The rockefeller university

We report two unrelated adults with homozygous (P1) or compound heterozygous (P2) private loss- of- function variants of V-Rel Reticuloendotheliosis Viral Oncogene Homolog B ( RELB ) . The resulting deficiency of functional RelB impairs the induction of NFKB2 mRNA and NF-kappa B2 (p100/p52) protein by lymphotoxin in the fibroblasts of the patients. These defects are rescued by transduction with wild- type RELB complementary DNA (cDNA). By contrast, the response of RelB- deficient fibroblasts to Tumor Necrosis Factor (TNF) or IL- 1 beta via the canonical NF-kappa B pathway remains intact. P1 and P2 have low proportions of na & iuml;ve CD4+ and CD8+ T cells and of memory B cells. Moreover, their na & iuml;ve B cells cannot differentiate into immunoglobulin G (IgG)- or immunoglobulin A (IgA)- secreting cells in response to CD40L/IL-21, and the development of IL- 17A/F- producing T cells is strongly impaired in vitro. Finally, the patients produce neutralizing autoantibodies against type I interferons (IFNs), even after hematopoietic stem cell transplantation, attesting to a persistent dysfunction of thymic epithelial cells in T cell selection and central tolerance to some autoantigens. Thus, inherited human RelB deficiency disrupts the alternative NF-kappa B pathway, underlying a T- and B cell immunodeficiency, which, together with neutralizing autoantibodies against type I IFNs, confers a predisposition to viral, bacterial, and fungal infections.

Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and there has been progress in structural studies on recombinant subassemblies. However, there is limited structural information on native kinetochore architecture. To address this, we purified functional, native kinetochores from the thermophilic yeast Kluyveromyces marxianus and examined them by electron microscopy (EM), cryoelectron tomography (cryo-ET), and atomic force microscopy (AFM). The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with micro- tubules and locating the microtubule binder, Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies and provides the foundation to study the global architecture and functions of kinetochores at a structural level.

A search for collective effects inside jets produced in proton-proton collisions is performed via correlation measurements of charged particles using the CMS detector at the CERN LHC. The analysis uses data collected at a center-of-mass energy of root s = 13 TeV, corresponding to an integrated luminosity of 138 fb(-1). Jets are reconstructed with the anti-kT algorithm with a distance parameter of 0.8 and are required to have transverse momentum greater than 550 GeV and pseudorapidity vertical bar eta(jet)vertical bar < 1.6. Two-particle correlations among the charged particles within the jets are studied as functions of the particles' azimuthal angle and pseudorapidity separations (Delta phi* and Delta eta*) in a jet coordinate basis, where particles' eta*, phi* are defined relative to the direction of the jet. The correlation functions are studied in classes of in-jet chargedparticle multiplicity up to N-ch(J) approximate to 100. Fourier harmonics are extracted from long-range azimuthal correlation functions to characterize azimuthal anisotropy for vertical bar Delta eta*vertical bar > 2. For low-N-ch(j) jets, the long-range elliptic anisotropic harmonic, v(2)*, is observed to decrease with N-ch(j). This trend is well described by Monte Carlo event generators. However, a rising trend for v(2)* emerges at N-ch(j) greater than or similar to 80, hinting at a possible onset of collective behavior, which is not reproduced by the models tested. This observation yields new insights into the dynamics of jet evolution in the vacuum.

Despite therapeutic efficacy observed with immune checkpoint blockade in advanced melanoma, many tumors do not respond to treatment, representing a need for new therapies. Here, we have generated chimeric antigen receptor (CAR) T cells targeting TYRP1, a melanoma differentiation antigen expressed on the surface of melanomas, including rare acral and uveal melanomas. TYRP1-targeted CART cells demonstrate antigen-specific activation and cytotoxic activity in vitro and in vivo against human melanomas independent of the MHC alleles and expression. In addition, the toxicity to pigmented normal tissues observed with T lymphocytes expressing TYRP1-targeted TCRs was not observed with TYRP1-targeted CAR T cells. Anti-TYRP1 CAR T cells provide a novel means to target advanced melanomas, serving as a platform for the development of similar novel therapeutic agents and as a tool to interrogate the immunobiology of melanomas.

