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The Arctic Ocean is currently undergoing significant transformations due to climate change, leading to profound changes in its microbial plankton communities, including photoautotrophic prokaryotes and eukaryotes (i.e. phytoplankton), as well as hetero-, phago-, and mixotrophic protistan species. Among these unicellular organisms, species of potentially toxic and/or harmful algae (hereafter referred to as HA) are of particular concern, as they pose a threat to human and ecosystem health if they potentially spread into Arctic waters. Despite their importance, the spatiotemporal distribution of these communities in the North American Arctic is poorly understood. To address this gap, we compiled and synthesized a large dataset from various sources, partitioned into nine regions based on the Large Marine Ecosystem classification. Our dataset contains 385 348 georeferenced data points and 18 268 unique sampling events (https://doi.org/10.5281/zenodo.10498858, Schiffrine et al., 2024), encompassing 1442 unique taxa, with Heterokontophyta (notably diatoms) and Dinoflagellata being the most dominant phyla. Our results indicate distinct spatial patterns of diversity, with the highest diversity observed in Atlantic-influenced regions of the North American Arctic. An analysis of the maximum latitude of HA species over time shows a gradual increase, with a notable rise towards the 1990s. However, this trend is likely influenced by increased research at higher latitudes, meaning no substantial spread of HA species into the North American part of the Arctic. Our study underscores the importance of extensive and long-term sampling efforts to understand the Arctic's biodiversity, particularly with respect to documenting the presence and distribution of HA species. While the occurrence of HA species in the Arctic is recognized, our findings highlight the need for further detailed investigations to fully grasp their ecological impacts and variability in the region. Overall, our results provide new insights into the spatial patterns and biodiversity of the microbial plankton communities in the North American Arctic and have implications for understanding the ecological functioning and response of this region to ongoing climate change.

We assessed antimicrobial resistance (AMR) in Neisseria gonorrhoeae in Nunavut, Canada, using remnant gonorrhea nucleic acid amplification test–positive urine specimens. This study confirms the feasibility of conducting N. gonorrhoeae AMR surveillance and highlights the diversity of gonococcal sequence types and geographic variation of AMR patterns in the territory.

Bimodal interactions between relevant visual and tactile inputs can facilitate attentional modulation at early stages in somatosensory cortices to achieve goal-oriented behaviors. However, the specific contribution of each sensory system during attentional processing and, importantly, how these interact with the required behavioural motor goals remains unclear. Here we used EEG and event-related potentials (ERPs) to test the hypothesis that activity from modality-specific somatosensory cortical regions would be enhanced with task-relevant bimodal (visual-tactile) stimuli and that the degree of modulation would depend on the difficulty of the associated sensory-motor task demands. Tactile stimuli were discrete vibrations to the index finger and visual stimuli were horizontal bars on a computer screen, both with random amplitudes. Streams of unimodal (tactile) and crossmodal (visual and tactile) stimuli were randomly presented and participants were instructed to attend to one type of stimulus (unimodal or crossmodal) and responses involved either an indication of the presence of an attended stimulus (detect), or the integration and summation of 2 stimulus amplitudes using a pressure-sensitive ball (grade). Force-amplitude associations were learned in a training session, and no feedback was provided during the task. ERPs were time-locked to tactile stimuli and extracted for early modality-specific components (P50, P100, N140). The P50 was enhanced with bimodal (visual-tactile) stimuli that were attended to. This was maximal when the motor requirements involved integration of the 2 stimuli in the grade task and when the visual stimulus occurred before (100 ms) the tactile stimulus. These results suggest that visual information relevant for movement modulates early somatosensory processing and that the motor behavioral context influences this likely through interaction of top-down attentional and motor preparatory systems with more bottom-up crossmodal influences.

The majority of naturally elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs) because they are unable to recognize the Env trimer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC-mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly neutralizing antibodies and even showed activity against HIV-1-infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies.IMPORTANCEThe elimination of HIV-1-infected cells remains an important medical goal. Although current antiretroviral therapy decreases viral loads below detection levels, it does not eliminate latently infected cells that form the viral reservoir. Here, we developed a cocktail of non-neutralizing antibodies targeting highly conserved Env regions and combined it with a potent indoline CD4mc. This combination exhibited potent ADCC activity against HIV-1-infected primary CD4 + T cells as well as monocyte-derived macrophages, suggesting its potential utility in decreasing the size of the viral reservoir., Competing Interests: The authors declare no conflict of interest.

