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1.
J Spec Oper Med ; 2024 Sep 23.
Article in English | MEDLINE | ID: mdl-39288031

ABSTRACT

Training needs of Special Operations Forces (SOF) medics were surveyed and new training initiatives have been created to meet their needs. SOF medics perform an array of medical procedures in austere environments with minimal supervision. Medical skills decay over time after initial training and the perceived training needs of active SOF medics were unclear. To fill this gap, active SOF medics (n=57) completed a survey that included confidence ratings and indications of whether additional training would make them more proficient in 70 medi cal knowledge and procedural skills, assembled into categories by a panel of experts (airway, trauma, neuro, differential di agnosis, administrative, infection, critical care, environmental, other). Data were analyzed with analysis of variance (ANOVA) and nonparametric statistics at P<.05. Confidence was highest in the trauma, administrative, and airway categories, and lowest in the infection, differential diagnosis, and neuro categories (P<.05 or less). Categories indicating the greatest need for additional training were environmental and critical care, while those indicating lowest need were the airway and trauma categories (P=.05). Additional training was endorsed by >75% of participants in each category. SOF medics also wanted additional training in all areas, preferably hands-on with live patients in realistic scenarios, taught by experienced medics. Findings highlight the training needs of SOF medics and demonstrate the value of bottom-up feedback toward op timizing sustainment training. Based on present findings, two TACMED (Tactical Medicine) Divisions at the SOF Echelon III level were created to meet the sustainment training needs of SOF medics.

2.
ChemistryOpen ; : e202400038, 2024 Sep 03.
Article in English | MEDLINE | ID: mdl-39226539

ABSTRACT

The aluminum content of concentrated (27 wt%) sodium chloride solutions could be crucial for large-scale chlor-alkali-based industries applying membrane cell electrolysis. Thus, a facile method which enables a fast and reliable protocol to determine the Al content of these solutions on ppb scale in industrial environments is fundamentally important. It was demonstrated that the increased sensitivity of colorful Al-ECR (eriochrome cyanine R) complex by the use of a cationic surfactant and specific biological buffers could effectively indicate the Al content in an extended pH interval of a concentrated saline medium under industrial conditions. The dependence of the analytical protocol on pH, temperature, time, wavelength, and the salinity of the medium was investigated. It was shown that the absorbance-based measurements of the solution should be performed at least 2-4 h after its preparation. By applying the selected two Good's buffers (HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, MOPS: 3-(N-morpholino)-propanesulfonic acid) and Tris (tris(hydroxymethyl)aminomethane), 32.8-38.1 % increase in the sensitivity was achieved for saturated NaCl solutions. Moreover, the limits of detection and quantification (LOD, LOQ) were also lowered by 19.0-29.8 %, and the salinity dependence of the calibration was also reduced.

3.
J Appl Clin Med Phys ; : e14503, 2024 Sep 05.
Article in English | MEDLINE | ID: mdl-39235683

ABSTRACT

INTRODUCTION: HyperArc (HA) auto-planning offers simplicity for the end user and consistently high-quality SRS plans. The "Ask For It" (AFI) optimization strategy offers a manual planning technique that, when coupled with R50%Analytic, can be guided to deliver a plan with an intermediate dose spill "as low as reasonably achievable" and high target dose conformity. A direct comparison of SRS plan quality obtained using the manual planning AFI strategy and HA has been performed. METHODS: Using a CT data set available from the Radiosurgery Society, 54 PTVs were created and used to generate 19 individual SRS/SRT cases. Case complexity ranged from single PTV plans to multiple PTV plans with a single isocenter. PTV locations ranged from relative isolation from critical structures to lesions within 1.5 mm of the optic apparatus and abutting the brainstem. All cases were planned using both the AFI and HA optimization strategies as implemented in the Varian Medical Systems Eclipse Treatment Planning System. A range of treatment plan quality metrics were obtained including Intermediate Dose Spill (R50%), Conformity Indices CIRTOG and CIPaddick, PTV Dose Coverage (Dn%), PTV Mean Dose, and Modulation Factor. The Wilcoxon Signed Rank Sum non-parametric statistical method was utilized to compare the obtained plan quality metrics. RESULTS: Statistically significant improvements were found for the AFI strategy for metrics R50%, CIRTOG, CIPaddick, and PTV Mean Dose (p < 0.001). HA achieved superior coverage for Dn% (p = 0.018), while the Modulation Factors were not significantly different for AFI compared to HA optimization (p = 0.13). CONCLUSION: This study provides evidence that the AFI manual planning strategy can produce high-quality planning metrics similar to the HA auto-planning method.

