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Vitamin D is known to affect the functions of pancreatic beta cells, but the effects of vitamin D deficiency on glucoregulatory mechanisms are still inconclusive. The aim of this study was to link vitamin D levels with parameters of insulin resistance and insulin secretion. The study included 70 male and female participants, 40 newly diagnosed patients with type 2 diabetes mellitus (T2DM) and 30 healthy controls. All participants were tested for fasting glucose, hemoglobin A1c, fasting insulin, vitamin D levels, and the HOMA indexes were calculated using HOMA2 calculator. Fasting glucose levels, insulinemia, hemoglobin A1c levels and HOMA IR were all significantly higher in the diabetic group (p

Individual susceptibility to develop acute respiratory distress syndrome is related to age and most frequent comorbidities. So far, it is known that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily infects the type II pneumocytes in humans, with the help of transmembrane serine protease type 2 (TMPRSS2). Up to now, the only known transcriptional promoters of genes coding TMPRSS2 are androgenic. Theoretically, the elevated level of androgens or androgen receptors would lead to a higher expression of TMPRSS2 and a higher level of viremia as a consequence.The aim of our research was to indirectly investigate if the severity of SARS-CoV-2 infection is dependent on the expression of androgen receptors.This observational study analysed male patients hospitalized for SARS-CoV-2 infection with respect to the length of hospitalisation, the outcome of the disease, the type of necessary oxygen support and the presence of comorbidities and hairiness. In hairiness estimation, we used an adapted version of the Hamilton-Norwood scale and the presence of the Gabrin sign.In total, 208 patients were enrolled in the study. There were statistically significant differences comparing the average age of patients with the different types of alopecia when groups were divided according to the presence of the Gabrin sign (t = 4.958, p 0.01). The outcomes and the type of needed minimal oxygen support, compared with the type of alopecia in the case of Gabrin + / - classification showed a statistically significant difference in the outcome of the disease (p = 0.027). There were no statistically significant differences in the distribution of comorbidities among alopecia groups, but hypertension was related to poor COVID-19 prognosis.Our findings suggest that the Gabrin sign and hypertension are related to a poor COVID-19 prognosis.

Introduction. Between 20 - 60% of patients with chronic hepatitis C present with elevated serum ferritin and iron, as well as increased transferrin saturation, yet without a significant liver iron burden. The objective of this article was to summarize current knowledge on interactions between hepatitis C virus infection and iron metabolism. Case Report. A 34-year-old female patient positive for hepatitis C virus genotype 3, underwent therapy of chronic hepatitis C with pegylated interferon/ribavirin. The therapy was discontinued after four weeks due to leukopenia, anemia and significantly increased ferritin levels. Liver biopsy (Perl?s stain) revealed iron deposition predominantly in periportal hepatocytes. The therapy of chronic hepatitis C was continued by direct-acting (sofosbuvir/antiviral/velpatasvir) drugs. During the course of the therapy, the patient presented with normal complete blood count, ferritin of 150 ?g/l, and ferremia at upper limit of normal. Twelve weeks after the end of the therapy, the patient showed a sustained virologic response. The additional diagnostics of hereditary hemochromatosis has been performed, and therapeutic phlebotomy procedures were initiated. Conclusion. In patients with chronic hepatitis C and significantly elevated serum ferritin and iron, the diagnosis should be extended to hereditary hemochromatosis.

