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Youth purpose was investigated using a two-phase embedded design with youth participating in Scouts BSA (N = 3,943), ages 9-20 (M = 14.0, SD = 1.9). Participating Scouts were mostly White (91%) and male (98%). In Phase 1, we conducted a two-step cluster analysis on Scouts' survey responses to three purpose dimensions (personal meaning, goal-directedness, beyond-the-self orientation). Four clusters emerged: "Purposeful", "Explorers", "Dreamers", "Nonpurposeful." In Phase 2, we explored qualities of purpose within each cluster and programmatic features and relationships within the scouting context fostering youth purpose with a Scout subsample (N = 30) who completed semi-structured interviews. Results demonstrated that adults supporting scouting, inspiration from older peers, and opportunities to help others and explore new activities supported youth purpose.

This paper focuses on data from a study of middle- to upper-middle class Scottish S2 ([approximately]seventh grade) students who participated in Inspiring Purpose, a school-based character development program, during the 2016 to 2017 school year. Directed by their teachers, participating youth reflected on values and future aspirations and researched and wrote about an inspiring person. We examine the hypotheses that youth who select a more proximal inspiring figure and/or make a personal connection with that person will demonstrate higher developmental outcomes in the areas of confidence, contribution, self-reflection, goal development, and self-concept. We connected 230 student posters of inspirational figures, 34 youth interviews, and 462 student surveys. Mixed-method analysis revealed students were more likely to describe proximal figures as inspiring and make explicit personal connections over the course of their engagement with the Inspiring Purpose program. Moreover, youth who made personal connections were more likely to score higher on confidence, general self-concept, and contribution. These results suggest that when youth see themselves in inspiring figures and firmly articulate this connection, this can spur beneficial developmental outcomes.

First-generation Bruton tyrosine kinase (BTK) inhibitor, ibrutinib, has been associated with an increased risk of cardiovascular toxicities. Zanubrutinib is a more selective, next-generation BTK inhibitor. In this analysis, incidence rates of atrial fibrillation, symptomatic (grade ≥2) ventricular arrhythmia, and hypertension were evaluated in a pooled analysis of 10 clinical studies with zanubrutinib monotherapy in patients (N = 1550) with B-cell malignancies and a pooled analysis of head-to-head studies comparing zanubrutinib with ibrutinib (ASPEN cohort 1; ALPINE). Among the 10 studies, most patients (median age, 67 years) were male (66.3%) and had CLL/SLL (60.5%). Overall incidence and exposure-adjusted incidence rates (EAIR) for atrial fibrillation, symptomatic ventricular arrhythmia, and hypertension were lower with zanubrutinib than ibrutinib. Despite a similar prevalence of preexisting cardiovascular events in ASPEN and ALPINE, atrial fibrillation/flutter incidence rates (6.1% vs 15.6%) and EAIR (0.2 vs 0.64 persons per 100 person-months; P < .0001) were lower with zanubrutinib than with ibrutinib. Symptomatic ventricular arrhythmia incidence was low for both zanubrutinib (0.7%) and ibrutinib (1.7%) with numerically lower EAIR (0.02 vs 0.06 persons per 100 person-months, respectively) for zanubrutinib. The hypertension EAIR was lower with zanubrutinib than ibrutinib in ASPEN but similar between treatment arms in ALPINE. The higher hypertension EAIR in ALPINE was inconsistent with other zanubrutinib studies. However, fewer discontinuations (1 vs 14) and deaths (0 vs 6) due to cardiac disorders occurred with zanubrutinib versus ibrutinib in ALPINE. These data support zanubrutinib as a treatment option with improved cardiovascular tolerability compared with ibrutinib for patients with B-cell malignancies in need of BTK inhibitors. These trials were registered at www.ClinicalTrials.gov as # NCT03053440, NCT03336333, NCT03734016, NCT04170283, NCT03206918, NCT03206970, NCT03332173, NCT03846427, NCT02343120, and NCT03189524.

