A novel formamidase is required for riboflavin biosynthesis in invasive bacteria.

Biosynthesis of riboflavin (RF), the precursor of the redox cofactors FMN and FAD, was thought to be well understood in bacteria, with all the pathway enzymes presumed to be known and essential. Our previous research has challenged this view by showing that, in the bacterium Sinorhizobium meliloti, deletion of the ribBA gene encoding the enzyme that catalyzes the initial steps on the RF biosynthesis pathway only causes a reduction in flavin secretion rather than RF auxotrophy. This finding led us to hypothesize that RibBA participates in the biosynthesis of flavins destined for secretion, whereas S. meliloti has another enzyme that performs this function for internal cellular metabolism. Here, we identify and biochemically characterize a novel formamidase (SMc02977) involved in the production of RF for intracellular functions in S. meliloti. This catalyst, which we named Sm-BrbF, releases formate from the early RF precursor 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate to yield 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate. We show that homologs of this enzyme are present in many bacteria, are highly abundant in the Rhizobiales order, and that sequence homologs from Brucella abortus and Liberobacter solanacearum complement the RF auxotrophy of the Sm1021ΔSMc02977 mutant. Furthermore, we show that the B. abortus enzyme (Bab2_0247, Ba-BrbF) is also an 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate formamidase, and that the bab2_0247 mutant is a RF auxotroph exhibiting a lower level of intracellular infection than the wildtype strain. Finally, we show that Sm-BrbF and Ba-BrbF directly interact with other RF biosynthesis pathway enzymes. Together, our results provide novel insight into the intricacies of RF biosynthesis in bacteria.

Inter- and intra-observer agreement of femoroacetabular impingement (FAI) parameters comparing plain radiographs and advanced, 3D computed tomographic (CT)-generated hip models in a surgical patient cohort.

PURPOSE: The purpose of our study was to investigate whether advanced, 3D computed tomographic (CT)-generated hip models improves inter-and intra-observer agreement when compared to plain radiographs in identifying femoroacetabular impingement (FAI) morphology. METHODS: Eight consecutive patients who underwent surgery for FAI pathology were selected for this study. Preoperative CT scan image data were used to create high resolution, 3D hip reconstruction models. Four observers (two attending hip surgeons and radiologists) performed a blinded review of preselected radiographs and 3D CT hip models. Alpha and lateral center-edge angle measurements, location of cam lesion and the presence of a "crossover sign" were assessed. Inter- and intra-observer agreement was determined by calculating the intra-class correlation coefficients (ICC) or kappa coefficients to evaluate agreement for categorical variables. RESULTS: The parameter that demonstrated the highest and poorest inter-observer agreement was the presence of a "crossover sign" using 3D CT-generated high resolution hip models (ICC = 0.76, p = 0.00) and anteroposterior pelvis radiography, respectively (ICC = 0.20, p = 0.02). Alpha angle values were significantly higher using plain radiographs when compared to 3D hip reconstruction models (61.1° ± 10.4° versus 55.4° ± 14.4°, p = 0.003). Furthermore, when compared to radiographs, 3D hip reconstruction models demonstrated significantly higher intra-observer agreement (ICC = 0.856 versus 0.405, p = 0.005) when determining the presence of a "crossover sign". CONCLUSIONS: Our findings were suggestive that for most commonly used FAI morphology parameters, CT-generated hip models demonstrated little benefit over plain radiographs in improving inter-observer agreement among providers. LEVEL OF EVIDENCE: III.

Metal-on-metal hip resurfacing compared with total hip arthroplasty: two to five year outcomes in men younger than sixty five years.

PURPOSE: There are limited studies examining the long-term survivorship for the current generation of metal-on-metal hip resurfacing (MOMHR) implants in the young male population, and fewer studies have been published on prospectively collected outcomes data for total hip resurfacing in the USA. The purpose of this study was to demonstrate the efficacy of MOMHR in comparison with total hip arthroplasty (THA) using validated outcome measures, survivorship and complication rates. METHODS: The study prospectively followed 136 implants in 123 male patients <65 years, all with a primary diagnosis of osteoarthritis and similar comorbidities as determined by the American Society of Anesthesiologists (ASA) score. A single-surgeon cohort of 89 MOMHRs was compared with a similar cohort of 47 THAs. Outcomes were prospectively assessed with the Short-Form Health Survey of 12 questions (SF-12) and Western Ontario and McMaster Universities (WOMAC) questionnaires pre- and postoperatively at yearly intervals. Minimum follow-up was two years, and average follow-up was 3.9 years. RESULTS: Diagnosis, body mass index (BMI), American Association of Anesthesiologists (ASA) and pre-operative pain and function scores were not significantly different between groups. There was no difference in SF-12 scores postoperatively. At one and two years postoperatively, the MOMHR group had better WOMAC scores than the THA group, but no difference was seen at three to five years postoperatively. There were no revisions in either group over the study period. CONCLUSIONS: This study demonstrated good results for hip resurfacing in men <65 years five years postoperatively and similar function to THA patients.

