Evaluation of TrpM and PsiD substrate promiscuity reveals new biocatalytic capabilities.

N-methylated tryptamines, such as the hallucinogenic natural products, psilocybin and N,N-dimethyltryptamine (DMT), are gaining interest from the medical community due to their potential as next generation treatments for mental health disorders. The clinical relevance of these compounds has driven scientists to develop biosynthetic production routes to a number of tryptamine drug candidates, and efforts are ongoing to expand and further develop these biosynthetic capabilities. To that end, we have further characterized the substrate preferences of two enzymes involved in tryptamine biosynthesis: TrpM, a tryptophan N-methyltransferase from Psilocybe serbica, and PsiD, the gateway decarboxylase of the psilocybin biosynthesis pathway. Here, we show that TrpM can N-methylate the non-native amino acid substrate, 4-hydroxytryptophan, a key intermediate in the Escherichia coli-based recombinant psilocybin biosynthesis pathway. However, the ability to incorporate TrpM into a functional psilocybin biosynthesis pathway was thwarted by PsiD's inability to use N,N-dimethyl-4-hydroxytryptophan as substrate, under the culturing conditions tested, despite demonstrating activity on N-methylated and 4-hydroxylated tryptophan derivatives individually. Taken together, this work expands upon the known substrates for TrpM and PsiD, further increasing the diversity of tryptamine biosynthetic products.

Surveillance of Vermont wildlife in 2021-2022 reveals no detected SARS-CoV-2 viral RNA.

Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes (Vulpes vulples and Urocyon cineroargentus, respectively), fishers (Martes pennati), river otters (Lutra canadensis), coyotes (Canis lantrans), bobcats (Lynx rufus rufus), black bears (Ursus americanus), and white-tailed deer (Odocoileus virginianus). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Surprisingly, we initially detected a number of N1 and/or N2 positive samples with high cycle threshold values, though after conducting environmental swabbing of the laboratory and verifying with a second independent primer set (WHO-E) and PCR without reverse transcriptase, we showed that these were false positives due to plasmid contamination from a construct expressing the N gene in the general laboratory environment. Our final results indicate that no sampled wildlife were positive for SARS-CoV-2 RNA, and highlight the importance of physically separate locations for the processing of samples for surveillance and experiments that require the use of plasmid DNA containing the target RNA sequence. These negative findings are surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American's wildlife populations.

Surveillance of Vermont wildlife in 2021-2022 reveals no detected SARS-CoV-2 viral RNA.

Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes ( Vulpes vulples and Urocyon cineroargentus , respectively), fishers ( Martes pennati ), river otters ( Lutra canadensis ), coyotes ( Canis lantrans ), bobcats ( Lynx rufus rufus ), black bears ( Ursus americanus ), and white-tailed deer ( Odocoileus virginianus ). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Our results indicate that no sampled wildlife were positive for SARS-CoV-2. This finding is surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American's wildlife populations.

"In vivo biosynthesis of N,N-dimethyltryptamine, 5-MeO-N,N-dimethyltryptamine, and bufotenine in E.coli".

N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and 5-hydroxy-N,N-dimethyltryptamine (bufotenine) are psychedelic tryptamines found naturally in both plants and animals and have shown clinical potential to help treat mental disorders, such as anxiety and depression. Advances in both metabolic and genetic engineering make it possible to engineer microbes as cell factories to produce DMT and its aforementioned derivatives to meet demand for ongoing clinical study. Here, we present the development of a biosynthetic production pathway for DMT, 5-MeO-DMT, and bufotenine in the model microbe Escherichia coli. Through the application of genetic optimization techniques and process optimization in benchtop fermenters, the in vivo production of DMT in E. coli was observed. DMT production with tryptophan supplementation reached maximum titers of 74.7 ± 10.5 mg/L under fed batch conditions in a 2-L bioreactor. Additionally, we show the first reported case of de novo production of DMT (from glucose) in E. coli at a maximum titer of 14.0 mg/L and report the first example of microbial 5-MeO-DMT and bufotenine production in vivo. This work provides a starting point for further genetic and fermentation optimization studies with the goal to increase methylated tryptamine production metrics to industrially competitive levels.

Considerations for Polymers Used in Ocular Drug Delivery.

