Porin-independent accumulation in Pseudomonas enables antibiotic discovery.

Gram-negative antibiotic development has been hindered by a poor understanding of the types of compounds that can accumulate within these bacteria1,2. The presence of efflux pumps and substrate-specific outer-membrane porins in Pseudomonas aeruginosa renders this pathogen particularly challenging3. As a result, there are few antibiotic options for P. aeruginosa infections4 and its many porins have made the prospect of discovering general accumulation guidelines seem unlikely5. Here we assess the whole-cell accumulation of 345 diverse compounds in P. aeruginosa and Escherichia coli. Although certain positively charged compounds permeate both bacterial species, P. aeruginosa is more restrictive compared to E. coli. Computational analysis identified distinct physicochemical properties of small molecules that specifically correlate with P. aeruginosa accumulation, such as formal charge, positive polar surface area and hydrogen bond donor surface area. Mode of uptake studies revealed that most small molecules permeate P. aeruginosa using a porin-independent pathway, thus enabling discovery of general P. aeruginosa accumulation trends with important implications for future antibiotic development. Retrospective antibiotic examples confirmed these trends and these discoveries were then applied to expand the spectrum of activity of a gram-positive-only antibiotic, fusidic acid, into a version that demonstrates a dramatic improvement in antibacterial activity against P. aeruginosa. We anticipate that these discoveries will facilitate the design and development of high-permeating antipseudomonals.

National trends in neoadjuvant chemotherapy utilization in patients with early-stage node-negative triple-negative breast cancer: the impact of the CREATE-X trial.

PURPOSE: Neoadjuvant chemotherapy (NAC) for triple-negative breast cancer (TNBC) allows for assessment of tumor pathological response and has survival implications. In 2017, the CREATE-X trial demonstrated survival benefit with adjuvant capecitabine in patients TNBC and residual disease after NAC. We aimed to assess national rates of NAC for cT1-2N0M0 TNBC before and after CREATE-X and examine factors associated with receiving NAC vs adjuvant chemotherapy (AC). METHODS: A retrospective cohort study of women with cT1-2N0M0 TNBC diagnosed from 2014 to 2019 in the National Cancer Database (NCDB) was performed. Variables were analyzed via ANOVA, Chi-squared, Fisher Exact tests, and a multivariate linear regression model was created. RESULTS: 55,633 women were included: 26.9% received NAC, 52.4% AC, and 20.7% received no chemotherapy (median ages 53, 59, and 71 years, p < 0.01). NAC utilization significantly increased over time: 19.5% in 2014-15 (n = 3,465 of 17,777), 27.1% in 2016-17 (n = 5,140 of 18,985), and 33.6% in 2018-19 (n = 6,337 of 18,871, p < 0.001). On multivariate analysis, increased NAC was associated with younger age (< 50), non-Hispanic white race/ethnicity, lack of comorbidities, cT2 tumors, care at an academic or integrated-network cancer program, and diagnosis post-2017 (p < 0.05 for all). Patients with government-provided insurance were less likely to receive NAC (p < 0.01). Women who traveled > 60 miles for treatment were more likely to receive NAC (p < 0.01). CONCLUSION: From 2014 to 2019, NAC utilization increased for patients with cT1-2N0M0 TNBC. Racial, socioeconomic, and access disparities were observed in who received NAC vs AC and warrants interventions to ensure equitable care.

The intensity-dependent effects of exercise and superimposing environmental heat stress on autophagy in peripheral blood mononuclear cells from older men.

