Toxoplasma gondii virulence factor ROP1 reduces parasite susceptibility to murine and human innate immune restriction.

Toxoplasma gondii is an intracellular parasite that can infect many host species and is a cause of significant human morbidity worldwide. T. gondii secretes a diverse array of effector proteins into the host cell which are critical for infection. The vast majority of these secreted proteins have no predicted functional domains and remain uncharacterised. Here, we carried out a pooled CRISPR knockout screen in the T. gondii Prugniaud strain in vivo to identify secreted proteins that contribute to parasite immune evasion in the host. We demonstrate that ROP1, the first-identified rhoptry protein of T. gondii, is essential for virulence and has a previously unrecognised role in parasite resistance to interferon gamma-mediated innate immune restriction. This function is conserved in the highly virulent RH strain of T. gondii and contributes to parasite growth in both murine and human macrophages. While ROP1 affects the morphology of rhoptries, from where the protein is secreted, it does not affect rhoptry secretion. Finally, we show that ROP1 co-immunoprecipitates with the host cell protein C1QBP, an emerging regulator of innate immune signaling. In summary, we identify putative in vivo virulence factors in the T. gondii Prugniaud strain and show that ROP1 is an important and previously overlooked effector protein that counteracts both murine and human innate immunity.

Divergent kinase regulates membrane ultrastructure of the Toxoplasma parasitophorous vacuole.

Apicomplexan parasites replicate within a protective organelle, called the parasitophorous vacuole (PV). The Toxoplasma gondii PV is filled with a network of tubulated membranes, which are thought to facilitate trafficking of effectors and nutrients. Despite being critical to parasite virulence, there is scant mechanistic understanding of the network's functions. Here, we identify the parasite-secreted kinase WNG1 (With-No-Gly-loop) as a critical regulator of tubular membrane biogenesis. WNG1 family members adopt an atypical protein kinase fold lacking the glycine rich ATP-binding loop that is required for catalysis in canonical kinases. Unexpectedly, we find that WNG1 is an active protein kinase that localizes to the PV lumen and phosphorylates PV-resident proteins, several of which are essential for the formation of a functional intravacuolar network. Moreover, we show that WNG1-dependent phosphorylation of these proteins is required for their membrane association, and thus their ability to tubulate membranes. Consequently, WNG1 knockout parasites have an aberrant PV membrane ultrastructure. Collectively, our results describe a unique family of Toxoplasma kinases and implicate phosphorylation of secreted proteins as a mechanism of regulating PV development during parasite infection.

Detectable High-Sensitivity Cardiac Troponin within the Population Reference Interval Conveys High 5-Year Cardiovascular Risk: An Observational Study.

BACKGROUND: Increased cardiac troponin I or T detected by high-sensitivity assays (hs-cTnI or hs-cTnT) confers an increased risk of adverse prognosis. We determined whether patients presenting with putatively normal, detectable cTn concentrations [> limit of detection and < upper reference limit (URL)] have increased risk of major adverse cardiovascular events (MACE) or all-cause mortality. METHODS: A prospective 5-year follow-up of patients recruited in the emergency department with possible acute coronary syndrome (ACS) and cTn concentrations measured with hs-cTnI (Abbott) and hs-cTnT (Roche) assays. Cox regression models were generated with adjustment for covariates in those without MACE on presentation. Hazard ratios (HRs) for hs-cTn were calculated relative to the HRs at the median concentration. RESULTS: Of 1113 patients, 836 were without presentation MACE. Of these, 138 incurred a MACE and 169 died during a median 5.8-year follow-up. HRs for MACE at the URLs were 2.3 (95% CI, 1.7-3.2) for hs-cTnI and 1.8 (95% CI, 1.3-2.4) for hs-cTnT. Corresponding HRs for mortality were 1.7 (95% CI, 1.2-2.2) for hs-cTnI and 2.3 (95 % CI, 1.7-3.1) for hs-cTnT. The HR for MACE increased with increasing hs-cTn concentration similarly for both assays, but the HR for mortality increased at approximately twice the rate for hs-cTnT than hs-cTnI. Patients with hs-cTnI ≥10 ng/L or hs-cTnT ≥16 ng/L had the same percentage of MACE at 5-year follow-up (33%) as patients with presentation MACE. CONCLUSIONS: Many patients with ACS ruled out and putatively normal but detectable hs-cTnI concentrations are at similar long-term risk as those with MACE. hs-cTnT concentrations are more strongly associated with 5-year mortality than hs-cTnI.

