Targeting RNA Polymerase Primary σ70 as a Therapeutic Strategy against Methicillin-Resistant Staphylococcus aureus by Antisense Peptide Nucleic Acid

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) causes threatening infection-related mortality worldwide. Currently, spread of multi-drug resistance (MDR) MRSA limits therapeutic options and requires new approaches to "druggable" target discovery, as well as development of novel MRSA-active antibiotics. RNA polymerase primary σ⁷⁰ (encoded by gene rpoD) is a highly conserved prokaryotic factor essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying potential for antisense inhibition. METHODOLOGY/PRINCIPAL FINDINGS: By synthesizing a serial of cell penetrating peptide conjugated peptide nucleic acids (PPNAs) based on software predicted parameters and further design optimization, we identified a target sequence (234 to 243 nt) within rpoD mRNA conserved region 3.0 being more sensitive to antisense inhibition. A (KFF)₃K peptide conjugated 10-mer complementary PNA (PPNA2332) was developed for potent micromolar-range growth inhibitory effects against four pathogenic S. aureus strains with different resistance phenotypes, including clinical vancomycin-intermediate resistance S. aureus and MDR-MRSA isolates. PPNA2332 showed bacteriocidal antisense effect at 3.2 fold of MIC value against MRSA/VISA Mu50, and its sequence specificity was demonstrated in that PPNA with scrambled PNA sequence (Scr PPNA2332) exhibited no growth inhibitory effect at higher concentrations. Also, PPNA2332 specifically interferes with rpoD mRNA, inhibiting translation of its protein product σ⁷⁰ in a concentration-dependent manner. Full decay of mRNA and suppressed expression of σ⁷⁰ were observed for 40 µM or 12.5 µM PPNA2332 treatment, respectively, but not for 40 µM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 (≥1 µM) essentially cleared lethal MRSA/VISA Mu50 infection in epithelial cell cultures, and eliminated viable bacterial cells in a time- and concentration- dependent manner, without showing any apparent toxicity at 10 µM. CONCLUSIONS: The present result suggested that RNAP primary σ⁷⁰ is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections

How to prevent ROP in preterm infants in Indonesia?

Background and Aims: Retinopathy of prematurity (ROP) is a severe disease in preterm infants. It is seen more frequently in Low-Middle Income Countries (LMIC) like Indonesia compared to High-Income Countries (HIC). Risk factors for ROP development are -extreme- preterm birth, use of oxygen, neonatal infections, respiratory problems, inadequate nutrition, and blood and exchange transfusions. In this paper, we give an overview of steps that can be taken in LMIC to prevent ROP and provide guidelines for screening and treating ROP. Methods: Based on the literature search and data obtained by us in Indonesia's studies, we propose guidelines for the prevention, screening, and treatment of ROP in preterm infants in LMIC. Results: Prevention of ROP starts before birth with preventing preterm labor, transferring a mother who might deliver <32 weeks to a perinatal center and giving corticosteroids to mothers that might deliver <34 weeks. Newborn resuscitation must be done using room air or, in the case of very preterm infants (<29-32 weeks) by using 30% oxygen. Respiratory problems must be prevented by starting continuous positive airway pressure (CPAP) in all preterm infants <32 weeks and in case of respiratory problems in more mature infants. If needed, the surfactant should be given in a minimally invasive manner, as ROP's lower incidence was found using this technique. The use of oxygen must be strictly regulated with a saturation monitor of 91-95%. Infections must be prevented as much as possible. Both oral and parenteral nutrition should be started in all preterm infants on day one of life with preferably mothers' milk. Blood transfusions can be prevented by reducing the amount of blood needed for laboratory analysis. Discussion: Preterm babies should be born in facilities able to care for them optimally. The use of oxygen must be strictly regulated. ROP screening is mandatory in infants born <34 weeks, and infants who received supplemental oxygen for a prolonged period. In case of progression of ROP, immediate mandatory treatment is required. Conclusion: Concerted action is needed to reduce the incidence of ROP in LMIC. "STOP - R1O2P3" is an acronym that can help implement standard practices in all neonatal intensive care units in LMIC to prevent development and progression