PURPOSE: Age-related eye diseases are becoming more prevalent. A notable increase has been seen in the most common causes including glaucoma, age-related macular degeneration (AMD), and cataract. Current clinical treatments vary from tissue replacement with polymers to topical eye drops and intravitreal injections. Research and development efforts have increased using polymers for sustained release to the eye to overcome treatment challenges, showing promise in improving drug release and delivery, patient experience, and treatment compliance. Polymers provide unique properties that allow for specific engineered devices to provide improved treatment options. Recent work has shown the utilization of synthetic and biopolymer derived biomaterials in various forms, with this review containing a focus on polymers Food and Drug Administration (FDA) approved for ocular use. METHODS: This provides an overview of some prevalent synthetic polymers and biopolymers used in ocular delivery and their benefits, brief discussion of the various types and synthesis methods used, and administration techniques. Polymers approved by the FDA for different applications in the eye are listed and compared to new polymers being explored in the literature. This article summarizes research findings using polymers for ocular drug delivery from various stages: laboratory, preclinical studies, clinical trials, and currently approved. This review also focuses on some of the challenges to bringing these new innovations to the clinic, including limited selection of approved polymers. RESULTS: Polymers help improve drug delivery by increasing solubility, controlling pharmacokinetics, and extending release. Several polymer classes including synthetic, biopolymer, and combinations were discussed along with the benefits and challenges of each class. The ways both polymer synthesis and processing techniques can influence drug release in the eye were discussed. CONCLUSION: The use of biomaterials, specifically polymers, is a well-studied field for drug delivery, and polymers have been used as implants in the eye for over 75 years. Promising new ocular drug delivery systems are emerging using polymers an innovative option for treating ocular diseases because of their tunable properties. This review touches on important considerations and challenges of using polymers for sustained ocular drug delivery with the goal translating research to the clinic.

A new nitinol stent for use in superior vena cava syndrome. Initial clinical experience.

AIM: The aim of the study was to assess the early clinical experience with the Zilver Vena stent in treating patients with malignant superior vena cava syndrome. METHODS: Demographic, procedural, and follow-up data of 12 patients (seven women; mean age 69 years) treated for superior vena cava syndrome with in all 21 Zilver Vena stents between March 2012 and October 2013 were retrospectively reviewed. All cavographies and contrast enhanced CT related to the treatment and during follow-up were evaluated and the patients had clinical follow-up until dead. They were all in terminal state at the time of stent deployment. RESULTS: All patients had superior vena cava obstruction and clinical superior vena cava syndrome caused by malignant expansive mediastinal disease (eight patients non-small cell lung cancer and four small cell lung cancer). The technical success with deployment of the stents in the intended position was 75%. Good clinical effect with resolution of superior vena cava syndrome was achieved in 11 of the 12 patients (92%). CONCLUSION: The Zilver Vena stent appeared to be safe, easy to deploy, and showed good clinical outcome. The high radial force of the stent might be of advantage in these patients with expanding masses around the superior vena cava.

Inflammatory response in patients undergoing uterine artery embolization as compared to patients undergoing conventional hysterectomy.

BACKGROUND: Uterine fibroids are benign tumors seen in 20-40% of women of childbearing age, and these fibroids are usually treated by hysterectomy. During the last decade, embolization of the uterine arteries with polyvinyl alcohol microparticles has become an alternative treatment. PURPOSE: To investigate whether uterine artery embolization generates a reduced inflammatory response as compared with conventional hysterectomy. MATERIAL AND METHODS: 40 women, 20 in each group, entered this prospective, non-randomized study. The two groups were comparable concerning age, comorbidity, and body-mass index (BMI). RESULTS: We found a significant difference between the inflammatory responses in women undergoing embolization compared with the inflammatory response in women having an abdominal hysterectomy. Women undergoing embolization were subjected to a much smaller inflammatory burden, their total morphine consumption was lower, and their return to work was faster than women subjected to conventional hysterectomy. CONCLUSION: Uterine artery embolization generates a reduced inflammatory response compared with conventional hysterectomy.

[Endovascular treatment of uterine fibromas]. / Endovaskulaer behandling af uterusfibromer.

Uterine artery embolisation represents a promising new method of treating fibroid-related menorrhagia and pelvic pain. The procedure is performed under local analgesia and intravenous sedation. Both uterine arteries are selectively catheterised under fluoroscopic control. Microparticles suspended in contrast medium are used to embolise the uterine vascular bed. Ischaemic pain during the first day is treated with intravenous morphine. Patients treated with embolisation can expect excellent results with respect to menorrhagia, pelvic pain, and reduction in the fibroid tumour volume. Women undergoing uterine embolisation retain their potential for future pregnancies. The procedure is well tolerated by patients, and possesses the advantages of shorter hospitalisation and recovery time, as compared to hysterectomy.

