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The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic biological chassis. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here, we report imitating methylation patterns rapidly in TXTL (IMPRINT), a generalized, rapid, and scalable approach based on cell-free transcription-translation (TXTL) to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from a bacterium's restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the bacterium's DNA methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also develop a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to screen a ribosome-binding site library in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling the use of sophisticated genetic manipulation tools across the bacterial world., Competing Interests: Declaration of interests J.M.V. and C.L.B. have filed a provisional patent application related to this work. C.L.B. is a co-founder of Leopard Biosciences, a co-founder and scientific advisory board member of Locus Biosciences, and a scientific advisory board member of Benson Hill., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Epilepsy is a common, and often genetic, neurological disorder. Few guidelines exist to help medical providers or insurance companies decide when to order or cover epilepsy panels for patients with epilepsy. The most recent guidelines were published by NSGC after this study's data collection. Since 2017, the Genetic Testing Stewardship Program (GTSP) at UPMC Children's Hospital of Pittsburgh (CHP) has been utilizing a set of internally developed epilepsy panel (EP) testing criteria to facilitate appropriate EP ordering practices. The purpose of this study was to assess these testing criteria by determining their sensitivities and positive predictive values (PPV). Retrospective chart review of the electronic medical record (EMR) was performed for 1242 CHP Neurology patients that were evaluated for a primary diagnosis of epilepsy between 2016 and 2018. One hundred and nine patients had EPs at various testing laboratories. Of the patients that met criteria, 17 had diagnostic EPs and 54 had negative EPs. Criteria were organized into category groupings (C1-C4), and analyzed alone for C1, in pairs for C2, etc. The highest sensitivity and PPV results in each category grouping were: C1 (64.7%, 60%); C2, (88%, 30.3%); C3, (94.1%, 27.1%); C4, (94.1%, 25.4%). Family history was crucial to increasing sensitivity. Confidence intervals (CIs) narrowed as category grouping level increased, though this was not statistically significant due to the considerable CI overlap across category groupings. The PPV from C4 was applied to the untested population cohort and predicted 121 patients with unidentified positive EPs. This study presents data supporting the predictive capabilities of EP testing criteria and suggests the addition of a family history criterion. This study impacts public health by encouraging the adoption of evidence-driven insurance policies and by suggesting guidelines to ease EP ordering and coverage decisions, which could potentially improve patient access to EP testing., (© 2023 National Society of Genetic Counselors.)

G protein-coupled receptors (GPCRs) enable cells to sense environmental cues and are indispensable for coordinating vital processes including quorum sensing, proliferation, and sexual reproduction. GPCRs comprise the largest class of cell surface receptors in eukaryotes, and for more than three decades the pheromone-induced mating pathway in baker's yeast Saccharomyces cerevisiae has served as a model for studying heterologous GPCRs (hGPCRs). Here we report transcriptome profiles following mating pathway activation in native and hGPCR-signaling yeast and use a model-guided approach to correlate gene expression to morphological changes. From this we demonstrate mating between haploid cells armed with hGPCRs and endogenous biosynthesis of their cognate ligands. Furthermore, we devise a ligand-free screening strategy for hGPCR compatibility with the yeast mating pathway and enable hGPCR-signaling in the probiotic yeast Saccharomyces boulardii. Combined, our findings enable new means to study mating, hGPCR-signaling, and cell-cell communication in a model eukaryote and yeast probiotics., (© 2022. The Author(s).)

Commensal bacteria are known to promote host growth. Such effect partly relies on the capacity of microbes to regulate the host's transcriptional response. However, these evidences mainly come from comparing the transcriptional response caused by commensal bacteria with that of axenic animals, making it difficult to identify the animal genes that are specifically regulated by beneficial microbes. Here, we employ Drosophila melanogaster associated with Lactiplantibacillus plantarum to understand the host genetic pathways regulated by beneficial bacteria and leading to improved host growth. We show that microbial benefit to the host relies on the downregulation of peptidoglycan-recognition proteins. Specifically, we report that bacterial proliferation triggers the lower expression of PGRP-SC1 in larval midgut, which ultimately leads to improved host growth and development. Our study helps elucidate the mechanisms underlying the beneficial effect exerted by commensal bacteria, defining the role of immune effectors in the relationship between Drosophila and its gut microbes., Competing Interests: The authors declared no competing interests., (© 2022 The Author(s).)

