A prospective, pragmatic non-inferiority study of emergency intubation success with the single-use i-view versus standard reusable video laryngoscope.

INTRODUCTION: Video laryngoscope (VL) technology improves first-pass success. The novel i-view VL device is inexpensive and disposable. We sought to determine the first-pass intubation success with the i-view VL device versus the standard reusable VL systems in routine use at each site. METHODS: We performed a prospective, pragmatic study at two major emergency departments (EDs) when VL was used. We rotated i-view versus reusable VL as the preferred device of the month based on an a priori schedule. An investigator-initiated interim analysis was performed. Our primary outcome was a first-pass success with a non-inferiority margin of 10% based on the per-protocol analysis. RESULTS: There were 93 intubations using the reusable VL devices and 81 intubations using the i-view. Our study was stopped early due to futility in reaching our predetermined non-inferiority margin. Operator and patient characteristics were similar between the two groups. The first-pass success rate for the i-view group was 69.1% compared to 84.3% for the reusable VL group. A non-inferiority analysis indicated that the difference (-15.1%) and corresponding 90% confidence limits (-25.3% to -5.0%) did not fall within the predetermined 10% non-inferiority margin. CONCLUSIONS: The i-view device failed to meet our predetermined non-inferiority margin when compared to the reusable VL systems with the study stopping early due to futility. Significant crossover occurred at the discretion of the intubating operator during the i-view month.

A prospective assessment of the time required to obtain one unit of fresh whole blood by civilian phlebotomists and Army laboratory technicians (68 K).

BACKGROUND: Resuscitation with blood products improves survival after major hemorrhage. Blood product administration at or near the point-of-injury (POI) amplifies this benefit. Size, weight, and cold-chain management challenges all limit the amount of blood medics can carry. Warm fresh whole blood (WFWB) transfusions from a pre-screened donor within the unit represent an alternative source of blood at the POI. We measured the time required for civilian and Army technicians performing phlebotomy frequently to obtain one unit of blood to serve as a goal metric for combat medics being trained in this skill. METHODS: We gathered demographic and experience data along with proportion of first intravenous cannulation attempt success, time to blood flow initiated, and time to unit draw complete. RESULTS: We prospectively enrolled 12 civilian phlebotomy technicians and 10 Army laboratory technicians performing whole blood collections on 50 and 68 donors respectively. The mean time from setup to needle insertion was 3.7 min for civilians versus 4.2 min for Army technicians. The mean time from blood flowing to the bag being full was 10.7 min versus 8.4 min for civilians versus Army technicians respectively. The mean bag weights were 514 g versus 522 g. First-pass intravenous cannulation success was 96% versus 98% respectively. CONCLUSIONS: We found a high first intravenous cannulation attempt success among both the civilian and Army technicians. Medians times were <5 min to obtain venipuncture and <11 min to obtain one unit. These findings provide time-based benchmarks for potential use during transfusion training among military medics.

A prospective assessment of the medic autologous blood transfusion skills for field transfusion preparation.

BACKGROUND: Data demonstrate the benefit of blood product administration near point-of-injury (POI). Fresh whole blood transfusion from a pre-screened donor provides a source of blood at the POI when resources are constrained. We captured transfusion skills data for medics performing autologous blood transfusion training. METHODS: We conducted a prospective, observational study of medics with varying levels of experience. Inexperienced medics were those with minimal or no reported experience learning the autologous transfusion procedures, versus reported experience among special operations medics. When available, medics were debriefed after the procedure for qualitative feedback. We followed them for up to 7 days for adverse events. RESULTS: The median number of attempts for inexperienced and experienced medics was 1 versus 1 (interquartile range 1-1 for both, p = .260). The inexperienced medics had a slower median time to needle venipuncture access for the donation of 7.3 versus 1.5 min, needle removal after clamping time of 0.3 versus 0.2 min, time to bag preparation of 1.9 versus 1.0 min, time to IV access for reinfusion of 6.0 versus 3.0 min, time to transfusion completion of 17.3 versus 11.0 min, and time to IV removal of 0.9 versus 0.3 min (all p < .05). We noted one administrative safety event in which an allogeneic transfusion occurred. No major adverse events occurred. Qualitative data saturated around the need for quarterly training. CONCLUSIONS: Inexperienced medics have longer procedure times when training autologous whole blood transfusion skills. This data will help establish training measures of performance for skills optimization when learning this procedure.