Uterine artery embolization of symptomatic uterine fibroida . Initial success and short-term results.

PURPOSE: To evaluate reduction in fibroid volume, the effect on clinical symptoms, adverse events and complications after percutaneous uterine artery embolization (UAE) as primary invasive treatment for symptomatic uterine fibroids. MATERIAL AND METHODS: Sixty-two patients entered the study. Indications for treatment were fibroid-induced menorrhagia, bulk symptoms, pain, and/or large fibroid size. The first 50 patients were evaluated by clinical examination and ultrasonography with measurement of fibroid volume before treament and 1, 6 and 12 months after UAE. The remaining 12 patients were followed 3 and 12 months after treatment. Embolization with microparticles was performed percutaneously in local analgesia by selective catheterization of both uterine arteries. RESULTS: A primary technical success with bilateral UAE was achieved in 60/62 (97%) of the patients. They were treated for postprocedural pain lasting up to 24 h. In 30 of the 62 patients with 6 months follow-up, the mean fibroid volume was reduced 68% 6 months after treatment. Twenty-nine (96%) of the patients experienced reduced bleeding, 21 (70%) reduced pain, and 18 (61%) reduced bulk symptoms at follow-up. CONCLUSION: UAE is a method with a high technical success rate. The treatment has good effect on fibroid volume reduction and clinical symptoms. Severe post-procedural pain occurs generally in successful bilateral embolizations, but complications and adverse events are otherwise few and minor. UAE represents a promising new method for treating uterine fibroid-related symptoms.

Fibroids treated by uterine artery embolization. A review.

An increasing number of reports indicate that uterine fibroids can be successfully treated with uterine artery embolization (UAE). UAE seems to be a promising treatment for women who want to retain their uterus. This review summarizes the technical considerations and the results. UAE is a radiological procedure using angiography for visualization of the blood circulation. Subsequently, the flow through the uterine arteries is blocked resulting in infarction of fibroids. Success rates of 87% have been achieved with an average 57% reduction of fibroid volume. Complications have been few compared to hysterectomy and patient satisfaction is high. However, none of the reports include controls. Further studies are needed to optimize patient selection and to evaluate long-term results.

Pulmonary arteriovenous malformations: screening procedures and pulmonary angiography in patients with hereditary hemorrhagic telangiectasia.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is a dominantly inherited disease with a high prevalence of pulmonary arteriovenous malformations (PAVMs). The first symptom of HHT may be stroke or fatal hemoptysis associated with the presence of PAVM. OBJECTIVE: To evaluate different screening methods applied for the identification of PAVMs. SETTING: Odense University Hospital. SUBJECTS: HHT patients with positive findings on contrast echocardiography (CE) who participated in a screening investigation and underwent pulmonary angiography (PA). METHODS: Different screening methods were evaluated against the results of PA. In a group of patients with positive findings on CE, we compared results of PA with the following: severity of dyspnea; results of pulse oximetry arterial oxygen saturation (SaO2) supine and upright; supine PaO2 in room air and while breathing 100% oxygen; size of arteriovenous shunt in supine position; chest radiograph; and intensity of contrast at CE. RESULTS: PA was performed in 25 HHT patients with positive findings on CE, 15 of whom had PAVM. Embolization therapy was recommended in 12 patients, and 3 patients had small PAVMs not accessible for therapy. In 10 patients, PAVM could not be demonstrated at PA. The sensitivity and specificity calculated for the screening procedures are as follows: 53% and 90%, respectively, for SaO2; 60% and 100%, respectively, for chest radiograph; 73% and 80%, respectively, for PaO2 in room air; 100% and 40%, respectively, for PaO2 breathing 100% oxygen; and 64% and 80%, respectively, for shunt measurement. CONCLUSION: Initial screening with CE followed by measurement of PaO2 while breathing 100% oxygen seemed to be the best screening procedure for identification of patients with PAVM. Screening with chest radiograph and pulse oximetry was shown to be insufficient.

Chest roentgenogram in the evaluation of heart defects in asymptomatic infants and children with a cardiac murmur: reproducibility and accuracy.

