Novel spirocyclic dimer, SpiD3, targets chronic lymphocytic leukemia survival pathways with potent preclinical effects.

Chronic lymphocytic leukemia (CLL) cell survival and growth is fueled by the induction of B-cell receptor (BCR) signaling within the tumor microenvironment (TME) driving activation of NF-κB signaling and the unfolded protein response (UPR). Malignant cells have higher basal levels of UPR posing a unique therapeutic window to combat CLL cell growth using pharmacological agents that induce accumulation of misfolded proteins. Frontline CLL therapeutics that directly target BCR signaling such as Bruton-tyrosine kinase (BTK) inhibitors (e.g., ibrutinib) have enhanced patient survival. However, resistance mechanisms wherein tumor cells bypass BTK inhibition through acquired BTK mutations, and/or activation of alternative survival mechanisms have rendered ibrutinib ineffective, imposing the need for novel therapeutics. We evaluated SpiD3, a novel spirocyclic dimer, in CLL cell lines, patient-derived CLL samples, ibrutinib-resistant CLL cells, and in the Eµ-TCL1 mouse model. Our integrated multi-omics and functional analyses revealed BCR signaling, NF-κB signaling, and endoplasmic reticulum stress among the top pathways modulated by SpiD3. This was accompanied by marked upregulation of the UPR and inhibition of global protein synthesis in CLL cell lines and patient-derived CLL cells. In ibrutinib-resistant CLL cells, SpiD3 retained its anti-leukemic effects, mirrored in reduced activation of key proliferative pathways (e.g., PRAS, ERK, MYC). Translationally, we observed reduced tumor burden in SpiD3-treated Eµ-TCL1 mice. Our findings reveal that SpiD3 exploits critical vulnerabilities in CLL cells including NF-κB signaling and the UPR, culminating in profound anti-tumor properties independent of TME stimuli.

'Something that helped the whole picture': Experiences of parents offered rapid prenatal exome sequencing in routine clinical care in the English National Health Service.

OBJECTIVES: In October 2020, rapid prenatal exome sequencing (pES) was introduced into routine National Health Service (NHS) care in England. This study aimed to explore parent experiences and their information and support needs from the perspective of parents offered pES and of health professionals involved in its delivery. METHODS: In this qualitative study, semi-structured interviews were conducted with 42 women and 6 male partners and 63 fetal medicine and genetic health professionals. Interviews were transcribed verbatim and analysed using thematic analysis. RESULTS: Overall views about pES were positive and parents were grateful to be offered the test. Highlighted benefits of pES included the value of the additional information for pregnancy management and planning for future pregnancies. An anxious wait for results was common, often associated with the need to make decisions near to 24 weeks in pregnancy when there are legal restrictions for late termination. Descriptions of dealing with uncertainty were also common, even when results had been returned. Many parents described pES results as informing decision-making around whether or not to terminate pregnancy. Some professionals were concerned that a non-informative result could be overly reassuring and highlighted that careful counselling was needed to ensure parents have a good understanding of what the result means for their pregnancy. Emotional support from professionals was valued; however, some parents felt that post-test support was lacking. CONCLUSION: Parents and professionals welcomed the introduction of pES. Results inform parents' decision-making around the termination of pregnancy. When there are no diagnostic findings or uncertain findings from pES, personalised counselling that considers scans and other tests are crucial. Directing parents to reliable online sources of information and providing emotional support throughout could improve their experiences of care.

Virtual BUILD Research Collaboratory: A biomedical data science training using innovative pedagogy to address structures of racism and inequitable stress for undergraduates of color.

