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Epigenetic mechanisms are processes that affect gene expression and cellular functions without involving changes in the DNA sequence. This abnormal or unstable expression of genes regulated by epigenetics can trigger cancer and other various diseases. The immune cells involved in anti-tumor responses and the immunogenicity of tumors may also be affected by epigenomic changes. This holds significant implications for the development and application of cancer immunotherapy, epigenetic therapy, and their combined treatments in the fight against cancer. We provide an overview of recent research literature focusing on how epigenomic changes in immune cells influence immune cell behavior and function, as well as the immunogenicity of cancer cells. And the combined utilization of epigenetic medications with immune checkpoint inhibitors that focus on immune checkpoint molecules [e.g., Programmed Death 1 (PD-1), Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA-4), T cell Immunoglobulin and Mucin Domain (TIM-3), Lymphocyte Activation Gene-3 (LAG-3)] present in immune cells and stromal cells associated with tumors. We highlight the potential of small-molecule inhibitors targeting epigenetic regulators to amplify anti-tumor immune responses. Moreover, we discuss how to leverage the intricate relationship between cancer epigenetics and cancer immunology to create treatment regimens that integrate epigenetic therapies with immunotherapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liang, Wang, Ma, Ju, Zhao and Shi.)

The ability to measure neutralizing antibodies on large scale can be important for understanding features of the natural history and epidemiology of infection, as well as an aid in determining the efficacy of interventions, particularly in outbreaks such as the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Because of the assay's rapid scalability and high efficiency, serology measurements that quantify the presence rather than function of serum antibodies often serve as proxies of immune protection. Here, we report the development of a high-throughput, automated fluorescence-based neutralization assay using SARS-CoV-2 virus to quantify neutralizing antibody activity in patient specimens. We performed large-scale testing of over 19,000 COVID-19 convalescent plasma (CCP) samples from patients who had been infected with SARS-CoV-2 between March and August 2020 across the United States. The neutralization capacity of the samples was moderately correlated with serological measurements of anti-receptor-binding domain (RBD) IgG levels. The neutralizing antibody levels within these convalescent-phase serum samples were highly variable against the original USA-WA1/2020 strain with almost 10% of individuals who had had PCR-confirmed SARS-CoV-2 infection having no detectable antibodies either by serology or neutralization, and ~1/3 having no or low neutralizing activity. Discordance between neutralization and serology measurements was mainly due to the presence of non-IgG RBD isotypes. Meanwhile, natural infection with the earliest SARS-CoV-2 strain USA-WA1/2020 resulted in weaker neutralization of subsequent B.1.1.7 (alpha) and the B.1.351 (beta) variants, with 88% of samples having no activity against the BA.1 (omicron) variant. IMPORTANCE The ability to directly measure neutralizing antibodies on live SARS-CoV-2 virus in individuals can play an important role in understanding the efficacy of therapeutic interventions or vaccines. In contrast to functional neutralization assays, serological assays only quantify the presence of antibodies as a proxy of immune protection. Here, we have developed a high-throughput, automated neutralization assay for SARS-CoV-2 and measured the neutralizing activity of ~19,000 COVID-19 convalescent plasma (CCP) samples collected across the United States between March and August of 2020. These data were used to support the FDA's interpretation of CCP efficacy in patients with SARS-CoV-2 infection and their issuance of emergency use authorization of CCP in 2020.

