Implementation of a delayed prescribing model to reduce antibiotic prescribing for suspected upper respiratory tract infections in a hospital outpatient department, Ghana

Background: High levels of antimicrobial resistance (AMR) in Ghana require the exploration of new approaches to optimise antimicrobial prescribing. This study aims to establish the feasibility of implementation of different delayed/back-up prescribing models on antimicrobial prescribing for upper respiratory tract infections (URTIs). Methods: This study was part of a quality improvement project at LEKMA Hospital, Ghana, (Dec 2019–Feb 2020). Patients meeting inclusion criteria were assigned to one of four groups (Group 0: No prescription given; Group 1; Patient received post-dated antibiotic prescription; Group 2: Offer of a rapid reassessment of patient by a nurse practitioner after 3 days; and Group 3: Post-dated prescription forwarded to hospital pharmacy). Patients were contacted 10 days afterwards to ascertain wellbeing and actions taken, and patients were asked rate the service on a Likert scale. Post-study informal discussions were conducted with hospital staff. Results: In total, 142 patients met inclusion criteria. Groups 0, 1, 2 and 3 had 61, 16, 44 and 21 patients, respectively. Common diagnosis was sore throat (73%). Only one patient took antibiotics after 3 days. Nearly all (141/142) patients were successfully contacted on day 10, and of these, 102 (72%) rated their experiences as good or very good. Informal discussions with staff revealed improved knowledge of AMR. Conclusions: Delayed/back-up prescribing can reduce antibiotic consumption amongst outpatient department patients with suspected URTIs. Delayed/back-up prescribing can be implemented safely in low and middle-income countries (LMICs)

Translational profiling of hypocretin neurons identifies candidate molecules for sleep regulation

Hypocretin (orexin; Hcrt)-containing neurons of the hypothalamus are essential for the normal regulation of sleep and wake behaviors and have been implicated in feeding, anxiety, depression, and reward. The absence of these neurons causes narcolepsy in humans and model organisms. However, little is known about the molecular phenotype of these cells; previous attempts at comprehensive profiling had only limited sensitivity or were inaccurate. We generated a Hcrt translating ribosome affinity purification (bacTRAP) line for comprehensive translational profiling of all ribosome-bound transcripts in these neurons in vivo. From this profile, we identified >6000 transcripts detectably expressed above background and 188 transcripts that are highly enriched in these neurons, including all known markers of the cells. Blinded analysis of in situ hybridization databases suggests that ∼60% of these are expressed in a Hcrt marker-like pattern. Fifteen of these were confirmed with double labeling and microscopy, including the transcription factor Lhx9. Ablation of this gene results in a >30% loss specifically of Hcrt neurons, without a general disruption of hypothalamic development. Polysomnography and activity monitoring revealed a profound hypersomnolence in these mice. These data provide an in-depth and accurate profile of Hcrt neuron gene expression and suggest that Lhx9 may be important for specification or survival of a subset of these cells

Understanding the Role of Cereblon in Hematopoiesis Through Structural and Functional Analyses