Introduction. Timely detection of insulin resistance is of great importance and a number of indices have been developed for its evaluation, among which the homeostasis model assessment of insulin resistance index is the most commonly used in clinical practice. However, it can be calculated via two different models - homeostasis model assessment 1 and homeostasis model assessment 2. Most studies determine the cut-off values of the study population using the homeostasis model assessment 1, while recently most physicians use homeostasis model assessment 2 in everyday clinical practice. The aim of our study was to examine whether there was a difference in the values of homeostasis model assessment of insulin resistance and homeostasis model assessment of panceratic beta cells function calculated using these two models. Material and Methods. Laboratory findings of 42 patients who were diagnosed with glycemia and insulinemia were used in this study. Fasting and postprandial glycemia and insulinemia were used to calculate homeostasis model assessment indices using homeostasis model assessment 1 and homeostasis model assessment 2. Results. When comparing the values of the homeostasis model assessment of insulin resistance and homeostasis model assessment B indices, calculated via homeostasis model assessment 1 and homeostasis model assessment 2, we found a statistically significant difference (p < 0.001) which was also obtained when comparing the values of the homeostasis model assessment B index. Linear correlation analysis showed a significant positive correlation between the measured values of the homeostasis model assessment of insulin resistance (calculated via both models) and postprandial insulinemia at 120 minutes (p < 0.005). Conclusion. The results indicate that homeostasis model assessment 2 yields significantly lower homeostasis model assessment of insulin resistance and homeostasis model assessment B index values than when calculated by the homeostasis model assessment, which may be a stumbling block in the use of homeostasis model assessment index. It is necessary to pay attention which homeostasis model assessment model was used to define the cut-off values of these indices, and to use the same model in the diagnosis of insulin resistance in each patient in everyday clinical practice.

Reliable and reproducible monitoring of the conformational state of therapeutic protein products remains an unmet technological need. This need is amplified by the increasing number of biosimilars entering the drug development pipeline as many branded biologics are reaching the end of their market exclusivity period. Availability of methods to better characterize protein conformation may improve detection of counterfit and unlicensed therapeutic proteins. In this study, we report the use of a set of modified DNA aptamers with enhanced chemical diversity to probe the conformational state of 12 recombinant human erythropoietin (rHuEPO) therapeutic protein products; one FDA-licensed rHuEPO originator biological product, three rHuEPO products that are approved for marketing in the US or EU as biosimilars, and eight rHuEPO products that are not approved for marketing in the US or EU. We show that several of these modified aptamers are able to distinguish rHuEPO reference products or approved biosimilars from non-licensed rHuEPO products on the basis of differences in binding kinetics and equilibrium affinity constants. These reagents exhibit sensitivity to the conformational integrity of various forms of rHuEPO and as such represent powerful, simple-to-use analytical tools to monitor the conformational integrity of therapeutic-proteins during manufacture and to screen for and identify both substandard and counterfeit products.

The modern version of the RNA World Hypothesis begins with activated ribonucleotides condensing (nonenzymatically) to make RNA molecules, some of which possess (perhaps slight) catalytic activity. We propose that noncanonical ribonucleotides, which would have been inevitable under prebiotic conditions, might decrease the RNA length required to have useful catalytic function by allowing short RNAs to possess a more versatile collection of folded motifs. We argue that modified versions of the standard bases, some with features that resemble cofactors, could have facilitated that first moment in which early RNA molecules with catalytic capability began their evolutionary path toward self-replication.

Introduction. Accidental hypothermia is an unintentional fall in core temperature to less than 35°C and it occurs when the body’s heat loss exceeds its heat production. The elderly population is particularly susceptible due to reduced adaptability to low temperatures and various comorbidities, which may be predisposing factors. Case Report. A 65-year-old Caucasian female was admitted to the hospital due to hypothermia (< 34°C), severe hypoglycemia (0.7 mmol/L), metabolic acidosis (6.970), elevated urea (42.4 mmol/L) and creatinine (686 μmol/L). The initial examination revealed shivering, dysarthria and general exhaustion. Hyperamylasemia (241 U/L) and hyperlipasemia (1920 U/L) were found on the second day after admission. Contrast enhanced computed tomography showed a focal asymmetric enlargement of the pancreatic head and body. Furthermore, a splenic infarction was also noticed. The complexity of human thermoregulatory mechanisms indicates the importance of precipitating factors in the development of hypothermia. Our patient presented with several precipitating factors such as age, sex, urinary tract infection, diabetes, and metformin-induced severe hypoglycemia. Conclusion. Based on the presented case and the relevant literature, it can be concluded that the clinical picture of hypothermia may be extremely complex. Knowledge of all the mechanisms involved in multi-organ failure due to hypothermia is of utmost importance, in order to conduct adequate and timely targeted diagnosis and apply appropriate treatment.

