Overcoming resolution attenuation during tilted cryo-EM data collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

Overcoming Resolution Attenuation During Tilted Cryo-EM Data Collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

Histo-morphologic and gravimetric changes of teeth exposed to high temperatures - An in-vitro study.

BACKGROUND: Fire intelligence is the multidisciplinary basis of reconnaissance, which includes determining the origin, cause, and identification of fire victims. Fire is a destructive force capable of inflicting significant damage. Destruction of soft tissue in fire disasters makes victim identification nearly impossible. Teeth are hard and resilient and withstand such conditions. Analyzing the precise morphological, stereomicroscopic, histological, and gravimetric findings can extract valuable information from dental evidence in forensic investigations. MATERIALS AND METHODS: Thirty-six mandibular premolar teeth extracted for therapeutic purposes were exposed to high-temperature gradients. Macroscopic, stereomicroscopic, histological, and dry weight analyses were performed at each temperature gradient. RESULTS: The colour of teeth changed from yellowish orange to metallic black bronze to chalky white. Stereomicroscopy showed intact teeth at 100°C, gradual micro-cracks at 500°C, and a fully fractured crown at 900°C. Decalcified sections revealed dilatation of dentinal tubular pattern at 300°C. Dentinal tubules showed appearance of vapour bubbles at 400°C, resulting in loss of typical architecture. In the ground sections, alterations in scalloping nature of dentino-enamel junction, coalescing radicular dentinal tubules, and sand cracking appearance of teeth were noted at 100°C, 300°C, and 900°C, respectively. Significant reductions in the weight of the teeth samples were observed with higher temperatures. CONCLUSION: From the morphological, histological, and gravimetric changes in a tooth caused by fire, it might be possible to determine the temperature and duration of fire exposure, and the cause of the fire.

An unusual case of a penetrating neck injury (PNI) illustrating the use of a "no zone" approach for the management of this injury and a review of the literature.

We present an unusual case of a young male with a penetrating neck injury (PNI) due to a work-related injury. A metallic foreign body traversed from entry at surgical Zone 2 to Zone 1 in the neck and resulted in a transection of the left thyrocervical trunk at the origin with the left subclavian artery. Computed Tomographic Angiography (CTA) of the aortic arch and major branch vessels demonstrated haemorrhage anterior to the left subclavian artery and left thyrocervical trunk. We describe some of the diagnostic and operative challenges which may occur in these rare and life-threatening injuries. We have also reviewed some of the recent key literature on this topic and have collated the recommendations of the review. In recent years, there has been a movement away from selective "zone-based" mandatory surgical exploration for Zone 2 injuries, as well as invasive and time-consuming investigations (such as digital subtraction angiography, contrast oesophageal swallow and bronchoscopy) for Zone 1 and 3 injuries due to the high number of negative surgical procedures and investigations. We demonstrate there is now an evidence-based algorithm which demonstrates that a "no zone" approach to the management of these patients is safe and effective. This requires an initial physical examination looking for the presence or absence of "hard", "soft" or "no" physical signs in these patients, and then deciding on subsequent management which would include immediate surgery, CTA of the aortic arch and branches (and subsequent surgical or other management) or observation only. Our aim in describing this case it to highlight that there is now good evidence-based guidance for the safe and effective management of patients with this infrequent but potentially fatal injury.

Over view of major traumatic injury in Australia--Implications for trauma system design.

BACKGROUND: Trauma registries are known to drive improvements and optimise trauma systems worldwide. This is the first reported comparison of the epidemiology and outcomes at major centres across Australia. METHODS: The Australian Trauma Registry was a collaboration of 26 major trauma centres across Australia at the time of this study and currently collects information on patients admitted to these centres who die after injury and/or sustain major trauma (Injury Severity Score (ISS) > 12). Data from 1 July 2016 to 30 June 2017 were analysed. Primary endpoints were risk adjusted length of stay and mortality (adjusted for age, cause of injury, arrival Glasgow coma scale (GCS), shock-index grouped in quartiles and ISS). RESULTS: There were 8423 patients from 24 centres included. The median age (IQR) was 48 (28-68) years. Median (IQR) ISS was 17 (14-25). There was a predominance of males (72%) apart from the extremes of age. Transport-related cases accounted for 45% of major trauma, followed by falls (35.1%). Patients took 1.42 (1.03-2.12) h to reach hospital and spent 7.10 (3.64-15.00) days in hospital. Risk adjusted length of stay and mortality did not differ significantly across sites. Primary endpoints across sites were also similar in paediatric and older adult (>65) age groups. CONCLUSION: Australia has the capability to identify national injury trends to target prevention and reduce the burden of injury. Quality of care following injury can now be benchmarked across Australia and with the planned enhancements to data collection and reporting, this will enable improved management of trauma victims.

