Assessment of a SARS-CoV-2 wastewater monitoring program in El Paso, Texas, from November 2020 to June 2022.

The border city of El Paso, Texas, and its water utility, El Paso Water, initiated a SARS-CoV-2 wastewater monitoring program to assess virus trends and the appropriateness of a wastewater monitoring program for the community. Nearly weekly sample collection at four wastewater treatment facilities (WWTFs), serving distinct regions of the city, was analyzed for SARS-CoV-2 genes using the CDC 2019-Novel coronavirus Real-Time RT-PCR diagnostic panel. Virus concentrations ranged from 86.7 to 268,000 gc/L, varying across time and at each WWTF. The lag time between virus concentrations in wastewater and reported COVID-19 case rates (per 100,00 population) ranged from 4-24 days for the four WWTFs, with the strongest trend occurring from November 2021 - June 2022. This study is an assessment of the utility of a geographically refined SARS-CoV-2 wastewater monitoring program to supplement public health efforts that will manage the virus as it becomes endemic in El Paso.

Morphine-induced osteolysis and hypersensitivity is mediated through toll-like receptor-4 in a murine model of metastatic breast cancer.

ABSTRACT: The propensity for breast cancer to metastasize to bone is coupled to the most common complaint among breast cancer patients: bone pain. Classically, this type of pain is treated using escalating doses of opioids, which lack long-term efficacy due to analgesic tolerance, opioid-induced hypersensitivity, and have recently been linked to enhanced bone loss. To date, the molecular mechanisms underlying these adverse effects have not been fully explored. Using an immunocompetent murine model of metastatic breast cancer, we demonstrated that sustained morphine infusion induced a significant increase in osteolysis and hypersensitivity within the ipsilateral femur through the activation of toll-like receptor-4 (TLR4). Pharmacological blockade with TAK242 (resatorvid) as well as the use of a TLR4 genetic knockout ameliorated the chronic morphine-induced osteolysis and hypersensitivity. Genetic MOR knockout did not mitigate chronic morphine hypersensitivity or bone loss. In vitro studies using RAW264.7 murine macrophages precursor cells demonstrated morphine-enhanced osteoclastogenesis that was inhibited by the TLR4 antagonist. Together, these data indicate that morphine induces osteolysis and hypersensitivity that are mediated, in part, through a TLR4 receptor mechanism.

A Retrospective, Observational Study of 12 Cases of Expanded-Access Customized Phage Therapy: Production, Characteristics, and Clinical Outcomes.

BACKGROUND: Antimicrobial resistance (AMR) is undermining modern medicine, a problem compounded by bacterial adaptation to antibiotic pressures. Phages are viruses that infect bacteria. Their diversity and evolvability offer the prospect of their use as a therapeutic solution. Reported are outcomes of customized phage therapy for patients with difficult-to-treat antimicrobial resistant infections. METHODS: We retrospectively assessed 12 cases of customized phage therapy from a phage production center. Phages were screened, purified, sequenced, characterized, and Food and Drug Administration-approved via the IND (investigational new drug) compassionate-care route. Outcomes were assessed as favorable or unfavorable by microbiologic and clinical standards. Infections were device-related or systemic. Other experiences such as time to treatment, antibiotic synergy, and immune responses were recorded. RESULTS: Fifty requests for phage therapy were received. Customized phages were generated for 12 patients. After treatment, 42% (5/12) of cases showed bacterial eradication and 58% (7/12) showed clinical improvement, with two-thirds of all cases (66%) showing favorable responses. No major adverse reactions were observed. Antibiotic-phage synergy in vitro was observed in most cases. Immunological neutralization of phages was reported in 5 cases. Several cases were complicated by secondary infections. Complete characterization of the phages (morphology, genomics, and activity) and their production (methods, sterility, and endotoxin tests) are reported. CONCLUSIONS: Customized phage production and therapy was safe and yielded favorable clinical or microbiological outcomes in two-thirds of cases. A center or pipeline dedicated to tailoring the phages against a patient's specific AMR bacterial infection may be a viable option where standard treatment has failed.