Changes in physiology and virulence during the selection of resistant Yersinia ruckeri mutants under subinhibitory cefotaxime concentrations.

Bacterial antibiotic resistance is one of the main healthcare problems currently. Apart from reducing antibiotic efficacy, it has awakened the interest of scientists due to its association with bacterial fitness and virulence. Interestingly, antibiotic resistance can be a source of both increased fitness and decreased fitness, even though the molecular basis of these relationships remains unknown. The aim of this work is to define the effects of sub-MIC concentrations of cefotaxime, an antibiotic extensively used in clinical practice, on the physiology and virulence of Yersinia ruckeri and to determine the importance of these sub-MIC concentrations for the selection of antibiotic-resistant mutants in the aquatic environment. Results indicated that exposure to sub-MIC concentrations of cefotaxime selected Y. ruckeri populations with irreversible alterations in the physiology, such as slow growth, aggregation in liquid cultures and modification of the colony morphology. These bacteria also displayed changes in the OMPs and LPS profiles and a full attenuation of virulence. An overexpression of the envelope stress regulator RpoE was also detected after exposure to the antibiotic. In conclusion, exposure to cefotaxime selected, at high frequency, Y. ruckeri strains that survive the antibiotic stress at the expense of a fitness cost and the loss of virulence.

Repeatability of the infant food reinforcement paradigm: Implications of individual and developmental differences.

The relative reinforcing value of food versus engagement in other behaviors may be related to the development of obesity, and interventions to reduce FRR may prevent the development of obesity. Our laboratory recently developed a paradigm to measure the reinforcing value of food versus an alternative behavior (i.e., playing with bubbles) in infants using a computerized laboratory task, during which infants press a button to earn reinforcers following a progressive ratio schedule of reinforcement. The primary purpose of this study was to examine the short-term (within 2 weeks) repeatability of this measure, specifically the outcome of infant food reinforcing ratio (FRR), or how hard infants will work for food relative to the alternative. The secondary aim was to examine whether infant age and temperament dimensions related to novelty responsiveness (high intensity pleasure and approach) moderated the repeatability of FRR. Thirty-seven infants aged 9-18 months completed this study. Repeated measures analysis of variance (ANOVA) showed no differences between time 1 and time 2 in responding for food (F = 0.463, p = 0.501), bubbles (F = 1.793, p = 0.189), or overall FRR (F = 0.797, p = 0.378). Regression models showed the association between BUB Pmax at time 1 and time 2 were moderated by infant age (p = 0.04), with greater repeatability in older infants. Linear regression models also demonstrated that the infant temperamental dimension of high intensity pleasure significantly predicted BUB Pmax at time 1 (ß = 2.89, p = 0.01), but not at time 2. Overall, our findings support the repeatability of this measure for food portion of the reinforcement task, but demonstrated that the measure of non-food portion of the task required modification, in particular among children younger than 13 months old.

The heat sensitive factor (HSF) of Yersinia ruckeri is produced by an alkyl sulphatase involved in sodium dodecyl sulphate (SDS) degradation but not in virulence.