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. Structural and conformational analyses reveal that CJF-III-288, in combination with anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation, This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

Natural Killer (NK) cells have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is tightly regulated by the engagement of its inhibitory and activating receptors. The activating receptor CD16 drives ADCC upon binding to the Fc portion of antibodies; NK cell activation is further sustained by the co-engagement of activating receptors NTB-A and 2B4. During HIV-1 infection, Nef and Vpu accessory proteins contribute to ADCC escape by downregulating the ligands of NTB-A and 2B4. HIV-1 also evades ADCC by keeping its envelope glycoproteins (Env) in a "closed" conformation which effectively masks epitopes recognized by non-neutralizing antibodies (nnAbs) which are abundant in the plasma of people living with HIV. To achieve this, the virus uses its accessory proteins Nef and Vpu to downregulate the CD4 receptor, which otherwise interacts with Env and exposes the epitopes recognized by nnAbs. Small CD4-mimetic compounds (CD4mc) have the capacity to expose these epitopes, thus sensitizing infected cells to ADCC. Given the central role of NK cell co-activating receptors NTB-A and 2B4 in Fc-effector functions, we studied their contribution to CD4mc-mediated ADCC. Despite the fact that their ligands are partially downregulated by HIV-1, we found that both co-activating receptors significantly contribute to CD4mc sensitization of HIV-1-infected cells to ADCC.

Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified 1-4 , the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Since the beginning of the SARS-CoV-2 pandemic, several variants of concern (VOCs), such as the Alpha, Beta, Gamma, Delta and Omicron variants, have arisen and spread worldwide. Today, the predominant circulating subvariants are sublineages of the Omicron variant, which have more than 30 mutations in their Spike glycoprotein compared to the ancestral strain. The Omicron subvariants were significantly less recognized and neutralized by antibodies from vaccinated individuals. This resulted in a surge in the number of infections, and booster shots were recommended to improve responses against these variants. While most studies mainly measured the neutralizing activity against variants, we and others previously reported that Fc-effector functions, including antibody-dependent cellular cytotoxicity (ADCC), play an important role in humoral responses against SARS-CoV-2. In this study, we analyzed Spike recognition and ADCC activity against several Omicron subvariants by generating cell lines expressing different Omicron subvariant Spikes. We tested these responses in a cohort of donors, who were recently infected or not, before and after a fourth dose of mRNA vaccine. We showed that ADCC activity is less affected than neutralization by the antigenic shift of the tested Omicron subvariant Spikes. Moreover, we found that individuals with a history of recent infection have higher antibody binding and ADCC activity against all Omicron subvariants than people who were not recently infected. With an increase in the number of reinfections, this study helps better understand Fc-effector responses in the context of hybrid immunity.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants have recently emerged, becoming the dominant circulating strains in many countries. These variants contain a large number of mutations in their spike glycoprotein, raising concerns about vaccine efficacy. In this study, we evaluate the ability of plasma from a cohort of individuals that received three doses of mRNA vaccine to recognize and neutralize these Omicron subvariant spikes. We observed that BA.4/5 and BQ.1.1 spikes are markedly less recognized and neutralized compared with the D614G and other Omicron subvariant spikes tested. Also, individuals who have been infected before or after vaccination present better humoral responses than SARS-CoV-2-naive vaccinated individuals, thus indicating that hybrid immunity generates better humoral responses against these subvariants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