The Physical Compatibility of Glycopyrrolate and Rocuronium.

OBJECTIVE: Scientific evidence has rarely, if at all, been reported in the literature demonstrating analytical confirmation of the physical compatibility and stability of glycopyrrolate and rocuronium combined. The purpose of this experiment was to determine if glycopyrrolate and rocuronium are physically compatible. METHODS: Glycopyrrolate and rocuronium were combined in various containers, observed over a 60-minute period, and compared against positive and negative controls. Measured metrics included color change, precipitate formation, Tyndall beam test, turbidity, and pH. Statistical analyses were used to assess significance of data trends. RESULTS: The combination of glycopyrrolate and rocuronium did not result in any color change, precipitate formation, a positive Tyndall beam test, or a significantly positive turbidity and did not result in any significant change in pH, regardless of container. CONCLUSION: Per the protocol used in this study, glycopyrrolate and rocuronium were determined to be physically compatible.

Genomic and Functional Characterization of Vancomycin-Resistant Enterococci-Specific Bacteriophages in the Galleria mellonella Wax Moth Larvae Model.

Phages are naturally occurring viruses that selectively kill bacterial species without disturbing the individual's normal flora, averting the collateral damage of antimicrobial usage. The safety and the effectiveness of phages have been mainly confirmed in the food industry as well as in animal models. In this study, we report on the successful isolation of phages specific to Vancomycin-resistant Enterococci, including Enterococcus faecium (VREfm) and Enterococcus faecalis from sewage samples, and demonstrate their efficacy and safety for VREfm infection in the greater wax moth Galleria mellonella model. No virulence-associated genes, antibiotic resistance genes or integrases were detected in the phages' genomes, rendering them safe to be used in an in vivo model. Phages may be considered as potential agents for therapy for bacterial infections secondary to multidrug-resistant organisms such as VREfm.

Impact of Frailty on Short-Term Outcomes After Laparoscopic and Open Hepatectomy.

BACKGROUND: Although laparoscopic hepatectomy (LH) is associated with improved short-term outcomes compared to open hepatectomy (OH), it is unknown whether frail patients also benefit from LH. The aim of this study was to evaluate the impact of frailty on post-operative outcomes after LH and OH. PATIENTS AND METHODS: Consecutive patients who underwent LH and OH between January 2011 and December 2018 were identified from a prospective database. Frailty was assessed using the modified Frailty Index (mFI), with patients scoring mFI ≥ 1 deemed to be frail. RESULTS: Of 1826 patients, 34.7% (N = 634) were frail and 18.6% (N = 340) were elderly (≥ 75 years). Frail patients had significantly higher 90-day mortality (6.6% vs. 2.9%, p < 0.001) and post-operative complications (36.3% vs. 26.1%, p < 0.001) than those who were not frail, effects that were independent of patient age on multivariate analysis. For those undergoing minor resections, the benefits of LH vs. OH were similar for frail and non-frail patients. Length of hospital stay was 53% longer in OH (vs. LH) in frail patients, compared to 58% longer in the subgroup of non-frail patients. CONCLUSIONS: Frailty is independently associated with inferior post-operative outcomes in patients undergoing hepatectomy. However, the benefits of laparoscopic (compared to open) hepatectomy are similar for frail and non-frail patients. Frailty should not be a contraindication to laparoscopic minor hepatectomy in carefully selected patients.

Cell surface photoengineering enables modeling of glycocalyx shedding dynamics.