Introduction: Many acutely ill children in low- and middle-income settings have a high risk of mortality both during and after hospitalisation despite guideline-based care. Understanding the biological mechanisms underpinning mortality may suggest optimal pathways to target for interventions to further reduce mortality. The Childhood Acute Illness and Nutrition (CHAIN) Network (www.chainnnetwork.org) Nested Case-Cohort Study (CNCC) aims to investigate biological mechanisms leading to inpatient and post-discharge mortality through an integrated multi-omic approach. Methods and analysis; The CNCC comprises a subset of participants from the CHAIN cohort (1278/3101 hospitalised participants, including 350 children who died and 658 survivors, and 270/1140 well community children of similar age and household location) from nine sites in six countries across sub-Saharan Africa and South Asia. Systemic proteome, metabolome, lipidome, lipopolysaccharides, haemoglobin variants, toxins, pathogens, intestinal microbiome and biomarkers of enteropathy will be determined. Computational systems biology analysis will include machine learning and multivariate predictive modelling with stacked generalization approaches accounting for the different characteristics of each biological modality. This systems approach is anticipated to yield mechanistic insights, show interactions and behaviours of the components of biological entities, and help develop interventions to reduce mortality among acutely ill children. Ethics and dissemination. The CHAIN Network cohort and CNCC was approved by institutional review boards of all partner sites. Results will be published in open access, peer reviewed scientific journals and presented to academic and policy stakeholders. Data will be made publicly available, including uploading to recognised omics databases. Trial registration NCT03208725.

Synonymous codons occur with different frequencies in different organisms, a phenomenon termed codon usage bias. Codon optimization, a common term for a variety of approaches used widely by the biopharmaceutical industry, involves synonymous substitutions to increase protein expression. It had long been presumed that synonymous variants, which, by definition, do not alter the primary amino acid sequence, have no effect on protein structure and function. However, a critical mass of reports suggests that synonymous codon variations may impact protein conformation. To investigate the impact of synonymous codons usage on protein expression and function, we designed an optimized coagulation factor IX (FIX) variant and used multiple methods to compare its properties to the wild-type FIX upon expression in HEK293T cells. We found that the two variants differ in their conformation, even when controlling for the difference in expression levels. Using ribosome profiling, we identified robust changes in the translational kinetics of the two variants and were able to identify a region in the gene that may have a role in altering the conformation of the protein. Our data have direct implications for codon optimization strategies, for production of recombinant proteins and gene therapies.

Introduction. Annual and periodized training protocols significantly affect the muscle adaptation in rowers. Considering that the main goal of the training period is increasing specific muscle strength and of detraining period increasing general strength and active rest, the aim of this study was to compare the strength of different muscle groups between training and detraining periods. Material and Methods. The study was conducted at the Department of Physiology, Faculty of Medicine Novi Sad, and it included 34 male and female rowers, 15 to 18 years of age. The muscle strength was measured using a Concept 2 DYNO dynamometer. The strength of the arm extensors and flexors, as well as the leg extensors was measured twice, at the end of the competition season (peak of performance) and before the beginning of the preparation season (after detraining). Results. A statistically significant decrease was found in absolute and relative muscle strength, flexor and arm extensor contraction rate, as well as relative leg extensor strength and contraction rate during the training and detraining periods (p < 0.05). No difference was found in the absolute leg extensor power between the two measurements (p > 0.05). Conclusion. Periodization of the annual training program in rowers has a higher impact on differences in the upper limb muscle adaptation, compared to lower limb muscles in terms of absolute strength.

