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Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. Here, we design a set of experiments using a diverse naïve synthetic camelid antibody fragment (nanobody) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally test our models' performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the models allow us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its functional properties. We provide a companion web-server that offers a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly compatible with many antigens. Here, we describe 'autonomous hypermutation yeast surface display' (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. By encoding antibody fragments on an error-prone orthogonal DNA replication system, surface-displayed antibody repertoires continuously mutate through simple cycles of yeast culturing and enrichment for antigen binding to produce high-affinity clones in as little as two weeks. We applied AHEAD to generate potent nanobodies against the SARS-CoV-2 S glycoprotein, a G-protein-coupled receptor and other targets, offering a template for streamlined antibody generation at large.

The ability to design functional sequences and predict effects of variation is central to protein engineering and biotherapeutics. State-of-art computational methods rely on models that leverage evolutionary information but are inadequate for important applications where multiple sequence alignments are not robust. Such applications include the prediction of variant effects of indels, disordered proteins, and the design of proteins such as antibodies due to the highly variable complementarity determining regions. We introduce a deep generative model adapted from natural language processing for prediction and design of diverse functional sequences without the need for alignments. The model performs state-of-art prediction of missense and indel effects and we successfully design and test a diverse 105-nanobody library that shows better expression than a 1000-fold larger synthetic library. Our results demonstrate the power of the alignment-free autoregressive model in generalizing to regions of sequence space traditionally considered beyond the reach of prediction and design.

Horizontal gene transfer (HGT) is the non-inherited acquisition of novel DNA sequences. HGT is common and important in bacteria because it enables the rapid generation of new phenotypes such as antibiotic resistance. Here we show that in vivo and in vitro DNA methylation patterns can be horizontally transferred into bacterial chromosomes to program cell phenotypes. The experiments were performed using a synthetic system in Escherichia coli where different DNA methylation patterns within the cis-regulatory sequence of the agn43 gene turn on or off a fluorescent reporter (CFP). With this system we demonstrated that DNA methylation patterns not only accompany the horizontal transfer of genes into the bacterial cytoplasm but can be transferred into chromosomes by: (i) bacteriophage P1 transduction; and (ii) transformation of extracellular synthetic DNA. We also modified the experimental system by replacing CFP with the SgrS small RNA, which regulates glucose and methyl α-D-glucoside uptake, and showed that horizontally acquired DNA methylation patterns can increase or decrease cell fitness. That is, horizontally acquired DNA methylation patterns can result in the selection for and against cells that have HGT. Findings from these proof-of-concept experiments have applications in synthetic biology and potentially broad implications for bacterial adaptation and evolution.

Small RNAs (sRNAs) are important regulators of gene expression during bacterial stress and pathogenesis. sRNAs act by forming duplexes with mRNAs to alter their translation and degradation. In some bacteria, duplex formation is mediated by the Hfq protein, which can bind the sRNA and mRNA in each pair in a random order. Here we investigate the consequences of this random-order binding and experimentally demonstrate that it can counterintuitively cause high Hfq concentrations to suppress rather than promote sRNA activity in Escherichia coli. As a result, maximum sRNA activity occurs when the Hfq concentration is neither too low nor too high relative to the sRNA and mRNA concentrations (‘Hfq set-point’). We further show with models and experiments that random-order binding combined with the formation of a dead-end mRNA–Hfq complex causes high concentrations of an mRNA to inhibit its own duplex formation by sequestering Hfq. In such cases, maximum sRNA activity requires an optimal mRNA concentration (‘mRNA set-point’) as well as an optimal Hfq concentration. The Hfq and mRNA set-points generate novel regulatory properties that can be harnessed by native and synthetic gene circuits to provide greater control over sRNA activity, generate non-monotonic responses and enhance the robustness of expression.

Objectives: To evaluate the therapeutic efficacy of the forced prolonged position (FPP) in patients with horizontal semicircular canal (HSCC) cupulolithiasis in whom the cupulolith repositioning maneuver (CuRM) failed. Methods: Fifty-four consecutive patients with HSCC cupulolithiasis were included, and immediate treatment efficacy of CuRM and short-term treatment efficacy of FPP were investigated. Results: We performed the CuRM in HSCC cupulolithiasis, and, if the CuRM did not show immediate success, instructed the patients to perform the FPP or the sham position (by random allocation) at home. The immediate therapeutic success of the CuRM was assessed by the absence of nystagmus and vertigo on positional testing after 30 minutes of the maneuver, which was 14.8% on the first visit day. And the resolution rate of HSCC cupulolithiasis was higher in the FPP group than in the sham position group at the second (78.3% vs 55.6%), third (75.0% vs 42.9%), and fourth visits (100% vs 25.0%). Conclusion: Although the CuRM has been considered to be a good therapeutic option for HSCC cupulolithiasis because it theoretically aims to detach otoconial particles attached both on the utricle and canal sides of the cupula, the immediate success rate was only 14.8% at the first visit. The FPP can be additionally recommended to improve the resolution rate in HSCC cupulolithiasis patients in whom the CuRM fails. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy, a lysosome-dependent self-degradative process, is a critical mechanism for the clearance of misfolded proteins and dysfunctional organelles in neurons. In the peripheral nervous system, autophagic stress is associated with the development of peripheral neuropathy. However, the molecular mechanism of axonal neuropathy induced by autophagic stress due to dysfunction of autophagy in peripheral neurons in vivo is still unclear. We found that dorsal root ganglion (DRG) neuron-specific atg7 (autophagy related 7) knockout (atg7-cKO) mice employing two different Cre recombinase systems exhibited sensory neuropathy approximately 2 months after birth. In electron microscopy analysis, axon degeneration was clearly observed in the myelinated fibers of the sciatic nerve before the appearance of neuronal cell death. Dystrophic axons filled with abnormal vesicular accumulations and amorphous inclusions were specifically localized in the myelinated axons within the DRG in atg7-cKO mice, indicating the presence of autophagic stress in proximal axons. In line with the EM findings, the mutant mice showed preferential induction of axonal injury-associated genes, including ATF3 (activating transcription factor 3), in large-size DRG neurons that constitute myelinated fibers without axotomy. SARM1 (sterile alpha and HEAT/Armadillo motif containing 1), the central executioner of Wallerian degeneration, was activated in the sciatic nerves of atg7-cKO mice, and axonal degeneration and sensory neuropathy in atg7-cKO mice were prevented via expression of a dominant-negative Sarm1 transgene. Our findings demonstrate the importance of SARM1-dependent axon degeneration in the development of peripheral sensory neuropathy induced by impairment of autophagy. AAV: adeno-associated virus; ATF3: activating transcription factor 3; ATG7: autophagy related 7; AVIL: advillin; cADPR: cyclic ADP ribose; CALC: calcitonin/calcitonin-related polypeptide; CMT: Charcot-Marie-Tooth disease; cKO: conditional knockout; DEG: differentially expressed gene; DRG: dorsal root ganglion; FE-SEM: field emission scanning electron microscopy; IF: immunofluorescence; NCV: nerve conduction velocity; PVALB: parvalbumin; RAG: regeneration-associated gene; ROS: reactive oxygen species; SARM1: sterile alpha and HEAT/Armadillo motif containing 1; SYN1: synapsin I [ABSTRACT FROM AUTHOR]

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