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Supplementary Figure S1. Computed Tomographic (CT) findings in patient CGLU116. Supplementary Figure S2. CT findings in patient CGLU127. Supplementary Figure S3. CT findings in patient CGLU161. Supplementary Figure S4. Tumor burden kinetics. Supplementary Figure S5. Neoantigen-specific TCR expansion in stimulated T cell cultures for patient CGLU127. Supplementary Figure S6. Neoantigen-specific TCR expansion in stimulated T cell cultures for patient CGLU161. Supplementary Figure S7. Loss of heterozygosity analyses for patient CGLU116. Supplementary Figure S8. Loss of heterozygosity analyses for patient CGLU117. Supplementary Figure S9. Loss of heterozygosity analyses for patient CGLU127. Supplementary Figure S10. Loss of heterozygosity analyses for patient CGLU161. Supplementary Figure S11. TCR clonality analyses for patients CGLU127 and CGLU161. Supplementary Figure S12. TCR clonality analyses for a responder and a non-responder to PD-1 blockade. Supplementary Figure S13. PD-L1 expression in responsive and resistant tumors. Supplementary Figure S14. Eliminated mutations for case CGHN2. Supplementary Figure S15. Comparison of five methods for estimation of tumor purity. Supplementary Figure S16. CD8+ T cell density in resistant tumors.

Supplementary Table S1. Summary of Patient and Sample Characteristics. Supplementary Table S2. Summary of Next-Generation Sequencing Analyses. Supplementary Table S3. Somatic Sequence Alterations*. Supplementary Table S4. Somatic Copy Number Alterations. Supplementary Table S5. Neoantigen Predictions. Supplementary Table S6. Characteristics of a Subset of Eliminated Candidate Neoantigens in the NSCLC patients*. Supplementary Table S7. Summary of Functionally Validated Eliminated, Gained, and Retained cMANAs. Supplementary Table S8. Eliminated MANA-specific T Cell Clonotypes in CGLU116, CGLU127 and CGLU161. Supplementary Table S9. Retained and Gained MANA-specific T Cell Clonotypes in CGLU116. Supplementary Table S10. Allelic Imbalance Analysis and Cellularity Estimates for CGLU116. Supplementary Table S11. Allelic Imbalance Analysis and Cellularity Estimates for CGLU117. Supplementary Table S12. Allelic Imbalance Analysis and Cellularity Estimates for CGLU127. Supplementary Table S13. Allelic Imbalance Analysis and Cellularity Estimates for CGLU161. Supplementary Table S14. Summary of Eliminated Neoantigens in NSCLC Cases. Supplementary Table S15. TCR-beta Sequencing Analysis. Supplementary Table S16. PD-L1 and CD8 Immunohistochemistry. Supplementary Table S17. Regions of Allelic Imbalance. Supplementary Table S18. Tumor Purity and Ploidy Estimates.

Supplementary Figure Legends

BACKGROUND: Excessive neutrophil inflammation is the hallmark of cystic fibrosis (CF) airway disease. Novel technologies for characterizing neutrophil dysfunction may provide insight into the nature of these abnormalities, revealing a greater mechanistic understanding and new avenues for CF therapies that target these mechanisms. METHODS: Blood was collected from individuals with CF in the outpatient clinic, CF individuals hospitalized for a pulmonary exacerbation, and non-CF controls. Using microfluidic assays and advanced imaging technologies, we characterized 1) spontaneous neutrophil migration using microfluidic motility mazes, 2) neutrophil migration to and phagocytosis of Staphylococcal aureus particles in a microfluidic arena, 3) neutrophil swarming on Candida albicans clusters, and 4) Pseudomonas aeruginosa-induced neutrophil transepithelial migration using micro-optical coherence technology (µOCT). RESULTS: Participants included 44 individuals: 16 Outpatient CF, 13 Hospitalized CF, and 15 Non-CF individuals. While no differences were seen with spontaneous migration, CF neutrophils migrated towards S. aureus particles more quickly than non-CF neutrophils (p

PURPOSE OF REVIEW Psychiatric illness is common in patients presenting for surgery. Overall health and surgical outcomes are adversely affected by the presence of psychiatric comorbidities. RECENT FINDINGS As new treatment modalities become available, their perioperative implications need to be evaluated. These implications include drug-drug interactions, hemodynamic effects, bleeding risk, and factors affecting perioperative exacerbation of the underlying psychiatric illness. SUMMARY From our review of the recent literature we continue to support the continuation of psychoactive agents in the perioperative period, taking into consideration the effects these agents have on concomitant drug use in the perioperative period; and the risks of withholding them at a high-stress time.