Abstract The tumor microenvironment (TME) is integral to cancer progression, impacting metastasis and treatment response. It consists of diverse cell types, extracellular matrix components, and signaling molecules that interact to promote tumor growth and therapeutic resistance. Elucidating the intricate interactions between cancer cells and the TME is crucial in understanding cancer progression and therapeutic challenges. A critical process induced by TME signaling is the epithelial-mesenchymal transition (EMT), wherein epithelial cells acquire mesenchymal traits, which enhance their motility and invasiveness and promote metastasis and cancer progression. By targeting various components of the TME, novel investigational strategies aim to disrupt the TME’s contribution to the EMT, thereby improving treatment efficacy, addressing therapeutic resistance, and offering a nuanced approach to cancer therapy. This review scrutinizes the key players in the TME and the TME's contribution to the EMT, emphasizing avenues to therapeutically disrupt the interactions between the various TME components. Moreover, the article discusses the TME’s implications for resistance mechanisms and highlights the current therapeutic strategies toward TME modulation along with potential caveats.

Abstract Background Opioid-related overdose is a leading cause of death for criminal legal-involved individuals and, although naloxone distribution and medications for opioid use disorder (MOUD) are effective means for reducing post-release overdose death risk, jail-based availability is limited. This case report describes the challenges faced by three Ohio communities as they implemented evidence-based practices (EBPs) in jails to combat post-release opioid overdose deaths. Method We present case examples of how barriers were overcome to implement jail-based EBPs in three Ohio communities (two urban and one rural) as part of the HEALing Communities Study (UM1DA049417; ClinicalTrials.gov Identifier: NCT04111939). Of the 18 participating Ohio HEALing Communities Study counties, we highlight 3 communities for the novelty of their EBPs implemented, the challenges that they faced, and their rural/urban status. We present descriptive data regarding the EBPs that they implemented and discuss the challenges identified by HEALing Communities Study staff with first-hand experience facilitating their implementation. Results Newly implemented interventions included overdose education and direct provision of naloxone to incarcerated individuals upon release (2 of 3 communities), initiating MOUD prior to release (3 of 3), linkage to ongoing MOUD treatment in the community (2 of 3), peer support-facilitated treatment retention efforts (2 of 3) and emergency housing (1 of 3) in the immediate post-incarceration period. Common challenges that emerged included skepticism about the need and feasibility of implementing EBPs to reduce overdose and death, lack of knowledge about the options available and whether external agencies may assist, and difficulty engaging stakeholders to overcome inertia. Conclusions Creative flexibility, calm persistence, technical facilitation, and collaboration with community service providers were assets that helped these Ohio jails implement evidence-based strategies that combat the opioid epidemic and reduce the likelihood of post-incarceration overdose and death in a high risk, formerly incarcerated population.

Abstract Background As the opioid epidemic continues to have a major negative impact across the US, community pharmacies have come under scrutiny from legal systems attempting to hold them accountable for their role in over dispensing and lack of patient intervention. While the most available tool for monitoring patients’ opioid use is Prescription Drug Monitoring Programs (PDMP), these do not provide pharmacists with actionable information and decision support. Our study addresses this gap through three objectives: [1] incorporate validated opioid risk metric thresholds into a PDMP platform to create the Opioid Risk Reduction Clinical Decision Support (ORRCDS) tool; [2] assess ORRCDS’ ability to reduce patient opioid risk; [3] assess ORRCDS’ sustainability and viability for broader dissemination in community pharmacy. Methods For objective 1, our team is partnering with leadership from the largest US PDMP organization and a top-five pharmacy chain to implement ORRCDS into the pharmacy chain’s workflow following the Guideline Implementation with Decision Support (GUIDES) framework. For objective 2, our team will conduct a type-1 implementation mixed methods study using a 2-arm parallel group clustered randomized design. We anticipate enrolling ~ 6,600 patients with moderate and high opioid use risk during the 6-month enrollment phase across 80 pharmacies. This sample size will provide 96.3% power to detect a 5% or greater difference in responder rate between the intervention and control arm. Responders are patients with moderate-risk at baseline who reduce to low-risk or those with high-risk at baseline who reduce to moderate or low-risk at 180 days post last intervention. To accomplish objective 3, we will use the Consolidated Framework for Implementation Research (CFIR) to develop and execute cross-sectional qualitative interviews with pharmacists (n = 15), pharmacy leaders (n = 15), and PDMP leaders (n = 15) regarding long term adoption and sustainability of the ORRCDS tool. Conclusions A PDMP tool that addresses moderate- and high-risk opioid use is not available in community pharmacy. This study will implement ORRCDS in a large retail pharmacy chain that will include additional screening and guidance to pharmacy staff to address risky opioid medication use. Our results will make critical advancements for protecting patient health and addressing the opioid epidemic.