OBJECTIVE: The unprecedented events of 2020 required a pivot in scientific training to better prepare the biomedical research workforce to address global pandemics, structural racism, and social inequities that devastate human health individually and erode it collectively. Furthermore, this pivot had to be accomplished in the virtual environment given the nation-wide lockdown. METHODS: These needs and context led to leveraging of the San Francisco Building Infrastructure Leading to Diversity (SF BUILD) theories of change to innovate a Virtual BUILD Research Collaboratory (VBRC). The purpose of VBRC was to train Black, Indigenous, and people of color (BIPOC) students to apply their unique perspectives to biomedical research. These training activities were evaluated using a pre-post survey design that included both validated and new psychosocial scales. A new scale was piloted to measure culturally relevant pedagogy. RESULTS: VBRC scholars increased science identity on two items: thinking of myself as a scientist (+1point, p = 0.006) and belonging to a community of scientists (+1point, p = 0.069). Overall, scholars perceived stress also decreased over VBRC (-2.35 points, p = 0.02). Post VBRC, scholars had high agency scores (µ = 11.02, Md = 12, range = 6-12, σ = 1.62) and cultural humility scores (µ = 22.11, Md = 23, range = 12-24, σ = 2.71). No notable race/ethnic differences were found in any measures. CONCLUSIONS: Taken together, our innovative approach to data science training for BIPOC in unprecedented times shows promise for better preparing the workforce critically needed to address the fundamental gaps in knowledge at the intersection of public health, structural racism, and biomedical sciences.

Heterogeneity in the progression of retinal pathologies in mice harboring patient mimicking Impg2 mutations.

Biallelic mutations in interphotoreceptor matrix proteoglycan 2 (IMPG2) in humans cause retinitis pigmentosa (RP) with early macular involvement, albeit the disease progression varies widely due to genetic heterogeneity and IMPG2 mutation type. There are currently no treatments for IMPG2-RP. To aid preclinical studies toward eventual treatments, there is a need to better understand the progression of disease pathology in appropriate animal models. Toward this goal, we developed mouse models with patient mimicking homozygous frameshift (T807Ter) or missense (Y250C) Impg2 mutations, as well as mice with a homozygous frameshift mutation (Q244Ter) designed to completely prevent IMPG2 protein expression, and characterized the trajectory of their retinal pathologies across postnatal development until late adulthood. We found that the Impg2T807Ter/T807Ter and Impg2Q244Ter/Q244Ter mice exhibited early onset gliosis, impaired photoreceptor outer segment maintenance, appearance of subretinal deposits near the optic disc, disruption of the outer retina, and neurosensorial detachment, whereas the Impg2Y250C/Y250C mice exhibited minimal retinal pathology. These results demonstrate the importance of mutation type in disease progression in IMPG2-RP and provide a toolkit and preclinical data for advancing therapeutic approaches.

Implementing school-based cognitive behavior therapy for anxiety in students with autism or suspected autism via a train-the-trainer approach: Results from a clustered randomized trial.

LAY ABSTRACT: Autistic youth frequently experience anxiety that can negatively affect them at home, with friends, and at school. Autistic youth have difficulty accessing mental health care, and this is particularly true for youth from traditionally underserved backgrounds. Providing mental health programs in schools may increase access to care for autistic youth with anxiety. The purpose of the study was to train interdisciplinary school providers to deliver school-based Facing Your Fears, a cognitive behavior therapy program for anxiety in autistic youth. Seventy-seven interdisciplinary school providers across 25 elementary/middle schools were trained by their colleagues and members of the research (train-the-trainer approach). Eighty-one students with autism or suspected autism, ages 8-14 years, were randomly assigned to either school-based Facing Your Fears or usual care. Students in school-based Facing Your Fears showed significant reductions in anxiety compared to students in usual care according to caregiver and student report. Other measures involved examining change in provider cognitive behavior therapy knowledge after training and determining how well interdisciplinary school providers were able to deliver school-based Facing Your Fears. Results indicated that interdisciplinary school providers showed significant improvements in cognitive behavior therapy knowledge after training. Interdisciplinary school providers were able to deliver most of school-based Facing Your Fears activities and with good quality. The positive outcomes in this study are encouraging. Training interdisciplinary school providers to deliver school-based Facing Your Fears may increase access to care for anxious autistic students. Future directions and limitations are discussed.

Accelerating Research Careers in Science Through Early Mentored Research Experiences for Undergraduates and Masters' Students at the University of California, San Francisco Center for AIDS Research.

