Comparative validation of the D. melanogaster modENCODE transcriptome annotation.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.

MouseVUER: video based open-source system for laboratory mouse home-cage monitoring.

Video monitoring of mice in the home-cage reveals behavior profiles without the disruptions caused by specialized test setups and makes it possible to quantify changes in behavior patterns continually over long time frames. Several commercial home-cage monitoring systems are available with varying costs and capabilities; however there are currently no open-source systems for home-cage monitoring. We present an open-source system for top-down video monitoring of research mice in a slightly modified home-cage. The system is designed for integration with Allentown NexGen ventilated racks and allows unobstructed view of up to three mice, but can also be operated outside the rack. The system has an easy to duplicate and assemble home-cage design along with a video acquisition solution. The system utilizes a depth video camera, and we demonstrate the robustness of depth video for home-cage mice monitoring. For researchers without access to Allentown NexGen ventilated racks, we provide designs and assembly instructions for a standalone non-ventilated rack solution that holds three systems for more compact and efficient housing. We make all the design files, along with detailed assembly and installation instructions, available on the project webpage ( https://github.com/NIH-CIT-OIR-SPIS/MouseVUER ).

To bleed or not to bleed? Case series and discussion of haemorrhage risk with enzymatic debridement in burn injuries.

Introduction: Surgical burn excision (along with skin grafting) carries the risk of blood loss. The use of enzymatic debridement agents such as Nexobrid® has gained increased popularity as an alternative to surgical debridement in the management of burns with its reported benefits of selective burn debridement, minimising blood loss and potentially reducing the need for skin grafting. However, there is limited evidence regarding its effects on bleeding. Currently, the manufacturer declares there is no evidence for increased risk of localised bleeding and its systemic effects upon coagulation are less clear. Methods: We present two clinical cases demonstrating the possible effects of Nexobrid® on coagulation and bleeding at the debridement site. Comparisons are drawn with the manufacturers' guidance as well as evaluating the current recommendations of its use. Discussion: Nexobrid® is a novel therapy and there are few adverse effects reported in the literature. The basis of its appeal is the reduced blood loss at the debridement site and the selectivity it possesses in preserving healthy dermis. However, our cases have demonstrated that haemorrhage can occur and that those using Nexobrid® should be mindful of the potential bleeding risk from varicosities within the burn wound. We have also illustrated that Nexobrid® can be used in patients with pre-existing clotting disorders without requiring the use of blood products. However, we emphasise the importance of haematological support for its safe administration. Lay Summary: Nexobrid®, a debriding agent that contains enzymes, has been developed as an alternative to surgery which for most surgeons is the traditional method of removing dead tissue following a burn injury. The active agent is bromelain and this is derived from the stems of pineapples. This novel treatment is increasingly being used in the management of middle to deep skin thickness burns and it seems to have a number of benefits such as reducing blood loss, reducing the need for skin grafting as well as being able to treat burns in certain areas of the body that would be technically challenging to remove in the standard fashion. It simply targets the dead tissue leaving viable remnants of the skin that would hopefully allow healing to occur without the need for surgical intervention. Being a relatively new concept, current evidence regarding the safety and value of Nexobrid® continues to develop. In 2020, an agreement guideline outlining best practice with the use of Nexobrid® was published. In this statement, it was advised that caution should be taken when using Nexobrid® in patients who have blood clotting disturbances as this could increase the likelihood of bleeding. However, they did not mention that excessive bleeding can potentially occur with this treatment.We present two clinical cases demonstrating the possible effects of Nexobrid® on the clotting system and bleeding at the application site. Comparisons are drawn with the manufacturers' guidance as well as assessing the current recommendations of its use. We illustrate that Nexobrid® can be safely used in patients with pre-existing clotting disturbances if the correct procedures are performed. We also highlight the potential complication of excessive bleeding if Nexobrid® is used in patients who have co-existing enlarged surface veins along with their burn injury. We feel the guidance should be updated to reflect these findings.

Positive outcomes for operatively managed self-harm burns.