Policies of dispersal are increasingly favoured internationally for the resettlement of refugees and asylum seekers. With forty percent of the world's forcibly displaced people being school-aged children, the dispersal of refugee-background people into regional areas means that rural schools are central sites of community response to refugees. Little is known in published research about how rural schools engage in refugee education within the policy context of 'dispersal'. This review of relevant literature examines the educational dimensions of dispersal policies, drawing on research in Australia, Canada, the United Kingdom, the United States and Sweden. Research linking refugee resettlement, refugee education and rurality shows a complex interplay between histories of exclusion and contemporary challenges in both the construction of rural spaces, and the deployment of humanitarian dispersal policies at national and international levels. This literature is thematically organised to show that in refugee education within a policyscape of dispersal, rural schools may be 1) operating in racialised community contexts; 2) working within poorly resourced infrastructure; 3) unfamiliar with refugee-background students; and, despite these challenges, they may become 4) key sites of resistance, creativity and support for refugee-background students and their families.

Concerns exist about how the landscape of school sports is changing, leaving many school coaches feeling unprepared to meet the challenges of this role today. This article explores the status of school sports coaching by drawing on recent findings from the National Coach Survey, the first-of-its-kind survey in the United States released in 2022.

Although chronic lymphocytic leukemia (CLL) is diagnosed by identifying a circulating B-cell clone that exceeds 5x106/μL, additional distinct clones (ADC) have been identified in various studies. Notably, the numbers of ADC documented in these studies has increased as the various technologies evolved. To better define the frequency and the characteristics of ADCs in CLL, we used a Next Generation Sequencing (NGS) platform that affords high sequencing depth along with steps that limit overcounting to analyze IGHV-IGHD-IGHJ gene rearrangements in circulating CD5+ B cells from 57 patients. Notably, all patients had at least one ADC, in addition to the clinically relevant clone (CRC)., in 46 patients for whom lymphocyte count data were available, 44 had at least one ADC above the threshold of 1 B cell/μL, and remarkably, the average number of ADCs was 12 per patient. Notably, in two patients, the predominant ADC (pADC) qualified clinically as a separate CLL clone and in the in the remaining cases as low/high-count monoclonal B-cell lymphocytosis (MBL) clones. Moreover, in 11 patients studied longitudinally, pADCs were persistent and often increased in number. ADCs in patients with CLL exhibited fourfold more stereotyped IGHV-IGHD-IGH rearrangements than found in CD5+ B cells from healthy individuals, and IGHV use, somatic mutations, and Ig isotype distribution was similar between pADCs and CRCs. Thus, finding multiple expanded clones within the CD5+ B cells is the rule in patients with CLL, indicating that leukemogenesis is a multiclonal process that likely involves competition among B cells with special BCR features.