The cellular glycocalyx, composed of membrane associated glycoproteins and glycolipids, is a complex and dynamic interface that facilitates interactions between cells and their environment. The glycocalyx composition is continuously changing through biosynthesis of new glycoconjugates and membrane turnover. Various glycocalyx components, such as mucins, can also be rapidly shed from the cell surface in response to acute events, such as pathogenic threat. Mucins, which are large extended glycoproteins, deliver important protective functions against infection by creating a physical barrier at the cell surface and by capturing and clearing pathogens through shedding. Evaluating these mucin functions may provide better understanding of early stages of pathogenesis; however, tools to tailor the composition and dynamics of the glycocalyx with precision are still limited. Here, we report a chemical cell surface engineering strategy to model the shedding behavior of mucins with spatial and temporal control. We generated synthetic mucin mimetic glycopolymers terminated with a photolabile membrane anchor, which could be introduced into the membranes of living cells and, subsequently, released upon exposure to UV light. By tuning the molecular density of the artificial glycocalyx we evaluated lectin crosslinking and its effect on shedding, showing that lectins can stabilize the glycocalyx and limit release of the mucin mimetics from the cell surface. Our findings indicate that endogenous and pathogen-associated lectins, which are known to interact with the host-cell glycocalyx, may alter mucin shedding dynamics and influence the protective properties of the mucosal barrier. More broadly, we present a method which enables photoengineering of the glycocalyx and can be used to facilitate the study of glycocalyx dynamics in other biological contexts.

Stem Cell Microarrays for Assessing Growth Factor Signaling in Engineered Glycan Microenvironments.

Extracellular glycans, such as glycosaminoglycans (GAGs), provide an essential regulatory component during the development and maintenance of tissues. GAGs, which harbor binding sites for a range of growth factors (GFs) and other morphogens, help establish gradients of these molecules in the extracellular matrix (ECM) and promote the formation of active signaling complexes when presented at the cell surface. As such, GAGs have been pursued as biologically active components for the development of biomaterials for cell-based regenerative therapies. However, their structural complexity and compositional heterogeneity make establishing structure-function relationships for this class of glycans difficult. Here, a stem cell array platform is described, in which chemically modified heparan sulfate (HS) GAG polysaccharides are conjugated to a gelatin matrix and introduced into a polyacrylamide hydrogel network. This array allowed for direct analysis of HS contributions to the signaling via the FGF2-dependent mitogen activated protein kinase (MAPK) pathway in mouse embryonic stem cells. With the recent emergence of powerful synthetic and recombinant technologies to produce well-defined GAG structures, a platform for analyzing both growth factor binding and signaling in response to the presence of these biomolecules will provide a powerful tool for integrating glycans into biomaterials to advance their biological properties and applications.

Metabolic profiling reveals nutrient preferences during carbon utilization in Bacillus species.

The genus Bacillus includes species with diverse natural histories, including free-living nonpathogenic heterotrophs such as B. subtilis and host-dependent pathogens such as B. anthracis (the etiological agent of the disease anthrax) and B. cereus, a cause of food poisoning. Although highly similar genotypically, the ecological niches of these three species are mutually exclusive, which raises the untested hypothesis that their metabolism has speciated along a nutritional tract. Here, we developed a pipeline for quantitative total assessment of the use of diverse sources of carbon for general metabolism to better appreciate the "culinary preferences" of three distinct Bacillus species, as well as related Staphylococcus aureus. We show that each species has widely varying metabolic ability to utilize diverse sources of carbon that correlated to their ecological niches. This approach was applied to the growth and survival of B. anthracis in a blood-like environment and find metabolism shifts from sugar to amino acids as the preferred source of energy. Finally, various nutrients in broth and host-like environments are identified that may promote or interfere with bacterial metabolism during infection.

Antiviral Resistance and Phage Counter Adaptation to Antibiotic-Resistant Extraintestinal Pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC), often multidrug resistant (MDR), is a leading cause of urinary tract and systemic infections. The crisis of emergent MDR pathogens has led some to propose bacteriophages as a therapeutic. However, bacterial resistance to phage is a concerning issue that threatens to undermine phage therapy. Here, we demonstrate that E. coli sequence type 131, a circulating pandemic strain of ExPEC, rapidly develops resistance to a well-studied and therapeutically active phage (ϕHP3). Whole-genome sequencing of the resisters revealed truncations in genes involved in lipopolysaccharide (LPS) biosynthesis, the outer membrane transporter ompA, or both, implicating them as phage receptors. We found ExPEC resistance to phage is associated with a loss of fitness in host microenvironments and attenuation in a murine model of systemic infection. Furthermore, we constructed a novel phage-bacterium bioreactor to generate an evolved phage isolate with restored infectivity to all LPS-truncated ExPEC resisters. This study suggests that although the resistance of pandemic E. coli to phage is frequent, it is associated with attenuation of virulence and susceptibility to new phage variants that arise by directed evolution.IMPORTANCE In response to the rising crisis of antimicrobial resistance, bacteriophage (phage) therapy has gained traction. In the United States, there have been over 10 cases of largely successful compassionate-use phage therapy to date. The resilience of pathogens allowing their broad antibiotic resistance means we must also consider resistance to therapeutic phages. This work fills gaps in knowledge regarding development of phage resisters in a model of infection and finds critical fitness losses in those resisters. We also found that the phage was able to rapidly readapt to these resisters.