Within healthcare generally, patients who self-harm can experience stigma and inequitable medical care. Previous studies have suggested that patients with small self-harm burn injuries may not be treated equally in comparison to non-intentional injuries. Furthermore, there is an absence of literature related to surgical outcomes for self-harm burn injuries. A retrospective cohort study of an adult burns service's outpatient attendances over a four-year period was completed. Self-harm burn injuries were identified and hospital medical records were used to extract demographic, burn injury, treatment and outcome information. 94 self-harm burn injuries in 58 patients presented over the study period. Of those who presented with self-harm burn injuries, 29 % (n = 17) of patients presented on more than one occasion, 54 % (n = 50) of wounds were managed surgically and 80 % (n = 36) of full thickness injuries were managed surgically. The post-operative course and healing time was similar to what would be expected after non-intentional burn injuries. In 93 % (n = 54) of all patients presenting with self-harm burn injuries, there was no reported tampering or non-compliance. There was no tampering or non-compliance in 94 % (n = 47) of those with self-harm burn injuries when wounds were treated surgically. The findings support the view that self-harm burn injuries should be treated in the same way as non-intentional burn injuries and that similar outcomes from treatment can be expected. However, further research is needed to explore this systematically.

Enantioselective desymmetrizing palladium catalyzed carbonylation reactions: the catalytic asymmetric synthesis of quaternary carbon center containing 1,3-dienes.

A desymmetrization protocol has been used to develop a palladium catalyzed enantioselective carbonylation process. Achiral cyclic bis-alkenyltriflates are converted to their corresponding monoester derivatives with selectivities of up to 96% ee.

Incisor degeneration in rats induced by vascular endothelial growth factor/fibroblast growth factor receptor tyrosine kinase inhibition.

BMS-645737, an inhibitor of vascular endothelial growth factor (VEGF) receptor-2 and fibroblast growth factor (FGF) receptor-1, has anti-angiogenic activity and was evaluated in nonclinical studies as a treatment for cancer. This article characterizes the BMS-645737-induced clinical, gross, and histologic lesions of incisor teeth in Sprague-Dawley (SD) rats. Rats received 0 800 mg/kg BMS-645737 in a single-dose study or consecutive daily doses of 0 20 mg/kg/day in a 1-month study. The reversibility of these effects was assessed in the 1-month study. White discoloration and fracture of incisors were observed clinically and grossly in the 1-month study. In both studies, dose-dependent histopathologic lesions of incisors were degeneration and/or necrosis of odontoblasts and ameloblasts; decreased mineralization of dentin; inflammation and necrosis of the dental pulp; and edema, congestion, and hemorrhage in the pulp and periodontal tissue adjacent to the enamel organ. Partial recovery was observed at lower doses after a two-week dose-free period in the one-month study. Drug-induced incisor lesions were considered to be related to the pharmacologic inhibitory effects on VEGF and FGF signaling, that is, inhibition of growth and maintenance of small-diameter vessels that support the formation of dentin and enamel in growing teeth and/or to perturbances of function of odontoblasts and ameloblasts or their precursors.

Successful treatment of mixed depth flame burns using enzymatic debridement with Nexobrid™ in a patient with aggressive systemic sclerosis (scleroderma).

INTRODUCTION: Enzymatic debridement for mixed depth and full thickness burns is gaining recognition as an important technique available to the burns surgeon. Reductions in blood loss, the need for autologous skin grafting and the number of wounds requiring surgical excision are among the benefits of this evolving treatment modality. We present a case of successful treatment using enzymatic debridement of mixed depth flame burns in a young patient with systemic sclerosis (scleroderma). METHODS: A 24-year-old man with rapidly progressive limited cutaneous systemic sclerosis suffered approximately 6% mixed depth flame burns to the right leg including areas of likely deep partial thickness burn totalling approximately 2% total body surface area (TBSA). Enzymatic debridement using Nexobrid™ was performed under spinal anaesthesia in accordance with unit protocol. Pain was well controlled and at 4 h the Nexobrid™ was removed and replaced with saline-soaked gauze initially then simple dressings. After liaison with Rheumatology, he was also started on iloprost infusions over five days. He was discharged home on day 9 and completely healed by day 31. Scarring was minimal, the skin was soft and full, and pain-free range of movement of the right knee was maintained. DISCUSSION AND CONCLUSION: This case demonstrates the safety and effectiveness of enzymatic debridement of mixed depth burns in a patient with very compromised wound healing. Enzymatic debridement combined with iloprost infusion provided maximum preservation of viable dermis. The authors recommend that this treatment strategy is considered in similar cases.

Adverse vacuolation in multiple tissues in cynomolgus monkeys following repeat-dose administration of a PEGylated protein.