"Skin Popping" and "Shooter's Patch" As Manifestations of Intradermal Drug Abuse.

Talc, a common adulterant in injectable opioids and filler in oral tablets, is frequently abused as crushed suspensions in injections. This review aims to recognize intradermal drug injection referred to colloquially as "skin popping" or "shooter's patch" as a cause of granulomatous disease and prevention of systemic complications from cutaneous cues.

Choroid plexus perfusion in sickle cell disease and moyamoya vasculopathy: Implications for glymphatic flow.

Cerebrospinal fluid (CSF) and interstitial fluid exchange have been shown to increase following pharmacologically-manipulated increases in cerebral arterial pulsatility, consistent with arterial pulsatility improving CSF circulation along perivascular glymphatic pathways. The choroid plexus (CP) complexes produce CSF, and CP activity may provide a centralized indicator of perivascular flow. We tested the primary hypothesis that elevated cortical cerebral blood volume and flow, present in sickle cell disease (SCD), is associated with fractionally-reduced CP perfusion relative to healthy adults, and the supplementary hypothesis that reduced arterial patency, present in moyamoya vasculopathy, is associated with elevated fractional CP perfusion relative to healthy adults. Participants (n = 75) provided informed consent and were scanned using a 3-Tesla arterial-spin-labeling MRI sequence for CP and cerebral gray matter (GM) perfusion quantification. ANOVA was used to calculate differences in CP-to-GM perfusion ratios between groups, and regression analyses applied to evaluate the dependence of the CP-to-GM perfusion ratio on group after co-varying for age and sex. ANOVA yielded significant (p < 0.001) group differences, with CP-to-GM perfusion ratios increasing between SCD (ratio = 0.93 ± 0.28), healthy (ratio = 1.04 ± 0.32), and moyamoya (ratio = 1.29 ± 0.32) participants, which was also consistent with regression analyses. Findings are consistent with CP perfusion being inversely associated with cortical perfusion.

Choroid plexus perfusion and intracranial cerebrospinal fluid changes after angiogenesis.

Recent studies have provided evidence that cortical brain ischemia may influence choroid plexus function, and such communication may be mediated by either traditional CSF circulation pathways and/or a possible glymphatic pathway. Here we investigated the hypothesis that improvements in arterial health following neoangiogenesis alter (i) intracranial CSF volume and (ii) choroid plexus perfusion in humans. CSF and tissue volume measurements were obtained from T1-weighted MRI, and cortical and choroid plexus perfusion were obtained from perfusion-weighted arterial spin labeling MRI, in patients with non-atherosclerotic intracranial stenosis (e.g. Moyamoya). Measurements were repeated after indirect surgical revascularization, which elicits cortical neoangiogenesis near the revascularization site (n = 23; age = 41.8 ± 13.4 years), or in a cohort of participants at two time points without interval surgeries (n = 10; age = 41.7 ± 10.7 years). Regression analyses were used to evaluate dependence of perfusion and volume on state (time 1 vs. 2). Post-surgery, neither CSF nor tissue volumes changed significantly. In surgical patients, cortical perfusion increased and choroid plexus perfusion decreased after surgery; in participants without surgeries, cortical perfusion reduced and choroid plexus perfusion increased between time points. Findings are discussed in the context of a homeostatic mechanism, whereby arterial health, paravascular flow, and/or ischemia can affect choroid plexus perfusion.

Tyrosine kinase inhibition: Ligand binding and conformational change in c-Kit and c-Abl.

The conformational flexibility exhibited by protein kinases poses an enormous challenge to the design of cancer therapeutics. Additionally the high degree of structural conservation within the kinase superfamily often leads to inhibitors that exhibit little selectivity and substantial cross reactivity. This work investigates the conformational changes that accompany the binding of Gleevec, or imatinib mesylate, to the tyrosine kinases c-Kit and c-Abl. Our analysis is that this fit is driven, at least in part, by the need to exclude water from solvent-exposed backbone hydrogen bonds. Both experimental and molecular modeling studies of the active state inhibitor of the tyrosine kinase c-Abl indicate that solvent exclusion also plays a role in this system.