Microbiota dictate T cell clonal selection to augment graft-versus-host disease after stem cell transplantation.

Allogeneic T cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor T cells interact with peptide-major histocompatibility complexes (MHCs), though clonal interrogation remains challenging due to the sparseness of the T cell repertoire. We developed a Bayesian model using donor and recipient T cell receptor (TCR) frequencies in murine stem cell transplant systems to define limited common expansion of T cell clones across genetically identical donor-recipient pairs. A subset of donor CD4+ T cell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific T cells augmented GVHD lethality and could target microbial antigens presented by gastrointestinal epithelium during an alloreactive response. The microbiota serves as a source of cognate antigens that contribute to clonotypic T cell expansion and the induction of GVHD independent of donor-recipient genetics.

Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.

The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

Stress response silencing by an E3 ligase mutated in neurodegeneration.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

Water splitting with silicon p-i-n superlattices suspended in solution.

Photoelectrochemical (PEC) water splitting to produce hydrogen fuel was first reported 50 years ago1, yet artificial photosynthesis has not become a widespread technology. Although planar Si solar cells have become a ubiquitous electrical energy source economically competitive with fossil fuels, analogous PEC devices have not been realized, and standard Si p-type/n-type (p-n) junctions cannot be used for water splitting because the bandgap precludes the generation of the needed photovoltage. An alternative paradigm, the particle suspension reactor (PSR), forgoes the rigid design in favour of individual PEC particles suspended in solution, a potentially low-cost option compared with planar systems2,3. Here we report Si-based PSRs by synthesizing high-photovoltage multijunction Si nanowires (SiNWs) that are co-functionalized to catalytically split water. By encoding a p-type-intrinsic-n-type (p-i-n) superlattice within single SiNWs, tunable photovoltages exceeding 10 V were observed under 1 sun illumination. Spatioselective photoelectrodeposition of oxygen and hydrogen evolution co-catalysts enabled water splitting at infrared wavelengths up to approximately 1,050 nm, with the efficiency and spectral dependence of hydrogen generation dictated by the photonic characteristics of the sub-wavelength-diameter SiNWs. Although initial energy conversion efficiencies are low, multijunction SiNWs bring the photonic advantages of a tunable, mesoscale geometry and the material advantages of Si-including the small bandgap and economies of scale-to the PSR design, providing a new approach for water-splitting reactors.

A somato-cognitive action network alternates with effector regions in motor cortex.

Motor cortex (M1) has been thought to form a continuous somatotopic homunculus extending down the precentral gyrus from foot to face representations1,2, despite evidence for concentric functional zones3 and maps of complex actions4. Here, using precision functional magnetic resonance imaging (fMRI) methods, we find that the classic homunculus is interrupted by regions with distinct connectivity, structure and function, alternating with effector-specific (foot, hand and mouth) areas. These inter-effector regions exhibit decreased cortical thickness and strong functional connectivity to each other, as well as to the cingulo-opercular network (CON), critical for action5 and physiological control6, arousal7, errors8 and pain9. This interdigitation of action control-linked and motor effector regions was verified in the three largest fMRI datasets. Macaque and pediatric (newborn, infant and child) precision fMRI suggested cross-species homologues and developmental precursors of the inter-effector system. A battery of motor and action fMRI tasks documented concentric effector somatotopies, separated by the CON-linked inter-effector regions. The inter-effectors lacked movement specificity and co-activated during action planning (coordination of hands and feet) and axial body movement (such as of the abdomen or eyebrows). These results, together with previous studies demonstrating stimulation-evoked complex actions4 and connectivity to internal organs10 such as the adrenal medulla, suggest that M1 is punctuated by a system for whole-body action planning, the somato-cognitive action network (SCAN). In M1, two parallel systems intertwine, forming an integrate-isolate pattern: effector-specific regions (foot, hand and mouth) for isolating fine motor control and the SCAN for integrating goals, physiology and body movement.

Biobanking animal genetic resources: critical infrastructure and growth opportunities.

National animal gene banks have acquired substantial quantities of germplasm that protect and preserve a wide range of livestock breeds. New challenges and growth opportunities are emerging. A key challenge will be increased gene bank use, but this requires increased characterization of phenotypes and genotypes for populations and collections.

BRCA1/BARD1 intrinsically disordered regions facilitate chromatin recruitment and ubiquitylation.

