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We present the investigation of a Burkholderia pseudomallei strain isolated from the urine of a 56-year-old male returning from a recreational trip to Thailand. The patient initially presented with fever and chills, and lobar as well as bladder and prostate involvement was demonstrated on imaging. Treatment first involved trimethoprim-sulfamethoxazole, the backbone of melioidosis therapy, but was discontinued upon the discovery of resistance to the drug through antimicrobial sensitivity testing via broth microdilution. Further analysis included genomic sequencing, immunotyping, and phenotypic assessment, including virulence testing in an animal model. Results indicated that this strain is a distinct, novel variant with antigenic similarity to other Thai strains displaying high virulence, with a significantly low murine intranasal lethal dose 50% inoculum. The emergence of such a strain, particularly if prevalent in tourist destinations in Thailand, could pose a substantial public health risk, highlighting the need for ongoing vigilance among infectious disease specialists and clinical microbiologists.

Organisms evolve mechanisms that regulate the properties of biogenic crystals to support a wide range of functions, from vision and camouflage to communication and thermal regulation. Yet, the mechanism underlying the formation of diverse intracellular crystals remains enigmatic. Here we unravel the biochemical control over crystal morphogenesis in zebrafish iridophores. We show that the chemical composition of the crystals determines their shape, particularly through the ratio between the nucleobases guanine and hypoxanthine. We reveal that these variations in composition are genetically controlled through tissue-specific expression of specialized paralogs, which exhibit remarkable substrate selectivity. This orchestrated combination grants the organism with the capacity to generate a broad spectrum of crystal morphologies. Overall, our findings suggest a mechanism for the morphological and functional diversity of biogenic crystals and may, thus, inspire the development of genetically designed biomaterials and medical therapeutics., (© 2024. The Author(s).)

Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change., Competing Interests: Competing interests statement:The authors declare no competing interest.

Objective: Burkholderia pseudomallei, the etiological cause of melioidosis, is a soil saprophyte endemic in South-East Asia, where it constitutes a public health concern of high-priority. Melioidosis cases are sporadically identified in nonendemic areas, usually associated with travelers or import of goods from endemic regions. Due to extensive intercontinental traveling and the anticipated climate change-associated alterations of the soil bacterial flora, there is an increasing concern for inadvertent establishment of novel endemic areas, which may expand the global burden of melioidosis. Rapid diagnosis, isolation and characterization of B. pseudomallei isolates is therefore of utmost importance particularly in non-endemic locations., Data Description: We report the genome sequences of two novel clinical isolates (MWH2021 and MST2022) of B. pseudomallei identified in distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The data includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of 131 B. pseudomallei strains documented in the NCBI database. Inspection of the genomic data revealed the presence or absence of loci encoding for several documented virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or chronic melioidosis established that both strains belong to the highly virulent class of B. pseudomalleii., (© 2024. The Author(s).)

Introduction: Brucellosis is often confused with other diseases or accompanies the conditions it imitates. It causes treatment delays, failure, relapse, and complications. This study aimed to investigate bacteremia and complication predictors in Brucellosis patients. Early detection may help reduce relapse rates, length of hospital stay, and surgical intervention rates by providing appropriate treatment., Methodology: We examined 220 adult patients diagnosed with Brucellosis in our tertiary care hospital in the Black Sea Region between January 01, 2010, and January 01, 2022. Patients with and without bacteremia and complications were compared regarding demographic characteristics, clinical features, and laboratory parameters., Results: The mean age was 46.4 ± 15.8 years (18-96 years), and 61% were male. Low back pain and absence of muscle pain were independent risk factors for predicting bacteremia (p = 0.049, p = 0.043, respectively). Weakness /fatigue, weight loss, and 1/320 Standard Tube Agglutination Test (STAT) or Brucella Coombs Gel Test (BCGT) titers were independent risk factors that reduced the risk of complications; in contrast, low back pain and splenomegaly were independent risk factors for development of complications. (p = 0.025, p = 0.007, p = 0.008, p = 0.003, p = 0.021 respectively). Thrombocytopenia was related to complications. When the platelet cut-off value was taken as 160,000/µL in predicting complications, the sensitivity was 31.30%, and the specificity was 97.73% (p = 0.011)., Conclusions: The risk of clinical progression and complications could be predicted with symptoms and signs such as myalgia, low back pain, weakness/fatigue, weight loss, splenomegaly, and easily accessible laboratory parameters such as serum STAT/BCGT titer and platelet level., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2023 Tuba Kuruoglu, Levent Sensoy, Aynur Atilla, Fatih Temocin, Demet Gur, Esra Tanyel.)

In a recent study, we demonstrated that vaccination with the polymeric F1 capsule antigen of the plague pathogen Yersinia pestis led to the rapid induction of a protective humoral immune response via the pivotal activation of innate-like B1b cells. Conversely, the monomeric version of F1 failed to promptly protect vaccinated animals in this model of the bubonic plague. In this study, we examined the ability of F1 to confer the rapid onset of protective immunity in the more challenging mouse model of the pneumonic plague. Vaccination with one dose of F1 adsorbed on aluminum hydroxide elicited effective protection against subsequent lethal intranasal exposure to a fully virulent Y. pestis strain within a week. Interestingly, the addition of the LcrV antigen shortened the time required for achieving such rapid protective immunity to 4-5 days after vaccination. As found previously, the polymeric structure of F1 was essential in affording the accelerated protective response observed by covaccination with LcrV. Finally, in a longevity study, a single vaccination with polymeric F1 induced a higher and more uniform humoral response than a similar vaccination with monomeric F1. However, in this setting, the dominant contribution of LcrV to long-lasting immunity against a lethal pulmonary challenge was reiterated.

Controlling the morphology of crystalline materials is challenging, as crystals have a strong tendency toward thermodynamically stable structures. Yet, organisms form crystals with distinct morphologies, such as the plate-like guanine crystals produced by many terrestrial and aquatic species for light manipulation. Regulation of crystal morphogenesis was hypothesized to entail physical growth restriction by the surrounding membrane, combined with fine-tuned interactions between organic molecules and the growing crystal. Using cryo-electron tomography of developing zebrafish larvae, we found that guanine crystals form via templated nucleation of thin leaflets on preassembled scaffolds made of 20-nm-thick amyloid fibers. These leaflets then merge and coalesce into a single plate-like crystal. Our findings shed light on the biological regulation of crystal morphogenesis, which determines their optical properties.

SARS-CoV-2 is evolving with increased transmission, host range, pathogenicity, and virulence. The original and mutant viruses escape host innate (Interferon) immunity and adaptive (Antibody) immunity, emphasizing unmet needs for high-yield, commercial-scale manufacturing to produce inexpensive vaccines/boosters for global/equitable distribution. We developed DYAI-100A85, a SARS-CoV-2 spike receptor binding domain (RBD) subunit antigen vaccine expressed in genetically modified thermophilic filamentous fungus, Thermothelomyces heterothallica C1, and secreted at high levels into fermentation medium. The RBD-C-tag antigen strongly binds ACE2 receptors in vitro. Alhydrogel ® '85'-adjuvanted RDB-C-tag-based vaccine candidate (DYAI-100A85) demonstrates strong immunogenicity, and antiviral efficacy, including in vivo protection against lethal intranasal SARS-CoV-2 (D614G) challenge in human ACE2-transgenic mice. No loss of body weight or adverse events occurred. DYAI-100A85 also demonstrates excellent safety profile in repeat-dose GLP toxicity study. In summary, subcutaneous prime/boost DYAI-100A85 inoculation induces high titers of RBD-specific neutralizing antibodies and protection of hACE2-transgenic mice against lethal challenge with SARS-CoV-2. Given its demonstrated safety, efficacy, and low production cost, vaccine candidate DYAI-100 received regulatory approval to initiate a Phase 1 clinical trial to demonstrate its safety and efficacy in humans.