Giant cell tumour of bone (GCTB) is viewed as a benign, locally aggressive primary bone tumour with metastatic potential. Current management is surgery with bone curettage or resection and systemic therapy with denosumab. Diagnosis is confirmed histologically prior to surgery, with staging for pulmonary disease, as pulmonary metastases (PM) reportedly occur in

Purpose The prospective Viral load Cohort North-East Lesotho (VICONEL) aims to support clinical management and generate scientific evidence to inform HIV care. Specifically, VICONEL allows for the monitoring of HIV treatment outcomes and health system performance, encompasses a biobank for further research with routinely collected blood plasma samples of consenting participants and provides a valuable framework for nested observational and interventional studies.Participants VICONEL captures routine viral load test results alongside associated demographic and treatment information among people in care for HIV in Lesotho, southern Africa. As of December 2023, it encompasses all viral load testing from 24 healthcare facilities in two districts of Lesotho.Findings to date From January 2016 to December 2023, 137 660 viral load test results were available for 29 380 participants. At the time of the last viral load test, median age was 42 years (IQR: 33–53); 18 511 (63%) were adult women, 10 029 (34%) adult men and 835 (3%) children

The PI3K/AKT/mTOR (PAM) signaling pathway is a highly conserved signal transduction network in eukaryotic cells that promotes cell survival, cell growth, and cell cycle progression. Growth factor signalling to transcription factors in the PAM axis is highly regulated by multiple cross-interactions with several other signaling pathways, and dysregulation of signal transduction can predispose to cancer development. The PAM axis is the most frequently activated signaling pathway in human cancer and is often implicated in resistance to anticancer therapies. Dysfunction of components of this pathway such as hyperactivity of PI3K, loss of function of PTEN, and gain-of-function of AKT, are notorious drivers of treatment resistance and disease progression in cancer. In this review we highlight the major dysregulations in the PAM signaling pathway in cancer, and discuss the results of PI3K, AKT and mTOR inhibitors as monotherapy and in co-administation with other antineoplastic agents in clinical trials as a strategy for overcoming treatment resistance. Finally, the major mechanisms of resistance to PAM signaling targeted therapies, including PAM signaling in immunology and immunotherapies are also discussed.

Of all cancers in female Canadians, the most rapidly increasing incidence is that of cervical cancer. The objective of this pilot study was to assess how HPV self-sampling might improve cervical cancer screening participation in both urban and rural settings in Saskatchewan, one of the most sparsely populated provinces in Canada. Study groups consisted of n = 250 participants to whom self-swabbing kits were mailed with instructions and n = 250 participants to whom kits were handed out in 6 urban and rural clinics. The inclusion criteria selected subjects aged 30–69 years who were Saskatchewan residents for at least 5 years with valid health coverage, had a cervix, and had no record of cervical cancer screening in 4 years. The returned samples were analyzed for specific HPV strains using the Roche Molecular Diagnostics Cobas 4800® System. The overall response rate was ~16%, with the response to the handout distribution being roughly double that of the mailout. While HPV positivity did not differ across the distribution groups, participants at a specific inner-city clinic reported significantly higher positivity to at least one HPV strain as compared to any other clinic and all mailouts combined. For this high-risk population, in-person handout of self-sampling kits may be the most effective means of improving screening.

RNA splicing dysregulation underlies the onset and progression of cancers. In chronic lymphocytic leukemia (CLL), spliceosome mutations leading to aberrant splicing occur in ∼20% of patients. However, the mechanism for splicing defects in spliceosome-unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis, we discover that proteins involved in RNA splicing are posttranscriptionally upregulated in CLL cells, resulting in splicing dysregulation. The abundance of splicing complexes is an independent risk factor for poor prognosis. Moreover, increased splicing factor expression is highly correlated with the abundance of METTL3, an RNA methyltransferase that deposits N6-methyladenosine (m6A) on mRNA. METTL3 is essential for cell growth in vitro and in vivo and controls splicing factor protein expression in a methyltransferase-dependent manner through m6A modification-mediated ribosome recycling and decoding. Our results uncover METTL3-mediated m6A modification as a novel regulatory axis in driving splicing dysregulation and contributing to aggressive CLL.SignificanceMETTL3 controls widespread splicing factor abundance via translational control of m6A-modified mRNA, contributes to RNA splicing dysregulation and disease progression in CLL, and serves as a potential therapeutic target in aggressive CLL. See related commentary by Janin and Esteller, p. 176. This article is highlighted in the In This Issue feature, p. 171.

