A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging.

Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7× increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca2+ responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca2+ concentration using fluorescence lifetime imaging microscopy (FLIM).

Extended-spectrum beta-lactamase (ESBL)- and non-ESBL producing Escherichia coli surveillance in surface water sources in Edo State, Nigeria: a public health concern.

This research explores the antimicrobial resistance (AMR) profiles and prevalence of extended-spectrum beta-lactamase (ESBL) and non-ESBL-producing Escherichia coli in Ojerame Dam and Ovokoto Spring, Edo State, Nigeria. Over 12 months, water was systematically sampled to accommodate seasonal variations and analyzed by employing an ESBL-selective medium for bacterial species. Additionally, bacterial isolates underwent identification and characterization using polymerase chain reaction (PCR) and disk diffusion methods to evaluate their susceptibility to antimicrobials. Results indicated significant prevalence of ESBL-producing E. coli, which exhibited complete resistance to common antimicrobials like ceftriaxone, ceftazidime, cefotaxime, and ampicillin while demonstrating 100% sensitivity to ertapenem, imipenem, meropenem, and nitrofurantoin. Non-ESBL-producing E. coli were resistant to ampicillin but sensitive to other antimicrobials mentioned earlier. Furthermore, both ESBL and non-ESBL-producing E. coli displayed multidrug resistance to varying degrees. Specific ESBL genes, including blaTEM, blaCTX-M-1, and blaCTX-M-15, were identified, alongside resistance genes like tetA, tetM, sul1, sul2, sul3, qnrA, qnrB, and qnrS in E. coli. This study pioneers the documentation of ESBL-producing E. coli in surface water in the region. This signals impending health risks associated with water being a reservoir of resistant genes while emphasizing the urgency for further research and public awareness concerning the quality of surface water.

Genomic insights into the evolution of secondary metabolism of Escovopsis and its allies, specialized fungal symbionts of fungus-farming ants.

The metabolic intimacy of symbiosis often demands the work of specialists. Natural products and defensive secondary metabolites can drive specificity by ensuring infection and propagation across host generations. But in contrast to bacteria, little is known about the diversity and distribution of natural product biosynthetic pathways among fungi and how they evolve to facilitate symbiosis and adaptation to their host environment. In this study, we define the secondary metabolism of Escovopsis and closely related genera, symbionts in the gardens of fungus-farming ants. We ask how the gain and loss of various biosynthetic pathways correspond to divergent lifestyles. Long-read sequencing allowed us to define the chromosomal features of representative Escovopsis strains, revealing highly reduced genomes composed of seven to eight chromosomes. The genomes are highly syntenic with macrosynteny decreasing with increasing phylogenetic distance, while maintaining a high degree of mesosynteny. An ancestral state reconstruction analysis of biosynthetic pathways revealed that, while many secondary metabolites are shared with non-ant-associated Sordariomycetes, 56 pathways are unique to the symbiotic genera. Reflecting adaptation to diverging ant agricultural systems, we observe that the stepwise acquisition of these pathways mirrors the ecological radiations of attine ants and the dynamic recruitment and replacement of their fungal cultivars. As different clades encode characteristic combinations of biosynthetic gene clusters, these delineating profiles provide important insights into the possible mechanisms underlying specificity between these symbionts and their fungal hosts. Collectively, our findings shed light on the evolutionary dynamic nature of secondary metabolism in Escovopsis and its allies, reflecting adaptation of the symbionts to an ancient agricultural system.IMPORTANCEMicrobial symbionts interact with their hosts and competitors through a remarkable array of secondary metabolites and natural products. Here, we highlight the highly streamlined genomic features of attine-associated fungal symbionts. The genomes of Escovopsis species, as well as species from other symbiont genera, many of which are common with the gardens of fungus-growing ants, are defined by seven chromosomes. Despite a high degree of metabolic conservation, we observe some variation in the symbionts' potential to produce secondary metabolites. As the phylogenetic distribution of the encoding biosynthetic gene clusters coincides with attine transitions in agricultural systems, we highlight the likely role of these metabolites in mediating adaptation by a group of highly specialized symbionts.

Characterization of resistance and virulence factors in livestock-associated methicillin-resistant Staphylococcus aureus.