OBJECTIVES: To evaluate the reproducibility and the accuracy of pediatric radiologists' assessments of chest radiographs with respect to the presence or absence of heart defects in children with an asymptomatic heart murmur. DESIGN: Ninety-eight children, ages 1 month to 15 years (median, 30.1 months), referred for evaluation of a heart murmur were consecutively included. They all had a standard chest radiograph and a color Doppler echocardiograph (CDE) performed. Six specialists in pediatric radiology evaluated the chest radiographs independently on two occasions 6 months apart. The radiologists were asked to classify each set of films into one of two categories: heart disease or no heart disease. The outcome of the CDE was considered the definite diagnosis. kappa statistics were used to analyze the reproducibility of the radiologic assessments. Sensitivity, specificity, and the predictive value of a positive and a negative test were used for evaluation of the accuracy of the radiologic assessments. RESULTS: Mean intra- and interobserver kappa values were all <0.6, and the majority were <0.4. Mean sensitivity was 0.3 (range: 0.17-0.52), mean predictive value of a positive test was 0.4, implying that 60% of the positive assessments were falsely positive. Mean specificity was 0.86 (range: 0.75-0.93) and the mean predictive value of a negative test was 0.80 implying that 20% of the negative assessments were falsely negative. CONCLUSION: We found a low reproducibility, as well as a low accuracy, of the radiologic assessments of the chest radiographs of children with an asymptomatic heart murmur with respect to the presence or absence of heart disease. A false-positive radiologic assessment of the chest radiograph with respect to heart defects causes unnecessary anxiety and further examinations, whereas a false-negative assessment might result in omission of relevant investigations and proper identification of the heart defect. We cannot recommend the use of chest radiographs in the initial evaluation of the asymptomatic child with a heart murmur. If a heart defect cannot be excluded by clinical examination a CDE must be performed.

Post-mortem radiological examination in infants: evidence of child abuse?

We examined the value of post-mortem radiological examination of infants who were brought in for medico-legal autopsy. Twenty children between the age of 1 month and 15 months died under the picture of SIDS. No radiological or other signs of previous child abuse were seen in our autopsy material. A fatal case of child abuse with several metaphyseal fractures is reported. Some fractures were not visible on gross examination, but could be demonstrated by radiography and histology. In our material no association between SIDS and child abuse was found. In suspected cases of child abuse, particularly rib fractures and metaphyseal fractures should be sought. We recommend that post-mortem radiography is performed in such cases. If fractures are demonstrated, they should be verified by histologic examination.

[Ultrasonically guided Biopty-biopsy of focal liver processes]. / Ultralydvejledt Biopty-biopsi af fokale leverprocesser.

During a period of three years, 51 patients with focal hepatic changes were submitted to 55 ultrasonically guided Biopty-biopsies. Histological diagnoses could be established in 45 patients at the first biopsy which corresponds to a diagnostic certainty of 88.2% Histological diagnoses were established in 49 patients which corresponds to a certainty of 96%. The frequency of complications was 3.9%. Biopty-biopsy is recommended as a good method because it is easy to carry out and provides just as good results as other methods of biopsy. In addition, it can provide more histological information than aspiration biopsies which are, however, somewhat cheaper.

Multicystic transformation of the kidneys in dialysis patients.

In a dialysis population patients who had been treated merely with haemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD) were examined with ultrasound. The occurrence of multicystic transformation of the kidney was 4/15 in HD patients and 8/25 in CAPD patients with no significant difference between the two groups. There was a significant association between the occurrence of multicystic transformation and the patient age while there was no significant association to the duration of dialysis or the duration of the uraemic state. No tumour or any other complication to cystic transformation was found. In 582 persons without renal disease examined as controls we found 44 with cystic change. This material does not support the recommendation of regular ultrasound examination of our dialysis patients. However, until these results can be confirmed by prospective studies we must recommend screening of all maintenance dialysis patients after a longer duration of dialysis.

Hypertrophic pyloric stenosis: determination of muscle dimensions by ultrasound.

To evaluate the usefulness of ultrasound in hypertrophic pyloric stenosis (HPS) and to analyse the correlation between the dimensions of the pyloric muscle and the age and the weight of the child, 34 children with suspected HPS and 34 controls were examined. An overlap between the dimensions of the pyloric muscle in the HPS group and in the controls stresses the need to assess the muscle length, the muscle diameter, and the muscle wall thickness in establishing the sonographic diagnosis of HPS. We found the following criteria useful: muscle length greater than or equal to 19 mm, muscle diameter greater than or equal to 10 mm, and muscle wall thickness greater than or equal to 4 mm. The results did not confirm previous reports of increasing dimensions of the pyloric muscle with age and weight.