The life-threatening disease tularemia is caused by Francisella tularensis, an intracellular Gram-negative bacterial pathogen. Due to the high mortality rates of the disease, as well as the low respiratory infectious dose, F. tularensis is categorized as a Tier 1 bioterror agent. The identification and isolation from clinical blood cultures of F. tularensis are complicated by its slow growth. Iron was shown to be one of the limiting nutrients required for F. tularensis metabolism and growth. Bacterial growth was shown to be restricted or enhanced in the absence or addition of iron. In this study, we tested the beneficial effect of enhanced iron concentrations on expediting F. tularensis blood culture diagnostics. Accordingly, bacterial growth rates in blood cultures with or without Fe 2+ supplementation were evaluated. Growth quantification by direct CFU counts demonstrated significant improvement of growth rates of up to 6 orders of magnitude in Fe 2+ -supplemented media compared to the corresponding nonmodified cultures. Fe 2+ supplementation significantly shortened incubation periods for successful diagnosis and isolation of F. tularensis by up to 92 h. This was achieved in a variety of blood culture types in spite of a low initial bacterial inoculum representative of low levels of bacteremia. These improvements were demonstrated with culture of either Francisella tularensis subsp. tularensis or subsp. holarctica in all examined commercial blood culture types routinely used in a clinical setup. Finally, essential downstream identification assays, such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), immunofluorescence, or antibiotic susceptibility tests, were not affected in the presence of Fe 2+ . To conclude, supplementing blood cultures with Fe 2+ enables a significant shortening of incubation times for F. tularensis diagnosis, without affecting subsequent identification or isolation assays. IMPORTANCE In this study, we evaluated bacterial growth rates of Francisella tularensis strains in iron (Fe)-enriched blood cultures as a means of improving and accelerating bacterial growth. The shortening of the culturing time should facilitate rapid pathogen detection and isolation, positively impacting clinical diagnosis and enabling prompt onset of efficient therapy.

The COVID-19 pandemic caused by the SARS-CoV-2 infection induced lung inflammation characterized by cytokine storm and fulminant immune response of both resident and migrated immune cells, accelerating alveolar damage. In this work we identified members of the matrix metalloprotease (MMPs) family associated with lung extra-cellular matrix (ECM) destruction using K18-hACE2-transgenic mice (K18-hACE2) infected intranasally with SARS-CoV-2. Five days post infection, the lungs exhibited overall alveolar damage of epithelial cells and massive leukocytes infiltration. A substantial pulmonary increase in MMP8, MMP9, and MMP14 in the lungs post SARS-CoV-2 infection was associated with degradation of ECM components including collagen, laminin, and proteoglycans. The process of tissue damage and ECM degradation during SARS-CoV-2 lung infection is suggested to be associated with activity of members of the MMPs family, which in turn may be used as a therapeutic intervention.

Longevity of the immune response following viral exposure is an essential aspect of SARS-CoV-2 infection. Mild SARS-CoV-2 infection of K18-hACE2 mice was implemented for evaluating the mounting and longevity of a specific memory immune response. We show that the infection of K18-hACE2 mice induced robust humoral and cellular immunity (systemic and local), which persisted for at least six months. Virus-specific T cells and neutralizing antibody titers decreased over time, yet their levels were sufficient to provide sterile immunity against lethal rechallenge six months post-primary infection. The study substantiates the role of naturally induced immunity against SARS-CoV-2 infection for preventing recurring morbidity.

Plague pandemics and outbreaks have killed millions of people during the history of humankind. The disease, caused by the bacteria Yersinia pestis , is currently treated effectively with antibiotics. However, in the case of multidrug-resistant (MDR) bacteria, alternative treatments are required. Bacteriophage (phage) therapy has shown efficient antibacterial activity in various experimental animal models and in human patients infected with different MDR pathogens. Here, we evaluated the efficiency of фA1122 and PST phage therapy, alone or in combination with second-line antibiotics, using a well-established mouse model of pneumonic plague. Phage treatment significantly delayed mortality and limited bacterial proliferation in the lungs. However, the treatment did not prevent bacteremia, suggesting that phage efficiency may decrease in the circulation. Indeed, in vitro phage proliferation assays indicated that blood exerts inhibitory effects on lytic activity, which may be the major cause of treatment inefficiency. Combining phage therapy and second-line ceftriaxone treatment, which are individually insufficient, provided protection that led to the survival of all infected animals-a synergistic protective effect that represents a proof of concept for efficient combinatorial therapy in an emergency event of a plague outbreak involving MDR Y. pestis strains.

The past decade has witnessed tremendous endeavors to deliver novel preclinical in vitro lung models for pulmonary research endpoints, including foremost with the advent of organ- and lung-on-chips . With growing interest in aerosol transmission and infection of respiratory viruses within a host, most notably the SARS-CoV-2 virus amidst the global COVID-19 pandemic, the importance of crosstalk between the different lung regions (i.e., extra-thoracic, conductive and respiratory), with distinct cellular makeups and physiology, are acknowledged to play an important role in the progression of the disease from the initial onset of infection. In the present Methods article, we designed and fabricated to the best of our knowledge the first multi-compartment human airway-on-chip platform to serve as a preclinical in vitro benchmark underlining regional lung crosstalk for viral infection pathways. Combining microfabrication and 3D printing techniques, our platform mimics key elements of the respiratory system spanning (i) nasal passages that serve as the alleged origin of infections, (ii) the mid-bronchial airway region and (iii) the deep acinar region, distinct with alveolated airways. Crosstalk between the three components was exemplified in various assays. First, viral-load (including SARS-CoV-2) injected into the apical partition of the nasal compartment was detected in distal bronchial and acinar components upon applying physiological airflow across the connected compartment models. Secondly, nebulized viral-like dsRNA, poly I:C aerosols were administered to the nasal apical compartment, transmitted to downstream compartments via respiratory airflows and leading to an elevation in inflammatory cytokine levels secreted by distinct epithelial cells in each respective compartment. Overall, our assays establish an in vitro methodology that supports the hypothesis for viral-laden airflow mediated transmission through the respiratory system cellular landscape. With a keen eye for broader end user applications, we share detailed methodologies for fabricating, assembling, calibrating, and using our multi-compartment platform, including open-source fabrication files. Our platform serves as an early proof-of-concept that can be readily designed and adapted to specific preclinical pulmonary research endpoints., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nof, Zidan, Artzy-Schnirman, Mouhadeb, Beckerman, Bhardwaj, Elias-Kirma, Gur, Beth-Din, Levenberg, Korin, Ordentlich and Sznitman.)