Rapid Rule-out of Acute Myocardial Infarction With a Single High-Sensitivity Cardiac Troponin T Measurement Below the Limit of Detection: A Collaborative Meta-analysis.

Background: High-sensitivity assays for cardiac troponin T (hs-cTnT) are sometimes used to rapidly rule out acute myocardial infarction (AMI). Purpose: To estimate the ability of a single hs-cTnT concentration below the limit of detection (<0.005 µg/L) and a nonischemic electrocardiogram (ECG) to rule out AMI in adults presenting to the emergency department (ED) with chest pain. Data Sources: EMBASE and MEDLINE without language restrictions (1 January 2008 to 14 December 2016). Study Selection: Cohort studies involving adults presenting to the ED with possible acute coronary syndrome in whom an ECG and hs-cTnT measurements were obtained and AMI outcomes adjudicated during initial hospitalization. Data Extraction: Investigators of studies provided data on the number of low-risk patients (no new ischemia on ECG and hs-cTnT measurements <0.005 µg/L) and the number who had AMI during hospitalization (primary outcome) or a major adverse cardiac event (MACE) or death within 30 days (secondary outcomes), by risk classification (low or not low risk). Two independent epidemiologists rated risk of bias of studies. Data Synthesis: Of 9241 patients in 11 cohort studies, 2825 (30.6%) were classified as low risk. Fourteen (0.5%) low-risk patients had AMI. Sensitivity of the risk classification for AMI ranged from 87.5% to 100% in individual studies. Pooled estimated sensitivity was 98.7% (95% CI, 96.6% to 99.5%). Sensitivity for 30-day MACEs ranged from 87.9% to 100%; pooled sensitivity was 98.0% (CI, 94.7% to 99.3%). No low-risk patients died. Limitation: Few studies, variation in timing and methods of reference standard troponin tests, and heterogeneity of risk and prevalence of AMI across studies. Conclusion: A single hs-cTnT concentration below the limit of detection in combination with a nonischemic ECG may successfully rule out AMI in patients presenting to EDs with possible emergency acute coronary syndrome. Primary Funding Source: Emergency Care Foundation.

Validating the Manchester Acute Coronary Syndromes (MACS) and Troponin-only Manchester Acute Coronary Syndromes (T-MACS) rules for the prediction of acute myocardial infarction in patients presenting to the emergency department with chest pain.

BACKGROUND: The Manchester Acute Coronary Syndromes (MACS) rule and the Troponin-only MACS (T-MACS) rule risk stratify patients with suspected acute coronary syndrome (ACS). This observational study sought to validate and compare the MACS and T-MACS rules for assessment of acute myocardial infarction (AMI). METHODS: Prospectively collected data from twoEDs in Australia and New Zealand were analysed. Patients were assigned a probability of ACS based on the MACS and T-MACS rules, incorporating high-sensitivity troponin T, heart-type fatty acid-binding protein, ECG results and clinical symptoms. Patients were then deemed very low risk, low risk, intermediate or high risk if their MACS probability was less than 2%, between 2% and 5%, between 5% and 95% and greater than 95%, respectively. The primary endpoint was 30-day diagnosis of AMI. The secondary endpoint was 30-day major adverse cardiac event (MACE) including AMI, revascularisation or coronary stenosis (>70%). Sensitivity, specificity and predictive values were calculated to assess the accuracy of the MACS and T-MACS rules. RESULTS: Of the 1244 patients, 114 (9.2%) were diagnosed with AMI and 163 (13.1%) with MACE. The MACS and T-MACS rules categorised 133 (10.7%) and 246 (19.8%) patients, respectively, as very low risk and potentially suitable for early discharge from the ED. There was one false negative case for both rules making sensitivity 99.1% (95.2%-100%). CONCLUSIONS: MACS and T-MACS accurately risk stratify very low risk patients. The T-MACS rule would allow for more patients to be discharged early. The potential for missed MACE events means that further outpatient testing for coronary artery disease may be required for patients identified as very low risk.

The Type III Secretion System Effector SeoC of Salmonella enterica subsp. salamae and S. enterica subsp. arizonae ADP-Ribosylates Src and Inhibits Opsonophagocytosis.