We introduce BacDrop, a highly scalable technology for bacterial single-cell RNA sequencing that has overcome many challenges hindering the development of scRNA-seq in bacteria. BacDrop can be applied to thousands to millions of cells from both gram-negative and gram-positive species. It features universal ribosomal RNA depletion and combinatorial barcodes that enable multiplexing and massively parallel sequencing. We applied BacDrop to study Klebsiella pneumoniae clinical isolates and to elucidate their heterogeneous responses to antibiotic stress. In an unperturbed population presumed to be homogeneous, we found within-population heterogeneity largely driven by the expression of mobile genetic elements that promote the evolution of antibiotic resistance. Under antibiotic perturbation, BacDrop revealed transcriptionally distinct subpopulations associated with different phenotypic outcomes including antibiotic persistence. BacDrop thus can capture cellular states that cannot be detected by bulk RNA-seq, which will unlock new microbiological insights into bacterial responses to perturbations and larger bacterial communities such as the microbiome., Competing Interests: Declaration of interests D.T.H., P.M., and H.M.A. have filed an U.S. Patent Application (application no. 17/819,034) based on this work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Unbiased assessment of the risks associated with acquisition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to informing mitigation efforts during pandemics. The objective of our study was to understand the risk factors for acquiring coronavirus disease 2019 (COVID-19) in a large prospective cohort of adult residents in a large US metropolitan area., Methods: We designed a fully remote longitudinal cohort study involving monthly at-home SARS-CoV-2 polymerase chain reaction (PCR) and serology self-testing and monthly surveys., Results: Between October 2020 and January 2021, we enrolled 10 289 adults reflective of the Boston metropolitan area census data. At study entry, 567 (5.5%) participants had evidence of current or prior SARS-CoV-2 infection. This increased to 13.4% by June 15, 2021. Compared with Whites, Black non-Hispanic participants had a 2.2-fold greater risk of acquiring COVID-19 (hazard ratio [HR], 2.19; 95% CI, 1.91-2.50; P < .001), and Hispanics had a 1.5-fold greater risk (HR, 1.52; 95% CI, 1.32-1.71; P < .016). Individuals aged 18-29, those who worked outside the home, and those living with other adults and children were at an increased risk. Individuals in the second and third lowest disadvantaged neighborhood communities were associated with an increased risk of acquiring COVID-19. Individuals with medical risk factors for severe disease were at a decreased risk of SARS-CoV-2 acquisition., Conclusions: These results demonstrate that race/ethnicity and socioeconomic status are the biggest determinants of acquisition of infection. This disparity is significantly underestimated if based on PCR data alone, as noted by the discrepancy in serology vs PCR detection for non-White participants, and points to persistent disparity in access to testing. Medical conditions and advanced age, which increase the risk for severity of SARS-CoV-2 disease, were associated with a lower risk of COVID-19 acquisition, suggesting the importance of behavior modifications. These findings highlight the need for mitigation programs that overcome challenges of structural racism in current and future pandemics., Competing Interests: Potential conflicts of interest. D.H. is a founder, consultant to, equity holder in, and inventor of technology licensed to Sherlock Biosciences and serves on the Scientific Advisory Board for Proof Diagnostics; both are infectious disease diagnostic companies, but neither company’s technologies was used in this work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel preamplification strategy that further enhances sensitivity and specificity. We demonstrate bCARMEN is capable of detecting and discriminating 52 clinically relevant bacterial species and several key antibiotic resistance genes. We further develop a simple proof of principle workflow using stabilized reagents and cell phone camera optical readout, opening up the possibility of a rapid point-of-care multiplexed bacterial pathogen identification and antibiotic susceptibility testing., (© The Author(s) 2022. Published by Oxford University Press on behalf of the National Academy of Sciences.)

Background: Carbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole-genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms., Methods: To overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem-susceptible Enterobacterales, isolated from patients from four US hospitals over nearly 5 years., Results: Our CRE collection comprised a diverse range of species, lineages, and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment., Conclusions: Collectively, the framework we developed provides a clearer, high-resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks., (© 2022. The Author(s).)

