Rapid Antibiotic Susceptibility Testing by Deuterium Labeling of Bacterial Lipids in On-Target Microdroplet Cultures.

Antimicrobial resistance is a serious challenge facing human and veterinary health. Current methods of detecting resistance are limited in turn-around time or universal detection. In this work, a new antimicrobial susceptibility test is developed and validated, which utilizes deuterium labeling of membrane lipids to track the growth of bacterial cells. We hypothesize that deuterium uptake and subsequent labeling of lipids can be detected using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Additionally, bacteria growth is performed on the MALDI target, minimizing sample preparation materials and time. When two Escherichia coli strains are grown in the presence of deuterium oxide, labeling can be detected in as little as 30 min to 2 h. The labeling efficiency, or the ratio of labeled to unlabeled lipid peaks, provides information about the growth rate of bacteria. This growth ratio can differentiate between resistant and susceptible strains of bacteria as a resistant strain will maintain ∼50% labeling efficiency between untreated and treated cultures. In comparison, a susceptible strain will see a decrease in fractional abundance of deuterium from ∼50% in the untreated to ∼10% in the treated. This approach is applied to measure the minimum inhibitory concentration (MIC) of the resistant and susceptible strains from on-target microdroplet culture in a range of antibiotic concentrations. The first antibiotic concentration with a significant decrease in fractional abundance of deuterium correlates well with a traditionally obtained MIC using broth dilution, indicating the clinical relevance of the results.

Effective antiviral coatings for deactivating SARS-CoV-2 virus on N95 respirator masks or filters.

The application of antiviral coatings to masks and respirators is a potential mitigating step toward reducing viral transmission during the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. The use of appropriate masks, social distancing, and vaccines is the immediate solution for limiting the viral spread and protecting people from this virus. N95 respirator masks are effective in filtering the virus particles, but they cannot kill or deactivate the virus. We report a possible approach to deactivating SARS-CoV-2 by applying an antimicrobial coating (Goldshield 75) to masks and respirators, rendering them suitable for repeated use. Masks coated with Goldshield 75 demonstrated continuous inactivation of the Alpha and Beta variants of the SARS-CoV-2 over a 3-day period and no loss of inactivation when stored at temperatures at 50 °C.

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors.

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, "humanized") mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2RγC-/- NODSIRPα) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; "hot" tumors exhibit large immune infiltration while "cold" tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

Antibacterial treatment of lumpfish (Cyclopterus lumpus) experimentally challenged with Vibrio anguillarum, atypical Aeromonas salmonicida and Pasteurella atlantica.

Lumpfish is a novel farmed species used as cleaner fish for the removal of lice from farmed salmon. As often with new, farmed species, there are challenges with bacterial infections. The frequency of prescription of antibiotic agents to lumpfish is increasing, despite the lack of knowledge about appropriate doses, duration of treatment and application protocols for the various antibacterial agents. In the current study, we have tested the effect of medicated feed with florfenicol (FFC), oxolinic acid (OA) and flumequine (FLU) on lumpfish experimentally challenged with Vibrio anguillarum, atypical Aeromonas salmonicida and Pasteurella atlantica. We found that all three antibacterial agents efficiently treated lumpfish with vibriosis using 10 and 20 mg kg-1  day-1 of FFC, 25 mg kg-1  day-1 of OA and 25 mg kg-1  day-1 FLU, whereas only FFC (20 mg kg-1  day-1 ) had good effect on lumpfish with pasteurellosis. None of the antibacterial agents were efficient to treat lumpfish with atypical furunculosis. FFC 20 mg kg-1  day-1 showed promising results in the beginning of the experiment, but the mortality increased rapidly 14 days post-medication. Efficient treatment is important for the welfare of lumpfish and for reducing the risk of development of antibiotic-resistant bacteria. To our knowledge, this is the first study to establish protocols for antibacterial treatment of lumpfish.

Psychometric properties for the Norwegian translations of two revised APHAB-subscales and an adapted IOI-HA (IOI-CI) in patients with cochlear implants.