Salmonella species utilize type III secretion systems (T3SSs) to translocate effectors into the cytosol of mammalian host cells, subverting cell signaling and facilitating the onset of gastroenteritis. In this study, we compared a draft genome assembly of Salmonella enterica subsp. salamae strain 3588/07 against the genomes of S. enterica subsp. enterica serovar Typhimurium strain LT2 and Salmonella bongori strain 12419. S. enterica subsp. salamae encodes the Salmonella pathogenicity island 1 (SPI-1), SPI-2, and the locus of enterocyte effacement (LEE) T3SSs. Though several key S Typhimurium effector genes are missing (e.g., avrA, sopB, and sseL), S. enterica subsp. salamae invades HeLa cells and contains homologues of S. bongori sboK and sboC, which we named seoC SboC and SeoC are homologues of EspJ from enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), which inhibit Src kinase-dependent phagocytosis by ADP-ribosylation. By screening 73 clinical and environmental Salmonella isolates, we identified EspJ homologues in S. bongori, S. enterica subsp. salamae, and Salmonella enterica subsp. arizonae The ß-lactamase TEM-1 reporter system showed that SeoC is translocated by the SPI-1 T3SS. All the Salmonella SeoC/SboC homologues ADP-ribosylate Src E310 in vitro Ectopic expression of SeoC/SboC inhibited phagocytosis of IgG-opsonized beads into Cos-7 cells stably expressing green fluorescent protein (GFP)-FcγRIIa. Concurrently, S. enterica subsp. salamae infection of J774.A1 macrophages inhibited phagocytosis of beads, in a seoC-dependent manner. These results show that S. bongori, S. enterica subsp. salamae, and S. enterica subsp. arizonae share features of the infection strategy of extracellular pathogens EPEC and EHEC and shed light on the complexities of the T3SS effector repertoires of Enterobacteriaceae.

Effectiveness of EDACS Versus ADAPT Accelerated Diagnostic Pathways for Chest Pain: A Pragmatic Randomized Controlled Trial Embedded Within Practice.

STUDY OBJECTIVE: A 2-hour accelerated diagnostic pathway based on the Thrombolysis in Myocardial Infarction score, ECG, and troponin measures (ADAPT-ADP) increased early discharge of patients with suspected acute myocardial infarction presenting to the emergency department compared with standard care (from 11% to 19.3%). Observational studies suggest that an accelerated diagnostic pathway using the Emergency Department Assessment of Chest Pain Score (EDACS-ADP) may further increase this proportion. This trial tests for the existence and size of any beneficial effect of using the EDACS-ADP in routine clinical care. METHODS: This was a pragmatic randomized controlled trial of adults with suspected acute myocardial infarction, comparing the ADAPT-ADP and the EDACS-ADP. The primary outcome was the proportion of patients discharged to outpatient care within 6 hours of attendance, without subsequent major adverse cardiac event within 30 days. RESULTS: Five hundred fifty-eight patients were recruited, 279 in each arm. Sixty-six patients (11.8%) had a major adverse cardiac event within 30 days (ADAPT-ADP 29; EDACS-ADP 37); 11.1% more patients (95% confidence interval 2.8% to 19.4%) were identified as low risk in EDACS-ADP (41.6%) than in ADAPT-ADP (30.5%). No low-risk patients had a major adverse cardiac event within 30 days (0.0% [0.0% to 1.9%]). There was no difference in the primary outcome of proportion discharged within 6 hours (EDACS-ADP 32.3%; ADAPT-ADP 34.4%; difference -2.1% [-10.3% to 6.0%], P=.65). CONCLUSION: There was no difference in the proportion of patients discharged early despite more patients being classified as low risk by the EDACS-ADP than the ADAPT-ADP. Both accelerated diagnostic pathways are effective strategies for chest pain assessment and resulted in an increased rate of early discharges compared with previously reported rates.

Heart Fatty Acid Binding Protein and cardiac troponin: development of an optimal rule-out strategy for acute myocardial infarction.