Background: Schizophrenia is a serious mental illness affecting approximately 20 million individuals globally. Both genetic and environmental factors contribute to the illness. If left undiagnosed and untreated, schizophrenia results in impaired social function, repeated hospital admissions, reduced quality of life and decreased life expectancy. Clinical diagnosis largely relies on subjective evidence, including self-reported experiences, and reported behavioural abnormalities followed by psychiatric evaluation. In addition, psychoses may occur along with other conditions, and the symptoms are often episodic and transient, posing a significant challenge to the precision of diagnosis. Therefore, objective, specific tests using biomarkers are urgently needed for differential diagnosis of schizophrenia in clinical practice., Aims: We aimed to provide evidence-based and consensus-based recommendations, with a summary of laboratory measurements that could potentially be used as biomarkers for schizophrenia, and to discuss directions for future research., Methods: We searched publications within the last 10 years with the following keywords: 'schizophrenia', 'gene', 'inflammation', 'neurotransmitter', 'protein marker', 'gut microbiota', 'pharmacogenomics' and 'biomarker'. A draft of the consensus was discussed and agreed on by all authors at a round table session., Results: We summarised the characteristics of candidate diagnostic markers for schizophrenia, including genetic, inflammatory, neurotransmitter, peripheral protein, pharmacogenomic and gut microbiota markers. We also proposed a novel laboratory process for diagnosing schizophrenia in clinical practice based on the evidence summarised in this paper., Conclusions: Further efforts are needed to identify schizophrenia-specific genetic and epigenetic markers for precise diagnosis, differential diagnosis and ethnicity-specific markers for the Chinese population. The development of novel laboratory techniques is making it possible to use these biomarkers clinically to diagnose disease., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Activation of hepatic stellate cells (HSC) is associated with hepatic fibrogenesis, which is one of complications of diabetes mellitus. Captopril possesses potent anti-inflammation, oxidative stress and fibrosis effects. However, the specific molecular mechanism of captopril in high glucose (HG)-induced hepatic stellate cells has not been elucidated. Following the treatment of HG or captopril treatment for rat hepatic stellate cells (HSC-T6), cell activities were detected by Cell Counting Kit-8 (CCK8) assay. Reactive oxygen species (ROS) levels were determined by ROS staining. The expression of inflammation-related proteins (Interleukin (IL)-1β, IL-6 and IL-8) and fibrosis-related proteins (fibronectin (FN), collagen I, collagen III, collagen IV, matrix metallopeptidase (MMP-2 and MMP-9) were determined by Western blot. Captopril significantly decreased HSC-T6 cell viability induced by HG in a dose-dependent manner, as well as decreased levels of malondialdehyde (MDA), ROS, pro-inflammatory markers and fibrosis-related proteins, while upregulated superoxide dismutase (SOD) activities. We further found that captopril decreased the ratio of p-IκBα/IκBα and the ratio of p-p65/p65. Intriguing, phorbol myristate acetate (PMA) or LiCl was able to significantly reverse the captopril-induced alteration of oxidative stress-, inflammation- and fibrosis-marker levels. In conclusion, in HG-stimulated HSC-T6 cells, captopril displayed a potent ability to inhibit oxidative stress, inflammation and hepatic fibrogenesis via NF-kappaB or wnt3α/β-catenin. These results demonstrated the mechanism of captopril as well as the role of the NF-kappaB or wnt3α/β-catenin on HSC-T6 activation induced by HG.

Background: Antibody response duration following severe acute respiratory syndrome coronavirus 2 infection tends to be variable and depends on severity of disease and method of detection., Study Design and Methods: COVID-19 convalescent plasma from 18 donors was collected longitudinally for a maximum of 63-129 days following resolution of symptoms. All the samples were initially screened by the Ortho total Ig test to confirm positivity and subsequently tested with seven additional direct sandwich or indirect binding assays (Ortho, Roche, Abbott, Broad Institute) directed against a variety of antigen targets (S1, receptor binding domain, and nucleocapsid [NC]), along with two neutralization assays (Broad Institute live virus PRNT and Vitalant Research Institute [VRI] Pseudovirus reporter viral particle neutralization [RVPN])., Results: The direct detection assays (Ortho total Ig total and Roche total Ig) showed increasing levels of antibodies over the time period, in contrast to the indirect IgG assays that showed a decline. Neutralization assays also demonstrated declining responses; the VRI RVPN pseudovirus had a greater rate of decline than the Broad PRNT live virus assay., Discussion: These data show that in addition to variable individual responses and associations with disease severity, the detection assay chosen contributes to the heterogeneous results in antibody stability over time. Depending on the scope of the research, one assay may be preferable over another. For serosurveillance studies, direct, double Ag-sandwich assays appear to be the best choice due to their stability; in particular, algorithms that include both S1- and NC-based assays can help reduce the rate of false-positivity and discriminate between natural infection and vaccine-derived seroreactivity., (© 2021 AABB.)