COVID-19 convalescent plasmas (CCPs) are chosen for plasma therapy based on neutralizing titers and anti-Spike immunoglobulin levels. However, CCP characteristics that promote SARS-CoV-2 control are complex and incompletely defined. Using an in vivo imaging approach, we demonstrate that CCPs with low neutralizing (ID 50  ≤ 1:250), but moderate to high Fc-effector activity, in contrast to those with poor Fc function, delay mortality and/or improve survival of SARS-CoV-2-challenged K18-hACE2 mice. The impact of innate immune cells on CCP efficacy depended on their residual neutralizing activity. Fractionation of a selected CCP revealed that IgG and Ig(M + A) were required during therapy, but the IgG fraction alone sufficed during prophylaxis. Finally, despite reduced neutralization, ancestral SARS-CoV-2-elicited CCPs significantly delayed Delta and Beta-induced mortality suggesting that Fc-effector functions contribute to immunity against VOCs. Thus, Fc activity of CCPs provide a second line of defense when neutralization is compromised and can serve as an important criterion for CCP selection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ethanol is a known vertebrate teratogen that causes craniofacial defects as a component of fetal alcohol syndrome (FAS). Our results show that sea urchin embryos treated with ethanol similarly show broad skeletal patterning defects, potentially analogous to the defects associated with FAS. The sea urchin larval skeleton is a simple patterning system that involves only two cell types: the primary mesenchymal cells (PMCs) that secrete the calcium carbonate skeleton and the ectodermal cells that provide migratory, positional, and differentiation cues for the PMCs. Perturbations in RA biosynthesis and Hh signaling pathways are thought to be causal for the FAS phenotype in vertebrates. Surprisingly, our results indicate that these pathways are not functionally relevant for the teratogenic effects of ethanol in developing sea urchins. We found that developmental morphology as well as the expression of some ectodermal and PMC genes was delayed by ethanol exposure. Temporal transcriptome analysis revealed significant impacts of ethanol on signaling and metabolic gene expression, and a disruption in the timing of GRN gene expression that includes both delayed and precocious gene expression throughout the specification network. We conclude that the skeletal patterning perturbations in ethanol-treated embryos likely arise from a loss of temporal synchrony within and between the instructive and responsive tissues., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Emerging data highlight the interactions of preadmission glycemia, reflected by admission HbA1c levels, glycemic control during critical illness, and mortality. The association of preadmission insulin treatment with outcomes is unknown., Methods: This observational cohort study includes 5245 patients admitted to the medical-surgical intensive care unit of a university-affiliated teaching hospital. Three groups were analyzed: patients with diabetes with prior insulin treatment (DM-INS, n  = 538); patients with diabetes with no prior insulin treatment (DM-No-INS, n  = 986); no history of diabetes (NO-DM, n  = 3721). Groups were stratified by HbA1c level: <6.5%; 6.5%-7.9% and >8.0%., Results: Among the three strata of HbA1c, mean blood glucose (BG), coefficient of variation (CV), and hypoglycemia increased with increasing HbA1c, and were higher for DM-INS than for DM-No-INS. Among patients with HbA1c < 6.5%, mean BG ≥ 180 mg/dL and CV > 30% were associated with lower severity-adjusted mortality in DM-INS compared to patients with mean BG 80-140 mg/dL and CV < 15%, ( P  = .0058 and < .0001, respectively), but higher severity-adjusted mortality among DM-No-INS ( P  = .0001 and < .0001, respectively) and NON-DM ( P  < .0001 and < .0001, respectively). Among patients with HbA1c ≥ 8.0%, mean BG ≥ 180 mg/dL was associated with lower severity-adjusted mortality for both DM-INS and DM-No-INS than was mean BG 80-140 mg/dL ( p  < 0.0001 for both comparisons)., Conclusions: Significant differences in mortality were found among patients with diabetes based on insulin treatment and HbA1c at home and post-admission glycemic control. Prospective studies need to confirm an individualized approach to glycemic control in the critically ill.

Lorlatinib is currently the most advanced, potent and selective anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor for the treatment of ALK-positive non-small cell lung cancer in the clinic; however, diverse compound ALK mutations driving therapy resistance emerge. Here, we determine the spectrum of lorlatinib-resistant compound ALK mutations in patients, following treatment with lorlatinib, the majority of which involve ALK G1202R or I1171N/S/T. We further identify structurally diverse lorlatinib analogs that harbor differential selective profiles against G1202R versus I1171N/S/T compound ALK mutations. Structural analysis revealed increased potency against compound mutations through improved inhibition of either G1202R or I1171N/S/T mutant kinases. Overall, we propose a classification of heterogenous ALK compound mutations enabling the development of distinct therapeutic strategies for precision targeting following sequential tyrosine kinase inhibitors., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

We assessed antimicrobial resistance (AMR) in Neisseria gonorrhoeae in Nunavut, Canada, using remnant gonorrhea nucleic acid amplification test-positive urine specimens. This study confirms the feasibility of conducting N. gonorrhoeae AMR surveillance and highlights the diversity of gonococcal sequence types and geographic variation of AMR patterns in the territory.

In B lymphocytes, the uracil N-glycosylase (UNG) excises genomic uracils made by activation-induced deaminase (AID), thus underpinning antibody gene diversification and oncogenic chromosomal translocations, but also initiating faithful DNA repair. Ung -/- mice develop B-cell lymphoma (BCL). However, since UNG has anti- and pro-oncogenic activities, its tumor suppressor relevance is unclear. Moreover, how the constant DNA damage and repair caused by the AID and UNG interplay affects B-cell fitness and thereby the dynamics of cell populations in vivo is unknown. Here, we show that UNG specifically protects the fitness of germinal center B cells, which express AID, and not of any other B-cell subset, coincident with AID-induced telomere damage activating p53-dependent checkpoints. Consistent with AID expression being detrimental in UNG-deficient B cells, Ung -/- mice develop BCL originating from activated B cells but lose AID expression in the established tumor. Accordingly, we find that UNG is rarely lost in human BCL. The fitness preservation activity of UNG contingent to AID expression was confirmed in a B-cell leukemia model. Hence, UNG, typically considered a tumor suppressor, acquires tumor-enabling activity in cancer cell populations that express AID by protecting cell fitness., (© The Author(s) 2020. Published by Oxford University Press on behalf of NAR Cancer.)

