Use of a Golden Gate Plasmid Set Enabling Scarless MoClo-Compatible Transcription Unit Assembly.

Golden Gate cloning has become a powerful and widely used DNA assembly method. Its modular nature and the reusability of standardized parts allow rapid construction of transcription units and multi-gene constructs. Importantly, its modular structure makes it compatible with laboratory automation, allowing for systematic and highly complex DNA assembly. Golden Gate cloning relies on type IIS enzymes that cleave an adjacent undefined sequence motif at a defined distance from the directed enzyme recognition motif. This feature has been used to define hierarchical Golden Gate assembly standards with defined overhangs ("fusion sites") for defined part libraries. The simplest Golden Gate standard would consist of three-part libraries, namely promoter, coding and terminator sequences, respectively. Each library would have defined fusion sites, allowing a hierarchical Golden Gate assembly to generate transcription units. Typically, type IIS enzymes are used, which generate four nucleotide overhangs. This results in small scar sequences in hierarchical DNA assemblies, which can affect the functionality of transcription units. However, there are enzymes that generate three nucleotide overhangs, such as SapI. Here we provide a step-by-step protocol on how to use SapI to assemble transcription units using the start and stop codon for scarless transcription unit assembly. The protocol also provides guidance on how to perform multi-gene Golden Gate assemblies with the resulting transcription units using the Modular Cloning standard. The transcription units expressing fluorophores are used as an example.

Reducing Blood Draws in Pediatric Patients With Solid Organ Injury Through Protocolized Transcutaneous Hemoglobin Monitoring.

BACKGROUND: Management of pediatric solid organ injuries continues to evolve, decreasing the need for serial hemoglobin measurements, repeat imaging, and operative intervention. Transcutaneous continuous hemoglobin monitoring (TCHM) has been shown to effectively monitor hemoglobin levels in children with solid organ trauma. METHODS: A 6-year, single-center, retrospective chart review was conducted of pediatric solid organ injury patients aged 30 days to <18 years admitted to a quaternary children's hospital following implementation of a highly protocolized TCHM system. A laboratory hemoglobin measurement was obtained at the time of diagnosis and additional measurements were determined based on injury grading. Adverse events were tracked and included: central or arterial line placement, blood product(s) administration, percutaneous embolization procedures, transfer to the pediatric ICU and operative intervention. RESULTS: A total of 97 patients met the inclusion criteria. Blood draws were significantly reduced following TCHM protocol implementation (3.0 [IQR 2.0-5.5] vs 2.0 [IQR 1.0-4.5], p 0.01) without a significant increase in blood product administration (p = 0.30), central or arterial line placement (p = 1), or operative intervention (p = 0.29). Length of stay was not impacted (p = 0.36). The rate of unplanned ICU transfers and percutaneous embolization procedures were too low for statistical evaluation. CONCLUSION: TCHM safely reduces the need for serial blood draws in pediatric trauma patients when utilized within a well-defined protocol for solid organ injury. Further studies are needed to evaluate the role of TCHM in shortening or eliminating hospital admission for low-grade solid organ injuries in children. LEVEL OF EVIDENCE: Level III. TYPE OF STUDY: Single-center, retrospective chart review cohort study.

Silent severe aortic regurgitation due to blunt chest trauma: ignore it at your peril-a case report.

Background: Blunt chest trauma (BCT) presenting to the emergency department is common and may cause life-threatening cardiac complications. Whilst complications causing haemodynamic instability are generally detected promptly, others may present late with long-term consequences. We describe a rare, serious complication of BCT presenting five years after a road traffic accident (RTA). Case summary: A 23-year-old man was incidentally found to have a murmur. Past history was notable only for BCT with rib fracture sustained in a RTA 5 years prior. Examination revealed a hyperdynamic pulse, loud decrescendo diastolic murmur, and Duroziez's sign over the femoral arteries. Echocardiography showed severe valvular aortic regurgitation (AR) from a hole in the left coronary cusp and holodiastolic flow reversal in the descending aorta. The left ventricle (LV) showed marked dilatation in diastole, mild dilatation in systole, and preserved systolic function. The aorta was normal. Severe AR was attributed to his previous BCT, with AR causing subsequent LV dilatation. He underwent aortic valve replacement (AVR) with rapid recovery. He remains well, and his echo shows a well-functioning AVR with normalization of LV dimensions. Discussion: Aortic regurgitation following BCT is rare but well-recognized, most often resulting from RTAs. Only a third of cases are diagnosed acutely. In others, lack of haemodynamic instability means that emergency echocardiography is not routinely performed, such that this may go unrecognized with long-term consequences. Clinicians should be aware of possible valve damage following BCT. Prompt echocardiography should be routinely performed for all BCT at initial presentation, even without haemodynamic instability.

Blunt Chest Wall Trauma Leading to Sudden Cardiac Arrest.

