Throughout the last 2 decades, several scholars observed that present day research into human genes rarely turns toward genes that had not already been extensively investigated in the past. Guided by hypotheses derived from studies of science and innovation, we present here a literature-wide data-driven meta-analysis to identify the specific scientific and organizational contexts that coincided with early-stage research into human genes throughout the past half century. We demonstrate that early-stage research into human genes differs in team size, citation impact, funding mechanisms, and publication outlet, but that generalized insights derived from studies of science and innovation only partially apply to early-stage research into human genes. Further, we demonstrate that, presently, genome biology accounts for most of the initial early-stage research, while subsequent early-stage research can engage other life sciences fields. We therefore anticipate that the specificity of our findings will enable scientists and policymakers to better promote early-stage research into human genes and increase overall innovation within the life sciences.

Erratum in PLoS Biol. 2022 Sep 21;20(9):e3001818.

The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylum Halobacterota, none of which have been isolated in pure culture. Here, we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features that separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor.

Biological functions arise from protein interactions, which are reflected in the natural variation of proteome configurations across individual cells. Emerging single-cell proteomics methods may decode this variation and empower inference of biological mechanisms with minimal assumptions.

The principal biological function of bacterial and archaeal CRISPR systems is RNA-guided adaptive immunity against viruses and other mobile genetic elements (MGEs). These systems show remarkable evolutionary plasticity and functional versatility at multiple levels, including both the defense mechanisms that lead to direct, specific elimination of the target DNA or RNA and those that cause programmed cell death (PCD) or induction of dormancy. This flexibility is also evident in the recruitment of CRISPR systems for nondefense functions. Defective CRISPR systems or individual CRISPR components have been recruited by transposons for RNA-guided transposition, by plasmids for interplasmid competition, and by viruses for antidefense and interviral conflicts. Additionally, multiple highly derived CRISPR variants of yet unknown functions have been discovered. A major route of innovation in CRISPR evolution is the repurposing of diverged repeat variants encoded outside CRISPR arrays for various structural and regulatory functions. The evolutionary plasticity and functional versatility of CRISPR systems are striking manifestations of the ubiquitous interplay between defense and "normal" cellular functions.

Psychological and neural distinctions between the technical concepts of "liking" and "wanting" pose important problems for motivated choice for goods. Why could we "want" something that we do not "like," or "like" something but be unwilling to exert effort to acquire it? Here, we suggest a framework for answering these questions through the medium of reinforcement learning. We consider "liking" to provide immediate, but preliminary and ultimately cancellable, information about the true, long-run worth of a good. Such initial estimates, viewed through the lens of what is known as potential-based shaping, help solve the temporally complex learning problems faced by animals.

Studies of neuronal oscillations have contributed substantial insight into the mechanisms of visual, auditory, and somatosensory perception. However, progress in such research in the human olfactory system has lagged behind. As a result, the electrophysiological properties of the human olfactory system are poorly understood, and, in particular, whether stimulus-driven high-frequency oscillations play a role in odor processing is unknown. Here, we used direct intracranial recordings from human piriform cortex during an odor identification task to show that 3 key oscillatory rhythms are an integral part of the human olfactory cortical response to smell: Odor induces theta, beta, and gamma rhythms in human piriform cortex. We further show that these rhythms have distinct relationships with perceptual behavior. Odor-elicited gamma oscillations occur only during trials in which the odor is accurately perceived, and features of gamma oscillations predict odor identification accuracy, suggesting that they are critical for odor identity perception in humans. We also found that the amplitude of high-frequency oscillations is organized by the phase of low-frequency signals shortly following sniff onset, only when odor is present. Our findings reinforce previous work on theta oscillations, suggest that gamma oscillations in human piriform cortex are important for perception of odor identity, and constitute a robust identification of the characteristic electrophysiological response to smell in the human brain. Future work will determine whether the distinct oscillations we identified reflect distinct perceptual features of odor stimuli.