PEGylation is considered a safe mechanism to enhance the pharmacokinetics (PK) and pharmacodynamics (PD) of biotherapeutics. Previous studies using PEGylation as a PK enhancement tool have reported benign PEG-related vacuolation in multiple tissues. This paper establishes a threshold for PEG burden beyond which there are alterations in tissue architecture that could potentially lead to dysfunction. As part of the nonclinical safety assessment of Compound A, a 12 kDa protein conjugated to a 40 kDa branched PEG molecule, monkeys were dosed subcutaneously twice weekly for 3 months at protein doses resulting in weekly PEG doses of 8, 24, 120, or 160 mg/kg. Consistent with previous reports with PEGylated biomolecules, Compound A administration resulted in intracellular vacuoles attributed to the PEG moiety in macrophages in numerous tissues and epithelial cells in the choroid plexus and kidney. Vacuolation occurred at all doses with dose-dependent severity and no evidence of recovery up to 2 months after dosing cessation. The vacuolation was considered nonadverse at PEG doses ≤120 mg/kg/week. However, at 160 mg/kg/week PEG, the vacuolation in choroid plexus, pituitary gland, kidney, and choroid of the eye was considered adverse due to significant alterations of tissue architecture that raised concern for the possibility of compromised tissue function. To our knowledge, this is the first report of potentially adverse cellular consequences of PEG accumulation in tissues other than kidney. Furthermore, the lack of reversibility of vacuolation coupled with the lack of a biomarker for intracellular PEG accumulation highlights a potential risk that should be weighed against the benefits of PK/PD enhancement for long-term administration of PEGylated compounds at high doses.

Biotransformation of [14C]dasatinib: in vitro studies in rat, monkey, and human and disposition after administration to rats and monkeys.

This study describes the in vitro metabolism of [(14)C]dasatinib in liver tissue incubations from rat, monkey, and human and the in vivo metabolism in rat and monkey. Across species, dasatinib underwent in vitro oxidative metabolism to form five primary oxidative metabolites. In addition to the primary metabolites, secondary metabolites formed from combinations of the oxidative pathways and conjugated metabolites of dasatinib and its oxidative metabolites were also observed in hepatocytes incubations. In in vivo studies in rats and monkeys, the majority of the radioactive dose was excreted in the bile and feces. In bile duct-cannulated monkeys after an i.v. dose, 13.7% of the radioactive dose was excreted in the feces through direct secretion. Dasatinib comprised 56 and 26% of the area under the curve (AUC) (0-8 h) of total radioactivity (TRA) in plasma, whereas multiple metabolites accounted for the remaining 44 and 74% of the AUC (0-8 h) of TRA for rats and monkeys, respectively. In rat and monkey bile, dasatinib accounted for < 12% of the excreted dose, suggesting that dasatinib was extensively metabolized before elimination. The metabolic profiles in bile were similar to the hepatocyte profiles. In both species, a large portion of the radioactivity excreted in bile (> or = 29% of the dose) was attributed to N-oxides and conjugated metabolites. In rat and monkey feces, only the oxidative metabolites and their further oxidation products were identified. The absence of conjugative or N-oxide metabolites in the feces suggests hydrolysis or reduction, respectively, in the gastrointestinal tract before elimination.

Towards Personalized Medicine: An Improved De Novo Assembly Procedure for Early Detection of Drug Resistant HIV Minor Quasispecies in Patient Samples.

The third-generation sequencing technology, PacBio, has shown an ability to sequence the HIV virus amplicons in their full length. The long read of PaBio offers a distinct advantage to comprehensively understand the virus evolution complexity at quasispecies level (i.e. maintaining linkage information of variants) comparing to the short reads from Illumina shotgun sequencing. However, due to the highnoise nature of the PacBio reads, it is still a challenge to build accurate contigs at high sensitivity. Most of previously developed NGS assembly tools work with the assumption that the input reads are fairly accurate, which is largely true for the data derived from Sanger or Illumina technologies. When applying these tools on PacBio high-noise reads, they are largely driven by noise rather than true signal eventually leading to poor results in most cases. In this study, we propose the de novo assembly procedure, which comprises a positivefocused strategy, and linkage-frequency noise reduction so that it is more suitable for PacBio high-noise reads. We further tested the unique de novo assembly procedure on HIV PacBio benchmark data and clinical samples, which accurately assembled dominant and minor populations of HIV quasispecies as expected. The improved de novo assembly procedure shows potential ability to promote PacBio technology in the field of HIV drug-resistance clinical detection, as well as in broad HIV phylogenetic studies.

Palladium-catalysed N-annulation routes to indoles: the synthesis of indoles with sterically demanding N-substituents, including demethylasterriquinone A1.

Tandem palladium-catalysed aryl and alkenyl C-N bond formation allows the synthesis of a variety of indoles bearing sterically demanding N-substituents, including the natural product demethylasterriquinone A1.

