How Violent Attacks Are Changing The Demands of Mass Casualty Incidents: A Review of The Challenges Associated with Intentional Mass Casualty Incidents

The article record as published may be found at https://www.hsaj.org/articles/16880Antagonistically induced mass casualty incidents (MCI) introduce unique conditions that are rarely addressed in current MCI policies. Many of today’s MCIs are intentional acts of violence, such as mass shootings, improvised explosions, mass stabbings, vehicle rammings, and other similar assault tactics.Sponsored the U. S. Department of Homeland Security’s National Preparedness Directorate, FEMA, CHDS is part of the Naval Postgraduate School (NPS)

ESCHERICHIA COLI PLASMID DNA FERMENTATION: STRAIN AND PROCESS-SPECIFIC EFFECTS ON VECTOR YIELD, QUALITY, AND TRANSGENE EXPRESSION

To commercialize DNA medicines, industrial plasmid DNA manufacturing processes are needed which meet the quality, economy, and scale requirements projected for future products. NTC has developed an inducible fed-batch fermentation process that incorporates novel cell bank and fermentation process innovations that reduce plasmid mediated metabolic burden. This process also incorporates a scalable plasmid induction profile that, in combination with vector backbone modifications that double fermentation productivity compared to existing high copy vectors such as pVAX1 and gWIZ, form a generic plasmid DNA production platform driving high plasmid yields up to 2.6 g/L, with specific yields of 5% of total dry cell weight. We have investigated the effect of various epigenetic modifiers on plasmid performance in this process. For example, the dcm gene encodes a DNA methylase that methylates the internal cytosine residues in the recognition sequence 5‘-CC*AGG-3’ or 5‘-CC*TGG-3’. This creates 5-methyl-cytosine (5mC), a common mammalian pattern, although dcm methylated cytosine is in a different sequence context in bacteria compared to mammals (CG sequence). While plasmid production yields and quality are similar between dcm+ and dcm- host strains, CMV promoter expression is reduced by dcm methylation. Surprisingly, despite improved expression, dcm- plasmid DNA is less immunogenic. Our results demonstrate that it is critical to lock the plasmid methylation pattern (i.e. production strain) early in product development and suggest that dcm+ strains may be superior for vaccine applications

RNA BASED PLASMID SELECTION SYSTEM FOR ANTIBIOTIC-FREE PLASMID DNA VECTOR PRODUCTION

Antibiotic resistance markers, typically kanamycin resistance (kanR), allow selective retention of plasmid DNA during bacterial fermentation and are the most commonly utilized selectable markers. However, to ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. The presence of an antibiotic resistance gene in the plasmid backbone is considered undesirable by regulatory agencies, due to: 1) the potential transfer of antibiotic resistance to endogenous microbial flora; and 2) the potential activation and transcription of the genes from mammalian promoters after cellular incorporation into the genome. Here, we describe the development and application of a novel antibiotic-free (AF) selection system. Vectors with this selection system incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a counter-selectable marker (SacB) from the host chromosome. SacB encodes a levansucrase, which is toxic in the presence of sucrose. Sucrose selectable DNA vaccine vectors combine antibiotic-free selection with highly productive fermentation manufacturing (\u3e1 g/L plasmid DNA yields), while improving in vivo expression of encoded proteins. The RNA-OUT selectable marker can be used to retrofit existing kanR DNA vaccine plasmids into antibiotic-free vectors. Interestingly, a minimum vector size for high yield plasmid production was identified; strategies to ensure high yield production of small plasmids are reported. These vectors are safer, more potent alternatives for DNA therapy or vaccination

Side Reactions of Nitroxide-Mediated Polymerization: N−O versus O−C Cleavage of Alkoxyamines