What makes cognition "advanced" is an open and not precisely defined question. One perspective involves increasing the complexity of associative learning, from conditioning to learning sequences of events ("chaining") to representing various cue combinations as "chunks." Here we develop a weighted graph model to study the mechanism enabling chunking ability and the conditions for its evolution and success, based on the ecology of the cleaner fish Labroides dimidiatus. In some environments, cleaners must learn to serve visitor clients before resident clients, because a visitor leaves if not attended while a resident waits for service. This challenge has been captured in various versions of the ephemeral reward task, which has been proven difficult for a range of cognitively capable species. We show that chaining is the minimal requirement for solving this task in its common simplified laboratory format that involves repeated simultaneous exposure to an ephemeral and permanent food source. Adding ephemeral-ephemeral and permanent-permanent combinations, as cleaners face in the wild, requires individuals to have chunking abilities to solve the task. Importantly, chunking parameters need to be calibrated to ecological conditions in order to produce adaptive decisions. Thus, it is the fine-tuning of this ability, which may be the major target of selection during the evolution of advanced associative learning.

The trade-off between offspring size and number is central to life history strategies. Both the evolutionary gain of parental care or more favorable habitats for offspring development are predicted to result in fewer, larger offspring. However, despite much research, it remains unclear whether and how different forms of care and habitats drive the evolution of the trade-off. Using data for over 800 amphibian species, we demonstrate that, after controlling for allometry, amphibians with direct development and those that lay eggs in terrestrial environments have larger eggs and smaller clutches, while different care behaviors and adaptations vary in their effects on the trade-off. Specifically, among the 11 care forms we considered at the egg, tadpole and juvenile stage, egg brooding, male egg attendance, and female egg attendance increase egg size; female tadpole attendance and tadpole feeding decrease egg size, while egg brooding, tadpole feeding, male tadpole attendance, and male tadpole transport decrease clutch size. Unlike egg size that shows exceptionally high rates of phenotypic change in just 19 branches of the amphibian phylogeny, clutch size has evolved at exceptionally high rates in 135 branches, indicating episodes of strong selection; egg and tadpole environment, direct development, egg brooding, tadpole feeding, male tadpole attendance, and tadpole transport explain 80% of these events. By explicitly considering diversity in parental care and offspring habitat by stage of offspring development, this study demonstrates that more favorable conditions for offspring development promote the evolution of larger offspring in smaller broods and reveals that the diversity of parental care forms influences the trade-off in more nuanced ways than previously appreciated.

Meta-research involves the interrogation of every stage of the research lifecycle, from conception to publication and dissemination. Looking back over the first six years of PLOS Biology Meta-Research Articles highlights the important insights that can be obtained from such "research on research".

The subiculum is positioned at a critical juncture at the interface of the hippocampus with the rest of the brain. However, the exact roles of the subiculum in most hippocampal-dependent memory tasks remain largely unknown. One obstacle to make comparisons of neural firing patterns between the subiculum and hippocampus is the broad firing fields of the subicular cells. Here, we used spiking phases in relation to theta rhythm to parse the broad firing field of a subicular neuron into multiple subfields to find the unique functional contribution of the subiculum while male rats performed a hippocampal-dependent visual scene memory task. Some of the broad firing fields of the subicular neurons were successfully divided into multiple subfields similar to those in the CA1 by using the theta phase precession cycle. The new paradigm significantly improved the detection of task-relevant information in subicular cells without affecting the information content represented by CA1 cells. Notably, we found that multiple fields of a single subicular neuron, unlike those in the CA1, carried heterogeneous task-related information such as visual context and choice response. Our findings suggest that the subicular cells integrate multiple task-related factors by using theta rhythm to associate environmental context with action.

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

We present a high-throughput optogenetic illumination system capable of simultaneous closed-loop light delivery to specified targets in populations of moving Caenorhabditis elegans. The instrument addresses three technical challenges: It delivers targeted illumination to specified regions of the animal's body such as its head or tail; it automatically delivers stimuli triggered upon the animal's behavior; and it achieves high throughput by targeting many animals simultaneously. The instrument was used to optogenetically probe the animal's behavioral response to competing mechanosensory stimuli in the the anterior and posterior gentle touch receptor neurons. Responses to more than 43,418 stimulus events from a range of anterior-posterior intensity combinations were measured. The animal's probability of sprinting forward in response to a mechanosensory stimulus depended on both the anterior and posterior stimulation intensity, while the probability of reversing depended primarily on the anterior stimulation intensity. We also probed the animal's response to mechanosensory stimulation during the onset of turning, a relatively rare behavioral event, by delivering stimuli automatically when the animal began to turn. Using this closed-loop approach, over 9,700 stimulus events were delivered during turning onset at a rate of 9.2 events per worm hour, a greater than 25-fold increase in throughput compared to previous investigations. These measurements validate with greater statistical power previous findings that turning acts to gate mechanosensory evoked reversals. Compared to previous approaches, the current system offers targeted optogenetic stimulation to specific body regions or behaviors with many fold increases in throughput to better constrain quantitative models of sensorimotor processing.