BACKGROUND: Investments early in the academic pathway are essential to increasing the diversity of the HIV research workforce. Applied mentored research experiences can advance research skills, self-efficacy, and retention in science among scholars considered to be underrepresented minorities. SETTING: The UCSF Center for AIDS Research Scholars Program is a 16-week program that pairs URM undergraduate and masters-level students from San Francisco State University with URM investigators from the UCSF. The program includes one-on-one mentorship on an independent research project, a core-curriculum on research methods and HIV-specific topics, and clinical shadowing. METHODS: We evaluated program outcomes and areas for improvement with preprogram and postprogram survey and qualitative data from scholars and mentors from June to October 2022. Outcomes included interest in HIV research and growth on a 20-item measure of research skills, knowledge, and confidence. RESULTS: Six URM scholars enrolled in the inaugural cohort and were paired with 6 mentors. Preprogram and postprogram evaluations showed gains in interest in HIV research, 0% preprogram and 100% of scholars postprogram reporting much or extensive interest in HIV research, and gains across all domains from self-confidence, skills, and clarification of a research career path. Qualitative findings noted the importance of peer support and interpersonal features of mentors, including treating them like equals. Areas for improvement included more opportunities for primary data collection and specific training around mentoring undergraduates for mentors. CONCLUSIONS: The UCSF Center for AIDS Research Scholars Program was successful in building foundational skills, knowledge, confidence, and interest in HIV research among URM undergraduates and masters-level students.

Micromolding-based encapsulation of mesenchymal stromal cells in alginate for intraarticular injection in osteoarthritis.

Osteoarthritis (OA) is an inflammatory joint disease that affects cartilage, subchondral bone, and joint tissues. Undifferentiated Mesenchymal Stromal Cells are a promising therapeutic option for OA due to their ability to release anti-inflammatory, immuno-modulatory, and pro-regenerative factors. They can be embedded in hydrogels to prevent their tissue engraftment and subsequent differentiation. In this study, human adipose stromal cells are successfully encapsulated in alginate microgels via a micromolding method. Microencapsulated cells retain their in vitro metabolic activity and bioactivity and can sense and respond to inflammatory stimuli, including synovial fluids from OA patients. After intra-articular injection in a rabbit model of post-traumatic OA, a single dose of microencapsulated human cells exhibit properties matching those of non-encapsulated cells. At 6 and 12 weeks post-injection, we evidenced a tendency toward a decreased OA severity, an increased expression of aggrecan, and a reduced expression of aggrecanase-generated catabolic neoepitope. Thus, these findings establish the feasibility, safety, and efficacy of injecting cells encapsulated in microgels, opening the door to a long-term follow-up in canine OA patients.

The Role of Maternal Anxiety in Treatment Response for Youth with ASD and Co-occurring Anxiety.

Anxiety disorders occur at higher rates in youth with ASD than in neurotypical youth. Although the efficacy of CBT for anxiety in children with ASD is widely supported, factors that influence treatment outcomes are not well understood. This study examined the role of maternal anxiety in treatment outcomes for youth with ASD. Youth with ASD and anxiety (ages 8 to 14), along with their mothers (n = 87), participated in a group CBT intervention. Results indicated that maternal anxiety did not improve over the course of treatment. However, findings suggest that high levels of maternal anxiety at pre-treatment predicted higher levels of youth anxiety post-treatment. Importantly, the relationship between parent anxiety and youth outcomes was moderated by child age. The findings of the present study may provide initial insight into the role that maternal anxiety plays in treatment outcomes for children with ASD and co-occurring anxiety, particularly when considering child age.

Effect of Co-occurring Psychiatric Disorders on Treatment of Children with Autism Spectrum Disorder and Anxiety.

Co-occurring psychiatric diagnoses are very common in individuals with ASD. Little is known about the effect that co-occurring psychiatric conditions may have on treatment response to CBT for children with ASD and anxiety. The present study examined the relationship between co-occurring psychiatric diagnoses and response to CBT for anxiety in ninety youth with ASD. Psychiatric complexity did not appear to differentially impact treatment response. A notable portion of youth with anxiety and externalizing disorders such as ADHD, no longer met criteria for those externalizing diagnoses following intervention. Results indicate that youth with ASD and anxiety present with complex psychiatric profiles and CBT for anxiety may positively affect co-occurring diagnoses. In addition, thorough and nuanced assessment of psychiatric symptoms in youth with ASD is needed to ensure the differentiation between diagnoses of anxiety and other co-occurring psychiatric symptoms.