Precooperative children and patients with intellectual disabilities often require intramuscular (IM) sedation prior to the induction of general anesthesia (GA). Ketamine is an effective preinduction sedative but can produce significant adverse side effects. Dexmedetomidine, a sedative with sympatholytic and analgesic properties, may provide advantages when used in combination with ketamine. This retrospective study evaluated the efficacy and safety of IM ketamine with dexmedetomidine for preoperative sedation. We conducted a chart review of all patients (n = 105) treated for dental rehabilitation who received either IM ketamine and dexmedetomidine (study group, n = 74) or IM ketamine and midazolam (control group, n = 31) prior to induction of GA. No significant difference (p = .14) was observed in the time interval from IM administration to operating room entry (median [interquartile range]) between the study and control groups (5 [4–8] vs 5 [2–7] minutes). Patients who received IM dexmedetomidine exhibited significantly lower mean arterial pressures throughout the induction (p = .004) and had lower heart rates (p = .01) throughout the intraoperative period compared with patients who did not receive dexmedetomidine. The combination of dexmedetomidine and ketamine may provide effective and safe IM sedation prior to the induction of GA.

Introduction Multimorbidity is found in around half of people with any long-term condition but is substantially less common in randomised controlled trials (‘trials’). Multimorbidity may diminish a participant’s ability to complete a trial. However, empirical estimates of the association between individual patient characteristics and early termination are lacking. Method Individual patient-level data were obtained from Phase 3/4 trials contained within two clinical trial repositories. Eligible trials for inclusion were identified according to pre-specified criteria (PROSPERO CRD42018048202). Within each trial, the association between morbidity count and early termination (failure for any reason to complete the final trial visit) was estimated in logistic regression models, adjusting for age and sex. These estimates were meta-analysed in Bayesian linear models, with partial pooling across index conditions and drug classes. Using these estimates, the impact of morbidity count on early termination was modelled for a set of notional trials. Results In 92 trials across 20 index conditions and 17 drug classes, the mean morbidity count ranged from 0.3–2.7. Neither age nor sex was associated with early termination (OR 1.04, 95% CI 0.98–1.11; OR 1.00, 95% PI 0.95–1.07 respectively). Morbidity count was associated with early termination (OR per additional morbidity: 1.11, 95% CI: 1.07 to 1.14). There was no evidence of non-linearity in the association between morbidity count and early termination, and there was minimal variation across drug classes and index conditions. For a notional trial with high level of early termination in individuals without multimorbidity, doubling the mean morbidity count from 1 to 2 increases risk of early termination from 29% to 31%. Conclusion Multimorbidity, irrespective of age and sex, is associated with a relatively modest increased odds of early termination of trial participation. The benefit of increased generalisability of trials by including patients with multimorbidity appears likely to outweigh the disadvantages of lower retention.

Introduction The applicability of randomised controlled trials of pharmacological agents to older people with frailty/multimorbidity is often uncertain, due to concerns that trials are not representative. However, assessing trial representativeness is challenging and complex. We explore an approach assessing trial representativeness by comparing rates of trial Serious Adverse Events (SAEs: most of which reflect hospitalisations/deaths) to rates of hospitalisation/death in routine care (which, in a trial setting, would be SAEs be definition). Method We identified 483 trials (n = 636,267) from clinicaltrials.gov across 21 index conditions. A routine care comparison was identified from SAIL databank (n = 2.3 M). SAIL data were used to derive the expected rate of hospitalisations/deaths by age, sex and index condition. We then calculated the expected number of SAEs for each trial compared to the observed number of SAEs (observed/expected SAE ratio). We then re-calculated the observed/expected SAE ratio additionally accounting for comorbidity count in 125 trials for which we accessed individual participant data. Results For 12/21 index conditions the observed/expected SAE ratio was Conclusion Trial participants experience fewer SAEs than expected based on age/sex/condition specific hospitalisation/death rates in routine care, confirming the predicted lack of representativeness. This difference is only partially explained by differences in multimorbidity. Assessing observed/expected SAE may help assess applicability of trial findings to older populations in whom multimorbidity and frailty are common.

Governments extol the virtues of “seamless” education systems, and prevalent in political discourse is the notion of pathways. Discussion involving pathways must inevitably consider their context—educational systems and labor markets. The first part draws on the international literature to examine the key themes of educational pathways, skills formation systems and labor markets. Most studies come from European perspectives, are school-based and concerned with young people's entrances into labor markets. The second part therefore focuses on Australian VET, as this system is largely post-secondary and focused more on whole of life and careers.