The study investigated the economic concerns associated with livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in livestock (cow), examining its connection to severe infections, antimicrobial resistance (AMR), and virulence factors. The research, conducted in Edo State, Nigeria, analyzed 400 samples (200 rectal and 200 nasal swabs) collected between March 2018 and February 2019. MRSA prevalence was identified using conventional culture-based methods and polymerase chain reaction (PCR) techniques, revealing 63.5% (n = 254) for Staphylococcus aureus and 55% (n = 220) for MRSA. Of the 76 mecA-positive MRSA isolates, 64.5% (n = 49) exhibited multidrug resistance (MDR) while the remaining were sensitive to specific antimicrobials. Key virulence genes, such as PVL (81.6%; n = 62) and tsst-1 (44.7%; n = 34), were prevalent, along with AMR genes like mecC, tetM, ermA, ermC, vanA, and vanC. Staphylococcal chromosomal cassette mec (SCCmec) typing identified different types, notably II, IVa, and IVb. Biofilm formation, a crucial virulence factor varied in strength, is associated with icaA and icaB genes (p < 0.01). The findings highlighted substantial AMR and biofilm-forming capacity within LA-MRSA isolates, emphasizing the importance of ongoing surveillance for informed treatment strategies, AMR policies, and control measures against MDR staphylococcal infections.

Near-infrared nanosensors enable optical imaging of oxytocin with selectivity over vasopressin in acute mouse brain slices.

Oxytocin plays a critical role in regulating social behaviors, yet our understanding of its function in both neurological health and disease remains incomplete. Real-time oxytocin imaging probes with spatiotemporal resolution relevant to its endogenous signaling are required to fully elucidate oxytocin's role in the brain. Herein, we describe a near-infrared oxytocin nanosensor (nIROXT), a synthetic probe capable of imaging oxytocin in the brain without interference from its structural analogue, vasopressin. nIROXT leverages the inherent tissue-transparent fluorescence of single-walled carbon nanotubes (SWCNT) and the molecular recognition capacity of an oxytocin receptor peptide fragment to selectively and reversibly image oxytocin. We employ these nanosensors to monitor electrically stimulated oxytocin release in brain tissue, revealing oxytocin release sites with a median size of 3 µm in the paraventricular nucleus of C57BL/6 mice, which putatively represents the spatial diffusion of oxytocin from its point of release. These data demonstrate that covalent SWCNT constructs, such as nIROXT, are powerful optical tools that can be leveraged to measure neuropeptide release in brain tissue.

Identifying and costing common gaps in Central and West Africa pharmaceutical regulation.

Background: Regulatory systems strengthening is crucial for catalyzing access to safe and effective medical products and health technologies (MPHT) for all. Identifying and addressing common regulatory gaps through regional approaches could be instrumental for the newly incepted African Medicine Agency. Aims: This original study sheds light on common gaps among 10 national regulatory authorities (NRAs) and ways to address them regionally. Objectives: The study used NRA self-assessment outcomes to identify common gaps in four critical regulatory pillars and estimate the cost of addressing them from regional perspectives that aimed at raising the maturity level of regulatory institutions. Methods: A cross-sectional study, using the WHO Global Benchmarking Tool (GBT), was conducted between 2020 and 2021 with five NRAs from ECCAS and ECOWAS member states that use French and Spanish as lingua franca. Results: The 10 NRAs operated in a non-formal-to-reactive approach (ML1-2), which hinders their ability to ensure the quality of MPHT and respond appropriately to public health emergencies. Common gaps were identified in four critical regulatory pillars-good regulatory practices, preparedness for public health emergencies, quality management systems, and substandard and falsified medical products-with overall cost to address gaps estimated at US$3.3 million. Contribution: We elaborated a reproducible method to strengthen regulatory systems at a regional level to improve equitable access to assured-quality MPHT. Our bottom-up approach could be utilized by RECs to address common gaps through common efforts.

Diversity and Evolution of Frog Visual Opsins: Spectral Tuning and Adaptation to Distinct Light Environments.