Purpose of the Review: Nowadays, the incidence of schizophrenia is noticeably increased. If left undiagnosed and untreated, it will lead to impaired social functions, repeated hospital admissions, decline in quality of life and life expectancy. However, the diagnosis of schizophrenia is complicated and challenging. Both genetic and environmental factors are considered as important contributors to the development and progression of this disorder. The environmental factors have been linked to changes in gene expression through epigenetic modulations, which have raised more and more research interests in recent years. This review article is to summarize the current findings and understanding of epigenetic modulation associated with pathogenesis of schizophrenia, aiming to provide useful information for further research in developing biomarkers for schizophrenia., Recent Findings: Three major types of epigenetic modulations have been described in this article. Firstly, both DNA hypermethylation and hypomethylated have been associated with schizophrenia via analyzing post-mortem brain tissues and peripheral blood of patients. Specific changes of non-coding RNAs, particularly microRNAs and long-chain non-coding RNAs, have been observed in central and peripheral samples of schizophrenia patients, indicating their significant diagnostic value for the disease, and may also potentially predict treatment response. The correlation between histone modification and schizophrenia, however, is largely unclear., Summary: Epigenetic modulations, including DNA methylation, ncRNA transcriptional regulation and histone modification, play an important role in the pathogenesis of schizophrenia. Therefore, tests of these epigenetic alterations may be utilized to assist in the diagnosis and determination of strategies of individualized treatment in clinical practice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Li, Jin, Shi, Li, Ma, Sun, Chen, Li and Lin.)

In this era of rising antibiotic resistance, in contrast to our increasing understanding of mechanisms that cause resistance, our understanding of mechanisms that influence the propensity to evolve resistance remains limited. Here, we identified genetic factors that facilitate the evolution of resistance to carbapenems, the antibiotic of 'last resort', in Klebsiella pneumoniae , the major carbapenem-resistant species. In clinical isolates, we found that high-level transposon insertional mutagenesis plays an important role in contributing to high-level resistance frequencies in several major and emerging carbapenem-resistant lineages. A broader spectrum of resistance-conferring mutations for select carbapenems such as ertapenem also enables higher resistance frequencies and, importantly, creates stepping-stones to achieve high-level resistance to all carbapenems. These mutational mechanisms can contribute to the evolution of resistance, in conjunction with the loss of systems that restrict horizontal resistance gene uptake, such as the CRISPR-Cas system. Given the need for greater antibiotic stewardship, these findings argue that in addition to considering the current efficacy of an antibiotic for a clinical isolate in antibiotic selection, considerations of future efficacy are also important. The genetic background of a clinical isolate and the exact antibiotic identity can and should also be considered as they are determinants of a strain's propensity to become resistant. Together, these findings thus provide a molecular framework for understanding acquisition of carbapenem resistance in K. pneumoniae with important implications for diagnosing and treating this important class of pathogens., Competing Interests: PM, LH, AP, HL, CE, AM, RB, AE, JL, DH No competing interests declared, (© 2021, Ma et al.)

Multidrug resistant organisms are a serious threat to human health 1,2 . Fast, accurate antibiotic susceptibility testing (AST) is a critical need in addressing escalating antibiotic resistance, since delays in identifying multidrug resistant organisms increase mortality 3,4 and use of broad-spectrum antibiotics, further selecting for resistant organisms. Yet current growth-based AST assays, such as broth microdilution 5 , require several days before informing key clinical decisions. Rapid AST would transform the care of patients with infection while ensuring that our antibiotic arsenal is deployed as efficiently as possible. Growth-based assays are fundamentally constrained in speed by doubling time of the pathogen, and genotypic assays are limited by the ever-growing diversity and complexity of bacterial antibiotic resistance mechanisms. Here we describe a rapid assay for combined genotypic and phenotypic AST through RNA detection, GoPhAST-R, that classifies strains with 94-99% accuracy by coupling machine learning analysis of early antibiotic-induced transcriptional changes with simultaneous detection of key genetic resistance determinants to increase accuracy of resistance detection, facilitate molecular epidemiology and enable early detection of emerging resistance mechanisms. This two-pronged approach provides phenotypic AST 24-36 h faster than standard workflows, with <4 h assay time on a pilot instrument for hybridization-based multiplexed RNA detection implemented directly from positive blood cultures.