Lignocellulose forms plant cell walls, and its three constituent polymers, cellulose, hemicellulose and lignin, represent the largest renewable organic carbon pool in the terrestrial biosphere. Insights into biological lignocellulose deconstruction inform understandings of global carbon sequestration dynamics and provide inspiration for biotechnologies seeking to address the current climate crisis by producing renewable chemicals from plant biomass. Organisms in diverse environments disassemble lignocellulose, and carbohydrate degradation processes are well defined, but biological lignin deconstruction is described only in aerobic systems. It is currently unclear whether anaerobic lignin deconstruction is impossible because of biochemical constraints or, alternatively, has not yet been measured. We applied whole cell-wall nuclear magnetic resonance, gel-permeation chromatography and transcriptome sequencing to interrogate the apparent paradox that anaerobic fungi (Neocallimastigomycetes), well-documented lignocellulose degradation specialists, are unable to modify lignin. We find that Neocallimastigomycetes anaerobically break chemical bonds in grass and hardwood lignins, and we further associate upregulated gene products with the observed lignocellulose deconstruction. These findings alter perceptions of lignin deconstruction by anaerobes and provide opportunities to advance decarbonization biotechnologies that depend on depolymerizing lignocellulose.

Introduction The approval of Bruton tyrosine kinase (BTK) inhibitor ibrutinib and PI3K inhibitor idelalisib has revolutionized chronic lymphocytic leukemia (CLL) therapy (1-4). These B cell receptor (BCR) inhibitors induce transient [...], Bruton tyrosine kinase (BTK) inhibitor therapy induces peripheral blood lymphocytosis in chronic lymphocytic leukemia (CLL), which lasts for several months. It remains unclear whether nongenetic adaptation mechanisms exist, allowing CLL cells&apos; survival during BTK inhibitor-induced lymphocytosis and/or playing a role in therapy resistance. We show that in approximately 70% of CLL cases, ibrutinib treatment in vivo increases Akt activity above pretherapy levels within several weeks, leading to compensatory CLL cell survival and a more prominent lymphocytosis on therapy. Ibrutinib-induced Akt phosphorylation ([pAkt.sup.S473]) is caused by the upregulation of Forkhead box protein 01 (FoxO1) transcription factor, which induces expression of Rictor, an assembly protein for the mTORC2 protein complex that directly phosphorylates Akt at serine 473 (S473). Knockout or inhibition of FoxO1 or Rictor led to a dramatic decrease in Akt phosphorylation and growth disadvantage for malignant B cells in the presence of ibrutinib (or PI3K inhibitor idelalisib) in vitro and in vivo. The FoxO1/ Rictor/[pAkt.sup.S473] axis represents an early nongenetic adaptation to B cell receptor (BCR) inhibitor therapy not requiring PI3K[delta] or BTK kinase activity. We further demonstrate that FoxO1 can be targeted therapeutically and its inhibition induces CLL cells&apos; apoptosis alone or in combination with BTK inhibitors (ibrutinib, acalabrutinib, pirtobrutinib) and blocks their proliferation triggered by T cell factors (CD40L, IL-4, and IL-21).

Chronic lymphocytic leukemia (CLL) patients have an increased risk of secondary cancers, along with predisposition to CLL in their relatives. We have previously identified germline ATM variants as associated with CLL risk; here, we present their impact on predisposition to secondary neoplasms in CLL patients and their relatives. Patients enrolled in our tissue bank who had germline ATM status available were mailed a questionnaire between April 2022 and May 2023. Of the 333 patients who replied to the questionnaire, 283 patients (85%) reported at least one relative with a cancer history. The prevalence of family history of B-cell lymphoproliferative disorders was significantly higher (p=0.02) in patients with germline ATM variants (32%) compared to those without germline ATM variants (21%) including familial CLL (25% vs 18%) (p=0.04). No significant difference in the prevalence of secondary cancers was found between patients with and without germline ATM variants (p=0.73), although the role for individual ATM variants in other malignancies could not be excluded given the small sample size. Time to first CLL treatment (TTFT) was shorter in patients harboring somatic ATM events while no difference was observed in patients with germline ATM variants. In conclusion, we demonstrate a higher prevalence of B-cell lymphoproliferative disorders, including familial CLL, in relatives of CLL patients carrying germline ATM variants. The presence of these germline variants did not impact TTFT compared to patients harboring somatic ATM mutations.

