Formation of Trideuteromethylated Artifacts of Pyrrole-Containing Natural Products.

Pyrrole-containing natural products form a large group of structurally diverse compounds that occur in both terrestrial and marine organisms. In the present study the formation of trideuteromethylated artifacts of pyrrole-containing natural products was investigated, focusing on the discorhabdins. Three deuterated discorhabdins, 1, 3, and 5, were identified to be isolation procedure artifacts caused by the presence of DMSO-d6 during NMR sample preparation and handling. Three additional semisynthetic derivatives, 7-9, were made during the investigation of the mechanism of formation, which was shown to be driven by trideuteromethyl radicals in the presence of water, methanol, TFA, and traces of iron in the deuterated solvent. Generation of trideuteromethylated artifacts was also confirmed for other classes of pyrrole-containing metabolites, namely, makaluvamines, tambjamines, and dibromotryptamines, which had also been dissolved in DMSO-d6 during the structure elucidation process. Semisynthetic discorhabdins were assessed for antiproliferative activity against a panel of human tumor cell lines, and 14-trideuteromethyldiscorhabdin L (3) averaged low micromolar potency.

Digitally enabled hemovigilance allows real time response to transfusion reactions.

BACKGROUND: Transfusion carries a risk of transfusion reaction that is often underdiagnosed due to reliance on passive reporting. The study investigated the utility of digital methods to identify potential transfusion reactions, thus allowing real-time intervention for affected patients. METHOD: The hemovigilance unit monitored 3856 patients receiving 43,515 transfusions under the hemovigilance program. Retrospective comparison data included 298,498 transfusions. Transfusion medicine physicians designed and validated algorithms in the electronic health record that analyze discrete data, such as vital sign changes, to assign a risk score during each transfusion. Dedicated hemovigilance nurses remotely monitor all patients and perform real-time chart reviews prioritized by risk score. When a reaction is suspected, a hemovigilance trained licensed clinician responds to manage the patient and ensure data collection. Board-certified transfusion medicine physicians reviewed data and classified transfusion reactions under various categories according to the Centers for Disease Control hemovigilance definitions. RESULTS: Transfusion medicine physicians diagnosed 564 transfusion reactions (1.3% of transfusions)-a 524% increase compared to the previous passive reporting. The rapid response provider reached the bedside on average at 12.4 min demonstrating logistic feasibility. While febrile reactions were most diagnosed, recognition of transfusion-associated circulatory overload demonstrated the greatest relative increase. Auditing and education programs further enhanced transfusion reaction awareness. DISCUSSION: The model of digitally-enabled expert real-time review of clinical data that prompts rapid response improved recognition of transfusion reactions. This approach could be applied to other patient deterioration events such as early identification of sepsis.

Oncology Patients Who Develop Transfusion-Associated Circulatory Overload: An Observational Study.

BACKGROUND: Transfusion-associated circulatory overload (TACO) is a largely preventable transfusion complication that results in significant morbidity and mortality. Cancers, related treatments, and comorbidities are among the factors that can predispose patients to TACO, but currently there are limited data on this topic in the literature. METHODS: We collected data retrospectively from the electronic health records of 93 adult patients with cancer who met Centers for Disease Control and Prevention (CDC) criteria for TACO from July 1, 2019, through October 31, 2020. The parameters we studied included demographics, comorbidities, treatment modalities, transfusion practices, and outcomes. We summarized data by means and ranges for continuous variables, and proportions for categorical variables. RESULTS: During the study period, the incidence of TACO among oncology patients was 0.84 per 1000 transfusions (95% CI, 0.68-1.02), representing 6.6% of all reactions. This percentage is high, compared with 1%-6% among other populations. Unique characteristics such as hematology malignancy (75.3%), receipt of cardiotoxic chemotherapy (87.1%), pneumonia (57.0%), preexisting oxygen use (59.1%), dyspnea (62.4%), hypertension (55.9%), renal insufficiency (46.2%), daily use of corticosteroids (43.0%), daily use of diuretics (40.9%), daily use of beta-blockers (36.6%), and elevated NT-proBNP (33.3%) were frequently observed in these group of oncology patients. CONCLUSIONS: Our study indicates that oncology patients have unique factors that may lead to diagnosis of TACO. Developing appropriate guidelines that apply to oncology patients, in addition to those set forth by the CDC, should be considered. Implementation by ordering healthcare providers of a tools that can predict TACO can help in early recognition and mitigation of TACO.