BACKGROUND: The heat sensitive factor (HSF) of the fish pathogen Yersinia ruckeri was previously identified as an unusual band on SDS-PAGE. According to this, Y. ruckeri strains were classified in HSF+ and HSF - in terms of the presence/absence of the factor. Experiments carried out by injection challenge with HSF + strains caused high mortalities in rainbow trout. In contrast, HSF - strains did not cause mortality. In conclusion, HSF appeared to be a relevant virulence factor in Y. ruckeri. RESULTS: We report here the identification and study of the gene coding for the enzyme involved in the production of HSF. Culture medium containing SDS and Coomassie brilliant blue dye was used to screen a mini-Tn5 Km2 mutant library of Y. ruckeri 150. Blue colonies lacking a surrounding creamy deposit, a phenotype described in former studies as HSF - , were identified. DNA sequence analysis of a selected mutant revealed that this had a transposon interruption in a chromosome-located gene which codes for a heat sensitive alkyl sulphatase of 78.7 kDa (YraS; Yersinia ruckeri alkyl sulphatase) which is able to degrade SDS to 1-dodecanol. As it was expected, the introduction of the yraS gene into an HSF - strain turned this into HSF + . Surprisingly, although the protein allows Y. ruckeri to degrade SDS, the bacterium could not use this compound as the sole carbon source. Moreover, the yraS mutant showed a similar level of SDS resistance to the parental strain. It was the interruption of the acrA gene which made Y. ruckeri sensitive to this compound. LD50 experiments showed a similar virulence of the yraS mutant and parental strain. CONCLUSIONS: The HSF of Y. ruckeri is the product of the alkyl sulphatase YraS, able to degrade SDS to 1-dodecanol. This degradation is not linked to the utilization of SDS as a carbon source and surprisingly, the enzyme is not involved in bacterial virulence or in the high SDS resistance displayed by the bacterium. This role is played by the AcrAB-TolC system.

Delivery of chemically glycosylated cytochrome c immobilized in mesoporous silica nanoparticles induces apoptosis in HeLa cancer cells.

Cytochrome c (Cyt c) is a small mitochondrial heme protein involved in the intrinsic apoptotic pathway. Once Cyt c is released into the cytosol, the caspase mediated apoptosis cascade is activated resulting in programmed cell death. Herein, we explore the covalent immobilization of Cyt c into mesoporous silica nanoparticles (MSN) to generate a smart delivery system for intracellular drug delivery to cancer cells aiming at affording subsequent cell death. Cyt c was modified with sulfosuccinimidyl-6-[3'-(2-pyridyldithio)-propionamido] hexanoate (SPDP) and incorporated into SH-functionalized MSN by thiol-disulfide interchange. Unfortunately, the delivery of Cyt c from the MSN was not efficient in inducing apoptosis in human cervical cancer HeLa cells. We tested whether chemical Cyt c glycosylation could be useful in overcoming the efficacy problems by potentially improving Cyt c thermodynamic stability and reducing proteolytic degradation. Cyt c lysine residues were modified with lactose at a lactose-to-protein molar ratio of 3.7 ± 0.9 using mono(lactosylamido)-mono(succinimidyl) suberate linker chemistry. Circular dichroism (CD) spectra demonstrated that part of the activity loss of Cyt c was due to conformational changes upon its modification with the SPDP linker. These conformational changes were prevented in the glycoconjugate. In agreement with the unfolding of Cyt c by the linker, a proteolytic assay demonstrated that the Cyt c-SPDP conjugate was more susceptible to proteolysis than Cyt c. Attachment of the four lactose molecules reversed this increased susceptibility and protected Cyt c from proteolytic degradation. Furthermore, a cell-free caspase-3 assay revealed 47% and 87% of relative caspase activation by Cyt c-SPDP and the Cyt c-lactose bioconjugate, respectively, when compared to Cyt c. This again demonstrates the efficiency of the glycosylation to improve maintaining Cyt c structure and thus function. To test for cytotoxicity, HeLa cells were incubated with Cyt c loaded MSN at different Cyt c concentrations (12.5, 25.0, and 37.5 µg/mL) for 24-72 h and cellular metabolic activity determined by a cell proliferation assay. While MSN-SPDP-Cyt c did not induced cell death, the Cyt c-lactose bioconjugate induced significant cell death after 72 h, reducing HeLa cell viability to 67% and 45% at the 25 µg/mL and 37.5 µg/mL concentrations, respectively. Confocal microscopy confirmed that the MSN immobilized Cyt c-lactose bioconjugate was internalized by HeLa cells and that the bioconjugate was capable of endosomal escape. The results clearly demonstrate that chemical glycosylation stabilized Cyt c upon formulation of a smart drug delivery system and upon delivery into cancer cells and highlight the general potential of chemical protein glycosylation to improve the stability of protein drugs.