Free energies for the homolysis of the NO−C and N−OC bonds were compared for a large number of alkoxyamines at 298 and 393 K, both in the gas phase and in toluene solution. On this basis, the scope of the N−OC homolysis side reaction in nitroxide-mediated polymerization was determined. It was found that the free energies of NO−C and N−OC homolysis are not correlated, with NO−C homolysis being more dependent upon the properties of the alkyl fragment and N−OC homolysis being more dependent upon the structure of the aminyl fragment. Acyclic alkoxyamines and those bearing the indoline functionality have lower free energies of N−OC homolysis than other cyclic alkoxyamines, with the five-membered pyrrolidine and isoindoline derivatives showing lower free energies than the six-membered piperidine derivatives. For most nitroxides, N−OC homolysis is normally favored above NO−C homolysis only when a heteroatom that is α to the NOC carbon center stabilizes the NO−C bond and/or the released alkyl radical is not sufficiently stabilized. As part of this work, accurate methods for the calculation of free energies for the homolysis of alkoxyamines were determined. Accurate thermodynamic parameters to within 4.5 kJ mol−1 of experimental values were found using an ONIOM approximation to G3(MP2)-RAD combined with PCM solvation energies at the B3-LYP/6-31G(d) level

Using smartwatches to observe changes in activity during recovery from critical illness following COVID-19:a 1 year multi-centre observational study

BACKGROUND: As a sequela of the COVID-19 pandemic, a large cohort of critical illness survivors have had to recover in the context of ongoing societal restrictions. OBJECTIVE: We aimed to use smartwatches (Fitbit Charge 3; Fitbit LLC) to assess changes in the step counts and heart rates of critical care survivors following hospital admission with COVID-19, use these devices within a remote multidisciplinary team (MDT) setting to support patient recovery, and report on our experiences with this. METHODS: We conducted a prospective, multicenter observational trial in 8 UK critical care units. A total of 50 participants with moderate or severe lung injury resulting from confirmed COVID-19 were recruited at discharge from critical care and given a smartwatch (Fitbit Charge 3) between April and June 2020. The data collected included step counts and daily resting heart rates. A subgroup of the overall cohort at one site—the MDT site (n=19)—had their smartwatch data used to inform a regular MDT meeting. A patient feedback questionnaire and direct feedback from the MDT were used to report our experience. Participants who did not upload smartwatch data were excluded from analysis. RESULTS: Of the 50 participants recruited, 35 (70%) used and uploaded data from their smartwatch during the 1-year period. At the MDT site, 74% (14/19) of smartwatch users uploaded smartwatch data, whereas 68% (21/31) of smartwatch users at the control sites uploaded smartwatch data. For the overall cohort, we recorded an increase in mean step count from 4359 (SD 3488) steps per day in the first month following discharge to 7914 (SD 4146) steps per day at 1 year (P=.003). The mean resting heart rate decreased from 79 (SD 7) beats per minute in the first month to 69 (SD 4) beats per minute at 1 year following discharge (P<.001). The MDT subgroup’s mean step count increased more than that of the control group (176% increase vs 42% increase, respectively; +5474 steps vs +2181 steps, respectively; P=.04) over 1 year. Further, 71% (10/14) of smartwatch users at the MDT site and 48% (10/21) of those at the control sites strongly agreed that their Fitbit motivated them to recover, and 86% (12/14) and 48% (10/21), respectively, strongly agreed that they aimed to increase their activity levels over time. CONCLUSIONS: This is the first study to use smartwatch data to report on the 1-year recovery of patients who survived a COVID-19 critical illness. This is also the first study to report on smartwatch use within a post–critical care MDT. Future work could explore the role of smartwatches as part of a randomized controlled trial to assess clinical and economic effectiveness. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.12968/ijtr.2020.010

KDM4B is a master regulator of the estrogen receptor signalling cascade

The importance of the estrogen receptor (ER) in breast cancer (BCa) development makes it a prominent target for therapy. Current treatments, however, have limited effectiveness, and hence the definition of new therapeutic targets is vital. The ER is a member of the nuclear hormone receptor superfamily of transcription factors that requires co-regulator proteins for complete regulation. Emerging evidence has implicated a small number of histone methyltransferase (HMT) and histone demethylase (HDM) enzymes as regulators of ER signalling, including the histone H3 lysine 9 tri-/di-methyl HDM enzyme KDM4B. Two recent independent reports have demonstrated that KDM4B is required for ER-mediated transcription and depletion of the enzyme attenuates BCa growth in vitro and in vivo. Here we show that KDM4B has an overarching regulatory role in the ER signalling cascade by controlling expression of the ER and FOXA1 genes, two critical components for maintenance of the estrogen-dependent phenotype. KDM4B interacts with the transcription factor GATA-3 in BCa cell lines and directly co-activates GATA-3 activity in reporter-based experiments. Moreover, we reveal that KDM4B recruitment and demethylation of repressive H3K9me3 marks within upstream regulatory regions of the ER gene permits binding of GATA-3 to drive receptor expression. Ultimately, our findings confirm the importance of KDM4B within the ER signalling cascade and as a potential therapeutic target for BCa treatment