Rapid bacterial identification remains a critical challenge in infectious disease diagnostics. We developed a novel molecular approach to detect and identify a wide diversity of bacterial pathogens in a single, simple assay, exploiting the conservation, abundance, and rich phylogenetic content of ribosomal RNA in a rapid fluorescent hybridization assay that requires no amplification or enzymology. Of 117 isolates from 64 species across 4 phyla, this assay identified bacteria with >89% accuracy at the species level and 100% accuracy at the family level, enabling all critical clinical distinctions. In pilot studies on primary clinical specimens, including sputum, blood cultures, and pus, bacteria from 5 different phyla were identified.

Carbapenem resistance is mainly mediated by carbapenemases or extended-spectrum β-lactamases (ESBL) plus a loss of porins. However, we have identified a Klebsiella pneumoniae clinical isolate that contains neither carbapenemases nor ESBLs. Instead, we found that high-level expression of a novel bla OXA-10 -derived β-lactamase gene, bla OXA-663 , in conjunction with OmpK36 deficiency results in high-level carbapenem resistance. This finding demonstrates the combinatorial complexity of factors, including β-lactamase activity, its expression levels, and porin activity, that yield carbapenem resistance., (Copyright © 2018 American Society for Microbiology.)

Tracking micro-objects in the noisy microscopy image sequences is important for the analysis of dynamic processes in biological objects. In this paper, an automated tracking framework is proposed to extract the trajectories of micro-objects. This framework uses a probability hypothesis density particle filtering (PF-PHD) tracker to implement a recursive state estimation and trajectories association. In order to increase the efficiency of this approach, an elliptical target model is presented to describe the micro-objects using shape parameters instead of point-like targets which may cause inaccurate tracking. A novel likelihood function, not only covering the spatiotemporal distance but also dealing with geometric shape function based on the Mahalanobis norm, is proposed to improve the accuracy of particle weight in the update process of the PF-PHD tracker. Using this framework, a larger number of tracks are obtained. The experiments are performed on simulated data of microtubule movements and real mouse stem cells. We compare the PF-PHD tracker with the nearest neighbor method and the multiple hypothesis tracking method. Our PF-PHD tracker can simultaneously track hundreds of micro-objects in the microscopy image sequence.

Circadian (daily) rhythms are a fundamental and ubiquitous property of eukaryotic organisms. However, cyanobacteria are the only prokaryotic group for which bona fide circadian properties have been persuasively documented, even though homologs of the cyanobacterial kaiABC central clock genes are distributed widely among Eubacteria and Archaea. We report the purple non-sulfur bacterium Rhodopseudomonas palustris (that harbors homologs of kaiB and kaiC) only poorly sustains rhythmicity in constant conditions-a defining characteristic of circadian rhythms. Moreover, the biochemical characteristics of the Rhodopseudomonas homolog of the KaiC protein in vivo and in vitro are different from those of cyanobacterial KaiC. Nevertheless, R. palustris cells exhibit adaptive kaiC-dependent growth enhancement in 24-h cyclic environments, but not under non-natural constant conditions. Therefore, our data indicate that Rhodopseudomonas does not have a classical circadian rhythm, but a novel timekeeping mechanism that does not sustain itself in constant conditions. These results question the adaptive value of self-sustained oscillatory capability for daily timekeepers and establish new criteria for circadian-like systems that are based on adaptive properties (i.e., fitness enhancement in rhythmic environments), rather than upon observations of persisting rhythms in constant conditions. We propose that the Rhodopseudomonas system is a "proto" circadian timekeeper, as in an ancestral system that is based on KaiC and KaiB proteins and includes some, but not necessarily all, of the canonical properties of circadian clocks. These data indicate reasonable intermediate steps by which bona fide circadian systems evolved in simple organisms.

