of M. truncatula. Transcript accumulation for both genes was highest in young seeds and flowers, consistent with accumulation of condensed tannins and leucoanthocyanidins in these tissues. MtDFR1 transcript levels in developing leaves closely paralleled leaf anthocyanin accumulation. Overexpression of MtDFR1 in transgenic tobacco (Nicotiana tabacum) resulted in visible increases in anthocyanin accumulation in flowers, whereas MtDFR2 did not. The data reveal unexpected properties and differences in two DFR proteins from a single species.

Trafficking the NGF signal: implications for normal and degenerating neurons.

Nerve growth factor (NGF) activates TrkA to trigger signaling events that promote the survival, differentiation and maintenance of neurons. The mechanism(s) that controls the retrograde transport of the NGF signal from axon terminals to neuron cell bodies is not known. The 'signaling endosome' hypothesis stipulates that NGF, TrkA and signaling proteins are retrogradely transported on endocytic vesicles. Here, we provide evidence for the existence of signaling endosomes. Following NGF treatment, clathrin-coated vesicles (CCVs) contain NGF bound to TrkA together with activated signaling proteins of the Ras/pErk1/2 pathway. NGF signals from isolated CCVs through the Erk1/2 pathway. Early endosomes appear to represent a second type of signaling endosomes. We found that NGF induced a sustained activation of Rap1, a small monomeric GTP-binding protein of the Ras family, and that this activation occurred in early endosomes that contain key elements of Rap1/pErk1/2 pathway. We discuss the possibility that the failure of retrograde NGF signaling in a mouse model of Down syndrome (Ts65Dn) may be due to the failure to retrograde transport signaling endosomes. It is important to define further the significance of signaling endosomes in the biology of both normal and degenerating neurons.

Profiling isoflavonoids found in legume root extracts using capillary electrophoresis.

Legumes such as alfalfa (Medicago sativa L.), barrel medic (Medicago truncatula), white sweet clover (Melilotus alba) and fenugreek (Trigonella graecum), normally accumulate (-)-medicarpin and its malonated glucose conjugate as natural inhibitors of fungal pathogens. These plants also accumulate the biosynthetic precursor formononetin as well as the malonated glycoside. We were interested in developing a robust high-throughput method to quantitate the levels of these two isoflavonoids, both free and conjugated, in legume root extracts, for use in screening for mutant plants accumulating altered levels of these compounds. Capillary electrophoresis was examined as an alternative to current high-performance liquid chromatography (HPLC) methods to generate isoflavonoid profiles. The developed assay used micellar electrokinetic capillary chromatography (MEKC) to provide the required selectivity in complex root extracts. The addition of 1,2-hexanediol to the sodium dodecyl sulfate (SDS) electrolyte provided improved resolution of adjacent isoflavonoids. We examined the role of several factors including sample preparation, buffer composition, buffer pH, and organic component in the injected sample. The use of capillaries with longer path lengths were also examined to increase sensitivity. A comparison of results obtained using MEKC and HPLC showed good correlation in the relative amounts of the isoflavonoids studied.