Autophagy is a vital cellular process, essential to maintaining cellular function during acute physiological stressors including exercise and heat stress. We previously showed that autophagy occurs during exercise in an intensity-dependent manner in peripheral blood mononuclear cells (PBMCs) from young men, with elevated responses in the heat. However, given autophagy declines with age, it is unclear whether a similar pattern of response occurs in older adults. Therefore, we evaluated autophagy and the cellular stress response [i.e., apoptosis, inflammation, and the heat shock response (HSR)] in PBMCs from 10 healthy older men [mean (SD): aged 70 yr (5)] in response to 30 min of semirecumbent cycling at low, moderate, and vigorous intensities [40, 55, and 70% maximal oxygen consumption (V̇o2max), respectively] in a temperate (25°C) environment, with an additional vigorous-intensity bout (70% of V̇o2max) performed in a hot environment (40°C). Responses were evaluated before and after exercise, as well as throughout a 6-h seated recovery period performed in the same environmental conditions as the respective exercise bout. Proteins were assessed via Western blot. Although we observed elevations in mean body temperature with each increase in exercise intensity, autophagy was only stimulated during vigorous-intensity exercise, where we observed elevations in LC3-II (P < 0.05). However, when the same exercise was performed in the heat, the LC3-II response was attenuated, which was accompanied by significant p62 accumulation (P < 0.05). Altogether, our findings demonstrate that older adults exhibit autophagic impairments when the same vigorous-intensity exercise is performed in hot environments, potentially underlying heat-induced cellular vulnerability in older men.NEW & NOTEWORTHY We demonstrate that autophagic stimulation occurs in response to short-duration (30-min) vigorous-intensity exercise in peripheral blood mononuclear cells from older adults; however, no changes in autophagy occur during low- or moderate-intensity exercise. Moreover, older adults exhibit autophagic impairments when the same vigorous-intensity exercise is performed in hot ambient conditions. When paired with an attenuated heat shock response, as well as elevated apoptotic responses, older men may exhibit greater cellular vulnerability to exertional heat stress.

Does Neoadjuvant Chemotherapy in Clinical T1-T2 N0 Triple-Negative Breast Cancer Increase the Extent of Axillary Surgery?

BACKGROUND: Current management strategies for early-stage triple-negative breast cancer (TNBC) include upfront surgery to determine pathologic stage to guide chemotherapy recommendations, or neoadjuvant chemotherapy (NAC) to de-escalate surgery, elucidate tumor response, and determine the role of adjuvant chemotherapy. However, patients who receive NAC with residual pathological nodal (pN) involvement require axillary lymph node dissection (ALND) as they are Z11/AMAROS ineligible. We aimed to evaluate the impact of NAC compared with upfront surgery on pN status and ALND rates in cT1-2N0 TNBC. METHODS: The National Cancer Database (NCDB) was queried for women with operable cT1-2N0 TNBC from 2014 to 2019. Demographic, clinicopathologic, and treatment data were collected. Multivariable linear regression analysis was performed to assess the odds of pN+ disease and undergoing ALND. RESULTS: Overall, 55,624 women were included: 26.9% (n = 14,942) underwent NAC and 73.1% (n = 40,682) underwent upfront surgery. The NAC cohort was younger (mean age 52.9 vs. 61.3 years; p < 0.001) with more cT2 tumors (71.6% vs. 31.0%; p < 0.001), and had lower ALND rates (4.3% vs. 5.5%; p < 0.001). The upfront surgery cohort was more likely to have one to three pathologically positive nodes (12.1% vs. 6.5%; odds ratio [OR] 2.37, 95% confidence interval (CI) 2.17-2.58; p < 0.001) but there was no difference in the likelihood of ALND (OR 1.1, 95% CI 0.99-1.24; p = 0.08). CONCLUSION: Patients who underwent upfront surgery were more likely to be pN+; however, ALND rates were similar between the two cohorts. Thus, the use of NAC does not result in a higher odds of ALND and the decision for NAC should be individualized and based on modern guidelines and systemic therapy benefits.

Elevated Electron Temperature Coincident with Observed Fusion Reactions in a Sheared-Flow-Stabilized Z Pinch.