Recifin A, Initial Example of the Tyr-Lock Peptide Structural Family, Is a Selective Allosteric Inhibitor of Tyrosyl-DNA Phosphodiesterase I.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the FDA-approved topoisomerase inhibitors topotecan and irinotecan. High-throughput screening of natural product extract and fraction libraries for inhibitors of TDP1 activity resulted in the discovery of a new class of knotted cyclic peptides from the marine sponge Axinella sp. Bioassay-guided fractionation of the source extract resulted in the isolation of the active component which was determined to be an unprecedented 42-residue cysteine-rich peptide named recifin A. The native NMR structure revealed a novel fold comprising a four strand antiparallel ß-sheet and two helical turns stabilized by a complex disulfide bond network that creates an embedded ring around one of the strands. The resulting structure, which we have termed the Tyr-lock peptide family, is stabilized by a tyrosine residue locked into three-dimensional space. Recifin A inhibited the cleavage of phosphodiester bonds by TDP1 in a FRET assay with an IC50 of 190 nM. Enzyme kinetics studies revealed that recifin A can specifically modulate the enzymatic activity of full-length TDP1 while not affecting the activity of a truncated catalytic domain of TDP1 lacking the N-terminal regulatory domain (Δ1-147), suggesting an allosteric binding site for recifin A on the regulatory domain of TDP1. Recifin A represents both the first of a unique structural class of knotted disulfide-rich peptides and defines a previously unseen mechanism of TDP1 inhibition that could be productively exploited for potential anticancer applications.

Transfusion reactions in pediatric and adolescent young adult haematology oncology and immune effector cell patients.

BACKGROUND: Active surveillance for transfusion reactions is critically important among pediatric patients undergoing chemotherapy. Among pediatric-adolescent-young-adult (AYA) hematology/oncology patients, who have been typically excluded from transfusion reaction studies, this profile remains poorly characterized. METHODS: We assessed the incidence and clinical characteristics of transfusion reactions (n = 3246 transfusions) in this population (n = 201 patients) at our center. FINDINGS: The incidence of adjudicated transfusion reactions was 2·04%. The incidence was higher for platelet (2·78%) compared to packed red blood cell transfusions (1·49%) (p = 0·0149). The majority (61·4%) of all reactions were classified as febrile non-haemolytic transfusion, while 35·7% were considered allergic, and 2·9% were classified as transfusion-associated circulatory overload. The incidence of transfusion reactions in patients who were pre-medicated was higher (2·51%) than in patients who were not (1·52%) (p = 0·0406). Sub-set analysis revealed a 3·95% incidence of adjudicated transfusion reactions among recipients of immune effector cells (IECs) (n = 3), all of which occurred during the potential window for cytokine release syndrome; two-thirds of these reactions were severe/potentially life-threatening. INTERPRETATION: The incidence of transfusion reactions among pediatric-AYA hematology/oncology patients may be lower than the general pediatric population. Patients with a prior history of transfusion reactions and those receiving platelet transfusions may be at higher risk for reaction. From our limited sample, IEC recipients may be at risk for severe transfusion reactions. Large multi-center prospective studies are needed to characterize transfusion reactions in this population. Appropriate characterization of reactions in this population may inform risk stratification and mitigate missed opportunities for prompt recognition and appropriate management. FUNDING: None.

Multiphase Assembly of Small Molecule Microcrystalline Peptide Hydrogel Allows Immunomodulatory Combination Therapy for Long-Term Heart Transplant Survival.