4.
JAMA Oncol ; 2024 Sep 05.
Article in English | MEDLINE | ID: mdl-39235788

ABSTRACT

This essay describes a physician's shift to patient-focused language in treatment of patients with cancer.

5.
Molecules ; 29(17)2024 Aug 24.
Article in English | MEDLINE | ID: mdl-39274851

ABSTRACT

A surfactant's equilibrium spreading pressure (ESP) is the maximum decrease in surface tension achievable at equilibrium below the Krafft point. Difficulties in measuring the ESP have been noted previously but no well-established experimental protocols to overcome them exist. We present a case study of three solid amphiphiles with different propensities to spread on the air-water interface. Starting with the partially water soluble n-dodecanol (C12H25OH), which spreads instantaneously. The strong Marangoni flows associated with the spreading result in the dislocating of the Wilhelmy plate or crystals attaching to it. A temporary mechanical barrier in front of the spreading crystals mitigates the flows disturbing the plate. Presaturating the subphase with the amphiphile prevents the establishment of dynamic steady states, reduces the standard error by a factor of three and causes faster equilibration. The perfluoroalkylated analog of dodecanol (11:1 fluorotelomer alcohol, C11F23CH2OH) is slow spreading. With surfactant crystals on the interface, the surface pressure reaches a pre-equilibrium plateau within an hour, followed by equilibration on day-long timescales. We show that it is better to estimate the ESP by averaging the values of multiple pre-equilibrium plateaus rather than waiting for equilibrium to be established. Finally, the nonspreading amphiphile DPPC exhibits a large barrier for the mass transfer from the DPPC crystal to the aqueous surface. This was overcome by introducing a volatile, water-immiscible solvent deposited on the surface next to the crystals to facilitate the spreading process and leave behind a monolayer.

6.
Zookeys ; 1206: 327-342, 2024.
Article in English | MEDLINE | ID: mdl-39034989

ABSTRACT

In recent years, several endemic species of Loxosceles, violin spiders, have been described from the North-Central Chile biodiversity hotspot, some of which have ambiguous placement within the species groups of the genus. In a recent expedition to the Atacama region, we collected male specimens representing new records of two recently described species: Loxoscelesvicentei Taucare-Ríos, Brescovit & Villablanca, 2022 and Loxoscelesvallenar Brescovit, Taucare-Ríos, Magalhaes & Santos, 2017 (Araneae, Sicariidae). Males of the latter are hitherto unknown and are here described for the first time. Examination of the morphology of these species revealed characters such as an embolic keel and digitiform median receptacles, which suggest they do not belong in the laeta species group, but rather in the spadicea species group, which is briefly re-diagnosed. With carapace lengths smaller than 2 mm, the newly discovered males of L.vallenar are the tiniest members of the genus. In addition, males of this species bear strong macrosetae in the clypeus, a sexually dimorphic character not previously reported in Loxosceles.

7.
PLoS One ; 19(7): e0307185, 2024.
Article in English | MEDLINE | ID: mdl-38985807

ABSTRACT

[This corrects the article DOI: 10.1371/journal.pone.0257527.].

8.
Lancet Reg Health West Pac ; 47: 101114, 2024 Jun.
Article in English | MEDLINE | ID: mdl-39022747

ABSTRACT

Cancer is a leading cause of death among children in the Philippines, a low-middle-income country of over 110 million people. In this Comment, we describe how financial toxicity affects families of pediatric patients with cancer in the Philippines. We explore direct costs of care, indirect costs such as transportation and lodging, and psychosocial sequelae, in the Filipino medical system and sociocultural contexts. We present examples of successful interventions in the Philippines and in similarly resourced settings, with the goal of galvanizing further research, clinical interventions, and policy-level changes, aimed at mitigating family financial toxicity for pediatric patients with cancer in the Philippines and globally.