Competing Interests: Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

We examined categorical agreement between automated mycobacterial susceptibility testing methods (Mycobacterial Growth Indicator Tube [MGIT] 960 System and the VersaTREK Mycobacteria Detection and Susceptibility System) which are based on single critical concentration (CC) "breakpoints" and a commercial microbroth dilution method (Sensititre Mycobacterium tuberculosis MIC Plate [MYCOTB]) which provides an MIC value. Mycobacterium tuberculosis isolates (n=355) were tested against three first-line antimycobacterial agents (ethambutol [EMB], isoniazid [INH], rifampin [RIF]) using the MYCOTB plate and either the MGIT 960 (site 1, n=142) or VersaTREK (site 2, n=213) systems. Overall categorical agreement was 96.8%. When stratified by drug and CC-defined susceptible and resistant isolates, concordance ranged from 75% to 100%. Interpretation of MIC-based results versus established CC-based results was challenging for drugs whose CC was not represented by an exactly equivalent concentration in the manufacturer-defined dilutions on the MYCOTB plate (EMB, INH). We propose interpretations of MYCOTB plate MICs using the currently available plate configuration., (Copyright © 2018. Published by Elsevier Inc.)

Background: Orally administered sucrose is effective and safe in reducing pain intensity during single, tissue-damaging procedures in neonates, and is commonly recommended in neonatal pain guidelines. However, there is wide variability in sucrose doses examined in research, and more than a 20-fold variation across neonatal care settings. The aim of this study was to determine the minimally effective dose of 24% sucrose for reducing pain in hospitalized neonates undergoing a single skin-breaking heel lance procedure., Methods: A total of 245 neonates from 4 Canadian tertiary neonatal intensive care units (NICUs), born between 24 and 42 weeks gestational age (GA), were prospectively randomized to receive one of three doses of 24% sucrose, plus non-nutritive sucking/pacifier, 2 min before a routine heel lance: 0.1 ml (Group 1; n = 81), 0.5 ml (Group 2; n = 81), or 1.0 ml (Group 3; n = 83). The primary outcome was pain intensity measured at 30 and 60 s following the heel lance, using the Premature Infant Pain Profile-Revised (PIPP-R). The secondary outcome was the incidence of adverse events. Analysis of covariance models, adjusting for GA and study site examined between group differences in pain intensity across intervention groups., Results: There was no difference in mean pain intensity PIPP-R scores between treatment groups at 30 s (P = .97) and 60 s (P = .93); however, pain was not fully eliminated during the heel lance procedure. There were 5 reported adverse events among 5/245 (2.0%) neonates, with no significant differences in the proportion of events by sucrose dose (P = .62). All events resolved spontaneously without medical intervention., Conclusions: The minimally effective dose of 24% sucrose required to treat pain associated with a single heel lance in neonates was 0.1 ml. Further evaluation regarding the sustained effectiveness of this dose in reducing pain intensity in neonates for repeated painful procedures is warranted., Trial Registration: ClinicalTrials.gov : NCT02134873. Date: May 5, 2014 (retrospectively registered).

Collaborative leadership and management structures are critical to transforming care delivery. Both nurse and physician managers are uniquely positioned to co-lead. However, little is known on how to prepare and support individuals for these co-leader arrangements. The re-design of healthcare professional education focuses on interprofessional collaboration, mutual learning, and a competency-based approach. While competencies for interprofessional collaboration have been delineated, competencies for collaborative management practice have yet to be addressed. An integrative review of empirical studies on existing educational interventions was conducted to critically appraise and synthesise the results regarding collaborative competence among nurse and physician leaders. We reviewed how these interventions have been designed, implemented, and evaluated within workplace settings in order to inform our understanding of what components are effective or ineffective for the future development of an educational programme. This review reports on key characteristics of nine empirical studies and emphasises that: a uniprofessional approach to leadership development is predominant within educational programmes and that the assessment of shared learning experiences are not addressed; there are inconsistency in terms used to describe competencies by individual researchers and limitations within the competency frameworks used in the studies reviewed; and there is a lack of suitable instruments available to assess whether competencies have been achieved through the educational programmes. None of the studies discussed the process of how individuals learned specific competencies or whether learning outcome were achieved. Educational programmes were developed based on a perceived lack of leadership preparation and orientation programmes for leaders in formal management positions and used multiple interventions. Only two of the programmes involved organisational or systems level competencies. Interprofessional co-leading requires enhanced capabilities and capacity for managers. There is a need for developing an in-action education intervention that addresses the unique learning needs of co-leader arrangements particularly among nurses and physicians who are new to their role.

In response to the Ebola outbreak in 2014, many hospitals designated specific areas to care for patients with Ebola and other highly infectious diseases. The safe handling of category A infectious substances is a unique challenge in this environment. One solution is on-site waste treatment with a steam sterilizer or autoclave. The Johns Hopkins Hospital (JHH) installed two pass-through autoclaves in its biocontainment unit (BCU). The JHH BCU and The Johns Hopkins biosafety level 3 (BSL-3) clinical microbiology laboratory designed and validated waste-handling protocols with simulated patient trash to ensure adequate sterilization. The results of the validation process revealed that autoclave factory default settings are potentially ineffective for certain types of medical waste and highlighted the critical role of waste packaging in successful sterilization. The lessons learned from the JHH validation process can inform the design of waste management protocols to ensure effective treatment of highly infectious medical waste., (Copyright © 2017 American Society for Microbiology.)