The Harpy Eagle (Harpia harpyja) is an iconic species that inhabits forested landscapes in Neotropical regions, with decreasing population trends mainly due to habitat loss, and currently classified as vulnerable. Here, we report on a chromosome-scale genome assembly for a female individual combining long reads, optical mapping, and chromatin conformation capture reads. The final assembly spans 1.35 Gb, with N50scaffold equal to 58.1 Mb and BUSCO completeness of 99.7%. We built the first extensive transposable element (TE) library for the Accipitridae to date and identified 7,228 intact TEs. We found a burst of an unknown TE similar to 13-22 million years ago (MYA), coincident with the split of the Harpy Eagle from other Harpiinae eagles. We also report a burst of solo-LTRs and CR1 retrotransposons similar to 31-33 MYA, overlapping with the split of the ancestor to all Harpiinae from other Accipitridae subfamilies. Comparative genomics with other Accipitridae, the closely related Cathartidae and Galloanserae revealed major chromosome-level rearrangements at the basal Accipitriformes genome, in contrast to a conserved ancient genome architecture for the latter two groups. A historical demography reconstruction showed a rapid decline in effective population size over the last 20,000 years. This reference genome serves as a crucial resource for future conservation efforts towards the Harpy Eagle.

In eukaryotes, the leading strand DNA is synthesized by Pol epsilon and the lagging strand by Pol delta. These replicative polymerases have higher processivity when paired with the DNA clamp PCNA. While the structure of the yeast Pol epsilon catalytic domain has been determined, how Pol epsilon interacts with PCNA is unknown in any eukaryote, human or yeast. Here we report two cryo-EM structures of human Pol epsilon-PCNA-DNA complex, one in an incoming nucleotide bound state and the other in a nucleotide exchange state. The structures reveal an unexpected three-point interface between the Pol epsilon catalytic domain and PCNA, with the conserved PIP (PCNA interacting peptide)-motif, the unique P-domain, and the thumb domain each interacting with a different protomer of the PCNA trimer. We propose that the multi-point interface prevents other PIP-containing factors from recruiting to PCNA while PCNA functions with Pol epsilon. Comparison of the two states reveals that the finger domain pivots around the [4Fe-4S] cluster-containing tip of the P-domain to regulate nucleotide exchange and incoming nucleotide binding. In eukaryotes, the leading strand DNA polymerase Pol epsilon synthesises the DNA with higher processivity when in complex with the DNA clamp PCNA. Here, the authors report two cryo-EM structures of human Pol epsilon bound to the PCNA clamp and a DNA substate, revealing the conformational changes associated with incoming nucleotide binding.

Judy Lieberman is a professor of pediatrics and adjunct professor of genetics at Harvard Medical School and an endowed chair in cellular and molecular medicine. Her lab studies cytotoxic T lymphocytes (CTL), key cells in the immune defense against viral infection and cancer, as well as molecular pathways activated by the granzymes, and how RNA interference (RNAi) regulates cell differentiation in health and disease states. We spoke to Judy about advice for early career researchers, how she first become interested in cytotoxic T lymphocytes, and key people who have provided mentorship across her career.

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences single molecule real-time sequencing. By using reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second-strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second-strand synthesis. Deletion and insertion rates increase for all RTs during second-strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.

Upon antigenic stimulation, naive CD4(+) T cells can give rise to phenotypically distinct effector T helper cells and long-lived memory T cells. We computationally reconstructed the in vivo trajectory of CD4(+) T cell differentiation during a type I inflammatory immune response and identified two distinct differentiation paths for effector and precursor central memory T cells arising directly from naive CD4(+) T cells. Unexpectedly, our studies revealed heterogeneity among naive CD4(+) T cells, which are typically considered homogeneous save for their diverse T cell receptor usage. Specifically, a previously unappreciated population of naive CD4(+) T cells sensing environmental type I IFN exhibited distinct activation thresholds, suggesting that naive CD4(+) T cell differentiation potential may be influenced by environmental cues. This population was expanded in human viral infection and type I IFN response-lined autoimmunity. Understanding the relevance of naive T cell heterogeneity to beneficial and maladaptive T cell responses may have therapeutic implications for adoptive T cell therapies in cancer immunotherapy and vaccination.