A prospective, randomized controlled trial comparing the efficacy and safety of sotalol, amiodarone, and digoxin for the reversion of new-onset atrial fibrillation.

STUDY OBJECTIVE: A prospective, randomized controlled trial of new-onset atrial fibrillation was conducted to compare the efficacy and safety of sotalol and amiodarone (active treatment) with rate control by digoxin alone for successful reversion to sinus rhythm at 48 hours. METHODS: We prospectively randomly assigned 120 patients with atrial fibrillation of less than 24 hours' duration to treatment with sotalol, amiodarone, or digoxin using a single intravenous dose followed by 48 hours of oral treatment. Patients had ECG monitoring for 48 hours, and time of reversion, adequacy of rate control, and numbers of adverse events were compared. After 48 hours, those still in atrial fibrillation underwent cardioversion according to a standardized protocol. After 48 hours of therapy and attempted cardioversion, the number of patients whose rhythms had successfully reverted were compared. RESULTS: There was a significant reduction in the time to reversion with both sotalol (13. 0+/-2.5 hours, P <.01) and amiodarone (18.1+/-2.9 hours, P <.05) treatment compared with digoxin only (26.9+/-3.4 hours). By 48 hours, the active treatment group was significantly more likely to have reverted to sinus rhythm than the rate control group (95% versus 78%, P <.05; risk ratio 5.4, 95% confidence interval [CI] 1.5 to 19.2 ). In those patients whose rhythms did not revert to sinus rhythm, there was superior ventricular rate control in the sotalol group at both 24 and 48 hours compared with those who received either amiodarone or digoxin. There were also fewer adverse events in the active treatment group compared with the rate control group. CONCLUSION: Immediate pharmacologic therapy for new-onset atrial fibrillation with class III antiarrhythmic drugs (sotalol or amiodarone) improves complication-free 48-hour reversion rates compared with rate control with digoxin.

Can the distance fallen predict serious injury after a fall from a height?

BACKGROUND: After a fall, the distance fallen is sometimes used to predict the injury severity. We aimed to examine how distance fallen performs as a predictor of major injury. METHOD: A cohort of trauma victims attending our emergency department after having fallen from a height was identified retrospectively, and data were collected regarding the fall and injuries sustained. Performance of threshold heights, ranging from 2 meters (6.6 feet) to 10 meters (32.8 feet), as a diagnostic test for major injury was assessed. RESULTS: Height fallen performed poorly over the range of thresholds used. At low thresholds, sensitivity was inadequate to rule out major trauma, whereas the low prevalence meant that, despite impressive specificity at higher thresholds, positive predictive value was poor. At the optimal threshold of 5 meters (16.4 feet), the positive predictive value was 0.17 and sensitivity was 0.33. CONCLUSION: Height of fall is a poor predictor of major injury.

An aid to blind nasal intubation.

Blind nasal intubation remains an important technique in the management of the difficult airway. Many aids to this technique have been described, but unfortunately, these often require additional expense, training, and equipment. Methods that involve listening at the end of the endotracheal tube and observing moisture condensation in the tube are relatively insensitive and may expose the operator to the patient's bodily fluids. Our aim was to devise an aid to blind nasal intubation that was effective, inexpensive, simple to learn, and easy to assemble. It is essential that breath sounds can be monitored through the endotracheal tube, as it is felt that this helps to minimize retropharyngeal perforation caused by the tube abutting the posterior pharyngeal wall. We describe the use of an endotracheal tube stethoscope in conjunction with either 'inline' or 'sidestream' capnometry and present a case of successful nasotracheal intubation using this device. The total cost of this aid is less than $10 Australian.

An unusual case of central pontine myelinolysis.

Central pontine myelinolysis (CPM) is a rare complication of alcoholism. There have been recent reports that hyponatremia and its rapid correction are of aetiological significance in the development of CPM. We describe the case of a 47-year-old alcoholic who developed CPM and subsequently recovered. Alcoholic and psychiatric patients are at risk for CPM and clinicians need to have a high index of suspicion to diagnose this condition.

Acute spinal epidural hematoma.

A 17-year-old female presented to the Emergency Department with a history of recent onset of severe upper thoracic back pain, urinary retention, weakness and paraesthesia in the lower limbs. Magnetic Resonance Imaging (MRI) Scan demonstrated a spinal epidural hematoma at the C7-T2 level. This report highlights a rare, but eminently treatable condition; the use of MRI Scan as a specific diagnostic tool; and the need for urgent surgical decompression to ensure good recovery of function.

Evaluation of a fluorinated 2-nitroimidazole binding to hypoxic cells in tumor-bearing rats by 19F magnetic resonance spectroscopy and immunohistochemistry.

