Umpolung Flow Chemistry for the Synthesis of a 3-Oxo-3H-spiro[benzofuran-2,4'-piperidine] Building Block.

An efficient and scalable route to tert-butyl 3-oxo-3H-spiro[benzofuran-2,4'-piperidine]-1'-carboxylate, a central prochiral intermediate in the synthesis of SHP2 inhibitor GDC-1971 (migoprotafib), was achieved. Preparation of the title compound from readily available 2-fluorobenzaldehyde included formation of a modified Katritzky benzotriazole hemiaminal, which, upon deprotonation by n-butyllithium, participated in umpolung reactivity via 1,2-addition to tert-butyl 4-oxopiperidine-1-carboxylate (N-Boc-4-piperidone). Most notably, this reaction was developed as a robust plug-flow process that could be executed on multiple kilograms without the need for pilot-scale reaction vessels operating at low cryogenic temperatures. Treatment of the resulting tetrahedral intermediate with oxalic acid resulted in collapse to the corresponding 4-(2-fluorobenzoyl)-4-hydroxypiperidine, which was isolated as a solid via crystallization. The synthesis concluded with an optimized intramolecular SNAr reaction and final crystallization to generate tert-butyl 3-oxo-3H-spiro[benzofuran-2,4'-piperidine]-1'-carboxylate as a stable, high-quality intermediate suitable for further functionalization toward GDC-1971.

Meta-analysis of primary care delivered buprenorphine treatment retention outcomes.

Background: Currently, the capacity to provide buprenorphine treatment (BT) is not sufficient to treat the growing number of people in the United States with opioid use disorder (OUD). We sought to examine participant retention in care rates of primary care delivered BT programs and to describe factors associated with retention/attrition for participants receiving BT in this setting.Objectives: A PRISMA-guided search of various databases was performed to identify the articles focusing on efficacy of BT treatment and OUD.Method: A systematic literature search identified 15 studies examining retention in care in the primary care setting between 2002 and 2020. Random effects meta-regression were used to identify retention rates across studies.Results: Retention rates decreased across time with a mean 0.52 rate at one year. Several factors were found to be related to retention, including: race, use of other drugs, receipt of counseling, and previous treatment with buprenorphine.Conclusions: While we only investigate BT through primary care, our findings indicate retention rates are equivalent to the rates reported in the specialty care literature. More work is needed to examine factors that may impact primary care delivered BT specifically and differentiate participants that may benefit from care delivered in specialty over primary care as well as the converse.

Syntheses of Square Planar Gallium Complexes and a Proton NMR Correlation Probing Metalloaromaticity.

Syntheses of square planar (SP) coordination complexes of gallium(III) are reported herein. Using the pyridine diimine ligand (PDI), we prepared both (PDI2-)GaH (4) and (PDI2-)GaCl (5), which were spectroscopically and structurally characterized. Reduction of PDI using Na metal afforded "Na2PDI", which reacts with in situ-prepared "GaHCl2" or GaCl3 to afford the SP 4 and 5. The planar geometry of these and previously reported SP Al(III) complexes is attributed to energetic stabilization derived from a ring-current effect, or metalloaromaticity. Typically, aromaticity in metal-containing ring systems can be difficult to characterize or confirm experimentally. An experimental approach employing proton NMR spectroscopy and described here provided an estimate of a downfield chemical shift promoted by a small ring-current associated with metalloaromaticity. Near infrared spectroscopic analyses display ligand-metal charge transfer bands which support the assignment of aromaticity. The SP complexes (PDI2-)AlH (1), (PDI2-)AlCl (2), (PDI2-)AlI (3), 4, and 5 are all discussed in this report, using aromaticity as a model for their electronic structure and reactivity properties.

Vitamin D insufficiency in neonatal hypoxic-ischemic encephalopathy.

