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Infant maltreatment increases vulnerability to physical and mental disorders, yet specific mechanisms embedded within this complex infant experience that induce this vulnerability remain elusive. To define critical features of maltreatment-induced vulnerability, rat pups were reared from postnatal day 8 (PN8) with a maltreating mother, which produced amygdala and hippocampal deficits and decreased social behavior at PN13. Next, we deconstructed the maltreatment experience to reveal sufficient and necessary conditions to induce this phenotype. Social behavior and amygdala deficits (volume, neurogenesis, c-Fos, local field potential) required combined chronic high corticosterone and maternal presence (not maternal behavior). Hippocampal deficits were induced by chronic high corticosterone regardless of social context. Causation was shown by blocking corticosterone during maltreatment and suppressing amygdala activity during social behavior testing. These results highlight (1) that early life maltreatment initiates multiple pathways to pathology, each with distinct causal mechanisms and outcomes, and (2) the importance of social presence on brain development.

TRAAK is a membrane tension-activated K+ channel that has been associated through behavioral studies to mechanical nociception. We used specific monoclonal antibodies in mice to show that TRAAK is localized exclusively to nodes of Ranvier, the action potential propagating elements of myelinated nerve fibers. Approximately 80 percent of myelinated nerve fibers throughout the central and peripheral nervous system contain TRAAK in what is likely an all-nodes or no-nodes per axon fashion. TRAAK is not observed at the axon initial segment where action potentials are first generated. We used polyclonal antibodies, the TRAAK inhibitor RU2 and node clamp amplifiers to demonstrate the presence and functional properties of TRAAK in rat nerve fibers. TRAAK contributes to the 'leak' K+ current in mammalian nerve fiber conduction by hyperpolarizing the resting membrane potential, thereby increasing Na+ channel availability for action potential propagation. We speculate on why nodes of Ranvier contain a mechanosensitive K+ channel.

DNA is both a fundamental building block of life and a fascinating natural polymer. The advent of single-molecule manipulation tools made it possible to exert controlled force on individual DNA molecules and measure their mechanical response. Such investigations elucidated the elastic properties of DNA and revealed its distinctive structural configurations across force regimes. In the meantime, a detailed understanding of DNA mechanics laid the groundwork for single-molecule studies of DNA-binding proteins and DNA-processing enzymes that bend, stretch, and twist DNA. These studies shed new light on the metabolism and transactions of nucleic acids, which constitute a major part of the cell's operating system. Furthermore, the marriage of single-molecule fluorescence visualization and force manipulation has enabled researchers to directly correlate the applied tension to changes in the DNA structure and the behavior of DNA-templated complexes. Overall, experimental exploitation of DNA mechanics has been and will continue to be a unique and powerful strategy for understanding how molecular machineries recognize and modify the physical state of DNA to accomplish their biological functions.

Protein-surface interactions play a pivotal role in processes as diverse as biomineralization, biofouling, and the cellular response to medical implants. In biomineralization processes, biomacromolecules control mineral deposition and architecture via complex and often unknown mechanisms. For studying these mechanisms, the formation of magnetite nanoparticles in magnetotactic bacteria has become an excellent model system. Most interestingly, nanoparticle morphologies have been discovered that defy crystallographic rules (e.g., in the species Desulfamplus magnetovallimortis strain BW-1). In certain conditions, this strain mineralizes bullet-shaped magnetite nanoparticles, which exhibit defined (111) crystal faces and are elongated along the [100] direction. We hypothesize that surface-specific protein interactions break the nanoparticle symmetry, inhibiting the growth of certain crystal faces and thereby favoring the growth of others. Screening the genome of BW-1, we identified Mad10 (Magnetosome-associated deep-branching) as a potential magnetite-binding protein. Using atomic force microscope (AFM)-based single-molecule force spectroscopy, we show that a Mad10-derived peptide, which represents the most conserved region of Mad10, binds strongly to (100)- and (111)-oriented single-crystalline magnetite thin films. The peptide-magnetite interaction is thus material- but not crystal-face-specific. It is characterized by broad rupture force distributions that do not depend on the retraction speed of the AFM cantilever. To account for these experimental findings, we introduce a three-state model that incorporates fast rebinding. The model suggests that the peptide-surface interaction is strong in the absence of load, which is a direct result of this fast rebinding process. Overall, our study sheds light on the kinetic nature of peptide-surface interactions and introduces a new magnetite-binding peptide with potential use as a functional coating for magnetite nanoparticles in biotechnological and biomedical applications.