Combination therapies that target multiple pathways involved in immune rejection of transplants hold promise for patients in need of restorative surgery. Herein, a noninteracting multiphase molecular assembly approach is developed to crystallize tofacitinib, a potent JAK1/3 inhibitor, within a shear-thinning self-assembled fibrillar peptide hydrogel network. The resulting microcrystalline tofacitinib hydrogel (MTH) can be syringe-injected directly to the grafting site during surgery to locally deliver the small molecule. The rate of drug delivered from MTH is largely controlled by the dissolution of the encapsulated microcrystals. A single application of MTH, in combination with systemically delivered CTLA4-Ig, a co-stimulation inhibitor, affords significant graft survival in mice receiving heterotopic heart transplants. Locoregional studies indicate that the local delivery of tofacitinib at the graft site enabled by MTH is required for the observed enhanced graft survival.

Overview of COVID-19 testing and implications for otolaryngologists.

BACKGROUND: Testing for SARS-CoV-2 is important for decision making prior to surgery in otolaryngology. An understanding of current and developing testing methods is important for interpreting test results. METHODS: We performed a literature review of current evidence surrounding SARS-CoV-2 diagnostic testing highlighting its utility, limitations, and implications for otolaryngologists. RESULTS: The currently accepted RT-PCR test for SARS-CoV-2 has varying sensitivity according to which subsite of the aerodigestive tract is sampled. Nasal swab sensitivities appear to be about 70%. Chest CT imaging for screening purposes is not currently recommended. CONCLUSION: Due to the current sensitivity of RT-PCR based testing for SARS-CoV-2, a negative test cannot rule out COVID-19. Full PPE should be worn during high-risk procedures such as aerosol generating procedures even if testing is negative. Patients who test positive during screening should have their surgeries postponed if possible until asymptomatic and have tested negative for SARS-CoV-2.

Fifteen-Year Journey to High Reliability in Pathology and Laboratory Medicine.

Many high-reliability organizations in industries outside of health care have sustained high levels of excellence and prevention of harm while managing complex systems and risk. To date, no health care organizations has organized its efforts to achieve highly reliable results despite several decades of improvement science. Laboratorians were early adopters of quality initiatives and process improvements. In the late 1990s, the Division of Pathology and Laboratory Medicine at The University of Texas MD Anderson Cancer Center embarked on a major effort to improve quality and patient safety and to reduce waste. This article describes the institution's journey toward approaching high reliability with the intent to share not only the tools and best practices, but also the ongoing reassessment of the problems detected on the journey. The authors hope that their experience will help the reader develop interventions to adapt in their own environment to facilitate more optimal patient care.

Preoperative autologous blood collection before bone marrow harvests in haploidentical related donors: is it justified?

BACKGROUND: With the increasing safety of allogeneic blood supply and declining need for transfusion due to patient blood management, the practice of preoperative autologous donation (PAD) continues to decline. The practice gained popularity during the 1980s and 1990s with the emergence of transfusion-transmitted human immunodeficiency virus and hepatitis C. At the peak of this public concern, the National Marrow Donor Program recommended that marrow donors have 1 to 3 autologous units of blood collected before their marrow harvest to minimize the likelihood of allogeneic transfusion. After three decades, the practice remains prevalent in marrow donors. We aimed to study the efficacy of PAD in healthy marrow donors. STUDY DESIGN AND METHODS: PADs performed before marrow harvest in healthy donors at our center between January 2013 and July 2015 were reviewed. The utilization of autologous units and decrease in hemoglobin levels due to PAD and marrow harvest were studied. Similar practices were assessed in the rest of the United States through a brief survey. RESULTS: Of a total of 262 autologous units collected from 136 donors, 25.2% were wasted. Ninety-nine percent of the marrow donors received at least 1 unit of blood irrespective of the need. PAD contributed to preoperative anemia, exposing three donors to allogeneic blood transfusion. The survey results showed a mixed response with some institutions continuing and others not practicing PAD. CONCLUSION: PADs are not justified in healthy marrow donors as they expose them to a risk of preoperative anemia and hence a greater risk of transfusion.

False-positive HIV nucleic acid amplification testing during CAR T-cell therapy.