Activation of caspase-dependent apoptosis by intracellular delivery of Cytochrome c-based nanoparticles.

BACKGROUND: Cytochrome c is an essential mediator of apoptosis when it is released from the mitochondria to the cytoplasm. This process normally takes place in response to DNA damage, but in many cancer cells (i.e., cancer stem cells) it is disabled due to various mechanisms. However, it has been demonstrated that the targeted delivery of Cytochrome c directly to the cytoplasm of cancer cells selective initiates apoptosis in many cancer cells. In this work we designed a novel nano-sized smart Cytochrome c drug delivery system to induce apoptosis in cancer cells upon delivery. RESULTS: Cytochrome c was precipitated with a solvent-displacement method to obtain protein nanoparticles. The size of the Cytochrome c nanoparticles obtained was 100-300 nm in diameter depending on the conditions used, indicating good potential to passively target tumors by the Enhanced Permeability and Retention effect. The surface of Cytochrome c nanoparticles was decorated with poly (lactic-co-glycolic) acid-SH via the linker succinimidyl 3-(2-pyridyldithio) propionate to prevent premature dissolution during delivery. The linker connecting the polymer to the protein nanoparticle contained a disulfide bond thus allowing polymer shedding and subsequent Cytochrome c release under intracellular reducing conditions. A cell-free caspase-3 assay revealed more than 80% of relative caspase activation by Cytochrome c after nanoprecipitation and polymer modification when compared to native Cytochrome c. Incubation of HeLa cells with the Cytochrome c based-nanoparticles showed significant reduction in cell viability after 6 hours while native Cytochrome c showed none. Confocal microscopy confirmed the induction of apoptosis in HeLa cells when they were stained with 4',6-diamidino-2-phenylindole and propidium iodide after incubation with the Cytochrome c-based nanoparticles. CONCLUSIONS: Our results demonstrate that the coating with a hydrophobic polymer stabilizes Cytochrome c nanoparticles allowing for their delivery to the cytoplasm of target cells. After smart release of Cytochrome c into the cytoplasm, it induced programmed cell death.

Comparative Analysis of Whole Blood Infusion Effects: Assessing LifeFlow Versus Pressure Bag in a Sus scrofa Model.

BACKGROUND: To improve survival for hemorrhagic shock treatment, guidelines emphasize two patient care priorities: (1) immediate hemorrhage control and (2) early resuscitation with whole blood or blood products. The LifeFlow device is designed to rapidly infuse blood products. However, the effects of using this device compared to pressure-bag systems remain unclear. We hypothesize that there will be no laboratory-measured difference with the blood when infused through the LifeFlow versus the current standard pressure bag system. METHODS: Two units of fresh whole blood were obtained from a sus scrofa model. One unit was "infused" using the LifeFlow with the other unit used as a control through a standard pressure bag system into an empty bag. The "before" measurements were obtained from blood samples from a standard fresh whole blood collection bag. The blood was "infused" into a whole blood bag devoid of storage solution from which the "after" measurements were obtained. RESULTS: This study utilized 22 clinically healthy sus scrofa. Blood units were primarily obtained from a left subclavian central line (50.0%). The median time to acquire and administer a unit of blood was similar for both the LifeFlow device (8.4 min and 8.1 min) and the pressure bag (8.7 min and 7.4 min). No significant differences were found in the total time to acquire or administer blood between the two devices. The median volume of blood acquired was 500 mL for both groups. While no significant differences in blood parameters were observed between the two devices, significant differences were noted when comparing pre- and post-transfusion values within each device. For the LifeFlow device, an increase in hemoglobin and chloride levels and a decrease in thromboplastin time and glucose levels were observed. With the pressure bag, only a decrease in blood urea nitrogen was observed. CONCLUSIONS: In comparing the LifeFlow to the pressure bag, there were no significant differences noted in the total time to acquire or administer a whole unit of blood. However, there were differences with several laboratory parameters of unclear clinical significance.

