Dynamics of Antibacterial Drone Establishment in Staphylococcus aureus: Unexpected Effects of Antibiotic Resistance Genes.

The antibacterial drone (ABD) system is based on repurposing the phage-inducible staphylococcal pathogenicity islands (SaPIs) for use as antibacterial agents that are indifferent to antibiotic resistance. The ABDs were constructed by inserting tetM for tetracycline resistance (Tcr) selection, replacing the SaPI virulence genes with bactericidal or bacteriostatic genes such as CRISPR/cas9/agrA, whose expression kills by double-strand cleavage of agrA, or CRISPR/dcas9/agrP2P3, whose expression blocks the target organism's virulence. ABD DNA is packaged in phage-like particles that attack their staphylococcal targets in vivo as well as in vitro. We determine ABD titers by transfer frequency, enumerate surviving cells as a function of multiplicity, and analyze the fate of ABD DNA with green fluorescent protein. An initial study revealed surprisingly that many more cells were killed by the ABD than were measured by transduction. Our study of this phenomenon has revealed several important features of the ABD system: (i) a significant number of entering ABD DNA molecules do not go on to establish stable transductants (i.e., are abortive); (ii) ABD cargo genes are expressed immediately following entry, even by the abortive ABDs; (iii) immediate plating on Tc-containing agar seriously underestimates particle numbers, partly owing to Tc inhibition of protein synthesis; (iv) replacement of tetM with cadA (conferring resistance to CdCl2) provides more accurate particle enumeration; (v) ABDs expressing CRISPR/cas9/agrA kill ∼99.99% of infected cells and provide the most accurate measurement of particle numbers as well as proof of principle for the system; and (vi) surprisingly, TetM interferes with stable establishment of ABD DNA independently of Tcr. IMPORTANCE For a particulate therapeutic agent, such as the ABD, accurate enumeration of particles is critical to enable evaluation of preparative procedures and calculation of therapeutic dosages. It is equally important that a selective marker used for these two purposes be biologically inert. We have long used tetM for these purposes but show here that tetM not only underestimates particle titers, by over 20-fold in some experiments, but also seriously impedes stable establishment of the therapeutic particle DNA. Given that tetM is a very convenient and widely used selective marker, publication of these findings is of considerable importance to the microbiological community as well as an interesting illustration of the unpredictable biological effects of genes taken out of their native context.

Sheath fluid impacts the depletion of cellular metabolites in cells afflicted by sorting induced cellular stress (SICS).

Flow cytometrists have long observed a spectrum of cell-type-specific changes ranging from minor functional defects to outright cell destruction after purification of cells using conventional droplet cell sorters. We have described this spectrum of cell perturbations as sorter induced cellular stress, or SICS (Lopez and Hulspas, Cytometry, 2020, 97, 105-106). Despite the potential impact of this issue and ubiquitous anecdotes, little has been reported about this phenomenon in the literature, and the underlying mechanism has been elusive. Inspired by others' observations (Llufrio et al., Redox Biology, 2018, 16, 381-387 and Binek et al., Journal of Proteome Research, 2019, 18, 169-181), we set out to examine SICS at the metabolic level and use this information to propose a working model. Using representative suspension (Jurkat) and adherent (NIH/3T3) cell lines we observed broad and consistent metabolic perturbations after sorting using a high-speed droplet cell sorter. Our results suggest that the SICS metabolic phenotype is a common cell-type-independent manifestation and may be the harbinger of a wide-range of functional defects either directly related to metabolism, or cell stress response pathways. We further demonstrate a proof of concept that a modification to the fluidic environment (complete media used as sheath fluid) in a droplet cell sorter can largely rescue the intracellular markers of SICS, and that this rescue is not due to a contribution of metabolites found in media. Future studies will focus on characterizing the potential electro-physical mechanisms inherent to the droplet cell sorting process to determine the major contributors to the SICS mechanism.

Diversity of Functionally Distinct Clonal Sets of Human Conventional Memory B Cells That Bind Staphylococcal Protein A.