Advancements in immunotherapy have opened a new era in oncology, to include genetic modification of human T-cells to express a chimeric antigen receptor (CAR) that enables targeted tumor recognition (Kochenderfer et al., 2015; Lee et al., 2015; Maus and Levine 2016; Rosenberg et al., 2008). Herein, we report a false-positive HIV testing in a patient who had undergone CAR T-cell therapy created with a lentiviral vector.

Exploring the influence of indololactone structure on selectivity for binding to the C1 domains of PKCα, PKCε, and RasGRP.

C1 domain-containing proteins, such as protein kinase C (PKC), have a central role in cellular signal transduction. Their involvement in many diseases, including cancer, cardiovascular disease, and immunological and neurological disorders has been extensively demonstrated and has prompted a search for small molecules to modulate their activity. By employing a diacylglycerol (DAG)-lactone template, we have been able to develop ultra potent analogs of diacylglycerol with nanomolar binding affinities approaching those of complex natural products such as phorbol esters and bryostatins. One current challenge is the development of selective ligands capable of discriminating between different protein family members. Recently, structure-activity relationship studies have shown that the introduction of an indole ring as a DAG-lactone substituent yielded selective Ras guanine nucleotide-releasing protein (RasGRP1) activators when compared to PKCα and PKCε. In the present work, we examine the effects of ligand selectivity relative to the orientation of the indole ring and the nature of the DAG-lactone template itself. Our results show that the indole ring must be attached to the lactone moiety through the sn-2 position in order to achieve RasGRP1 selectivity.

Cancer type predicts alloimmunization following RhD-incompatible RBC transfusions.

BACKGROUND: Immunosuppressed, RhD-negative oncology patients tend to have lower rates of sensitization to the D antigen when they receive transfusion with RhD-positive blood components. Clinical factors associated with alloimmunization to the D antigen in RhD-negative oncology patients when they receive transfusion with RhD-positive red blood cells (RBCs) have not been well defined. STUDY DESIGN AND METHODS: This was a 4-year, retrospective analysis identifying RhD-negative oncology patients who received RhD-positive RBCs and were not previously alloimmunized to the D antigen. Age, sex, race, ABO group, primary oncology diagnosis, and numbers of RhD-incompatible RBC transfusions were recorded. The association between antibody formation and clinical factors was studied. The incidence of alloanti-D was calculated from a subsequent antibody-detection test performed at least 28 days after receipt of the first transfusion of RhD-positive RBCs. RESULTS: In total, 545 RhD-negative oncology patients received 4295 RhD-positive RBC transfusions. Of these, 76 (14%) became alloimmunized to the D antigen. Diagnosis type was the only factor significantly associated with responder status. The logistic regression model indicated that patients who had myelodysplastic syndrome or solid malignancies were more likely to be responders than those who had acute leukemia. CONCLUSION: We measured a 14% sensitization rate to the D antigen in our RhD-negative oncology population. The rate of alloimmunization was higher in patients who had solid cancers (22.6%) or myelodysplastic syndrome (23%) compared with those who had other hematologic malignancies (7%). Knowledge of diagnoses that predispose to RhD alloimmunization enables better utilization of RhD-negative RBCs during times of shortage.

A young male runner with a posterior knee mass--not just your typical Baker's cyst.

We present the case of a 28-year-old man with a growing mass in his right popliteal fossa causing pain on exertion. The differential diagnosis included Baker's cyst, entrapment syndrome of the popliteal artery, as well as a benign or malignant neoplasm. An ultrasound was non-specific. Follow-up MRI of the knee demonstrated cystic adventitial disease (CAD). With only about 500 cases reported in the literature since its discovery in 1947, CAD is a rare entity. The disease is characterised by mucinous or gelatinous cysts in the arterial or venous adventitia. The disease is predominantly seen in the popliteal artery and typically affects otherwise healthy males in the fourth to fifth decade of life. It presents clinically as intermittent exertional claudication. Examination of our case and a review of the literature will highlight the importance of considering CAD in patients who report of a popliteal mass and intermittent claudication.