Enhancing grant-writing expertise in BUILD institutions: Building infrastructure leading to diversity.

BACKGROUND: The lack of race/ethnic and gender diversity in grants funded by the National Institutes of Health (NIH) is a persistent challenge related to career advancement and the quality and relevance of health research. We describe pilot programs at nine institutions supported by the NIH-sponsored Building Infrastructure Leading to Diversity (BUILD) program aimed at increasing diversity in biomedical research. METHODS: We collected data from the 2016-2017 Higher Education Research Institute survey of faculty and NIH progress reports for the first four years of the program (2015-2018). We then conducted descriptive analyses of data from the nine BUILD institutions that had collected data and evaluated which activities were associated with research productivity. We used Poisson regression and rate ratios of the numbers of BUILD pilots funded, students included, abstracts, presentations, publications, and submitted and funded grant proposals. RESULTS: Teaching workshops were associated with more abstracts (RR 4.04, 95% CI 2.21-8.09). Workshops on grant writing were associated with more publications (RR 2.64, 95% CI 1.64-4.34) and marginally with marginally more presentations. Incentives to develop courses were associated with more abstracts published (RR 4.33, 95% CI 2.56-7.75). Workshops on research skills and other incentives were not associated with any positive effects. CONCLUSIONS: Pilot interventions show promise in supporting diversity in NIH-level research. Longitudinal modeling that considers time lags in career development in moving from project development to grants submissions can provide more direction for future diversity pilot interventions.

A Novel Triple-Action Inhibitor Targeting B-Cell Receptor Signaling and BRD4 Demonstrates Preclinical Activity in Chronic Lymphocytic Leukemia.

B-cell chronic lymphocytic leukemia (CLL) results from intrinsic genetic defects and complex microenvironment stimuli that fuel CLL cell growth through an array of survival signaling pathways. Novel small-molecule agents targeting the B-cell receptor pathway and anti-apoptotic proteins alone or in combination have revolutionized the management of CLL, yet combination therapy carries significant toxicity and CLL remains incurable due to residual disease and relapse. Single-molecule inhibitors that can target multiple disease-driving factors are thus an attractive approach to combat both drug resistance and combination-therapy-related toxicities. We demonstrate that SRX3305, a novel small-molecule BTK/PI3K/BRD4 inhibitor that targets three distinctive facets of CLL biology, attenuates CLL cell proliferation and promotes apoptosis in a dose-dependent fashion. SRX3305 also inhibits the activation-induced proliferation of primary CLL cells in vitro and effectively blocks microenvironment-mediated survival signals, including stromal cell contact. Furthermore, SRX3305 blocks CLL cell migration toward CXCL-12 and CXCL-13, which are major chemokines involved in CLL cell homing and retention in microenvironment niches. Importantly, SRX3305 maintains its anti-tumor effects in ibrutinib-resistant CLL cells. Collectively, this study establishes the preclinical efficacy of SRX3305 in CLL, providing significant rationale for its development as a therapeutic agent for CLL and related disorders.

Binding of the SARS-CoV-2 envelope E protein to human BRD4 is essential for infection.

Emerging new variants of SARS-CoV-2 and inevitable acquired drug resistance call for the continued search of new pharmacological targets to fight the potentially fatal infection. Here, we describe the mechanisms by which the E protein of SARS-CoV-2 hijacks the human transcriptional regulator BRD4. We found that SARS-CoV-2 E is acetylated in vivo and co-immunoprecipitates with BRD4 in human cells. Bromodomains (BDs) of BRD4 bind to the C-terminus of the E protein, acetylated by human acetyltransferase p300, whereas the ET domain of BRD4 recognizes the unmodified motif of the E protein. Inhibitors of BRD4 BDs, JQ1 or OTX015, decrease SARS-CoV-2 infectivity in lung bronchial epithelial cells, indicating that the acetyllysine binding function of BDs is necessary for the virus fitness and that BRD4 represents a potential anti-COVID-19 target. Our findings provide insight into molecular mechanisms that contribute to SARS-CoV-2 pathogenesis and shed light on a new strategy to block SARS-CoV-2 infection.