Plague, caused by the human pathogen Yersinia pestis , is a severe and rapidly progressing lethal disease that has caused millions of deaths globally throughout human history and still presents a significant public health concern, mainly in developing countries. Owing to the possibility of its malicious use as a bio-threat agent, Y. pestis is classified as a tier-1 select agent. The prompt administration of an effective antimicrobial therapy, essential for a favorable patient prognosis, requires early pathogen detection, identification and isolation. Although the disease rapidly progresses and the pathogen replicates at high rates within the host, Y. pestis exhibits a slow growth in vitro under routinely employed clinical culturing conditions, complicating the diagnosis and isolation. In the current study, the in vitro bacterial growth in blood cultures was accelerated by the addition of nutritional supplements. We report the ability of calcium (Ca +2 )- and iron (Fe +2 )-enriched aerobic blood culture media to expedite the growth of various virulent Y. pestis strains. Using a supplemented blood culture, a shortening of the doubling time from ~110 min to ~45 min could be achieved, resulting in increase of 5 order of magnitude in the bacterial loads within 24 h of incubation, consequently allowing the rapid detection and isolation of the slow growing Y. pestis bacteria. In addition, the aerobic and anaerobic blood culture bottles used in clinical set-up were compared for a Y. pestis culture in the presence of Ca +2 and Fe +2 . The comparison established the superiority of the supplemented aerobic cultures for an early detection and achieved a significant increase in the yields of the pathogen. In line with the accelerated bacterial growth rates, the specific diagnostic markers F1 and LcrV (V) antigens could be directly detected significantly earlier. Downstream identification employing MALDI-TOF and immunofluorescence assays were performed directly from the inoculated supplemented blood culture, resulting in an increased sensitivity and without any detectable compromise of the accuracy of the antibiotic susceptibility testing (E-test), critical for subsequent successful therapeutic interventions.

Objective: Irisin is a myokine thought to be involved in the pathophysiological process of atherosclerosis with its' cardiovascular protective effects. Patients with diabetes mellitus (DM) have lower levels of irisin. Therefore, we investigated whether there is a connection between irisin, DM, coronary collateral circulation (CCC), and SYNTAX scores representing coronary artery disease (CAD) severity., Methods: This study evaluated 86 patients who have at least one epicardial coronary artery with chronic total occlusion. We included Rentrop 0-1 into the poor CCC group (n=45) and Rentrop 2-3 into the good CCC group (n=41) and measured serum irisin levels., Results: Irisin levels did not differ (17585 [882-37741] pg/ml and (17504 [813-47683] pg/ml, p=0.772) between the two groups. Irisin levels were lower in patients with diabetes (n=41; 14485 [813-29398] pg/ml) than those without diabetes (n=45; 19724 [865-47683] pg/ml (p=0.002). Irisin was not correlated with SYNTAX scores. In multivariate analysis, DM (OR=0.463; CI: 0.184-0.783; p=0.012) was a negative predictor of good CCC development., Conclusion: Although its level is decreased in patients with diabetes, serum irisin levels have no role in the pathophysiology of collateral development and CAD severity., Competing Interests: Conflict of Interest: No conflict of interest was declared by the authors., (Copyright © by by Istanbul Provincial Directorate of Health - Available online at www.northclinist.com.)

The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in-vivo neutralization of SARS-CoV-2 is not yet clear, and it is of high importance to delineate the role this process plays in antibody-mediated protection. Toward this aim, we have chosen two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, MD65 and BLN1 that target distinct domains of the spike (RBD and NTD, respectively). The Fc of these antibodies was engineered to include the triple mutation N297G/S298G/T299A that eliminates glycosylation and the binding to FcγR and to the complement system activator C1q. As expected, the virus neutralization activity ( in-vitro ) of the engineered antibodies was retained. To study the role of Fc-mediated functions, the protective activity of these antibodies was tested against lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice, when treatment was initiated either before or two days post-exposure. Antibody treatment with both Fc-variants similarly rescued the mice from death reduced viral load and prevented signs of morbidity. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in MD65 and BLN1 antibody-mediated protection, which should aid in the future design of effective antibody-based therapies.

Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing lethal infection. The various phases of pneumonic plague are yet to be fully understood. A well-established way to address the pathology of infectious diseases in general, and pneumonic plague in particular, is to conduct concomitant transcriptomic analysis of the bacteria and the host. The analysis of dual RNA by RNA sequencing technology is challenging, due the difficulties of extracting bacterial RNA, which is overwhelmingly outnumbered by the host RNA, especially at the critical early time points post-infection (prior to 48 h). Here, we describe a novel technique that employed the infusion of an RNA preserving reagent (RNAlater) into the lungs of the animals, through the trachea, under deep anesthesia. This method enabled the isolation of stable dual mRNA from the lungs of mice infected with Y. pestis , as early as 24 h post-infection. The RNA was used for transcriptomic analysis, which provided a comprehensive gene expression profile of both the host and the pathogen.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The continued spread of SARS-CoV-2 increases the probability of influenza/SARS-CoV-2 coinfection, which may result in severe disease. In this study, we examine the disease outcome of influenza A virus (IAV) and SARS-CoV-2 coinfection in K18-hACE2 mice. Our data indicate enhance susceptibility of IAV-infected mice to developing severe disease upon coinfection with SARS-CoV-2 two days later. In contrast to nonfatal influenza and lower mortality rates due to SARS-CoV-2 alone, this coinfection results in severe morbidity and nearly complete mortality. Coinfection is associated with elevated influenza viral loads in respiratory organs. Remarkably, prior immunity to influenza, but not to SARS-CoV-2, prevents severe disease and mortality. This protection is antibody-dependent. These data experimentally support the necessity of seasonal influenza vaccination for reducing the risk of severe influenza/COVID-19 comorbidity during the COVID-19 pandemic., (© 2021. The Author(s).)

The COVID-19 pandemic initiated a worldwide race toward the development of treatments and vaccines. Small animal models included the Syrian golden hamster and the K18-hACE2 mice infected with SARS-CoV-2 to display a disease state with some aspects of human COVID-19. A group activity of animals in their home cage continuously monitored by the HCMS100 (Home cage Monitoring System 100) was used as a sensitive marker of disease, successfully detecting morbidity symptoms of SARS-CoV-2 infection in hamsters and in K18-hACE2 mice. COVID-19 convalescent hamsters rechallenged with SARS-CoV-2 exhibited minor reduction in group activity compared to naive hamsters. To evaluate the rVSV-ΔG-spike vaccination efficacy against SARS-CoV-2, we used the HCMS100 to monitor the group activity of hamsters in their home cage. A single-dose rVSV-ΔG-spike vaccination of the immunized group showed a faster recovery than the nonimmunized infected hamsters, substantiating the efficacy of rVSV-ΔG-spike vaccine. HCMS100 offers nonintrusive, hands-free monitoring of a number of home cages of hamsters or mice modeling COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Melamed, Politi, Grauer, Achdout, Aftalion, Gur, Tamir, Yahalom-Ronen, Maimon, Yitzhak, Weiss, Rosner, Erez, Yitzhaki, Shapira, Paran, Mamroud, Vagima and Israely.)