10.
Microorganisms ; 12(7)2024 Jul 09.
Article in English | MEDLINE | ID: mdl-39065162

ABSTRACT

Clostridioides difficile is a Gram-positive pathogen known for its toxin production and spore formation. It is primarily responsible for most cases of antibiotic-associated diarrhea. Bacterial persisters are a small subset of the population that exhibits transient tolerance to bactericidal substances, and they are of significant medical concern due to their association with the emergence of antibiotic resistance and difficult-to-treat chronic or recurrent infections. Vancomycin, the predominant antibiotic utilized in the management of C. difficile infection, is extensively applied in the realm of clinical practice. Previous studies have demonstrated a persister-like phenotype with treatments involving this antibiotic. However, the mechanism in C. difficile remains largely unknown, primarily due to the challenge of isolating this small population at any given time. To better characterize C. difficile persister cells, we present a study that enables the enrichment and characterization of persister cells from bacterial cultures in both the exponential and stationary phases. Moreover, we could differentiate between triggered (induced using antibiotics such as vancomycin) and spontaneous (stochastic) persister cells. Additionally, we observed the involvement of toxin-antitoxin systems and Clp proteases in persister cell formation.

11.
Cladistics ; 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38861251

ABSTRACT

The Neotropical region is the most diverse on the planet, largely owing to its mosaic of tropical rainforests. Multiple tectonic and climatic processes have been hypothesized to contribute to generating this diversity, including Andean orogeny, the closure of the Isthmus of Panama, the GAARlandia land bridge and historical connections among currently isolated forests. Micrathena spiders are diverse and widespread in the region, and thus a complete phylogeny of this genus allows the testing of hypotheses at multiple scales. We estimated a complete, dated phylogeny using morphological data for 117 Micrathena species and molecular data of up to five genes for a subset of 79 species. Employing eventc-based approaches and biogeographic stochastic mapping while considering phylogenetic uncertainty, we estimated ancestral distributions, the timing and direction of dispersal events and diversification rates among areas. The phylogeny is generally robust, with uncertainty in the position of some of the species lacking sequences. Micrathena started diversifying around 25 Ma. Andean cloud forests show the highest in-situ speciation, while the Amazon is the major dispersal source for adjacent areas. The Dry Diagonal generated few species and is a sink of diversity. Species exchange between Central and South America involved approximately 23 dispersal events and started ~20 Ma, which is consistent with a Miocene age for the Isthmus of Panama closure. We inferred four dispersal events from Central America to the Antilles in the last 20 Myr, indicating the spiders did not reach the islands through the GAARlandia land bridge. We identified important species exchange routes among the Amazon, Andean cloud forests and Atlantic forests during the Plio-Pleistocene. Sampling all species of the genus was fundamental to the conclusions above, especially in identifying the Andean forests as the area that generated the majority of species. This highlights the importance of complete taxonomic sampling in biogeographic studies.

12.
Folia Med (Plovdiv) ; 66(2): 235-242, 2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38690819

ABSTRACT

INTRODUCTION: Tooth extraction is still one of the most common dental procedures, routinely performed for a variety of reasons. Tooth extraction forceps and elevators are well-known extraction instruments which have been the standard in tooth extraction procedures for well over a hundred years. Physics forceps are one possible alternative, aiming to perform less traumatic and more predictable extractions.


Subject(s)
Surgical Instruments , Tooth Extraction , Humans , Tooth Extraction/instrumentation , Minimally Invasive Surgical Procedures/instrumentation , Minimally Invasive Surgical Procedures/methods , Female , Male , Adult , Middle Aged , Young Adult , Aged
13.
J Pharm Sci ; 113(8): 2651-2655, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38750935

ABSTRACT

Characterization and understanding of protein higher order structure (HOS) is essential at all stages of biologics development. Here, two folding variants of a bispecific monoclonal antibody, the correctly folded form and an alternative configuration with reduced potency, were characterized by several HOS characterization techniques. Specifically, differential scanning calorimetry (DSC), circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), Raman and Raman optical activity (ROA) spectroscopy were used together to elucidate the impacts of disulfide bond scrambling in the fused scFv domains on the structure and thermal stability of the antibody. This study illustrates the importance of selecting appropriate biophysical characterization techniques based on the nature and magnitude of the HOS change.