BACKGROUND: Vitamin D has neuroprotective and immunomodulatory properties, and deficiency is associated with worse stroke outcomes. Little is known about effects of hypoxia-ischemia or hypothermia treatment on vitamin D status in neonates with hypoxic-ischemic encephalopathy (HIE). We hypothesized vitamin D metabolism would be dysregulated in neonatal HIE altering specific cytokines involved in Th17 activation, which might be mitigated by hypothermia. METHODS: We analyzed short-term relationships between 25(OH) and 1,25(OH)2 vitamin D, vitamin D binding protein, and cytokines related to Th17 function in serum samples from a multicenter randomized controlled trial of hypothermia 33 °C for 48 h after HIE birth vs. normothermia in 50 infants with moderate to severe HIE. RESULTS: Insufficiency of 25(OH) vitamin D was observed after birth in 70% of infants, with further decline over the first 72 h, regardless of treatment. 25(OH) vitamin D positively correlated with anti-inflammatory cytokine IL-17E in all HIE infants. However, Th17 cytokine suppressor IL-27 was significantly increased by hypothermia, negating the IL-27 correlation with vitamin D observed in normothermic HIE infants. CONCLUSION: Serum 25(OH) vitamin D insufficiency is present in the majority of term HIE neonates and is related to lower circulating anti-inflammatory IL-17E. Hypothermia does not mitigate vitamin D deficiency in HIE.

ATM Regulation of the Cohesin Complex Is Required for Repression of DNA Replication and Transcription in the Vicinity of DNA Double-Strand Breaks.

IMPLICATIONS: Multiple members of the cohesin complex are involved in the regulation of DNA replication and transcription in the vicinity of DNA double-strand breaks and their role(s) are regulated by the ATM kinase.

Participation of ATM, SMG1, and DDX5 in a DNA Damage-Induced Alternative Splicing Pathway.

Altered cellular responses to DNA damage can contribute to cancer development, progression, and therapeutic resistance. Mutations in key DNA damage response factors occur across many cancer types, and the DNA damage-responsive gene, TP53, is frequently mutated in a high percentage of cancers. We recently reported that an alternative splicing pathway induced by DNA damage regulates alternative splicing of TP53 RNA and further modulates cellular stress responses. Through damage-induced inhibition of the SMG1 kinase, TP53 pre-mRNA is alternatively spliced to generate TP53b mRNA and p53b protein is required for optimal induction of cellular senescence after ionizing radiation-induced DNA damage. Herein, we confirmed and extended these observations by demonstrating that the ATM protein kinase is required for repression of SMG1 kinase activity after ionizing radiation. We found that the RNA helicase and splicing factor, DDX5, interacts with SMG1, is required for alternative splicing of TP53 pre-mRNA to TP53b and TP53c mRNAs after DNA damage, and contributes to radiation-induced cellular senescence. Interestingly, the role of SMG1 in alternative splicing of p53 appears to be distinguishable from its role in regulating nonsense-mediated RNA decay. Thus, ATM, SMG1, and DDX5 participate in a DNA damage-induced alternative splicing pathway that regulates TP53 splicing and modulates radiation-induced cellular senescence.

Classical complement pathway inhibition reduces brain damage in a hypoxic ischemic encephalopathy animal model.

Perinatal hypoxic ischemic encephalopathy (HIE) remains a major contributor of infant death and long-term disability worldwide. The role played by the complement system in this ischemia-reperfusion injury remains poorly understood. In order to better understand the role of complement activation and other modifiable mechanisms of injury in HIE, we tested the dual-targeting anti-inflammatory peptide, RLS-0071 in an animal model of HIE. Using the well-established HIE rat pup model we measured the effects of RLS-0071 during the acute stages of the brain injury and on long-term neurocognitive outcomes. Rat pups subject to hypoxia-ischemia insult received one of 4 interventions including normothermia, hypothermia and RLS-0071 with and without hypothermia. We measured histopathological effects, brain C1q levels and neuroimaging at day 1 and 21 after the injury. A subset of animals was followed into adolescence and evaluated for neurocognitive function. On histological evaluation, RLS-0071 showed neuronal protection in combination with hypothermia (P = 0.048) in addition to reducing C1q levels in the brain at 1hr (P = 0.01) and at 8 hr in combination with hypothermia (P = 0.005). MRI neuroimaging demonstrated that RLS-0071 in combination with hypothermia reduced lesion volume at 24 hours (P<0.05) as well as decreased T2 signal at day 21 in combination with hypothermia (P<0.01). RLS-0071 alone or in combination with hypothermia improved both short-term and long-term memory. These findings suggest that modulation by RLS-0071 can potentially decrease brain damage resulting from HIE.

Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1.

The ATR kinase controls cell cycle transitions and the DNA damage response. ATR activity is regulated through two ATR-activating proteins, ETAA1 and TOPBP1. To examine how each activator contributes to ATR signaling, we used quantitative mass spectrometry to identify changes in protein phosphorylation in ETAA1- or TOPBP1-deficient cells. We identified 724, 285, and 118 phosphosites to be regulated by TOPBP1, ETAA1, or both ATR activators, respectively. Gene ontology analysis of TOPBP1- and ETAA1-dependent phosphoproteins revealed TOPBP1 to be a primary ATR activator for replication stress, while ETAA1 regulates mitotic ATR signaling. Inactivation of ATR or ETAA1, but not TOPBP1, results in decreased Aurora B kinase activity during mitosis. Additionally, ATR activation by ETAA1 is required for proper chromosome alignment during metaphase and for a fully functional spindle assembly checkpoint response. Thus, we conclude that ETAA1 and TOPBP1 regulate distinct aspects of ATR signaling with ETAA1 having a dominant function in mitotic cells.

An intrinsic S/G2 checkpoint enforced by ATR.

The cell cycle is strictly ordered to ensure faithful genome duplication and chromosome segregation. Control mechanisms establish this order by dictating when a cell transitions from one phase to the next. Much is known about the control of the G1/S, G2/M, and metaphase/anaphase transitions, but thus far, no control mechanism has been identified for the S/G2 transition. Here we show that cells transactivate the mitotic gene network as they exit the S phase through a CDK1 (cyclin-dependent kinase 1)-directed FOXM1 phosphorylation switch. During normal DNA replication, the checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is activated by ETAA1 to block this switch until the S phase ends. ATR inhibition prematurely activates FOXM1, deregulating the S/G2 transition and leading to early mitosis, underreplicated DNA, and DNA damage. Thus, ATR couples DNA replication with mitosis and preserves genome integrity by enforcing an S/G2 checkpoint.

ETAA1 acts at stalled replication forks to maintain genome integrity.

The ATR checkpoint kinase coordinates cellular responses to DNA replication stress. Budding yeast contain three activators of Mec1 (the ATR orthologue); however, only TOPBP1 is known to activate ATR in vertebrates. We identified ETAA1 as a replication stress response protein in two proteomic screens. ETAA1-deficient cells accumulate double-strand breaks, sister chromatid exchanges, and other hallmarks of genome instability. They are also hypersensitive to replication stress and have increased frequencies of replication fork collapse. ETAA1 contains two RPA-interaction motifs that localize ETAA1 to stalled replication forks. It also interacts with several DNA damage response proteins including the BLM/TOP3α/RMI1/RMI2 and ATR/ATRIP complexes. It binds ATR/ATRIP directly using a motif with sequence similarity to the TOPBP1 ATR-activation domain; and like TOPBP1, ETAA1 acts as a direct ATR activator. ETAA1 functions in parallel to the TOPBP1/RAD9/HUS1/RAD1 pathway to regulate ATR and maintain genome stability. Thus, vertebrate cells contain at least two ATR-activating proteins.

Analysis of cost outliers after gastric bypass surgery: what can we learn?