BACKGROUND: Improved ability to rapidly rule-out Acute Myocardial Infarction (AMI) in patients presenting with chest pain will promote decongestion of the Emergency Department (ED) and reduce unnecessary hospital admissions. We assessed a new commercial Heart Fatty Acid Binding Protein (H-FABP) assay for additional diagnostic value when combined with cardiac troponin (using a high sensitivity assay). METHODS: H-FABP and high-sensitivity troponins I (hs-cTnI) and T (hs-cTnT) were measured in samples taken on-presentation from patients, attending the ED, with symptoms triggering investigation for possible acute coronary syndrome. The optimal combination of H-FABP with each hs-cTn was defined as that which maximized the proportion of patients with a negative test (low-risk) whilst maintaining at least 99 % sensitivity for AMI. A negative test comprised both H-FABP and hs-cTn below the chosen threshold in the absence of ischemic changes on the ECG. RESULTS: One thousand seventy-nine patients were recruited including 248 with AMI. H-FABP < 4.3 ng/mL plus hs-cTnI < 10.0 ng/L together with a negative ECG maintained >99 % sensitivity for AMI whilst classifying 40.9 % of patients as low-risk. The combination of H-FABP < 3.9 ng/mL and hs-cTnT < 7.6 ng/L with a negative ECG maintained the same sensitivity whilst classifying 32.1 % of patients as low risk. CONCLUSIONS: In patients requiring rule-out of AMI, the addition of H-FABP to hs-cTn at presentation (in the absence of new ischaemic ECG findings) may accelerate clinical diagnostic decision making by identifying up to 40 % of such patients as low-risk for AMI on the basis of blood tests performed on presentation. If implemented this has the potential to significantly accelerate triaging of patients for early discharge from the ED.

Structures of the compact helical core domains of feline calicivirus and murine norovirus VPg proteins.

We report the solution structures of the VPg proteins from feline calicivirus (FCV) and murine norovirus (MNV), which have been determined by nuclear magnetic resonance spectroscopy. In both cases, the core of the protein adopts a compact helical structure flanked by flexible N and C termini. Remarkably, while the core of FCV VPg contains a well-defined three-helix bundle, the MNV VPg core has just the first two of these secondary structure elements. In both cases, the VPg cores are stabilized by networks of hydrophobic and salt bridge interactions. The Tyr residue in VPg that is nucleotidylated by the viral NS7 polymerase (Y24 in FCV, Y26 in MNV) occurs in a conserved position within the first helix of the core. Intriguingly, given its structure, VPg would appear to be unable to bind to the viral polymerase so as to place this Tyr in the active site without a major conformation change to VPg or the polymerase. However, mutations that destabilized the VPg core either had no effect on or reduced both the ability of the protein to be nucleotidylated and virus infectivity and did not reveal a clear structure-activity relationship. The precise role of the calicivirus VPg core in virus replication remains to be determined, but knowledge of its structure will facilitate future investigations.

Derivation and Validation of Thresholds Using Synthetic Data Methods for Single-Test Screening of Emergency Department Patients with Possible Acute Myocardial Infarction Using a Point-of-Care Troponin Assay.

BACKGROUND: Single-sample (screening) rule-out of acute myocardial infarction (AMI) with troponin requires derivation of a single-test screening threshold. In data sets with small event numbers, the lowest one or two concentrations of myocardial infarction (MI) patients dictate the threshold. This is not optimal. We aimed to demonstrate a process incorporating both real and synthetic data for deriving such thresholds using a novel pre-production high-precision point-of-care assay. METHODS: cTnI concentrations were measured from thawed plasma using the Troponin I Next (TnI-Nx) assay (i-STAT; Abbott) in adults on arrival to the emergency department with symptoms suggestive of AMI. The primary outcome was an AMI or cardiac death within 30 days. We used internal-external validation with synthetic data production based on clinical and demographic data, plus the measured TnI-Nx concentration, to derive and validate decision thresholds for TnI-Nx. The target low-risk threshold was a sensitivity of 99% and a high-risk threshold specificity of >95%. RESULTS: In total, 1356 patients were included, of whom 191 (14.1%) had the primary outcome. A total of 500 synthetic data sets were constructed. The mean low-risk threshold was determined to be 5 ng/L. This categorized 38% (95% CI, 6%-68%) to low-risk with a sensitivity of 99.0% (95% CI, 98.6%-99.5%) and a negative predictive value of 99.4% (95% CI, 97.6%-99.8%). A similarly derived high-risk threshold of 25 ng/L had a specificity of 95.0% (95% CI, 94.8%-95.1%) and a positive predictive value of 74.8% (95% CI, 71.5%-78.0%). CONCLUSIONS: With the TnI-Nx assay, we successfully demonstrated an approach using synthetic data generation to derive low-risk thresholds for safe and effective screening.