OBJECTIVE: The objective was to examine the psychometric properties of two questionnaires in Norwegian for the self-assessment of satisfaction following cochlear implantation. DESIGN: The International Outcome Inventory for Hearing Aids adapted for cochlear implants (IOI-CI) and two revised subscales from the Norwegian translation of the Abbreviated Profile of Hearing Aid Benefit (APHAB) were applied. Internal consistency was tested using Cronbach's α. Testing of psychometric properties included the calculation of inter-item correlations and corrected item-total correlations. Spearman's rho was used to investigate associations. Exploratory principal component analyses with Kaiser normalisation were performed. STUDY SAMPLE: One hundred and twenty-one patients (51 males) with cochlear implants (27-88 years, M = 59). RESULTS: For IOI-CI, Cronbach's α was 0.79. Corrected item-total correlations ranged from 0.29 to 0.69. The factor analysis revealed two components. For APHAB, Cronbach's α was 0.91. Two components were revealed by the factor analysis. Corrected item-total correlations for the two subscales "communication in quiet situations" and "communication in adverse situations" were 0.47-0.81 and 0.58-0.80, respectively. The convergent validity of the questionnaires was adequate as reflected by a Spearman's coefficient of 0.67. CONCLUSION: Both questionnaires show good psychometric properties.

Single-dose combination nanovaccine induces both rapid and durable humoral immunity and toxin neutralizing antibody responses against Bacillus anthracis.

Bacillus anthracis, the causative agent of anthrax, continues to be a prominent biological warfare and bioterrorism threat. Vaccination is likely to remain the most effective and user-friendly public health measure to counter this threat in the foreseeable future. The commercially available AVA BioThrax vaccine has a number of shortcomings where improvement would lead to a more practical and effective vaccine for use in the case of an exposure event. Identification of more effective adjuvants and novel delivery platforms is necessary to improve not only the effectiveness of the anthrax vaccine, but also enhance its shelf stability and ease-of-use. Polyanhydride particles have proven to be an effective platform at adjuvanting the vaccine-associated adaptive immune response as well as enhancing stability of encapsulated antigens. Another class of adjuvants, the STING pathway-targeting cyclic dinucleotides, have proven to be uniquely effective at inducing a beneficial inflammatory response that leads to the rapid induction of high titer antibodies post-vaccination capable of providing protection against bacterial pathogens. In this work, we evaluate the individual contributions of cyclic di-GMP (CDG), polyanhydride nanoparticles, and a combination thereof towards inducing neutralizing antibody (nAb) against the secreted protective antigen (PA) from B. anthracis. Our results show that the combination nanovaccine elicited rapid, high titer, and neutralizing IgG anti-PA antibody following single dose immunization that persisted for at least 108 DPI.

Immediate Bacille Calmette-Guérin Vaccination to Neonates Requiring Perinatal Treatment at the Maternity Ward in Guinea-Bissau: A Randomized Controlled Trial.

BACKGROUND: Randomized controlled trials (RCTs) indicate that bacille Calmette-Guérin (BCG) vaccination provides broad beneficial "nonspecific" protection against infections. We investigated the effect on in-hospital mortality of providing BCG immediately upon admission to a neonatal intensive care unit (NICU), rather than BCG-at-discharge. The pretrial NICU mortality was 13% and we hypothesized that BCG would reduce mortality by 40%. METHODS: Parallel-group, open-label RCT was initiated in 2013 in Guinea-Bissau. Neonatal intensive care unit-admitted neonates were randomized 1:1 to BCG + oral polio vaccine (OPV) immediately (intervention) versus BCG + OPV at hospital discharge (control; usual practice). The trial was discontinued due to decreasing in-hospital mortality and major NICU restructuring. We assessed overall and disease-specific mortality by randomization allocation in cox proportional hazards models providing mortality rate ratios (MRRs). RESULTS: We recruited 3353 neonates, and the overall mortality was 3.1% (52 of 1676) for BCG-vaccinated neonates versus 3.3% (55 of 1677) for controls (MRR = 0.94; 0.64-1.36). For noninfectious causes of death, the MRR was 1.20 (0.70-2.07), and there tended to be fewer deaths from infections in the BCG group (N = 14) than among controls (N = 21) (MRR = 0.65; 0.33-1.28). CONCLUSIONS: Providing BCG + OPV to frail neonates was safe and might protect against fatal infection in the immediate newborn period. Deaths due to prematurity and perinatal complications were unaffected by BCG.

Reduced grey- and white matter volumes due to unilateral hearing loss following treatment for vestibular schwannoma.