The bacterial genus Lactobacillus comprises a vast range of strains with varying metabolic and probiotic traits, with genome editing representing an essential tool to probe genotype-phenotype relationships and enhance their beneficial properties. Currently, one of the most effective means of genome editing in bacteria couples low-efficiency recombineering with high-efficiency counterselection by nucleases from CRISPR-Cas systems. In lactobacilli, several CRISPR-based genome editing methods exist that have shown varying success in different strains. Here, we detail a fast and simple approach using two shuttle vectors encoding a recombineering template as well as the Streptococcus pyogenes Cas9, a trans-activating RNA, and a CRISPR array. We provide a step-by-step procedure for cloning the shuttle vectors, sequentially transforming the vectors into lactobacilli, screening for the desired edit, and finally clearing the shuttle vectors from the mutant strain. As CRISPR-based genome editing in bacteria can fail for various reasons, we also lay out instructions for probing mechanisms of escape. Finally, we include practical notes along the way to facilitate each stage of genome editing, and we illustrate the technique using a representative edit in a strain of Lactobacillus plantarum. Overall, this method should serve as a complete guide to performing genome editing in lactobacilli., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

A prominent feature of lactic acid bacteria (LAB) is their ability to inhibit growth of spoilage organisms in food, but hitherto research efforts to establish the mechanisms underlying bioactivity focused on the production of antimicrobial compounds by LAB. We show, in this study, that competitive exclusion, i.e., competition for a limited resource by different organisms, is a major mechanism of fungal growth inhibition by lactobacilli in fermented dairy products. The depletion of the essential trace element manganese by two Lactobacillus species was uncovered as the main mechanism for growth inhibition of dairy spoilage yeast and molds. A manganese transporter (MntH1), representing one of the highest expressed gene products in both lactobacilli, facilitates the exhaustive manganese scavenging. Expression of the mntH1 gene was found to be strain dependent, affected by species coculturing and the growth phase. Further, deletion of the mntH1 gene in one of the strains resulted in a loss of bioactivity, proving this gene to be important for manganese depletion. The presence of an mntH gene displayed a distinct phylogenetic pattern within the Lactobacillus genus. Moreover, assaying the bioprotective ability in fermented milk of selected lactobacilli from 10 major phylogenetic groups identified a correlation between the presence of mntH and bioprotective activity. Thus, manganese scavenging emerges as a common trait within the Lactobacillus genus, but differences in expression result in some strains showing more bioprotective effect than others. In summary, competitive exclusion through ion depletion is herein reported as a novel mechanism in LAB to delay the growth of spoilage contaminants in dairy products. IMPORTANCE In societies that have food choices, conscious consumers demand natural solutions to keep their food healthy and fresh during storage, simultaneously reducing food waste. The use of "good bacteria" to protect food against spoilage organisms has a long, successful history, even though the molecular mechanisms are not fully understood. In this study, we show that the depletion of free manganese is a major bioprotective mechanism of lactobacilli in dairy products. High manganese uptake and intracellular storage provide a link to the distinct, nonenzymatic, manganese-catalyzed oxidative stress defense mechanism, previously described for certain lactobacilli. The evaluation of representative Lactobacillus species in our study identifies multiple relevant species groups for fungal growth inhibition via manganese depletion. Hence, through the natural mechanism of nutrient depletion, the use of dedicated bioprotective lactobacilli constitutes an attractive alternative to artificial preservation., (Copyright © 2020 Siedler et al.)

Genome editing is essential for probing genotype-phenotype relationships and for enhancing chemical production and phenotypic robustness in industrial bacteria. Currently, the most popular tools for genome editing couple recombineering with DNA cleavage by the CRISPR nuclease Cas9 from Streptococcus pyogenes. Although successful in some model strains, CRISPR-based genome editing has been slow to extend to the multitude of industrially relevant bacteria. In this review, we analyze existing barriers to implementing CRISPR-based editing across diverse bacterial species. We first compare the efficacy of current CRISPR-based editing strategies. Next, we discuss alternatives when the S. pyogenes Cas9 does not yield colonies. Finally, we describe different ways bacteria can evade editing and how elucidating these failure modes can improve CRISPR-based genome editing across strains. Together, this review highlights existing obstacles to CRISPR-based editing in bacteria and offers guidelines to help achieve and enhance editing in a wider range of bacterial species, including non-model strains.