Visual systems adapt to different light environments through several avenues including optical changes to the eye and neurological changes in how light signals are processed and interpreted. Spectral sensitivity can evolve via changes to visual pigments housed in the retinal photoreceptors through gene duplication and loss, differential and coexpression, and sequence evolution. Frogs provide an excellent, yet understudied, system for visual evolution research due to their diversity of ecologies (including biphasic aquatic-terrestrial life cycles) that we hypothesize imposed different selective pressures leading to adaptive evolution of the visual system, notably the opsins that encode the protein component of the visual pigments responsible for the first step in visual perception. Here, we analyze the diversity and evolution of visual opsin genes from 93 new eye transcriptomes plus published data for a combined dataset spanning 122 frog species and 34 families. We find that most species express the four visual opsins previously identified in frogs but show evidence for gene loss in two lineages. Further, we present evidence of positive selection in three opsins and shifts in selective pressures associated with differences in habitat and life history, but not activity pattern. We identify substantial novel variation in the visual opsins and, using microspectrophotometry, find highly variable spectral sensitivities, expanding known ranges for all frog visual pigments. Mutations at spectral-tuning sites only partially account for this variation, suggesting that frogs have used tuning pathways that are unique among vertebrates. These results support the hypothesis of adaptive evolution in photoreceptor physiology across the frog tree of life in response to varying environmental and ecological factors and further our growing understanding of vertebrate visual evolution.

Carbon Nanomaterial Fluorescent Probes and Their Biological Applications.

Fluorescent carbon nanomaterials have broadly useful chemical and photophysical attributes that are conducive to applications in biology. In this review, we focus on materials whose photophysics allow for the use of these materials in biomedical and environmental applications, with emphasis on imaging, biosensing, and cargo delivery. The review focuses primarily on graphitic carbon nanomaterials including graphene and its derivatives, carbon nanotubes, as well as carbon dots and carbon nanohoops. Recent advances in and future prospects of these fields are discussed at depth, and where appropriate, references to reviews pertaining to older literature are provided.

Synthesis and Evaluation of Antifungal and Antibacterial Abilities of Carbon Nanotubes Grafted to Poly(2-hydroxyethyl methacrylate) Nanocomposites.

Developing nanomaterials with the capacity to restrict the growth of bacteria and fungus is of current interest. In this study, nanocomposites of poly(2-hydroxyethyl methacrylate) (PHEMA) and carbon nanotubes (CNTs) functionalized with primary amine, hydroxyl, and carboxyl groups were prepared and characterized. An analysis by Fourier-transform infrared (FT-IR) spectroscopy showed that PHEMA chains were grafted to the functionalized CNTs. X-ray photoelectron spectroscopy suggested that the grafting reaction was viable. The morphology of the prepared nanocomposites studied by field-emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) showed significant changes with respect to the observed for pure PHEMA. The thermal behavior of the nanocomposites studied by differential scanning calorimetry (DSC) revealed that the functionalized CNTs strongly affect the mobility of the PHEMA chains. Tests carried out by thermogravimetric analysis (TGA) were used to calculate the degree of grafting of the PHEMA chains. The ability of the prepared nanocomposites to inhibit the growth of the fungus Candida albicans and the bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was evaluated. A reduced antifungal and antibacterial capacity of the prepared nanocomposites was determined.

Successful repair of left ventricular rupture with pseudoaneurysm: a case report.

Ventricular rupture with pseudoaneurysm is a rare phenomenon that usually occurs after myocardial infarction, previous cardiac surgery and infectious or inflammatory conditions. To prevent rupture of the pseudoaneurysm, urgent repair is recommended. We report successful open surgical repair of a 46-year-old man, who presented with pseudoaneurysm communicating with left ventricle.

A modular chemigenetic calcium indicator enables in vivo functional imaging with near-infrared light.

Genetically encoded fluorescent calcium indicators have revolutionized neuroscience and other biological fields by allowing cellular-resolution recording of physiology during behavior. However, we currently lack bright, genetically targetable indicators in the near infrared that can be used in animals. Here, we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains that can be genetically targeted to specific cell populations. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several dye-ligands that efficiently label the central nervous system in animals. When bound to a near-infrared dye-ligand, WHaloCaMP1a is more than twice as bright as jGCaMP8s, and shows a 7× increase in fluorescence intensity and a 2.1 ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a with near-infrared fluorescence emission to image Ca2+ responses in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae, and to quantitate calcium concentration using fluorescence lifetime imaging microscopy (FLIM).

Design and Validation of a Soft Robotic Simulator for Transseptal Puncture Training.