Objective/background: Mycobacterium cosmeticum, first described in 2004, was recovered from a patient undergoing a cosmetic procedure. Subsequently, this species was associated with an outbreak in a nail salon. In all cases, the isolates were susceptible to all antibiotics tested. Recently, however, we recovered a strain of M. cosmeticum from the Chesapeake Bay, resistant to 11 of 14 antimicrobials. The objective of this work was to present our findings on the resistance and susceptibility of this isolate to various antibiotics., Materials and Methods: Surface water samples were collected from 10 sites in the Chesapeake Bay and upper tributaries to assess microbial diversity and antibiotic resistance. Site selection was based on proximity to agricultural runoff, industrial contaminants, and sewage effluents. Samples were processed and recovered organisms were identified and subjected to antimicrobial-susceptibility testing., Results: One nontuberculous species, identified as M. cosmeticum, was recovered from Sandy Point State Park. Resistance was detected to several antibiotics: doxycycline (16 μg/mL), tigecycline (≥4 μg/mL), clarithromycin (8 μg/mL), trimethoprim/sulfamethoxazole (≥8/152 μg/mL), imipenem (32 μg/mL), cefoxitin (32 μg/mL), ethionamide (≥20 μg/mL), and streptomycin (16 μg/mL). Of the 14 antibiotics tested, only the fluoroquinolones, linezolid, and amikacin demonstrated potent activity with susceptible minimum inhibitory concentrations., Conclusion: The antimicrobial resistance identified in this M. cosmeticum isolates from the Chesapeake Bay raises some important concerns: (a) why is the susceptibility pattern in this isolate so different from the previously published reports, (b) how did resistance emerge in this isolate,, ((c) is there a source of environmental exposure to antibiotics, (d) is it a human isolate transferred to the watershed, or (e) is it the result of lateral gene transfer with other resistant organisms in the Bay?)

Mycobacterial identification using MALDI-ToF MS (MALDI) has been hindered by inadequate extraction methods. Adaptive Focused Acoustics™ uses concentrated ultrasonic energy to achieve cellular disruption. Using this technology, we developed a rapid mycobacterial inactivation/protein extraction method for MALDI-based identification. Agreement for identification to the species level versus conventional identification was stratified by log confidence cut-offs of ≥2.0, ≥ 1.8, or ≥1.7. A total of 182 mycobacterial isolates were tested. Complete inactivation of all species/strains was achieved after 2min. Using a log confidence cut-off of ≥2.0, overall agreement for the commercial method (CM) was 41.7% versus 66.7% for the novel method (NM). For the CM, agreement increased to 66.7% and 83.3% using log confidence cut-offs of ≥1.8 and ≥1.7, respectively; for the NM, agreement was 100% for both cut-offs with all isolates. With no alteration to the existing database, overall agreement for the NM was 83.4%, largely due to low scores for clinical isolates of M. chelonae and M. mucogenicum. Addition of spectra from a single clinical strain of each species to the existing database increased overall agreement to 93.1%., (Copyright © 2016. Published by Elsevier Inc.)

In response to the 2014-2015 Ebola virus disease outbreak in West Africa, Johns Hopkins Medicine created a biocontainment unit to care for patients infected with Ebola virus and other high-consequence pathogens. The unit team examined published literature and guidelines, visited two existing U.S. biocontainment units, and contacted national and international experts to inform the design of the physical structure and patient care activities of the unit. The resulting four-bed unit allows for unidirectional flow of providers and materials and has ample space for donning and doffing personal protective equipment. The air-handling system allows treatment of diseases spread by contact, droplet, or airborne routes of transmission. An onsite laboratory and an autoclave waste management system minimize the transport of infectious materials out of the unit. The unit is staffed by self-selected nurses, providers, and support staff with pediatric and adult capabilities. A telecommunications system allows other providers and family members to interact with patients and staff remotely. A full-time nurse educator is responsible for staff training, including quarterly exercises and competency assessment in the donning and doffing of personal protective equipment. The creation of the Johns Hopkins Biocontainment Unit required the highest level of multidisciplinary collaboration. When not used for clinical care and training, the unit will be a site for research and innovation in highly infectious diseases. The lessons learned from the design process can inform a new research agenda focused on the care of patients in a biocontainment environment.

Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain (Fc domain) regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.  .

