Rarely acquired type II-A CRISPR-Cas spacers mediate anti-viral immunity through the targeting of a non-canonical PAM sequence.

The Streptococcus pyogenes type II-A CRISPR-Cas systems provides adaptive immunity through the acquisition of short DNA sequences from invading viral genomes, called spacers. Spacers are transcribed into short RNA guides that match regions of the viral genome followed by a conserved NGG DNA motif, known as the PAM. These RNA guides, in turn, are used by the Cas9 nuclease to find and destroy complementary DNA targets within the viral genome. While most of the spacers present in bacterial populations that survive phage infection target protospacers flanked by NGG sequences, there is a small fraction that target non-canonical PAMs. Whether these spacers originate through accidental acquisition of phage sequences and/or provide efficient defense is unknown. Here we found that many of them match phage target regions flanked by an NAGG PAM. Despite being scarcely present in bacterial populations, NAGG spacers provide substantial immunity in vivo and generate RNA guides that support robust DNA cleavage by Cas9 in vitro; with both activities comparable to spacers that target sequences followed by the canonical AGG PAM. In contrast, acquisition experiments showed that NAGG spacers are acquired at very low frequencies. We therefore conclude that discrimination against these sequences occurs during immunization of the host. Our results reveal unexpected differences in PAM recognition during the spacer acquisition and targeting stages of the type II-A CRISPR-Cas immune response.

Improved Water and Waste Management Practices Reduce Diarrhea Risk in Children under Age Five in Rural Tanzania: A Community-Based, Cross-Sectional Analysis.

Diarrhea remains a significant cause of morbidity and mortality among children in developing countries. Water, sanitation, and hygiene practices (WASH) have demonstrated improved diarrhea-related outcomes but may have limited implementation in certain communities. This study analyzes the adoption and effect of WASH-based practices on diarrhea in children under age five in the rural Busiya chiefdom in northwestern Tanzania. In a cross-sectional analysis spanning July-September 2019, 779 households representing 1338 under-five children were surveyed. Among households, 250 (32.1%) reported at least one child with diarrhea over a two-week interval. Diarrhea prevalence in under-five children was 25.6%. In per-household and per-child analyses, the strongest protective factors against childhood diarrhea included dedicated drinking water storage (OR 0.25, 95% CI 0.18−0.36; p < 0.001), improved waste management (OR 0.37, 95% CI 0.27−0.51; p < 0.001), and separation of drinking water (OR 0.38, 95% CI 0.24−0.59; p < 0.001). Improved water sources were associated with decreased risk of childhood diarrhea in per-household analysis (OR 0.72, 95% CI 0.52−0.99, p = 0.04), but not per-child analysis (OR 0.83, 95% CI 0.65−1.05, p = 0.13). Diarrhea was widely treated (87.5%), mostly with antibiotics (44.0%) and oral rehydration solution (27.3%). Targeting water transportation, storage, and sanitation is key to reducing diarrhea in rural populations with limited water access.

A cross-domain charge interaction governs the activity of NO synthase.

NO synthase (NOS) enzymes perform interdomain electron transfer reactions during catalysis that may rely on complementary charge interactions at domain-domain interfaces. Guided by our previous results and a computer-generated domain-docking model, we assessed the importance of cross-domain charge interactions in the FMN-to-heme electron transfer in neuronal NOS (nNOS). We reversed the charge of three residues (Glu-762, Glu-816, and Glu-819) that form an electronegative triad on the FMN domain and then individually reversed the charges of three electropositive residues (Lys-423, Lys-620, and Lys-660) on the oxygenase domain (NOSoxy), to potentially restore a cross-domain charge interaction with the triad, but in reversed polarity. Charge reversal of the triad completely eliminated heme reduction and NO synthesis in nNOS. These functions were partly restored by the charge reversal at oxygenase residue Lys-423, but not at Lys-620 or Lys-660. Full recovery of heme reduction was probably muted by an accompanying change in FMN midpoint potential that made electron transfer to the heme thermodynamically unfavorable. Our results provide direct evidence that cross-domain charge pairing is required for the FMN-to-heme electron transfer in nNOS. The unique ability of charge reversal at position 423 to rescue function indicates that it participates in an essential cross-domain charge interaction with the FMN domain triad. This supports our domain-docking model and suggests that it may depict a productive electron transfer complex formed during nNOS catalysis.

Distinct conformational behaviors of four mammalian dual-flavin reductases (cytochrome P450 reductase, methionine synthase reductase, neuronal nitric oxide synthase, endothelial nitric oxide synthase) determine their unique catalytic profiles.

Multidomain enzymes often rely on large conformational motions to function. However, the conformational setpoints, rates of domain motions and relationships between these parameters and catalytic activity are not well understood. To address this, we determined and compared the conformational setpoints and the rates of conformational switching between closed unreactive and open reactive states in four mammalian diflavin NADPH oxidoreductases that catalyze important biological electron transfer reactions: cytochrome P450 reductase, methionine synthase reductase and endothelial and neuronal nitric oxide synthase. We used stopped-flow spectroscopy, single turnover methods and a kinetic model that relates electron flux through each enzyme to its conformational setpoint and its rates of conformational switching. The results show that the four flavoproteins, when fully-reduced, have a broad range of conformational setpoints (from 12% to 72% open state) and also vary 100-fold with respect to their rates of conformational switching between unreactive closed and reactive open states (cytochrome P450 reductase > neuronal nitric oxide synthase > methionine synthase reductase > endothelial nitric oxide synthase). Furthermore, simulations of the kinetic model could explain how each flavoprotein can support its given rate of electron flux (cytochrome c reductase activity) based on its unique conformational setpoint and switching rates. The present study is the first to quantify these conformational parameters among the diflavin enzymes and suggests how the parameters might be manipulated to speed or slow biological electron flux.

BYK191023 (2-[2-(4-methoxy-pyridin-2-yl)-ethyl]-3h-imidazo[4,5-b]pyridine) is an NADPH- and time-dependent irreversible inhibitor of inducible nitric-oxide synthase.

Imidazopyridine derivates were recently shown to be a novel class of selective and arginine-competitive inhibitors of inducible nitric-oxide synthase (iNOS), and 2-[2-(4-methoxypyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) was found to have very high selectivity in enzymatic and cellular models ( Mol Pharmacol 69: 328-337, 2006 ). Here, we show that BYK191023 irreversibly inactivates murine iNOS in an NADPH- and time-dependent manner, whereas it acts only as a reversible l-arginine-competitive inhibitor in the absence of NADPH or during anaerobic preincubation. Time-dependent irreversible inhibition by BYK191023 could also be demonstrated in intact cells using the RAW macrophage or iNOS-overexpressing human embryonic kidney 293 cell lines. The mechanism of BYK191023 inhibition in the presence of NADPH was studied using spectral, kinetic, chromatographic, and radioligand binding methods. BYK191023-bound iNOS was spectrally indistinguishable from l-arginine-bound iNOS, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. [(3)H]BYK191023 was recovered quantitatively from irreversibly inactivated iNOS, and no inhibitor metabolite was detected by high-performance liquid chromatography (HPLC). Size exclusion chromatography revealed only about 20% iNOS dissociation into monomers. Furthermore, HPLC and spectrophotometric analysis showed that the irreversible inhibition was associated with loss of heme from iNOS and a reduced ability to form the distinctive ferrous heme-CO complex (cytochrome P450). Thus, enzyme inactivation is mainly caused by heme loss, and it occurs in the inhibitor-bound enzyme in the presence of electron flux from NADPH.