OBJECTIVE: Transseptal puncture (TP) is the technique used to access the left atrium of the heart from the right atrium during cardiac catheterization procedures. Through repetition, electrophysiologists and interventional cardiologists experienced in TP develop manual skills to navigate the transseptal catheter assembly to their target on the fossa ovalis (FO). Cardiology fellows and cardiologists that are new to TP currently train on patients to develop this skill, resulting in increased risk of complications. The goal of this work was to create low-risk training opportunities for new TP operators. METHODS: We developed a Soft Active Transseptal Puncture Simulator (SATPS), designed to match the dynamics, static response, and visualization of the heart during TP. The SATPS includes three subsystems: (i) A soft robotic right atrium with pneumatic actuators mimics the dynamics of a beating heart. (ii) A fossa ovalis insert simulates cardiac tissue properties. (iii) A simulated intracardiac echocardiography environment provides live visual feedback. Subsystem performance was verified with benchtop tests. Face and content validity were evaluated by experienced clinicians. RESULTS: Subsystems accurately represented atrial volume displacement, tenting and puncture force, and FO deformation. Passive and active actuation states were deemed suitable for simulating different cardiac conditions. Participants rated the SATPS as realistic and useful for training cardiology fellows in TP. CONCLUSION: The SATPS can help improve catheterization skills of novice TP operators. SIGNIFICANCE: The SATPS could provide novice TP operators the opportunity to improve their TP skills before operating on a patient for the first time, reducing the likelihood of complications.

Prevalence, multiple antibiotic resistance and virulence profile of methicillin-resistant Staphylococcus aureus (MRSA) in retail poultry meat from Edo, Nigeria.

Introduction: Staphylococcus aureus causes staphylococcal food poisoning and several difficult-to-treat infections. The occurrence and dissemination of methicillin-resistance S. aureus (MRSA) in Nigeria is crucial and well documented in hospitals. However, findings on MRSA from meat in the country are yet to be adequately reported. The current study determined the prevalence, virulence profile and antibiogram characteristics of MRSA from a raw chicken product from retail outlets within Edo. Methods: A total of 368 poultry meat samples were assessed for MRSA using a standard culture-based approach and characterized further using a molecular method. The antimicrobial susceptibility profile of the isolates was determined using the disc diffusion method. The biofilm profile of the isolates was assayed via the crystal violet microtitre-plate method. Virulence and antimicrobial resistance genes were screened using polymerase chain reaction via specific primers. Results: Of the samples tested, 110 (29.9%) were positive for MRSA. All the isolates were positive for deoxyribonuclease (DNase), coagulase and beta-hemolysis production. Biofilm profile revealed 27 (24.55%) weak biofilm formers, 18 (16.36%) moderate biofilm formers, and 39 (35.45%) strong biofilm formers. The isolates harboured 2 and ≤17 virulence genes. Enterotoxin gene profiling revealed that 100 (90.9%) isolates harboured one or more genes. Resistance against the tested antibiotics followed the order: tetracycline 64(58.2%), ciprofloxacin 71(64.6%), trimethoprim 71(64.6%) and rifampin 103(93.6%). A total of 89 isolates were multidrug-resistant, while 3 isolates were resistant to all 22 antibiotics tested. The isolates harboured antimicrobial-resistant determinants such as methicillin-resistant gene (mecA), tetracycline resistance genes (tetK, tetL), erythromycin resistance genes (ermA, ermC), trimethoprim resistance gene (dfrK). All the staphylococcal cassette chromosome mec (SCCmec) IVa and SCCmec V positive isolates harboured the Panton-Valentine Leukocidin Gene (PVL). Conclusion: In conclusion, S. aureus was resistant to commonly used antibiotics; a concern to public health concerning the transmission of these pathogens after consuming these highlight the significance of antimicrobial and enterotoxigenic monitoring of S. aureus in food chains.