Expert Consensus Panel Recommendations for Selection of the Optimal Supraglottic Airway Device for Inclusion to the Medic's Aid Bag.

INTRODUCTION: Airway obstruction is the second leading cause of potentially survivable death on the battlefield. The Committee on Tactical Combat Casualty Care (CoTCCC) has evolving recommendations for the optimal supraglottic airway (SGA) device for inclusion to the medics' aid bag. METHODS: We convened an expert consensus panel consisting of a mix of 8 prehospital specialists, emergency medicine experts, and experienced combat medics, with the intent to offer recommendations for optimal SGA selection. Prior to meeting, we independently reviewed previously published studies conducted by our study team, conducted a virtual meeting, and summarized the findings to the panel. The studies included an analysis of end-user after action reviews, a market analysis, engineering testing, and prospective feedback from combat medics. The panel members then made recommendations regarding their top 3 choices of devices including the options of military custom design. Simple descriptive statistics were used to analyze panel recommendations. RESULTS: The preponderance (7/8, 88%) of panel members recommended the gel-cuffed SGA, followed by the self-inflating-cuff SGA (5/8, 62%) and laryngeal tube SGA (5/8, 62%). Panel members expressed concerns primarily related to the (1) devices' tolerance for the military environment, and (2) ability to effectively secure the gel-cuffed SGA and the self-inflating-cuff SGA during transport. CONCLUSIONS: A preponderance of panel members selected the gel-cuff SGA with substantial feedback highlighting the need for military-specific customizations to support the combat environment needs.

Clinical Assessment of Low Calcium In traUMa (CALCIUM).

Major trauma frequently occurs in the deployed, combat setting and is especially applicable in the recent conflicts with explosives dominating the combat wounded. In future near-peer conflicts, we will likely face even more profound weapons including mortars and artillery. As such, the number of severely wounded will likely increase. Hypocalcemia frequently occurs after blood transfusions, secondary to the preservatives in the blood products; however, recent data suggests major trauma in and of itself is a risk factor for hypocalcemia. Calcium is a major ion involved in heart contractility; thus, hypocalcemia can lead to poor contractility. Smaller studies have linked hypocalcemia to worse outcomes, but it remains unclear what causes hypocalcemia and if intervening could potentially save lives. The objective of this study is to determine the incidence of hypocalcemia on hospital arrival and the association with survival. We are seeking to address the following scientific questions, (1) Is hypocalcemia present following traumatic injury prior to transfusion during resuscitation? (2) Does hypocalcemia influence the amount of blood products transfused? (3) To what extent is hypocalcemia further exacerbated by transfusion? (4) What is the relationship between hypocalcemia following traumatic injury and mortality? We will conduct a multicenter, prospective, observational study. We will gather ionized calcium levels at 0, 3, 6, 12, 18, and 24 hours as part of scheduled calcium measurements. This will ensure we have accurate data to assess the early and late effects of hypocalcemia throughout the course of resuscitation and hemorrhage control. These data will be captured by a trained study team at every site. Our findings will inform clinical practice guidelines and optimize the care delivered in the combat and civilian trauma setting. We are seeking 391 patients with complete data to meet our a priori inclusion criteria. Our study will have major immediate short-term findings including risk prediction modeling to assess who is at risk for hypocalcemia, data assessing interventions associated with the incidence of hypocalcemia, and outcome data including mortality and its link to early hypocalcemia.