Purpose: The purpose of this study is to explore the role of the adaptive immune system in patients with aqueous-deficient dry eye (ADDE) and lipid-deficient dry eye (LDDE)., Methods: Patients (n = 29) with moderate to severe dry eye (dry eye workshop [DEWS] severity grading scheme) were enrolled in a cross-sectional study and classified as ADDE (Schirmer < 10), LDDE (abnormal meibum), combined (meeting both criteria), or generic (meeting neither criterion). Tears were collected by Schirmer strips, and samples for both eyes were pooled for each subject. Thirty micrograms of total protein was used in a normalized volume for microarray analysis (Quantibody Human Inflammation Array 3; RayBiotech). Six markers of TH1 cells (interferon [IFN]γ, interleukin [IL]-2), TH2 cells (IL-4, IL-5, IL-13), and TH17 cells (IL-17) were assessed., Results: ADDE demonstrated the highest total cytokine concentration, followed by the LDDE, combined, and generic groups. IFNγ and IL-2 were detectable in all subgroups. IL-4, -5, and -13 were detectable in ADDE and LDDE, but only IL-13 was detected in both the combined and generic groups. IL-17 was present in the ADDE, LDDE, and combined groups., Conclusions: TH1 cells seem to be involved in all forms of dry eye. ADDE and LDDE seem to be mediated by TH1, TH2, and TH17 cells. The combined-mechanism group is mediated by TH1 and TH17 cells, and generic dry eye seems to be mediated by TH1 cells only. ADDE has the greatest overall T-cell-mediated pathophysiology compared with the other subgroups, which is consistent with previous reports of improved efficacy with antiinflammatory therapy in these patients.

Purpose: Inflammatory mediators have been shown to modulate dry eye (DE) disease and may correlate with disease severity, yet the methods used and the associated findings vary significantly in the literature. The goal of this research was to compare two methods, the quantitative microarray and the magnetic bead assay, for detecting cytokine levels in extracted tear samples across three subject groups., Methods: Tears were collected from Schirmer strips of the right and left eyes of 20 soft contact lens wearers (CL), 20 normal non-contact lens wearers (NOR), and 20 DE subjects and stored at -80 °C. Tear proteins were eluted and precipitated using ammonium bicarbonate and acetone. The right and left eye samples were combined for each subject. Following the Bradford protein quantitation method, 10 µg of total protein was used for each of the two analyses, Quantibody® Human Inflammation Array 3 (RayBiotech) and High Sensitivity Human Cytokine Magnetic Bead Kit (Millipore). The assays were run using the GenePix® 4000B Scanner (Molecular Devices) or the Luminex MagPix® plate reader (Luminex), respectively. The data were then compared between the two instruments and the three subject groups., Results: Of the 40 proteins on the Quantibody® microarray, seven had average expression levels above the lower limit of detection: ICAM-1, MCP-1, MIG, MCSF, TIMP-1, TIMP-2, and TNF-RI. Significant differences in expression levels (p<0.05) were detected between the CL and DE groups for MCSF, TIMP-1, and TNF R1, between the NOR and DE groups for ICAM-1, and between the CL and NOR groups for ICAM-1, MCP-1, MCSF, TIMP-1, TIMP-2, and TNF-R1 when using the Student t test. Of the 13 proteins tested with Luminex, IL-1β, IL-4, IL-6, IL-7, and IL-8 had expression levels above the minimum detectable level, and these were most often detected using the Luminex assay compared to the Quantibody® microarray. Contrarily, IL-2, IL-12, IL-13, INF-g, and GM-CSF were detected more frequently using the Quantibody® microarray than the Luminex assay. Significant differences in expression levels (p<0.05) were only detected between the CL and DE groups for IL-7 and IL-8 and between the CL and NOR subjects for IL-8., Conclusions: In addition to detecting more significant differences between the subject groups, the Quantibody® microarray detected more inflammatory cytokines in total within the range of detection than the Luminex assay. Differences were also noted in the types of cytokines each assay could detect from the limited protein samples. Both methods offer advantages and disadvantages; therefore, these factors should be considered when determining the appropriate assay for analyzing tear protein samples.

Commercial extraction methods for identification of mycobacteria using MALDI-ToF MS are laborious and time consuming. We have developed a novel extraction method which utilizes a bead beater and zirconia/silica beads to significantly shorten the existing protocol. This novel method provides a more rapid extraction of mycobacteria versus the commercial standard., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: To determine the resistance of Mycobacterium tuberculosis to first- and second-line agents in adult pulmonary tuberculosis (TB) patients in Cameroon using a novel phenotypic assay., Setting: Samples were collected from TB patients at Bamenda Hospital in Bamenda, Cameroon., Design: Samples were collected consecutively from adult pulmonary TB patients over a 2-month period. TREK Sensititre(TM) MYCOTB panels were used to perform phenotypic drug susceptibility testing (DST). Susceptibility/resistance was determined by comparing minimum inhibitory concentrations to standard critical concentrations established for first- and second-line anti-tuberculosis drugs., Results: Of 103 sputum samples processed, growth on Löwenstein-Jensen media was confirmed in 78 samples, 65 of which were suitable for DST. Thirty-nine strains (60%) were susceptible to all first- and second-line drugs. Five strains (8%) were categorized as multidrug-resistant TB. Two strains (3%) were classified as pre-extensively drug-resistant TB. Of those isolates susceptible to first-line drugs, 20% were resistant to at least one second-line drug., Conclusion: Antimicrobial resistance may be higher than assumed in TB strains in Cameroon, especially with regard to second-line drugs. There remains a need for rapid, comprehensive DST.