Insect cell expression and purification of recombinant SARS-COV-2 spike proteins that demonstrate ACE2 binding.

The COVID-19 pandemic caused by SARS-CoV-2 infection has led to socio-economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID-19. SARS-CoV-2 spike is a target of interest for the development of therapeutic targets. We developed a robust SARS-CoV-2 S spike expression and purification protocol from insect cells and studied four recombinant SARS-CoV-2 spike protein constructs based on the original SARS-CoV-2 sequence using a baculovirus expression system: a spike protein receptor-binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S-RBD-eGFP), spike ectodomain coupled to a fluorescent tag (S-Ecto-eGFP), spike ectodomain with six proline mutations and a foldon domain (S-Ecto-HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S-Ecto-HexaPro(-F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S-Ecto-eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation).

Combinatorial inhibition of BTK, PI3K-AKT and BRD4-MYC as a strategy for treatment of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a subtype of non-Hodgkin's lymphoma characterized by poor prognosis. The complexity of MCL pathogenesis arises from aberrant activities of diverse signaling pathways, including BTK, PI3K-AKT-mTOR and MYC-BRD4. Here, we report that MCL-related signaling pathways can be altered by a single small molecule inhibitor, SRX3305. Binding and kinase activities along with resonance changes in NMR experiments reveal that SRX3305 targets both bromodomains of BRD4 and is highly potent in inhibition of the PI3K isoforms α, γ and δ, as well as BTK and the drug-resistant BTK mutant. Preclinical investigations herein reveal that SRX3305 perturbs the cell cycle, promotes apoptosis in MCL cell lines and shows dose dependent anti-proliferative activity in both MCL and drug-resistant MCL cells. Our findings underscore the effectiveness of novel multi-action small molecule inhibitors for potential treatment of MCL.

Environmental Justice in the American Public Health Context: Trends in the Scientific Literature at the Intersection Between Health, Environment, and Social Status.

Although various governmental entities in the USA are required to consider environmental justice (EJ) impacts of their actions during decision-making, socially vulnerable groups continue to be disproportionately exposed to environmental hazards. Tools and programs to quantify and mitigate environmental injustices are limited by existing data, which may not capture the full range of health disparities exacerbated by the complex interactions between environmental exposures and social stressors. In this study, we analyzed how the scientific literature approaches EJ issues in the USA. We searched PubMed for journal articles discussing at least one sociodemographic or environmental variable in the context of cumulative impacts and analyzed the relative frequency with which various EJ topics were studied. Our findings indicate that demographic variables are commonly used in epidemiologic studies, though some areas (e.g., age) are better studied than others. Similarly, occupational exposure and ambient air pollution were more studied than other types of exposures. Word frequency analyses revealed which toxicants and health outcomes are the most frequently studied. In addition, temporality analyses showed that the rate of occupational publications rose rapidly in the 1970-1980s and has since plateaued, while other publication rates increased two decades later and are still on the rise. Cumulative impacts are considered in a relatively small portion of journal articles across all topics; nevertheless, they have seen an exponential climb in the last decade. A more equitable distribution of scientific efforts might be needed for a better distribution of funding, policy-making efforts, and other resources to socially and environmentally vulnerable communities.

Delayed Presentation and Mortality in Children With Sepsis in a Public Tertiary Care Hospital in Tanzania.

