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We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.The unfolded protein response (UPR) and the heat shock response (HSR) are two evolutionarily conserved systems that protect plants from heat stress. The UPR and HSR occur in different cellular compartments and both responses are elicited by misfolded proteins that accumulate under adverse environmental conditions such as heat stress. While the UPR and HSR appear to operate independently, we have found a link between them in maize (Zea mays) involving the production of the BASIC LEUCINE ZIPPER60 (bZIP60) transcription factor, a pivotal response of the UPR to heat stress. Surprisingly, a mutant (bzip60-2) knocking down bZIP60 expression blunted the HSR at elevated temperatures and prevented the normal upregulation of a group of heat shock protein genes in response to elevated temperature. The expression of a key HEAT SHOCK FACTOR TRANSCRIPTION FACTOR13 (HSFTF13, a HEAT SHOCK FACTOR A6B [HSFA6B] family member) was compromised in bzip60-2, and the HSFTF13 promoter was shown to be a target of bZIP60 in maize protoplasts. In addition, the upregulation by heat of genes involved in chlorophyll catabolism and chloroplast protein turnover were subdued in bzip60-2, and these genes were also found to be targets of bZIP60. Thus, the UPR, an endoplasmic-reticulum-associated response, quite unexpectedly contributes to the nuclear/cytoplasmic HSR in maize.CONSTANS, CONSTANS-LIKE, and TIMING OF CAB EXPRESSION1 (CCT) domain-containing proteins are a large family unique to plants. They transcriptionally regulate photoperiodic flowering, circadian rhythms, vernalization, and other related processes. Through their CCT domains, CONSTANS and HEADING DATE1 (HD1) coordinate with the NUCLEAR FACTOR Y (NF-Y) B/C dimer to specifically target a conserved ‘CCACA’ motif within the promoters of their target genes. However, the mechanism underlying DNA recognition by the CCT domain remains unclear. Here we determined the crystal structures of the rice (Oryza sativa) NF-YB/YC dimer and the florigen gene Heading date 3a (Hd3a)-bound HD1CCT/NF-YB/YC trimer with resolutions of 2.0 Å and 2.55 Å, respectively. The CCT domain of HD1 displays an elongated structure containing two α-helices and two loops, tethering Hd3a to the NF-YB/YC dimer. Helix α2 and loop 2 are anchored into the minor groove of the ‘CCACA’ motif, which determines the specific base recognition. Our structures reveal the interaction mechanism among the CCT domain, NF-YB/YC dimer, and the target DNA. These results not only provide insight into the network between the CCT proteins and NF-Y subunits, but also offer potential approaches for improving productivity and global adaptability of crops by manipulating florigen expression.Pancreatic acinar cell carcinoma (PAC) is a rare disease with a poor prognosis. Treatment options for metastatic PAC are limited and often follow chemotherapeutic regimens for pancreatic ductal adenocarcinoma. Although recurrent genomic alterations, such as BRAF fusions and defects in genes involved in homologous recombination DNA repair, have been described in PAC, data on the clinical efficacy of molecularly guided, targeted treatment are scarce. Here we describe the case of a 27-yr-old patient with BRAFV600E-mutated PAC who was successfully treated with a combination of BRAF and MEK inhibitors. The patient presented to our clinic with abdominal pain and weight loss. Imaging showed extensive retroperitoneal disease as well as mediastinal lymphadenopathy. Because of elevated α-fetoprotein (AFP) levels and inconclusive histologic findings, a germ cell tumor was suspected; however, PEI chemotherapy was unsuccessful. A repeat biopsy yielded the diagnosis of PAC and treatment with FOLFIRINOX was initiated. Comprehensive molecular profiling within the MASTER (Molecularly Aided Stratification for Tumor Eradication Research) precision oncology program revealed a somatic BRAFV600E mutation and a germline PALB2 stop-gain mutation. Therapy was therefore switched to BRAF/MEK inhibition, resulting in almost complete remission and disease control for 12 mo and a remarkable improvement in the patient’s general condition. These results indicate that BRAF alterations are a valid therapeutic target in PAC that should be routinely assessed in this patient population.Relatively little is known about phenotypic variability in nonsyndromic nephropathy associated with the gene encoding the WT1 transcription factor. We report a 12-mo-old female who presented with vomiting, diarrhea, and fatigue in the setting of renal failure and malignant hypertension. Trio ultra-rapid whole-genome sequencing identified a novel, likely pathogenic, de novo missense variant (c.485T > A, p.Val162Asp) in WT1 in 46 h, consistent with a diagnosis of nephrotic syndrome type 4 (NPHS4; OMIM 256370). This disorder typically presents with nephrotic syndrome (gross proteinuria, hypoalbuminemia, and edema). Rapid diagnosis had an immediate impact on her clinical management in the pediatric intensive care unit. Diagnostic renal biopsy was avoided, and placement of permanent dialysis access, a gastrostomy tube, and bilateral nephrectomy were accelerated. This report expands the presenting phenotype of nonsyndromic nephrotic syndrome and/or renal failure due to heterozygous variants in WT1 (NPHS4). It also highlights the relationship between time to genomic diagnosis and clinical utility in critically ill infants.Although cutaneous squamous cell carcinoma (cSCC) is treatable in the majority of cases, deadly invasive and metastatic cases do occur. To date there are neither reliable predictive biomarkers of