Objective: The induction of general anesthesia for children and patients with special needs frequently requires preinduction sedation, especially when anxiety and agitation lead to violent or combative behavior. In these situations, preoperative intramuscular (IM) sedation may facilitate patient transfer, intravenous cannulation, and/or mask induction. This survey aimed to capture data regarding the current preoperative IM sedation practices of dentist anesthesiologists. Methods: An electronic survey was distributed in 2020 to all members of the American Society of Dentist Anesthesiologists regarding the administration of preoperative IM sedation. It included questions about the demographics of respondents and their patients who require IM sedation, the most common drug regimens used, decision-making criteria regarding ketamine dosing, the intended level of sedation, sequence of anesthetic management following IM sedation, and observed outcomes. Results: A total of 193 responses (43%) were received; of those, 162 reported using preoperative IM sedation. Ketamine was included in 98.7% of reported IM drug regimens. The most common IM sedation regimen was combined ketamine and midazolam (median 2.5 mg/kg and 0.1 mg/kg, respectively). Of the respondents who use preoperative IM sedation, 87% reported using the same drug regimen in at least 80% of cases. Conclusion: The most frequently reported drug regimen used by dentist anesthesiologists in North America for preoperative IM sedation was a combination of ketamine and midazolam.

The recent Translations article by Bishop et al.1 draws much-needed attention to social communication (SC) in autism spectrum disorder (ASD) and to the need in autism research for treatment-sensitive measures of this key domain. In this context, the authors define SC ability as "the appropriate use and modulation of verbal and nonverbal behaviors during interactions with others"1(p. 555). "Appropriate" is defined relative to normative behaviors for developmental age and language level based on parent report. This stirred us to share our concern that clinicians, too, need ways to assess SC. Historically, observation of a patient's SC has not been part of the routine psychiatric mental status examination (MSE); clinicians lack even a common basic vocabulary for describing this vital domain. The DSM-52 does not explicitly define SC or distinguish it from social interaction (SI) or language, important terms also used in the criteria for ASD. All three terms are used interchangeably and inconsistently across the literature. Here we offer a definition of SC, distinguish it from SI and language, and propose a schema, or conceptual model, for observing and documenting an impression of a patient's SC.

Although systemic progesterone (PROG) treatment has been shown to be neuroprotective by many laboratories and in multiple animal models of brain injury including traumatic brain injury (TBI), PROG's poor aqueous solubility limits its potential for use as a therapeutic agent. The problem of solubility presents challenges for an acute intervention for neural injury, when getting a neuroprotectant to the brain quickly is crucial. Native PROG (nPROG) is hydrophobic and does not readily dissolve in an aqueous-based medium, so this makes it harder to give under emergency field conditions. An agent with properties similar to those of PROG but easier to store, transport, formulate, and administer early in emergency trauma situations could lead to better and more consistent clinical outcomes following TBI. At the same time, the engineering of a new molecule designed to treat a complex systemic injury must anticipate a range of translational issues including solubility and bioavailability. Here we describe the development of EIDD-1723, a novel, highly stable PROG analog with >104-fold higher aqueous solubility than that of nPROG. We think that, with further testing, EIDD-1723 could become an attractive candidate use as a field-ready treatment for TBI patients. This article is part of the Special Issue entitled "Novel Treatments for Traumatic Brain Injury".

Psychiatric illness is common in patients presenting for surgery. Overall health and surgical outcomes are adversely affected by the presence of psychiatric comorbidities.As new treatment modalities become available, their perioperative implications need to be evaluated. These implications include drug-drug interactions, hemodynamic effects, bleeding risk, and factors affecting perioperative exacerbation of the underlying psychiatric illness.From our review of the recent literature we continue to support the continuation of psychoactive agents in the perioperative period, taking into consideration the effects these agents have on concomitant drug use in the perioperative period; and the risks of withholding them at a high-stress time.