Lactic-acid bacteria such as Lactobacillus plantarum are commonly used for fermenting foods and as probiotics, where increasingly sophisticated genome-editing tools are employed to elucidate and enhance these microbes' beneficial properties. The most advanced tools to date utilize an oligonucleotide or double-stranded DNA donor for recombineering and Cas9 for targeted DNA cleavage. As the associated methods are often developed in isolation for one strain, it remains unclear how different Cas9-based editing methods compare across strains. Here, this work directly compares two methods in different strains of L. plantarum: one utilizing a plasmid-encoded recombineering template and another utilizing an oligonucleotide donor and an inducible DNA recombinase. This comparison reveals one instance in which only the recombineering-template method generates desired edits and another instance in which only the oligo method generates desired edits. It is further found that both methods exhibit highly variable success editing the same site across multiple L. plantarum strains. Finally, failure modes are identified for the recombineering-template method, including a consistent genomic deletion and reversion of a point mutation in the recombineering template. This study therefore highlights surprising differences for Cas9-mediated genome editing between methods and related strains, arguing for the need for multiple, distinct methods when performing CRISPR-based editing in bacteria., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mitochondrial diseases are a genetically and clinically diverse group of disorders that arise as a result of dysfunction of the mitochondria. Mitochondrial disorders can be caused by alterations in nuclear DNA and/or mitochondrial DNA. Although some mitochondrial syndromes have been described clearly in the literature many others present as challenging clinical cases with multisystemic involvement at variable ages of onset. Given the clinical variability and genetic heterogeneity of these conditions, patients and their families often experience a lengthy and complicated diagnostic process. The diagnostic journey may be characterized by heightened levels of uncertainty due to the delayed diagnosis and the absence of a clear prognosis, among other factors. Uncertainty surrounding issues of family planning and genetic testing may also affect the patient. The role of the genetic counselor is particularly important to help explain these complexities and support the patient and family's ability to achieve effective coping strategies in dealing with increased levels of uncertainty.

Primary mitochondrial disorders are clinically and genetically heterogeneous, caused by an alteration(s) in either mitochondrial DNA or nuclear DNA, and affect the respiratory chain's ability to undergo oxidative phosphorylation, leading to decreased production of adenosine triphosphophate and subsequent energy failure. These disorders may present at any age, but children tend to have an acute onset of disease compared with subacute or slowly progressive presentation in adults. Varying organ involvement also contributes to the phenotypic spectrum seen in these disorders. The childhood presentation of primary mitochondrial disease is mainly due to nuclear DNA mutations, with mitochondrial DNA mutations being less frequent in childhood and more prominent in adulthood disease. The clinician should be aware of the pediatric presentation of mitochondrial disease and have an understanding of the myriad of nuclear genes responsible for these disorders. The nuclear genes can be best understood by utilizing a classification system of location and function within the mitochondria.