The sheared-flow-stabilized Z pinch concept has been studied extensively and is able to produce fusion-relevant plasma parameters along with neutron production over several microseconds. We present here elevated electron temperature results spatially and temporally coincident with the plasma neutron source. An optical Thomson scattering apparatus designed for the FuZE device measures temperatures in the range of 1-3 keV on the axis of the device, 20 cm downstream of the nose cone. The 17-fiber system measures the radial profiles of the electron temperature. Scanning the laser time with respect to the neutron pulse time over a series of discharges allows the reconstruction of the T_{e} temporal response, confirming that the electron temperature peaks simultaneously with the neutron output, as well as the pinch current and inductive voltage generated within the plasma. Comparison to spectroscopic ion temperature measurements suggests a plasma in thermal equilibrium. The elevated T_{e} confirms the presence of a plasma assembled on axis, and indicates limited radiative losses, demonstrating a basis for scaling this device toward net gain fusion conditions.

Prehospital Assaults Against Asian Americans, Native Hawaiians, and Pacific Islanders During COVID-19.

INTRODUCTION: Anti-Asian sentiment increased when the SARS-CoV-2 virus reached the United States in 2020. Trends in national assaults occurring during the COVID-19 pandemic in the Asian American, Native Hawaiian, and Pacific Islander (AANHPI) community were evaluated. METHODS: Patients treated for assaults by emergency medical services between January 2019 and December 2021 were extracted from ImageTrend Collaborate, a national database. Multivariable logistic regression models, adjusting for age, sex, and urbanicity, were used to compare assault rates overall and in the AANHPI population. RESULTS: There were 84,825 assaults (8.5% of injury incidents) in 2019; 96,314 (9.2%) in 2020; and 97,841 (8.4%) in 2021. Assaults against AANHPI increased from 870 (7.1 assaults per 100 injuries) to 987 (8.3) and 1150 (7.9) between 2019 and 2021, respectively. Compared to 2019, risk of assaults in 2020 in all races increased (OR 1.08; 1.07, 1.10) but decreased in 2021 (OR 0.97; 0.96, 0.98). However, among AANHPI, risk of assaults increased in both 2020 (OR 1.22; 1.10, 1.35) and 2021 (OR 1.13; 1.03, 1.25). Most AANHPI assault victims were between 25 and 34 y old (11.8% in 2019) with an increase in 2020 (15.6%) and 2021 (14.4%). Assaults against AANHPI with blunt and sharp objects increased annually from 2019 to 2021. CONCLUSIONS: Despite national decreases of assaults in 2021 to pre-COVID baseline, the rate of assaults treated by emergency medical services for the AANHPI population remained elevated. Further studies analyzing in-hospital assault trends will allow for better understanding and will quantify the impact the pandemic and surrounding social influences had on minorities across the United States.

Barriers to Participation in a Structured Quality Improvement Initiative to Reduce Avoidable Emergency Department Visits-A Qualitative Study.

INTRODUCTION: Barriers to quality improvement (QI) initiatives in multi-institutional hospital settings are understudied. Here we describe a qualitative investigation of factors negatively affecting a QI initiative focused on reducing avoidable emergency department (ED) visits after bariatric surgery across 17 hospitals. Our goal was to explore participant perspectives and identify themes describing why the program was not effectively implemented or why the program may have been ineffective when correctly implemented. METHODS: We performed semistructured group interviews with 17 sites (42 interviews) participating in a statewide bariatric QI program. We used descriptive content analysis to identify challenges, facilitators, and barriers to implementation of the QI program. All analyses were conducted using MAXQDA software. RESULTS: Results revealed barriers across hospitals related to four themes: buy-in, provider accessibility, resources at participating hospitals, and patient barriers to care. In particular, the initiative faced difficulty if it was not well-matched to the factors driving increasing ED visits at a particular site, such as lack of patient access to outpatient or primary care. Additional challenges occurred if the initiative was not adapted and customized to the working systems in place at each site, involving employees, surgeons, support staff, and leadership. CONCLUSIONS: Overall, findings can direct future focused efforts aimed at site-specific interventions to reduce unnecessary postoperative ED visits. Results demonstrated a need for a nuanced approach that can be adapted based on facility needs and resources.

Impact of Estrogen and Progesterone on Immune Cells and Host-Pathogen Interactions in the Lower Female Reproductive Tract.