Subject(s)
Antibodies, Bispecific , Calorimetry, Differential Scanning , Circular Dichroism , Disulfides , Disulfides/chemistry , Antibodies, Bispecific/chemistry , Spectroscopy, Fourier Transform Infrared/methods , Protein Stability , Protein Folding , Spectrum Analysis, Raman/methods , Antibodies, Monoclonal/chemistry , Protein Conformation
14.
Molecules ; 29(9)2024 Apr 25.
Article in English | MEDLINE | ID: mdl-38731451

ABSTRACT

A novel second-generation blue fluorescent polyamidoamine dendrimer peripherally modified with sixteen 4-N,N-dimethylaninoethyloxy-1,8-naphthalimide units was synthesized. Its basic photophysical characteristics were investigated in organic solvents of different polarity. It was found that in these solvents, the dendrimer is colorless and emitted blue fluorescence with different intensities depending on their polarity. The effect of the pH of the medium on the fluorescence intensity was investigated and it was found that in the acidic medium, the fluorescence is intense and is quenched in the alkaline medium. The ability of the dendrimer to detect metal ions (Pb2+, Zn2+, Mg2+, Sn2+, Ba2+, Ni2+, Sn2+, Mn2+, Co2+, Fe3+, and Al3+) was also investigated, and it was found that in the presence of Fe3+, the fluorescent intensity was amplified more than 66 times. The antimicrobial activity of the new compound has been tested in vitro against Gram-positive B. cereus and Gram-negative P. aeruginosa. The tests were performed in the dark and after irradiation with visible light. The antimicrobial activity of the compound enhanced after light irradiation and B. cereus was found slightly more sensitive than P. aeruginosa. The increase in antimicrobial activity after light irradiation is due to the generation of singlet oxygen particles, which attack bacterial cell membranes.


Subject(s)
Dendrimers , Microbial Sensitivity Tests , Naphthalimides , Polyamines , Naphthalimides/chemistry , Naphthalimides/pharmacology , Dendrimers/chemistry , Dendrimers/pharmacology , Polyamines/chemistry , Polyamines/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Fluorescence , Pseudomonas aeruginosa/drug effects , Hydrogen-Ion Concentration , Bacillus cereus/drug effects , Light , Fluorescent Dyes/chemistry , Spectrometry, Fluorescence
15.
Molecules ; 29(7)2024 Mar 22.
Article in English | MEDLINE | ID: mdl-38611701

ABSTRACT

Amphiphilic fluorocarbon substances are a trending topic of research due to their wide range of applications accompanied by an alarming environmental and health impact. In order to predict their fate in the environment, use them more economically, develop new water treatment methods, etc., a better understanding of their physicochemical behavior is required. Their hydrophobicity in water/oil systems is particularly sensitive to one key thermodynamic parameter: the free energy of transfer of a perfluoromethylene group from oil to water. However, for the -CF2- moiety, the transfer energy values reported in the literature vary by more than ±25%. Due to the exponential relationship between this energy and the adsorption constants or the partition coefficients, such an uncertainty can lead to orders of magnitude error in the predicted distribution of fluorinated species. We address this problem by presenting an experimental determination of the hydrophobic effect of a -CF2- moiety with a greater certainty than currently available. The transfer energy is determined by measuring the interfacial tension of water|hexane for aqueous solutions of short-chained fluorotelomer alcohols. The obtained results for the free energy of transfer of a -CF2- moiety from oil to water are 1.68±0.02×RT0, 1.75±0.02×RT0, and 1.88±0.02×RT0 at 288.15 K, 293.15 K, and 303.15 K, respectively.

16.
Proc Natl Acad Sci U S A ; 121(16): e2304704121, 2024 Apr 16.
Article in English | MEDLINE | ID: mdl-38593073

ABSTRACT

Childhood maltreatment (CM) leads to a lifelong susceptibility to mental ill-health which might be reflected by its effects on adult brain structure, perhaps indirectly mediated by its effects on adult metabolic, immune, and psychosocial systems. Indexing these systemic factors via body mass index (BMI), C-reactive protein (CRP), and rates of adult trauma (AT), respectively, we tested three hypotheses: (H1) CM has direct or indirect effects on adult trauma, BMI, and CRP; (H2) adult trauma, BMI, and CRP are all independently related to adult brain structure; and (H3) childhood maltreatment has indirect effects on adult brain structure mediated in parallel by BMI, CRP, and AT. Using path analysis and data from N = 116,887 participants in UK Biobank, we find that CM is related to greater BMI and AT levels, and that these two variables mediate CM's effects on CRP [H1]. Regression analyses on the UKB MRI subsample (N = 21,738) revealed that greater CRP and BMI were both independently related to a spatially convergent pattern of cortical effects (Spearman's ρ = 0.87) characterized by fronto-occipital increases and temporo-parietal reductions in thickness. Subcortically, BMI was associated with greater volume, AT with lower volume and CPR with effects in both directions [H2]. Finally, path models indicated that CM has indirect effects in a subset of brain regions mediated through its direct effects on BMI and AT and indirect effects on CRP [H3]. Results provide evidence that childhood maltreatment can influence brain structure decades after exposure by increasing individual risk toward adult trauma, obesity, and inflammation.