BACKGROUND: A clinical pathway for gastric bypass surgery (GBS) implemented at our institution in 1999 resulted in reduced costs and decreased variability in patient care. However, a reanalysis of GBS hospital costs identified a 16% incidence of "cost outliers". We hypothesized that analysis of clinical variables would identify factors associated with increased hospital costs following GBS. METHODS: Medical records and financial data for 91 GBS patients from November 2000 to July 2001 were reviewed. Patients with costs >1 SD above the total hospital cost mean comprised the cost outlier (CO) group, while the remaining patients were considered the normal cost (NC) group. Potential etiologies for COs included patient demographics, the number and severity of medical co-morbidities, surgical factors, and major postoperative complications. RESULTS: There were 15 patients in the CO group, and 76 patients in the NC group. Patient demographics were similar in both groups. Diabetes mellitus and severe medical co-morbidities, especially sleep apnea and degenerative joint disease were more common in the CO group (60% vs 9.2%, P < 0.05 vs NC). The incidence of major complications (33% vs 8%) was significantly increased in the CO group (P < 0.05 vs NC). CONCLUSIONS: Despite utilization of a clinical pathway for GBS, 16% of patients were "cost outliers". Factors associated with increased hospital costs after GBS included severe medical co-morbidities (especially diabetes mellitus and sleep apnea) and the occurrence of major postoperative complications. Prospective identification of "high risk" GBS patients may allow hospitals with bariatric surgery programs to modify perioperative care and eliminate potential cost outliers.

Comparison of carbon dioxide and iodinated contrast for cavography prior to inferior vena cava filter placement.

BACKGROUND: The use of iodinated contrast in the critically ill trauma patient has been associated with the development of acute renal failure. The low incidence of nephrotoxicity associated with carbon dioxide (CO(2)) makes it an ideal contrast agent for cavography. However, the use of CO(2) has been limited, because reportedly it underestimates the diameter of the inferior vena cava (IVC). METHODS: During a 6-month period (January 2000 through June 2000), 25 adult trauma patients required IVC filter placement. Bedside cavagrams using CO(2) followed by iodinated contrast were employed to determine the diameter of the IVC and the anatomy of the renal veins. RESULTS: Using CO(2) injection for cavography, we were able to determine the diameter of the IVC and the anatomy of the renal veins in all patients. Furthermore, when CO(2) cavography was compared with the results obtained with iodinated contrast, the difference in diameter of the IVC was within 1 mm. CONCLUSIONS: Based on these data, it was determined that CO(2) cavagrams accurately reflect the diameter of the IVC and the anatomy of the renal veins. Additionally, CO(2) cavagrams can be safely performed in the intensive care unit during bedside placement of IVC filters.

Laparoscopic management of traumatic hemorrhagic cholecystitis.

BACKGROUND AND OBJECTIVES: Blunt trauma to the gallbladder is a rare entity, particularly when no other organ is injured. In isolated blunt traumatic injury to the gallbladder, treatment options vary depending on the specific injury. The types of blunt trauma injuries to the gallbladder and their appropriate management are discussed. In addition, a case successfully managed with minimally invasive techniques is presented. METHODS: A passenger admitted after a high-speed front-end motor vehicle crash was safely managed with laparoscopic surgery for a rare case of isolated gallbladder trauma. The preoperative and operative management are discussed as well as the application of minimally invasive surgery for this rare process. RESULTS: Laparoscopic cholecystectomy was performed successfully. The patient did well postoperatively with no complications. No other injuries were identified at the time of laparoscopy. CONCLUSION: Minimally invasive techniques may be safely applied to blunt trauma of the gallbladder in certain circumstances.

Reliability and validity of Endotower, a virtual reality trainer for angled endoscope navigation.

We hypothesized that a simulator designed to train surgical novices angled laparoscopic navigation would show an improvement in subjects' performance, a high test re-test reliability and high internal validity as measured by standardized coefficient alpha. It was also predicted that simulator performance would be strongly related to objectively assess perceptual and visuospatial ability. EndoTower has good face validity in that it mimics precisely the performance of an angled laparoscope and previous studies suggest construct validity. In this study, EndoTower is shown to be a trainer intended for laparoscopic skill than can test for inherent perceptual and visuospatial ability.