Baylor College of Medicine

Li S., Nordick K.V., Elsenousi A.E., Bhattacharya R., Kirby R.P., Hassan A.M., Hochman-Mendez C., Rosengart T.K., Liao K.K., Mondal N.K.

We investigated ferroptosis, a type of programmed cell death mechanism, in human hearts donated after brain death (DBD) and those donated after circulatory death (DCD), focusing on warm ischemia time (WIT) and cold storage. A total of twenty-four hearts were procured, with six from the DBD group and eighteen from the DCD group. The DCD group was divided into three subgroups, each containing six hearts, based on different WITs of 20, 40, and 60 minutes. All procured hearts were placed in cold storage for up to 6 hours. Left ventricular biopsies were performed at 0, 2, 4, and 6 hours. We measured ferroptosis regulators (GPX4, ACSL4, and transferrin receptor), iron content (Fe2+ and Fe3+), and lipid peroxidation (MDA) in the cardiac tissue. Modulation of ferroptosis was observed in both DBD and DCD hearts. Warm ischemia injury increased myocardial vulnerability to ferroptotic cell death. For DBD hearts, up to 6 hours of cold storage increases cardiac levels of MDA, iron content and ACSL4, thereby increasing vulnerability to ferroptotic cell death. In contrast, for DCD hearts with a WIT of 40 minutes or more, warm ischemia injury was identified as the primary factor contributing to increased myocardial susceptibility to ferroptotic cell death. Ferroptosis may serve as a promising target to optimize cold preservation for DBD hearts. For DCD hearts, strategies to inhibit ferroptosis should focus on the early warm ischemia phase to assess donor heart quality and suitability for transplantation.

Green A.L., Minard C.G., Liu X., Safgren S.L., Pinkney K., Harris L., Link G., DeSisto J., Voss S., Nelson M.D., Reid J.M., Fox E., Weigel B.J., Glade Bender J.

Abstract Purpose: Selinexor is a first-in-class, central nervous system (CNS)–penetrant, oral inhibitor of exportin 1 (XPO1), the main nuclear exporter of many key tumor suppressors. We report a phase I trial of selinexor in children and adolescents with recurrent CNS and solid tumors (NCT02323880). Patients and Methods: A rolling six design was used to evaluate the maximum tolerated dose (MTD) and first dose pharmacokinetics of selinexor administered once (35–45 mg/m2) or twice (20–35 mg/m2) weekly during a 28-day cycle (part A). Ten additional patients with high-grade glioma (HGG) were treated at the MTD administered once weekly (part B). Results: In part A, 49 patients were enrolled. Continuous twice weekly dosing was limited by extended hematologic toxicity. The MTD on a twice weekly schedule for 3 weeks on/1 week off (twice weekly 3/1) was 20 mg/m2/dose. Dose-limiting toxicities (DLTs) on this schedule included fatigue, acute reversible neurologic changes, neutropenia, thrombocytopenia, and aspartate aminotransferase/alanine aminotransferase increase. On a continuous once weekly schedule, the MTD was 35 mg/m2/dose; DLTs included seizure and thrombocytopenia. In part B (HGG expansion), there were no additional DLTs observed. Non-DLTs included lymphopenia, leukopenia, neutropenia, thrombocytopenia, anorexia, fatigue, hypophosphatemia, nausea, and vomiting. There were no objective responses. The median number of cycles received was 1 (range, 1–9); eight of 59 patients (13.5%) received 5 to 9 cycles, five of whom had HGG. Conclusions: Selinexor-related toxicities were primarily hematologic and neurologic, requiring dose or dose-frequency reduction. The MTD and recommended initial phase II dose of selinexor in children and adolescents with recurrent solid and CNS tumors is 35 mg/m2/dose once weekly.

Rakhlin N.V., Li N., Aljughaiman A., Grigorenko E.L.