Influence of non-alcoholic fatty liver disease on non-variceal upper gastrointestinal bleeding: A nationwide analysis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disease globally with an estimated prevalence of 25%, with the clinical and economic burden expected to continue to increase. In the United States, non-variceal upper gastrointestinal bleeding (NVUGIB) has an estimated incidence of 61-78 cases per 100000 people with a mortality rate of 2%-15% based on co-morbidity burden. AIM: To identify the outcomes of NVUGIB in NAFLD hospitalizations in the United States. METHODS: We utilized the National Inpatient Sample from 2016-2019 to identify all NVUGIB hospitalizations in the United States. This population was divided based on the presence and absence of NAFLD. Hospitalization characteristics, outcomes and complications were compared. RESULTS: The total number of hospitalizations for NVUGIB was 799785, of which 6% were found to have NAFLD. NAFLD and GIB was, on average, more common in younger patients, females, and Hispanics than GIB without NAFLD. Interestingly, GIB was less common amongst blacks with NAFLD. Multivariate logistic regression analysis was conducted, controlling for the multiple covariates. The primary outcome of interest, mortality, was found to be significantly higher in patients with NAFLD and GIB [adjusted odds ratio (aOR) = 1.018 (1.013-1.022)]. Secondary outcomes of interest, shock [aOR = 1.015 (1.008-1.022)], acute respiratory failure [aOR = 1.01 (1.005-1.015)] and acute liver failure [aOR = 1.016 (1.013-1.019)] were all more likely to occur in this cohort. Patients with NAFLD were also more likely to incur higher total hospital charges (THC) [$2148 ($1677-$2618)]; however, were less likely to have a longer length of stay [0.27 d (0.17-0.38)]. Interestingly, in our study, the patients with NAFLD were less likely to suffer from acute myocardial infarction [aOR = 0.992 (0.989-0.995)]. Patients with NAFLD were not more likely to suffer acute kidney injury, sepsis, blood transfusion, intubation, or dialysis. CONCLUSION: NVUGIB in NAFLD hospitalizations had higher inpatient mortality, THC, and complications such as shock, acute respiratory failure, and acute liver failure compared to those without NAFLD.

Detection of colorectal adenomas using artificial intelligence models in patients with chronic hepatitis C.

BACKGROUND: Hepatitis C virus is known for its oncogenic potential, especially in hepatocellular carcinoma and non-Hodgkin lymphoma. Several studies have shown that chronic hepatitis C (CHC) has an increased risk of the development of colorectal cancer (CRC). AIM: To analyze this positive relationship and develop an artificial intelligence (AI)-based tool using machine learning (ML) algorithms to stratify these patient populations into risk groups for CRC/adenoma detection. METHODS: To develop the AI automated calculator, we applied ML to train models to predict the probability and the number of adenomas detected on colonoscopy. Data sets were split into 70:30 ratios for training and internal validation. The Scikit-learn standard scaler was used to scale values of continuous variables. Colonoscopy findings were used as the gold standard and deep learning architecture was used to train six ML models for prediction. A Flask (customizable Python framework) application programming interface (API) was used to deploy the trained ML model with the highest accuracy as a web application. Finally, Heroku was used for the deployment of the web-based API to https://adenomadetection.herokuapp.com. RESULTS: Of 415 patients, 206 had colonoscopy results. On internal validation, the Bernoulli naive Bayes model predicted the probability of adenoma detection with the highest accuracy of 56%, precision of 55%, recall of 55%, and F1 measure of 54%. Support vector regressor predicted the number of adenomas with the least mean absolute error of 0.905. CONCLUSION: Our AI-based tool can help providers stratify patients with CHC for early referral for screening colonoscopy. Along with providing a numerical percentage, the calculator can also comment on the number of adenomatous polyps a gastroenterologist can expect, prompting a higher adenoma detection rate.

Identification and characterization of MDR virulent Salmonella spp isolated from smallholder poultry production environment in Edo and Delta States, Nigeria.

Salmonella is responsible for some foodborne disease cases worldwide. It is mainly transmitted to humans through foods of animal origin through the consumption of poultry products. The increased international trade and the ease of transboundary movement could propel outbreaks of local origin to translate into severe global threats. The present study aimed to characterize Salmonella serovars isolated from poultry farms in Edo and Delta States, Nigeria. A total of 150 samples (faecal, water and feed) were collected from ten poultry farms between January and August 2020 and analyzed for Salmonella characterization using standard bacteriological and molecular methods. Salmonella serovars identified include: Salmonella Enteritidis [n = 17 (39.5%)], Salmonella Typhimurium [n = 13 (30.2%)] and other Salmonella serovars [n = 13 (30.2%)]. All Salmonella serovars were cefotaxime and ampicillin resistant. The presence of the invA gene ranged from 9(69.2%) to 15(88.2%). The spvC gene ranged from 2(14.4%) to 10(58.8%). All Salmonella serovars had sdiA gene. The Salmonella isolates produced some extracellular virulence factors (such as protease, lipase, ß-hemolytic activity, and gelatinase), while 13(30.2%) of the overall isolates formed strong biofilms. In conclusion, the detection of multiple antibiotic-resistant Salmonella serovars in faecal sources, which also exhibited virulence determinants, constituted a public health risk as these faecal samples have the potential as manure in the growing of crops. These pathogens can be transmitted to humans nearby and through poultry products, resulting in difficult-to-treat infections and economic loss.