Wastewater Analysis of SARS-CoV-2 as a Predictive Metric of Positivity Rate for a Major Metropolis

Wastewater monitoring for SARS-CoV-2 has been suggested as an epidemiological indicator of community infection dynamics and disease prevalence. We report wastewater viral RNA levels of SARS-CoV-2 in a major metropolis serving over 3.6 million people geographically spread over 39 distinct sampling sites. Viral RNA levels were followed weekly for 22 weeks, both before, during, and after a major surge in cases, and simultaneously by two independent laboratories. We found SARS-CoV-2 RNA wastewater levels were a strong predictive indicator of trends in the nasal positivity rate two-weeks in advance. Furthermore, wastewater viral RNA loads demonstrated robust tracking of positivity rate for populations served by individual treatment plants, findings which were used in real-time to make public health interventions, including deployment of testing and education strike teams.

Phage-Antibiotic Synergy Is Driven by a Unique Combination of Antibacterial Mechanism of Action and Stoichiometry.

The continued rise in antibiotic resistance is precipitating a medical crisis. Bacteriophage (phage) has been hailed as one possible therapeutic option to augment the efficacy of antibiotics. However, only a few studies have addressed the synergistic relationship between phage and antibiotics. Here, we report a comprehensive analysis of phage-antibiotic interaction that evaluates synergism, additivism, and antagonism for all classes of antibiotics across clinically achievable stoichiometries. We combined an optically based real-time microtiter plate readout with a matrix-like heat map of treatment potencies to measure phage and antibiotic synergy (PAS), a process we term synography. Phage-antibiotic synography was performed against a pandemic drug-resistant clonal group of extraintestinal pathogenic Escherichia coli (ExPEC) with antibiotic levels blanketing the MIC across seven orders of viral titers. Our results suggest that, under certain conditions, phages provide an adjuvating effect by lowering the MIC for drug-resistant strains. Furthermore, synergistic and antagonistic interactions are highly dependent on the mechanism of bacterial inhibition by the class of antibiotic paired to the phage, and when synergism is observed, it suppresses the emergence of resistant cells. Host conditions that simulate the infection environment, including serum and urine, suppress PAS in a bacterial growth-dependent manner. Lastly, two different related phages that differed in their burst sizes produced drastically different synograms. Collectively, these data suggest lytic phages can resuscitate an ineffective antibiotic for previously resistant bacteria while also synergizing with antibiotics in a class-dependent manner, processes that may be dampened by lower bacterial growth rates found in host environments.IMPORTANCE Bacteriophage (phage) therapy is a promising approach to combat the rise of multidrug-resistant bacteria. Currently, the preferred clinical modality is to pair phage with an antibiotic, a practice thought to improve efficacy. However, antagonism between phage and antibiotics has been reported, the choice of phage and antibiotic is not often empirically determined, and the effect of the host factors on the effectiveness is unknown. Here, we interrogate phage-antibiotic interactions across antibiotics with different mechanisms of action. Our results suggest that phage can lower the working MIC for bacterial strains already resistant to the antibiotic, is dependent on the antibiotic class and stoichiometry of the pairing, and is dramatically influenced by the host microenvironment.

Heat shock protein 90 inhibitors block the antinociceptive effects of opioids in mouse chemotherapy-induced neuropathy and cancer bone pain models.