Microbial infections are a threat to women's reproductive health. Although reproductive cycles and pregnancy are controlled by sex hormones, the impact of hormones on host-pathogen interactions and immune function in the female reproductive tract are understudied. Furthermore, the changing endocrine environment throughout pregnancy may influence how and when women are susceptible to ascending infection. Because most intrauterine microbial infections originate in the lower reproductive tract, it is vital that future studies determine how different hormonal conditions influence the lower reproductive tract's susceptibility to infection to understand temporal components of infection susceptibilities across pregnancy. These studies should also extend to nonpregnant women, as it is critical to establish how hormonal fluctuations across the menstrual cycle and hormonal contraceptives may influence disease susceptibility. This review summarizes current knowledge of how estrogen and progesterone impact vaginal and cervical mucosal immunity, barrier function, and interactions with microbial communities.

Human biology and the study of precarity: How the intersection of uncertainty and inequality is taking us to new extremes.

Inequality represents an extreme environment to which humans must respond. One phenomenon that contributes to this growing extreme is precarity or the intersection of uncertainty and some form of inequality. While precarity has an important intellectual history in the fields of sociology and sociocultural anthropology, it has not been well studied in the field of human biology. Rather human biologists have engaged with the study of closely related concepts such as uncertainty and resource insecurity. In this article, we propose that human biology take on the study of precarity as a novel way of investigating inequality. We first provide a brief intellectual history of precarity which is followed by a review of research on uncertainty and resource security in human biology which, while not exhaustive, illustrates some key gaps that precarity may aid us in addressing. We then review some of the pathways through which precarity comes to affect human biology and health and some of the evidence for why the unpredictable nature of precarity may make it a unique physiological stress. A case study based on research in Nuñoa, Peru provides an important example of how precarity can elucidate the influences of health in an extreme setting, albeit with insights that apply more broadly. We conclude that precarity holds important potential for the study of human biology, including helping us more effectively operationalize and study uncertainty, encouraging us to explore the predictability of resources and stressors, and reminding us to think about the intersectional nature of stressors.

Parous women perform less moderate to vigorous physical activity than their nulliparous peers: a population-based study in Denmark.

OBJECTIVES: The World Health Organization (WHO) highlights parous women as a key population for monitoring trends of physical activity (PA). We aimed to estimate the proportion of Danish women non-adhering to WHO PA guidelines in parous women compared with nulliparous women and to describe leisure-time PA intensity in each of these groups. STUDY DESIGN: Cross-sectional study. METHODS: This population-based study builds on a sample of 27,668 women aged 16-40 years from the Danish National Health Survey 2021. These data were linked with childbirth data from the Danish National Birth Registry. The primary outcome was self-reported weekly hours of moderate to vigorous leisure-time PA (MVPA) dichotomized into: (i) adhering to WHO guidelines for MVPA or (ii) not adhering to WHO guidelines for MVPA. Binomial regression analysis was used to calculate prevalence proportions (PP) and prevalence proportion ratios (PPR). RESULTS: Of the 27,668 women, a total of 20,022 were included; 9338 (46.6%) parous women and 10,684 (53.4%) nulliparous women. The PP of women non-adhering to WHO PA guidelines was 63.8% (95% CI 62.9-64.8) for parous and 51.3% (95% CI 50.4-52.3) for nulliparous women, corresponding to a PPR of 1.24 (95% CI 1.21; 1.27). CONCLUSIONS: The proportion of parous women who did not adhere to WHO PA guidelines for MVPA was 24% higher than that of nulliparous women. This highlights parous women as a subgroup of the adult population at increased risk of non-adherence to WHO PA guidelines. These findings call for future research to inform new strategies aiming to promote PA in parous women.

Impaired autophagy following ex vivo cooling of simulated hypothermic temperatures in peripheral blood mononuclear cells from young and older adults.