Extremely low gestational age infants (<28 weeks at birth) experience significant pain from repeated therapeutic procedures while hospitalized in the neonatal intensive care unit. As part of a program of research examining pain in preterm infants, we conducted a qualitatively driven mixed-methods design, supplemented with a qualitative and quantitative component, to understand how health care professionals (HCPs) assess and manage procedural pain for tiny and underdeveloped preterm infants. Fifty-nine HCPs from different disciplines across four tertiary-level neonatal units in Canada participated in individual or focus group interviews and completed a brief questionnaire. Four themes from the content analysis were (a) subtlety and unpredictability of pain indicators, (b) infant and caregiver attributes and contextual factors that influence pain response and practices, (c) the complex nature of pain assessment, and (d) uncertainty in the management of pain. The information gleaned from this study can assist in identifying gaps in knowledge and informing unit-based and organizational knowledge translation strategies for this vulnerable population., (© The Author(s) 2015.)

The N-glycan of the IgG constant region (Fc) plays a central role in tuning and directing multiple antibody functions in vivo, including antibody-dependent cellular cytotoxicity, complement deposition, and the regulation of inflammation, among others. However, traditional methods of N-glycan analysis, including HPLC and mass spectrometry, are technically challenging and ill suited to handle the large numbers of low concentration samples analyzed in clinical or animal studies of the N-glycosylation of polyclonal IgG. Here we describe a capillary electrophoresis-based technique to analyze plasma-derived polyclonal IgG-glycosylation quickly and accurately in a cost-effective, sensitive manner that is well suited for high-throughput analyses. Additionally, because a significant fraction of polyclonal IgG is glycosylated on both Fc and Fab domains, we developed an approach to separate and analyze domain-specific glycosylation in polyclonal human, rhesus and mouse IgGs. Overall, this protocol allows for the rapid, accurate, and sensitive analysis of Fc-specific IgG glycosylation, which is critical for population-level studies of how antibody glycosylation may vary in response to vaccination or infection, and across disease states ranging from autoimmunity to cancer in both clinical and animal studies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The complex structure and function of the cerebral cortex critically depend on the balance of excitation and inhibition provided by the pyramidal projection neurons and GABAergic interneurons, respectively. The calretinin-expressing (CalR(+)) cell is a subtype of GABAergic cortical interneurons that is more prevalent in humans than in rodents. In rodents, CalR(+) interneurons originate in the caudal ganglionic eminence (CGE) from Gsx2(+) progenitors, but in humans it has been suggested that a subpopulation of CalR(+) cells can also be generated in the cortical ventricular/subventricular zone (VZ/SVZ). The progenitors for cortically generated CalR(+) subpopulation in primates are not yet characterized. Hence, the aim of this study was to identify patterns of expression of the transcription factors (TFs) that commit cortical stem cells to the CalR fate, with a focus on Gsx2. First, we studied the expression of Gsx2 and its downstream effectors, Ascl1 and Sp8 in the cortical regions of the fetal human forebrain at midgestation. Next, we established that a subpopulation of cells expressing these TFs are proliferating in the cortical SVZ, and can be co-labeled with CalR. The presence and proliferation of Gsx2(+) cells, not only in the ventral telencephalon (GE) as previously reported, but also in the cerebral cortex suggests cortical origin of a subpopulation of CalR(+) neurons in humans. In vitro treatment of human cortical progenitors with Sonic hedgehog (Shh), an important morphogen in the specification of interneurons, decreased levels of Ascl1 and Sp8 proteins, but did not affect Gsx2 levels. Taken together, our ex-vivo and in vitro results on human fetal brain suggest complex endogenous and exogenous regulation of TFs implied in the specification of different subtypes of CalR(+) cortical interneurons.