Tandem palladium-catalyzed urea arylation-intramolecular ester amidation: regioselective synthesis of 3-alkylated 2,4-quinazolinediones.

o-Halo benzoates can be combined with monoalkyl ureas in a tandem palladium-catalyzed arylation-ester amidation sequence to deliver quinazolinedione products. The reactions are regioselective for formation of the 3-N-alkyl isomers. Significant variation of both coupling partners is possible, allowing the synthesis of a diverse array of substituted quinazolinediones, exemplified by the preparation of a simple unsymmetric-dialkylated natural product. [reaction: see text]

Occurrence of spontaneous pancreatic lesions in normal and diabetic rats: a potential confounding factor in the nonclinical assessment of GLP-1-based therapies.

Glucagon-like peptide 1-based therapies, collectively described as incretins, produce glycemic benefits in the treatment of type 2 diabetes. Recent publications raised concern for a potential increased risk of pancreatitis and pancreatic cancer with incretins based in part on findings from a small number of rodents. However, extensive toxicology assessments in a substantial number of animals dosed up to 2 years at high multiples of human exposure do not support these concerns. We hypothesized that the lesions being attributed to incretins are commonly observed background findings and endeavored to characterize the incidence of spontaneous pancreatic lesions in three rat strains (Sprague-Dawley [S-D] rats, Zucker diabetic fatty [ZDF] rats, and rats expressing human islet amyloid polypeptide [HIP]; n = 36/group) on a normal or high-fat diet over 4 months. Pancreatic findings in all groups included focal exocrine degeneration, atrophy, inflammation, ductular cell proliferation, and/or observations in large pancreatic ducts similar to those described in the literature, with an incidence of exocrine atrophy/inflammation seen in S-D (42-72%), HIP (39%), and ZDF (6%) rats. These data indicate that the pancreatic findings attributed to incretins are common background findings, observed without drug treatment and independent of diet or glycemic status, suggesting a need to exercise caution when interpreting the relevance of some recent reports regarding human safety.

Reconstruction for time-domain in vivo EPR 3D multigradient oximetric imaging--a parallel processing perspective.

Three-dimensional Oximetric Electron Paramagnetic Resonance Imaging using the Single Point Imaging modality generates unpaired spin density and oxygen images that can readily distinguish between normal and tumor tissues in small animals. It is also possible with fast imaging to track the changes in tissue oxygenation in response to the oxygen content in the breathing air. However, this involves dealing with gigabytes of data for each 3D oximetric imaging experiment involving digital band pass filtering and background noise subtraction, followed by 3D Fourier reconstruction. This process is rather slow in a conventional uniprocessor system. This paper presents a parallelization framework using OpenMP runtime support and parallel MATLAB to execute such computationally intensive programs. The Intel compiler is used to develop a parallel C++ code based on OpenMP. The code is executed on four Dual-Core AMD Opteron shared memory processors, to reduce the computational burden of the filtration task significantly. The results show that the parallel code for filtration has achieved a speed up factor of 46.66 as against the equivalent serial MATLAB code. In addition, a parallel MATLAB code has been developed to perform 3D Fourier reconstruction. Speedup factors of 4.57 and 4.25 have been achieved during the reconstruction process and oximetry computation, for a data set with 23 x 23 x 23 gradient steps. The execution time has been computed for both the serial and parallel implementations using different dimensions of the data and presented for comparison. The reported system has been designed to be easily accessible even from low-cost personal computers through local internet (NIHnet). The experimental results demonstrate that the parallel computing provides a source of high computational power to obtain biophysical parameters from 3D EPR oximetric imaging, almost in real-time.

Synthesis of heteroarenes using cascade radical cyclisation via iminyl radicals.

Cascade radical cyclisation involving homolytic aromatic substitution has been used to synthesise new tetracycles. Treatment of vinyl iodide radical precursors with Me(3)Sn. radicals (from hexamethylditin) yielded intermediate vinyl radicals which undergo 5-exo cyclisation onto suitably placed nitrile groups to yield intermediate iminyl radicals. The iminyl radicals undergo aromatic homolytic substitution via 6-endo cyclisation (or 5-exo cyclisation followed by neophyl rearrangement) with loss of hydrogen (H.) in a H-abstraction step. We propose that this abstraction was facilitated by tert-butoxyl (t-BuO.) radicals from di-tert-butyl peroxide or methyl radicals, generated from breakdown of trimethylstannyl radicals (Me(3)Sn.). The biologically active alkaloids mappicine and luotonin A were synthesised using the new methodology. A novel radical conversion of nitriles to primary amides is proposed.