Characteristics of COVID-19 and mortality-associated factors during the first year of the pandemic in southeast Mexico.

Aim: The present study was designed to evaluate the clinical characteristics of COVID-19 and mortality-associated factors during the first year of the pandemic in patients from southeastern Mexico. Patient & methods: A total of 953 records from patients with COVID-19 were cross-sectionally studied in a primary care hospital in southeast Mexico between 2020 and 2021. Results: The prevalent symptoms were fever (78.6%), cough (80.5%) and headache (82.8%) and dyspnea reached 13.5%. The mortality rate was 7.63% and the clinical variables associated with it were age >60 years, hypertension, severe disease, radiographic pneumonia, days to diagnosis and having two Mayan surnames. Conclusion: Future health strategies should consider age, comorbidities, disease severity, clinical manifestations and possessing ethnicity of risk (i.e., Mayan genetic background).

Stimulus-responsive controlled release system by covalent immobilization of an enzyme into mesoporous silica nanoparticles.

Mesoporous silica nanoparticles (MSN) have emerged as an attractive class of drug delivery carriers for therapeutic agents. Herein, we explored the covalent immobilization of proteins into MSN to generate a stimulus-responsive controlled release system. First, MSN were functionalized with thiol groups using (mercaptopropyl)-trimethoxysilane (MPTMS). Functionalization was verified by X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared (FTIR) spectroscopy, and dynamic light scattering. The model enzyme carbonic anhydrase (CA) was coupled to sulfosuccinimidyl 6-[3'(2-pyridyldithio)-propionamido]hexanoate (Sulfo-LC-SPDP) at a low ratio of 1:1 to prevent enzyme inactivation and subsequently covalently immobilized into MSN via thiol-disulfide interchange. The enzyme could be released from MSN with 10 mM glutathione, which represents intracellular redox conditions, while it remained bound to the MSN at extracellular redox conditions represented by 1 µM glutathione. The activity of the released enzyme was >80% demonstrating that the enzyme was still largely functional and active after immobilization and release. Human cervical cancer (HeLa) cells were incubated with the MSN-CA bioconjugates at various concentrations for 24 h and the data show good biocompatibility. In summary, we demonstrate the potential of MSN as drug delivery systems for proteins.

Simulation-Based Assessment of the Novel, Disposable i-view Video Laryngoscope by Emergency Medicine Physicians.

INTRODUCTION: Airway obstruction is the second leading cause of potentially preventable death on the battlefield. Previous studies demonstrate the most frequent airway intervention is intubation. Currently, the US Army has advanced video laryngoscopy (VL) in its sets, kits, and outfits (SKOs) at a cost of approximately $12,000 and generally only deploys them to the forward resuscitative surgical detachments and field hospitals. The i-view is a disposable video laryngoscope that costs approximately $120. The purpose of this study was to assess operator performance with this device and survey user opinions. METHODS: We conducted a prospective, observational study with emergency medicine residents and attending physicians using a synthetic cadaver model. Placement success, time-to-cannulation, number of attempts, and number and type of complications were recorded, followed by surveys. RESULTS: We enrolled 31 participants. One was missing data and was excluded, leaving 30 for analysis. The median age was 29, most (66%) were male, most were Air Force (57%), in-training residents (77%) with few reporting previous deployment experience (13%). Almost all had real patient experience with both direct (93%) and video laryngoscopy (90%). Most (90%) were able to get a grade 1 view with all achieving airway cannulation on first-pass attempt. The median time to cannulation was 11.6 seconds. On the post-procedure survey, most strongly agreed they would use this in the deployed setting (77%). Most reported they found it easy to use (77%). CONCLUSION: Our simulation-based study demonstrates the device has strong potential use for the clinical setting with all achieving rapid first-pass success for intubation. This study lays the foundation for validation of this device in the clinical setting.

A mixed methods end-user assessment to determine the ideal supraglottic airway device for inclusion into the medic's aid bag.