Failure of a Single Varicella Vaccination to Protect Children With Cancer From Life-Threatening Breakthrough Varicella.

We report 2 children with life-threatening breakthrough varicella. Both had received 1 varicella vaccination before onset of cancer. Despite treatment with intravenous acyclovir, 1 child died of disseminated varicella. Because similar fatal cases have been reported, high-risk immunocompromised children with 1 varicella vaccination may warrant the same varicella prophylaxis as immunocompromised children who have never been vaccinated.

Molecular genotyping of human papillomavirus l1 gene in low-risk and high-risk populations in Bangkok.

BACKGROUND: Human papillomavirus (HPV) infections in Thailand are a public health concern, but information on HPV infection in sex workers and men who have sex with men (MSM) is limited. The aim of this study was to measure the prevalence and genotype distribution of HPV among low- and high-risk, HIV-negative populations. METHODS: A total of 300 participants were categorized as general women, female sex workers, MSM, and MSM sex workers. Human papillomavirus infections were identified by the Papanicolaou test and nested polymerase chain reaction. A phylogenetic analysis of partial HPV L1 genes was performed. RESULTS: Abnormal cytology was found in 5% of general women, 10% of female sex workers, 24% of MSM, and 28% of MSM sex workers. Human papillomavirus was detected in 9% of general women, 13% of female sex workers, and 30% in both MSM and the MSM sex workers. The prevalence of HPV high-risk genotypes was significantly higher in female sex workers and MSM, whereas low-risk genotypes and genital warts were significantly higher in MSM sex workers. Significantly more patients with genital warts and cervical intraepithelial neoplasia I/anal intraepithelial neoplasia I harbored low-risk genotypes, whereas those with cervical intraepithelial neoplasia II/anal intraepithelial neoplasia II harbored high-risk genotypes. CONCLUSIONS: High- and low-risk HPV genotypes persist in high-risk groups in Bangkok. Some genotypes infecting at-risk populations are not vaccine preventable. These findings may help to elucidate the prevalence of HPV infections in Thailand and serve as the basis for additional investigations into risk factors for these populations.

Mono-anionic phosphopeptides produced by unexpected histidine alkylation exhibit high Plk1 polo-box domain-binding affinities and enhanced antiproliferative effects in HeLa cells.

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics.

Design and synthesis of Fmoc-Thr[PO(OH)(OPOM)] for the preparation of peptide prodrugs containing phosphothreonine in fully protected form.

The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported. This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols. It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl protection. The methodology allows the first reported synthesis of pThr-containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.

Synthesis, biological, and biophysical studies of DAG-indololactones designed as selective activators of RasGRP.

The development of selective agents capable of discriminating between protein kinase C (PKC) isoforms and other diacylglycerol (DAG)-responsive C1 domain-containing proteins represents an important challenge. Recent studies have highlighted the role that Ras guanine nucleotide-releasing protein (RasGRP) isoforms play both in immune responses as well as in the development of prostate cancer and melanoma, suggesting that the discovery of selective ligands could have potential therapeutic value. Thus far, the N-methyl-substituted indololactone 1 is the agonist with the highest reported potency and selectivity for RasGRP relative to PKC. Here we present the synthesis, binding studies, cellular assays and biophysical analysis of interactions with model membranes of a family of regioisomers of 1 (compounds 2-5) that differ in the position of the linkage between the indole ring and the lactone moiety. These structural variations were studied to explore the interaction of the active complex (C1 domain-ligand) with cellular membranes, which is believed to be an important factor for selectivity in the activation of DAG-responsive C1 domain containing signaling proteins. All compounds were potent and selective activators of RasGRP when compared to PKCα with selectivities ranging from 6 to 65 fold. However, the parent compound 1 was appreciably more selective than any of the other isomers. In intact cells, modest differences in the patterns of translocation of the C1 domain targets were observed. Biophysical studies using giant vesicles as model membranes did show substantial differences in terms of molecular interactions impacting lipid organization, dynamics and membrane insertion. However, these differences did not yield correspondingly large changes in patterns of biological response, at least for the parameters examined.