Bacterial droplet-based single-cell RNA-seq reveals antibiotic-associated heterogeneous cellular states.

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.

SARS-CoV-2 antibody persistence in COVID-19 convalescent plasma donors: Dependency on assay format and applicability to serosurveillance.

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.

Non-optimal codon usage is a mechanism to achieve circadian clock conditionality.

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.

Evolution of KaiC-Dependent Timekeepers: A Proto-circadian Timing Mechanism Confers Adaptive Fitness in the Purple Bacterium Rhodopseudomonas palustris.

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.

Carbapenem Resistance Caused by High-Level Expression of OXA-663 ß-Lactamase in an OmpK36-Deficient Klebsiella pneumoniae Clinical Isolate.

Carbapenem resistance is mainly mediated by carbapenemases or extended-spectrum ß-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 blaOXA-10-derived ß-lactamase gene, blaOXA-663, in conjunction with OmpK36 deficiency results in high-level carbapenem resistance. This finding demonstrates the combinatorial complexity of factors, including ß-lactamase activity, its expression levels, and porin activity, that yield carbapenem resistance.

Micro-object motion tracking based on the probability hypothesis density particle tracker.

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.

An Evolutionary Fitness Enhancement Conferred by the Circadian System in Cyanobacteria.

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.

Enhancing anti-tumor immune responses through combination therapies: epigenetic drugs and immune checkpoint inhibitors.

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.

A comparison between children and adolescents with autism spectrum disorders and healthy controls in biomedical factors, trace elements, and microbiota biomarkers: a meta-analysis.

Introduction: Autism spectrum disorder (ASD) is a multifaceted developmental condition that commonly appears during early childhood. The etiology of ASD remains multifactorial and not yet fully understood. The identification of biomarkers may provide insights into the underlying mechanisms and pathophysiology of the disorder. The present study aimed to explore the causes of ASD by investigating the key biomedical markers, trace elements, and microbiota factors between children with autism spectrum disorder (ASD) and control subjects. Methods: Medline, PubMed, ProQuest, EMBASE, Cochrane Library, PsycINFO, Web of Science, and EMBSCO databases have been searched for publications from 2012 to 2023 with no language restrictions using the population, intervention, control, and outcome (PICO) approach. Keywords including "autism spectrum disorder," "oxytocin," "GABA," "Serotonin," "CRP," "IL-6," "Fe," "Zn," "Cu," and "gut microbiota" were used for the search. The Joanna Briggs Institute (JBI) critical appraisal checklist was used to assess the article quality, and a random model was used to assess the mean difference and standardized difference between ASD and the control group in all biomedical markers, trace elements, and microbiota factors. Results: From 76,217 records, 43 studies met the inclusion and exclusion criteria and were included in this meta-analysis. The pooled analyses showed that children with ASD had significantly lower levels of oxytocin (mean differences, MD = -45.691, 95% confidence interval, CI: -61.667, -29.717), iron (MD = -3.203, 95% CI: -4.891, -1.514), and zinc (MD = -6.707, 95% CI: -12.691, -0.722), lower relative abundance of Bifidobacterium (MD = -1.321, 95% CI: -2.403, -0.238) and Parabacteroides (MD = -0.081, 95% CI: -0.148, -0.013), higher levels of c-reactive protein, CRP (MD = 0.401, 95% CI: 0.036, 0.772), and GABA (MD = 0.115, 95% CI: 0.045, 0.186), and higher relative abundance of Bacteroides (MD = 1.386, 95% CI: 0.717, 2.055) and Clostridium (MD = 0.281, 95% CI: 0.035, 0.526) when compared with controls. The results of the overall analyses were stable after performing the sensitivity analyses. Additionally, no substantial publication bias was observed among the studies. Interpretation: Children with ASD have significantly higher levels of CRP and GABA, lower levels of oxytocin, iron, and zinc, lower relative abundance of Bifidobacterium and Parabacteroides, and higher relative abundance of Faecalibacterium, Bacteroides, and Clostridium when compared with controls. These results suggest that these indicators may be a potential biomarker panel for the diagnosis or determining therapeutic targets of ASD. Furthermore, large, sample-based, and randomized controlled trials are needed to confirm these results.

High-Throughput Neutralization and Serology Assays Reveal Correlated but Highly Variable Humoral Immune Responses in a Large Population of Individuals Infected with SARS-CoV-2 in the US between March and August 2020.

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.