A model of non-elemental olfactory learning in Drosophila.

The pathways for olfactory learning in the fruitfly Drosophila have been extensively investigated, with mounting evidence that that the mushroom body is the site of the olfactory associative memory trace (Heisenberg, Nature 4:266-275, 2003; Gerber et al., Curr Opin Neurobiol 14:737-744, 2004). Heisenberg's description of the mushroom body as an associative learning device is a testable hypothesis that relates the mushroom body's function to its neural structure and input and output pathways. Here, we formalise a relatively complete computational model of the network interactions in the neural circuitry of the insect antennal lobe and mushroom body, to investigate their role in olfactory learning, and specifically, how this might support learning of complex (non-elemental; Giurfa, Curr Opin Neuroethol 13:726-735, 2003) discriminations involving compound stimuli. We find that the circuit is able to learn all tested non-elemental paradigms. This does not crucially depend on the number of Kenyon cells but rather on the connection strength of projection neurons to Kenyon cells, such that the Kenyon cells require a certain number of coincident inputs to fire. As a consequence, the encoding in the mushroom body resembles a unique cue or configural representation of compound stimuli (Pearce, Psychol Rev 101:587-607, 1994). Learning of some conditions, particularly negative patterning, is strongly affected by the assumption of normalisation effects occurring at the level of the antennal lobe. Surprisingly, the learning capacity of this circuit, which is a simplification of the actual circuitry in the fly, seems to be greater than the capacity expressed by the fly in shock-odour association experiments (Young et al. 2010).

Pharmacokinetic comparison of a generic coenzyme Q10 solubilizate and a formulation with soybean phytosterols.

Coenzyme Q10 (CoQ10) is essential for all cells, and deficiency has been implicated in cardiovascular disease. Plant phytosterols inhibit cholesterol absorption, and may thereby also reduce cardiovascular risk. This study compared the relative bioavailability of CoQ10 solubilized in low-dose soybean phytosterols (SterolQ10) with a generic CoQ10 solubilizate. In a randomized, cross-over design, 36 healthy males received a single 100 mg dose of CoQ10, as SterolQ10 or generic CoQ10, with a two-week washout between treatments. Plasma CoQ10 was analysed at baseline, and at 2, 4, 6, 8 and 10 h after supplement ingestion. Subjects were then administered either 100 mg/day of generic CoQ10 or SterolQ10 for 4 weeks. Fasting plasma CoQ10 levels were measured at baseline and following supplementation. The two preparations were bioequivalent in regard to the area under the curve (AUC(0-10h) ) and maximum increase in concentration (C(max) ), with geometric mean ratios of 0.89 (CI 0.81-0.98) and 0.88 (CI 0.80-0.96), respectively. Four-weeks of CoQ10 resulted in a comparable twofold increase in CoQ10 levels for both formulations (p < 0.001), which was similar between preparations (p = 0.74). The combined CoQ10 and phytosterol formulation, SterolQ10, showed bioequivalence to the generic CoQ10 following a single CoQ10 dose, and demonstrated comparable bioavailability following multiple dose administration.

Outpatient synacthen testing in a large metropolitan region: a clinical audit.

AIM: To audit short synacthen tests (SSTs) performed at a single laboratory within the greater Auckland area. METHODS: Two hundred and eighty-seven SSTs conducted in 286 individuals between September 2016 and September 2019 were assessed. Test requests were not triaged. We assessed source of referrals, indications for testing, adequacy of pre-test information and test outcomes. RESULTS: Seventy-one percent of referrals were for women. Fifty-six percent were from primary care and 18% from rheumatology. One-hundred and fifteen (40%) of those referred had been taking corticosteroids within the previous three months: this information was only provided in 49 referrals. In 32% of referrals, no serum cortisol measurement had been undertaken within the previous six months. In 20% of referrals, no indication was provided. Thirty-three (11%) SSTs were abnormal. Of these, 29 were in patients taking corticosteroids. No SSTs were abnormal among 64 patients with pre-test serum cortisol >300nmol/L or >400nmol/L according to the cortisol assay in use. CONCLUSIONS: Referrals for SSTs often lack important information, such as the indication for testing and recent corticosteroid exposure. Up to one quarter of SSTs could be avoided if a serum cortisol was routinely measured prior to referral. Adopting a structured referral form that mandates provision of important clinical and biochemical data might reduce unnecessary testing.