Purpose To determine, using an aptamer-based technology in patients with intermediate age-related macular degeneration (AMD), (1) if there is a difference in plasma levels of 4979 proteins in patients with and without reticular pseudodrusen (RPD), and (2) if plasma levels of proteins are related to time to conversion to advanced AMD. Methods Patients with intermediate AMD and RPD were identified from an AMD registry. Relative concentrations of each protein were log (base 2) transformed and compared between patients with and without RPD using linear regression. A Cox proportional hazards survival model was fit to each aptamer to quantify associations with time to conversion. A pathway analysis was conducted in converters versus non-converters using the Reactome database. Results Of the 109 intermediate AMD patients, 39 had bilateral RPD (36%). Two proteins, TCL1A and CNDP1, were lower in patients in the intermediate AMD group with RPD. Twenty-one patients converted to advanced AMD with a median time to conversion of 25.2 months (range, 2.3-48.5 months) and median follow-up time in non-converters of 26.4 months (range, 0.03-49.7 months). Several proteins (lysozyme C, TFF3, RNAS6, and SAP3) distinguished patients who converted from those who did not convert to advanced AMD. The top conversion pathways included tumor necrosis factors bind their physiological receptors, digestion and absorption, signaling by activin, and signaling by TGF-β family members. Conclusions We identified a protein signature related to RPD, as well as to conversion to advanced AMD. The pathway analysis suggests that dysfunction of critical systemic pathways may have links to conversion to advanced AMD. Translational relevance Biomarkers identified in plasma likely reflect systemic alterations in protein expression in patients with intermediate AMD.

Genome-wide association studies have identified many individual genetic loci associated with multiple complex traits and common diseases. There are, however, few examples where the molecular basis of such pleiotropy has been elucidated. To address this challenge, we describe an integrative approach, focusing on the p.Ser219Gly (rs867186 A>G) variant in thePROCRgene (encoding the endothelial protein C receptor, EPCR), which has been associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. In a phenome scan of 12 cardiometabolic diseases and 24 molecular factors, we found thatPROCR-219Gly associated with higher plasma levels of zymogenic and activated protein C as well as coagulation factor VII. Using statistical colocalization and Mendelian randomization analyses, we uncovered shared genetic etiology across activated protein C, factor VII, CAD and VTE, identifying p.S219G as the likely causal variant at the locus. In a recall-by-genotype study of 52 healthy volunteers stratified by p.S219G, we detected 2.5-fold higher soluble EPCR levels and 1.2-fold higher protein C levels in plasma per effect allele, suggesting the allele induces EPCR shedding from the membrane of endothelial cells. Finally, in cell adhesion assays, we found that increasing concentrations of activated protein C, but not soluble EPCR, reduced leukocyte–endothelial cell adhesion, a marker for vascular inflammation. These results support a role for protein C as a causal factor in arterial and venous diseases, suggesting thatPROCR-219Gly protects against CAD through anti-inflammatory mechanisms while it promotes VTE risk through pro-thrombotic mechanisms. Overall, our study illustrates a multi-modal approach that can help reveal molecular underpinnings of cross-disease associations.

The pathogenesis of predominantly neurological decompression sickness (DCS) is multifactorial. In SCUBA diving, besides gas bubbles, DCS has been linked to microparticle release, impaired endothelial function, and platelet activation. This study focused on vascular damage and its potential role in the genesis of DCS in breath-hold diving. Eleven breath-hold divers participated in a field study comprising eight deep breath-hold dives with short surface periods and repetitive breath-hold dives lasting for 6 h. Endothelium-dependent vasodilation of the brachial artery, via flow-mediated dilation (FMD), and the number of microparticles (MPs) were assessed before and after each protocol. All measures were analyzed by two-way within-subject ANOVA (2 × 2 ANOVA; factors: time and protocol). Absolute FMD was reduced following both diving protocols (p < 0.001), with no interaction (p = 0.288) or main effect of protocol (p = 0.151). There was a significant difference in the total number of circulating MPs between protocols (p = 0.007), where both increased post-dive (p = 0.012). The number of CD31+/CD41– and CD66b+ MP subtypes, although different between protocols (p < 0.001), also increased by 41.0% ± 56.6% (p = 0.050) and 60.0% ± 53.2% (p = 0.045) following deep and repetitive breath-hold dives, respectively. Both deep and repetitive breath-hold diving lead to endothelial dysfunction that may play an important role in the genesis of neurological DCS.