BRCA1/BARD1 is a tumor suppressor E3 ubiquitin (Ub) ligase with roles in DNA damage repair and in transcriptional regulation. BRCA1/BARD1 RING domains interact with nucleosomes to facilitate mono-ubiquitylation of distinct residues on the C-terminal tail of histone H2A. These enzymatic domains constitute a small fraction of the heterodimer, raising the possibility of functional chromatin interactions involving other regions such as the BARD1 C-terminal domains that bind nucleosomes containing the DNA damage signal H2A K15-Ub and H4 K20me0, or portions of the expansive intrinsically disordered regions found in both subunits. Herein, we reveal novel interactions that support robust H2A ubiquitylation activity mediated through a high-affinity, intrinsically disordered DNA-binding region of BARD1. These interactions support BRCA1/BARD1 recruitment to chromatin and sites of DNA damage in cells and contribute to their survival. We also reveal distinct BRCA1/BARD1 complexes that depend on the presence of H2A K15-Ub, including a complex where a single BARD1 subunit spans adjacent nucleosome units. Our findings identify an extensive network of multivalent BARD1-nucleosome interactions that serve as a platform for BRCA1/BARD1-associated functions on chromatin.

Rusfertide, a Hepcidin Mimetic, for Control of Erythrocytosis in Polycythemia Vera.

BACKGROUND: Polycythemia vera is a chronic myeloproliferative neoplasm characterized by erythrocytosis. Rusfertide, an injectable peptide mimetic of the master iron regulatory hormone hepcidin, restricts the availability of iron for erythropoiesis. The safety and efficacy of rusfertide in patients with phlebotomy-dependent polycythemia vera are unknown. METHODS: In part 1 of the international, phase 2 REVIVE trial, we enrolled patients in a 28-week dose-finding assessment of rusfertide. Part 2 was a double-blind, randomized withdrawal period in which we assigned patients, in a 1:1 ratio, to receive rusfertide or placebo for 12 weeks. The primary efficacy end point was a response, defined by hematocrit control, absence of phlebotomy, and completion of the trial regimen during part 2. Patient-reported outcomes were assessed by means of the modified Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) patient diary (scores range from 0 to 10, with higher scores indicating greater severity of symptoms). RESULTS: Seventy patients were enrolled in part 1 of the trial, and 59 were assigned to receive rusfertide (30 patients) or placebo (29 patients) in part 2. The estimated mean (±SD) number of phlebotomies per year was 8.7±2.9 during the 28 weeks before the first dose of rusfertide and 0.6±1.0 during part 1 (estimated difference, 8.1 phlebotomies per year). The mean maximum hematocrit was 44.5±2.2% during part 1 as compared with 50.0±5.8% during the 28 weeks before the first dose of rusfertide. During part 2, a response was observed in 60% of the patients who received rusfertide as compared with 17% of those who received placebo (P = 0.002). Between baseline and the end of part 1, rusfertide treatment was associated with a decrease in individual symptom scores on the MPN-SAF in patients with moderate or severe symptoms at baseline. During parts 1 and 2, grade 3 adverse events occurred in 13% of the patients, and none of the patients had a grade 4 or 5 event. Injection-site reactions of grade 1 or 2 in severity were common. CONCLUSIONS: In patients with polycythemia vera, rusfertide treatment was associated with a mean hematocrit of less than 45% during the 28-week dose-finding period, and the percentage of patients with a response during the 12-week randomized withdrawal period was greater with rusfertide than with placebo. (Funded by Protagonist Therapeutics; REVIVE ClinicalTrials.gov number, NCT04057040.).

Telomerase reactivation following telomere dysfunction yields murine prostate tumors with bone metastases.

To determine the role of telomere dysfunction and telomerase reactivation in generating pro-oncogenic genomic events and in carcinoma progression, an inducible telomerase reverse transcriptase (mTert) allele was crossed onto a prostate cancer-prone mouse model null for Pten and p53 tumor suppressors. Constitutive telomerase deficiency and associated telomere dysfunction constrained cancer progression. In contrast, telomerase reactivation in the setting of telomere dysfunction alleviated intratumoral DNA-damage signaling and generated aggressive cancers with rearranged genomes and new tumor biological properties (bone metastases). Comparative oncogenomic analysis revealed numerous recurrent amplifications and deletions of relevance to human prostate cancer. Murine tumors show enrichment of the TGF-ß/SMAD4 network, and genetic validation studies confirmed the cooperative roles of Pten, p53, and Smad4 deficiencies in prostate cancer progression, including skeletal metastases. Thus, telomerase reactivation in tumor cells experiencing telomere dysfunction enables full malignant progression and provides a mechanism for acquisition of cancer-relevant genomic events endowing new tumor biological capabilities.

Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism.

Tumor maintenance relies on continued activity of driver oncogenes, although their rate-limiting role is highly context dependent. Oncogenic Kras mutation is the signature event in pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible Kras(G12D)-driven PDAC mouse model establishes that advanced PDAC remains strictly dependent on Kras(G12D) expression. Transcriptome and metabolomic analyses indicate that Kras(G12D) serves a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis and pentose phosphate pathways (PPP). These studies also reveal that oncogenic Kras promotes ribose biogenesis. Unlike canonical models, we demonstrate that Kras(G12D) drives glycolysis intermediates into the nonoxidative PPP, thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in PDAC.

Dietary fructose improves intestinal cell survival and nutrient absorption.

Fructose consumption is linked to the rising incidence of obesity and cancer, which are two of the leading causes of morbidity and mortality globally1,2. Dietary fructose metabolism begins at the epithelium of the small intestine, where fructose is transported by glucose transporter type 5 (GLUT5; encoded by SLC2A5) and phosphorylated by ketohexokinase to form fructose 1-phosphate, which accumulates to high levels in the cell3,4. Although this pathway has been implicated in obesity and tumour promotion, the exact mechanism that drives these pathologies in the intestine remains unclear. Here we show that dietary fructose improves the survival of intestinal cells and increases intestinal villus length in several mouse models. The increase in villus length expands the surface area of the gut and increases nutrient absorption and adiposity in mice that are fed a high-fat diet. In hypoxic intestinal cells, fructose 1-phosphate inhibits the M2 isoform of pyruvate kinase to promote cell survival5-7. Genetic ablation of ketohexokinase or stimulation of pyruvate kinase prevents villus elongation and abolishes the nutrient absorption and tumour growth that are induced by feeding mice with high-fructose corn syrup. The ability of fructose to promote cell survival through an allosteric metabolite thus provides additional insights into the excess adiposity generated by a Western diet, and a compelling explanation for the promotion of tumour growth by high-fructose corn syrup.

Non-canonical mTORC2 Signaling Regulates Brown Adipocyte Lipid Catabolism through SIRT6-FoxO1.

mTORC2 controls glucose and lipid metabolism, but the mechanisms are unclear. Here, we show that conditionally deleting the essential mTORC2 subunit Rictor in murine brown adipocytes inhibits de novo lipid synthesis, promotes lipid catabolism and thermogenesis, and protects against diet-induced obesity and hepatic steatosis. AKT kinases are the canonical mTORC2 substrates; however, deleting Rictor in brown adipocytes appears to drive lipid catabolism by promoting FoxO1 deacetylation independently of AKT, and in a pathway distinct from its positive role in anabolic lipid synthesis. This facilitates FoxO1 nuclear retention, enhances lipid uptake and lipolysis, and potentiates UCP1 expression. We provide evidence that SIRT6 is the FoxO1 deacetylase suppressed by mTORC2 and show an endogenous interaction between SIRT6 and mTORC2 in both mouse and human cells. Our findings suggest a new paradigm of mTORC2 function filling an important gap in our understanding of this more mysterious mTOR complex.

The thermoneutral zone in women takes an "arctic" shift compared to men.

Conventionally, women are perceived to feel colder than men, but controlled comparisons are sparse. We measured the response of healthy, lean, young women and men to a range of ambient temperatures typical of the daily environment (17 to 31 °C). The Scholander model of thermoregulation defines the lower critical temperature as threshold of the thermoneutral zone, below which additional heat production is required to defend core body temperature. This parameter can be used to characterize the thermoregulatory phenotypes of endotherms on a spectrum from "arctic" to "tropical." We found that women had a cooler lower critical temperature (mean ± SD: 21.9 ± 1.3 °C vs. 22.9 ± 1.2 °C, P = 0.047), resembling an "arctic" shift compared to men. The more arctic profile of women was predominantly driven by higher insulation associated with more body fat compared to men, countering the lower basal metabolic rate associated with their smaller body size, which typically favors a "tropical" shift. We did not detect sex-based differences in secondary measures of thermoregulation including brown adipose tissue glucose uptake, muscle electrical activity, skin temperatures, cold-induced thermogenesis, or self-reported thermal comfort. In conclusion, the principal contributors to individual differences in human thermoregulation are physical attributes, including body size and composition, which may be partly mediated by sex.

BRCA1/BARD1 is a nucleosome reader and writer.