Implementation and evaluation of a rural general practice assessment pathway for possible cardiac chest pain using point-of-care troponin testing: a pilot study.

OBJECTIVES: To assess the feasibility and acceptability, and additionally to preliminarily evaluate, the effectiveness and safety of an accelerated diagnostic chest pain pathway in rural general practice using point-of-care troponin to identify patients at low risk of acute myocardial infarction, avoiding unnecessary patient transfer to hospital and enabling early discharge home. DESIGN: A prospective observational pilot evaluation. SETTING: Twelve rural general (family) practices in the Midlands region of New Zealand. PARTICIPANTS: Patients aged ≥18 years who presented acutely to rural general practice with suspected ischaemic chest pain for whom the doctor intended transfer to hospital for serial troponin measurement. OUTCOME MEASURES: The proportion of patients managed using the low-risk pathway without transfer to hospital and without 30-day major adverse cardiac event (MACE); pathway adherence; rate of 30-day MACE; patient satisfaction with care; and agreement between point-of-care and laboratory measured troponin concentrations. RESULTS: A total of 180 patients were assessed by the pathway. The pathway classified 111 patients (61.7%) as low-risk and all were managed in rural general practice with no 30-day MACE (0%, 95% CI 0.0% to 3.3%). Adherence to the low-risk pathway was 95.5% (106 out of 111). Of the 56 patients classified as non-low-risk and referred to hospital, 9 (16.1%) had a 30-day MACE. A further 13 non-low-risk patients were not transferred to hospital, with no events. The sensitivity of the pathway for 30-day MACE was 100.0% (95% CI 70.1% to 100%). Of low-risk patients, 94% reported good to excellent satisfaction with care. Good concordance was observed between point-of-care and duplicate laboratory measured troponin concentrations. CONCLUSIONS: The use of an accelerated diagnostic chest pain pathway incorporating point-of-care troponin in a rural general practice setting was feasible and acceptable, with preliminary results suggesting that it may safely and effectively reduce the urgent transfer of low-risk patients to hospital.

A prospective multi-centre study assessing the safety and effectiveness following the implementation of an accelerated chest pain pathway using point-of-care troponin for use in New Zealand rural hospital and primary care settings.

AIMS: Most rural hospitals and general practices in New Zealand (NZ) are reliant on point-of-care troponin. A rural accelerated chest pain pathway (RACPP), combining an electrocardiogram (ECG), a structured risk score (Emergency Department Assessment of Chest Pain Score), and serial point-of-care troponin, was designed for use in rural hospital and primary care settings across NZ. The aim of this study was to evaluate the safety and effectiveness of the RACPP. METHODS AND RESULTS: A prospective multi-centre evaluation following implementation of the RACPP was undertaken from 1 July 2018 to 31 December 2020 in rural hospitals, rural and urban general practices, and urgent care clinics. The primary outcome measure was the presence of 30-day major adverse cardiac events (MACEs) in low-risk patients. The secondary outcome was the percentage of patients classified as low-risk that avoided transfer or were eligible for early discharge. There were 1205 patients enrolled in the study. 132 patients were excluded. Of the 1073 patients included in the primary analysis, 474 (44.0%) patients were identified as low-risk. There were no [95% confidence interval (CI): 0-0.3%] MACE within 30 days of the presentation among low-risk patients. Most of these patients (91.8%) were discharged without admission to hospital. Almost all patients who presented to general practice (99%) and urgent care clinics (97.6%) were discharged to home directly. CONCLUSION: The RACPP is safe and effective at excluding MACEs in NZ rural hospital and primary care settings, where it can identify a group of low-risk patients who can be safely discharged home without transfer to hospital.

Targeted Treatment of Triple-Negative Breast Cancer.

ABSTRACT: Triple-negative breast cancer is increasingly recognized as a heterogeneous entity that can be categorized according to histologic, molecular, and clinical subtypes. While chemotherapy remains the backbone of treatment for this disease, there are now several available targeted agents including immunotherapy, poly(adenosine diphosphate-ribose) polymerase inhibitors, and most recently a Food and Drug Administration-approved antibody-drug conjugate sacituzumab govitecan-hziy as a third-line treatment of metastatic triple-negative breast cancer. We review several actionable targets for triple-negative breast cancer and describe promising nonimmunotherapeutic agents including cyclin-dependent kinase inhibitors, androgen receptor inhibitors, mitogen-activated protein kinase inhibitors, phosphoinositide 3-kinase inhibitors, AKT (also known as protein kinase B) inhibitors, and antibody-drug conjugates.

