Coordinated DNA polymerization by Polγ and the region of LonP1 regulated proteolysis.

The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polγ regulation and its interactions with regulatory proteins, which are essential for fine-tuning polymerase function, remain poorly understood. Misregulation of the Polγ heterotrimer, consisting of (i) PolG, the polymerase catalytic subunit and (ii) PolG2, the accessory subunit, ultimately results in mitochondrial diseases. Here, we used single particle cryo-electron microscopy to resolve the structure of PolG in its apoprotein state and we captured Polγ at three intermediates within the catalytic cycle: DNA bound, engaged, and an active polymerization state. Chemical crosslinking mass spectrometry, and site-directed mutagenesis uncovered the region of LonP1 engagement of PolG, which promoted proteolysis and regulation of PolG protein levels. PolG2 clinical variants, which disrupted a stable Polγ complex, led to enhanced LonP1-mediated PolG degradation. Overall, this insight into Polγ aids in an understanding of mitochondrial DNA replication and characterizes how machinery of the replication fork may be targeted for proteolytic degradation when improperly functioning.

Diagnostic Utility of Cerebrospinal Fluid White Blood Cell Components for the Identification of Bacterial Meningitis in Infants.

BACKGROUND: To evaluate the diagnostic and predictive utility of cerebrospinal fluid (CSF) white blood cell (WBC) components in the diagnosis of bacterial meningitis in infants discharged from the neonatal intensive care unit (NICU). METHODS: We identified a cohort of infants discharged from a Pediatrix NICU between 1997 and 2020 who did not have an immunodeficiency, had at least 1 CSF culture collected within the first 120 days of life, and at least 1 CSF laboratory specimen obtained on the day of culture collection. We only included an infant's first CSF culture and excluded cultures from CSF reservoirs and those growing contaminants or nonbacterial organisms. We examined the utility of CSF WBC components to diagnose or predict bacterial meningitis by calculating sensitivity, specificity, positive and negative predictive values, likelihood ratios, and area under the receiver operating curve (AUC) at different cutoff values for each parameter. We performed subgroup analysis excluding infants treated with antibiotics the day before CSF culture collection. RESULTS: Of the 20 756 infants that met the study inclusion criteria, 320 (2%) were diagnosed with bacterial meningitis. We found (AUC [95% CI]) CSF WBC count (0.76 [0.73-0.79]), CSF neutrophil count (0.74 [0.70-0.78]), and CSF neutrophil percent (0.71 [0.67-0.75]) had the highest predictive values for bacterial meningitis, even when excluding infants with early antibiotic administration. CONCLUSIONS: No single clinical prediction rule had the optimal discriminatory power for predicting culture-proven bacterial meningitis, and clinicians should be cautious when interpreting CSF WBC parameters in infants with suspected meningitis.