Bruton tyrosine kinase inhibitors (BTKi) have transformed the treatment of B-cell malignancies, but intolerance has often led to their discontinuation. The phase 1/2 BRUIN study evaluated pirtobrutinib, a highly selective non-covalent (reversible) BTKi, in patients with R/R B-cell malignancies (NCT03740529). Pirtobrutinib was investigated in 127 patients with intolerance to at least one prior BTKi therapy in the absence of progressive disease. The most common adverse event (AE) leading to BTKi discontinuation was cardiac disorders (n=40, 31.5%), specifically atrial fibrillation (n=30, 23.6%). The median follow-up was 17.4 months and the median time on pirtobrutinib was 15.3 months. The most common reasons for pirtobrutinib discontinuation were progressive disease (26.8%), AE (10.2%), or death (5.5%). The most frequent treatment-emergent AEs were fatigue (39.4%) and neutropenia (37.0%). Among patients who discontinued a prior BTKi for a cardiac issue, 75% had no recurrence of their cardiac AE. No patient discontinued pirtobrutinib for the same AE that led to discontinuation of the prior BTKi. In 78 CLL/SLL and 21 MCL patients intolerant to prior BTKi, ORR to pirtobrutinib was 76.9% and 81.0%, respectively. Median PFS for CLL/SLL was 28.4 months and was not estimable for MCL. These results suggest that pirtobrutinib was safe, well-tolerated, and an efficacious option in patients with prior BTKi-intolerance.

A system for co-cultivation of anaerobic fungi with anaerobic bacteria was established based on lactate cross-feeding to produce butyrate and butanol from plant biomass. Several co-culture formulations were assembled that consisted of anaerobic fungi (Anaeromyces robustus, Neocallimastix californiae, or Caecomyces churrovis) with the bacterium Clostridium acetobutylicum. Co-cultures were grown simultaneously (e.g., 'one pot'), and compared to cultures where bacteria were cultured in fungal hydrolysate sequentially. Fungal hydrolysis of lignocellulose resulted in 7-11 mM amounts of glucose and xylose, as well as acetate, formate, ethanol, and lactate to support clostridial growth. Under these conditions, one-stage simultaneous co-culture of anaerobic fungi with C. acetobutylicum promoted the production of butyrate up to 30 mM. Alternatively, two-stage growth slightly promoted solventogenesis and elevated butanol levels (∼4-9 mM). Transcriptional regulation in the two-stage growth condition indicated that this cultivation method may decrease the time required to reach solventogenesis and induce the expression of cellulose-degrading genes in C. acetobutylicum due to relieved carbon-catabolite repression. Overall, this study demonstrates a proof of concept for biobutanol and bio-butyrate production from lignocellulose using an anaerobic fungal-bacterial co-culture system.

