Loose patch clamp membrane current measurements in cornus ammonis 1 neurons in murine hippocampal slices.

Hippocampal pyramidal neuronal activity has been previously studied using conventional patch clamp in isolated cells and brain slices. We here introduce the loose patch clamping study of voltage-activated currents from in situ pyramidal neurons in murine cornus ammonis 1 hippocampal coronal slices. Depolarizing pulses of 15-ms duration elicited early transient inward, followed by transient and prolonged outward currents in the readily identifiable junctional region between the stratum pyramidalis (SP) and oriens (SO) containing pyramidal cell somas and initial segments. These resembled pyramidal cell currents previously recorded using conventional patch clamp. Shortening the depolarizing pulses to >1-2 ms continued to evoke transient currents; hyperpolarizing pulses to varying voltages evoked decays whose time constants could be shortened to <1 ms, clarifying the speed of clamping in this experimental system. The inward and outward currents had distinct pharmacological characteristics and voltage-dependent inactivation and recovery from inactivation. Comparative recordings from the SP, known to contain pyramidal cell somas, demonstrated similar current properties. Recordings from the SO and stratum radiatum demonstrated smaller inward and outward current magnitudes and reduced transient outward currents, consistent with previous conventional patch clamp results from their different interneuron types. The loose patch clamp method is thus useful for in situ studies of neurons in hippocampal brain slices.

Rethinking 13C-metabolic flux analysis - The Bayesian way of flux inference.

Metabolic reaction rates (fluxes) play a crucial role in comprehending cellular phenotypes and are essential in areas such as metabolic engineering, biotechnology, and biomedical research. The state-of-the-art technique for estimating fluxes is metabolic flux analysis using isotopic labelling (13C-MFA), which uses a dataset-model combination to determine the fluxes. Bayesian statistical methods are gaining popularity in the field of life sciences, but the use of 13C-MFA is still dominated by conventional best-fit approaches. The slow take-up of Bayesian approaches is, at least partly, due to the unfamiliarity of Bayesian methods to metabolic engineering researchers. To address this unfamiliarity, we here outline similarities and differences between the two approaches and highlight particular advantages of the Bayesian way of flux analysis. With a real-life example, re-analysing a moderately informative labelling dataset of E. coli, we identify situations in which Bayesian methods are advantageous and more informative, pointing to potential pitfalls of current 13C-MFA evaluation approaches. We propose the use of Bayesian model averaging (BMA) for flux inference as a means of overcoming the problem of model uncertainty through its tendency to assign low probabilities to both, models that are unsupported by data, and models that are overly complex. In this capacity, BMA resembles a tempered Ockham's razor. With the tempered razor as a guide, BMA-based 13C-MFA alleviates the problem of model selection uncertainty and is thereby capable of becoming a game changer for metabolic engineering by uncovering new insights and inspiring novel approaches.

Carving Nature at Its Joints: A Comparison of CEMI Field Theory with Integrated Information Theory and Global Workspace Theory.

The quest to comprehend the nature of consciousness has spurred the development of many theories that seek to explain its underlying mechanisms and account for its neural correlates. In this paper, I compare my own conscious electromagnetic information field (cemi field) theory with integrated information theory (IIT) and global workspace theory (GWT) for their ability to 'carve nature at its joints' in the sense of predicting the entities, structures, states and dynamics that are conventionally recognized as being conscious or nonconscious. I go on to argue that, though the cemi field theory shares features of both integrated information theory and global workspace theory, it is more successful at carving nature at its conventionally accepted joints between conscious and nonconscious systems, and is thereby a more successful theory of consciousness.

Razor sharp: The role of Occam's razor in science.

Occam's razor-the principle of simplicity-has recently been attacked as a cultural bias without rational foundation. Increasingly, belief in pseudoscience and mysticism is growing. I argue that inclusion of Occam's razor is an essential factor that distinguishes science from superstition and pseudoscience. I also describe how the razor is embedded in Bayesian inference and argue that science is primarily the means to discover the simplest descriptions of our world.

Anti-persister efficacy of colistin and meropenem against uropathogenic Escherichia coli is dependent on environmental conditions.