Heat shock protein 90 (Hsp90) is a ubiquitous signal transduction regulator, and Hsp90 inhibitors are in clinical development as cancer therapeutics. However, there have been very few studies on the impact of Hsp90 inhibitors on pain or analgesia, a serious concern for cancer patients. We previously found that Hsp90 inhibitors injected into the brain block opioid-induced antinociception in tail flick, paw incision, and HIV neuropathy pain. This study extended from that initial work to test the cancer-related clinical impact of Hsp90 inhibitors on opioid antinociception in cancer-induced bone pain in female BALB/c mice and chemotherapy-induced peripheral neuropathy in male and female CD-1 mice. Mice were treated with Hsp90 inhibitors (17-AAG, KU-32) by the intracerebroventricular, intrathecal, or intraperitoneal routes, and after 24 hours, pain behaviors were evaluated after analgesic drug treatment. Heat shock protein 90 inhibition in the brain or systemically completely blocked morphine and oxymorphone antinociception in chemotherapy-induced peripheral neuropathy; this effect was partly mediated by decreased ERK and JNK MAPK activation and by increased protein translation, was not altered by chronic treatment, and Hsp90 inhibition had no effect on gabapentin antinociception. We also found that the Hsp90 isoform Hsp90α and the cochaperone Cdc37 were responsible for the observed changes in opioid antinociception. By contrast, Hsp90 inhibition in the spinal cord or systemically partially reduced opioid antinociception in cancer-induced bone pain. These results demonstrate that Hsp90 inhibitors block opioid antinociception in cancer-related pain, suggesting that Hsp90 inhibitors for cancer therapy could decrease opioid treatment efficacy.

Tailored Antibacterials and Innovative Laboratories for Phage (Φ) Research: Personalized Infectious Disease Medicine for the Most Vulnerable At-Risk Patients.

Mutation is the most powerful driver of change for life on Earth. Pathogenic bacteria utilize mutation as a means to survive strong live-die selective pressures generated by chemical antibiotics. As such, the traditional drug-making pipeline, characterized by significant financial and time investment, is insufficient to keep pace with the rapid evolution of bacterial resistance to structurally fixed and chemically unmalleable antibacterial compounds. In contrast, the genetic diversity and adaptive mutability of the bacteriophage can be leveraged to not only overcome resistance but also used for the development of enhanced traits that increase lytic potential and therapeutic efficacy in relevant host microenvironments. This is the fundamental premise behind Baylor College of Medicine's Tailored Antibacterials and Innovative Laboratories for Phage (Φ) Research (TAILΦR) initiative. In this perspective, we outline the concept, structure, and process behind TAILΦR's attempt to generate a personalized therapeutic phage that addresses the most clinically challenging of bacterial infections.

Tunable pH-Sensitive 2-Carboxybenzyl Phosphoramidate Cleavable Linkers.

We previously described a pH-sensitive phosphoramidate linker scaffold that can be tuned to release amine-containing drugs at various pH values. In these previous studies it was determined that the tunability of this linker was dependent upon the proximity of an acidic group (e.g., carboxylic acid or pyridinium). In this study, we confirmed that the tunability of pH-triggered amine-release was also dependent upon the pKa of the proximal acidic group. A series of 2-carboxybenzyl phosphoramidates was prepared in which the pKa of the proximal benzoic acid was predictably attenuated by substituents on the benzoate ring consistent with their σ-values.

Constructing and Characterizing Bacteriophage Libraries for Phage Therapy of Human Infections.

Phage therapy requires libraries of well-characterized phages. Here we describe the generation of phage libraries for three target species: Escherichia coli, Pseudomonas aeruginosa, and Enterobacter cloacae. The basic phage characteristics on the isolation host, sequence analysis, growth properties, and host range and virulence on a number of contemporary clinical isolates are presented. This information is required before phages can be added to a phage library for potential human use or sharing between laboratories for use in compassionate use protocols in humans under eIND (emergency investigational new drug). Clinical scenarios in which these phages can potentially be used are discussed. The phages presented here are currently being characterized in animal models and are available for eINDs.

Silencing glycosaminoglycan functions in mouse embryonic stem cells with small molecule antagonists.