Mutations in BRCA1 and BARD1 predispose carriers to breast and ovarian cancers. The BRCA1 and BARD1 proteins form a heterodimeric complex (BRCA1/BARD1) that regulates many biological processes, including transcription and DNA double-stranded break repair. These functions are mediated by the only known enzymatic activity of BRCA1/BARD1 in its capacity as an E3 ubiquitin ligase and its role as a central hub for many large protein complexes. But the mechanisms by which BRCA1/BARD1 interfaces with chromatin, where it exerts its major functions, have remained unknown. Here, we review recent advancements in structural and cellular biology that have provided critical insights into how BRCA1/BARD1 serves as both a nucleosome reader and writer to facilitate transcriptional regulation and DNA repair by homologous recombination.

Spastin is an essential regulator of male meiosis, acrosome formation, manchette structure and nuclear integrity.

The development and function of male gametes is dependent on a dynamic microtubule network, yet how this is regulated remains poorly understood. We have recently shown that microtubule severing, via the action of the meiotic AAA ATPase protein clade, plays a crucial role in this process. Here, we sought to elucidate the roles of spastin, an as-yet-unexplored member of this clade in spermatogenesis. Using a SpastKO/KO mouse model, we reveal that spastin loss resulted in a complete loss of functional germ cells. Spastin plays a crucial role in the assembly and function of the male meiotic spindle. Consistent with meiotic failure, round spermatid nuclei were enlarged, indicating aneuploidy, but were still able to enter spermiogenesis. During spermiogenesis, we observed extreme abnormalities in manchette structure, acrosome biogenesis and, commonly, a catastrophic loss of nuclear integrity. This work defines an essential role for spastin in regulating microtubule dynamics during spermatogenesis, and is of potential relevance to individuals carrying spastin variants and to the medically assisted reproductive technology industry.

Regulatory T cells suppress myeloma-specific immunity during autologous stem cell mobilization and transplantation.

ABSTRACT: Autologous stem cell transplantation (ASCT) is the standard of care consolidation therapy for eligible patients with myeloma but most patients eventually progress, an event associated with features of immune escape. Novel approaches to enhance antimyeloma immunity after ASCT represent a major unmet need. Here, we demonstrate that patient-mobilized stem cell grafts contain high numbers of effector CD8 T cells and immunosuppressive regulatory T cells (Tregs). We showed that bone marrow (BM)-residing T cells are efficiently mobilized during stem cell mobilization (SCM) and hypothesized that mobilized and highly suppressive BM-derived Tregs might limit antimyeloma immunity during SCM. Thus, we performed ASCT in a preclinical myeloma model with or without stringent Treg depletion during SCM. Treg depletion generated SCM grafts containing polyfunctional CD8 T effector memory cells, which dramatically enhanced myeloma control after ASCT. Thus, we explored clinically tractable translational approaches to mimic this scenario. Antibody-based approaches resulted in only partial Treg depletion and were inadequate to recapitulate this effect. In contrast, a synthetic interleukin-2 (IL-2)/IL-15 mimetic that stimulates the IL-2 receptor on CD8 T cells without binding to the high-affinity IL-2Ra used by Tregs efficiently expanded polyfunctional CD8 T cells in mobilized grafts and protected recipients from myeloma progression after ASCT. We confirmed that Treg depletion during stem cell mobilization can mitigate constraints on tumor immunity and result in profound myeloma control after ASCT. Direct and selective cytokine signaling of CD8 T cells can recapitulate this effect and represent a clinically testable strategy to improve responses after ASCT.

The ecological and genomic basis of explosive adaptive radiation.

Speciation rates vary considerably among lineages, and our understanding of what drives the rapid succession of speciation events within young adaptive radiations remains incomplete1-11. The cichlid fish family provides a notable example of such variation, with many slowly speciating lineages as well as several exceptionally large and rapid radiations12. Here, by reconstructing a large phylogeny of all currently described cichlid species, we show that explosive speciation is solely concentrated in species flocks of several large young lakes. Increases in the speciation rate are associated with the absence of top predators; however, this does not sufficiently explain explosive speciation. Across lake radiations, we observe a positive relationship between the speciation rate and enrichment of large insertion or deletion polymorphisms. Assembly of 100 cichlid genomes within the most rapidly speciating cichlid radiation, which is found in Lake Victoria, reveals exceptional 'genomic potential'-hundreds of ancient haplotypes bear insertion or deletion polymorphisms, many of which are associated with specific ecologies and shared with ecologically similar species from other older radiations elsewhere in Africa. Network analysis reveals fundamentally non-treelike evolution through recombining old haplotypes, and the origins of ecological guilds are concentrated early in the radiation. Our results suggest that the combination of ecological opportunity, sexual selection and exceptional genomic potential is the key to understanding explosive adaptive radiation.