Transplant-associated thrombotic microangiopathy: theoretical considerations and a practical approach to an unrefined diagnosis.

Transplant-associated thrombotic microangiopathy (TA-TMA) is an increasingly recognized complication of hematopoietic stem cell transplant (HSCT) with high morbidity and mortality. The triad of endothelial cell activation, complement dysregulation, and microvascular hemolytic anemia has the potential to cause end organ dysfunction, multiple organ dysfunction syndrome and death, but clinical features mimic other disorders following HSCT, delaying diagnosis. Recent advances have implicated complement as a major contributor and the therapeutic potential of complement inhibition has been explored. Eculizumab has emerged as an effective therapy and narsoplimab (OMS721) has been granted priority review by the FDA. Large studies performed mostly in pediatric patients suggest that earlier recognition and treatment may lead to improved outcomes. Here we present a clinically focused summary of recently published literature and propose a diagnostic and treatment algorithm.

Evidence for a lineage of virulent bacteriophages that target Campylobacter.

BACKGROUND: Our understanding of the dynamics of genome stability versus gene flux within bacteriophage lineages is limited. Recently, there has been a renewed interest in the use of bacteriophages as 'therapeutic' agents; a prerequisite for their use in such therapies is a thorough understanding of their genetic complement, genome stability and their ecology to avoid the dissemination or mobilisation of phage or bacterial virulence and toxin genes. Campylobacter, a food-borne pathogen, is one of the organisms for which the use of bacteriophage is being considered to reduce human exposure to this organism. RESULTS: Sequencing and genome analysis was performed for two Campylobacter bacteriophages. The genomes were extremely similar at the nucleotide level (> or = 96%) with most differences accounted for by novel insertion sequences, DNA methylases and an approximately 10 kb contiguous region of metabolic genes that were dissimilar at the sequence level but similar in gene function between the two phages. Both bacteriophages contained a large number of radical S-adenosylmethionine (SAM) genes, presumably involved in boosting host metabolism during infection, as well as evidence that many genes had been acquired from a wide range of bacterial species. Further bacteriophages, from the UK Campylobacter typing set, were screened for the presence of bacteriophage structural genes, DNA methylases, mobile genetic elements and regulatory genes identified from the genome sequences. The results indicate that many of these bacteriophages are related, with 10 out of 15 showing some relationship to the sequenced genomes. CONCLUSIONS: Two large virulent Campylobacter bacteriophages were found to show very high levels of sequence conservation despite separation in time and place of isolation. The bacteriophages show adaptations to their host and possess genes that may enhance Campylobacter metabolism, potentially advantaging both the bacteriophage and its host. Genetic conservation has been shown to extend to other Campylobacter bacteriophages, forming a highly conserved lineage of bacteriophages that predate upon campylobacters and indicating that highly adapted bacteriophage genomes can be stable over prolonged periods of time.

Implementation of a Pediatric Pharmacy Education Program at a Community Regional Medical Center.

OBJECTIVES: This study aimed to implement a web-based pediatric education program designed for pharmacists who participate in neonatal and pediatric order verification at a community-based health system and to evaluate the success through measuring outcomes related to both comfort and competence of pharmacists in pediatric and neonatal pharmacotherapy. METHODS: This prospective quality improvement study assessed changes in confidence and competence from before to after education. Eight educational modules were designed to provide education based on the needs of this institution. All pharmacists who participate in neonatal and pediatric order verification were eligible for inclusion throughout the health system. Time in the verification queue for pediatric and neonatal medication orders was compared for before to after education as an objective surrogate marker for comfort and competence. A provider survey was conducted before and after education to assess the providers' perspective of the quality and necessity of pharmacist-provider interactions. RESULTS: All confidence scores showed statistical improvement from before to after education (p < 0.001). Before to after education competency scores significantly improved (median 77% [IQR, 69%-85%] to 100% [IQR, 92%-100%]; p < 0.01). The module with the lowest mean score (87%) was module 4 (Antibiotics Part 1), and the one with highest number of retakes (24 retakes from 16 different pharmacists) was module 5 (Antibiotics Part 2). CONCLUSIONS: Targeted web-based education effectively improved both confidence and competence among health-system pharmacists to provide pediatric and neonatal care in a community hospital.