A wide range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing monoclonal antibodies (mAbs) have been reported, most of which target the spike glycoprotein. Therapeutic implementation of these antibodies has been challenged by emerging SARS-CoV-2 variants harboring mutated spike versions. Consequently, re-assessment of previously identified mAbs is of high priority. Four previously selected mAbs targeting non-overlapping epitopes are now evaluated for binding potency to mutated RBD versions, reported to mediate escape from antibody neutralization. In vitro neutralization potencies of these mAbs, and two NTD-specific mAbs, are evaluated against two frequent SARS-CoV-2 variants of concern, the B.1.1.7 Alpha and the B.1.351 Beta. Furthermore, we demonstrate therapeutic potential of three selected mAbs by treatment of K18-human angiotensin-converting enzyme 2 (hACE2) transgenic mice 2 days post-infection with each virus variant. Thus, despite the accumulation of spike mutations, the highly potent MD65 and BL6 mAbs retain their ability to bind the prevalent viral mutants, effectively protecting against B.1.1.7 and B.1.351 variants., Competing Interests: Declaration of interests Patent application for the described antibodies was filed by the Israel Institute for Biological Research. None of the authors declared any additional competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

HLA transgenic mice are instrumental for evaluation of human-specific immune responses to viral infection. Mice do not develop COVID-19 upon infection with SARS-CoV-2 due to the strict tropism of the virus to the human ACE2 receptor. The aim of the current study was the implementation of an adenovirus-mediated infection protocol for human ACE2 expression in HLA transgenic mice. Transient pulmonary expression of the human ACE2 receptor in these mice results in their sensitisation to SARS-CoV-2 infection, consequently providing a valuable animal model for COVID-19. Infection results in a transient loss in body weight starting 3 days post-infection, reaching 20-30% loss of weight at day 7 and full recovery at days 11-13 post-infection. The evolution of the disease revealed high reproducibility and very low variability among individual mice. The method was implemented in two different strains of HLA immunized mice. Infected animals developed strong protective humoral and cellular immune responses specific to the viral spike-protein, strictly depending on the adenovirus-mediated human ACE2 expression. Convalescent animals were protected against a subsequent re-infection with SARS-CoV-2, demonstrating that the model may be applied for assessment of efficacy of anti-viral immune responses.

According to the WHO, 75% of the world's plague cases are found in Madagascar, with an average of 200 to 700 cases suspected annually (mainly bubonic plague). In 2017, a pneumonic plague epidemic of unusual proportions occurred, which raised several challenges for laboratory confirmation of cases, pointing to the need for the development of Yersinia pestis isolation procedures, especially those that can be performed in remote areas. As the WHO gold standard for plague diagnosis is bacterial culture, we sought to develop a simple method to prepare a highly selective medium, fit for use in remote areas where plague is endemic. The performance of the new medium, named improved BIN, was examined in terms of growth support and selectivity with spiked samples as well in isolating Y. pestis from clinical specimens, and it was compared to the results obtained with commercially available selective media. The preparation of the new medium is less complex and its performance was found to be superior to that of first-generation BIN medium. The growth support of the medium is higher, there is no batch diversity, and it maintains high selectivity properties. In 55 clinical specimens obtained from patients suspected to be infected with Y. pestis, approximately 20% more Y. pestis-positive isolates were identified by the improved BIN medium than were identified by commercially available selective media. The improved BIN medium is notably advantageous for the isolation of Y. pestis from clinical specimens obtained from plague patients, thus offering better surveillance tools and proper promotion of medical treatment to more patients suspected of being infected with Y. pestis.

Rapid antibiotic susceptibility tests, performed directly on whole blood samples, will offer great clinical advantages. This issue is of considerable importance when it comes to bioterror pathogens where prompt antibiotic treatment should be offered to infected patients as well as prophylaxis to suspected exposed individuals. Herein, we describe a novel and rapid method, named MAPt, that is based on the direct application of a blood sample onto solid agar that has been embedded with different concentrations of the tested antibiotic. Following a short incubation, bacterial growth is monitored by qPCR. The method was applied on blood cultures and whole blood samples inoculated with the Tier-1 pathogens Bacillus anthracis , Yersinia pestis , and Francisella tularensis . The use of agar medium, which better supports the growth of bacteria at low concentrations, together with the use of qPCR, which provides sensitivity and specificity, allowed minimal inhibitory concentration (MIC) determination to a wide range of bacterial concentrations, ranging from ∼5 × 10 2 cfu/ml up to 10 8 cfu/ml. The omission of the enrichment procedure in blood culture and the isolation step, both required in standard antibiotic susceptibility tests (ASTs), allowed a dramatic reduction in time to answer, from a few days to a few hours. The total time required for MIC determination was ∼6 h for fast-growing bacteria, such as B. anthracis , and 12-16 h for slow-growing bacteria, represented by Y. pestis and F. tularensis . Accordingly, MAPt may offer health authorities means for public preparedness in the case of a bioterror attack as well as prompt clinical treatment options in common blood stream infections., Competing Interests: Patent application (IL270342) for the described antibiotic susceptibility test (MAPt) was filed by the Israel Institute for Biological Research. The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest., (Copyright © 2021 Rotem, Shifman, Aftalion, Gur, Aminov and Aloni-Grinstein.)

Mice are normally unaffected by SARS coronavirus 2 (SARS-CoV-2) infection since the virus does not bind effectively to the murine version of the angiotensin-converting enzyme 2 (ACE2) receptor molecule. Here, we report that induced mild pulmonary morbidities rendered SARS-CoV-2-refractive CD-1 mice susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low doses of the acute lung injury stimulants bleomycin or ricin caused severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates greater than 50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart, and serum of low-dose ricin-pretreated mice compared with non-pretreated mice. Furthermore, lung extracts prepared 2-3 days after viral infection contained subgenomic mRNA and virus particles capable of replication only when derived from the pretreated mice. The deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against the SARS-CoV-2 receptor binding domain (RBD). Thus, viral cell entry in the sensitized mice seems to depend on viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. This unique mode of viral entry, observed over a mildly injured tissue background, may contribute to the exacerbation of coronavirus disease 2019 (COVID-19) pathologies in patients with preexisting morbidities.

The COVID-19 pandemic led to development of mRNA vaccines, which became a leading anti-SARS-CoV-2 immunization platform. Preclinical studies are limited to infection-prone animals such as hamsters and monkeys in which protective efficacy of vaccines cannot be fully appreciated. We recently reported a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs). BALB/c mice demonstrated specific immunologic responses following RBD-hFc mRNA vaccination. Now, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model. Administration of an RBD-hFc mRNA vaccine to K18-hACE2 mice resulted in robust humoral responses comprising binding and neutralizing antibodies. In correlation with this response, 70% of vaccinated mice withstood a lethal SARS-CoV-2 dose, while all control animals succumbed to infection. To the best of our knowledge, this is the first nonreplicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.