BACKGROUND: Many advancements in supraglottic airway technology have occurred since the start of the Global War on Terrorism. While the Tactical Combat Casualty Care guidelines previously recommend the i-gel device, this is based on little data and minimal end-user input. OBJECTIVE: We sought to use a mixed methods approach to investigate the properties of an ideal device for inclusion into the medic's aid bag. METHODS: We performed prospective, serial qualitative studies to uncover and articulate themes relative to airway device usability with 68W-combat medics. 68W are trained roughly to the level of a civilian advanced emergency medical technician with a heavier focus on trauma care. Physicians with airway expertise demonstrated the use of each device and provided formal training on all the presented devices. We then administered performed focus groups to solicit end-user feedback along with survey data. RESULTS: We enrolled 250 medics during the study. The preponderance of medics were of the rank E4 (28%) and E5 (44%). Only 35% reported ever placing a supraglottic airway in a real human. When reporting on usability, the i-gel had the highest median score, ease of manipulation, grip comfort and ease of insertion while also scoring the best in regard to requiring minimal training. The other compared devices had no clear highest score. Qualitative data saturated around a strong preference for the BaskaMask and/or the i-gel airway device, with the least favorite being the AirQ and the LMA Fastrach airway devises. There was a strong qualitative alignment in how both the BaskaMask and i-gel provided ease of use and simplicity of training. CONCLUSION: There were strong qualitative preferences for two specific airway devices: Baska Mask and i-gel. However, many medics commented on their previous experience with the i-gel compared with the other devices, which may have biased them toward the i-gel. The overall data suggest that medics would prefer a device engineered with features from several devices. LEVEL OF EVIDENCE: Therapeutic/care management; Level V.

A novel cdsAB operon is involved in the uptake of L-cysteine and participates in the pathogenesis of Yersinia ruckeri.

Application of in vivo expression technology (IVET) to Yersinia ruckeri, an important fish pathogen, allowed the identification of two adjacent genes that represent a novel bacterial system involved in the uptake and degradation of l-cysteine. Analysis of the translational products of both genes showed permease domains (open reading frame 1 [ORF1]) and amino acid position identities (ORF2) with the l-cysteine desulfidase from Methanocaldococcus jannaschii, a new type of enzyme involved in the breakdown of l-cysteine. The operon was named cdsAB (cysteine desulfidase) and is found widely in anaerobic and facultative bacteria. cdsAB promoter analysis using lacZY gene fusion showed highest induction in the presence of l-cysteine. Two cdsA and cdsB mutant strains were generated. The limited toxic effect and the low utilization of l-cysteine observed in the cdsA mutant, together with radiolabeled experiments, strongly suggested that CdsA is an l-cysteine permease. Fifty percent lethal dose (LD(50)) and competence index experiments showed that both the cdsA and cdsB loci were involved in the pathogenesis of the bacteria. In conclusion, this study has shown for the first time in bacteria the existence of an l-cysteine uptake system that together with an additional l-cysteine desulfidase-encoding gene constitutes a novel operon involved in bacterial virulence.

Comparative analysis and mutation effects of fpp2-fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum.

Flavobacterium psychrophilum is a very significant fish pathogen that secretes two biochemically characterized extracellular proteolytic enzymes, Fpp1 and Fpp2. The genes encoding these enzymes are organized as an fpp2-fpp1 tandem in the genome of strain F. psychrophilum THC02/90. Analysis of the corresponding encoded proteins showed that they belong to two different protease families. For gene function analysis, new genetic tools were developed in F. psychrophilum by constructing stable isogenic fpp1 and fpp2 mutants via single-crossover homologous recombination. RT-PCR analysis of wild-type and mutant strains suggested that both genes are transcribed as a single mRNA from the promoter located upstream of the fpp2 gene. Phenotypic characterization of the fpp2 mutant showed lack of caseinolytic activity and higher colony spreading compared with the wild-type strain. Both characteristics were recovered in the complemented strain. One objective of this work was to assess the contribution to virulence of these proteolytic enzymes. LD(50) experiments using the wild-type strain and mutants showed no significant differences in virulence in a rainbow trout challenge model, suggesting instead a possible nutritional role. The gene disruption procedure developed in this work, together with the knowledge of the complete genome sequence of F. psychrophilum, open new perspectives for the study of gene function in this bacterium.