Intrinsically disordered regions (IDRs) of proteins are implicated in key macromolecular interactions. However, the molecular forces underlying IDR function within multicomponent assemblies remain elusive. By combining thermodynamic and structural data, we have discovered an allostery-based mechanism regulating the soluble core region of the nuclear pore complex (NPC) composed of nucleoporins Nup53, Nic96, and Nup157. We have identified distinct IDRs in Nup53 that are functionally coupled when binding to partner nucleoporins and karyopherins (Kaps) involved in NPC assembly and nucleocytoplasmic transport. We show that the Nup53.Kap121 complex forms an ensemble of structures that destabilize Nup53 hub interactions. Our study provides a molecular framework for understanding how disordered and folded domains communicate within macromolecular complexes.

Clostridium perfringens epsilon toxin (ETX) is responsible for causing the economically devastating disease, enterotoxaemia, in livestock. It is well accepted that ETX causes blood brain barrier (BBB) permeability, however the mechanisms involved in this process are not well understood. Using in vivo and in vitro methods, we determined that ETX causes BBB permeability in mice by increasing caveolae-dependent transcytosis in brain endothelial cells. When mice are intravenously injected with ETX, robust ETX binding is observed in the microvasculature of the central nervous system (CNS) with limited to no binding observed in the vasculature of peripheral organs, indicating that ETX specifically targets CNS endothelial cells. ETX binding to CNS microvasculature is dependent on MAL expression, as ETX binding to CNS microvasculature of MAL-deficient mice was not detected. ETX treatment also induces extravasation of molecular tracers including 376Da fluorescein salt, 60kDA serum albumin, 70kDa dextran, and 155kDA IgG. Importantly, ETX-induced BBB permeability requires expression of both MAL and caveolin-1, as mice deficient in MAL or caveolin-1 did not exhibit ETX-induced BBB permeability. Examination of primary murine brain endothelial cells revealed an increase in caveolae in ETX-treated cells, resulting in dynamin and lipid raft-dependent vacuolation without cell death. ETX-treatment also results in a rapid loss of EEA1 positive early endosomes and accumulation of large, RAB7-positive late endosomes and multivesicular bodies. Based on these results, we hypothesize that ETX binds to MAL on the apical surface of brain endothelial cells, causing recruitment of caveolin-1, triggering caveolae formation and internalization. Internalized caveolae fuse with early endosomes which traffic to late endosomes and multivesicular bodies. We believe that these multivesicular bodies fuse basally, releasing their contents into the brain parenchyma.

The ability to determine full-length nucleotide composition of individual RNA molecules is essential for understanding the architecture and function of a transcriptome. However, experimental approaches capable of capturing the sequences of both 5' and 3' termini of the same transcript remain scarce. In the present study, simultaneous 5' and 3' end sequencing (SEnd-seq)-a high-throughput and unbiased method that simultaneously maps transcription start and termination sites with single-nucleotide resolution-is presented. Using this method, a comprehensive view of the Escherichia coli transcriptome was obtained, which displays an unexpected level of complexity. SEnd-seq notably expands the catalogue of transcription start sites and termination sites, defines unique transcription units and detects prevalent antisense RNA. Strikingly, the results of the present study unveil widespread overlapping bidirectional terminators located between opposing gene pairs. Furthermore, it has been shown that convergent transcription is a major contributor to highly efficient bidirectional termination both in vitro and in vivo. This finding highlights an underappreciated role of RNA polymerase conflicts in shaping transcript boundaries and suggests an evolutionary strategy for modulating transcriptional output by arranging gene orientation.