Antibiotic persistence is a phenomenon observed when genetically susceptible cells survive long-term exposure to antibiotics. These 'persisters' are an intrinsic component of bacterial populations and stem from phenotypic heterogeneity. Persistence to antibiotics is a concern for public health globally, as it increases treatment duration and can contribute to treatment failure. Furthermore, there is a growing array of evidence that persistence is a 'stepping-stone' for the development of genetic antimicrobial resistance. Urinary tract infections (UTIs) are a major contributor to antibiotic consumption worldwide, and are known to be both persistent (i.e. affecting the host for a prolonged period) and recurring. Currently, in clinical settings, routine laboratory screening of pathogenic isolates does not determine the presence or the frequency of persister cells. Furthermore, the majority of research undertaken on antibiotic persistence has been done on lab-adapted bacterial strains. In the study presented here, we characterized antibiotic persisters in a panel of clinical uropathogenic Escherichia coli isolates collected from hospitals in the UK and Australia. We found that a urine-pH mimicking environment not only induces higher levels of antibiotic persistence to meropenem and colistin than standard laboratory growth conditions, but also results in rapid development of transient colistin resistance, regardless of the genetic resistance profile of the isolate. Furthermore, we provide evidence for the presence of multiple virulence factors involved in stress resistance and biofilm formation in the genomes of these isolates, whose activities have been previously shown to contribute to the formation of persister cells.

Development of a loop-mediated isothermal amplification (LAMP)-based electrochemical test for rapid detection of SARS-CoV-2.

Rapid, reliable, sensitive, portable, and accurate diagnostics are required to control disease outbreaks such as COVID-19 that pose an immense burden on human health and the global economy. Here we developed a loop-mediated isothermal amplification (LAMP)-based electrochemical test for the detection of SARS-CoV-2 that causes COVID-19. The test is based on the oxidation-reduction reaction between pyrophosphates (generated from positive LAMP reaction) and molybdate that is detected by cyclic voltammetry using inexpensive and disposable carbon screen printed electrodes. Our test showed higher sensitivity (detecting as low as 5.29 RNA copies/µL) compared to the conventional fluorescent reverse transcriptase (RT)-LAMP. We validated our tests using human serum and saliva spiked with SARS-CoV-2 RNA and clinical (saliva and nasal-pharyngeal) swab samples demonstrating 100% specificity and 93.33% sensitivity. Our assay provides a rapid, specific, and sensitive test with an electrochemical readout in less than 45 min that could be adapted for point-of-care settings.

Dysregulated NAD(H) homeostasis associated with ciprofloxacin tolerance in Escherichia coli investigated on a single-cell level with the Peredox [NADH:NAD+] biosensor.

Introduction: Antibiotic persistence (subpopulation tolerance) occurs when a subpopulation of antibiotic sensitive cells survives prolonged exposure to a bactericidal concentration of an antibiotic, and is capable of regrowth once the antibiotic is removed. This phenomenon has been shown to contribute to prolonged treatment duration, infection recurrence, and accelerated development of genetic resistance. Currently, there are no biomarkers which would allow for segregation of these antibiotic-tolerant cells from the bulk population prior to antibiotic exposure, limiting research on this phenomenon to retrograde analyses. However, it has been previously shown that persisters often have a dysregulated intracellular redox homeostasis, warranting its investigation as a potential marker for antibiotic tolerance. Furthermore, it is currently unknown whether another antibiotic tolerant subpopulation - viable but non-culturable cells (VBNCs), are simply persisters with extreme lag phase, or are formed through separate pathways. VBNCs similarly to persisters remain viable following antibiotic exposure, however, are not capable of regrowth in standard conditions. Methods: In this article we employed an NADH:NAD+ biosensor (Peredox) to investigate NADH homeostasis of ciprofloxacin-tolerant E. coli cells on a single-cell level. [NADH:NAD+] was used as a proxy for measuring intracellular redox homeostasis and respiration rate. Results and Discussion: First, we demonstrated that ciprofloxacin exposure results in a high number of VBNCs, several orders of magnitude higher than persisters. However, we found no correlation in the frequencies of persister and VBNC subpopulations. Ciprofloxacin-tolerant cells (persisters & VBNCs) were actively undergoing respiration, although at a significantly lower rate on average when compared to the bulk population. We also noted significant heterogeneity on a single-cell level within the subpopulations, however were unable to segregate persisters from VBNCs based on these observations alone. Finally, we showed that in the highly-persistent strain of E. coli, E. coli HipQ, ciprofloxacin-tolerant cells have a significantly lower [NADH:NAD+] ratio than tolerant cells of its parental strain, providing further link between disturbed NADH homeostasis and antibiotic tolerance.

One-shot 13 C15 N-metabolic flux analysis for simultaneous quantification of carbon and nitrogen flux.