Glycosylation is a ubiquitous post-translational modification that decorates proteins and lipids with glycans. These glycans can play critical roles in regulating biological events, and therefore, the discovery of strategies that target these molecules represent an important advancement toward understanding and controlling glycan-mediated cellular phenotypes. We describe the use of a small molecule, surfen, to temporarily silence the functions mediated by heparan sulfate glycosaminoglycans in mouse embryonic stem cells. Surfen binds heparan sulfate to antagonize growth factor interactions, thereby inhibiting signal transduction events that lead to differentiation. The strategies outlined in this chapter allow the characterization of resulting antagonistic effects caused by glycan-small molecule binding events toward maintaining embryonic stem cell pluripotency, curbing differentiation, and inhibiting signaling events.

Determination of Gas-Phase Ion Mobility Coefficients Using Voltage Sweep Multiplexing.

In a standard single averaged, drift tube ion mobility spectrometry (IMS) experiment, typically less than 1% of the ions are utilized, with the rest of the ions neutralizing on a closed ion gate or ion optic element. Though some efforts at lower pressures (e.g., 4 Torr) have been made to address this issue by concentrating ions prior to release into a drift cell, the ion current reaching the detector during an IMS experiment is often diminished due to this lower duty cycle. Additionally, when considering the temporal nature of the drift tube IMS experiment and the trajectory of IMS towards higher resolution separations and lower duty cycles, increased detector sampling rates are another factor also which further necessitates new modes of conducting the IMS experiment. Placing this trend in context with ion mobility-mass spectrometry instruments (IM-MS), there are numerous types of mass spectrometers that are simply incompatible with the single averaged ion mobility spectrometry experiments due to timing incompatibilities (i.e., ion traps are an order of magnitude slower than the IMS experiment). However, by utilizing a dual gate ion mobility spectrometer for ion multiplexing, ion utilization efficiency can be significantly increased while creating a measurement signal that can be recorded at low sampling rates. In this work, we present the fundamental theory and first results from proof-of-concept measurements using a new type of ion multiplexing that relies on changing the electric field within the drift cell during the course of an experiment while simultaneously opening the ion gates at a constant frequency. For brevity, this mode is termed voltage sweep multiplexing (VSM). Key variables for this type of experiment are discussed and verified with measurements from traditional signal averaged experiments. Graphical Abstract .

Animal Models for the Study of Bone-Derived Pain.

Bone pain is a prevalent issue in society today and also is one of the hardest types of pain to control. Pain originating in the bone can be caused by many different entities including metastatic and primary neoplasm, fracture, osteoarthritis as well as numerous other metabolic disorders. In this chapter we describe the methods and protocols that currently are accepted and validated for the study of bone pain in models of metastatic cancer, bicortical fracture and osteoarthritis. These animal models provide invaluable information as to the nature of bone pain and give rise to potential new targets for its treatment and management.

Optimized Reconstruction Techniques for Multiplexed Dual-Gate Ion Mobility Mass Spectrometry Experiments.

When coupling drift-tube gas-phase ion mobility separations with ion trapping mass analyzers an integrative, stepped approach to spectral reconstruction is a logical, yet highly inefficient means to determine gas-phase mobility coefficients. This experimental mode is largely predicated on the respective time scales of the two techniques each requiring tens of milliseconds to complete under routine conditions. Multiplexing techniques, such as Fourier and Hadamard based techniques, are a potential solution but still require extended experimental times that are not fully compatible with modern front-end separation schemes. Using a basis pursuit denoising (BPDN) approach to deconvolute Fourier transform ion mobility mass spectrometry (FT-IMMS) drift time spectra, we demonstrate significant time savings while maintaining a high degree of spectral resolution and signal-to-noise ratio. Under ideal conditions, the FT-IMMS operates with increased ion transmission (up to 25%); however, the linear chirp that spans into the kHz range often leads to significant levels of ion gate depletion, which limit both resolving power and ion transmission. The method proposed in this manuscript demonstrates the potential to reduce IMS acquisition time while simultaneously maximizing spectral resolution at longer effective gate pulse widths compared to the traditional set of multiplexing and signal averaging experiments.