The discovery and implementation of immunomodulatory drugs (IMiD®s) has revolutionized the treatment of many hematological malignancies due to the plethora of IMiD®-induced clinical responses that include anti-angiogenesis, anti-inflammation, and anti-tumor effects, as well as enhanced erythropoiesis, immune modulation and improved metabolism. More ground-breaking was the identification of cereblon as the target of IMiD®s. Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction of neo-substrates by engaging the DDB1-CUL4A-Roc1-RBX1 E3-ubiquitin ligase in human cells but not in mouse cells suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN’s activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN’s non-primate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants of different species retain their drug binding properties to both classical immunomodulatory drugs and to dBET1, a chemical compound and targeting ligand (i.e. a PROTAC) designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN’s E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight provides evidence that mouse CRBN is functional and provides a new avenue of exploring CRBN’s endogenous substrate recruiting function in hematopoietic cells. Hematopoietic stem cell (HSC) ontogeny is governed by extrinsic and intrinsic programs to control proliferation, localization, differentiation and self-renewal. The E3 ubiquitin ligase substrate receptor CRBN is expressed in hematopoietic lineages, including long-term HSCs (CD48-CD150+Lineage-Sca-1+c-Kit+ (LSK)), but plays an undefined role in hematopoiesis. As CRBN is the only confirmed target of IMiD®s and since IMiD®s have substantial clinical activity in a number of hematological malignancies, CRBN’s involvement in hematopoiesis was investigated. In these diseases, the IMiD®-CRBN interaction is associated with tumor cell death and other cellular responses through at least two major proposed mechanisms including, 1) drug-induced activation of neomorphic protein degradation, and 2) inhibition of endogenous substrate recruitment leading to toxic protein accumulation[3]. To gain insights into the role of CRBN in hematopoiesis, we assessed adult steady state and transplant-associated hematopoiesis and evaluated HSC development during embryogenesis using germline Crbn-/- mice. Compared to Crbn+/+ mice, CRBN deficiency was associated with a 2-3-fold reduction in stem cells in adult bone marrow and a similar reduction in HSCs in the fetal liver at embryonic stage 14.5 when the HSC pool is initiated from several anatomical sites including the yolk sac, aorta gonad mesonephros and fetal liver. Postnatal bone marrow HSCs are maintained throughout life by a process of self-renewal and differentiation. Interestingly, mature lymphoid and myeloid progeny were moderately expanded in peripheral blood of Crbn-/- mice under homeostatic conditions and following serial bone marrow transplantation suggesting that CRBN is critical for stem cell maintenance. RNA-seq analyses and qRT-PCR validation of BM-derived Crbn-/- LSKs and HSCs showed a marked suppression in a “stemness”-related gene signature consisting of Tie2, Hif1α, and Notch1 suggesting that CRBN is required to maintain HSCs in the undifferentiated state. Moreover, high amounts of proliferation within the LT-HSCs at steady-state, increased mature cells at steady-state and following serial transplantation, and increased lethality to repeated 5-fluorouracil (5-FU) challenge in Crbn-/- mice indicate that CRBN maintains HSC quiescence. Collectively our data suggest CRBN functions to maintain HSC quiescence required to maintain the undifferentiated properties of the HSC cells in the adult bone marrow. HSCs reside in highly specialized bone marrow niches where multiple cooperative networks of intrinsic and extrinsic factors are required for HSC homeostasis. Transplantation of Crbn+/+ HSCs into Crbn-/- recipients failed to generate a defined phenotype. However, HSC adhesion (Vcam1, Cxcr4)-associated genes were significantly suppressed. Particularly, the G protein-coupled chemokine receptor CXCR4-CXCL12 interaction which controls fetal and mature BM colonization and regulates homeostatic and regenerative HSC maintenance. These results indicate that CRBN is a functional protein in rodents and demonstrates a novel role for CRBN in the control of hematopoiesis with important implications in understanding how IMiD®s exert their therapeutic actions in hematological cancers. Our results demonstrate a novel role for CRBN in the control of hemopoiesis with important implications in understanding how IMiD®s exert their therapeutic actions in hematological cancers

Clonal hematopoiesis of indeterminate potential and its impact on patient trajectories after stem cell transplantation.

Clonal hematopoiesis of indeterminate potential (CHIP) is a recently identified process where older patients accumulate distinct subclones defined by recurring somatic mutations in hematopoietic stem cells. CHIP's implications for stem cell transplantation have been harder to identify due to the high degree of mutational heterogeneity that is present within the genetically distinct subclones. In order to gain a better understanding of CHIP and the impact of clonal dynamics on transplantation outcomes, we created a mathematical model of clonal competition dynamics. Our analyses highlight the importance of understanding competition intensity between healthy and mutant clones. Importantly, we highlight the risk that CHIP poses in leading to dominance of precancerous mutant clones and the risk of donor derived leukemia. Furthermore, we estimate the degree of competition intensity and bone marrow niche decline in mice during aging by using our modeling framework. Together, our work highlights the importance of better characterizing the ecological and clonal composition in hematopoietic donor populations at the time of stem cell transplantation

Ligand-mediated Protein Degradation reveals Functional Conservation among Sequence Variants of the CUL4-type E3 Ligase Substrate Receptor Cereblon

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1–CUL4A–Roc1–RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN\u27s activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN\u27s nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN\u27s E3 ubiquitin–conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN\u27s substrate-recruiting function