disease progression nor FDA-approved targeted therapies as standard of care. To address these issues, we screened patient-derived primary cultured cells from invasive/metastatic cSCC with 107 small-molecule inhibitors. In-house bioinformatics tools were used to cross-analyze drug responses and DNA mutations in tumors detected by whole-exome sequencing (WES). Aberrations in molecular pathways with evidence of potential drug targets were identified, including the Eph-ephrin and neutrophil degranulation signaling pathways. Using a screening panel of siRNAs, we identified EPHA6 and EPHA7 as targets within the Eph-ephrin pathway responsible for mitigating decreased cell viability. These studies form a plausible foundation for detecting biomarkers of high-risk progressive disease applicable in dermatopathology and for patient-specific therapeutic options for invasive/metastatic cSCC.Parkes Weber syndrome is associated with autosomal dominant inheritance, caused by germline heterozygous inactivating changes in the RASA1 gene, characterized by multiple micro arteriovenous fistulas and segmental overgrowth of soft tissue and skeletal components. The focal nature and variable expressivity associated with this disease has led to the hypothesis that somatic “second hit” inactivating changes in RASA1 are necessary for disease development. We report a 2-yr-old male with extensive capillary malformation and segmental overgrowth of his lower left extremity. Ultrasound showed subcutaneous phlebectasia draining the capillary malformation; magnetic resonance imaging showed overgrowth of the extremity with prominence of fatty tissues, fatty infiltration, and enlargement of all the major muscle groups. Germline RASA1 testing was normal. Later somatic testing from affected tissue showed two pathogenic variants in RASA1 consistent with the c.934_938del, p.(Glu312Argfs*14) and the c.2925del, p.(Asn976Metfs*20) with variant allele fractions of 3.6% and 4.2%, respectively. The intrafamilial variability of Parkes Weber syndrome involving segmental overgrowth of soft tissue, endothelium, and bone is strongly suggestive of a somatic second-hit model. There are at least two reports of confirmed second somatic hits in RASA1 To our knowledge, this is the first report of an individual with two somatic pathogenic variants in the RASA1 gene in DNA from a vascular lesion.Pathogenic variants in the XPC complex subunit, DNA damage recognition, and repair factor (XPC) are the cause of xeroderma pigmentosum, group C (MIM 278720). Xeroderma pigmentosum is an inherited condition characterized by hypersensitivity to ultraviolet (UV) irradiation and increased risk of skin cancer due to a defect in nucleotide excision repair (NER). Here we describe an individual with a novel missense variant and deletion of exons 14-15 in XPC presenting with a history of recurrent melanomas. The proband is a 39-yr-old female evaluated through the Mayo Clinic Department of Clinical Genomics. Prior to age 36, she had more than 60 skin biopsies that showed dysplastic nevi, many of which had atypia. At age 36 she presented with her first melanoma in situ, and since then has had more than 10 melanomas. The proband underwent research whole-exome sequencing (WES) through the Mayo Clinic’s Center for Individualized Medicine and a novel heterozygous variant of uncertain significance (VUS) in XPC (c.1709T > G, p.Val570Gly) was identified. Clinical confirmation pursued via XPC gene sequencing and deletion/duplication analysis of XPC revealed a pathogenic heterozygous deletion of ∼1 kb within XPC, including exons 14 and 15. Research studies determined the alterations to be in trans Although variants in XPC generally result in early-onset skin cancer in childhood, the proband is atypical in that she did not present with her first melanoma until age 36. 3-TYP supplier Review of the patient’s clinical, pathological, and genetic findings points to a diagnosis of delayed presentation of xeroderma pigmentosum.Within histone H3, lysine 27 (H3K27) is one of the residues that functions as a molecular switch, by virtue of being subject to mutually exclusive post-translational modifications that have reciprocal effects on gene expression. Whereas acetylation of H3K27 is associated with transcriptional activation, methylation at this residue causes transcriptional silencing; these two modifications are mutually exclusive. Establishment of these epigenetic marks is important in defining cellular identity and for maintaining normal cell function, as evidenced by rare genetic disorders of epigenetic writers involved in H3K27 post-translational modification. Polycomb repressive complex (PRC2)-related overgrowth and Rubinstein-Taybi syndrome (RSTS) are respectively associated with impaired H3K27 methylation and acetylation. Whereas these syndromes share commonalities like intellectual disability and susceptibility to cancers, they are generally divergent in their skeletal growth phenotypes, potentially through dysregulation of their opposing H3K27 writer functions. In this review, we discuss the requirement of H3K27 modifications for successful embryogenesis, highlighting data from relevant mouse knockout studies. Although such gene ablation studies are integral for defining fundamental biological roles of methyl- and acetyltransferase function in vivo, studies of partial loss-of-function models are likely to yield more meaningful translational insight into progression of PRC2-related overgrowth or RSTS. Thus, modeling of rare human PRC2-related overgrowth and RSTS variants in mice is needed to fully understand the causative role of aberrant H3K27 modification in the pathophysiology of these syndromes.