Staphylococcus aureus, a common cause of serious and often fatal infections, is well-armed with secreted factors that disarm host immune defenses. Highly expressed in vivo during infection, Staphylococcal protein A (SpA) is reported to also contribute to nasal colonization that can be a prelude to invasive infection. Co-evolution with the host immune system has provided SpA with an Fc-antibody binding site, and a Fab-binding site responsible for non-immune superantigen interactions via germline-encoded surfaces expressed on many human BCRs. We wondered whether the recurrent exposures to S. aureus commonly experienced by adults, result in the accumulation of memory B-cell responses to other determinants on SpA. We therefore isolated SpA-specific class-switched memory B cells, and characterized their encoding VH : VL antibody genes. In SpA-reactive memory B cells, we confirmed a striking bias in usage for VH genes, which retain the surface that mediates the SpA-superantigen interaction. We postulate these interactions reflect co-evolution of the host immune system and SpA, which during infection results in immune recruitment of an extraordinarily high prevalence of B cells in the repertoire that subverts the augmentation of protective defenses. Herein, we provide the first evidence that human memory responses are supplemented by B-cell clones, and circulating-antibodies, that bind to SpA determinants independent of the non-immune Fc- and Fab-binding sites. In parallel, we demonstrate that healthy individuals, and patients recovering from S. aureus infection, both have circulating antibodies with these conventional binding specificities. These findings rationalize the potential utility of incorporating specially engineered SpA proteins into a protective vaccine.

Evaluating the Effects of Cell Sorting on Gene Expression.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

Evaluating the Effects of Cell Sorting on Gene Expression.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments.

Poor adherence to best practices, insufficient training, and pressure to produce data quickly may lead to publications of suboptimal biomedical research flow cytometry data, which contributes to the body of irreproducible research findings. In addition, documentation of compliance with best flow cytometry practices for submission, visualization, and publication of flow cytometry data is currently endorsed by very few scientific journals, which is particularly concerning as numerous peer-reviewed flow cytometry publications emphasize instrumentation, experimental design, and data analysis as important sources of variability. Guidelines and resources for adequate reporting, annotation and deposition of flow cytometry experiments are provided by MIFlowCyt and the FlowRepository database, and comprehensive expert recommendations covering principles and techniques of field-specific flow cytometry applications have been published. To facilitate the integration of quality-defining parameters into manuscript and grant submission and publication requirements across biomedical fields that rely on the use of flow-cytometry-based techniques, a single comprehensive yet easily and universally applicable document is needed. To produce such a list of gold-standard parameters that assess whether a research flow cytometry experiment has been planned, conducted, interpreted, and reported at the highest standard, a new initiative defining the minimum set of standards a robust and rigorous research flow experiment must fulfill (MiSet RFC Standards) was proposed at CYTO 2019. MiSet RFC Standards will integrate and simplify existing resources to provide a universal benchmark a flow cytometry experiment can easily be measured against. The goal of MiSET RFC Standards is its integration into peer-review and publication procedures through partnership with stakeholders, journals and publishers in biomedical and translational research. This article introduces the aims and anticipated timeline and discusses strategies for interdisciplinary consensus and implementation. A single-resource broadly applicable guideline will harmonize standards across different fields of biomedical research and lead to publication of more robust research findings. © 2019 International Society for Advancement of Cytometry.

Survey on Scientific Shared Resource Rigor and Reproducibility.

Shared scientific resources, also known as core facilities, support a significant portion of the research conducted at biomolecular research institutions. The Association of Biomolecular Resource Facilities (ABRF) established the Committee on Core Rigor and Reproducibility (CCoRRe) to further its mission of integrating advanced technologies, education, and communication in the operations of shared scientific resources in support of reproducible research. In order to first assess the needs of the scientific shared resource community, the CCoRRe solicited feedback from ABRF members via a survey. The purpose of the survey was to gain information on how U.S. National Institutes of Health (NIH) initiatives on advancing scientific rigor and reproducibility influenced current services and new technology development. In addition, the survey aimed to identify the challenges and opportunities related to implementation of new reporting requirements and to identify new practices and resources needed to ensure rigorous research. The results revealed a surprising unfamiliarity with the NIH guidelines. Many of the perceived challenges to the effective implementation of best practices (i.e., those designed to ensure rigor and reproducibility) were similarly noted as a challenge to effective provision of support services in a core setting. Further, most cores routinely use best practices and offer services that support rigor and reproducibility. These services include access to well-maintained instrumentation and training on experimental design and data analysis as well as data management. Feedback from this survey will enable the ABRF to build better educational resources and share critical best-practice guidelines. These resources will become important tools to the core community and the researchers they serve to impact rigor and transparency across the range of science and technology.

Inspiratory Capacity and Vital Capacity of Healthy Subjects 9-81 Years of Age at Moderate-High Altitude.