Purpose: We investigated the role of spoken language in the acquisition of literacy in the context of Arabic diglossia, where the written language, Standard Arabic, deviates substantially from the spoken language, Colloquial Arabic, children acquire naturally from birth. Method: The participants ( N = 110; 40 girls) were Saudi Arabic–speaking children in Grades 2–4. Children completed assessments of oral paragraph reading and word decoding using a vowelized script. They also completed three spoken assessments of Colloquial Arabic, which include sentence comprehension, sentence completion, and pragmatic knowledge, as well as a test of phonological awareness. We used path analysis to investigate the contributions of each of the spoken language indicators, decoding, and phonological awareness to reading comprehension (RC) in single and multiple serial mediator models. Results: We found that spoken language (i.e., sentence comprehension, sentence completion, and pragmatic knowledge) and word decoding uniquely contributed to RC. Moreover, word decoding mediated the association between spoken language and RC. The path from spoken language to phonological awareness, then to word decoding, and finally to RC was inconclusive. Conclusions: While most studies on literacy acquisition in the context of Arabic diglossia focus on the linguistic distance between the colloquial (spoken) and the standard (written) language varieties, our results highlight the important contribution of spoken language skills to reading skills in Arabic despite this linguistic gap. The important implication of these findings is that spoken language interventions aimed at boosting children's narrative language skills in their home language (Colloquial Arabic) are an important tool for building a foundation for literacy in diglossia contexts.

Piedra F., Salazar M.A., Abouelniaj S., Rahman R., Clark J.C., Han Y., Wang Z., Maresso A.

ABSTRACT The scale of plastic pollution boggles the mind. Nearly 400 megatons of virgin plastics are produced annually, with an environmental release rate of 80%, and plastic waste, including microplastics and nanoplastics, is associated with a plethora of problems. The naturally evolved abilities of plastic-degrading microbes offer a starting point for generating sustainable and eco-centric solutions to plastic pollution—a field of endeavor we term eco-microbiology. Here, we developed an iterative discovery procedure coupling faster polyethylene terephthalate (PET)-dependent bioactivity screens with longer-term PET biodegradation assays to find biochemical boosters of PET consumption by the bacterium Piscinibacter sakaiensis . We discovered multiple hits supporting the enhancement of PET biodegradation, with a 0.39% dilution of growth medium #802, a rich medium similar to Luria-Bertani broth, on average more than doubling the rate of PET biodegradation both alone and in combination with 0.125% ethylene glycol. In addition, we identified other chemical species (sodium phosphate, L-serine, GABA) worth further exploring, especially in combination with growth medium #802, for enhanced PET biodegradation by P. sakaiensis . This work represents an important step toward the creation of a low-cost PET fermentation process needed to help solve PET plastic pollution. IMPORTANCE Plastic pollution is an urgent issue. Adding to the well-known problems of bulk plastic litter, shed microplastics and nanoplastics are globally distributed, found in diverse organisms including human foodstuffs and tissues, and increasingly associated with chronic disease. Solutions are needed and the microbial world offers abundant help via naturally evolved consumers of plastic waste. We are working to accelerate polyethylene terephthalate (PET) plastic biodegradation by Piscinibacter sakaiensis , a recently described bacterium that evolved to slowly but completely consume PET, one of the most common types of plastic pollution. We used a combination of PET-dependent bioactivity screens and biodegradation tests to find stimulators of PET biodegradation. Out of hundreds, we found a small number of biochemical conditions that more than double the PET biodegradation rate. Our work provides a foundation for further studies to realize a fermentation process needed to help solve PET plastic pollution.

Midani F.S., Danhof H.A., Mathew N., Ardis C.K., Garey K.W., Spinler J.K., Britton R.A.