Metabolic flux is the final output of cellular regulation and has been extensively studied for carbon but much less is known about nitrogen, which is another important building block for living organisms. For the tuberculosis pathogen, this is particularly important in informing the development of effective drugs targeting the pathogen's metabolism. Here we performed 13 C15 N dual isotopic labeling of Mycobacterium bovis BCG steady state cultures, quantified intracellular carbon and nitrogen fluxes and inferred reaction bidirectionalities. This was achieved by model scope extension and refinement, implemented in a multi-atom transition model, within the statistical framework of Bayesian model averaging (BMA). Using BMA-based 13 C15 N-metabolic flux analysis, we jointly resolve carbon and nitrogen fluxes quantitatively. We provide the first nitrogen flux distributions for amino acid and nucleotide biosynthesis in mycobacteria and establish glutamate as the central node for nitrogen metabolism. We improved resolution of the notoriously elusive anaplerotic node in central carbon metabolism and revealed possible operation modes. Our study provides a powerful and statistically rigorous platform to simultaneously infer carbon and nitrogen metabolism in any biological system.

Challenges for the development of a universal vaccine against leptospirosis revealed by the evaluation of 22 vaccine candidates.

Leptospirosis is a neglected disease of man and animals that affects nearly half a million people annually and causes considerable economic losses. Current human vaccines are inactivated whole-cell preparations (bacterins) of Leptospira spp. that provide strong homologous protection yet fail to induce a cross-protective immune response. Yearly boosters are required, and serious side-effects are frequently reported so the vaccine is licensed for use in humans in only a handful of countries. Novel universal vaccines require identification of conserved surface-exposed epitopes of leptospiral antigens. Outer membrane ß-barrel proteins (ßb-OMPs) meet these requirements and have been successfully used as vaccines for other diseases. We report the evaluation of 22 constructs containing protein fragments from 33 leptospiral ßb-OMPs, previously identified by reverse and structural vaccinology and cell-surface immunoprecipitation. Three-dimensional structures for each leptospiral ßb-OMP were predicted by I-TASSER. The surface-exposed epitopes were predicted using NetMHCII 2.2 and BepiPred 2.0. Recombinant constructs containing regions from one or more ßb-OMPs were cloned and expressed in Escherichia coli. IMAC-purified recombinant proteins were adsorbed to an aluminium hydroxide adjuvant to produce the vaccine formulations. Hamsters (4-6 weeks old) were vaccinated with 2 doses containing 50 - 125 µg of recombinant protein, with a 14-day interval between doses. Immunoprotection was evaluated in the hamster model of leptospirosis against a homologous challenge (10 - 20× ED50) with L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130. Of the vaccine formulations, 20/22 were immunogenic and induced significant humoral immune responses (IgG) prior to challenge. Four constructs induced significant protection (100%, P < 0.001) and sterilizing immunity in two independent experiments, however, this was not reproducible in subsequent evaluations (0 - 33.3% protection, P > 0.05). The lack of reproducibility seen in these challenge experiments and in other reports in the literature, together with the lack of immune correlates and commercially available reagents to characterize the immune response, suggest that the hamster may not be the ideal model for evaluation of leptospirosis vaccines and highlight the need for evaluation of alternative models, such as the mouse.

Thapsigargin blocks electromagnetic field-elicited intracellular Ca2+ increase in HEK 293 cells.

Biological effects of electromagnetic fields (EMFs) have previously been identified for cellular proliferation and changes in expression and conduction of diverse types of ion channels. The major effect elicited by EMFs seems to be directed toward Ca2+ homeostasis. This is particularly remarkable since Ca2+ acts as a central modulator in various signaling pathways, including, but not limited to, cell differentiation and survival. Despite this, the mechanisms underlying this modulation have yet to be unraveled. Here, we assessed the effect of EMFs on intracellular [Ca2+ ], by exposing HEK 293 cells to both radio-frequency electromagnetic fields (RF-EMFs) and static magnetic fields (SMFs). We detected a constant and significant increase in [Ca2+ ] subsequent to exposure to both types of fields. Strikingly, the increase was nulled by administration of 10 µM Thapsigargin, a blocker of sarco/endoplasmic reticulum Ca2+ -ATPases (SERCAs), indicating the involvement of the endoplasmic reticulum (ER) in EMF-related modulation of Ca2+ homeostasis.

Consciousness: Matter or EMF?