BackgroundThere continues to be a great need for better biomarkers and host-directed treatment targets for community-acquired pneumonia (CAP). Alterations in phospholipid metabolism may constitute a source of small molecule biomarkers for acute infections including CAP. Evidence from animal models of pulmonary infections and sepsis suggests that inhibiting acid sphingomyelinase (which releases ceramides from sphingomyelins) may reduce end-organ damage.MethodsWe measured concentrations of 105 phospholipids, 40 acylcarnitines, and 4 ceramides, as well as acid sphingomyelinase activity, in plasma from patients with CAP (n=29, sampled on admission and 4 subsequent time points), chronic obstructive pulmonary disease exacerbation with infection (COPD, n=13) as a clinically important disease control, and 33 age- and sex-matched controls.ResultsPhospholipid concentrations were greatly decreased in CAP and normalized along clinical improvement. Greatest changes were seen in phosphatidylcholines, followed by lysophosphatidylcholines, sphingomyelins and ceramides (three of which were upregulated), and were least in acylcarnitines. Changes in COPD were less pronounced, but also differed qualitatively, e.g. by increases in selected sphingomyelins. We identified highly accurate biomarkers for CAP (AUC <= 0.97) and COPD (AUC <= 0.93) vs. Controls, and moderately accurate biomarkers for CAP vs. COPD (AUC <= 0.83), all of which were phospholipids. Phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins were also markedly decreased in S. aureus-infected human A549 and differentiated THP1 cells. Correlations with C-reactive protein and procalcitonin were predominantly negative but only of mild-to-moderate extent, suggesting that these markers reflect more than merely inflammation. Consistent with the increased ceramide concentrations, increased acid sphingomyelinase activity accurately distinguished CAP (fold change=2.8, AUC=0.94) and COPD (1.75, 0.88) from Controls and normalized with clinical resolution.ConclusionsThe results underscore the high potential of plasma phospholipids as biomarkers for CAP, begin to reveal differences in lipid dysregulation between CAP and infection-associated COPD exacerbation, and suggest that the decreases in plasma concentrations are at least partially determined by changes in host target cells. Furthermore, they provide validation in clinical blood samples of acid sphingomyelinase as a potential treatment target to improve clinical outcome of CAP.

Methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage is a major risk factor for infection, namely among populations in the community with inherent prompting factors, such as the homeless. In Portugal, there are no data on S. aureus/MRSA nasal carriage among the homeless community. A total of 84 homeless individuals living in Lisbon (34 with no permanent address and 50 living in shelter) were nasally screened for S. aureus/ MRSA. All isolates were characterized to determine antimicrobial susceptibility and clonal type. A total of 43 (51.2%) S. aureus carriers were identified, including a single individual colonized with MRSA (1.2%). S. aureus carriage rate was higher among individuals with no permanent address (58.8% versus 46%), younger (45.7 +/- 12.7 versus 52.5 +/- 10.8 years), and with diagnosis of asthma (9% versus 0%). The single MRSA belonged to the EMRSA-15 clone (PFGE D, ST15-SCCmec IVh, and spa type t790). Almost half of the methicillin-susceptible S. aureus (MSSA) isolates (41.9%, n = 18) belonged to two major clones, ST398-t1451 (n = 13) and ST30-t399/t11980/t12808 associated with PFGE I (n = 5). A high proportion of isolates showed non-susceptibility to mupirocin (64%), erythromycin (45%), and fusidic acid (20%) and induced resistance to clindamycin (39%). None of the isolates harboured PVL. Our results suggest that the homeless population of Lisbon does not constitute a reservoir of MRSA in the community, but harbour the highly transmissible ST398-t1451 MSSA lineage.