Parallel purification of microscale libraries via automated solid phase extraction.

High-throughput experimentation (HTE) has become more widely utilized in drug discovery for rapid reaction optimization and generation of large synthetic compound arrays. While this has accelerated medicinal chemistry design, make, test (DMT) iterations, the bottleneck of purification persists, consuming time and resources. Herein we describe a general parallel purification approach based on solid phase extraction (SPE) that provides a more efficient and sustainable workflow producing compound libraries with significantly upgraded purity. This robust, user-friendly workflow is fully automated and integrated with HTE library synthesis, as demonstrated by its application to a diverse parallel library compound array generated via amide-bond coupling in HTE microscale format.

The impact of the COVID-19 pandemic on young children and their caregivers.

BACKGROUND: The COVID-19 pandemic has adversely impacted child development and the well-being of caregivers, and such evidence ought to be used to inform public policy decisions. This study investigated the impact of COVID-19 on children's behaviours and their caregivers' needs. METHODS: A cross-sectional study was conducted with 153 caregivers of children (from 0 to 5 years old) from three public daycare centres in Brazil. The Nurturing Care Framework of the World Health Organization was used to guide the assessment of caregivers' needs. Online data collection using a questionnaire was conducted from June to July 2020. RESULTS: The COVID-19 pandemic increased stressors such as low family income, unemployment, sadness, depression and anxiety of caregivers. Their most commonly reported needs were related to offering age-appropriate playful activities (49.7%), organizing the care routine of children at home (41.8%) and educating children when they do something wrong (39.9%). Additionally, the results showed that misbehaviour, aggressiveness and agitation occurred more frequently among preschoolers than infants or toddlers (p ≤ 0.05). CONCLUSION: During the COVID-19 pandemic, public policies should provide mental health support to caregivers, as well as information about security, safety and early learning opportunities for childcare at home.

A national experiment reveals where a growth mindset improves achievement.

A global priority for the behavioural sciences is to develop cost-effective, scalable interventions that could improve the academic outcomes of adolescents at a population level, but no such interventions have so far been evaluated in a population-generalizable sample. Here we show that a short (less than one hour), online growth mindset intervention-which teaches that intellectual abilities can be developed-improved grades among lower-achieving students and increased overall enrolment to advanced mathematics courses in a nationally representative sample of students in secondary education in the United States. Notably, the study identified school contexts that sustained the effects of the growth mindset intervention: the intervention changed grades when peer norms aligned with the messages of the intervention. Confidence in the conclusions of this study comes from independent data collection and processing, pre-registration of analyses, and corroboration of results by a blinded Bayesian analysis.

Mapping the dark space of chemical reactions with extended nanomole synthesis and MALDI-TOF MS.

Understanding the practical limitations of chemical reactions is critically important for efficiently planning the synthesis of compounds in pharmaceutical, agrochemical, and specialty chemical research and development. However, literature reports of the scope of new reactions are often cursory and biased toward successful results, severely limiting the ability to predict reaction outcomes for untested substrates. We herein illustrate strategies for carrying out large-scale surveys of chemical reactivity by using a material-sparing nanomole-scale automated synthesis platform with greatly expanded synthetic scope combined with ultrahigh-throughput matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

Teacher Math Anxiety Relates to Adolescent Students' Math Achievement.

Elementary school teachers' math anxiety has been found to play a role in their students' math achievement. The current study addresses the role of teacher math anxiety on ninth-grade students' math achievement and the mediating factors underlying this relationship. Using data from the National Mindset Study, we find that higher teacher math anxiety is associated with lower math achievement. This relationship is partially mediated by the students' perception that their teacher believes not everyone can be good at math and is not explainable by teachers' usable knowledge to teach mathematics. In subsequent analyses, we find that higher teacher math anxiety relates to a reduction in process-oriented (as opposed to ability-oriented) teaching practices, which in turn predict students' perception of teacher mindset. We argue that math anxious teachers and their use of particular teaching strategies have the potential to shape students' math achievement and their perceptions of what their teacher believes about math.

The design and synthesis of novel spirocyclic heterocyclic sulfone ROMK inhibitors as diuretics.

