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We report the chemical synthesis of pseudouridimycin (1), an antimicrobial natural product that potently and selectively inhibits bacterial RNA polymerase. Chemical stability studies revealed intramolecular hydroxamate bond scission to be a major decomposition pathway for 1 in aqueous buffer. Replacement of the hydroxamate bond with a tertiary amide, as in 16, afforded a conformational isostere resistant to degradation. These studies pave the way for the design and synthesis of analogues with improved chemical stability and biological activity.

The statistical validation of peptide and protein identifications in mass spectrometry proteomics is a critical step in the analytical workflow. This is particularly important in discovery experiments to ensure only confident identifications are accumulated for downstream analysis and biomarker consideration. However, the inherent nature of discovery proteomics experiments leads to scenarios where the search space will inflate substantially due to the increased number of potential proteins that are being queried in each sample. In these cases, issues will begin to arise when the machine learning algorithms that are trained on an experiment specific basis cannot accurately distinguish between correct and incorrect identifications and will struggle to accurately control the false discovery rate. Here, we propose an alternative validation algorithm trained on a curated external data set of 2.8 million extracted peakgroups that leverages advanced machine learning techniques to create a generalizable peakgroup scoring (GPS) method for data independent acquisition (DIA) mass spectrometry. By breaking the reliance on the experimental data at hand and instead training on a curated external dataset, GPS can confidently control the false discovery rate while increasing the number of identifications and providing more accurate quantification in different search space scenarios. To first test the performance of GPS in a standard experimental environment and to provide a benchmark against other methods, a novel spike-in data set with known varying concentrations was analyzed. When compared to existing methods GPS increased the nunmber of identifications by 5-18% and was able to provide more accurate quantification by increasing the number of ratio validated identifications by 24-74%. To evaluate GPS in a larger search space, a novel data set of 141 blood plasma samples from patients developing acute kidney injury after sepsis was searched with a human tissue spectral library (10000+ proteins). Using GPS, we were able to provide a 207-377% increase in the number of candidate differentially abundant proteins compared to the existing methods while maintaining competitive numbers of global identifications. Finally, using an optimized human tissue library and workflow we were able to identify 1205 proteins from the 141 plasma samples and increase the number of candidate differentially abundant proteins by 70.87%. With the addition of machine learning aided differential expression, we were able to identify potential new biomarkers for stratifying subphenotypes of acute kidney injury in sepsis. These findings suggest that by using a generalized model such as GPS in tandem with a massive scale spectral library it is possible to expand the boundaries of discovery experiments in DIA proteomics. GPS is open source and freely available on github at (https://github.com/InfectionMedicineProteomics/gscore).

Correction for ‘Total synthesis and chemical stability of pseudouridimycin’ by Christopher F. Cain et al., Chem. Commun., 2022, DOI: 10.1039/d1cc07059b.

Epidural anesthesia as a perioperative adjunct has been shown to provide superior pain control and has been implicated in more rapid ileus resolution after major abdominal surgery, possibly through a sympatholytic mechanism. Studies suggest that the vertebral level of epidural administration influences these parameters.One hundred seventy-nine patients (120 male, 59 female; average age, 36 years) underwent restorative proctocolectomy for ulcerative colitis or familial polyposis between 1989 and 1995. Patients were grouped according to type of anesthesia. Group THO (n = 53) received thoracic (T6 to T10) epidurals. Group LUM (n = 51) received lumbar (L2 to L4) epidurals, and group PCA (n = 75) received patient-controlled intravenous narcotic analgesia. Patients were compared for complications, perioperative risk factors, postoperative pain, and ileus resolution.Epidural narcotics, alone or combined with local anesthetics, were administered for an average of 2 (LUM) to 4 (THO) days without significant complications. Infrequent problems related to the epidural catheters included self-limited headaches or back pain (four) and site infections (two). Epidural failure, as measured by conversion to PCA for inadequate pain control, was not significantly greater for LUM (25%) than THO (23%). Average pain scores, rated daily on a visual analog scale, were significantly higher (indicating more pain) for PCA patients (4.2) during postoperative days 1 through 5 than for LUM (3.5) (p0.05) and for THO (2.4) (p0.05). Ileus resolution, as determined by stool output and return of bowel sounds, was significantly faster in THO than in LUM or PCA (p0.05). Resolution of ileus was not significantly different between PCA and LUM (p0.05).Thoracic epidural analgesia has distinct advantages over both lumbar epidural or traditional patient-controlled analgesia in shortening parameters measuring postoperative ileus and in reducing surgical pain. The procedure is safe and associated with low morbidity. Thoracic epidural anesthesia is also economically justifiable and may prove to impact significantly on future postoperative management by reducing length of hospitalization. Our data and those of others are most striking in these regards for patients with thoracic catheters, indicating the importance of vertebral level in epidural drug administration.

PURPOSE: Medical management of severe ulcerative colitis has used cyclosporine with increasing frequency as an adjuvant to systemic steroids and mercaptopurine. However, the effects of combined management with cyclosporine and prednisone may lead to significant immune compromise and adversely affect operative morbidity in the event urgent surgery is required. METHODS: A case is reported of a 43-year-old white male who presented with severe ulcerative colitis. The patient had been initially treated with prednisone and cyclosporine for six weeks before surgical intervention. The intractability of his ulcerative colitis caused the patient to present to surgery, where he underwent restorative proctocolectomy. RESULTS: On initial presentation, the patient manifested systemic signs of severe ulcerative colitis with hypoalbuminemia, anemia, and weight loss, despite continuous prednisone and cyclosporine management. Before surgical intervention, a chest x-ray and the patient's respiratory status were normal. A total abdominal colectomy with ileal pouch reconstruction and temporary loop ileostomy were performed without incident. On the fifth postoperative day, the patient developed respiratory failure, which was subsequently diagnosed asPneumocystis carinii pneumonia. Although ventilator support and both aggressive medical and surgical management eventually resulted in successful outcome, significant perioperative morbidity occurred. CONCLUSIONS: In the era of aggressive medical management for ulcerative colitis with both steroids and cyclosporine, the complications of immunosuppression may be significant, including opportunistic pneumonia. Prophylaxis againstP. carinii pneumonia with sulfa antibiotics should be considered, especially in patients for whom proctocolectomy is a potential end point.