Objective: For breast US interpretation, to assess impact of computer-aided diagnosis (CADx) in original mode or with improved sensitivity or specificity., Methods: In this IRB approved protocol, orthogonal-paired US images of 319 lesions identified on screening, including 88 (27.6%) cancers (median 7 mm, range 1-34 mm), were reviewed by 9 breast imaging radiologists. Each observer provided BI-RADS assessments (2, 3, 4A, 4B, 4C, 5) before and after CADx in a mode-balanced design: mode 1, original CADx (outputs benign, probably benign, suspicious, or malignant); mode 2, artificially-high-sensitivity CADx (benign or malignant); and mode 3, artificially-high-specificity CADx (benign or malignant). Area under the receiver operating characteristic curve (AUC) was estimated under each modality and for standalone CADx outputs. Multi-reader analysis accounted for inter-reader variability and correlation between same-lesion assessments., Results: AUC of standalone CADx was 0.77 (95% CI: 0.72-0.83). For mode 1, average reader AUC was 0.82 (range 0.76-0.84) without CADx and not significantly changed with CADx. In high-sensitivity mode, all observers' AUCs increased: average AUC 0.83 (range 0.78-0.86) before CADx increased to 0.88 (range 0.84-0.90), P < 0.001. In high-specificity mode, all observers' AUCs increased: average AUC 0.82 (range 0.76-0.84) before CADx increased to 0.89 (range 0.87-0.92), P < 0.0001. Radiologists responded more frequently to malignant CADx cues in high-specificity mode (42.7% vs 23.2% mode 1, and 27.0% mode 2, P = 0.008)., Conclusion: Original CADx did not substantially impact radiologists' interpretations. Radiologists showed improved performance and were more responsive when CADx produced fewer false-positive malignant cues., (© Society of Breast Imaging 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Neutralizing antibodies represent a valuable therapeutic approach to countermeasure the current COVID-19 pandemic. Emergence of SARS-CoV-2 variants emphasizes the notion that antibody treatments need to rely on highly neutralizing monoclonal antibodies (mAbs), targeting several distinct epitopes for circumventing therapy escape mutants. Previously, we reported efficient human therapeutic mAbs recognizing epitopes on the spike receptor-binding domain (RBD) of SARS-CoV-2. Here we report the isolation, characterization, and recombinant production of 12 neutralizing human mAbs, targeting three distinct epitopes on the spike N-terminal domain of the virus. Neutralization mechanism of these antibodies involves receptors other than the canonical hACE2 on target cells, relying both on amino acid and N -glycan epitope recognition, suggesting alternative viral cellular portals. Two selected mAbs demonstrated full protection of K18-hACE2 transgenic mice when administered at low doses and late post-exposure, demonstrating the high potential of the mAbs for therapy of SARS-CoV-2 infection., Competing Interests: Patent application for the described antibodies was filed by the Israel Institute for Biological Research. None of the authors declared any additional competing interests., (© 2021 The Author(s).)

Rapid determination of bacterial antibiotic susceptibility is important for proper treatment of infections. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has recently published guidelines for rapid antimicrobial susceptibility testing (RAST) performed directly from positive blood culture vials. These guidelines, however, were only published for a limited number of common pathogenic bacteria. In this study, we evaluated the applicability of these guidelines to three Tier 1 bioterror agents ( Bacillus anthracis , Yersinia pestis and Francisella tularensis ) that require prompt antibiotic treatment to mitigate morbidity and mortality. We used spiked-in human blood incubated in a BACTEC™ FX40 system to determine the proper conditions for RAST using disc-diffusion and Etest assays. We found that reliable disc-diffusion inhibition diameters and Etest MIC values could be obtained in remarkably short times. Compared to the EUCAST-recommended disc-diffusion assays that will require adjusted clinical breakpoint tables, Etest-based RAST was advantageous, as the obtained MIC values were similar to the standard MIC values, enabling the use of established category breakpoint tables. Our results demonstrate the promising applicability of the EUCAST RAST for B. anthracis -, Y. pestis - or F. tularensis -positive blood cultures, which can lead to shorter diagnostics and prompt antibiotic treatment of these dangerous pathogens.

Mouse monoclonal antibodies were raised against plague disease biomarkers: the bacterial capsular protein fraction 1 (F1) and the low-calcium response-LcrV virulence factor (Vag). A novel tandem assay, employing BioLayer Interferometry (BLI), enabled the isolation of antibodies against four different epitopes on Vag. The tandem assay was carried out with hybridoma supernatants, circumventing the need for antibody purification. The BioLayer assay was further adopted for characterization of epitope-repetitive antigens, enabling the discovery of two unique epitopes on F1. The selected antibodies were purified and applied as "oligo-clonal" reagents for the immuno-detection of both biomarkers. The developed Homogenous Time Resolved Fluorescence (HTRF) tests were short (10 min) and simple (no washing steps), allowing for detection of 10 ng/mL F1 and 2.5 ng/mL Vag. The tests were successfully applied for detection of disease biomarkers produced by various Y. pestis strains during growth in blood culture vials.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibits high levels of mortality and morbidity and has dramatic consequences on human life, sociality and global economy. Neutralizing antibodies constitute a highly promising approach for treating and preventing infection by this novel pathogen. In the present study, we characterize and further evaluate the recently identified human monoclonal MD65 antibody for its ability to provide protection against a lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice. Eighty percent of the untreated mice succumbed 6-9 days post-infection, while administration of the MD65 antibody as late as 3 days after exposure rescued all infected animals. In addition, the efficiency of the treatment is supported by prevention of morbidity and ablation of the load of infective virions in the lungs of treated animals. The data demonstrate the therapeutic value of human monoclonal antibodies as a life-saving treatment for severe COVID-19 infection.

Yersinia pestis is a Gram-negative pathogen that causes plague, a devastating disease that kills millions worldwide. Although plague is efficiently treatable by recommended antibiotics, the time of antibiotic therapy initiation is critical, as high mortality rates have been observed if treatment is delayed for longer than 24 h after symptom onset. To overcome the emergence of antibiotic resistant strains, we attempted a systematic screening of Food and Drug Administration (FDA)-approved drugs to identify alternative compounds which may possess antibacterial activity against Y. pestis . Here, we describe a drug-repurposing approach, which led to the identification of two antibiotic-like activities of the anticancer drugs bleomycin sulfate and streptozocin that have the potential for designing novel antiplague therapy approaches. The inhibitory characteristics of these two drugs were further addressed as well as their efficiency in affecting the growth of Y. pestis strains resistant to doxycycline and ciprofloxacin, antibiotics recommended for plague treatment.

Objective: The pathophysiology of non-dipper hypertension has not been clarified. The relationship between salusins with atherosclerosis and hypertension has gained attention in recent years. The aim of this paper is to investigate whether salusins are associated with circadian blood pressure, left ventricular mass index, and diastolic functions in newly diagnosed hypertensives., Methods: The study included 88 newly diagnosed hypertensive individuals. Twenty-four-hour ambulatory blood pressure monitoring and echocardiographic examinations were performed. The patients were assigned to dipper hypertension (n = 41) and non-dipper hypertension (n = 47) groups based on the ambulatory blood pressure monitoring results according to the presence of ≥ a 10% decrease in nighttime blood pressure values or not. Serum salusin α and β levels were determined by electrochemiluminescence immunological test method., Results: Compared to dipper hypertension, non-dipper hypertension group demonstrated lower salusin α levels (1818.71 ± 221.67 vs 1963 ± 200.75 pg/mL, p = .002), mitral E/A, septal E'/A' and higher salusin β levels (576.24 ± 68.15 vs 516.13 ± 90.7 pg/ml, p = .001) and left ventricular mass index. Multivariate logistic regression analysis revealed salusin-α (OR 0.474, 95% CI 0.262 to 0.986, p = .001), salusin-β (OR 2.550, 95% CI 2.123 to 2.991, p = .018), and left ventricular mass index (OR 2.620, 95% CI 2.124 to 2.860, p = .011) as independent predictors of non-dipper hypertension. As candidate markers to predict non-dipper hypertension, decreased salusin α, and increased salusin β levels may mediate crosstalk between sympathetic and parasympathetic systems and indicate poor cardiovascular prognosis in hypertension.