A 54-year-old hockey player survived sudden cardiac arrest after a chest slapshot, receiving immediate resuscitation and defibrillation of ventricular fibrillation. Examinations revealed chest trauma and subclinical single-vessel disease; a coronary dissection could not be ruled out. The patient recovered without complications, underscoring the importance of rescue equipment in sports facilities.

Wolf in sheep's clothing: a case of primary lung adenosquamous carcinoma mimicking traumatic pulmonary pseudocyst.

BACKGROUND: Traumatic pulmonary pseudocyst is a rare "cystlike" lung lesion that typically develops following blunt chest trauma. It differs from lung cancer associated with cystic airspaces in terms of pathogenic mechanisms, clinical manifestations, and radiological features. Furthermore, there are few reports of the diagnostic bias between traumatic pulmonary pseudocyst and lung cancer associated with cystic airspaces. Here, we present a rare case of lung cancer associated with cystic airspaces that mimicks traumatic pulmonary pseudocyst. CASE PRESENTATION: A 61-year-old man with no chest medical or surgical history, no chest radiologic examination within the last five years, and no smoking history had an air-filled "cystlike" lesion surrounded by solid components and ground-glass opacities in the middle third of the right upper lobe of the lung during a computed tomography evaluation following blunt chest trauma. He was initially diagnosed with traumatic pulmonary pseudocyst and treated conservatively. On the third post-trauma day, he experienced hemoptysis, which was successfully treated with intravenous hemostatic medication. On the ninth post-trauma day, he exhibited a significant hemoptysis and a moderate dyspnea. A subsequent chest computed tomography scan demonstrated that the solid components had entered the lesion's cavity and significantly expanded, and the surrounding ground-glass opacities had slightly enlarged. A contrast-enhanced chest computed tomography scan and a three-dimensional reconstruction computed tomography image confirmed that the solid components were a hematoma caused by damage to the right upper pulmonary vein. A right upper lobectomy was performed based on the concern about severe intrapulmonary bleeding. An intraoperative frozen section analysis showed significant bleeding in the lung parenchyma. Adenosquamous carcinoma was unexpectedly identified during the postoperative pathological examination of the resected specimen. A diagnosis of primary lung adenosquamous carcinoma was made. He was discharged on the seventh postoperative day and followed up for two years without any recurrence. CONCLUSIONS: The potential of lung cancer associated with cystic airspaces should be considered for "cystlike" lung lesions discovered in elderly patients after blunt chest trauma. A comprehensive review of the medical history, meticulous analysis of the radiological findings, and close monitoring can help clinicians reduce the risk of diagnostic bias.

Traumatic chylothorax following blunt thoracic trauma.

Background: Chylothorax occurs when chyle from the thoracic duct leaks into the pleural space. While majority of cases are iatrogenic, traumatic chylothorax can occur when rib or vertebral fractures disrupt the thoracic duct. These occurrences are exceedingly rare, particularly following blunt traumatic insult. Methods: We performed a retrospective review of a case of chylothorax following blunt trauma. Data was extracted from the electronic medical record. Case: A 60-year-old female presented to the trauma bay after a motor vehicle crash as a restrained driver with bilateral chest pain. Of note, patient had three left rib fractures from fall five days prior. She was neurologically and hemodynamically normal on arrival. Physical exam was notable for chest wall tenderness. Computed tomography revealed the following: bilateral hemopneumothoraces, pneumomediastinum, manubrium fracture, retrosternal hematoma, left 2-10 and right 1-2 rib fractures along with multiple orthopedic injuries. Left tube thoracostomy yielded 150 mL of blood. She was admitted to the intensive care unit. Patient had a 48-h period of cardiogenic shock requiring vasopressors and aggressive fluid resuscitation. On post-injury day (PID) 2, the chest tube drained milky fluid. Pleural fluid sampling was significant for triglyceride levels of 1292 mg/dL. Hemodynamics then improved. Due to low output (<500 mL/day), patient was managed conservatively a fat-restricted diet supplemented with medium-chain fatty acids. Chest tube was removed PID-7 once chyle leak resolved. Repeat chest radiograph PID-10 was negative for effusion. She was discharged to rehabilitation PID-13. At one-week follow-up, repeat CXR showed a small, loculated left lateral pleural effusion. Patient had no complaints and was maintaining adequate oxygen saturations on room air. Discussion: We present a case of delayed chylothorax after blunt trauma precipitated by increased central venous pressure secondary to right heart failure, aggressive fluid resuscitation and vasopressor use. Traumatic chylothorax should be considered in patients with pleural effusion in the setting of blunt chest trauma as sudden hyperextension of the spine can disrupt the thoracic duct. Delayed diagnosis is not uncommon due to an average latency period of 2-10 days. Pleural fluid with triglyceride level > 110 mg/dL and chylomicrons is diagnostic. Initial management consists of chyle reduction through diet modification (high protein/restricted fat diet). Octreotide can be used as a pharmacological adjunct. Refractory or high-output cases (>1000 mL/day) may require surgical ligation of the thoracic duct. Early identification and intervention are paramount as untreated chylothorax is associated with significant morbidity and mortality rates up to 50 %.