A spirocyclic class of ROMK inhibitors was developed containing a structurally diverse heterocyclic sulfone moiety and spirocyclic core starting from lead 1. These compounds not only displayed exquisite ROMK potency but significantly improved selectivity over hERG. The lead compounds were found to have favorable pharmacokinetic properties and displayed robust diuretic, natriuretic and blood pressure lowering effects in spontaneously hypertensive rats.

Extending SME to Handle Large-Scale Cognitive Modeling.

Analogy and similarity are central phenomena in human cognition, involved in processes ranging from visual perception to conceptual change. To capture this centrality requires that a model of comparison must be able to integrate with other processes and handle the size and complexity of the representations required by the tasks being modeled. This paper describes extensions to Structure-Mapping Engine (SME) since its inception in 1986 that have increased its scope of operation. We first review the basic SME algorithm, describe psychological evidence for SME as a process model, and summarize its role in simulating similarity-based retrieval and generalization. Then we describe five techniques now incorporated into the SME that have enabled it to tackle large-scale modeling tasks: (a) Greedy merging rapidly constructs one or more best interpretations of a match in polynomial time: O(n2 log(n)); (b) Incremental operation enables mappings to be extended as new information is retrieved or derived about the base or target, to model situations where information in a task is updated over time; (c) Ubiquitous predicates model the varying degrees to which items may suggest alignment; (d) Structural evaluation of analogical inferences models aspects of plausibility judgments; (e) Match filters enable large-scale task models to communicate constraints to SME to influence the mapping process. We illustrate via examples from published studies how these enable it to capture a broader range of psychological phenomena than before.

Discovery of NMS-E973 as novel, selective and potent inhibitor of heat shock protein 90 (Hsp90).

Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a fragment based drug discovery approach (FBDD) and subsequent optimization with a combination of structure guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovarian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile.

Discovery and optimization of pyrrolo[1,2-a]pyrazinones leads to novel and selective inhibitors of PIM kinases.

A novel series of PIM inhibitors was derived from a combined effort in natural product-inspired library generation and screening. The novel pyrrolo[1,2-a]pyrazinones initial hits are inhibitors of PIM isoforms with IC50 values in the low micromolar range. The application of a rational optimization strategy, guided by the determination of the crystal structure of the complex in the kinase domain of PIM1 with compound 1, led to the discovery of compound 15a, which is a potent PIM kinases inhibitor exhibiting excellent selectivity against a large panel of kinases, representative of each family. The synthesis, structure-activity relationship studies, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Furthermore, the cellular activities including inhibition of cell growth and modulation of downstream targets are also described.

Autologous umbilical cord blood infusion followed by oral docosahexaenoic acid and vitamin D supplementation for C-peptide preservation in children with Type 1 diabetes.

We sought to determine if autologous umbilical cord blood (UCB) infusion followed by 1 year of supplementation with vitamin D and docosahexaenoic acid (DHA) can preserve C-peptide in children with type 1 diabetes. We conducted an open-label, 2:1 randomized study in which 15 type 1 diabetes subjects with stimulated C-peptide > .2 pmol/mL received either (1) autologous UCB infusion, 1 year of daily oral vitamin D (2000 IU), and DHA (38 mg/kg) and intensive diabetes management or (2) intensive diabetes management alone. Primary analyses were performed 1 year after UCB infusion. Treated (N = 10) and control (N = 5) subjects had median ages of 7.2 and 6.6 years, respectively. No severe adverse events were observed. Although the absolute rate of C-peptide decline was slower in treated versus control subjects, intergroup comparisons failed to reach significance (P = .29). Area under the curve C-peptide declined and insulin use increased in both groups (P < .01). Vitamin D levels remained stable in treated subjects but declined in control subjects (P = .01). DHA levels rose in treated subjects versus control subjects (P = .003). CD4/CD8 ratio remained stable in treated subjects but declined in control subjects (P = .03). No changes were seen in regulatory T cell frequency, total CD4 counts, or autoantibody titers. Autologous UCB infusion followed by daily supplementation with vitamin D and DHA was safe but failed to preserve C-peptide. Lack of significance may reflect small sample size. Future efforts will require expansion of specific immunoregulatory cell subsets, optimization of combined immunoregulatory and anti-inflammatory agents, and larger study cohorts.

Histopathology and outcome of acute humoral rejection in renal allografts.