BACKGROUND: Measurements of inspiratory capacity (IC) and vital capacity (VC) are used to recognize dynamic hyperinflation, but appropriate reference values are required to achieve accurate clinical interpretations. Altitude above sea level is a potential determining factor for lung volumes, including IC and VC. OBJECTIVE: To describe IC and VC for healthy people who live in Mexico City at an altitude of 2,240 m above sea level. METHODS: Healthy subjects ages 9-81 y completed slow spirometry by following 2005 American Thoracic Society/European Respiratory Society standards. Once associations were explored, linear regression models were constructed and values were compared with those from previously published equations. RESULTS: A total of 441 healthy subjects (55.1% women) participated. The mean age was 32 y (minimum age, 9 y; maximum age, 81 y). IC and VC measurements were associated with sex, age, height, and weight. An accelerated increase in IC and VC was evident from 9 to 20 y of age, followed by a gradual decrease in both sexes. In general, IC was higher in our population than predicted by previously published reference equations. CONCLUSIONS: IC in healthy people at 2,240 m above sea level was higher than that of previous reports about European and Latin-American subjects of the same height, sex, and age who were at sea level. The present study provided robust reference values for persons who lived at a moderate altitude.

Nascent Induced Pluripotent Stem Cells Efficiently Generate Entirely iPSC-Derived Mice while Expressing Differentiation-Associated Genes.

The ability of induced pluripotent stem cells (iPSCs) to differentiate into all adult cell types makes them attractive for research and regenerative medicine; however, it remains unknown when and how this capacity is established. We characterized the acquisition of developmental pluripotency in a suitable reprogramming system to show that iPSCs prior to passaging become capable of generating all tissues upon injection into preimplantation embryos. The developmental potential of nascent iPSCs is comparable to or even surpasses that of established pluripotent cells. Further functional assays and genome-wide molecular analyses suggest that cells acquiring developmental pluripotency exhibit a unique combination of properties that distinguish them from canonical naive and primed pluripotency states. These include reduced clonal self-renewal potential and the elevated expression of differentiation-associated transcriptional regulators. Our observations close a gap in the understanding of induced pluripotency and provide an improved roadmap of cellular reprogramming with ramifications for the use of iPSCs.

Risk Factors for Dysphagia Following a Cervical Fusion in a Trauma Population.

Background Dysphagia following a cervical fusion is a known complication; however, this has not been examined in the trauma population. We sought to identify risk factors that can be optimized in this population. Methods We performed a retrospective chart review on consecutive trauma patients who underwent a cervical fusion from 2014 to 2017 at a single institution with multiple surgeons. We included patients more than 18-years-old who were admitted through the emergency department with a diagnosis of acute cervical injury and underwent a cervical fusion during the same admission. We excluded patients who remained intubated postoperatively or underwent a tracheostomy. The primary outcome was dysphagia as evaluated by a bedside swallow test on postoperative day one by the nursing staff. This was followed by a standardized assessment performed by a speech therapist on postoperative day two in some cases. Variables of interest included sex, age, mechanism of injury, surgical approach, cervical levels, and Charlson comorbidity index. Univariate analysis was also utilized. Results Sixty patients met the study criteria. Nineteen patients (31.7%) developed dysphagia postoperatively. Mechanical falls were the most common injury mechanism (80%) and most surgical procedures were performed on the subaxial cervical spine (68.3%). Comparing the dysphagia groups, there was no significant difference among the confounding variables. Patients with dysphagia had an increased length of stay (10.6 ± 6.7 vs. 7.4 ± 3.1, p = 0.056) and were more likely to have had an anterior vs. posterior cervical fusion (63.2% vs. 34.1%, p = 0.056). Conclusions We found no statistically significant risk factors leading to postoperative dysphagia. The objective of this pilot is to find the baseline dysphagia rate and the potential modifiable factors in this unique patient population undergoing cervical fusion procedures.

Maximum Voluntary Ventilation in a Population Residing at 2,240 Meters Above Sea Level.

BACKGROUND: Measured maximum voluntary ventilation (MVV) correlates with maximum ventilatory capacity during exercise. As a shortcut, MVV is often estimated by multiplying measured FEV1 times 35 or 40, but this index varies with altitude due to reduced air density. The objective was to describe MVV in healthy individuals residing at 2,240 m above sea level and compare it with the reference values customarily employed. METHODS: We recruited a convenience sample of respiratory-healthy, non-obese volunteers >10 y of age who had resided for >2 y in Mexico City. All participants performed forced spirometry and MVV according to current standards. Multiple regression models were fitted, including age, height, and measured FEV1, separately for males and females to obtain reference values. The impact of lower air density on MVV at this elevation was estimated from the reported increase in peak flow in relation to altitude. RESULTS: We studied 381 individuals (210 females [55.1%]) age 10-80 y with a mean MVV of 145.6 ± 48 L/min. Both FEV1 × 35 and FEV1 × 40 underestimated the MVV observed: in males by approximately 26% and in females by approximately 10%. MVV for our population approached FEV1 × 45 (98 ± 15.6% of real MVV). Multiple regression models including height, weight, and measured FEV1 explained 70% of residual variability once sex was taken into account. CONCLUSIONS: At an altitude of 2,240 m, MVV is about 45 times the measured FEV1, and it can be estimated for other altitudes. The best predicting equations for MVV were calculated separately for females and males and included the following predictors: age, age2, and measured FEV1. The study found that reference values for MVV from studies conducted at sea level are inaccurate at this altitude.