ATI-2173 is a novel liver-targeted molecule designed to deliver the 5′-monophosphate of clevudine for the treatment of chronic hepatitis B infection. Unlike other nucleos(t)ides, the active clevudine-5′-triphosphate is a noncompetitive, non-chain-terminating inhibitor of hepatitis B virus (HBV) polymerase that delivers prolonged reduction of viremia in both a woodchuck HBV model and in humans for up to 6 months after cessation of treatment. However, long-term clevudine treatment was found to exhibit reversible skeletal myopathy in a small subset of patients and was subsequently discontinued from development., ATI-2173 is a novel liver-targeted molecule designed to deliver the 5′-monophosphate of clevudine for the treatment of chronic hepatitis B infection. Unlike other nucleos(t)ides, the active clevudine-5′-triphosphate is a noncompetitive, non-chain-terminating inhibitor of hepatitis B virus (HBV) polymerase that delivers prolonged reduction of viremia in both a woodchuck HBV model and in humans for up to 6 months after cessation of treatment. However, long-term clevudine treatment was found to exhibit reversible skeletal myopathy in a small subset of patients and was subsequently discontinued from development. ATI-2173 was designed by modifying clevudine with a 5′-phosphoramidate to deliver the 5′-monophosphate to the liver. Bypassing the first phosphorylation step of clevudine, the 5′-monophosphate is converted to the active 5′-triphosphate in the liver. ATI-2173 is a selective inhibitor of HBV with an anti-HBV 50% effective concentration (EC50) of 1.31 nM in primary human hepatocytes, with minimal to no toxicity in hepatocytes, skeletal muscle, liver, kidney, bone marrow, and cardiomyocytes. ATI-2173 activity was decreased by viral polymerase mutations associated with entecavir, lamivudine, and adefovir resistance, but not capsid inhibitor resistance mutations. A single oral dose of ATI-2173 demonstrated 82% hepatic extraction, no food effect, and greatly reduced peripheral exposure of clevudine compared with equimolar oral dosing of clevudine. Despite reduced plasma clevudine exposure, liver concentrations of the 5′-triphosphate were equivalent following ATI-2173 versus clevudine administration. By selectively delivering the 5′-monophosphate to the liver, while retaining the unique anti-HBV activity of the 5′-triphosphate, ATI-2173 may provide an improved pharmacokinetic profile for clinical use, reducing systemic exposure of clevudine and potentially eliminating skeletal myopathy.

Tracheopulmonary complications following placement of a nasogastric (NG) feeding tube are uncommon but can cause significant morbidity and mortality. In this case report, an 83-year-old woman of American Society of Anesthesiologists class IV with underlying pulmonary disease required placement of an NG feeding tube after surgical treatment of primary squamous cell carcinoma of the tongue. Malpositioning of the NG feeding tube into the right pleural space was confirmed by computed tomography. Removal of the NG feeding tube resulted in a tension pneumothorax that necessitated chest tube placement. Because of the difficulty of blind NG feeding tube placement in this patient, the subsequently placed NG feeding tube was successfully positioned with the aid of a video laryngoscope. This case report illustrates the risk of NG feeding tube malpositioning in a nasally intubated patient undergoing head and neck surgery and discusses improvements in techniques for proper NG feeding tube placement.

N 4-Hydroxycytidine (NHC) is an antiviral ribonucleoside analog that acts as a competitive alternative substrate for virally encoded RNA-dependent RNA polymerases. It exhibits measurable levels of cytotoxicity, with 50% cytotoxic concentration values ranging from 7.5 μM in CEM cells and up to >100 μM in other cell lines. The mitochondrial DNA-dependent RNA polymerase (POLRMT) has been shown to incorporate some nucleotide analogs into mitochondrial RNAs, resulting in substantial mitochondrial toxicity. NHC was tested in multiple assays intended to determine its potential to cause mitochondrial toxicity. NHC showed similar cytotoxicity in HepG2 cells incubated in a glucose-free and glucose-containing media, suggesting that NHC does not impair mitochondrial function in this cell line based on the Crabtree effect. We demonstrate that the 5'-triphosphate of NHC can be used by POLRMT for incorporation into nascent RNA chain but does not cause immediate chain termination. In PC-3 cells treated with NHC, the 50% inhibitory concentrations of mitochondrial protein expression inhibition were 2.7-fold lower than those for nuclear-encoded protein expression, but this effect did not result in selective mitochondrial toxicity. A 14-day incubation of HepG2 cells with NHC had no effect on mitochondrial DNA copy number or extracellular lactate levels. In CEM cells treated with NHC at 10 μM, a slight decrease (by ∼20%) in mitochondrial DNA copy number and a corresponding slight increase in extracellular lactate levels were detected, but these effects were not enhanced by an increase in NHC treatment concentration. In summary, the results indicate that mitochondrial impairment by NHC is not the main contributor to the compound's observed cytotoxicity in these cell lines.