Biochemical and biomedical applications of graphene are critically dependent on the interaction between biomolecules and the nanomaterial. In this work, we developed a graphene-based signal-amplification nanoprobe by combining anti-immunoglobulin G (anti-IgG) and horseradish peroxidase (HRP) with graphene oxide (GO). The structure and function of HRP in the nano-interface of GO were firstly investigated, which demonstrated that the enzyme retained 78% of its native activity and 77% of its native α-helix content. HRP and anti-IgG were then co-adsorbed onto GO to form bifunctional nanoprobes. The nanoprobes provide both improved binding ability and signal-amplification ability. Comparing with conventional bioconjugates such as enzyme-linked antibody, co-adsorption could avoid chemical conjugation between biomolecules, keeping their bioactivity well. As an example for their application, the nanoprobes were used to obtain amplified signals in a sandwich-type immunoassay for cancer marker, instead of conventional colorimetric conjugates. This approach provided a detection limit of 10 pg/mL alpha-fetoprotein (AFP), which was much more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) methods. The easily fabricated GO-based nanoprobes have the potential to become universal probes for molecular diagnostics., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Circadian clocks are found in a wide variety of organisms from cyanobacteria to mammals. Many believe that the circadian clock system evolved as an adaption to the daily cycles in light and temperature driven by the rotation of the earth. Studies on the cyanobacterium, Synechococcus elongatus PCC 7942, have confirmed that the circadian clock in resonance with environmental cycles confers an adaptive advantage to cyanobacterial strains with different clock properties when grown in competition under light-dark cycles. The results thus far suggest that in a cyclic environment, the cyanobacterial strains whose free running periods are closest to the environmental period are the most fit and the strains lacking a functional circadian clock are at a competitive disadvantage relative to strains with a functional clock. In contrast, the circadian system provides little or no advantage to cyanobacteria grown in competition in constant light. To explain the potential mechanism of this clock-mediated enhancement in fitness in cyanobacteria, several models have been proposed; these include the limiting resource model, the diffusible inhibitor model and the cell-to-cell communication model. None of these models have been excluded by the currently available experimental data and the mechanistic basis of clock-mediated fitness enhancement remains elusive.

Circadian rhythms are oscillations in biological processes that function as a key adaptation to the daily rhythms of most environments. In the model cyanobacterial circadian clock system, the core oscillator proteins are encoded by the gene cluster kaiABC. Genes with high expression and functional importance, such as the kai genes, are usually encoded by optimal codons, yet the codon-usage bias of the kaiBC genes is not optimized for translational efficiency. We discovered a relationship between codon usage and a general property of circadian rhythms called conditionality; namely, that endogenous rhythmicity is robustly expressed under some environmental conditions but not others. Despite the generality of circadian conditionality, however, its molecular basis is unknown for any system. Here we show that in the cyanobacterium Synechococcus elongate, non-optimal codon usage was selected as a post-transcriptional mechanism to switch between circadian and non-circadian regulation of gene expression as an adaptive response to environmental conditions. When the kaiBC sequence was experimentally optimized to enhance expression of the KaiB and KaiC proteins, intrinsic rhythmicity was enhanced at cool temperatures that are experienced by this organism in its natural habitat. However, fitness at those temperatures was highest in cells in which the endogenous rhythms were suppressed at cool temperatures as compared with cells exhibiting high-amplitude rhythmicity. These results indicate natural selection against circadian systems in cyanobacteria that are intrinsically robust at cool temperatures. Modulation of circadian amplitude is therefore crucial to its adaptive significance. Moreover, these results show the direct effects of codon usage on a complex phenotype and organismal fitness. Our work also challenges the long-standing view of directional selection towards optimal codons, and provides a key example of natural selection against optimal codons to achieve adaptive responses to environmental changes.