Directed Evolution of Aptamer Discovery Technologies

Although antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of non-nattural aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

The Anti-Bureaucratic Ghost in China's Bureaucratic Machine

The Chinese Communist Party's (CCP) ideology, rooted in its foundational struggles, explicitly denounces “bureaucratism” (guanliaozhuyi) as an intrinsic ailment of bureaucracy. Yet while the revolutionary Party has blasted bureaucratism, its revolutionary regime has had to find a way to coexist with bureaucracy, which is a requisite for effective governance. An anti-bureaucratic ghost thus dwells in the machinery of China's bureaucratic state. We analyse the CCP's anti-bureaucratism through two steps. First, we perform a historical analysis of the Party's anti-bureaucratic ideology, teasing out its substance and emphasizing its roots in and departures from European Marxism and Leninism. Second, we trace both the continuity and evolution in the Party's anti-bureaucratic rhetoric, taking an interactive approach that combines close reading with computational analysis of the entire corpus of the People's Daily (1947–2020). We find striking endurance as well as subtle shifts in the substance of the CCP's anti-bureaucratic ideology. We show that bureaucratism is an umbrella term that expresses the revolutionary Party's anxiety about losing its popular legitimacy. Yet the substance of the Party's concern evolved from commandism and revisionism under Mao, to corruption and formalism during reform. The Party's ongoing critiques of bureaucratism and formalism unfold in parallel fashion with its efforts to standardize, regularize and institutionalize the state.

San’ani Arabic Stress in Harmonic Serialism

The word stress system in San’ani Arabic exhibits patterns of stress placement that associate some level of prominence with syllables with long vowels and syllables that end in the left-leg of a geminate. The fact that such syllables always succeed in attracting stress away from other non-final CVC syllables, even beyond the final trisyllabic window, clearly indicates the role that underlying moraicity plays in the stress algorithm. The proposed account, offered in this paper for the word stress system in San’ani, is couched in Harmonic Serialism, as a serial version of Optimality Theory. Key to the analyses presented is the assumption of gradual prosodification. The distinction drawn between faithful and unfaithful prosodic operations allows for applying some in a parallel fashion, but confines others to serialism. Central to the analysis, as well, is the exceptional case of final stress, which is mainly attributed to the intrinsic prominence of syllables with underlying bimoraic sequences.

Signal Classification Algorithms over Time Selective Channels

In this work, we propose a general framework to design a signal classification algorithm over time selective channels for wireless communications applications. We derive an upper bound on the maximum number of observation samples over which the channel response is an essential invariant. The proposed framework relies on dividing the received signal into blocks, and each of them has a length less than the mentioned bound. Then, these blocks are fed into a number of classifiers in a parallel fashion. A final decision is made through a well-designed combiner and detector. As a case study, we employ the proposed framework on a space-time block-code classification problem by developing two combiners and detectors. Monte Carlo simulations show that the proposed framework is capable of achieving excellent classification performance over time selective channels compared to the conventional algorithms.

An Analytical Review of the Structural Features of Pentatricopeptide Repeats: Strategic Amino Acids, Repeat Arrangements and Superhelical Architecture

Tricopeptide repeats are common in natural proteins, and are exemplified by 34- and 35-residue repeats, known respectively as tetratricopeptide repeats (TPRs) and pentatricopeptide repeats (PPRs). In both classes, each repeat unit forms an antiparallel bihelical structure, so that multiple such units in a polypeptide are arranged in a parallel fashion. The primary structures of the motifs are nonidentical, but amino acids of similar properties occur in strategic positions. The focus of the present work was on PPR, but TPR, its better-studied cousin, is often included for comparison. The analyses revealed that critical amino acids, namely Gly, Pro, Ala and Trp, were placed at distinct locations in the higher order structure of PPR domains. While most TPRs occur in repeats of three, the PPRs exhibited a much greater diversity in repeat numbers, from 1 to 30 or more, separated by spacers of various sequences and lengths. Studies of PPR strings in proteins showed that the majority of PPR units are single, and that the longer tandems (i.e., without space in between) occurred in decreasing order. The multi-PPR domains also formed superhelical vortices, likely governed by interhelical angles rather than the spacers. These findings should be useful in designing and understanding the PPR domains.

Bootstrapping of integer concepts: the stronger deviant-interpretation challenge (and how to solve it)

Beck (Cognition 158:110–121, 2017) presents an outline of the procedure of bootstrapping of integer concepts, with the purpose of explicating the account of Carey (The Origin of Concepts, 2009). According to that theory, integer concepts are acquired through a process of inductive and analogous reasoning based on the object tracking system (OTS), which allows individuating objects in a parallel fashion. Discussing the bootstrapping theory, Beck dismisses what he calls the "deviant-interpretation challenge"—the possibility that the bootstrapped integer sequence does not follow a linear progression after some point—as being general to any account of inductive learning. While the account of Carey and Beck focuses on the OTS, in this paper I want to reconsider the importance of another empirically well-established cognitive core system for treating numerosities, namely the approximate number system (ANS). Since the ANS-based account offers a potential alternative for integer concept acquisition, I show that it provides a good reason to revisit the deviant-interpretation challenge. Finally, I will present a hybrid OTS-ANS model as the foundation of integer concept acquisition and the framework of enculturation as a solution to the challenge.