Chronic inflammation is often associated with the development of tissue fibrosis, but how mesenchymal cell responses dictate pathological fibrosis versus resolution and healing remains unclear. Defining stromal heterogeneity and identifying molecular circuits driving extracellular matrix deposition and remodeling stands to illuminate the relationship between inflammation, fibrosis, and healing. We performed single-cell RNA-sequencing of colon-derived stromal cells and identified distinct classes of fibroblasts with gene signatures that are differentially regulated by chronic inflammation, including IL-11-producing inflammatory fibroblasts. We further identify a transcriptional program associated with trans-differentiation of mucosa-associated fibroblasts and define a functional gene signature associated with matrix deposition and remodeling in the inflamed colon. Our analysis supports a critical role for the metalloprotease Adamdec1 at the interface between tissue remodeling and healing during colitis, demonstrating its requirement for colon epithelial integrity. These findings provide mechanistic insight into how inflammation perturbs stromal cell behaviors to drive fibroblastic responses controlling mucosal matrix remodeling and healing.

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1β (IL-1β), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB, important for AMP induction, and found that while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects.

The sympathetic nervous system (SNS) contributes to immune balance by promoting anti-inflammatory B cells. However, whether B cells possess a self-regulating mechanism by which they modulate regulatory B cell (Breg) function is not well understood. In this study, we investigated the ability of B cells to synthesize their own catecholamines upon stimulation with different B cell activators and found that expression of the enzyme tyrosine hydroxylase (TH), required to generate catecholamines, is up-regulated by Toll-like receptor (TLR)9. This TLR9-dependent expression of TH correlated with up-regulation of adrenergic receptors (ADRs), enhanced interleukin (IL)-10 production, and overexpression of the co-inhibitory ligands programmed death ligand 1 (PD-L1) and Fas ligand (FasL). Moreover, concomitant stimulation of ß1-3-ADRs together with a B cell receptor (BCR)/TLR9 stimulus clearly enhances the anti-inflammatory potential of Bregs to suppress CD4 T cells, a crucial population in the pathogenesis of autoimmune diseases, like rheumatoid arthritis (RA). Furthermore, TH up-regulation was also demonstrated in B cells during the course of collagen-induced arthritis (CIA), a mouse model for the investigation of RA. In conclusion, our data show that B cells possess an autonomous mechanism to modulate their regulatory function in an autocrine and/or paracrine manner. These findings help to better understand the function of B cells in the regulation of autoimmune diseases and the interplay of SNS.

Nonalcoholic fatty liver disease (NAFLD) affects approximately a quarter of the population worldwide, and persistent overnutrition is one of the major causes. However, the underlying molecular basis has not been fully elucidated, and no specific drug has been approved for this disease. Here, we identify a regulatory mechanism that reveals a novel function of Rab2A in the progression of NAFLD based on energy status and PPARγ. The mechanistic analysis shows that nutrition repletion suppresses the phosphorylation of AMPK-TBC1D1 signaling, augments the level of GTP-bound Rab2A, and then increases the protein stability of PPARγ, which ultimately promotes the hepatic accumulation of lipids in vitro and in vivo. Furthermore, we found that blocking the AMPK-TBC1D1 pathway in TBC1D1S231A-knock-in (KI) mice led to a markedly increased GTP-bound Rab2A and subsequent fatty liver in aged mice. Our studies also showed that inhibition of Rab2A expression alleviated hepatic lipid deposition in western diet-induced obesity (DIO) mice by reducing the protein level of PPARγ and the expression of PPARγ target genes. Our findings not only reveal a new molecular mechanism regulating the progression of NAFLD during persistent overnutrition but also have potential implications for drug discovery to combat this disease.

Bacteria have membrane-spanning efflux pumps to secrete toxic compounds ranging from heavy metal ions to organic chemicals, including antibiotic drugs. The overall architecture of these efflux pumps is highly conserved: with an inner membrane energy-transducing subunit coupled via an adaptor protein to an outer membrane conduit subunit that enables toxic compounds to be expelled into the environment. Here, we map the distribution of efflux pumps across bacterial lineages to show these proteins are more widespread than previously recognised. Complex phylogenetics support the concept that gene cassettes encoding the subunits for these pumps are commonly acquired by horizontal gene transfer. Using TolC as a model protein, we demonstrate that assembly of conduit subunits into the outer membrane uses the chaperone TAM to physically organise the membrane-embedded staves of the conduit subunit of the efflux pump. The characteristics of this assembly pathway have impact for the acquisition of efflux pumps across bacterial species and for the development of new antimicrobial compounds that inhibit efflux pump function.