PurposeZanubrutinib is a potent, irreversible next-generation Bruton tyrosine kinase (BTK) inhibitor designed to maximize BTK occupancy and minimize off-target kinase inhibition. We hypothesized that complete/sustained BTK occupancy may improve efficacy outcomes and increased BTK specificity may minimize off-target inhibition-related toxicities.Patients and methodsALPINE (ClinicalTrials.gov identifier: NCT03734016) is a global, randomized, open-label phase III study of zanubrutinib versus ibrutinib in patients with relapsed/refractory chronic lymphocytic leukemia. The primary end point was investigator-assessed overall response rate (ORR). The preplanned interim analysis was scheduled approximately 12 months after the first 415 patients were enrolled.ResultsBetween November 1, 2018, and December 14, 2020, 652 patients were enrolled. We present the interim analysis of the first 415 enrolled patients randomly assigned to receive zanubrutinib (n = 207) or ibrutinib (n = 208). At 15 months of median follow-up, ORR (partial or complete response) was significantly higher with zanubrutinib (78.3%; 95% CI, 72.0 to 83.7) versus ibrutinib (62.5%; 95% CI, 55.5 to 69.1; two-sided P < .001). ORR was higher with zanubrutinib versus ibrutinib in subgroups with del(17p)/TP53 mutations (80.5% v 50.0%) and del(11q) (83.6% v 69.1%); 12-month progression-free survival in all patients was higher with zanubrutinib (94.9%) versus ibrutinib (84.0%; hazard ratio, 0.40; 95% CI, 0.23 to 0.69). Atrial fibrillation rate was significantly lower with zanubrutinib versus ibrutinib (2.5% v 10.1%; two-sided P = .001). Rates of cardiac events, major hemorrhages, and adverse events leading to treatment discontinuation/death were lower with zanubrutinib.ConclusionZanubrutinib had a significantly higher ORR, lower atrial fibrillation rate, and improved progression-free survival and overall cardiac safety profile versus ibrutinib. These data support improved efficacy/safety outcomes with selective BTK inhibition.

Anaerobic fungi produce biomass-degrading enzymes and natural products that are important to harness for several biotechnology applications. Although progress has been made in the development of methods for extracting nucleic acids for genomic and transcriptomic sequencing of these fungi, most studies are limited in that they do not sample multiple fungal growth phases in batch culture. In this study, we establish a method to harvest RNA from fungal monocultures and fungal-methanogen co-cultures, and also determine an optimal time frame for high-quality RNA extraction from anaerobic fungi. Based on RNA quality and quantity targets, the optimal time frame in which to harvest anaerobic fungal monocultures and fungal-methanogen co-cultures for RNA extraction was 2-5 days of growth post-inoculation. When grown on cellulose, the fungal strain Anaeromyces robustus cocultivated with the methanogen Methanobacterium bryantii upregulated genes encoding fungal carbohydrate-active enzymes and other cellulosome components relative to fungal monocultures during this time frame, but expression patterns changed at 24-hour intervals throughout the fungal growth phase. These results demonstrate the importance of establishing methods to extract high-quality RNA from anaerobic fungi at multiple time points during batch cultivation.

Richter syndrome (RS) arising from chronic lymphocytic leukemia (CLL) exemplifies an aggressive malignancy that develops from an indolent neoplasm. To decipher the genetics underlying this transformation, we computationally deconvoluted admixtures of CLL and RS cells from 52 patients with RS, evaluating paired CLL-RS whole-exome sequencing data. We discovered RS-specific somatic driver mutations (including IRF2BP2, SRSF1, B2M, DNMT3A and CCND3), recurrent copy-number alterations beyond del(9p21)(CDKN2A/B), whole-genome duplication and chromothripsis, which were confirmed in 45 independent RS cases and in an external set of RS whole genomes. Through unsupervised clustering, clonally related RS was largely distinct from diffuse large B cell lymphoma. We distinguished pathways that were dysregulated in RS versus CLL, and detected clonal evolution of transformation at single-cell resolution, identifying intermediate cell states. Our study defines distinct molecular subtypes of RS and highlights cell-free DNA analysis as a potential tool for early diagnosis and monitoring.

Immune cell function varies tremendously between individuals, posing a major challenge to emerging cellular immunotherapies. This report pursues the use of cell morphology as an indicator of high-level T cell function. Short-term spreading of T cells on planar, elastic surfaces was quantified by 11 morphological parameters and analyzed to identify effects of both intrinsic and extrinsic factors. Our findings identified morphological features that varied between T cells isolated from healthy donors and those from patients being treated for Chronic Lymphocytic Leukemia (CLL). This approach also identified differences between cell responses to substrates of different elastic modulus. Combining multiple features through a machine learning approach such as Decision Tree or Random Forest provided an effective means for identifying whether T cells came from healthy or CLL donors. Further development of this approach could lead to a rapid assay of T cell function to guide cellular immunotherapy.