We developed a simplified microarray test for detecting and identifying mutations in rpoB, katG, inhA, embB, and rpsL and compared the analytical performance of the test to that of phenotypic drug susceptibility testing (DST). The analytical sensitivity was estimated to be at least 110 genome copies per amplification reaction. The microarray test correctly detected 95.2% of mutations for which there was a sequence-specific probe on the microarray and 100% of 96 wild-type sequences. In a blinded analysis of 153 clinical isolates, microarray sensitivity for first-line drugs relative to phenotypic DST (true resistance) was 100% for rifampin (RIF) (14/14), 90.0% for isoniazid (INH) (36/40), 70% for ethambutol (EMB) (7/10), and 89.1% (57/64) combined. Microarray specificity (true susceptibility) for first-line agents was 95.0% for RIF (132/139), 98.2% for INH (111/113), and 98.6% for EMB (141/143). Overall microarray specificity for RIF, INH, and EMB combined was 97.2% (384/395). The overall positive and negative predictive values for RIF, INH, and EMB combined were 84.9% and 98.3%, respectively. For the second-line drug streptomycin (STR), overall concordance between the agar proportion method and microarray analysis was 89.5% (137/153). Sensitivity was 34.8% (8/23) because of limited microarray coverage for STR-conferring mutations, and specificity was 99.2% (129/130). All false-susceptible discrepant results were a consequence of DNA mutations that are not represented by a specific microarray probe. There were zero invalid results from 220 total tests. The simplified microarray system is suitable for detecting resistance-conferring mutations in clinical M. tuberculosis isolates and can now be used for prospective trials or integrated into an all-in-one, closed-amplicon consumable., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Objectives: To examine the construct validity, inter-rater reliability, and feasibility of the Premature Infant Pain Profile-Revised in infants of varying gestational ages, diagnoses, and procedures., Methods: A prospective cross-over study with infants in three gestational age groups (26-31, 32-36, and ≥37 weeks) at three university-affiliated Neonatal Intensive Care Units in Canada. One hundred and ninety five bedside nurses and expert raters rated 202 hospitalized infants' pain during scheduled procedures using the measure. An expert rater and a nurse independently assessed infants' pain scores, using the Premature Infant Pain Profile-Revised, during 246 scheduled pairs of painful and non-painful procedures in the 202 infants. Nurses also completed a feasibility survey on using the measure in a clinical setting. To establish construct validity, pain scores were computed during painful and non-painful procedures. Inter-rater reliability between pain experts and nurses was calculated. A 5-point Likert scale was used to measure feasibility in terms of clarity, ease of use, and time to complete., Results: Irrespective of gestational age, Premature Infant Pain Profile-Revised scores were significantly higher during painful procedures (mean 6.7 [SD 3.0]) compared to non-painful procedures (mean 4.8 [SD 2.9]). There was a high degree of correlation between nurses' and experts' ratings for painful (all R(2)=0.92, p<0.001) and non-painful (all R(2)=0.87, p<0.001) procedures. Mean scores on all feasibility indicators were equal to or higher than 3.8., Discussion: The Premature Infant Pain Profile Revised has beginning construct validation, inter-rater reliability, and is considered feasible by clinicians. Concurrent validation studies should be considered., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objectives: To describe revisions to the Premature Infant Pain Profile (PIPP) and initial construct validation and feasibility of the Premature Infant Pain Profile-Revised (PIPP-R)., Methods: The PIPP was revised to enhance validity and feasibility. To validate the PIPP-R, data from 2 randomized cross-over studies were utilized to: (1) calculate and compare PIPP and PIPP-R scores in extremely low gestational age infants undergoing a painful and nonpainful event (N=52; dataset #1) and (2) calculate and compare PIPP and PIPP-R scores in assessing the effectiveness of (a) sucrose, (b) non-nutritive sucking (NNS)+sucrose, and (c) facilitated tucking+NNS+sucrose during heel lance (N = 85; dataset #2). Pearson correlations between PIPP and PIPP-R scores were calculated, and Student t tests and 1-way analysis of variance were used to determine construct validity during painful and nonpainful events. To establish feasibility, a survey of 31 Neonatal Intensive Care Unit nurses was conducted., Results: PIPP-R scores were significantly lower during nonpainful (mean, 8.3; SD = 2.9) compared with painful (mean, 9.9; SD=3.1; t95 = 4.51, P = 0.036) events in extremely low gestational age infants in dataset #1. In dataset #2, PIPP-R scores were significantly lower in infants 25 to 41 weeks gestation in the group receiving NNS+sucrose compared with the other 2 groups (F2,79 = 2.9, P<0.05). Overall, nurses rated the PIPP-R as feasible., Discussion: Initial construct validation and feasibility of the PIPP-R was demonstrated. Further testing with infants of varying gestational ages, diagnoses, and pain conditions is required; as is exploration of PIPP-R in relation to other types of physiological and cognitive responses.

A live, attenuated form of Mycobacterium bovis, bacillus Calmette-Guérin (BCG), is commonly used as intravesical immunotherapy for non-invasive urothelial bladder carcinoma. While complications are rare, dissemination can occur. A case of mycotic aortic aneurysm following BCG administration with recovery of Mycobacterium bovis in culture is reported. A review of the published experience with this problem is also presented., (Copyright © 2013 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.)