Arrested Development: Infantile Hemangioma and the Stem Cell Teratogenic Hypothesis.

BACKGROUND: Early-life programming is defined by the adaptive changes made by the fetus in response to an adverse in utero environment. Infantile hemangioma (IH), a vascular anomaly, is the most common tumor of infancy. Here we take IH as the tumor model to propose the stem cell teratogenic hypothesis of tumorigenesis and the potential involvement of the immune system. OBJECTIVES: Teratogenic agents include chemicals, heavy metals, pathogens, and ionizing radiation. To investigate the etiology and pathogenesis of IH, we hypothesized that they result from a teratogenic mechanism. Immature, incompletely differentiated, dysregulated progenitor cells (multipotential stem cells) are arrested in development with vasculogenic, angiogenic, and tumorigenic potential due to exposure to teratogenic agents such as extrinsic factors that disrupt intrinsic factors via molecular mimicry. During the critical period of immunological tolerance, environmental exposure to immunotoxic agents may harness the teratogenic potential in the developing embryo or fetus and modify the early-life programming algorithm by altering normal fetal development, causing malformations, and inducing tumorigenesis. Specifically, exposure to environmental agents may interfere with physiological signaling pathways and contribute to the generation of IH, by several mechanisms. DISCUSSION: An adverse in utero environment no longer serves as a sustainable environment for proper embryogenesis and normal development. Targeted disruption of stem cells by extrinsic factors can alter the genetic program. CONCLUSIONS: This article offers new perspectives to stimulate discussion, explore novel experimental approaches (such as immunotoxicity/vasculotoxicity assays and novel isogenic models), and to address the questions raised to convert the hypotheses into nontoxic, noninvasive treatments.

Repeat Head CT? Not Necessary for Patients with a Negative Initial Head CT on Anticoagulation or Antiplatelet Therapy Suffering Low-Altitude Falls.

Anticoagulation and antiplatelet (ACAP) medications are increasingly prescribed to patients at high risk for falls. Many trauma centers have developed protocols for obtaining repeat head CT (HCT) for patients with low-altitude falls on ACAP therapy. We assess the need for routine scheduled repeat HCT in this population. Prospective, observational analysis of all low-altitude fall (<6 feet) patients on ACAP therapy evaluated at a Level II trauma center. All low-altitude fall patients with visible or suspected head trauma received an initial HCT. Patients were admitted and repeat HCT was obtained 12 hours later or earlier if acute neurologic decline developed. Chi-squared, Fischer exact, t, and Wilcoxon rank-sum tests were used. Statistical significance was defined as P < 0.05. Total of 1501 patients enrolled suffering low-altitude falls with initial HCT. Among them 1379(91.2%) were negative and 122(8.1%) were initially positive for intracranial hemorrhage. Mean age was 79.9 ± 11.4 years, 61 per cent were female and 85 per cent had visible head trauma at presentation. One hundred ninety-nine were excluded secondary to not receiving repeat HCT. Of the 1180 patients with normal initial HCT who underwent repeat HCT, only 7 (0.51%) had delayed intracranial hemorrhage. None of these patients required surgery, major changes in medical management or suffered head trauma-related mortality; 69 per cent were taking aspirin (acetylsalicylic acid, ASA), 19 per cent warfarin, 17 per cent clopidogrel, 6 per cent other anticoagulants, and 11 per cent were on combination therapy. Repeat HCT for patients on any ACAP therapy after low-altitude fall with a negative initial HCT is not necessary. Thorough neurologic examination and close monitoring is as effective as obtaining a repeat HCT.

Infantile Hemangioma Originates From A Dysregulated But Not Fully Transformed Multipotent Stem Cell.

Infantile hemangioma (IH) is the most common tumor of infancy. Its cellular origin and biological signals for uncontrolled growth are poorly understood, and specific pharmacological treatment is unavailable. To understand the process of hemangioma-genesis we characterized the progenitor hemangioma-derived stem cell (HemSC) and its lineage and non-lineage derivatives. For this purpose we performed a high-throughput (HT) phenotypic and gene expression analysis of HemSCs, and analyzed HemSC-derived tumorspheres. We found that IH is characterized by high expression of genes involved in vasculogenesis, angiogenesis, tumorigenesis and associated signaling pathways. These results show that IH derives from a dysregulated stem cell that remains in an immature, arrested stage of development. The potential biomarkers we identified can afford the development of diagnostic tools and precision-medicine therapies to "rewire" or redirect cellular transitions at an early stage, such as signaling pathways or immune response modifiers.