The rockefeller university

Most eukaryotes do not use a consensus DNA sequence as binding sites for the origin recognition complex (ORC) to initiate DNA replication, however budding yeast do. Here the authors show S. cerevisiae ORC can bind nucleosomes near nucleosome-free regions and recruit replicative helicases to form a pre-replication complex independent of the DNA sequence. The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

Over the recent years, the treatment of multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) has evolved very rapidly and a large number of disease-modifying treatments (DMTs) are now available. However, most DMTs are associated with adverse events, the most frequent of which being infections. Consideration of all DMT-associated risks facilitates development of risk mitigation strategies. An international focused workshop with expert-led discussions was sponsored by the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) and was held in April 2021 to review our current knowledge about the risk of infections associated with the use of DMTs for people with MS and NMOSD and corresponding risk mitigation strategies. The workshop addressed DMT-associated infections in specific populations, such as children and pregnant women with MS, or people with MS who have other comorbidities or live in regions with an exceptionally high infection burden. Finally, we reviewed the topic of DMT-associated infectious risks in the context of the current SARS-CoV-2 pandemic. Herein, we summarize available evidence and identify gaps in knowledge which justify further research.

The melanocortin receptors are G-protein-coupled receptors, which are essential components of the hypothalamic-pituitary-adrenal axis, and they mediate the actions of melanocortins (melanocyte-stimulating hormones: alpha-MSH, beta-MSH, and gamma-MSH) as well as the adrenocorticotropin hormone (ACTH) in skin pigmentation, adrenal steroidogenesis, and stress response. Three melanocortin receptor genes (MC1R, MC2R, and MC5R) contribute to the risk of major depressive disorder (MDD), and one melanocortin receptor gene (MC4R) contributes to the risk of type 2 diabetes (T2D). MDD increases T2D risk in drug-naive patients; thus, MDD and T2D commonly coexist. The five melanocortin receptor genes might confer risk for both disorders. However, they have never been investigated jointly to evaluate their potential contributing roles in the MDD-T2D comorbidity, specifically within families. In 212 Italian families with T2D and MDD, we tested 11 single nucleotide polymorphisms (SNPs) in the MC1R gene, 9 SNPs in MC2R, 3 SNPs in MC3R, 4 SNPs in MC4R, and 2 SNPs in MC5R. The testing used 2-point parametric linkage and linkage disequilibrium (LD) (i.e., association) analysis with four models (dominant with complete penetrance (D1), dominant with incomplete penetrance (D2), recessive with complete penetrance (R1), and recessive with incomplete penetrance (R2)). We detected significant (p <= 0.05) linkage and/or LD (i.e., association) to/with MDD for one SNP in MC2R (rs111734014) and one SNP in MC5R (rs2236700), and to/with T2D for three SNPs in MC1R (rs1805007 and rs201192930, and rs2228479), one SNP in MC2R (rs104894660), two SNPs in MC3R (rs3746619 and rs3827103), and one SNP in MC4R genes (Chr18-60372302). The linkage/LD/association was significant across different linkage patterns and different modes of inheritance. All reported variants are novel in MDD and T2D. This is the first study to report risk variants in MC1R, MC2R, and MC3R genes in T2D. MC2R and MC5R genes are replicated in MDD, with one novel variant each. Within our dataset, only the MC2R gene appears to confer risk for both MDD and T2D, albeit with different risk variants. To further clarity the role of the melanocortin receptor genes in MDD-T2D, these findings should be sought among other ethnicities as well.

Fibrolamellar carcinoma (FLC) is a rare, often lethal, liver cancer affecting adolescents and young adults, for which there are no approved therapeutics. The development of therapeutics is hampered by a lack of in vitro models. Organoids have shown utility as a model system for studying many diseases. In this study, tumor tissue and the adjacent non-tumor liver were obtained at the time of surgery. The tissue was dissociated and grown as organoids. We developed 21 patient-derived organoid lines: 12 from metastases, three from the liver tumor and six from adjacent non-tumor liver. These patient-derived FLC organoids recapitulate the histologic morphology, immunohistochem-istry, and transcriptome of the patient tumor. Patient-derived FLC organoids were used in a preliminary high-throughput drug screen to show proof of concept for the identification of therapeutics. This model system has the potential to improve our understanding of this rare cancer and holds significant promise for drug testing and development.