N4-Hydroxycytidine (NHC) is an antiviral ribonucleoside analog that acts as a competitive alternative substrate for virally encoded RNA-dependent RNA polymerases. It exhibits measurable levels of cytotoxicity, with 50% cytotoxic concentration values ranging from 7.5 μM in CEM cells and up to >100 μM in other cell lines., N4-Hydroxycytidine (NHC) is an antiviral ribonucleoside analog that acts as a competitive alternative substrate for virally encoded RNA-dependent RNA polymerases. It exhibits measurable levels of cytotoxicity, with 50% cytotoxic concentration values ranging from 7.5 μM in CEM cells and up to >100 μM in other cell lines. The mitochondrial DNA-dependent RNA polymerase (POLRMT) has been shown to incorporate some nucleotide analogs into mitochondrial RNAs, resulting in substantial mitochondrial toxicity. NHC was tested in multiple assays intended to determine its potential to cause mitochondrial toxicity. NHC showed similar cytotoxicity in HepG2 cells incubated in a glucose-free and glucose-containing media, suggesting that NHC does not impair mitochondrial function in this cell line based on the Crabtree effect. We demonstrate that the 5′-triphosphate of NHC can be used by POLRMT for incorporation into nascent RNA chain but does not cause immediate chain termination. In PC-3 cells treated with NHC, the 50% inhibitory concentrations of mitochondrial protein expression inhibition were 2.7-fold lower than those for nuclear-encoded protein expression, but this effect did not result in selective mitochondrial toxicity. A 14-day incubation of HepG2 cells with NHC had no effect on mitochondrial DNA copy number or extracellular lactate levels. In CEM cells treated with NHC at 10 μM, a slight decrease (by ∼20%) in mitochondrial DNA copy number and a corresponding slight increase in extracellular lactate levels were detected, but these effects were not enhanced by an increase in NHC treatment concentration. In summary, the results indicate that mitochondrial impairment by NHC is not the main contributor to the compound’s observed cytotoxicity in these cell lines.

Background: High blood pressure is common in acute stroke and is a predictor of poor outcome; however, large trials of lowering blood pressure have given variable results, and the management of high blood pressure in ultra-acute stroke remains unclear. We investigated whether transdermal glyceryl trinitrate (GTN; also known as nitroglycerin), a nitric oxide donor, might improve outcome when administered very early after stroke onset.Methods: We did a multicentre, paramedic-delivered, ambulance-based, prospective, randomised, sham-controlled, blinded-endpoint, phase 3 trial in adults with presumed stroke within 4 h of onset, face-arm-speech-time score of 2 or 3, and systolic blood pressure 120 mm Hg or higher. Participants were randomly assigned (1:1) to receive transdermal GTN (5 mg once daily for 4 days; the GTN group) or a similar sham dressing (the sham group) in UK-based ambulances by paramedics, with treatment continued in hospital. Paramedics were unmasked to treatment, whereas participants were masked. The primary outcome was the 7-level modified Rankin Scale (mRS; a measure of functional outcome) at 90 days, assessed by central telephone follow-up with masking to treatment. Analysis was hierarchical, first in participants with a confirmed stroke or transient ischaemic attack (cohort 1), and then in all participants who were randomly assigned (intention to treat, cohort 2) according to the statistical analysis plan. This trial is registered with ISRCTN, number ISRCTN26986053.Findings: Between Oct 22, 2015, and May 23, 2018, 516 paramedics from eight UK ambulance services recruited 1149 participants (n=568 in the GTN group, n=581 in the sham group). The median time to randomisation was 71 min (IQR 45–116). 597 (52%) patients had ischaemic stroke, 145 (13%) had intracerebral haemorrhage, 109 (9%) had transient ischaemic attack, and 297 (26%) had a non-stroke mimic at the final diagnosis of the index event. In the GTN group, participants' systolic blood pressure was lowered by 5·8 mm Hg compared with the sham group (pvs 3 [2–5]; n=558, in the sham group; 1·04 [0·84–1·29]; p=0·69). We found no difference in secondary outcomes, death (treatment-related deaths: 36 in the GTN group vs 23 in the sham group [p=0·091]), or serious adverse events (188 in the GTN group vs 170 in the sham group [p=0·16]) between treatment groups.Interpretation: Prehospital treatment with transdermal GTN does not seem to improve functional outcome in patients with presumed stroke. It is feasible for UK paramedics to obtain consent and treat patients with stroke in the ultra-acute prehospital setting.Funding: British Heart Foundation.