Nailfold Capillaroscopy in Systemic Diseases: Short Overview for Internal Medicine

Nailfold capillaroscopy (NFC) is now one of the main imaging tools in systemic sclerosis and imposed over time as an easy, non-invasive method for the nailfold microvascular bed assessment. In qualitative NFC normal pattern is characterized by homogeneous, parallel fashion arrangement of the last capillaries row as well as by capillaries with hairpin or non-specific variations like tortuous and/ or crossing shape. Nailfold capillaroscopy is strongly recommended for evaluation of all patients with Raynaud phenomenon. Appearance of giant capillaries is chronologically the first finding relevant for scleroderma spectrum disorders development (systemic sclerosis, dermatomyositis, undifferentiated and mixed connective tissue disease). Collapses of the giant loops generate microhemorrhages and further capillary loss with subsequent hypoxia, and neoangiogenesis seen as ramified/ bushy capillaries. Nailfold capillaroscopy is indicated especially in systemic sclerosis, being also included in the classification criteria. Based on these major NFC pathologic findings (giant capillaries, microhemorrhages, avascularity and neoangiogenesis), three evolutive stages were described in systemic sclerosis, namely the early, active, and late scleroderma pattern. In other connective tissue diseases than those scleroderma-related, like systemic lupus erythematosus, psoriatic arthritis, or antiphospholipid syndrome, the interest for capillaroscopy is growing, but the attempts of defining specific characteristics failed until now. Besides qualitative NFC, semiquantitative and quantitative capillaroscopic assessments were proposed for more accurate evaluation. Lately, automated systems are under development. There is still need of more studies to sustain the nailfold capillaroscopy validity as diagnostic and prognostic test.

Continuous-Time Programming of Floating-Gate Transistors for Nonvolatile Analog Memory Arrays

Floating-gate (FG) transistors are a primary means of providing nonvolatile digital memory in standard CMOS processes, but they are also key enablers for large-scale programmable analog systems, as well. Such programmable analog systems are often designed for battery-powered and resource-constrained applications, which require the memory cells to program quickly and with low infrastructural overhead. To meet these needs, we present a four-transistor analog floating-gate memory cell that offers both voltage and current outputs and has linear programming characteristics. Furthermore, we present a simple programming circuit that forces the memory cell to converge to targets with 13.0 bit resolution. Finally, we demonstrate how to use the FG memory cell and the programmer circuit in array configurations. We show how to program an array in either a serial or parallel fashion and demonstrate the effectiveness of the array programming with an application of a bandpass filter array.

Exploring autoantibody signatures in brain tissue from patients with severe mental illness

In recent years, studies have shown higher prevalence of autoantibodies in patients with schizophrenia compared to healthy individuals. This study applies an untargeted and a targeted affinity proteomics approach to explore and characterize the autoantibody repertoire in brain tissues from 73 subjects diagnosed with schizophrenia and 52 control subjects with no psychiatric or neurological disorders. Selected brain tissue lysates were first explored for IgG reactivity on planar microarrays composed of 11,520 protein fragments representing 10,820 unique proteins. Based on these results of ours and other previous studies of autoantibodies related to psychosis, we selected 226 fragments with an average length of 80 amino acids, representing 127 unique proteins. Tissue-based analysis of IgG reactivities using antigen suspension bead arrays was performed in a multiplex and parallel fashion for all 125 subjects. Among the detected autoantigens, higher IgG reactivity in subjects with schizophrenia, as compared to psychiatrically healthy subjects, was found against the glutamate ionotropic receptor NMDA type subunit 2D (anti-GluN2D). In a separate cohort with serum samples from 395 young adults with a wider spectrum of psychiatric disorders, higher levels of serum autoantibodies targeting GluN2D were found when compared to 102 control individuals. By further validating GluN2D and additional potential autoantigens, we will seek insights into how these are associated with severe mental illnesses.

Culture in the service of politics. The German question and relations between the German Democratic Republic and the Polish People’s Republic

The politics of memory and culture of the Polish People’s Republic alludes to Ideologiekritik in its idiosyncratic way characterized by Marxist utopianism. In reality, this leads to reversing this theory. The causes of fascism are seen in “German imperialism”. For political and strategic reasons the German Democratic Republic becomes one of the main partners of the Polish People’s Republic, with regard to which it is capable of accomplishing the guidelines of its policy in the cultural dimension. This is accompanied by an intensive cultural exchange between the two countries. In the language of official politics manifested, for instance, in the strategy of publishing houses of the Polish People’s Republic the culture of the two countries develop in a parallel fashion, which is an obvious product of the propaganda.