Hypothermia is a critical consequence of extreme cold exposure that increases the risk of cold-related injury and death in humans. While the initiation of cytoprotective mechanisms including the process of autophagy and the heat shock response (HSR) is crucial to cellular survival during periods of stress, age-related decrements in these systems may underlie cold-induced cellular vulnerability in older adults. Moreover, whether potential sex-related differences in autophagic regulation influence the human cold stress response remain unknown. We evaluated the effect of age and sex on mechanisms of cytoprotection (autophagy and the HSR) and cellular stress (apoptotic signaling and the acute inflammatory response) during ex vivo hypothermic cooling. Venous blood samples from 20 healthy young (10 females; mean [SD]: 22 [2] years) and 20 healthy older (10 females; 66 [5] years) adults were either isolated immediately (baseline) for peripheral blood mononuclear cells (PBMCs) or exposed to water bath temperatures maintained at 37, 35, 33, 31, or 4 °C for 90 min before PBMC isolation. Proteins associated with autophagy, apoptosis, the HSR, and inflammation were analyzed via Western blotting. Indicators of autophagic initiation and signaling (LC3, ULK1, and beclin-2) and the HSR (HSP90 and HSP70) increased when exposed to hypothermic temperatures in young and older adults (all p ≤ 0.007). Sex-related differences were only observed with autophagic initiation (ULK1; p = 0.015). However, despite increases in autophagic initiators ULK1 and beclin-2 (all p < 0.001), this was paralleled by autophagic dysfunction (increased p62) in all groups (all p < 0.001). Further, apoptotic (cleaved-caspase-3) and inflammatory (IL-6 and TNF-α) signaling increased in all groups (all p < 0.001). We demonstrated that exposure to hypothermic conditions is associated with autophagic dysfunction, irrespective of age or sex, although there may exist innate sex-related differences in cytoprotection in response to cold exposure as evidenced through altered autophagic initiation.

Investigating and Treating a Corneal Ulcer Due to Extensively Drug-Resistant Pseudomonas aeruginosa.

Resistant Gram-negative bacteria are a growing concern in the United States, leading to significant morbidity and mortality. We identified a 72-year-old female patient who presented with unilateral vision loss. She was found to have a large corneal ulcer with hypopyon. Culture of corneal scrapings grew extensively drug-resistant Pseudomonas aeruginosa. Treatment involved a combination of systemic and topical antibiotics. Whole genome sequencing revealed the presence of blaVIM-80, blaGES-9, and other resistance determinants. This distinctive organism was linked to an over-the-counter artificial tears product.

Nonionic, Cleavable Surfactant for Top-Down Proteomics.

Nonionic surfactants are often used as general reagents for cell lysis enabling protein extraction, stabilization, and purification under nondenaturing conditions for downstream analysis in structural biology. However, the presence of surfactants in the sample matrix often has a deleterious effect on electrospray ionization (ESI)-mass spectrometry (MS) analysis of proteins and complexes. Here, we report a nonionic, cleavable surfactant, n-decyl-disulfide-ß-D-maltoside (DSSM), for top-down proteomics. DSSM was designed to mimic the properties of one of the most common surfactants used in structural biology, n-dodecyl-ß-D-maltoside (DDM), but contains a disulfide bond that allows for facile cleavage and surfactant removal before or during MS analysis. We have shown that DSSM is compatible with direct electrospray ionization (ESI)-MS analysis and reversed-phase liquid chromatography (RPLC)-MS analysis of proteins and protein complexes. We have demonstrated that DSSM can facilitate top-down proteomic characterization of membrane proteins such as a model ion channel protein and a G protein-coupled receptor as well as endogenous proteins from cell lysates for the determination of sequence variations and posttranslational modifications (PTMs). Conceivably, DSSM could serve as a general replacement for DDM in proteomic experiments and structural biology studies.

National Trends in "Going Flat" After Mastectomy.