Application of suppressive subtractive hybridization to the identification of genetic differences between two Lactococcus garvieae strains showing distinct differences in virulence for rainbow trout and mouse.

Lactococcus garvieae is the causative microbial agent of lactococcosis, an important and damaging fish disease in aquaculture. This bacterium has also been isolated from vegetables, milk, cheese, meat and sausages, from cow and buffalo as a mastitis agent, and even from humans, as an opportunistic infectious agent. In this work pathogenicity experiments were performed in rainbow trout and mouse models with strains isolated from human (L. garvieae HF) and rainbow trout (L. garvieae UNIUDO74; henceforth referred to as 074). The mean LD(50) value in rainbow trout obtained for strain 074 was 2.1 × 10(2) ± 84 per fish. High doses of the bacteria caused specific signs of disease as well as histological alterations in mice. In contrast, strain HF did not prove to be pathogenic either for rainbow trout or for mice. Based on these virulence differences, two suppressive subtractive hybridizations were carried out to identify unique genetic sequences present in L. garvieae HF (SSHI) and L. garvieae 074 (SSHII). Differential dot-blot screening of the subtracted libraries allowed the identification of 26 and 13 putative ORFs specific for L. garvieae HF and L. garvieae 074, respectively. Additionally, a PCR-based screening of 12 of the 26 HF-specific putative ORFs and the 13 074-specific ones was conducted to identify their presence/absence in 25 L. garvieae strains isolated from different origins and geographical areas. This study demonstrates the existence of genetic heterogeneity within L. garvieae isolates and provides a more complete picture of the genetic background of this bacterium.

The yctCBA operon of Yersinia ruckeri, involved in in vivo citrate uptake, is not required for virulence.

A three-gene operon, named yctCBA (Yersinia citrate transporter), induced by citrate and repressed by glucose was identified from a previously selected in vivo-induced (ivi) clone in the fish pathogen Yersinia ruckeri. Interestingly, despite being an ivi clone, the drastic growth reduction of the yctC mutant in the presence of citrate, and the relatively high content of this compound in rainbow trout serum, the operon was not required for virulence.

A Prospective, Feasibility Assessment of a Novel, Disposable Video Laryngoscope With Special Operations Medical Personnel in a Mobile Helicopter Simulation Setting.

BACKGROUND: Video laryngoscopy (VL) is shown to improve first-pass success rates and decrease complications in intubations, especially in novice proceduralists. However, the currently fielded VL devices are cost-prohibitive for dispersion across the battlespace. The novel i-view VL is a low-cost, disposable VL device that may serve as a potential solution. We sought to perform end-user performance testing and solicit feedback. METHODS: We prospectively enrolled Special Operations flight medics with the 160th Special Operations Aviation Regiment at Hunter Army Airfield, Savannah, Georgia. We asked them to perform an intubation using a synthetic cadaver model while in a mobile helicopter simulation setting. We surveyed their feedback afterward. RESULTS: The median age of participants was 30 and all were male. Of those, 60% reported previous combat deployments, with a median of 20 months of deployment time. Of the 10, 90% were successful with intubation, with 60% on first-pass success with an average of 83 seconds time to intubation. Most had a grade 1 view. Most agreed or strongly agreed that it was easy to use (70%), with half (50%) reporting they would use it in the deployed setting. Several made comments about the screen not being bright enough and would prefer one with a rotating display. CONCLUSIONS: We found a high proportion of success for intubation in the mobile simulator and a high satisfaction rate for this device by Special Operations Forces medics.