The addition of novel side chains at the 5-position of uracil is an effective means to increase chemical diversity of aptamers and hence the success rate for discovery of high-affinity ligands to protein targets. Such modifications also increase nuclease resistance, which is useful in a range of applications, especially for therapeutics. In this study, we assess the impact of these side chains on plasma pharmacokinetics of modified aptamers conjugated to a 40 kDa polyethylene glycol. We show that clearance from plasma depends on relative hydrophobicity: side chains with a negative cLogP (more hydrophilic) result in slower plasma clearance compared with side chains with a positive cLogP (more hydrophobic). We show that clearance increases with the number of side chains in sequences of ≥28 synthons, but this effect is dramatically diminished in shorter sequences. These results serve as a guide for the design of new therapeutic aptamers with diversity-enhancing side chains.

Vertebrate organisms express a diversity of protein receptors that recognize and respond to the presence of pathogenic molecules, functioning as an early warning system for infection. As a result of mutation or dysregulated metabolism, these same innate immune receptors can be inappropriately activated, leading to inflammation and disease. One of the most important receptors for detection and response to RNA viruses is called RIG-I, and dysregulation of this protein is linked with a variety of disease states. Despite its central role in inflammatory responses, antagonists for RIG-I are underdeveloped. In this study, we use invitro selection from a pool of modified DNA aptamers to create a high affinity RIG-I antagonist. A high resolution crystal structure of the complex reveals molecular mimicry between the aptamer and the 5′-triphosphate terminus of viral ligands, which bind to the same amino acids within the CTD recognition platform of the RIG-I receptor. Our study suggests a powerful, generalizable strategy for generating immunomodulatory drugs and mechanistic tool compounds.

Direct pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity for Mycobacterium tuberculosis proteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to native M. tuberculosis proteins was confirmed by using M. tuberculosis culture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant ( P < 0.0001) differences in the median M. tuberculosis signals and in specific pathogen markers, such as antigen 85B. Samples where many M. tuberculosis aptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals ( P < 0.0276), particularly in TB patients with HIV coinfection. In conclusion, direct M. tuberculosis antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of M. tuberculosis SOMAmers using other platforms and sample types is warranted.

IL-1α is an essential cytokine that contributes to inflammatory responses and is implicated in various forms of pathogenesis and cancer. Here we report a naphthyl modified DNA aptamer that specifically binds IL-1α and inhibits its signaling pathway. By solving the crystal structure of the IL-1α/aptamer, we provide a high-resolution structure of this critical cytokine and we reveal its functional interaction interface with high-affinity ligands. The non-helical aptamer, which represents a highly compact nucleic acid structure, contains a wealth of new conformational features, including an unknown form of G-quadruplex. The IL-1α/aptamer interface is composed of unusual polar and hydrophobic elements, along with an elaborate hydrogen bonding network that is mediated by sodium ion. IL-1α uses the same interface to interact with both the aptamer and its cognate receptor IL-1RI, thereby suggesting a novel route to immunomodulatory therapeutics., The cytokine interleukin 1α (IL-1α) plays an important role in inflammatory processes. Here the authors use SELEX to generate a modified DNA aptamer which specifically binds IL-1α, present the structure of the IL-1α/aptamer complex and show that this aptamer inhibits the IL-1α signaling pathway.