Surviving severe COVID-19: Interviews with patients, informal carers and health professionals

Background:The COVID-19 pandemic has been associated with an unprecedentednumber of critical care survivors. Their experiences through illness and recovery arelikely to be complex, but little is known about how best to support them. This studyaimed to explore experiences of illness and recovery from the perspective of survi-vors, their relatives and professionals involved in their care.Study design:In-depth qualitative interviews were conducted with three stakeholdergroups during the first wave of the pandemic. A total of 23 participants (12 profes-sionals, 6 survivors and 5 relatives) were recruited from 5 acute hospitals in Englandand interviewed by telephone or video call. Data analysis followed the principles ofReflexive Thematic Analysis.Findings:Three themes were generated from their interview data: (1) Deterioratingfast—a downhill journey from symptom onset to critical care; (2) Facing a new virusin a hospital—a remote place; and (3) Returning home as a survivor, maintaining nor-mality and recovering slowly.Conclusions:Our findings highlight challenges in accessing care and communicationbetween patients, hospital staff and relatives. Following hospital discharge, patientsadopted a reframed‘survivor identity’to cope with their experience of illness andslow recovery process. The concept of survivorship in this patient group may be ben-eficial to promote and explore further.Relevance to clinical practice:All efforts should be made to continue to improvecommunication between patients, relatives and health professionals during criticalcare admissions, particularly while hospital visits are restricted. Adapting to life aftercritical illness may be more challenging while health services are restricted by theimpacts of the pandemic. It may be beneficial to promote the concept of survivorship,following admission to critical care due to severe COVID-19

Effects of chewing gum on nitric oxide metabolism, markers of cardiovascular health and neurocognitive performance after a nitrate-rich meal

Objectives: Cardiovascular and neurocognitive responses to chewing gum have been reported, but the mechanisms are not well understood. Chewing gum after a nitrate-rich meal may upregulate the reduction of oral nitrate to nitrite and increase nitric oxide (NO), a molecule important to cardiovascular and neurocognitive health. We aimed to explore effects of chewing gum after a nitrate-rich meal on nitrate metabolism (through the enterosalivary nitrate-nitrite-NO pathway), endothelial function, blood pressure (BP), neurocognitive performance, mood and anxiety. Methods: Twenty healthy men (n=6) and women (n=14) with a mean age of 48 years (range: 23-69) were recruited to a randomized controlled cross-over trial. After consumption of a nitrate-rich meal (180 mg of nitrate), we assessed the acute effects of chewing gum, compared to no gum chewing, on (i) salivary nitrate, nitrite and the nitrate reductase ratio (100 x [nitrite] / ([nitrate] + [nitrite]); (ii) plasma nitrite, S-nitrosothiols and other nitroso species (RXNO); (iii) endothelial function (measured by flow mediated dilatation); (iv) BP; (v) neurocognitive performance; (vi) mood; and (vii) anxiety. Results: Consumption of the nitrate-rich meal resulted in a significant increase in markers of nitrate metabolism. A significantly higher peak flow mediated dilatation was observed with chewing compared to no chewing (baseline adjusted mean difference: 1.10%, 95% CI: 0.06, 2.14; p=0.038) after the nitrate-rich meal. A significant small increase in systolic BP, diastolic BP and heart rate were observed with chewing compared to no chewing after the nitrate-rich meal. The study did not observe increased oral reduction of nitrate to nitrite and NO, or improvements in neurocognitive performance, mood or anxiety with chewing compared to no chewing. Conclusion: Chewing gum after a nitrate-rich meal resulted in an acute improvement in endothelial function and a small increase in BP but did not result in acute effects on neurocognitive function, mood or anxiety