Definitive demonstration by synthesis of genome annotation completeness.

We develop a method for completing the genetics of natural living systems by which the absence of expected future discoveries can be established. We demonstrate the method using bacteriophage øX174, the first DNA genome to be sequenced. Like many well-studied natural organisms, closely related genome sequences are available-23 Bullavirinae genomes related to øX174. Using bioinformatic tools, we first identified 315 potential open reading frames (ORFs) within the genome, including the 11 established essential genes and 82 highly conserved ORFs that have no known gene products or assigned functions. Using genome-scale design and synthesis, we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved but cryptic ORFs. The resulting genome is not viable. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with ∼50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation that modulates expression of gene H, a known essential gene, and fully suppresses the fitness defect. Taken together, we conclude that the annotation of currently functional ORFs for the øX174 genome is formally complete. More broadly, we show that sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations.

Pre-Operative Antithyroid Antibodies in Differentiated Thyroid Cancer.

OBJECTIVE: To evaluate the significance of antithyroglobulin and antithyroid peroxidase antibody levels associated with locoregional metastatic disease in patients with well-differentiated thyroid cancer. METHODS: Patients underwent initial treatment for well-differentiated thyroid cancer at our institution between 2014 and 2018. The following variables were collected: age, sex, pre-operative thyroid-stimulating hormone, thyroglobulin, antithyroglobulin antibody (TgAb), antithyroid peroxidase antibody (TPOAb), the extent of surgery, T-stage, N-stage, extrathyroidal extension (ETE), extranodal extension (ENE), lymphovascular invasion, and multifocal disease. The relationships between disease status and pre-operative TPOAb, TgAb, thyroglobulin, and thyroid-stimulating hormone were analyzed. RESULTS: A total of 405 patients (mean age, 52 years) were included in the study, of which 66.4% were women. Elevated TgAb was associated with the presence of lymph node metastases (LNM) in both the central and lateral neck (P < .01), with a stronger correlation to N1b versus N1a disease (P = .03). The presence of ETE was inversely related to the TgAb titer (P = .03). TPOAb was associated with a lower T-stage (P = .04), fewer LNM (P = .04), and a lower likelihood of ETE (P = .02). From multivariable analysis, TgAb ≥40 IU/mL was an independent predictive factor for a higher N-stage (P < .01 for N0 vs N1; P = .01 for N1a vs N1b), and ENE (P < .01). TPOAb ≥60 IU/mL was associated with a lower T-stage (P = .04 for T <3) and absence of ETE (P = .01). CONCLUSION: Elevated pre-operative TgAb was an independent predictor of nodal metastases and ENE, while elevated TPOAb was associated with a lower pathologic T- and N-stage. Pre-operative antithyroid antibody titers may be useful to inform the disease extent and features.

Trends and inpatient outcomes for palliative care services in major burn patients: A 10-year analysis of the nationwide inpatient sample.

INTRODUCTION: Despite advances in critical care and the surgical management of major burns, highly moribund patients are unlikely to survive. Little is known regarding the utilization and effects of palliative care services in this population. METHODS: All major burn hospitalizations were identified within the Nationwide Inpatient Sample. Patients were characterized by burn, demographic, facility, and diseases factors. Palliative care services were identified with International Classification Disease 9th edition code V6.67. Temporal trends were assessed with Poisson modeling. Inpatient mortality and death without surgical intervention were assessed with logistic regression. Outcomes were stratified by modified Baux scores. RESULTS: 7424 major burns were included; 1.9% received palliative care services. Patients receiving palliation had a mean age of 63.6 years (SD 19.6), mean total body surface area of 62.2% (SD 24.9%), and mean modified Baux score of 127.1 (SD 26.7). Adjusting for covariates, the incidence rate ratio was 1.42 over the 10-year period (95% CI, 1.31-1.54, p<0.001). Independent predictors of palliative consultations included older age, larger burns, deeper burns, and higher Elixhauser comorbidity score. Among patients with modified Baux scores between 100-153, those receiving palliative care services were significantly more likely to die without surgery, OR 3.24 (95% CI 1.13-10.39, p=0.029), with no significant difference in mortality, OR 11.72 (95% CI 0.87-22.57, p=0.051) CONCLUSION AND RELEVANCE: Palliative care services were increasingly used during the study period. Palliative care services in highly moribund burn patients do not impact survival and may decrease the likelihood of surgical intervention in select patients.