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Lipid droplets (LDs) have increasingly been recognized as an essential organelle for eukaryotes. Although the biochemistry of lipid synthesis and degradation is well characterized, the regulation of LD dynamics, including its formation, maintenance, and secretion, is poorly understood. Here, we report that mice lacking Occludin (Ocln) show defective lipid metabolism. We show that LDs were larger than normal along its biogenesis and secretion pathway in Ocln null mammary cells. This defect in LD size control did not result from abnormal lipid synthesis or degradation; rather, it was because of secretion failure during the lactation stage. We found that OCLN was located on the LD membrane and was bound to essential regulators of lipid secretion, including BTN1a1 and XOR, in a C-terminus-dependent manner. Finally, OCLN was a phosphorylation target of Src kinase, whose loss causes lactation failure. Together, we demonstrate that Ocln is a downstream target of Src kinase and promotes LD secretion by binding to BTN1a1 and XOR.

In the clinic, most cases of congenital heart valve defects are thought to arise through errors that occur after the endothelial-mesenchymal transition (EndoMT) stage of valve development. Although mechanical forces caused by heartbeat are essential modulators of cardiovascular development, their role in these later developmental events is poorly understood. To address this question, we used the zebrafish superior atrioventricular valve (AV) as a model. We found that cellularized cushions of the superior atrioventricular canal (AVC) morph into valve leaflets via mesenchymal-endothelial transition (MEndoT) and tissue sheet delamination. Defects in delamination result in thickened, hyperplastic valves, and reduced heart function. Mechanical, chemical, and genetic perturbation of cardiac forces showed that mechanical stimuli are important regulators of valve delamination. Mechanistically, we show that forces modulate Nfatc activity to control delamination. Together, our results establish the cellular and molecular signature of cardiac valve delamination in vivo and demonstrate the continuous regulatory role of mechanical forces and blood flow during valve formation.

Anopheles gambiae melanization-based refractoriness to the human malaria parasite Plasmodium falciparum has rarely been observed in either laboratory or natural conditions, in contrast to the rodent model malaria parasite Plasmodium berghei that can become completely melanized by a TEP1 complement-like system-dependent mechanism. Multiple studies have shown that the rodent parasite evades this defense by recruiting the C-type lectins CTL4 and CTLMA2, while permissiveness to the human malaria parasite was not affected by partial depletion of these factors by RNAi silencing. Using CRISPR/Cas9-based CTL4 knockout, we show that A. gambiae can mount melanization-based refractoriness to the human malaria parasite, which is independent of the TEP1 complement-like system and the major anti-Plasmodium immune pathway Imd. Our study indicates a hierarchical specificity in the control of Plasmodium melanization and proves CTL4 as an essential host factor for P. falciparum transmission and one of the most potent mosquito-encoded malaria transmission-blocking targets.

Prokaryotes have numerous mobile genetic elements (MGEs) that mediate horizontal gene transfer (HGT) between cells. These elements can be costly, even deadly, and cells use numerous defense systems to filter, control, or inactivate them. Recent studies have shown that prophages, conjugative elements, their parasites (phage satellites and mobilizable elements), and other poorly described MGEs encode defense systems homologous to those of bacteria. These constitute a significant fraction of the repertoire of cellular defense genes. As components of MGEs, these defense systems have presumably evolved to provide them, not the cell, adaptive functions. While the interests of the host and MGEs are aligned when they face a common threat such as an infection by a virulent phage, defensive functions carried by MGEs might also play more selfish roles to fend off other antagonistic MGEs or to ensure their maintenance in the cell. MGEs are eventually lost from the surviving host genomes by mutational processes and their defense systems can be co-opted when they provide an advantage to the cell. The abundance of defense systems in MGEs thus sheds new light on the role, effect, and fate of the so-called "cellular defense systems," whereby they are not only merely microbial defensive weapons in a 2-partner arms race, but also tools of intragenomic conflict between multiple genetic elements with divergent interests that shape cell fate and gene flow at the population level.

Comment on PLoS Biol. 2022 Jan 10;20(1):e3001469.

Comment in PLoS Biol. 2022 Jan 11;20(1):e3001504.