Conventional theories of consciousness (ToCs) that assume that the substrate of consciousness is the brain's neuronal matter fail to account for fundamental features of consciousness, such as the binding problem. Field ToC's propose that the substrate of consciousness is the brain's best accounted by some kind of field in the brain. Electromagnetic (EM) ToCs propose that the conscious field is the brain's well-known EM field. EM-ToCs were first proposed only around 20 years ago primarily to account for the experimental discovery that synchronous neuronal firing was the strongest neural correlate of consciousness (NCC). Although EM-ToCs are gaining increasing support, they remain controversial and are often ignored by neurobiologists and philosophers and passed over in most published reviews of consciousness. In this review I examine EM-ToCs against established criteria for distinguishing between ToCs and demonstrate that they outperform all conventional ToCs and provide novel insights into the nature of consciousness as well as a feasible route toward building artificial consciousnesses.

Dissecting Host-Pathogen Interactions in TB Using Systems-Based Omic Approaches.

Tuberculosis (TB) is a devastating infectious disease that kills over a million people every year. There is an increasing burden of multi drug resistance (MDR) and extensively drug resistance (XDR) TB. New and improved therapies are urgently needed to overcome the limitations of current treatment. The causative agent, Mycobacterium tuberculosis (Mtb) is one of the most successful pathogens that can manipulate host cell environment for adaptation, evading immune defences, virulence, and pathogenesis of TB infection. Host-pathogen interaction is important to establish infection and it involves a complex set of processes. Metabolic cross talk between the host and pathogen is a facet of TB infection and has been an important topic of research where there is growing interest in developing therapies and drugs that target these interactions and metabolism of the pathogen in the host. Mtb scavenges multiple nutrient sources from the host and has adapted its metabolism to survive in the intracellular niche. Advancements in systems-based omic technologies have been successful to unravel host-pathogen interactions in TB. In this review we discuss the application and usefulness of omics in TB research that provides promising interventions for developing anti-TB therapies.

Lactate-Induced Dispersal of Neisseria meningitidis Microcolonies Is Mediated by Changes in Cell Density and Pilus Retraction and Is Influenced by Temperature Change.

Neisseria meningitidis is the etiologic agent of meningococcal meningitis and sepsis. Initial colonization of meningococci in the upper respiratory tract epithelium is crucial for disease development. The colonization occurs in several steps and expression of type IV pili (Tfp) is essential for both attachment and microcolony formation of encapsulated bacteria. Previously, we have shown that host-derived lactate induces synchronized dispersal of meningococcal microcolonies. In this study, we demonstrated that lactate-induced dispersal is dependent on bacterial concentration but not on the quorum-sensing system autoinducer-2 or the two-component systems NarP/NarQ, PilR/PilS, NtrY/NtrX, and MisR/MisS. Further, there were no changes in expression of genes related to assembly, elongation, retraction, and modification of Tfp throughout the time course of lactate induction. By using pilT and pptB mutants, however, we found that lactate-induced dispersal was dependent on PilT retraction but not on phosphoglycerol modification of Tfp even though the PptB activity was important for preventing reaggregation postdispersal. Furthermore, protein synthesis was required for lactate-induced dispersal. Finally, we found that at a lower temperature, lactate-induced dispersal was delayed and unsynchronized, and bacteria reformed microcolonies. We conclude that lactate-induced microcolony dispersal is dependent on bacterial concentration, PilT-dependent Tfp retraction, and protein synthesis and is influenced by environmental temperature.

Effects of electromagnetic fields on neuronal ion channels: a systematic review.

Many aspects of chemistry and biology are mediated by electromagnetic field (EMF) interactions. The central nervous system (CNS) is particularly sensitive to EMF stimuli. Studies have explored the direct effect of different EMFs on the electrical properties of neurons in the last two decades, particularly focusing on the role of voltage-gated ion channels (VGCs). This work aims to systematically review published evidence in the last two decades detailing the effects of EMFs on neuronal ion channels as per the PRISM guidelines. Following a predetermined exclusion and inclusion criteria, 22 papers were included after searches on three online databases. Changes in calcium homeostasis, attributable to the voltage-gated calcium channels, were found to be the most commonly reported result of EMF exposure. EMF effects on the neuronal landscape appear to be diverse and greatly dependent on parameters, such as the field's frequency, exposure time, and intrinsic properties of the irradiated tissue, such as the expression of VGCs. Here, we systematically clarify how neuronal ion channels are particularly affected and differentially modulated by EMFs at multiple levels, such as gating dynamics, ion conductance, concentration in the membrane, and gene and protein expression. Ion channels represent a major transducer for EMF-related effects on the CNS.

Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis.

The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.

Development of Loop-Mediated Isothermal Amplification Rapid Diagnostic Assays for the Detection of Klebsiella pneumoniae and Carbapenemase Genes in Clinical Samples.

Klebsiella pneumoniae is an important pathogenic bacterium commonly associated with human healthcare and community-acquired infections. In recent years, K. pneumoniae has become a significant threat to global public and veterinary health, because of its high rates of antimicrobial resistance (AMR). Early diagnosis of K. pneumoniae infection and detection of any associated AMR would help to accelerate directed therapy and reduce the risk of the emergence of multidrug-resistant isolates. In this study, we identified three target genes (yhaI, epsL, and xcpW) common to K. pneumoniae isolates from both China and Europe and designed loop-mediated isothermal amplification (LAMP) assays for the detection of K. pneumoniae in clinical samples. We also designed LAMP assays for the detection of five AMR genes commonly associated with K. pneumoniae. The LAMP assays were validated on a total of 319 type reference strains and clinical isolates of diverse genetic backgrounds, in addition to 40 clinical human sputum samples, and were shown to be reliable, highly specific, and sensitive. For the K. pneumoniae-specific LAMP assay, the calculated sensitivity, specificity, and positive and negative predictive values (comparison with culture and matrix-assisted laser desorption/ionization-time of flight mass spectrometry) were all 100% on clinical isolates and, respectively, of 100%, 91%, and 90%, and 100% when tested on clinical sputum samples, while being significantly faster than the reference methods. For the bla KPC and other carbapenemases' LAMP assays, the concordance between the LAMP results and the references methods (susceptibility tests) was 100%, on both pure cultures (n = 125) and clinical samples (n = 18). In conclusion, we developed highly sensitive and specific LAMP assays for the clinical identification of K. pneumoniae and detection of carbapenem resistance.

Author Correction: Development of a diagnostic compatible BCG vaccine against Bovine tuberculosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Integrating information in the brain's EM field: the cemi field theory of consciousness.

A key aspect of consciousness is that it represents bound or integrated information, prompting an increasing conviction that the physical substrate of consciousness must be capable of encoding integrated information in the brain. However, as Ralph Landauer insisted, 'information is physical' so integrated information must be physically integrated. I argue here that nearly all examples of so-called 'integrated information', including neuronal information processing and conventional computing, are only temporally integrated in the sense that outputs are correlated with multiple inputs: the information integration is implemented in time, rather than space, and thereby cannot correspond to physically integrated information. I point out that only energy fields are capable of integrating information in space. I describe the conscious electromagnetic information (cemi) field theory which has proposed that consciousness is physically integrated, and causally active, information encoded in the brain's global electromagnetic (EM) field. I here extend the theory to argue that consciousness implements algorithms in space, rather than time, within the brain's EM field. I describe how the cemi field theory accounts for most observed features of consciousness and describe recent experimental support for the theory. I also describe several untested predictions of the theory and discuss its implications for the design of artificial consciousness. The cemi field theory proposes a scientific dualism that is rooted in the difference between matter and energy, rather than matter and spirit.

Genetic screening for the protective antigenic targets of BCG vaccination.

Bovine tuberculosis is an important animal health problem and the predominant cause of zoonotic tuberculosis worldwide. It results in serious economic burden due to losses in productivity and the cost of control programmes. Control could be greatly improved by the introduction of an efficacious cattle vaccine but the most likely candidate, BCG, has several limitations including variable efficacy. Augmentation of BCG with a subunit vaccine booster has been shown to increase protection but the selection of antigens has hitherto been left largely to serendipity. In the present study, we take a rational approach to identify the protective antigens of BCG, selecting a BCG transposon mutant library in naïve and BCG-vaccinated cattle. Ten mutants had increased relative survival in vaccinated compared to naïve cattle, consistent with loss of protective antigen targets making the mutants less visible to the BCG immune response. The immunogenicity of three putative protective antigens, BCG_0116, BCG_0205 (YrbE1B) and BCG_1448 (PPE20) was investigated using peptide pools and PBMCs from BCG vaccinated cattle. BCG vaccination induced PBMC to release elevated levels of IP10, IL-17a and IL-10 in response to all three antigens. Taken together, the data supports the further study of these antigens for use in subunit vaccines.