ABSTRACT Clostridioides difficile is a gram-positive spore-forming pathogen that commonly causes diarrheal infections in the developed world. Although C. difficile is a genetically diverse species, certain ribotypes are overrepresented in human infections, and it is unclear if metabolic adaptations are essential for the emergence of these epidemic ribotypes. To identify ribotype-specific metabolic differences, we therefore tested carbon substrate utilization by 88 C . difficile isolates and looked for differences in growth between 22 ribotypes. As expected, C. difficile was capable of growing on a variety of carbon substrates. Further, C. difficile strains clustered by phylogenetic relationship and displayed ribotype-specific and clade-specific metabolic capabilities. Surprisingly, we observed that two emerging lineages, ribotypes 023 and 255, have divergent metabolic phenotypes. In addition, although C. difficile Clade 5 is the most evolutionary distant clade and often detected in animals, it displayed robust growth on simple sugars similar to Clades 1–4. Altogether, our results corroborate the generalist metabolic strategy of C. difficile but also demonstrate lineage-specific metabolic capabilities. IMPORTANCE The gut pathogen Clostridioides difficile utilizes a wide range of carbon sources. Microbial communities can be rationally designed to combat C. difficile by depleting its preferred nutrients in the gut. However, C. difficile is genetically diverse with hundreds of identified ribotypes, and most of its metabolic studies were performed with lab-adapted strains. To identify ribotype-specific metabolic differences, we profiled carbon metabolism by a myriad of C. difficile clinical isolates. While the metabolic capabilities of these isolates clustered by their genetic lineage, we observed surprising metabolic divergence between two emerging lineages. We also found that genetically newer and older clades grew to a similar level on simple sugars, which contrasts with recent findings that newer clades experienced positive selection on genes involved in simple sugar metabolism. Altogether, our results underscore the importance of considering the metabolic diversity of pathogens in the study of their evolution and the rational design of therapeutic interventions.

Fiorica P.N., Golmard L., Kim J., Bao R., Lin F.Y., Roy A., Pribnow A., Perrino M.R., Masliah-Planchon J., Michalak-Provost S., Wong J., Filser M., Stoppa-Lyonnet D., Bourdeaut F., Brahimi A., et. al.

Abstract Purpose: DROSHA, DGCR8, and DICER1 regulate miRNA biogenesis and are commonly mutated in cancer. Although DGCR8 and DICER1 germline pathogenic variants (GPV) cause autosomal dominant tumor predisposition, no association between DROSHA GPVs and clinical phenotypes has been reported. Experimental Design: After obtaining informed consent, sequencing was performed on germline and tumor samples from all patients. The occurrence of germline DROSHA GPVs was investigated in large pediatric and adult cancer datasets. The population prevalence of DROSHA GPVs was investigated in the UK Biobank and Geisinger DiscovEHR cohorts. Results: We describe nine children from eight families with heterozygous DROSHA GPVs and a diagnosis of pineoblastoma (n = 8) or Wilms tumor (n = 1). A somatic second hit in DROSHA was detected in all eight tumors analyzed. All pineoblastoma tumors analyzed were classified as miRNA processing–altered 1 subtype. We estimate the population prevalence of germline DROSHA loss-of-function variants to be 1:3,875 to 1:4,843 but find no evidence for increased adult cancer risk. Conclusions: This is the first report of DROSHA-related tumor predisposition. As pineoblastoma and Wilms tumor are also associated with DICER1 GPVs, our results suggest that the tissues of origin for these tumors are uniquely tolerant of general miRNA loss. The miRNA processing–altered 1 pineoblastoma subtype is associated with older age of diagnosis and better outcomes than other subtypes, suggesting DROSHA GPV status may have important clinical and prognostic significance. We suggest that genetic testing for DROSHA GPVs be considered for patients with pineoblastoma, Wilms tumor, or other DICER1-/DGCR8-related conditions and propose surveillance recommendations through research studies for individuals with DROSHA GPVs.

Fletcher F.E.

In this narrative medicine essay, a bioethicist deliberates about whether she should consent to an autopsy following her mother’s sudden death to advance science or decline to honor, respect, and protect her.