We have examined a hexafluorinated 2-nitroimidazole, CCI-103F, as a probe for hypoxic tumor cells by in vivo 19F magnetic resonance spectroscopy (MRS). Following initial intraperitoneal injections of the drug in tumor-bearing (Dunning R3327-AT1-Matlylu) rats, 19F spectra were obtained on an Otsuka 2.0T Vivospec spectrometer using a 1.5-cm surface coil. Signal at 1- and 2-h time points indicated initial biodistribution of drug in the tumor. At 4 and 8 h, a progressive increase in signal intensity was observed, indicating retention of drug within the tumor. Tumor signal remained detectable in 4 of 10 rats at 24 h, indicating possible nitroreductive bioactivation by hypoxic cells. Immunohistochemistry of these tumors revealed a staining pattern consistent with labeling of hypoxic cells. No detectable 19F signal was found at 24 h for the other rats, indicating complete washout of unbound drug. Immunohistochemical assessment of these tumors revealed some staining for bound drug at the periphery of necrotic zones. 31P-MRS of the tumors showed good correlation with the presence or absence of hypoxia as evaluated by 19F-MRS, T1- and T2-weighted images, and immunohistochemistry. These results provide the groundwork for further studies using this misonidazole analog for noninvasive identification of hypoxic tumor cells in vivo by MRS.

19F nuclear magnetic resonance analysis of trifluoroethanol metabolites in the urine of the Sprague-Dawley rat.

2,2,2-Trifluoroethanol (TFE) is a common industrial solvent and a known metabolite of the inhalation anesthetics fluroxene (2,2,2-trifluoroethyl vinyl ether) and halothane (2-bromo-2-chloro-1,1,1-trifluoroethane). The water-soluble metabolites of TFE were identified in the urine of Sprague-Dawley rats using 19F NMR spectroscopy. In rats dosed with 0.21 g TFE/kg body weight, approximately one-half of the administered TFE was excreted as the trifluoroethyl glucuronide. The remaining TFE was oxidized, primarily to trifluoroacetaldehyde hydrate, with a small percentage of the aldehyde oxidized further to trifluoroacetate. One additional fluorinated compound was found; after investigation, this was identified as a Schiff's base compound resulting from the addition of trifluoroacetaldehyde to urea. The time-dependent excretion of TFE metabolites was measured as a function of ethanol induction of hepatic enzymes. This study demonstrates the utility of 19F NMR for the analysis of drug metabolism in laboratory animals. In addition, the resistance of trifluoroacetaldehyde hydrate to further oxidation, coupled with its reactivity with common cellular amines, indicates the potential toxicity of this metabolite to mammalian tissues.

Studies in healthy human T-cell-leukemia lymphoma virus (HTLV-I) carriers from the Caribbean.

Six healthy relatives of 3 adult T-cell leukemia lymphoma (ATLL) patients and 6 members of a Caribbean family immigrant to the UK have been investigated for the presence of HTLV-I and expression of interleukin 2 (IL-2) receptors. Serum antibodies to HTLV-I were detected in all but 4 samples. Four to 10% of circulating cells from 3/4 seropositive donors studied displayed IL-2 receptors (anti-Tac+) and were shown to be convoluted lymphocytes by light microscopy morphology and immunoelectronmicroscopy. After 5 to 28 days in culture, cells from 4 seropositive donors reacted with monoclonal antibodies (MAbs) against the HTLV-I core proteins, p19 and p24, and released retrovirus particles. Similar experiments with blood from 3 seronegative donors from the same families and 4 normal controls proved negative. Our findings indicate that seropositive individuals harbour the virus in a population of T-lymphocytes which may then acquire receptors for IL-2. These individuals are at risk of developing ATLL.

Studies in healthy human T-cell-leukemia lymphoma virus (HTLV-I) carriers from the Caribbean

Six healthy relatives of 3 adult T-cell leukemia lymphoma (ATLL) patients and 6 members of a Caribbean family immigrant to the UK have been investigated for the presence of HTLV-I and expression of interleukin 2(IL-2) receptors. Serum antibodies to HTLV-I were detected in all but 4 samples. Four to 10 percent of circulating cells from 3/4 seropositive donors studied displayed IL-2 receptors (anti-Tac+) and were shown to be convoluted lymphocytes by light microscopy morphology and immunoelectromicroscopy. After 5 to 28 days in culture, cells from 4 seropositive donors reacted with monoclonal antibodies (MAbs) against the HTLV-I core proteins, p19 and p24, and released retrovirus particles. Simmilar experiments with blood from 3 seronegative donors from the same families and 4 normal controls proved negative. Our findings indicate that seropositive individuals harbour the virus in a population of T-lymphocytes which may then acquire receptors for IL-2. These individuals are at risk of developing ATLL. (AU)