INTRODUCTION: The "Going Flat" movement became widely publicized in 2016 and provides information and support to women who choose to forego post-mastectomy breast reconstruction (PMBR). The objectives of this study were to evaluate temporal trends in PMBR to ascertain the potential impact of this movement and assess which factors are associated with going flat. METHODS: A retrospective cohort analysis was performed using the NCDB of women with non-metastatic breast cancer who underwent mastectomy between 2004 and 2019. Trends in going flat after mastectomy were examined and stratified by age (< 50, 50-69, ≥ 70). A multivariate logistic regression model was used to identify factors associated with going flat. RESULTS: 650,983 patients met the inclusion criteria: 244,201 (37.5%) underwent PMBR and 406,782 (62.5%) went flat. Among women < 70, rates of going flat steadily decreased from 2004 to 2015 and then stabilized after 2015, coinciding with the rise of the "Going Flat" movement. In multivariate analysis, non-White race, older age, increasing comorbidities, government provided insurance, treatment at a community program, radiotherapy, and adjuvant chemotherapy were associated with a higher likelihood of going flat (p < 0.001). CONCLUSION: In the first 2 years after the "Going Flat" movement, the number of women going flat after mastectomy has stabilized in women < 70 for the first time in over a decade. These trends suggest that the social and cultural impact of this movement may have contributed to the stabilization of PMBR rates.

Proof-of-Principle Experiment for Testing Strong-Field Quantum Electrodynamics with Exotic Atoms: High Precision X-Ray Spectroscopy of Muonic Neon.

To test bound-state quantum electrodynamics (BSQED) in the strong-field regime, we have performed high precision x-ray spectroscopy of the 5g-4f and 5f- 4d transitions (BSQED contribution of 2.4 and 5.2 eV, respectively) of muonic neon atoms in the low-pressure gas phase without bound electrons. Muonic atoms have been recently proposed as an alternative to few-electron high-Z ions for BSQED tests by focusing on circular Rydberg states where nuclear contributions are negligibly small. We determined the 5g_{9/2}- 4f_{7/2} transition energy to be 6297.08±0.04(stat)±0.13(syst) eV using superconducting transition-edge sensor microcalorimeters (5.2-5.5 eV FWHM resolution), which agrees well with the most advanced BSQED theoretical prediction of 6297.26 eV.

Synthesis, Self-Assembly Properties, and Degradation Characterization of a Nonionic Photocleavable Azo-Sulfide Surfactant Family.

We report the synthesis and characterization of a new family of maltose-derived nonionic surfactants that contain a photocleavable azo-sulfide linker (mAzo). The self-assembly properties of these surfactants were investigated using surface tension measurements to determine the critical micelle concentration (CMC), dynamic light scattering (DLS) to reveal the hydrodynamic radius of their self-assemblies, and transmission electron microscopy (TEM) to elucidate the micelle morphology. Ultraviolet-visible (UV-visible) spectroscopy confirmed the rapid photodegradation of these surfactants, but surface tension measurements of the surfactant solutions before and after degradation showed unusual degradation products. The photodegradation process was further studied using online liquid chromatography coupled with mass spectrometry (LC-MS),which revealed that these surfactants can form another photo-stable surfactant post-degradation. Finally, traditionally challenging proteins from heart tissue were solubilized using the mAzo surfactants to demonstrate their potential in biological applications.

Reasons for Receiving or Not Receiving Bivalent COVID-19 Booster Vaccinations Among Adults - United States, November 1-December 10, 2022.

Bivalent COVID-19 booster vaccines, developed to protect against both ancestral and Omicron BA.4/BA.5 variants, are recommended to increase protection against SARS-CoV-2 infection and severe disease* (1,2). However, relatively few eligible U.S. adults have received a bivalent booster dose (3), and reasons for low coverage are unclear. An opt-in Internet survey of 1,200 COVID-19-vaccinated U.S. adults was conducted to assess reasons for receiving or not receiving a bivalent booster dose. Participants could select multiple reasons from a list of suggested reasons to report why they had or had not received a bivalent booster dose. The most common reasons cited for not receiving the bivalent booster dose were lack of awareness of eligibility for vaccination (23.2%) or of vaccine availability (19.3%), and perceived immunity against infection (18.9%). After viewing information about eligibility and availability, 67.8% of participants who had not received the bivalent booster dose indicated that they planned to do so; in a follow-up survey 1 month later, 28.6% of these participants reported having received the dose. Among those who had planned to receive the booster dose but had not yet done so, 82.6% still intended to do so. Participants who had still not received the booster dose most commonly reported being too busy to get vaccinated (35.6%). To help increase bivalent booster dose coverage, health care and public health professionals should use evidence-based strategies to convey information about booster vaccination recommendations and waning immunity (4), while also working to increase convenient access.