OBJECTIVE: Previous studies of the consequences of unilateral hearing loss (UHL) on the functional-structural organization of the brain has included subjects with various degrees of UHL. We suggest that the consequences of a total loss of hearing in one ear might differ from those seen in subjects with residual hearing in the affected ear. Thus, the main aim of the present study was to compare the structural properties of auditory and non-auditory brain regions in persons with complete UHL to those of normal hearing controls. We hypothesize that the consequences of complete UHL following treatment for vestibular schwannoma will differ between ipsi- and contralateral structures, as well as between right- and left side deafness. DESIGN: A 3T Siemens Prisma MR-scanner was used. Anatomical images were acquired using a high-resolution T1-weighted sequence. Grey- and white matter volumes were assessed using voxel-based morphometry. STUDY SAMPLE: Twenty-two patients with left- or right-side unilateral hearing loss. Fifty normal hearing controls. RESULTS: Reductions in grey- and white matter volumes were seen in cortical and sub-cortical regions, mainly in the right hemisphere including the auditory cortex, lingual gyrus, cuneus, middle temporal gyrus, occipital fusiform gyrus, middle cingulate gyrus and the superior temporal gyrus. Patients displayed reduced grey- and white matter volumes in cerebellar exterior structures ipsilateral to the tumor side. CONCLUSION: When compared to controls, right side hearing loss yields more widespread reduction of grey matter volume than left side hearing loss. The findings of reduced grey- and white matter volumes in auditory and non-auditory brain regions could be related to problems with speech perception in adverse listening conditions, increased listening effort and reduced quality of life reported by persons with unilateral hearing loss despite normal hearing in the unaffected ear.

BCG vaccination-induced emergency granulopoiesis provides rapid protection from neonatal sepsis.

Death from sepsis in the neonatal period remains a serious threat for millions. Within 3 days of administration, bacille Calmette-Guérin (BCG) vaccination can reduce mortality from neonatal sepsis in human newborns, but the underlying mechanism for this rapid protection is unknown. We found that BCG was also protective in a mouse model of neonatal polymicrobial sepsis, where it induced granulocyte colony-stimulating factor (G-CSF) within hours of administration. This was necessary and sufficient to drive emergency granulopoiesis (EG), resulting in a marked increase in neutrophils. This increase in neutrophils was directly and quantitatively responsible for protection from sepsis. Rapid induction of EG after BCG administration also occurred in three independent cohorts of human neonates.

Glycosylation is important for legumain localization and processing to active forms but not for cystatin E/M inhibitory functions.

The asparaginyl endopeptidase legumain and its inhibitor cystatin E/M are endogenously glycosylated. However, little is known about the nature of the carbohydrate groups and whether they affect the functions of these proteins. In this study both glycosylated and unglycosylated forms of legumain and cystatin E/M were studied. HEK293 cell lines stably over-expressing legumain or cystatin E/M, and HCT116 cells were used as cell models, and mature legumain was purified from bovine kidneys. To obtain unglycosylated proteins, cells were treated with tunicamycin, an inhibitor of N-linked glycosylation, whereas PNGase F and Endo H were used to characterize the glycosylation types. Cells were incubated with glycosylated, unglycosylated proteins and/or legumain selective activity-based probe, and legumain and/or cystatin E/M was studied by activity measurement, ELISA or immunoblotting in cell lysates or conditioned media. Legumain and probe in whole cells were studied by immunofluorescence. The carbohydrates on legumain were shown to be of the hybrid or high mannose type, whereas cystatin E/M was characterized as complex mannose-linked. While glycosylated prolegumain was able to autoactivate, the unglycosylated form was not, and addition of glycosaminoglycans did not facilitate autoactivation of unglycosylated prolegumain. Glycosylated prolegumain was internalized and processed to the mature active form, but no internalization of unglycosylated prolegumain was observed. A Cy5-labelled legumain specific activity-based probe (MP-L09) was synthesized and shown to be a novel tool to study intracellular legumain. Also, internalization of mature legumain (36 kDa) was visualized both alone and complexed with probe. Contrary to the importance of legumain glycosylation, both glycosylated and unglycosylated cystatin E/M showed similar capacity to inhibit legumain. In conclusion, glycosylation of prolegumain is necessary for correct processing to active forms and internalization, whereas the inhibitory property of cystatin E/M is independent of the glycosylation status.