Development of an Amplicon Nanopore Sequencing Strategy for Detection of Mutations Conferring Intermediate Resistance to Vancomycin in Staphylococcus aureus Strains.

Staphylococcus aureus is a major cause of bacteremia and other hospital-acquired infections. The cell-wall active antibiotic vancomycin is commonly used to treat both methicillin-resistant (MRSA) and sensitive (MSSA) infections. Vancomycin intermediate S. aureus (VISA) variants can arise through de novo mutations. Here, we performed pilot experiments to develop a combined PCR/long-read sequencing-based method for detection of previously known VISA-causing mutations. Primers were designed to generate 10 amplicons covering 16 genes associated with the VISA phenotype. We sequenced amplicon pools as long reads with Oxford Nanopore adapter ligation on Flongle flow cells. We then detected mutations by mapping reads against a parental consensus or known reference sequence and comparing called variants against a database of known VISA mutations from laboratory selection. Each amplicon in the pool was sequenced to high (>1,000×) coverage, and no relationship was found between amplicon length and coverage. We also were able to detect the causative mutation (walK 646C>G) in a VISA mutant derived from the USA300 strain (N384-3 from parental strain N384). Mixing mutant (N384-3) and parental (N384) DNA at various ratios from 0 to 1 mutant suggested a mutation detection threshold of the average minor allele frequency (6.5%) at 95% confidence (two standard errors above mean mutation frequency). The study lays the groundwork for direct S. aureus antibiotic resistance genotype inference using rapid nanopore sequencing from clinical samples. IMPORTANCE Bacteremia mortality is known to increase rapidly with time after infection, making rapid diagnostics and treatment necessary. Successful treatment depends on correct administration of antibiotics based on knowledge of strain antibiotic susceptibility. Staphylococcus aureus is a major causative agent of bacteremia that is also commonly antibiotic resistant. In this work, we develop a method to accelerate detection of a complex, polygenic antibiotic resistance phenotype in S. aureus, vancomycin-intermediate resistance (VISA), through long-read genomic sequencing of amplicons representing genes most commonly mutated in VISA selection. This method both rapidly identifies VISA genotypes and incorporates the most comprehensive database of VISA genetic determinants known to date.

Nonlinear coherent heat machines.

We propose heat machines that are nonlinear, coherent, and closed systems composed of few field (oscillator) modes. Their thermal-state input is transformed by nonlinear Kerr interactions into nonthermal (non-Gaussian) output with controlled quantum fluctuations and the capacity to deliver work in a chosen mode. These machines can provide an output with strongly reduced phase and amplitude uncertainty that may be useful for sensing or communications in the quantum domain. They are experimentally realizable in optomechanical cavities where photonic and phononic modes are coupled by a Josephson qubit or in cold gases where interactions between photons are transformed into dipole-dipole interacting Rydberg atom polaritons. This proposed approach is a step toward the bridging of quantum and classical coherent and thermodynamic descriptions.

In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

Visualizing synaptic dopamine efflux with a 2D composite nanofilm.

Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional composite nanofilm that facilitates visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ≈ 3.2 µm (full width at half maximum [FWHM]) and are observed with a mean spatial frequency of one hotspot per ≈ 7.5 µm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots co-localized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals.

To form the vast and complex network necessary for an organism to sense and react to the world, neurons must connect at highly specialized junctions. Individual cells communicate at these 'synapses' by releasing chemical signals (or neurotransmitters) such as dopamine, a molecule involved in learning and motivation. Despite the central role that synapses play in the brain, it remains challenging to measure exactly where neurotransmitters are released and how far they travel from their release site. Currently, most tools available to scientists only allow bulk measurements of neurotransmitter release. To tackle this limitation, Bulumulla et al. developed a new way to measure neurotransmitter release from neurons, harnessing a technique which uses fluorescent nanosensors that glow brighter when exposed to dopamine. These sensors form a very thin film upon which neurons can grow; when the cells release dopamine, the sensors 'light up' as they encounter the molecule. Dubbed DopaFilm, the technology reveals exactly where the neurotransmitter comes from and how it spreads between cells in real time. In particular, the approach showed that dopamine emerges from 'hot spots' at specific sites in cells; it also helped Bulumulla et al. study how dopamine is released from subcellular compartments that have previously not been well characterized. Improving the sensors so that the film could detect other neurotransmitters besides dopamine would broaden the use of this approach. In the future, combining this technology with other types of imaging should enable studies of individual synapses with intricate detail.