Calculation of effective orifice area (EOA) is crucial for the evaluation of prosthetic valve (PV) function and there is lack of data on the best method, particularly in obese patients, in whom two-dimensional (2D) transthoracic echocardiography (TTE) is cumbersome. We sought to compare two methods of calculating EOA through Continuity equation; one using standard 2D-TTE and other three-dimensional (3D) stoke volume (SV), in patients with bileaflet mechanical PV stratified by body mass index (BMI). On conventional TTE, SV mas measured using standard 2D derived data and 3D derived SV in 38 aortic and 62 mitral PV patients who were referred for further evaluation for mild/moderate symptoms of dyspnea. Patients were categorized with regard to transprosthetic flow into 'normal-flow' and 'high-flow' groups and several echocardiographic data including 2D and 3D EOA were compared. Rates of obesity (BMI ≥ 30) were similar within high and normal flow groups of mitral and aortic PV patients. Correlation and agreement of 2D and 3D EOA was sought in patients with and without obesity. After identifying patients with possible severe obstruction, ROC analysis was carried out to identify whether 2D and 3D derived EOA could discriminate those with obstruction. There was good correlation and agreement between two methods in patients without obesity in both mitral and aortic PV. In obese individuals, however, there was no correlation between 2D and 3D EOA; in whom echocardiographic criteria showing severe obstruction revealed that 3D EOA measurements were more accurate. ROC analysis supported that 3D EOA performs better to identify patients with obstructive characteristics. In patients with bileaflet PV, measurement of EAO by 3D derived SV yields more accurate results irrespective of BMI.

Skin color patterns are ubiquitous in nature, impact social behavior, predator avoidance, and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the zebrafish; its alternating blue stripes and yellow interstripes depend on light-reflecting cells called iridophores. It was suggested that the zebrafish's color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. However, here we find that iridophores do not migrate between stripes and interstripes but instead differentiate and proliferate in-place, based on their micro-environment. RNA-sequencing analysis further reveals that stripe and interstripe iridophores have different transcriptomic states, while cryogenic-scanning-electron-microscopy and micro-X-ray diffraction identify different crystal-arrays architectures, indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present an alternative model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, organelles arise in stripe and interstripe by in-situ differentiation.

Various respiratory viral infections in general and seasonal influenza in particular may increase the susceptibility to bacterial infections. Plague caused by Yersinia pestis endangers large populations during outbreaks or bioterrorism attacks. Recommended antibiotic countermeasures include well-established protocols based on animal studies and corroborated by effective treatment of human cases. Until now, prior exposure to viral respiratory infections was not taken into consideration when selecting the appropriate treatment for plague. Here, we show that as late as 25 days after exposure to influenza virus, convalescent mice still exhibited an increased susceptibility to sublethal doses of Y. pestis, presented with aberrant cytokine expression, and impaired neutrophil infiltration in the lungs. Increased levels of M2 alveolar macrophages and type II epithelial cells, as well as induction in metalloproteases expression and collagen and laminin degradation, suggested that the previous viral infection was under resolution, correlating with enhanced susceptibility to plague. Surprisingly, postexposure prophylaxis treatment with the recommended drugs revealed that ciprofloxacin was superior to doxycycline in mice recovering from influenza infection. These results suggest that after an influenza infection, the consequences, such as impaired immunity and lung tissue remodeling and damage, should be considered when treating subsequent Y. pestis exposure.

Antibiotic resistance of bio-threat agents holds major concerns especially in light of advances in methods for engineering pathogens with antibiotic resistance. Preparedness means for rapid identification and prompt proper medical treatment are of need to contain the event and prevent morbidity and spreading of the disease by properly treating exposed individuals before symptoms appearance. Herein, we describe a novel, rapid, simple, specific, and sensitive method named Micro-Agar-PCR-test (MAPt), which determines antibiotic susceptibility of bio-terror pathogens, directly from environmental samples, with no need for any prior isolation, quantification, or enrichment steps. As proof of concept, we have used this approach to obtain correct therapeutic antibiotic minimal inhibitory concentration (MIC) values for the Tier-1 select agents, Bacillus anthracis , Yersinia pestis , and Francisella tularensis , spiked in various environmental samples recapitulating potential bioterror scenarios. The method demonstrated efficiency for a broad dynamic range of bacterial concentrations, both for fast-growing as well as slow-growing bacteria and most importantly significantly shortening the time for accurate results from days to a few hours. The MAPt allows us to address bioterror agents-contaminated environmental samples, offering rational targeted prophylactic treatment, before the onset of morbidity in exposed individuals. Hence, MAPt is expected to provide data for decision-making personal for treatment regimens before the onset of symptoms in infected individuals., (Copyright © 2020 Aloni-Grinstein, Shifman, Gur, Aftalion and Rotem.)

Large numbers of rodents are often used in the study of disease progression and in the evaluation of its potential treatments. To avoid subjective observation and to minimize home cage interference, we developed a computerized home cage monitoring system (HCMS100) based on a standard cage rack adapted with a single laser beam and a detector mounted on each cage, enabling to monitor mice movements based on laser beam interruptions. This retrofit system provided continuous and uninterrupted monitoring of spontaneous movement of a group of mice in a home cage. Validity was evaluated using disease state induced by LPS modelling bacterial infection and by influenza virus. RESULTS: Spontaneous activity of different number of mice (2-8) per cage showed the expected circadian rhythm with increased activity during the night, and its extent dependent on the number of mice in the cage. Females and males show similar circadian rhythm. Intranasal LPS administration and pulmonary infection with live influenza virus resulted in major reduction of mice activity along disease progression. Increase in activity over time was a good indicator of the recovery process from both LPS exposure and the flu infection. CONCLUSIONS: HCMS100 was shown to be a reliable, inexpensive, easy to use system that requires no changes in the common housing of various experimental animals (mice, hamsters, rats etc.). With minimal intervention, HCMS100 provides a continuous record of group activity with clear pattern of circadian rhythm, allowing long term recording of home cage activity even in restricted access environments., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Provisional Patent pending US62977333 for the HCMS100 was filed by the Israel Institute for Biological Research., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Escherichia coli ST131 clone and H30-R/H30-Rx subclones are the most common multidrug-resistant high-risk clones in UTIs. Antimicrobial susceptibility of fosfomycin was compared to five other agents in consecutively collected 299 urinary isolates using the agar dilution method. Prevalence of the ST131 clone and the occurrence of blaCTX-M were also investigated. Overall resistance to fosfomycin, cefuroxime, and ceftriaxone were 2.7%, 35.4%, and 30.1% respectively. fosA, fosA3, and fosC2 genes were not detected. In isolates resistant to ciprofloxacin (34.7%), the prevalence of ST131 clone was 31.7%, of which 81.8% belonged to H30-R and 66.7% to H30-Rx subclones. None of the isolates of the ST131 clone were resistant to fosfomycin. However, bla CTX-M occurred in 57.6% of the isolates among this clone, 62.9% in H30-R and 68.2% in H30-Rx subclones. The results of this study suggest that fosfomycin resistance is not prevalent in urinary isolates, however, bla CTX-M positive ST131 clone is quite common., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rationale and Objectives: To preliminarily asses if Contrast Enhanced Digital Mammography (CEDM) can accurately reduce biopsy rates for soft tissue BI-RADS 4A or 4B lesions., Materials and Methods: Eight radiologists retrospectively and independently reviewed 60 lesions in 54 consenting patients who underwent CEDM under Health Insurance Portability and Accountability Act compliant institutional review board-approved protocols. Readers provided Breast Imaging Reporting & Data System ratings sequentially for digital mammography/digital breast tomosynthesis (DM/DBT), then with ultrasound, then with CEDM for each lesion. Area under the curve (AUC), true positive rates and false positive rates, positive predictive values and negative predictive values were calculated. Statistical analysis accounting for correlation between lesion-examinations and between-reader variability was performed using OR/DBM (for SAS v.3.0), generalized linear mixed model for binary data (proc glimmix, SAS v.9.4, SAS Institute, Cary North Carolina), and bootstrap., Results: The cohort included 49 benign, two high-risk and nine cancerous lesions in 54 women aged 34-74 (average 50) years. Reader-averaged AUC for CEDM was significantly higher than DM/DBT alone (0.85 versus 0.66, p < 0.001) or with US (0.85 versus 0.75, p = 0.001). CEDM increased true positive rates from 0.74 under DB/DBT, and 0.89 with US, to 0.90 with CEDM, (p = 0.019 DM/DBT versus CEDM, p = 0.78 DM/DBT + US versus CEDM) and decreased false positive rates from 0.47 using DM/DBT and 0.61 with US to 0.39 with CEDM (p = 0.017 DM/DBT versus CEDM, p = 0.001 DM/DBT+ US versus CEDM). For an expected cancer rate of 10%, CEDM positive predictive values was 20.5% (95% CI: 16%-27%) and negative predictive values 98.3% (95% CI: 96%-100%)., Conclusion: Addition of CEDM for evaluation of low-moderate suspicion soft tissue breast lesions can substantially reduce biopsy of benign lesions without compromising cancer detection., (Copyright © 2019 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.)