Subject(s)
Brain , Child Abuse , Adult , Humans , Child , Brain/diagnostic imaging , Brain/metabolism , C-Reactive Protein/metabolism , Inflammation/metabolism , Obesity/complications , Child Abuse/psychology
17.
Front Microbiol ; 15: 1389663, 2024.
Article in English | MEDLINE | ID: mdl-38591031

ABSTRACT

The rise of multidrug-resistant bacteria is a global concern, leading to a renewed reliance on older antibiotics like polymyxins as a last resort. Polymyxins, cationic cyclic peptides synthesized nonribosomally, feature a hydrophobic acyl tail and positively charged residues. Their antimicrobial mechanism involves initial interaction with Gram-negative bacterial outer-membrane components through polar and hydrophobic interactions. Outer membrane vesicles (OMVs), nano-sized proteoliposomes secreted from the outer membrane of Gram-negative bacteria, play a crucial role in tolerating harmful molecules, including cationic peptides such as polymyxins. Existing literature has documented environmental changes' impact on modulating OMV properties in Salmonella Typhimurium. However, less information exists regarding OMV production and characteristics in Salmonella Typhi. A previous study in our laboratory showed that S. Typhi ΔmrcB, a mutant associated with penicillin-binding protein (PBP, a ß-lactam antibiotic target), exhibited hypervesiculation. Consequently, this study investigated the potential impact of ß-lactam antibiotics on promoting polymyxin tolerance via OMVs in S. Typhi. Our results demonstrated that sub-lethal doses of ß-lactams increased bacterial survival against polymyxin B in S. Typhi. This phenomenon stems from ß-lactam antibiotics inducing hypervesiculation of OMVs with higher affinity for polymyxin B, capturing and diminishing its biologically effective concentration. These findings suggest that ß-lactam antibiotic use may inadvertently contribute to decreased polymyxin effectivity against S. Typhi or other Gram-negative bacteria, complicating the effective treatment of infections caused by these pathogens. This study emphasizes the importance of evaluating the influence of ß-lactam antibiotics on the interaction between OMVs and other antimicrobial agents.

18.
Front Public Health ; 12: 1234584, 2024.
Article in English | MEDLINE | ID: mdl-38450141

ABSTRACT

Introduction: Self-immolation is an uncommon way of attempting and committing a suicide, with a fatality rate of 80%. The risk factors in self-immolation victims vary depending on demographic characteristics, socio-economic and cultural factors as well as religious beliefs. Whether the COVID-19 pandemic was a potentially important stressor for self-immolation is still unknown, with insufficient studies examining this issue. Therefore, in this study, we aimed to examine the trend of self-immolation in a 13-year timeline, and the potential association of COVID-19 pandemic with the increase in the incidence and severity of self-immolation injuries in Serbia in 2021. Materials and methods: The study included hospitalized patients due to intentional burns caused by self-immolation in the period from January 1, 2008 to December 31, 2021. Joinpoint regression analysis was used for the analysis of continuous linear trends of self-immolation cases with change points. Results: While a rising trend was observed in the 2008-2013 time segment, followed by a decline in the upcoming 2013-2016 time segment, a significant increase reached its maximum during COVID-19 pandemic (2021), with annual percent change of 37.1% (p = 0.001). A significant increase in the median number of cases per year was observed during 2021 compared to the previous periods (7.5 vs. 2). Frequency of patients with a psychiatric diagnosis vs. those without a psychiatric diagnosis was significantly higher during than before the COVID-19 period (66.7 vs. 36.1%, p = 0.046). Conclusion: In our study, a significant increase in the frequency of suicide attempts by self-immolation during COVID-19 pandemic was noticed. There was also an increased frequency of pre-existing psychiatric illness among patients during the pandemic period. With limited high-quality data available, the study adds to a rising body of evidence for assessment of outcomes of the pandemic on mental health and recognition of stressors for self-immolation.