The epidemiology of spinal fractures: A nationwide data-based study in Iran.

Background: Blunt trauma is a physical injury to a part of the body, mainly caused by road accidents, direct blows, attacks, sports injuries, and falls in elderly people. Spinal fractures are observed only in a small percentage of injured patients. Accordingly, the present study was conducted on collected data between 2018 and 2022 to determine the frequency of spinal fractures in blunt trauma in Iran while also considering the mechanism of injury as a secondary outcome of interest. Methods: In this retrospective study, blunt trauma patients with spinal fractures, regardless of age were included by the census sampling method. Data were obtained from the National Trauma Registry of Iran. Means and standard deviations were used for continuous variables, and the chi-square test was used to assess the relationship between the variables. Results: Among 25,986 cases of all-cause trauma patients, 1,167 cases (4.5%) of blunt trauma and spinal fracture were included in the study. Gender, the severity of injury, and the cause of trauma showed a significant difference among different age groups (p < 0.05). Significant differences were found in the injury mechanisms across various spine regions (p < 0.05). The majority of patients (68.2%) had lumbar spinal fractures. Road traffic collisions were the most common cause of spinal cord injuries, accounting for 58.3% of cases, followed by falls (36.1%). The injury severity score was higher in younger patients (under 18 years old), with a mean of 4.4 ± 3.5, and in patients with cervical injuries. The majority of injuries occurred in the lumbar area (68.2%), followed by the thoracic area. Furthermore, notable variations existed in Emergency Room (ER) stay duration, overall hospitalization, Intensive Care Unit (ICU) stay duration, and injury severity levels, all influenced by the spinal regions (p < 0.05). Distinctively, ICU stay durations and ER stay duration showed significant differences, particularly in relation to injuries in the lumbar and thoracic regions (p < 0.05). Conclusion: According to the results of the present study, trauma is more severe, and cervical injuries are more common in young people, which is a critical finding that underscores the need for targeted interventions to mitigate the severity of trauma in this age group. Additionally, the majority of cervical injuries occurred in young people, which is a particularly concerning finding given the potential for long-term disability and impact on quality of life. Our findings suggest that strategies to reduce cervical injuries, such as speed control, seat belt use, and phone-free driving, are crucial interventions for mitigating the severity of trauma and promoting patient outcomes in young people.

Iliac transposition technique for reconstruction of aortic bifurcation in pediatric blunt abdominal aortic injury.

Blunt abdominal aortic injuries, especially in pediatric patients, represents a rare and critical challenge. We report a unique case of a 10-year-old boy who presented after a high-speed motor vehicle collision resulting in injury to the aortic bifurcation, alongside other intra-abdominal trauma. A novel surgical approach for aortoiliac reconstruction in a contaminated field was used. This technique consisted of the mobilization of the distal aorta and common iliac arteries, ligation of right internal iliac artery, and creation of aortoiliac and common to internal iliac artery anastomoses. This method demonstrates a potentially lifesaving technique in select patients.

Performance of Machine Learning Algorithms in Predicting Prolonged Mechanical Ventilation in Patients with Blunt Chest Trauma.

Purpose: Mechanical ventilation (MV) is one of the most common treatments for patients with blunt chest trauma (BCT) admitted to the intensive care unit (ICU). Our study aimed to investigate the performance of machine learning algorithms in predicting the prolonged duration of mechanical ventilation (PDMV) in patients with BCT. Methods: In this single-center observational study, patients with BCT who were treated with MV through nasal or oral intubation were selected. PDMV was defined as the duration of mechanical ventilation ≥7 days after endotracheal intubation (normal vs prolonged MV; dichotomous outcomes). K-means was used to cluster data from the original cohort by an unsupervised learning method. Multiple machine learning algorithms were used to predict DMV categories. The most significant predictors were identified by feature importance analysis. Finally, a decision tree based on the chi-square automatic interaction detection (CHAID) algorithm was developed to study the cutoff points of predictors in clinical decision-making. Results: A total of 426 patients and 35 characteristics were included. K-means clustering divided the cohort into two clusters (high risk and low risk). The area under the curve (AUC) of the DMV classification algorithms ranged from 0.753 to 0.923. The importance analysis showed that the volume of pulmonary contusion (VPC) was the most important feature to predict DMV. The prediction accuracy of the decision tree based on CHAID reached 86.4%. Conclusion: Machine learning algorithms can predict PDMV in patients with BCT. Therefore, limited medical resources can be more appropriately allocated to BCT patients at risk for PDMV.