Venezuelan equine encephalitis virus (VEEV) is a representative member of the New World alphaviruses. It is transmitted by mosquito vectors and causes highly debilitating disease in humans, equids, and other vertebrate hosts. Despite a continuous public health threat, very few compounds with anti-VEEV activity in cell culture and in mouse models have been identified to date, and rapid development of virus resistance to some of them has been recorded. In this study, we investigated the possibility of using a modified nucleoside analog, β- d - N 4 -hydroxycytidine (NHC), as an anti-VEEV agent and defined the mechanism of its anti-VEEV activity. The results demonstrate that NHC is a very potent antiviral agent. It affects both the release of genome RNA-containing VEE virions and their infectivity. Both of these antiviral activities are determined by the NHC-induced accumulation of mutations in virus-specific RNAs. The antiviral effect is most prominent when NHC is applied early in the infectious process, during the amplification of negative- and positive-strand RNAs in infected cells. Most importantly, only a low-level resistance of VEEV to NHC can be developed, and it requires acquisition and cooperative function of more than one mutation in nsP4. These adaptive mutations are closely located in the same segment of nsP4. Our data suggest that NHC is more potent than ribavirin as an anti-VEEV agent and likely can be used to treat other alphavirus infections. IMPORTANCE Venezuelan equine encephalitis virus (VEEV) can cause widespread epidemics among humans and domestic animals. VEEV infections result in severe meningoencephalitis and long-term sequelae. No approved therapeutics exist for treatment of VEEV infections. Our study demonstrates that β- d - N 4 -hydroxycytidine (NHC) is a very potent anti-VEEV compound, with the 50% effective concentration being below 1 μM. The mechanism of NHC antiviral activity is based on induction of high mutation rates in the viral genome. Accordingly, NHC treatment affects both the rates of particle release and the particle infectivity. Most importantly, in contrast to most of the anti-alphavirus drugs that are under development, resistance of VEEV to NHC develops very inefficiently. Even low levels of resistance require acquisition of multiple mutations in the gene of the VEEV-specific RNA-dependent RNA polymerase nsP4.

Venezuelan equine encephalitis virus (VEEV) is a representative member of the New World alphaviruses. It is transmitted by mosquito vectors and causes highly debilitating disease in humans, equids, and other vertebrate hosts. Despite a continuous public health threat, very few compounds with anti-VEEV activity in cell culture and in mouse models have been identified to date, and rapid development of virus resistance to some of them has been recorded. In this study, we investigated the possibility of using a modified nucleoside analog, β-d-N4-hydroxycytidine (NHC), as an anti-VEEV agent and defined the mechanism of its anti-VEEV activity. The results demonstrate that NHC is a very potent antiviral agent. It affects both the release of genome RNA-containing VEE virions and their infectivity. Both of these antiviral activities are determined by the NHC-induced accumulation of mutations in virus-specific RNAs. The antiviral effect is most prominent when NHC is applied early in the infectious process, during the amplification of negative- and positive-strand RNAs in infected cells. Most importantly, only a low-level resistance of VEEV to NHC can be developed, and it requires acquisition and cooperative function of more than one mutation in nsP4. These adaptive mutations are closely located in the same segment of nsP4. Our data suggest that NHC is more potent than ribavirin as an anti-VEEV agent and likely can be used to treat other alphavirus infections.

The New World alphaviruses Venezuelan, Eastern, and Western equine encephalitis viruses (VEEV, EEEV and WEEV, respectively) commonly cause a febrile disease that can progress to meningoencephalitis, resulting in significant morbidity and mortality. To address the need for a therapeutic agent for the treatment of Alphavirus infections, we identified and pursued preclinical characterization of a ribonucleoside analog EIDD-1931 (β-D-N4-hydroxycytidine, NHC), which has shown broad activity against alphaviruses in vitro and has a very high genetic barrier for development of resistance. To be truly effective as a therapeutic agent for VEEV infection a drug must penetrate the blood brain barrier and arrest virus replication in the brain. High plasma levels of EIDD-1931 are rapidly achieved in mice after oral dosing. Once in the plasma EIDD-1931 is efficiently distributed into organs, including brain, where it is rapidly converted to its active 5′-triphosphate. EIDD-1931 showed a good safety profile in mice after 7-day repeated dosing with up to 1000 mg/kg/day doses. In mouse model studies, EIDD-1931 was 90–100% effective in protecting mice against lethal intranasal infection when therapeutic treatment was started as late as 24 h post-infection, and partial protection was achieved when treatment was delayed for 48 h post-infection. These results support further preclinical development of EIDD-1931 as a potential anti-alphavirus drug.