The field of neurogenetics has expanded dramatically over recent years. Neurogenetics has developed from a distinct subspecialty within neurology to something that transcends most of the common presenting conditions for neurologists. The importance of understanding the genetic contribution to conditions like epilepsy, neurodevelopmental disorders, and metabolic diseases has become very evident in neurology. In the era of personalized medicine and genomic testing, we have just begun to understand how genetic factors contribute to the development of disease, influence its natural history and severity, and determine its response to therapy. Genetic risk analysis and interpretation have become central components for the modern clinical assessment, and a comprehensive family history analysis is essential for neurologists. The benefits of collecting and updating a detailed pedigree are important for diagnostic guidance, risk counseling, and test interpretation. A review of genetic inheritance patterns, pedigree construction, and factors that influence disease presentation is outlined for the application in neurology practice., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The majority of primary mitochondrial disorders are due to nuclear gene mutations, not aberrations within the mitochondrial genome. The nervous system is frequently involved due to its high-energy demands. Many nonspecific neurologic symptoms may be present in mitochondrial disease; however, there are well-recognized red flags that should alert the clinician to the possibility of mitochondrial disease. There is an ever increasing number of nuclear gene mutations discovered that play a role in primary mitochondrial disease and its neurologic symptomatology. Neurologists need to be aware of the wide neurologic presentation, the red-flag symptoms, and the nuclear gene mutations involved in the pathophysiology of mitochondrial disease to diagnose and manage this patient population., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Integration of genetic and genomic information into the clinical practice of child neurologists is revolutionizing how diagnosis, prognosis, and treatment of neurogenetic diseases are approached. Genetic testing technology, informatics, and interpretation are rapidly expanding. High-throughput next-generation sequencing has changed the landscape of many fields of medicine by enabling new diagnoses and gene/disease associations. Now more than ever, a comprehensive understanding of the risks, benefits, limitations, and nuances of testing in the pediatric population is essential for the child neurologist., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to explore anecdotal evidence for an increase in the prevalence of autoimmune diseases in family members of patients with Aicardi-Goutières syndrome (AGS)., Methods: Pedigrees of patients and controls were analyzed using chi-square and logistic regression to assess differences in reports of autoimmune disease among family members of cases and controls. Data was collected at Children's National Medical Center in Washington, DC, USA and at the International Aicardi-Goutières Syndrome Association Scientific Headquarters, C. Mondino National Institute of Neurology in Pavia, Italy., Results: The number of individuals with reported autoimmune disease is significantly related to having a family member with AGS (χ(2) = 6.25, P = 0.01); 10% (35/320) of relatives of patients with AGS had a reported autoimmune disease diagnosis compared to 5% (18/344) of relatives of controls. There was a greater percent of maternal relatives of patients with AGS reporting autoimmune disease (14.6%), compared to controls (6.8%), with the association being statistically significant. The association was not significant for paternal relatives., Conclusion: The prevalence of autoimmune disease in relatives of children with AGS is significantly increased compared to controls. More research is needed to better understand this association.

1,1,2-trifluoro-1-bromo-2-chloro ethane, an isomer of halothane, was tested for its anesthetic and toxic properties in rats. The isomer is about one-half as potent as halothane and is metabolized to a lethal compound.

Manipulation of prostaglandins (PG) in animal models of sepsis and acute respiratory failure (ARF) is promising. Prostacyclin (PGI2), a short-acting vasodilator, was evaluated in a porcine model of ARF produced by continuous infusion of live Pseudomonas aeruginosa (Ps.). Cardiopulmonary parameters were monitored in three groups of spontaneously breathing animals that received 0.1 micrograms PGI2/kg/min begun 20 min after baseline (Group I); 2 X 10(8) Ps./20 kg/min (Group II); identical Ps. infusion and then PGI2 begun at 20 min (Group III). The decrease in mean arterial blood pressure and cardiac index with Ps. infusion was improved by PGI2 treatment. In Groups II and III, mean pulmonary artery pressure (PAP) doubled (P less than 0.005) and pulmonary vascular resistance (PVR) tripled (P less than 0.01) by 15 min. Both PAP and PVR were decreased significantly with PGI2 treatment. In both Ps. groups, significant hypoxemia occurred. PGI2 improves cardiac output and acts as a pulmonary vasodilator, but does not improve oxygenation in this porcine model of severe ARF.

Oxygen delivery (DO2) in patients with acute respiratory failure has been correlated with total lung and chest wall compliance (CT), the optimal positive end-expiratory pressure (PEEP) level reportedly corresponds to maximal DO2. To test the validity of the relationship, we studied the correlation between CT and DO2 in 12 septic pigs with acute respiratory failure induced by continuous infusion of live Pseudomonas aeruginosa bacteria. Cardiac output, pulmonary and systemic arterial BP, blood gases, extravascular lung water, tidal volume, and airway pressure were measured serially in six control animals and six animals receiving increasing amounts of PEEP. There was no significant correlation between DO2 and CT in either group; however, animals receiving PEEP had less extravascular lung water.