Hwang E.S., Hyslop T., Lynch T., Ryser M.D., Weiss A., Wolf A., Norris K., Witten M., Grimm L., Schnitt S., Badve S., Factor R., Frank E., Collyar D., Basila D., et. al.

ImportanceActive monitoring for low-risk ductal carcinoma in situ (DCIS) of the breast has been proposed as an alternative to guideline-concordant care, but the safety of this approach is unknown.ObjectiveTo compare rates of invasive cancer in patients with low-risk DCIS receiving active monitoring vs guideline-concordant care.Design, Setting, and ParticipantsProspective, randomized noninferiority trial enrolling 995 women aged 40 years or older with a new diagnosis of hormone receptor–positive grade 1 or grade 2 DCIS without invasive cancer at 100 US Alliance Cancer Cooperative Group clinical trial sites from 2017 to 2023.InterventionsParticipants were randomized to receive active monitoring (follow-up every 6 months with breast imaging and physical examination; n = 484) or guideline-concordant care (surgery with or without radiation therapy; n = 473).Main Outcomes and MeasuresThe primary outcome was 2-year cumulative risk of ipsilateral invasive cancer diagnosis, according to planned intention-to-treat and per-protocol analyses, with a noninferiority bound of 0.05%.ResultsThe median age of the 957 participants analyzed was 63.6 (95% CI, 55.5-70.5) years in the guideline-concordant care group and 63.7 (95% CI, 60.0-71.6) years in the active monitoring group. Overall, 15.7% of participants were Black and 75.0% were White. In this prespecified primary analysis, median follow-up was 36.9 months; 346 patients had surgery for DCIS, 264 in the guideline-concordant care group and 82 in the active monitoring group. Forty-six women were diagnosed with invasive cancer, 19 in the active monitoring group and 27 in the guideline-concordant care group. The 2-year Kaplan-Meier cumulative rate of ipsilateral invasive cancer was 4.2% in the active monitoring group vs 5.9% in the guideline-concordant care group, a difference of −1.7% (upper limit of the 95% CI, 0.95%), indicating that active monitoring is not inferior to guideline-concordant care. Invasive tumor characteristics did not differ significantly between groups.Conclusions and RelevanceWomen with low-risk DCIS randomized to active monitoring did not have a higher rate of invasive cancer in the same breast at 2 years compared with those randomized to guideline-concordant care.Trial RegistrationClinicalTrials.gov Identifier: NCT02926911

Devine K.J., Schwartz L.F., El-Mallawany N.K.

Abstract Peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) is a rare mature T-cell non-Hodgkin lymphoma (NHL) seen in both children and adults. Although it is the most common nonanaplastic mature T-cell lymphoma of childhood, it is quite rare and, therefore, the standard of care remains largely undefined. It is a disease characterized by clinical and pathological heterogeneity and is generally associated with an aggressive clinical course and poor prognosis in adults. Retrospective reports on treatment outcomes for pediatric PTCL-NOS are limited by small cohorts, variable clinical presentations, and heterogeneous treatment regimens. Although published survival rates in children appear encouraging compared with those from prospective studies in adults, the prognosis is guarded, and relatively low curative outcomes are in stark contrast to more common pediatric NHL. Although recent landmark gene profiling studies have shed light on the molecular landscape of the disease in adults, identifying molecular subgroups with prognostic significance, the biology of PTCL-NOS remains unclear in children. Here, we review the clinical presentation and diagnosis, historical treatment approaches, current knowledge of the disease biology, and the role of hematopoietic stem cell transplant (HSCT) in PTCL-NOS in children to pursue a better understanding of this heterogeneous condition and empower physicians to use this information to best support our pediatric population. Studies focusing on pediatric PTCL-NOS are required to unravel the disease biology in children, improve risk stratification, and better define upfront treatment through the role of targeted agents and HSCT, as we look to future directions of the care of children with PTCL-NOS.