An observation-based, reduced-form model for oxidation in the remote marine troposphere.

The hydroxyl radical (OH) fuels atmospheric chemical cycling as the main sink for methane and a driver of the formation and loss of many air pollutants, but direct OH observations are sparse. We develop and evaluate an observation-based proxy for short-term, spatial variations in OH (ProxyOH) in the remote marine troposphere using comprehensive measurements from the NASA Atmospheric Tomography (ATom) airborne campaign. ProxyOH is a reduced form of the OH steady-state equation representing the dominant OH production and loss pathways in the remote marine troposphere, according to box model simulations of OH constrained with ATom observations. ProxyOH comprises only eight variables that are generally observed by routine ground- or satellite-based instruments. ProxyOH scales linearly with in situ [OH] spatial variations along the ATom flight tracks (median r2 = 0.90, interquartile range = 0.80 to 0.94 across 2-km altitude by 20° latitudinal regions). We deconstruct spatial variations in ProxyOH as a first-order approximation of the sensitivity of OH variations to individual terms. Two terms modulate within-region ProxyOH variations-water vapor (H2O) and, to a lesser extent, nitric oxide (NO). This implies that a limited set of observations could offer an avenue for observation-based mapping of OH spatial variations over much of the remote marine troposphere. Both H2O and NO are expected to change with climate, while NO also varies strongly with human activities. We also illustrate the utility of ProxyOH as a process-based approach for evaluating intermodel differences in remote marine tropospheric OH.

A recurrent de novo splice site variant involving DNM1 exon 10a causes developmental and epileptic encephalopathy through a dominant-negative mechanism.

Heterozygous pathogenic variants in DNM1 cause developmental and epileptic encephalopathy (DEE) as a result of a dominant-negative mechanism impeding vesicular fission. Thus far, pathogenic variants in DNM1 have been studied with a canonical transcript that includes the alternatively spliced exon 10b. However, after performing RNA sequencing in 39 pediatric brain samples, we find the primary transcript expressed in the brain includes the downstream exon 10a instead. Using this information, we evaluated genotype-phenotype correlations of variants affecting exon 10a and identified a cohort of eleven previously unreported individuals. Eight individuals harbor a recurrent de novo splice site variant, c.1197-8G>A (GenBank: NM_001288739.1), which affects exon 10a and leads to DEE consistent with the classical DNM1 phenotype. We find this splice site variant leads to disease through an unexpected dominant-negative mechanism. Functional testing reveals an in-frame upstream splice acceptor causing insertion of two amino acids predicted to impair oligomerization-dependent activity. This is supported by neuropathological samples showing accumulation of enlarged synaptic vesicles adherent to the plasma membrane consistent with impaired vesicular fission. Two additional individuals with missense variants affecting exon 10a, p.Arg399Trp and p.Gly401Asp, had a similar DEE phenotype. In contrast, one individual with a missense variant affecting exon 10b, p.Pro405Leu, which is less expressed in the brain, had a correspondingly less severe presentation. Thus, we implicate variants affecting exon 10a as causing the severe DEE typically associated with DNM1-related disorders. We highlight the importance of considering relevant isoforms for disease-causing variants as well as the possibility of splice site variants acting through a dominant-negative mechanism.

Formamide significantly enhances the efficiency of chemical adenylation of RNA sequencing ligation adaptors.

Preadenylated single-stranded DNA ligation adaptors are essential reagents in many next generation RNA sequencing library preparation protocols. These oligonucleotides can be adenylated enzymatically or chemically. Enzymatic adenylation reactions have high yield but are not amendable to scale up. In chemical adenylation, adenosine 5'-phosphorimidazolide (ImpA) reacts with 5' phosphorylated DNA. It is easily scalable but gives poor yields, requiring labor-intensive cleanup steps. Here, we describe an improved chemical adenylation method using 95% formamide as the solvent, which results in the adenylation of oligonucleotides with >90% yield. In standard conditions, with water as the solvent, hydrolysis of the starting material to adenosine monophosphate limits the yields. To our surprise, we find that rather than increasing adenylation yields by decreasing the rate of ImpA hydrolysis, formamide does so by increasing the reaction rate between ImpA and 5'-phosphorylated DNA by ∼10-fold. The method described here enables straightforward preparation of chemically adenylated adapters with higher than 90% yield, simplifying reagent preparation for NGS.