A 3D-printed multi-compartment device that enables dynamic PK/PD profiles of antibiotics.

Pathogens develop resistance to various drugs while under the selective pressure of antibiotics resulting in the emergence of bacterial strains that are resistant to multiple treatment options. Unfortunately, the resistance to antibiotics has also been accompanied by a reduction in the development of novel antibiotics to combat various pathogens. Current diagnostic tools, which are used in parts of the early developmental process of antibiotics, primarily consist of static susceptibility tests that do not resemble the pharmacokinetics of the therapy in vivo. Here, we designed and 3D-printed cubical inserts with membranes on two of the cube faces that allow diffusion of a molecule across two planes. These inserts are used with a 3D-printed device to create a two-compartment model to mimic the pharmacokinetics of a molecule in humans from multiple types of administration. Fluorescein was used to characterize the device and the diffusion of molecules from a flowing channel, through a membrane in the first plane (representing the primary compartment in vivo, or plasma), followed by measurement in the second compartment (that represents the interstitial fluid). The dynamic, two-compartment model was tested using both gram-positive and gram-negative bacterial strains in the secondary compartment. The ATP/OD600 (a measure of antibiotic activity) of a kanamycin-resistant E. coli strain challenged with the antibiotic levofloxacin increased after reaching an effective concentration of the antibiotic at 2 h, equating to a secondary compartment concentration of 3.5 ± 1.3 µM levofloxacin. The ATP/OD600 of a chloramphenicol-resistant B. subtilis strain challenged with the antibiotic levofloxacin remained steady or increased slightly after reaching an effective concentration of the antibiotic. The earliest statistical difference was detected 3 h after the start of the PK curve, which corresponds with a secondary compartment concentration of 4.8 ± 1.8 µM levofloxacin. Our results demonstrate that a fabricated 2-compartment model (1) provides realistic PK values to those published from in vivo studies and (2) can be used to determine antibiotic pharmacodynamics.

Non-Clinical Cell Therapy Development Using the NCG Mouse Model as a Test System.

The NCG triple immunodeficient mice on a NOD/Nju background lack functional/mature T, B, and NK cells, and have reduced macrophage and dendritic cell function. This study characterized the NCG mouse model for toxicity, engraftment and tumorigenicity assessments of cell therapies, using CD34+ hHSPC adult mobilized cells with two myeloablation regimens.Mice received sub-lethal irradiation or busulfan and were then injected intravenously with CD34+ hHSPCs (1.0 x 106 cells/mouse) or PBS (control), while positive control animals received 2 x 106 HL-60 cells/mouse. hCD34+ cell donors were treated with the mobilizing agent G-CSF prior to leukapheresis. Following injections, mouse blood samples were collected to assess engraftment rates by flow cytometry with body weights recorded periodically up to 20 weeks post-cell injection. No significant clinical signs or body weight changes were observed. At week 10 post-cell injection, the peripheral blood chimerism of hCD45+ cells was above 20%. While mCD45+ concentration was constant between week 10 and 17 in whole blood samples, hCD45+ concentration and chimerism slightly decreased at week 17. However, chimerism remained above 10%, with busulfan-treated mice presenting higher values. Chimerism was further assessed by quantifying human Alu sequences in blood and multiple organs using qPCR. Alu sequences were most abundant in the spleen and bone marrow, while lowest in the testes. In the positive control group, expected mortalities due to tumorigenesis were observed between days 27 and 40 post-cell injection. Overall, study results may be used to inform study design and potential toxicological endpoints relevant to non-clinical cell therapy development.