Our purpose was to see if histopathologic features of acute antibody-mediated rejection (AMR) in renal allografts have prognostic value; and to compare two-year graft survival with and without additional therapy with plasmapheresis and intravenous immunoglobulin (IVIG). We reviewed renal allograft biopsies taken within the first 6 months after transplant from patients with C4d positive AMR, performed between January 2000 to December 2005 (n=57). We formed two groups: Group 1: biopsied between 2003 and 2005 (n=26), when C4d staining was routinely performed and option for plasmapheresis and IVIG was available; Group 2: biopsied between 2000 and 2002 (n=31), retrospectively found to be C4d positive. Patients whose biopsies showed cortical necrosis or arterial fibrinoid necrosis had early graft loss. Other histopathologic features did not statistically correlate with graft loss. Overall, additional plasmapheresis/IVIG treatment did not show convincing improvement in graft survival or function at 2 years post-transplant, but all six patients with thrombotic microangiopathy (TMA) who received plasmapheresis/IVIG had functioning grafts at two-year follow-up.

Steroid minimization for sirolimus-treated renal transplant recipients.

Steroids are associated with a myriad of post-transplant side effects. Therefore, as new immunosuppressive drugs have been developed, attempts have been made to minimize steroid exposure. Sirolimus (SRL) has been demonstrated to have efficacy in early and late post-transplant immunosuppression. Accordingly, numerous trials have studied steroid minimization (early and late post-transplant) in the context of SRL-containing protocols (either with or without a calcineurin inhibitor). We herein review these trials and show that recent studies have determined that both late steroid withdrawal and early rapid discontinuation can be successful with SRL immunosuppression.

4,5-Dihydro-1H-pyrazolo[4,3-h]quinazolines as potent and selective Polo-like kinase 1 (PLK1) inhibitors.

A series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives was optimized as Polo-like kinase 1 inhibitors. Extensive SAR afforded a highly potent and selective PLK1 compound. The compound showed good antiproliferative activity when tested in a panel of tumor cell lines with PLK1 related mechanism of action and with good in vivo antitumor efficacy in two xenograft models after i.v. administration.

Identification of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as a new class of orally and selective Polo-like kinase 1 inhibitors.

Polo-like kinase 1 (Plk1) is a fundamental regulator of mitotic progression whose overexpression is often associated with oncogenesis and therefore is recognized as an attractive therapeutic target in the treatment of proliferative diseases. Here we discuss the structure-activity relationship of the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline class of compounds that emerged from a high throughput screening (HTS) campaign as potent inhibitors of Plk1 kinase. Furthermore, we describe the discovery of 49, 8-{[2-methoxy-5-(4-methylpiperazin-1-yl)phenyl]amino}-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide, as a highly potent and specific ATP mimetic inhibitor of Plk1 (IC(50) = 0.007 microM) as well as its crystal structure in complex with the methylated Plk1(36-345) construct. Compound 49 was active in cell proliferation against different tumor cell lines with IC(50) values in the submicromolar range and active in vivo in the HCT116 xenograft model where it showed 82% tumor growth inhibition after repeated oral administration.

Peritubular capillary C4d staining in late acute renal allograft rejection--is it relevant?

BACKGROUND: In the early post-transplant period, renal allograft rejection with diffuse peritubular capillary (PTC) C4d deposition predicts poor graft survival. In the late post-transplant setting, that is, one or more yr after transplantation, the implication of diffuse PTC C4d deposition is still a topic of debate. The purpose of our study was to see if diffuse PTC C4d deposition, in late acute rejection (LAR), occurring more than one yr post-transplant, has any impact on graft survival and function. METHODS: We selected cases, both cadaveric as well as living donor renal transplant recipients, in whom acute rejection with PTC C4d deposition was first detected after the first year post-transplant. Recipients with multiple acute rejection episodes during the first year post-transplant were excluded from the study. The first biopsy diagnosed with LAR was considered the index biopsy (n = 40). We formed two groups: group 1, C4d-positive LAR (n = 20), and group 2, C4d-negative LAR (n = 20). Groups were matched for maintenance and post-rejection immunosuppressive therapy, baseline serum creatinine levels before the time of the index biopsy, time from transplant to index biopsy, as well as chronic allograft damage index (CADI) score in the index biopsies. We compared the rate of graft loss, and the graft function of the surviving grafts at the end of the study period, as well as histologic parameters in the index biopsy specimens between the two groups. The mean follow-up period was 20 months. RESULTS: No significant differences in the rate of graft loss or graft function were found between groups 1 and 2 at the end of the follow-up period. Histologically, PTC margination and transplant glomerulopathy were more common in the C4d-positive group, and this difference was statistically significant. There was no statistically significant difference in the degree of plasma cell infiltrates. CONCLUSIONS: Unlike in the acute setting, the presence or absence of PTC C4d staining in renal allografts with LAR may not have a predictive value regarding graft outcome.