The tests for diagnosing latent tuberculosis infection (LTBI) are limited by a poor predictive value for identifying people at the highest risk for progressing to active tuberculosis (TB) and have various sensitivities and specificities in different populations. Identifying a more robust signature for LTBI is important for TB prevention and elimination. A pilot study was conducted with samples from immigrants to the United States that were screened for LTBI by the three commercially approved tests, namely, the tuberculin skin test (TST), the Quantiferon-TB Gold in-tube (QFT-GIT), and the T-SPOT.TB (T-SPOT). QFT-GIT supernatants from 13 people with concordant positive results and 26 people with concordant negative results were analyzed via the highly multiplexed SOMAscan proteomic assay. The proteins in the stimulated supernatants that distinguished LTBI from controls included interleukin-2 (IL-2), monocyte chemotactic protein 2 (MCP-2), interferon gamma inducible protein-10 (IP-10), interferon gamma (IFN-γ), tumor necrosis factor superfamily member 14 (TNFSF14, also known as LIGHT), monokine induced by gamma interferon (MIG), and granzyme B ( P P

The mature forms of the TGF-β family members GDF-11 and GDF-8 are highly similar 25 kDa homodimers with 90% amino acid sequence identity and 99% similarity. Cross-reactivity of GDF-11 and GDF-8 binding reagents is common, making it difficult to attribute distinct roles of these two proteins in biology. We report the selection of GDF-11 and GDF-8 specific SOMAmer (Slow Off-rate Modified Aptamer) reagents aided by a combination of positive selection for one protein coupled with counter-selection against the other. We identified GDF-11 specific SOMAmer reagents from four modified DNA libraries that showed a high affinity (Kd range 0.05-1.2 nM) for GDF-11 but did not bind to GDF-8 (Kd > 1 μM). Conversely, we identified one SOMAmer reagent for GDF-8 from one of the modified libraries that demonstrated excellent affinity (Kd = 0.23 nM) and specificity. In contrast, standard protocols that utilized only positive selection produced binding reagents with similar affinity for both proteins. High affinity and specificity were efficiently encoded in minimal sequences of 21 nucleotides for GDF-11 and 24 nucleotides for GDF-8. Further characterization in pull-down, competition, sandwich-binding, and kinetic studies revealed robust binding under a wide range of buffer and assay conditions. For highly similar proteins like GDF-11 and GDF-8, our method of selection coupled with counter-selection was essential for identification of high-affinity, specific reagents that have the potential to elucidate the fundamental distinction of these growth factors in biology.

CRS3123 is a novel small molecule that potently inhibits methionyl-tRNA synthetase of Clostridioides difficile, inhibiting C. difficile toxin production and spore formation. CRS3123 has been evaluated in a multiple-ascending-dose placebo-controlled phase 1 trial., CRS3123 is a novel small molecule that potently inhibits methionyl-tRNA synthetase of Clostridioides difficile, inhibiting C. difficile toxin production and spore formation. CRS3123 has been evaluated in a multiple-ascending-dose placebo-controlled phase 1 trial. Thirty healthy subjects, ages 18 to 45 years, were randomized into three cohorts of 10 subjects each, receiving either 200, 400, or 600 mg of CRS3123 (8 subjects per cohort) or placebo (2 subjects per cohort) by oral administration twice daily for 10 days. CRS3123 was generally safe and well tolerated, with no serious adverse events (SAEs) or severe treatment-emergent adverse events (TEAEs) reported. All subjects completed their assigned treatment and follow-up visits, and there were no trends in systemic, vital sign, or laboratory TEAEs. There were no QTcF interval changes or any clinically significant changes in other electrocardiogram (ECG) intervals or morphology. CRS3123 showed limited but detectable systemic uptake; although absorption increased with increasing dose, the increase was less than dose proportional. Importantly, the bulk of the oral dose was not absorbed, and fecal concentrations were substantially above the MIC90 value of 1 μg/ml at all dosages tested. Subjects receiving either of the two lower doses of CRS3123 exhibited minimal disruption of normal gut microbiota after 10 days of twice-daily dosing. CRS3123 was inactive against important commensal anaerobes, including Bacteroides, bifidobacteria, and commensal clostridia. Microbiome data showed favorable differentiation compared to other CDI therapeutics. These results support further development of CRS3123 as an oral agent for the treatment of CDI. (This study has been registered at Clinicaltrials.gov under identifier NCT02106338.)