Background: Over 40% of the global burden of sepsis occurs in children under 5 years of age, making pediatric sepsis the top cause of death for this age group. Prior studies have shown that outcomes in children with sepsis improve by minimizing the time between symptom onset and treatment. This is a challenge in resource-limited settings where access to definitive care is limited. Methods: A secondary analysis was performed on data from 1,803 patients (28 days-14 years old) who presented to the emergency department (ED) at Muhimbili National Hospital (MNH) from July 1, 2016 to June 30, 2017 with a suspected infection and ≥2 clinical systemic inflammatory response syndrome criteria. The objective of this study was to determine the relationship between delayed presentation to definitive care (>48 h between fever onset and presentation to the ED) and mortality, as well as the association between socioeconomic status (SES) and delayed presentation. Multivariable logistic regression models tested the two relationships of interest. We report both unadjusted and adjusted odds ratios and 95% confidence intervals. Results: During the study period, 11.3% (n = 203) of children who presented to MNH with sepsis died inhospital. Delayed presentation was more common in non-survivors (n = 90/151, 60%) compared to survivors (n = 614/1,353, 45%) (p ≤ 0.01). Children who had delayed presentation to definitive care, compared to those who did not, had an adjusted odds ratio for mortality of 1.85 (95% CI: 1.17-3.00). Conclusions: Delayed presentation was an independent risk factor for mortality in this cohort, emphasizing the importance of timely presentation to care for pediatric sepsis patients. Potential interventions include more efficient referral networks and emergency transportation systems to MNH. Additional clinics or hospitals with pediatric critical care may reduce pediatric sepsis mortality in Tanzania, as well as parental education programs for recognizing pediatric sepsis.

Use of whole genome sequencing to determine genetic basis of suspected mitochondrial disorders: cohort study.

OBJECTIVE: To determine whether whole genome sequencing can be used to define the molecular basis of suspected mitochondrial disease. DESIGN: Cohort study. SETTING: National Health Service, England, including secondary and tertiary care. PARTICIPANTS: 345 patients with suspected mitochondrial disorders recruited to the 100 000 Genomes Project in England between 2015 and 2018. INTERVENTION: Short read whole genome sequencing was performed. Nuclear variants were prioritised on the basis of gene panels chosen according to phenotypes, ClinVar pathogenic/likely pathogenic variants, and the top 10 prioritised variants from Exomiser. Mitochondrial DNA variants were called using an in-house pipeline and compared with a list of pathogenic variants. Copy number variants and short tandem repeats for 13 neurological disorders were also analysed. American College of Medical Genetics guidelines were followed for classification of variants. MAIN OUTCOME MEASURE: Definite or probable genetic diagnosis. RESULTS: A definite or probable genetic diagnosis was identified in 98/319 (31%) families, with an additional 6 (2%) possible diagnoses. Fourteen of the diagnoses (4% of the 319 families) explained only part of the clinical features. A total of 95 different genes were implicated. Of 104 families given a diagnosis, 39 (38%) had a mitochondrial diagnosis and 65 (63%) had a non-mitochondrial diagnosis. CONCLUSION: Whole genome sequencing is a useful diagnostic test in patients with suspected mitochondrial disorders, yielding a diagnosis in a further 31% after exclusion of common causes. Most diagnoses were non-mitochondrial disorders and included developmental disorders with intellectual disability, epileptic encephalopathies, other metabolic disorders, cardiomyopathies, and leukodystrophies. These would have been missed if a targeted approach was taken, and some have specific treatments.

Bromodomain and Extraterminal Protein Inhibitor, Apabetalone (RVX-208), Reduces ACE2 Expression and Attenuates SARS-Cov-2 Infection In Vitro.

Effective therapeutics are urgently needed to counter infection and improve outcomes for patients suffering from COVID-19 and to combat this pandemic. Manipulation of epigenetic machinery to influence viral infectivity of host cells is a relatively unexplored area. The bromodomain and extraterminal (BET) family of epigenetic readers have been reported to modulate SARS-CoV-2 infection. Herein, we demonstrate apabetalone, the most clinical advanced BET inhibitor, downregulates expression of cell surface receptors involved in SARS-CoV-2 entry, including angiotensin-converting enzyme 2 (ACE2) and dipeptidyl-peptidase 4 (DPP4 or CD26) in SARS-CoV-2 permissive cells. Moreover, we show that apabetalone inhibits SARS-CoV-2 infection in vitro to levels comparable to those of antiviral agents. Taken together, our study supports further evaluation of apabetalone to treat COVID-19, either alone or in combination with emerging therapeutics.