Introduction: Infections caused by Carbapenemase-producing Enterobacterales (CPE) are an important public health issue. Intravenous fosfomycin can be considered as an alternative for the treatment of serious infections caused by CPE. In this study, in vitro activity of fosfomycin was investigated among CPE isolates., Methodology: Overall, 158 clinically relevant isolates obtained from 18 hospitals of 13 cities in Turkey with predetermined carbapenemase types were evaluated in the study, including Escherichia coli (n = 19) and Klebsiella spp. (n = 139). In vitro activity of fosfomycin was determined with agar dilution method. Among Klebsiella spp., 104 harbored blaOXA-48, 15 isolates carried both blaOXA-48 and blaNDM; three had both blaOXA-48 and blaVIM and nine isolates had blaNDM alone. Four isolates carried only blaVIM and two isolates harbored blaIMP alone. One isolate co-harbored blaVIM and blaNDM. Among E. coli isolates, blaOXA-48 and blaNDM were carried by 18 and one isolates, respectively., Results: Resistance to fosfomycin was detected in 43.7% of the isolates. Among Klebsiella spp. and E. coli, these rates were 46.8% and 21.1%, respectively. In Klebsiella spp. resistance to fosfomycin was 49.5% in blaOXA-48 carriers; 26.7% in isolates co-harbouring blaOXA-48 and blaNDM and 66.7% in blaNDM carriers. In E. coli, fosfomycin resistance was detected among 16.7% of the blaOXA-48 carriers., Conclusions: High level of fosfomycin resistance in these isolates may be attributable to the fact that these isolates are multidrug resistant. The genetic background of resistance should also be investigated in order to understand the co-occurrence and transfer of resistance among the CPE., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2020 Selay Demirci-Duarte, Tugce Unalan-Altintop, Zeynep Gulay, Ayse Nur Sari Kaygisiz, Asli Cakar, Deniz Gur.)

High-throughput DNA sequencing (HTS) of pathogens in whole blood samples is hampered by the high host/pathogen nucleic acids ratio. We describe a novel and rapid bacterial enrichment procedure whose implementation is exemplified in simulated bacteremic human blood samples. The procedure involves depletion of the host DNA, rapid HTS and bioinformatic analyses. Following this procedure, Y. pestis , F. tularensis and B. anthracis spiked-in samples displayed an improved host/pathogen DNA ratio of 2.5-5.9 orders of magnitude, in samples with bacteria spiked-in at 10 3 -10 5 CFU/ml. The procedure described in this study enables rapid and detailed metagenomic profiling of pathogens within 8-9 h, circumventing the challenges imposed by the high background present in the bacteremic blood and by the unknown nature of the sample., Competing Interests: Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript., (© 2020 Ofir Israeli.)

Objective: To assess prospectively the interpretative performance of automated breast ultrasound (ABUS) as a supplemental screening after digital breast tomosynthesis (DBT) or as a standalone screening of women with dense breast tissue., Methods: Under an IRB-approved protocol (written consent required), women with dense breasts prospectively underwent concurrent baseline DBT and ABUS screening. Examinations were independently evaluated, in opposite order, by two of seven Mammography Quality Standards Act-qualified radiologists, with the primary radiologist arbitrating disagreements and making clinical management recommendations. We report results for 1111 screening examinations (598 first year and 513 second year) for which all diagnostic workups are complete. Imaging was also retrospectively reviewed for all cancers. Statistical assessments used a 0.05 significance level and accounted for correlation between participants' examinations., Results: Of 1111 women screened, primary radiologists initially "recalled" based on DBT alone (6.6%, 73/1111, CI: 5.2%-8.2%), of which 20 were biopsied, yielding 6/8 total cancers. Automated breast ultrasound increased recalls overall to 14.4% (160/1111, CI: 12.4%-16.6%), with 27 total biopsies, yielding 1 additional cancer. Double reading of DBT alone increased the recall rate to 10.7% (119/1111), with 21 biopsies, with no improvement in cancer detection. Double reading ABUS increased the recall rate to 15.2% (169/1111, CI: 13.2%-17.5%) of women, of whom 22 were biopsied, yielding the detection of 7 cancers, including one seen only on double reading ABUS. Inter-radiologist agreement was similar for recall recommendations from DBT (κ = 0.24, CI: 0.14-0.34) and ABUS (κ = 0.23, CI: 0.15-0.32). Integrated assessments from both readers resulted in a recall rate of 15.1% (168/1111, CI: 13.1%-17.4%)., Conclusion: Supplemental or standalone ABUS screening detected cancers not seen on DBT, but substantially increased noncancer recall rates., (© Society of Breast Imaging 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Most animal handling procedures are associated with injuries among veterinary staff and laboratory animal researchers. However, much of the currently available animal handling equipment is inadequate, limiting access to the treated animal or making workflow cumbersome. Moreover, restraining animals to perform procedures, such as blood collection or injection, elicits stress in both the animal and the worker. Herein, we present 4 home-built restraint and blood collection devices in extensive use in our institute., Methods: Animal laboratory workers and experienced veterinarians regularly using the devices (n = 14) were asked to complete a survey ranking the contribution of the devices to worker safety and procedural efficiency., Results: The overwhelming majority of responders (≥75%) associated all 4 devices with substantial improvements in worker safety and procedural efficiency. There were no reports of impaired workflow or safety when using the devices., Discussion: Infection and exposure control may be implemented on various levels, including use of safer procedures, such as injection and blood collection devices. The presented intuitive handling and restraint devices allow the animal worker/researcher to perform various procedures safely and efficiently while eliciting less animal and worker stress. The devices can be easily adjusted to accommodate animal size and disease status., Conclusion: The current devices will serve as prototypes for design of devices for larger laboratory animals., (© ABSA International 2019.)