Background Autosomal recessive (AR) PKC delta deficiency is a rare inborn error of immunity (IEI) characterized by autoimmunity and susceptibility to bacterial, fungal, and viral infections. PKC delta is involved in the intracellular production of reactive oxidative species (ROS). Material and Methods We studied a 5-year old girl presenting with a history of Burkholderia cepacia infection. She had no history of autoimmunity, lymphocyte counts were normal, and no auto-antibodies were detected in her plasma. We performed a targeted panel analysis of 407 immunity-related genes and immunological investigations of the underlying genetic condition in this patient. Results Consistent with a history suggestive of chronic granulomatous disease (CGD), oxidative burst impairment was observed in the patient's circulating phagocytes in a dihydrorhodamine 123 (DHR) assay. However, targeted genetic panel analysis identified no candidate variants of known CGD-causing genes. Two heterozygous candidate variants were detected in PRKCD: c.285C > A (p.C95*) and c.376G > T (p.D126Y). The missense variant was also predicted to cause abnormal splicing, as it is located at the splice donor site of exon 5. TOPO-TA cloning confirmed that exon 5 was completely skipped, resulting in a truncated protein. No PKC delta protein was detected in the patient's neutrophils and monocyte-derived macrophages. The monocyte-derived macrophages of the patient produced abnormally low levels of ROS, as shown in an Amplex Red assay. Conclusion PKC delta deficiency should be considered in young patients with CGD-like clinical manifestations and abnormal DHR assay results, even in the absence of clinical and biological manifestations of autoimmunity.

Protein kinases play a vital role in biology and deregulation of kinases is implicated in numerous diseases ranging from cancer to neurodegenerative diseases, making them a major target class for the pharmaceutical industry. However, the high degree of conservation that exists between ATP-binding sites among kinases makes it difficult for current inhibitors to be highly specific. In the context of neurodegeneration, several groups including ours, have linked different kinases such as CK1 and Alzheimer's disease for example. Strictly CK1-isoform specific regulators do not exist and known CK1 inhibitors are inhibiting the enzymatic activity, targeting the ATP-binding site. Here we review compounds known to target CK1, as well as other inhibitory types that could benefit CK1. We introduce the DNA-encoded library (DEL) technology that might represent an interesting approach to uncover allosteric modulators instead of ATP competitors. Such a strategy, taking into account known allosteric inhibitors and mechanisms, might help designing modulators that are more specific towards a specific kinase, and in the case of CK1, toward specific isoforms.

Global population immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is accumulating through heterogeneous combinations of infection and vaccination. Vaccine distribution in low- and middle-income countries has been variable and reliant on diverse vaccine platforms. We studied B-cell immunity in Mexico, a middle-income country where five different vaccines have been deployed to populations with high SARS-CoV-2 incidences. Levels of antibodies that bound a stabilized prefusion spike trimer, neutralizing antibody titers, and memory B-cell expansion correlated with each other across vaccine platforms. Nevertheless, the vaccines elicited variable levels of B-cell immunity, and the majority of recipients had undetectable neutralizing activity against the recently emergent omicron variant. SARS-CoV-2 infection, experienced before or after vaccination, potentiated B-cell immune responses and enabled the generation of neutralizing activity against omicron and SARS-CoV for all vaccines in nearly all individuals. These findings suggest that broad population immunity to SARS-CoV-2 will eventually be achieved but by heterogeneous paths. IMPORTANCE The majority of studies on SARS-CoV-2 vaccine-elicited immunity and immune evasion have focused on single vaccines corresponding to those distributed in high-income countries. However, in low- and middle-income countries, vaccine deployment has been far less uniform. It is therefore important to determine the levels of immunity elicited by vaccines that have been deployed globally. Such data should help inform policy. Thus, this paper is very much a "real-world" study that focuses on a middle-income country, Mexico, in which five different vaccines based on mRNA, adenovirus, and inactivated-virus platforms have been extensively deployed, while (as documented in our study) SARS-CoV-2 variants with increasing degrees of immune evasiveness have propagated in the Mexican population, culminating in the recent emergence of B.1.1.529 (omicron). The majority of studies on SARS-CoV-2 vaccine-elicited immunity and immune evasion have focused on single vaccines corresponding to those distributed in high-income countries. However, in low- and middle-income countries, vaccine deployment has been far less uniform.

The induction of broadly neutralizing antibodies (bNAbs) is a potential strategy for a vaccine against HIV-1. However, most bNAbs exhibit features such as unusually high somatic hypermutation, including insertions and deletions, which make their induction challenging. VRC01-class bNAbs not only exhibit extraordinary breadth and potency but also rank among the most highly somatically mutated bNAbs. Here, we describe a VRC01-class antibody isolated from a viremic controller, BG24, that is much less mutated than most relatives of its class while achieving comparable breadth and potency. A 3.8-angstrom x-ray crystal structure of a BG24-BG505 Env trimer complex revealed conserved contacts at the gp120 interface characteristic of the VRC01-class Abs, despite lacking common CDR3 sequence motifs. The existence of moderately mutated CD4-binding site (CD4bs) bNAbs such as BG24 provides a simpler blueprint for CD4bs antibody induction by a vaccine, raising the prospect that such an induction might be feasible with a germline-targeting approach.