Subject(s)
COVID-19 , Mental Disorders , Humans , COVID-19/epidemiology , Pandemics , Data Accuracy , Mental Health
19.
Res Q Exerc Sport ; 95(3): 730-739, 2024 Sep.
Article in English | MEDLINE | ID: mdl-38319611

ABSTRACT

Purpose: The aim of this study was to investigate the effects of moderate combined training (CT) on both the gene expression of pro- and anti-inflammatory markers and senescence in the immune system in peripheral blood mononuclear cells (PBMCs) and subcutaneous adipose tissue (SAT) of obese middle-aged individuals with type 2 diabetes (T2D). Methods: Thirty obese individuals (50.2 ± 9.4 years; body mass index: 31.8 ± 2.3 kg/m²) with T2D underwent 16 weeks of a CT group [CT; aerobic (50-60% of VO2max) plus resistance (50-75% of 1RM) training; 3 times/week, 70 min/session; n = 16)] or a control group (CG, n = 14). Nutritional patterns, muscle strength (1RM), cardiorespiratory fitness (VO2max), waist circumference (WC), body composition (Air Displacement Plethysmograph) and blood collections for biochemical (serum leptin, IL-2, IL-4, IL-6, IL-10, TNF-α and anti-CMV) and molecular (gene expression of leptin, IL-2, IL-4, IL-6, IL-10, TNF-α, PD-1, P16ink4a, CCR7, CD28 and CD27 in PBMCs and SAT) analyses were assessed before (Pre) and after (Post) the 16 weeks of the experimental period. Results: Significant decreases were observed in WC and IL4, TNF-α, PD-1 and CD27 expression in PBMCs for CT. Furthermore, significant increases were observed in 1RM and VO2max for CT after the experimental period. Conclusion: Moderate CT contributed to a reduction in the gene expression of markers associated to chronic inflammation and immunosenescence in PBMCs of obese middle-aged individuals with T2D.


Subject(s)
Diabetes Mellitus, Type 2 , Immunosenescence , Leukocytes, Mononuclear , Obesity , Resistance Training , Humans , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/immunology , Obesity/immunology , Obesity/genetics , Middle Aged , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/immunology , Male , Female , Subcutaneous Fat/metabolism , Adult , Muscle Strength/physiology , Cardiorespiratory Fitness/physiology , Waist Circumference , Gene Expression , Cytokines/genetics , Cytokines/blood , Body Composition , Biomarkers/blood
20.
Microorganisms ; 12(2)2024 Jan 29.
Article in English | MEDLINE | ID: mdl-38399687

ABSTRACT

Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves, contributing to their persistence in the aquatic environment. Detection methods for this and other fish pathogens can be time-consuming and lack specificity and sensitivity, limiting timely monitoring, the treatment of microbial infections, and effective control of their transmission in aquaculture settings. Rapid and sensitive detection methods for nucleic acids can be crucial for an appropriate surveillance of bacterial pathogens, and the CRISPR/Cas-based assays have emerged as a good alternative since it has been proven to be a useful tool for the rapid, specific, and sensitive detection of viruses and some bacteria. In this study, we explored the capability of the CRISPR/Cas13a system (SHERLOCK) to specifically detect both DNA and RNA (gene transcripts) from planktonic and biofilm samples of the bacterial fish pathogen Y. ruckeri. The assay was designed to detect the gyrA gene and the small noncoding RNAs (sRNAs) MicA and RprA from planktonic cultures and biofilm samples prepared in marine broth. The specific crRNA designed for these gene targets included a 28 nt specific gene sequence, and a scaffold sequence necessary for Cas13-binding. For all the assays, the nucleic acids obtained from samples were previously subjected to isothermal amplification with the recombinase polymerase amplification (RPA) method and the subsequent T7 transcription of the RPA amplicons. Finally, the detection of nucleic acids of Y. ruckeri was by means of a reporter signal released by the Cas13a collateral RNA cleavage triggered upon target recognition, measured by fluorescence- or lateral-flow-based readouts. This CRISPR/Cas13a-based assay was able to specifically detect both DNA and sRNAs from the Y. ruckeri samples, and the sensitivity was comparable to that obtained with qPCR analysis, highlighting the potential applicability of this CRISPR/Cas13a-based assay for fish pathogen surveillance.

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