Comparison of the portal vein and kidney subcapsule as sites for primate islet autotransplantation.

To date, the portal vein has been the primary site for clinical islet transplantation. Despite success, potential complications such as portal vein thrombosis still exist. The kidney subcapsule has been used successfully in rodent models of islet transplantation. We hypothesized that the kidney subcapsule as a site for islet transplantation in the nonhuman primate model would be as effective as the portal vein. Diabetes was induced in the primate Macaca fascicularis via a total pancreatectomy. Animals were kept under anesthesia during the isolation procedure. Islet isolation was performed using intraductal infusion with Liberase HI and mechanical digestion in the Ricordi chamber, and were purified using a continuous Ficoll gradient. Purified islets were autotransplanted either into the portal vein (n = 6) or the left kidney subcapsule (n = 5) of pancreatectomized animals. Intravenous glucose tolerance tests were performed prior to pancreatectomy and 10 days following transplantation. Three animals underwent pancreatectomy and served as diabetic controls. Of the six animals receiving islets in the portal vein, one developed portal vein thrombosis. All remaining autotransplanted animals in this group remained normoglycemic with glucose-induced insulin secretion that was not different from that prior to pancreatectomy. Of the five animals undergoing transplantation into the kidney subcapsule, only one maintained normoglycemia and elicited insulin secretion in response to glucose stimulation. The other four animals remained hyperglycemic. We conclude that the portal vein is superior to the kidney subcapsule as a site for islet transplantation in nonhuman primates 10 days posttransplantation.

Steroid-free maintenance immunosuppression with rapamune and low-dose neoral in pancreas transplant recipients.

BACKGROUND: Steroid-free immunosuppression is an attractive option because it avoids the many side effects of chronic corticosteroid use. It is especially attractive in pancreas recipients because it avoids the diabetogenic effects of steroids. METHODS: We evaluated the outcome of a steroid-free maintenance immunosuppressive protocol in pancreas transplant recipients. Between August 2003 and May 2006, a total of 97 pancreas transplant recipients received steroid-free maintenance immunosuppression, consisting of induction with thymoglobulin and prednisone for the first 5 days. Patients were maintained on sirolimus adjusted to a target rapamycin trough level and reduced-dose cyclosporine adjusted to target C2 levels. All pancreas transplants (n=124) performed in the previous 3 years and maintained on a steroid-based immunosuppressive protocol with cyclosporine and mycophenolate mofetil were used for comparison. RESULTS: One-year patient and death censored pancreas graft survival were 93.8% and 94.8% for the steroid free group versus 95.2% and 87.9% for the comparator group, respectively. The incidence of acute rejection was 9.3% in the steroid-free group versus 28.3% in the comparator group (P<0.01). No pancreas loss in the steroid-free group was caused by acute rejection, whereas seven (5.6%) patients in the comparator group lost their pancreases because of acute rejection (P<0.05). At 1 year after transplant, the mean serum glucose and creatinine levels were not different between the two groups. CONCLUSION: We conclude that excellent graft survival with a significantly lower incidence of acute rejection can be achieved using a steroid-free maintenance immunosuppressive protocol consisting of sirolimus and cyclosporine.

Impact of long-term therapy with FTY720 or mycophenolate mofetil on cardiac conduction and rhythm in stable adult renal transplant patients.