Tian G., Barragán G.A., Yu H., Martinez-Amador C., Adaikkalavan A., Rios X., Guo L., Drabek J.M., Pardias O., Xu X., Montalbano A., Zhang C., Li Y., Courtney A.N., Di Pierro E.J., et. al.

Abstract Natural killer T cells (NKTs) are a promising platform for cancer immunotherapy, but few genes involved in the regulation of NKT therapeutic activity have been identified. To find regulators of NKT functional fitness, we developed a CRISPR/Cas9-based mutagenesis screen that uses a guide RNA (gRNA) library targeting 1,118 immune-related genes. Unmodified NKTs and NKTs expressing a GD2-specific chimeric antigen receptor (GD2.CAR) were transduced with the gRNA library and exposed to CD1d+ leukemia or CD1d−GD2+ neuroblastoma cells, respectively, over six challenge cycles in vitro. Quantification of gRNA abundance revealed enrichment of PRDM1-specific gRNAs in both NKTs and GD2.CAR NKTs, a result that was validated through targeted PRDM1 knockout. Transcriptional, phenotypic, and functional analyses demonstrated that CAR NKTs with PRDM1 knockout underwent central memory–like differentiation and resisted exhaustion. However, these cells downregulated the cytotoxic mediator granzyme B and showed reduced in vitro cytotoxicity and only moderate in vivo antitumor activity in a xenogeneic neuroblastoma model. In contrast, short hairpin RNA-mediated PRDM1 knockdown preserved effector function while promoting central memory differentiation, resulting in GD2.CAR NKTs with potent in vivo antitumor activity. Thus, we have identified PRDM1 as a regulator of NKT memory differentiation and effector function that can be exploited to improve the efficacy of NKT-based cancer immunotherapies.

Zhu D., Wozniak K.J., Midani F., Wang S., Sun X., Britton R.A.

ABSTRACT Mutations affecting Clostridioides difficile flagellin (FliC) have been shown to be hypervirulent in animal models and display increased toxin production and alterations in central metabolism. The regulation of flagellin levels in bacteria is governed by a tripartite regulatory network involving fliC , fliW , and csrA , which creates a feedback system to regulate flagella production. Through genomic analysis of C. difficile clade 5 strains (non-motile), we identified they have jettisoned many of the genes required for flagellum biosynthesis yet retain the major flagellin gene fliC and regulatory gene fliW . We therefore investigated the roles of fliC , fliW , and csrA in the clade 5 ribotype 078 strain C. difficile 1015, which lacks flagella and is non-motile. Analysis of mutations in fliC , fliW , and csrA (and all combinations) on C. difficile pathogenesis indicated that FliW plays a central role in C. difficile virulence as animals infected with strains carrying a deletion of fliW showed decreased survival and increased disease severity. These in vivo findings were supported by in vitro studies showing that mutations impacting the activity of FliW showed increased toxin production. We further identified that FliW can interact with the toxin-positive regulator TcdR, indicating that modulation of toxin production via FliW occurs by sequestering TcdR from activating toxin transcription. Furthermore, disruption of the fliC-fliW-csrA network results in significant changes in carbon source utilization and sporulation. This work highlights that key proteins involved in flagellar biosynthesis retain their regulatory roles in C. difficile pathogenesis and physiology independent of their functions in motility. IMPORTANCE Clostridioides difficile is a leading cause of nosocomial antibiotic-associated diarrhea in developed countries with many known virulence factors. In several pathogens, motility and virulence are intimately linked by regulatory networks that allow coordination of these processes in pathogenesis and physiology. Regulation of C. difficile toxin production by FliC has been demonstrated in vitro and in vivo and has been proposed to link motility and virulence. Here, we show that clinically important, non-motile C. difficile strains have conserved FliC and regulatory partners FliW and CsrA, despite lacking the rest of the machinery to produce functional flagella. Our work highlights a novel role for flagellin outside of its role in motility and FliW in the pathogenesis and physiology of C. difficile .