Breath-hold diving is a ubiquitous activity, with recreational, fishing, military and competitive divers. During a dive, immersion and the increasing hydrostatic pressure of descent facilitate card...

For any new class of therapeutics, there are certain types of indications that represent a natural fit. For nucleic acid ligands in general, and aptamers in particular, the eye has historically been an attractive site for therapeutic intervention. In this review, we recount the discovery and early development of three aptamers designated for use in ophthalmology, one approved (Macugen), and two in late-stage development (Fovista and Zimura). Every one of these molecules was originally intended for other indications. Key improvements in technology, specifically with regard to libraries used for in vitro selection and subsequent chemical optimization of aptamers, have played an important role in allowing the identification of development candidates with suitable properties. The lessons learned from the selection of these molecules are valuable for informing us about the many remaining opportunities for aptamer-based therapeutics in ophthalmology as well as for identifying additional indications for which aptamers as a class of therapeutics have distinct advantages.

Understanding the structural rules that govern specific, high-affinity binding characteristic of aptamer–protein interactions is important in view of the increasing use of aptamers across many applications. From the modest number of 16 aptamer–protein structures currently available, trends are emerging. The flexible phosphodiester backbone allows folding into precise three-dimensional structures using known nucleic acid motifs as scaffolds that orient specific functional groups for target recognition. Still, completely novel motifs essential for structure and function are found in modified aptamers with diversity-enhancing side chains. Aptamers and antibodies, two classes of macromolecules used as affinity reagents with entirely different backbones and composition, recognize protein epitopes of similar size and with comparably high shape complementarity.

Interleukin-6 (IL-6) is a potent mediator of inflammatory and immune responses, and a validated target for therapeutic intervention of inflammatory diseases. Previous studies have shown that SL1026, a slow off-rate modified aptamer (SOMAmer) antagonist of IL-6, neutralizes IL-6 signaling in vitro. In the present study, we show that SL1026 delays the onset and reduces the severity of rheumatoid symptoms in a collagen-induced arthritis model in cynomolgus monkeys. SL1026 (1 and 10 mg/kg), administered q.i.d., delayed the progression of arthritis and the concomitant increase in serum IL-6 levels compared to the untreated control group. Furthermore, SL1026 inhibited IL-6-induced STAT3 phosphorylation ex vivo in T lymphocytes from human blood and IL-6-induced C-reactive protein and serum amyloid A production in human primary hepatocytes. Importantly, SOMAmer treatment did not elicit an immune response, as evidenced by the absence of anti-SOMAmer antibodies in plasma of treated monkeys. These results demonstrate that SOMAmer antagonists of IL-6 may be attractive agents for the treatment of IL-6-mediated diseases, including rheumatoid arthritis.

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson's disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity-together with the LRR domain-to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.

Although plasma proteins have important roles in biological processes and are the direct targets of many drugs, the genetic factors that control inter-individual variation in plasma protein levels are not well understood. Here we characterize the genetic architecture of the human plasma proteome in healthy blood donors from the INTERVAL study. We identify 1,927 genetic associations with 1,478 proteins, a fourfold increase on existing knowledge, including trans associations for 1,104 proteins. To understand the consequences of perturbations in plasma protein levels, we apply an integrated approach that links genetic variation with biological pathway, disease, and drug databases. We show that protein quantitative trait loci overlap with gene expression quantitative trait loci, as well as with disease-associated loci, and find evidence that protein biomarkers have causal roles in disease using Mendelian randomization analysis. By linking genetic factors to diseases via specific proteins, our analyses highlight potential therapeutic targets, opportunities for matching existing drugs with new disease indications, and potential safety concerns for drugs under development.