Background: Differences between mothers who do and do not succeed in breastfeeding are likely to confound associations of lactation with later maternal adiposity.Objective: We compared adiposity and blood pressure (BP) in women randomly assigned to an intervention to promote prolonged and exclusive breastfeeding or usual care.Design: We performed a cluster-randomized trial at 31 hospitals in Belarus in 1996–1997.Results: Of 17,046 women enrolled at delivery, we assessed 11,867 women (69.6%) at 11.5 y postpartum. The prevalence of exclusive breastfeeding ≥3 mo was 44.5% in 6321 women in the intervention group and 7.1% in 5546 women in the control group. At 11.5 y postpartum, mean (±SD) body mass index (BMI; in kg/m2) was 26.5 ± 5.5, the percentage of body fat was 33.6% ± 8.3%, and systolic BP was 124.6 ± 14.6 mm Hg. On intention-to-treat analysis (without imputation) with adjustment for clustering by hospital, mean outcomes were lower in intervention compared with control mothers for BMI (mean difference: −0.27; 95% CI: −0.91, 0.37), body fat (−0.49%; 95% CI: −1.25%, 0.27%), and systolic BP (−0.81 mm Hg; 95% CI: −3.33, 1.71 mm Hg), but effect sizes were small, CIs were wide, and results were attenuated further toward the null after adjustment for baseline characteristics. Results were similar in sensitivity analyses [ie, by using conventional observational analyses disregarding treatment assignment, instrumental variable analyses to estimate the causal effect of breastfeeding, and multiple imputation to account for missing outcome measures (n = 17,046)].Conclusion: In women who initiated breastfeeding, an intervention to promote longer breastfeeding duration did not result in an important lowering of adiposity or BP. This trial was registered at clinicaltrials.gov as NCT01561612 and at Current Controlled Trials as ISRCTN37687716.

ObjectiveFish consumption influences a number of health outcomes. Few studies have directly compared dietary assessment methods to determine the best approach to estimating intakes of fish and its component nutrients, including DHA, and toxicants, including methylmercury. Our objective was to compare three methods of assessing fish intake.DesignWe assessed 30 d fish intake using three approaches: (i) a single question on total fish consumption; (ii) a brief comprehensive FFQ that included four questions about fish; and (iii) a focused FFQ with thirty-six questions about different finfish and shellfish.SettingObstetrics practices in Boston, MA, USA.SubjectsFifty-nine pregnant women who consumed ≤2 monthly fish servings.ResultsEstimated intakes of fish, DHA and Hg were lowest with the one-question screener and highest with the thirty-six-item fish questionnaire. Estimated intake of DHA with the thirty-six-item questionnaire was 4·4-fold higher (97 v. 22 mg/d), and intake of Hg was 3·8-fold higher (1·6 v. 0·42 μg/d), compared with the one-question screener. Plasma DHA concentration was correlated with fish intake assessed with the one-question screener (Spearman r = 0·27, P = 0·04), but not with the four-item FFQ (r = 0·08, P = 0·54) or the thirty-six-item fish questionnaire (r = 0·01, P = 0·93). In contrast, blood and hair Hg concentrations were similarly correlated with fish and Hg intakes regardless of the assessment method (r = 0·35 to 0·52).ConclusionsA longer questionnaire provides no advantage over shorter questionnaires in ranking intakes of fish, DHA and Hg compared with biomarkers, but estimates of absolute intakes can vary by as much as fourfold across methods.

Background Gestational weight gain (GWG) is an important predictor of short and long-term pregnancy outcomes for both mother and child, and women who set a GWG goal are more likely to gain within recommended ranges. Little information is available regarding potentially modifiable factors that underlie a woman’s GWG goals. Our aims were to explore women’s perceptions regarding factors that affect GWG, their understanding of appropriate GWG, their goal-setting experiences including patient-health care provider (HCP) conversations, and supportive interventions they would most like to help them achieve the recommended GWG. Methods We conducted nine in-depth interviews and seven focus groups with a total of 33 Boston, Massachusetts (MA) area women who were pregnant and had delivered within the prior 6 months. We recorded and transcribed all interviews. Two investigators independently coded resulting transcripts. We managed data using MAXQDA2 and conducted a content analysis. Results Perceived factors that contributed to GWG goal-setting included the mother’s weight control behaviors concerning exercise and diet—including a “new way of eating for two” and “semblance of control”, experiences during prior pregnancies, conversations with HCPs, and influence from various information sources. Women focused on behaviors with consistent messaging across multiple sources of information, but mainly trusted their HCP, valued one-to-one conversations with them about GWG, preferred that the HCP initiate the conversation about GWG goals, and would be open to have the conversation started based on visual aid based on their own GWG progression. Conclusions Pregnant women highly value discussions with their HCP to set GWG goals. Pregnant women view their clinicians as the most reliable source of information and believe that clinicians should open weight-related discussions throughout pregnancy.