The novel immunomodulator FTY720 has been associated with a mild reduction in heart rate (HR) in clinical trials. A total of 421 patients (FTY720, n=94; mycophenolate mofetil [MMF], n=327) underwent 2-day electrocardiogram and 24-h Holter monitoring. Patients had been maintained on cyclosporine plus MMF or FTY720 (2.5 mg and 5.0 mg) for > or =12 months. No significant differences in mean hourly heart rate (HR) over 24 hrs were noted between groups. Bradycardia (HR 35-50 bpm) and sustained bradycardia (HR <50 bpm for >1 min) were more common with MMF than FTY720 (53% vs. 37% and 34% vs. 21%, respectively). Electrocardiogram parameters did not differ significantly between FTY720 and MMF groups, or between FTY720 groups, supporting the absence of a dose-dependent effect. The absence of any clinically significant effect of FTY720 on cardiac rhythm demonstrates that the reduction in HR seen after the first dose does not persist in the maintenance phase.

Excellent clinical outcomes in primary kidney transplant recipients treated with steroid-free maintenance immunosuppression.

Steroid-free maintenance immunosuppression is desirable to eliminate the side effects of chronic corticosteroid use. Complete steroid avoidance or rapid post-transplant steroid withdrawal has recently been used in renal transplant recipients with encouraging results. The present study evaluated the outcome of a steroid-free maintenance immunosuppressive protocol in kidney transplant recipients with at least one-yr follow up. Between April 2002 and October 2004, a total of 301 primary kidney transplant recipients received steroid-free maintenance immunosuppression. The regimen consisted of induction with thymogobulin and prednisone for the first five d. Patients were maintained on Sirolimus and Neoral. Neoral dose was adjusted to target C2 levels and the Sirolimus dose was adjusted to a target rapamycin trough level. All primary kidney transplants (n = 502) performed in the two yr (starting January 2000) prior to institution of the steroid-free regimen and thus maintained on a steroid-based immunosuppressive protocol were used for comparison. One-year patient and death censored graft survival were 93.1% and 98.1% for the steroid-free group vs. 95.2% and 95.2% for the comparator groups (p = ns). The incidence of biopsy-proven acute rejection was 4.9% in the steroid-free group vs. 9.4% in the comparator group (p < 0.01). Two (0.7%) of 301 patients in the steroid-free group lost their grafts because of acute rejection compared with nine (1.8%) patients in the comparator group (p < 0.05). At one-yr post-transplant the mean serum creatinine level was not different between the two groups. There were no significant differences in mean serum cholesterol and triglycerides levels as well as the percentage of patients on lipid lowering agents between the groups. White blood cell counts, daily doses of Neoral and weight gain were significantly lower in the steroid-free group vs. the comparator group. However, more patients in the steroid-free group required erythropoietin and iron therapy for anemia (p < 0.001). We conclude that excellent graft survival with a significantly lower incidence of acute rejection can be achieved using a steroid-free maintenance immunosuppressive protocol consisting of Neoral and Sirolimus.

Acute renal failure following kidney transplantation associated with myoglobinuria in patients treated with rapamycin.

BACKGROUND: Since using an immunosuppression regimen that includes rapamycin, we have occasionally encountered renal transplant patients who develop unexpected severe acute renal dysfunction. Biopsies obtained in these recipients demonstrate acute tubular necrosis (ATN) occasionally associated with tubular casts giving the classic appearance of myoglobin casts. METHODS: We retrospectively reviewed all biopsies from consecutively transplanted kidneys engrafted between April 9, 2002 and June 29, 2004 to determine the incidence of ATN, ATN with intratubular casts, and casts with the classic myoglobin appearance. The clinical setting, treatment, and outcomes of those patients with classic myoglobin-appearing casts are reviewed. RESULTS: Histological ATN as the principal finding in at least one biopsy occurred in 10.5% (57/543) of patients. About half of these patients (30/57) had tubular casts present in at least one biopsy and in 14 of these the casts had a classic appearance of myoglobin casts. These myoglobin-appearing casts were only noted in patients receiving rapamycin. A review of 28 ATN biopsies from an earlier prerapamycin era did not demonstrate similar myoglobin-appearing casts. Immunostaining for myoglobin was positive in all 14 recipient biopsies. This was confirmed by western blot analyses in three of five patient biopsies tested. Three of three recipients tested had elevated serum creatine phosphokinase levels and detectable serum myoglobin. All 14 patients slowly resolved their acute renal dysfunction and no grafts were lost. CONCLUSION: We conclude that myoglobinuria with myoglobin cast formation can occur following rapamycin administration, and may be a causative factor in the development of unexpected severe acute renal dysfunction.