To maintain body homeostasis, endocrine systems must detect and integrate blood-borne peripheral signals. This is mediated by fenestrae, specialized permeable pores in the endothelial membrane. Plasmalemma vesicle-associated protein (Plvap) is located in the fenestral diaphragm and is thought to play a role in the passage of proteins through the fenestrae. However, this suggested function has yet to be demonstrated directly. We studied the development of fenestrated capillaries in the hypophysis, a major neuroendocrine interface between the blood and brain. Using a transgenic biosensor to visualize the vascular excretion of the genetically tagged plasma protein DBP-EGFP, we show that the developmental acquisition of vascular permeability coincides with differential expression of zebrafish plvap orthologs in the hypophysis versus brain. Ultrastructural analysis revealed that plvapb mutants display deficiencies in fenestral diaphragms and increased density of hypophyseal fenestrae. Measurements of DBP-EGFP extravasation in plvapb mutants provided direct proof that Plvap limits the rate of blood-borne protein passage through fenestrated endothelia. We present the regulatory role of Plvap in the development of blood-borne protein detection machinery at a neuroendocrine interface through which hormones are released to the general circulation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background Staging newly diagnosed breast cancer by using dynamic contrast material-enhanced MRI is limited by access, high cost, and false-positive findings. The utility of contrast-enhanced mammography (CEM) and 99m Tc sestamibi-based molecular breast imaging (MBI) in this setting is largely unknown. Purpose To compare extent-of-disease assessments by using MRI, CEM, and MBI versus pathology in women with breast cancer. Materials and Methods In this HIPAA-compliant prospective study, women with biopsy-proven breast cancer underwent MRI, CEM, and MBI between October 2014 and April 2018. Eight radiologists independently interpreted each examination result prospectively and were blinded to interpretations of findings with the other modalities. Visibility of index malignancies, lesion size, and additional suspicious lesions (malignant or benign) were compared during pathology review. Accuracy of index lesion sizing and detection of additional lesions in women without neoadjuvant chemotherapy were compared. Results A total of 102 women were enrolled and 99 completed the study protocol (mean age, 51 years ± 11 [standard deviation]; range, 32-77 years). Lumpectomy or mastectomy was performed in 71 women (79 index malignancies) without neoadjuvant chemotherapy and in 28 women (31 index malignancies) with neoadjuvant chemotherapy. Of the 110 index malignancies, MRI, CEM, and MBI depicted 102 (93%; 95% confidence interval [CI]: 86%, 97%), 100 (91%; 95% CI: 84%, 96%), and 101 (92%; 95% CI: 85%, 96%) malignancies, respectively. In patients without neoadjuvant chemotherapy, pathologic size of index malignancies was overestimated with all modalities ( P = .02). MRI led to overestimation of 24% (17 of 72) of malignancies by more than 1.5 cm compared with 11% (eight of 70) with CEM and 15% (11 of 72) with MBI. MRI depicted more ( P = .007) nonindex lesions, with sensitivity similar to that of CEM or MBI, resulting in lower positive predictive value of additional biopsies (13 of 46 [28%; 95% CI: 17%, 44%] for MRI; 14 of 27 [52%; 95% CI: 32%, 71%] for CEM; and 11 of 25 [44%; 95% CI: 24%, 65%] for MBI (overall P = .01). Conclusion Contrast-enhanced mammography, molecular breast imaging, and MRI showed similar detection of all malignancies. MRI depicted more nonindex suspicious benign lesions than did contrast-enhanced mammography or molecular breast imaging, leading to lower positive predictive value of additional biopsies. All three modalities led to overestimation of index tumor size, particularly MRI. © RSNA, 2019 Online supplemental material is available for this article.

Objective: The pathophysiology of coronary slow flow (CSF) has not been clarified. Salusin-β is released predominantly from the atheroma plaques and influences the pathophysiologic processes of atherosclerosis. Therefore, this study aimed to determine serum salusin-β levels in CSF and its correlation with CSF., Methods: The study included 39 patients with CSF, and the control group (n=42) consisted of consecutive subjects with normal coronary arteriogram. We measured salusin-β and thrombolysis in myocardial infarction frame count (TFC)., Results: Age, body mass index (BMI), systolic blood pressure, diabetes, hyperlipidemia, and smoking rates were similar (p values>0.05) in both groups. High sensitive C-reactive protein (2.80±1.2 vs. 2.21±1.2 mg/dL, p=0.011), salusin-β [1205 (330-2092) vs. 162 (29-676), pg/ml, p<0.001], corrected TFC of left anterior descending coronary artery (29±9 vs. 19.7±3.7, p<0.001), circumflex artery TFC (25±10 vs. 15±3.2, p<0.001), right coronary artery TFC (28±7.1 vs. 13±3.3, p<0.001), and mean TFC (28±4.4 vs. 16±3.7, p<0.001) were significantly higher in the CSF group. In univariate and multivariate regression analysis, only BMI (unstandardized β±SE=0.178±0.08, p=0.036) and salusin-β levels (unstandardized β±SE=0.006±0.01, p<0.001) were determined as predictors of CSF. There was a good correlation between serum salusin-β and mean TFC values (r=0.564; p<0.001)., Conclusion: There is an association between serum salusin-β levels and CSF.

Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing contagious disease. In the plague mouse model, a single immunization with the EV76 live attenuated Y. pestis strain rapidly induced the expression of hemopexin and haptoglobin in the lung and serum, both of which are important in iron sequestration. Immunization against a concomitant lethal Y. pestis respiratory challenge was correlated with temporary inhibition of disease progression. Combining EV76-immunization and second-line antibiotic treatment, which are individually insufficient, led to a synergistic protective effect that represents a proof of concept for efficient combinational therapy in cases of infection with antibiotic-resistant strains., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Understanding genetic and cellular bases of adult form remains a fundamental goal at the intersection of developmental and evolutionary biology. The skin pigment cells of vertebrates, derived from embryonic neural crest, are a useful system for elucidating mechanisms of fate specification, pattern formation, and how particular phenotypes impact organismal behavior and ecology. In a survey of Danio fishes, including the zebrafish Danio rerio , we identified two populations of white pigment cells-leucophores-one of which arises by transdifferentiation of adult melanophores and another of which develops from a yellow-orange xanthophore or xanthophore-like progenitor. Single-cell transcriptomic, mutational, chemical, and ultrastructural analyses of zebrafish leucophores revealed cell-type-specific chemical compositions, organelle configurations, and genetic requirements. At the organismal level, we identified distinct physiological responses of leucophores during environmental background matching, and we showed that leucophore complement influences behavior. Together, our studies reveal independently arisen pigment cell types and mechanisms of fate acquisition in zebrafish and illustrate how concerted analyses across hierarchical levels can provide insights into phenotypes and their evolution., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)