Pose E., Jiménez C., Zaccherini G., Campion D., Piano S., Uschner F.E., de Wit K., Roux O., Gananandan K., Laleman W., Solé C., Alonso S., Cuyàs B., Ariza X., Juanola A., et. al.

ImportanceThere are no useful treatments to prevent the development of severe complications of liver cirrhosis. Simvastatin and rifaximin have shown beneficial effects in liver cirrhosis.ObjectiveTo assess whether simvastatin combined with rifaximin improves outcomes in patients with decompensated cirrhosis.Design, Setting, and ParticipantsDouble-blind, placebo-controlled, phase 3 trial conducted among patients with decompensated cirrhosis in 14 European hospitals between January 2019 and December 2022. The last date of follow-up was December 2022.InterventionsPatients were randomly assigned to receive simvastatin, 20 mg/d, plus rifaximin, 1200 mg/d (n = 117), or identical-appearing placebo (n = 120) for 12 months in addition to standard therapy, stratified according to Child-Pugh class B or C.Main Outcomes and MeasuresThe primary end point was incidence of severe complications of liver cirrhosis associated with organ failure meeting criteria for acute-on-chronic liver failure. Secondary outcomes included transplant or death and a composite end point of complications of cirrhosis (ascites, hepatic encephalopathy, variceal bleeding, acute kidney injury, and infection).ResultsAmong the 237 participants randomized (Child-Pugh class B: n = 194; Child-Pugh class C: n = 43), 72% were male and the mean age was 57 years. There were no differences between the 2 groups in terms of development of acute-on-chronic liver failure (21 [17.9%] vs 17 [14.2%] patients in the treatment and placebo groups, respectively; hazard ratio, 1.23; 95% CI, 0.65-2.34; P = .52); transplant or death (22 [18.8%] vs 29 [24.2%] patients in the treatment and placebo groups, respectively; hazard ratio, 0.75; 95% CI, 0.43-1.32; P = .32); or development of complications of cirrhosis (50 [42.7%] vs 55 [45.8%] patients in the treatment and placebo groups, respectively; hazard ratio, 0.93; 95% CI, 0.63-1.36; P = .70). Incidence of adverse events was similar in both groups (426 vs 419; P = .59), but 3 patients in the treatment group (2.6%) developed rhabdomyolysis.Conclusions and RelevanceThe addition of simvastatin plus rifaximin to standard therapy does not improve outcomes in patients with decompensated liver cirrhosis.Trial RegistrationClinicalTrials.gov Identifier: NCT03780673

O'Farrell C., Grimes A.B., Nakano T.A., Klaassen R.J., Lambert M.P., Neunert C.E., Rothman J.A., Shimano K.A., Grace R.F.

Mercado A.

Du H., Szafranski P., Gerard A., Azamian M.S., Bi W., Bekheirnia M.R., Stankiewicz P.

ABSTRACTClark–Baraitser syndrome is a rare neurodevelopmental disorder associated with the E3 ubiquitin‐protein ligase gene TRIP12. Using chromosomal microarray analysis (CMA), long‐range PCR, breakpoint sequencing, and RNA analyses, we studied a 6‐year‐old female presenting with developmental delay, aggressive behavior, attention‐deficit hyperactivity disorder, and mild dysmorphic features. CMA revealed a de novo ~87 kb copy‐number variant (CNV) duplication at 2q36.3, involving Exons 3–14 of TRIP12. Long‐range PCR and Sanger sequencing showed a head‐to‐tail tandem duplication with breakpoints in Introns 2 and 14. RNA analysis identified a novel splicing junction between the coding Exon 14 and the stop codon of the noncoding portion of Exon 3, resulting in a premature translation termination. This suggests the neo‐transcript undergoes nonsense‐mediated decay and/or produces a truncated protein lacking the critical E6AP‐type E3 ubiquitin–protein ligase domain. This case further highlights the challenges with the clinical interpretation of CNV gains and the usefulness of RNA sequencing in the clarification of the impacts of intragenic duplications.