The poor aqueous solubility of progesterone (PROG) limits its potential use as a therapeutic agent. We designed and tested EIDD-1723, a novel water-soluble analog of PROG with >100-fold higher solubility than that of native PROG, as candidate for development as a field-ready treatment for traumatic brain injury (TBI). The pharmacokinetic effects of EIDD-1723 on morphological and functional outcomes in rats with bilateral cortical impact injury were evaluated. Following TBI, 10-mg/kg doses of EIDD-1723 or PROG were given intramuscularly (i.m.) at 1, 6 and 24 h post-injury, then daily for the next 6 days, with tapering of the last 2 treatments. Rats were tested pre-injury to establish baseline performance on grip strength and sensory neglect, and then retested at 4, 9 and 21 days post-TBI. Spatial learning was evaluated from days 11–17 post-TBI. At 22 days post-injury, rats were perfused and brains extracted and processed for lesion size. For the edema assay the animals were killed and brains removed at 24 h post-injury. EIDD-1723 significantly reduced cerebral edema and improved recovery from motor, sensory and spatial learning deficits as well as, or better than, native PROG. Pharmacokinetic investigation after a single i.m. injection in rats revealed that EIDD-1723 was rapidly converted to the active metabolite EIDD-036, demonstrating first-order elimination kinetics and ability to cross the blood-brain barrier. Our results suggest that EIDD-1723 represents a substantial advantage over current PROG formulations because it overcomes storage, formulation and delivery limitations of PROG and can thereby reduce the time between injury and treatment.

Excessive gestational weight gain (GWG) predicts adverse pregnancy outcomes and later obesity risk for both mother and child. Women who receive GWG advice from their obstetric clinicians are more likely to gain the recommended amount, but many clinicians do not counsel their patients on GWG, pointing to the need for new strategies. Electronic medical records (EMRs) are a useful tool for tracking weight and supporting guideline-concordant care, but their use for care related to GWG has not been evaluated. We performed in-depth interviews with 16 obstetric clinicians from a multi-site group practice in Massachusetts that uses an EMR. We recorded, transcribed, coded, and analyzed the interviews using immersion-crystallization. Many respondents believed that GWG had "a lot" of influence on pregnancy and child health outcomes but that their patients did not consider it important. Most indicated that excessive GWG was a big or moderate problem in their practice, and that inadequate GWG was rarely a problem. All used an EMR feature that calculates total GWG at each visit. Many were enthusiastic about additional EMR-based supports, such as a reference for recommended GWG for each patient based on pre-pregnancy body mass index, a "growth chart" to plot actual and recommended GWG, and an alert to identify out-of-range gains, features which many felt would remind them to counsel patients about excessive weight gain. Additional decision support tools within EMRs would be well received by many clinicians and may help improve the frequency and accuracy of GWG tracking and counseling.

AIMS/HYPOTHESIS: Current drug labels for thiazolidinediones (TZDs) warn of increased fractures, predominantly for distal fractures in women. We examined whether exposure to TZDs affects hip fracture in women and men and compared the risk to that found with other drugs used in diabetes. METHODS: Using a nationwide database of prescriptions, hospital admissions and deaths in those with type 2 diabetes in Scotland we calculated TZD exposure among 206,672 individuals. Discrete-time failure analysis was used to model the effect of cumulative drug exposure on hip fracture during 1999-2008. RESULTS: There were 176 hip fractures among 37,479 exposed individuals. Hip fracture risk increased with cumulative exposure to TZD: OR per year of exposure 1.18 (95% CI 1.09, 1.28; p = 3 × 10(-5)), adjusted for age, sex and calendar month. Hip fracture increased with cumulative exposure in both men (OR 1.20; 95% CI 1.03, 1.41) and women (OR 1.18; 95% CI 1.07, 1.29) and risks were similar for pioglitazone (OR 1.18) and rosiglitazone (OR 1.16). The association was similar when adjusted for exposure to other drugs for diabetes and for other potential confounders. There was no association of hip fracture with cumulative exposure to sulfonylureas, metformin or insulin in this analysis. The 90-day mortality associated with hip fractures was similar in ever-users of TZD (15%) and in never-users (13%). CONCLUSIONS/INTERPRETATION: Hip fracture is a severe adverse effect with TZDs, affecting both sexes; labels should be changed to warn of this. The excess mortality is at least as much as expected from the reported association of pioglitazone with bladder cancer.

After more than 30 years of research and 30 failed clinical trials with as many different treatments, progesterone is the first agent to demonstrate robust clinical efficacy as a treatment for traumatic brain injuries. It is currently being investigated in two, independent phase III clinical trials in hospital settings; however, it presents a formidable solubility challenge that has so far prevented the identification of a formulation that would be suitable for emergency field response use or battlefield situations. Accordingly, we have designed and tested a novel series of water-soluble analogues that address this critical need. We report here the synthesis of C-20 oxime conjugates of progesterone as therapeutic agents for traumatic brain injuries with comparable efficacy in animal models of traumatic brain injury and improved solubility and pharmacokinetic profiles. Pharmacodynamic analysis reveals that a nonprogesterone steroidal analogue may be primarily responsible for the observed activity.