Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics.

Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

Engineering functional 3-dimensional patient-derived endocrine organoids for broad multiplatform applications.

BACKGROUND: Recent advancements in 3-dimensional patient-derived organoid models have revolutionized the field of cancer biology. There is an urgent need for development of endocrine tumor organoid models for medullary thyroid carcinoma, adrenocortical carcinoma, papillary thyroid carcinoma, and a spectrum of benign hyperfunctioning parathyroid and adrenal neoplasms. We aimed to engineer functionally intact 3-dimensional endocrine patient-derived organoids to expand the in vitro and translational applications for the advancement of endocrine research. METHODS: Using our recently developed fine needle aspiration-based methodology, we established patient-derived 3-dimensional endocrine organoid models using prospectively collected human papillary thyroid carcinoma (n = 6), medullary thyroid carcinoma (n = 3), adrenocortical carcinoma (n = 3), and parathyroid (n = 5). and adrenal (n = 5) neoplasms. Multiplatform analyses of endocrine patient-derived organoids and applications in oncoimmunology, near-infrared autofluorescence, and radiosensitization studies under 3-dimensional in vitro conditions were performed. RESULTS: We have successfully modeled and analyzed the complex endocrine microenvironment for a spectrum of endocrine neoplasms in 3-dimensional culture. The endocrine patient-derived organoids recapitulated complex tumor microenvironment of endocrine neoplasms morphologically and functionally and maintained cytokine production and near-infrared autofluorescence properties. CONCLUSION: Our novel engineered endocrine patient-derived organoid models of thyroid, parathyroid and adrenal neoplasms represent an exciting and elegant alternative to current limited 2-dimensional systems and afford future broad multiplatform in vitro and translational applications, including in endocrine oncoimmunology.

Efficacy and safety of the long-acting C5 inhibitor ravulizumab in patients with atypical hemolytic uremic syndrome triggered by pregnancy: a subgroup analysis.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) triggered by pregnancy is a rare disease caused by dysregulation of the alternative complement pathway that occurs in approximately 1 in 25,000 pregnancies. The 311 phase 3 trial (NCT02949128) showed that ravulizumab, a long-acting C5 inhibitor obtained through selective modifications to eculizumab, is efficacious in inhibiting complement-mediated thrombotic microangiopathy (TMA) in patients with aHUS. In this analysis, we report outcomes in a subgroup of patients from the 311 study who developed TMA postpartum. METHODS: This was a phase 3, multicenter trial evaluating efficacy and safety of ravulizumab in adults (≥18 years of age) with aHUS naïve to complement inhibitor treatment. The primary endpoint was complete TMA response (simultaneous platelet count normalization [≥150 × 109/L], lactate dehydrogenase normalization [≤246 U/L] and 25% improvement in serum creatinine) through the 183-day initial evaluation period. Additional efficacy endpoints included time to complete TMA response, hematologic normalization, and dialysis requirement status. RESULTS: Eight patients presenting with TMA postpartum (median age of 37.7 [range; 22.1-45.2] years) were diagnosed with aHUS and received ≥1 dose of ravulizumab. Five patients (63%) were on dialysis at baseline. Complete TMA response was achieved in 7/8 patients (87.5%) in a median time of 31.5 days. Hematologic normalization was observed in all patients. All patients on dialysis at baseline discontinued dialysis within 21 days after treatment with ravulizumab. All patients showed continued improvements in the estimated glomerular filtration rate from baseline to Day 183. Three possible treatment-related adverse events were observed in 2 patients (arthralgia and nasopharyngitis [both non-severe]; urinary tract infection). No deaths or meningococcal infections occurred. CONCLUSIONS: Treatment with ravulizumab provided immediate and complete C5 inhibition, resulting in rapid clinical and laboratory improvements and complete TMA response through 183 days in patients with aHUS triggered by pregnancy. The safety profile observed in this subset of patients analysed is consistent with the 311 study investigating ravulizumab in patients with aHUS naïve to complement treatment. TRIAL REGISTRATION: Clinical trial identifier: NCT02949128 .

FCRL, a novel member of the leukocyte Fc receptor family possesses unique structural features.

A novel conserved member of the leukocyte Fc receptor (FcR) family was identified in human and mouse. The presumably secreted protein, designated FCRL (FcR-like) is comprised of four domains. The three N-terminal domains are related to the extracellular region of FcgammaRI, with the second (35-37% residue identity) and the third (46-52%) domains showing highest similarity. The C-terminal domain is a unique sequence enriched with proline residues. In humans, alternative transcripts for six FCRL isoforms were revealed. Spleen and tonsils were found to be the major sources of FCRL mRNA in human tissues. Western blotting of tonsil cell lysate using FCRL-specific antibodies recognized a 44-kDa protein produced as a monomer containing free sulfhydryl groups. The monomer, however, was able to form disulfide-linked homo-oligomer upon oxidation. In COS-7 cells transiently transfected with two human FCRL isoforms, both resided intracellularly. Immunohistochemical staining of tonsil sections demonstrated the FCRL expression in germinal centers, suggesting that the protein may be implicated in germinal center-specific stages of B cell development. The phylogenetic analysis of the FCRL relationships with the leukocyte FcR supports a view that the three-domain structure was primordial in the evolution of the family.