Although current implementations of super-resolution microscopy are technically approaching true molecular-scale resolution, this has not translated to imaging of biological specimens, because of the large size of conventional affinity reagents. Here we introduce slow off-rate modified aptamers (SOMAmers) as small and specific labeling reagents for use with DNA points accumulation in nanoscale topography (DNA-PAINT). To demonstrate the achievable resolution, specificity, and multiplexing capability of SOMAmers, we labeled and imaged both transmembrane and intracellular targets in fixed and live cells.

Clostridium difficile causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against C. difficile and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for C. difficile infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.)

Proteins are the primary functional units of biology and the direct targets of most drugs, yet there is limited knowledge of the genetic factors determining inter-individual variation in protein levels. Here we reveal the genetic architecture of the human plasma proteome, testing 10.6 million DNA variants against levels of 2,994 proteins in 3,301 individuals. We identify 1,927 genetic associations with 1,478 proteins, a 4-fold increase on existing knowledge, including trans associations for 1,104 proteins. To understand consequences of perturbations in plasma protein levels, we introduce an approach that links naturally occurring genetic variation with biological, disease, and drug databases. We provide insights into pathogenesis by uncovering the molecular effects of disease-associated variants. We identify causal roles for protein biomarkers in disease through Mendelian randomization analysis. Our results reveal new drug targets, opportunities for matching existing drugs with new disease indications, and potential safety concerns for drugs under development.

The tests for diagnosing latent tuberculosis infection (LTBI) are limited by a poor predictive value for identifying people at the highest risk for progressing to active tuberculosis (TB) and have various sensitivities and specificities in different populations. Identifying a more robust signature for LTBI is important for TB prevention and elimination. A pilot study was conducted with samples from immigrants to the United States that were screened for LTBI by the three commercially approved tests, namely, the tuberculin skin test (TST), the Quantiferon-TB Gold in-tube (QFT-GIT), and the T-SPOT.TB (T-SPOT). QFT-GIT supernatants from 13 people with concordant positive results and 26 people with concordant negative results were analyzed via the highly multiplexed SOMAscan proteomic assay. The proteins in the stimulated supernatants that distinguished LTBI from controls included interleukin-2 (IL-2), monocyte chemotactic protein 2 (MCP-2), interferon gamma inducible protein-10 (IP-10), interferon gamma (IFN-γ), tumor necrosis factor superfamily member 14 (TNFSF14, also known as LIGHT), monokine induced by gamma interferon (MIG), and granzyme B (P

New non-sputum biomarker tests for active tuberculosis (TB) diagnostics are of the highest priority for global TB control. We performed in-depth proteomic analysis using the 4,000-plex SOMAscan assay on 1,470 serum samples from seven countries where TB is endemic. All samples were from patients with symptoms and signs suggestive of active pulmonary TB that were systematically confirmed or ruled out for TB by culture and clinical follow-up. HIV coinfection was present in 34% of samples, and 25% were sputum smear negative. Serum protein biomarkers were identified by stability selection using L1-regularized logistic regression and by Kolmogorov-Smirnov (KS) statistics. A naive Bayes classifier using six host response markers (HR6 model), including SYWC, kallistatin, complement C9, gelsolin, testican-2, and aldolase C, performed well in a training set (area under the sensitivity-specificity curve [AUC] of 0.94) and in a blinded verification set (AUC of 0.92) to distinguish TB and non-TB samples. Differential expression was also highly significant ( P < 10 −20 ) for previously described TB markers, such as IP-10, LBP, FCG3B, and TSP4, and for many novel proteins not previously associated with TB. Proteins with the largest median fold changes were SAA (serum amyloid protein A), NPS-PLA2 (secreted phospholipase A2), and CA6 (carbonic anhydrase 6). Target product profiles (TPPs) for a non-sputum biomarker test to diagnose active TB for treatment initiation (TPP#1) and for a community-based triage or referral test (TPP#2) have been published by the WHO. With 90% sensitivity and 80% specificity, the HR6 model fell short of TPP#1 but reached TPP#2 performance criteria. In conclusion, we identified and validated a six-marker signature for active TB that warrants diagnostic development on a patient-near platform.

The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid–like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications.