Cefepime versus carbapenems for treatment of AmpC beta-lactamase-producing Enterobacterales bloodstream infections.

PURPOSE: Cefepime is recommended for treating infections caused by AmpC beta-lactamase-producing Enterobacterales (AmpC-PE), though supporting evidence is limited. Therefore, this study compared outcomes associated with cefepime versus carbapenem therapy for bloodstream infections (BSIs) caused by AmpC-PE after phenotypic exclusion of ESBL-co-producing isolates. METHODS: This retrospective cohort study compared definite cefepime versus carbapenem treatment for AmpC-PE BSI in hospitalized patients of the University Hospital Basel, Switzerland, between 01/2015 and 07/2020. Primary outcomes included in-hospital death, renal impairment and neurologic adverse events; secondary outcomes included length of hospital stay and recurrent infection. RESULTS: Two hundred and seventy episodes of AmpC-PE BSI were included, 162, 77 and 31 were treated with a carbapenem, cefepime and other antibiotics, respectively. Patients treated with carbapenems were more likely to be transferred to the ICU on admission and more frequently had central venous catheter as a source of infection. In uni- and multivariable analyses, primary and secondary outcomes did not differ between the two treatment groups, except for more frequent occurrence of neurological adverse events among patients treated with carbapenems and shorter length of hospital stay among survivors treated with cefepime. CONCLUSION: After excluding isolates with phenotypic ESBL-co-production, cefepime was not associated with adverse outcomes compared to carbapenems when used to treat BSIs caused by AmpC-PE. Our study provides evidence to support the use of cefepime as a safe treatment strategy for AmpC-PE BSI, particularly in clinically stable patients without initial renal impairment or increased susceptibility to neurological adverse events.

Application of a Robust Thermoelectric Gas Sensor in Firewood Combustion Exhausts.

The quality of wood combustion processes can be effectively improved by achieving the automated control of the combustion air feed. For this purpose, continuous flue gas analysis using in situ sensors is essential. Besides the successfully introduced monitoring of the combustion temperature and the residual oxygen concentration, in this study, in addition, a planar gas sensor is suggested that utilizes the thermoelectric principle to measure the exothermic heat generated by the oxidation of unburnt reducing exhaust gas components such as carbon monoxide (CO) and hydrocarbons (CxHy). The robust design made of high-temperature stable materials is tailored to the needs of flue gas analysis and offers numerous optimization options. Sensor signals are compared to flue gas analysis data from FTIR measurements during wood log batch firing. In general, impressive correlations between both data were found. Discrepancies occur during the cold start combustion phase. They can be attributed to changes in the ambient conditions around the sensor housing.

The Angelman Syndrome Online Registry - A multilingual approach to support global research.

In collaboration with the German Angelman syndrome (AS) community, we developed a web-based AS Online Registry to congregate existing as well as future information and scientifically quantify observations made by parents, families and medical professionals. With its user-friendly design as well as its concise and multilingual questionnaire, the registry aims at families who had so far refrained from being recruited by other, more comprehensive and/or English-only, registries. Data can be entered by both parents/families and medical professionals. The study design allows for re-contacting individuals (e.g. to request additional information) enabling collection of longitudinal data. Since its launch in June 2020, more than 200 individuals with AS age 2 month to 83 years have registered and entered their clinical and genetic data. In addition to the German, Turkish, English, Dutch, Italian, Danish and Finnish versions of the registry, we aim for translation into further languages to enable international and user-friendly recruitment of AS individuals. This novel registry will allow for extensive genotype-phenotype correlations and facilitate sharing of de-identified information among clinicians, researchers as well as the Global AS Registry. Furthermore, the registry will allow for identification of individuals suitable for future clinical or pharmacologic trials according to particular genotypic and/or phenotypic properties.

CCSchool: a multicentre, prospective study on improving continuum of care in children and adolescents with mental health problems associated with school problems in Germany.

BACKGROUND: Most psychiatric disorders in childhood and adolescence cause impairment in academic performance. Early interventions in school are thought to reduce the burden of disorder and prevent chronicity of disorder, while a delay in reachable help may result in more severe symptoms upon first time presentation, often then causing upon first-time presentation immediate need of inpatient care. METHODS: The study aims at reducing hospitalization rates and increasing social participation and quality of life among children and adolescents by establishing collaborations between schools, mental health care services and youth welfare services. CCSchool offers children and adolescents, aged six to 18 years, who present with psychiatric problems associated to school problems, a standardized screening and diagnostic procedure as well as treatment in school if necessary. Students can participate in CCSchool in three federal states of Germany if they a) show symptoms vindicating a mental health diagnosis, b) present with confirmed school problems and c) have a level of general functioning below 70 on the children global assessment of Functioning (C-GAF). Intervention takes place in three steps: module A (expected n = 901, according to power calculation) with standardized diagnostic procedures; module B (expected n = 428) implies a school-based assessment followed by a first intervention; module C (expected n = 103) offering school-based interventions with either four to six sessions (basic, 80% of patients) or eight to 12 sessions (intensive, 20% of patients). Primary aim is to evaluate the effectiveness of CCSchool, in reducing the need of hospitalization in children with mental health problems. The analyses will be conducted by an independent institute using mainly data collected from patients and their caregivers during study participation. Additionally, claims data from statutory health insurances will be analysed. Relevant confounders will be controlled in all analyses. DISCUSSION: Evaluation may show if CCSchool can prevent hospitalizations, enhance social participation and improve quality of life of children and adolescents with mental health problems by providing early accessible interventions in the school setting. TRIAL REGISTRATION: Deutsches Register Klinischer Studien, Trial registration number: DRKS00014838 , registered on 6th of June 2018.

Big fish in big ponds: Contrast and assimilation effects on math and verbal self-concepts of students in within-school gifted tracks.

BACKGROUND: Ability grouping can lower students' academic self-perceptions through reference group effects of class-average achievement on academic self-concept (ASC) - the Big-fish-little-Pond-effect (BFLPE; Marsh & Parker, 1984, J. Pers. Soc. Psychol., 47, 213). Although the effect itself is well documented, many open questions remain. For instance, negative contrast effects of group average achievement and positive assimilation effects of group status are confounded in most BFLPE studies. For the verbal domain, no study has yet investigated contrast and assimilation effects simultaneously. Strong assimilation effects can be assumed for gifted ability grouping. AIMS: We aimed at disentangling contrast and assimilation effects of full-time within-school gifted tracking on verbal ASC and math ASC. SAMPLE: Students attended regular or gifted classes within the top track of German secondary schools. Our sample comprised 1,330 fifth-grade students (42 regular classes, n = 1,069, 48% female; 15 gifted classes, n = 261, 39% female). METHODS: Using multilevel regression analyses, we simultaneously modelled negative contrast effects of class ability and positive assimilation effects of class type (regular vs. gifted) on ASC in math and the verbal domain. RESULTS AND CONCLUSIONS: In the mathematical domain, the assimilation effect, caused by membership in gifted classes, compensated for negative contrast effects of class-average achievement on ASC even after controlling for previous ASC. In the verbal domain, we found neither a significant contrast effect nor an assimilation effect. Implications for gifted education are discussed.

A2E and lipofuscin distributions in macaque retinal pigment epithelium are similar to human.

The accumulation of lipofuscin, an autofluorescent aging marker, in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD). Lipofuscin contains several visual cycle byproducts, most notably the bisretinoid N-retinylidene-N-retinylethanolamine (A2E). Previous studies with human donor eyes have shown a significant mismatch between lipofuscin autofluorescence (AF) and A2E distributions. The goal of the current project was to examine this relationship in a primate model with a retinal anatomy similar to that of humans. Ophthalmologically naive young (<10 years., N = 3) and old (>10 years., N = 4) Macaca fascicularis (macaque) eyes, were enucleated, dissected to yield RPE/choroid tissue, and flat-mounted on indium-tin-oxide-coated conductive slides. To compare the spatial distributions of lipofuscin and A2E, fluorescence and mass spectrometric imaging were carried out sequentially on the same samples. The distribution of lipofuscin fluorescence in the primate RPE reflected previously obtained human results, having the highest intensities in a perifoveal ring. Contrarily, A2E levels were consistently highest in the periphery, confirming a lack of correlation between the distributions of lipofuscin and A2E previously described in human donor eyes. We conclude that the mismatch between lipofuscin AF and A2E distributions is related to anatomical features specific to primates, such as the macula, and that this primate model has the potential to fill an important gap in current AMD research.

Isoproterenol effects evaluated in heart slices of human and rat in comparison to rat heart in vivo.

Human response to isoproterenol induced cardiac injury was evaluated by gene and protein pathway changes in human heart slices, and compared to rat heart slices and rat heart in vivo. Isoproterenol (10 and 100µM) altered human and rat heart slice markers of oxidative stress (ATP and GSH) at 24h. In this in vivo rat study (0.5mg/kg), serum troponin concentrations increased with lesion severity, minimal to mild necrosis at 24 and 48h. In the rat and the human heart, isoproterenol altered pathways for apoptosis/necrosis, stress/energy, inflammation, and remodeling/fibrosis. The rat and human heart slices were in an apoptotic phase, while the in vivo rat heart exhibited necrosis histologically and further progression of tissue remodeling. In human heart slices genes for several heat shock 70kD members were altered, indicative of stress to mitigate apoptosis. The stress response included alterations in energy utilization, fatty acid processing, and the up-regulation of inducible nitric oxide synthase, a marker of increased oxidative stress in both species. Inflammation markers linked with remodeling included IL-1α, Il-1ß, IL-6 and TNFα in both species. Tissue remodeling changes in both species included increases in the TIMP proteins, inhibitors of matrix degradation, the gene/protein of IL-4 linked with cardiac fibrosis, and the gene Ccl7 a chemokine that induces collagen synthesis, and Reg3b a growth factor for cardiac repair. This study demonstrates that the initial human heart slice response to isoproterenol cardiac injury results in apoptosis, stress/energy status, inflammation and tissue remodeling at concentrations similar to that in rat heart slices.

Homo-oligomerization of the activating natural killer cell receptor NKp30 ectodomain increases its binding affinity for cellular ligands.

The natural cytotoxicity receptors, comprised of three type I membrane proteins NKp30, NKp44, and NKp46, are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. Among these, NKp30 is a major receptor targeting virus-infected cells, malignantly transformed cells, and immature dendritic cells. To date, only few cellular ligands of NKp30 have been discovered, and the molecular details of ligand recognition by NKp30 are poorly understood. Within the current study, we found that the ectodomain of NKp30 forms functional homo-oligomers that mediate high affinity binding to its corresponding cellular ligand B7-H6. Notably, this homo-oligomerization is strongly promoted by the stalk domain of NKp30. Based on these data, we suggest that homo-oligomerization of NKp30 in the plasma membrane of NK cells, which might be favored by IL-2-dependent up-regulation of NKp30 expression, provides a way to improve recognition and lysis of target cells by NK cells.

Effects of rapeseed and soybean oil dietary supplementation on bovine fat metabolism, fatty acid composition and cholesterol levels in milk.

The main goal of this experiment was to study the effect of milk fat depression, induced by supplementing diet with plant oils, on the bovine fat metabolism, with special interest in cholesterol levels. For this purpose 39 cows were divided in three groups and fed different rations: a control group (C) without any oil supplementation and two groups with soybean oil (SO) or rapeseed oil (RO) added to the partial mixed ration (PMR). A decrease in milk fat percentage was observed in both oil feedings with a higher decrease of -1·14 % with SO than RO with -0·98 % compared with the physiological (-0·15 %) decline in the C group. There was no significant change in protein and lactose yield. The daily milk cholesterol yield was lower in both oil rations than in control ration, while the blood cholesterol level showed an opposite variation. The milk fatty acid pattern showed a highly significant decrease of over 10 % in the amount of saturated fatty acids (SFA) in both oil feedings and a highly significant increase in mono (MUFA) and poly (PUFA) unsaturated fatty acids, conjugated linoleic acids (CLA) included. The results of this experiment suggest that the feeding of oil supplements has a high impact on milk fat composition and its significance for human health, by decreasing fats with a potentially negative effect (SFA and cholesterol) while simultaneously increasing others with positive (MUFA, PUFA, CLA).

A soluble fragment of the tumor antigen BCL2-associated athanogene 6 (BAG-6) is essential and sufficient for inhibition of NKp30 receptor-dependent cytotoxicity of natural killer cells.

Immunosurveillance of tumor cells depends on NKp30, a major activating receptor of human natural killer (NK) cells. The human BCL2-associated athanogene 6 (BAG-6, also known as BAT3; 1126 amino acids) is a cellular ligand of NKp30. To date, little is known about the molecular details of this receptor ligand system. Within the current study, we have located the binding site of NKp30 to a sequence stretch of 250 amino acids in the C-terminal region of BAG-6 (BAG-6(686-936)). BAG-6(686-936) forms a noncovalent dimer of 57-59 kDa, which is sufficient for high affinity interaction with NKp30 (KD < 100 nM). As our most important finding, BAG-6(686-936) inhibits NKp30-dependent signaling, interferon-γ release, and degranulation of NK cells in the presence of malignantly transformed target cells. Based on these data, we show for the first time that BAG-6(686-936) comprises a subdomain of BAG-6, which is sufficient for receptor docking and inhibition of NKp30-dependent NK cell cytotoxicity as part of a tumor immune escape mechanism. These molecular insights provide an access point to restore tumor immunosurveillance by NK cells and to increase the efficacy of cellular therapies.

The stalk domain and the glycosylation status of the activating natural killer cell receptor NKp30 are important for ligand binding.

The natural cytotoxicity receptors are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The human natural cytotoxicity receptor family comprises the three type I membrane proteins NKp30, NKp44, and NKp46. Especially NKp30 is critical for the cytotoxicity of NK cells against different targets including tumor, virus-infected, and immature dendritic cells. Although the crystal structure of NKp30 was recently solved (Li, Y., Wang, Q., and Mariuzza, R. A. (2011) J. Exp. Med. 208, 703-714; Joyce, M. G., Tran, P., Zhuravleva, M. A., Jaw, J., Colonna, M., and Sun, P. D. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 6223-6228), a key question, how NKp30 recognizes several non-related ligands, remains unclear. Therefore, we investigated the parameters that impact ligand recognition of NKp30. Based on various NKp30-hIgG1-Fc fusion proteins, which were optimized for minimal background binding to cellular Fcγ receptors, we identified the flexible stalk region of NKp30 as an important but so far neglected module for ligand recognition and related signaling of the corresponding full-length receptor proteins. Moreover, we found that the ectodomain of NKp30 is N-linked glycosylated at three different sites. Mutational analyses revealed differential binding affinities and signaling capacities of mono-, di-, or triglycosylated NKp30, suggesting that the degree of glycosylation could provide a switch to modulate the ligand binding properties of NKp30 and NK cell cytotoxicity.

Genetic evidence for a contribution of EphA:ephrinA reverse signaling to motor axon guidance.

Repulsive Eph forward signaling from limb-derived ephrins guides the axons of lateral motor column (LMC) motor neurons. LMC axons also express ephrinAs, while their EphA receptors are expressed in the limb mesenchyme. In vitro studies have suggested that reverse signaling from limb-derived EphA4 to axonal ephrinAs might result in attraction of LMC axons. However, genetic evidence for this function is lacking. Here we use the Dunn chamber turning assay to show that EphA proteins are chemoattractants and elicit fast turning responses in LMC neurons in vitro. Moreover, ectopic expression of EphA4 in chick hindlimb changes the limb trajectory of LMC axons. Nervous system-specific deletion of EphA4 in mice resulted in fewer LMC axon projection errors than the ubiquitous deletion of EphA4. Additionally, a signaling-incompetent EphA4 mutant partially rescued guidance errors in the hindlimb, suggesting that limb-derived EphA4 contributes to the establishment of LMC projections. In summary, we provide evidence for a role of EphA:ephrinA attractive reverse signaling in motor axon guidance and in vivo evidence of in-parallel forward Eph and reverse ephrin signaling function in the same neuronal population.

Influence of different CLA isomers on insulin resistance and adipocytokines in pre-diabetic, middle-aged men with PPARγ2 Pro12Ala polymorphism.

Conjugated linoleic acids (CLAs) are natural PPARγ ligands, which showed conflicting effects on metabolism in humans. We examined metabolic effects of different isomers of CLA in subjects with PPARγ2 Pro12Ala polymorphisms. A total of 35 men underwent four intervention periods in a crossover study design: subjects with either genotypes received c9, t11 CLA or t10, c12 CLA, a commercially available 1:1 mix of both isomers or reference oil (linoleic acid (LA)). Adipocytokines, insulin, glucose and triglycerides were assessed in the fasting state and after a standardized mixed meal. Across all genotypes, there was a significant (p = 0.025) CLA treatment effect upon postprandial (pp) HOMA-IR values, with c9, t11 CLA and CLA isomer mix improving, but t10, c12 CLA isomer worsening. In Ala12Ala subjects, the t10, c12 isomer caused weight gain (p = 0.03) and tended to increase postprandial insulin levels (p = 0.05). In Pro12Pro subjects, t10, c12 resulted in reduction in waist circumference (p = 0.03). The comparison of the different genotype groups revealed statistically different changes in fasting and postprandial insulin, HOMA-IR and leptin after intervention. c9, t11 CLA and the commercial CLA mix showed beneficial effects on insulin sensitivity compared with LA, while t10, c12 CLA adversely affects body weight and insulin sensitivity in different PPAR genotypes. CLA isomers have different effects on metabolism in Ala and Pro carriers.

Expression of fatty acid binding proteins 3 and 5 genes in rat pancreatic islets and INS-1E cells: regulation by fatty acids and glucose.

Fatty acids binding proteins (FABPs) are involved in uptake, binding, transport and metabolism of fatty acids (FAs). Although FAs are known to stimulate insulin secretion from pancreatic islets when transiently elevated, while contributing to islet loss of function, lipotoxicity and apoptosis when chronically elevated, almost nothing is known regarding the FABPs in this tissue. The present study aimed at exploring the expression pattern and regulation of FABPs in rat islets and the insulin-secreting INS-1E cells. Rat islets and INS-1E cells expressed the heart/muscle type (FABP3) and the epidermal type (FABP5) genes. Different FAs significantly enhanced the expression of both FABPs. High glucose concentration induced a similar elevation of both FABP mRNA levels, and similarly to its effect on insulin 1 mRNA. Addition of oleic or palmitic acids to glucose did not render a further effect. FABP3 gene expression increased in response to PPARα agonist, while FABP5 increased in response to PPARα and PPARγ agonists, and decreased in response to a PPARß agonist. Beta-oxidation of FAs is required for the gene expression of both FABP genes in INS-1E cells. Inhibition of CPT-1 by etomoxir inhibited the oleic acid-induced FABP 3 and 5 gene expression, while activation of AMPK by metformin amplified the oleic-induced expression of both FABPs. FABP3 and 5 gene transcription required de novo protein synthesis, since inhibition by cycloheximide significantly decreased both FABP mRNAs. These data show a complex interrelationship between glucose and FAs in the control of FABP gene expression and that FABP3 and 5 may play a role in insulin secretion.

Human intestinal fatty acid binding protein 2 expression is associated with fat intake and polymorphisms.

The intestinal fatty acid binding protein (FABP2) is involved in lipid metabolism whereby variations in the promoter (haplotypes A/B) and exon 2 (Ala54Thr) are associated with dyslipidemia and insulin resistance. To elucidate which factors determine FABP2 expression in human mucosa, we investigated the association between fat intake, genotypes, biochemical variables, and FABP2 expression. FABP2 gene expression was assessed in duodenal specimens from 100 participants who answered a FFQ and who were genotyped and characterized for traits of metabolic syndrome and further biochemical data. Homozygotes for haplotype A tended to have lower fat intake than B-allele carriers (P = 0.066). Searching for an explanation, we evaluated the orexigenic glucose-dependent insulinotropic polypeptide (GIP) in a subset from the Metabolic Intervention Cohort Kiel. AA homozygotes had lower postprandial GIP concentrations than BB homozygotes. Duodenal FABP2 expression was correlated with (n-3) fatty acid (FA) intake in AA homozygotes (r = 0.49; P = 0.021). It was higher in AA homozygotes than in B-allele carriers after adjustment for (n-3) FA intake (P = 0.049) and was negatively correlated with serum FFA (r = -0.41; P < 0.01). Our data indicate that FABP2 expression depends on (n-3) FA intake and FABP2 genotypes. FABP2 might be involved in regulating food intake and intestinal FA utilization.

[Attempted and completed homicide in Hamburg--a comparison of two six-year periods]. / Versuchte und vollendete Tötungsdelikte in Hamburg--eine vergleichende Untersuchung von zwei Sechsjahres-Zeiträumen.

The present study compared cases of attempted and completed homicide in Hamburg from 1984 to 1989 and from 1995 to 2000 (n = 887). Data collection was performed using the police records. Attempted homicide showed a significant increase (34.8% vs. 57.9%, P < 0.0001). The majority of the victims and offenders were male with the share of male victims increasing from 59.7% to 74.2% (P < 0.0001). The age of the victims and offenders ranged between 22 and 40 years in both periods. The share of persons with a nationality other than German increased both in the victims (23.1% vs. 37.2%, P < 0.0001) and in the offenders (26.8% vs. 37.2%, P < 0.0001). The most common motives were interpersonal conflicts and robbery. The most frequently used forms of violence were sharp force, blunt force and strangulation.

EphA4 deficient mice maintain astroglial-fibrotic scar formation after spinal cord injury.

One important aspect of recovery and repair after spinal cord injury (SCI) lies in the complex cellular interactions at the injury site that leads to the formation of a lesion scar. EphA4, a promiscuous member of the EphA family of repulsive axon guidance receptors, is expressed by multiple cell types in the injured spinal cord, including astrocytes and neurons. We hypothesized that EphA4 contributes to aspects of cell-cell interactions at the injury site after SCI, thus modulating the formation of the astroglial-fibrotic scar. To test this hypothesis, we studied tissue responses to a thoracic dorsal hemisection SCI in an EphA4 mutant mouse line. We found that EphA4 expression, as assessed by beta-galactosidase reporter gene activity, is associated primarily with astrocytes in the spinal cord, neurons in the cerebral cortex and, to a lesser extent, spinal neurons, before and after SCI. However, we did not observe any overt reduction of glial fibrillary acidic protein (GFAP) expression in the injured area of EphA4 mutants in comparison with controls following SCI. Furthermore, there was no evident disruption of the fibrotic scar, and the boundary between reactive astrocytes and meningeal fibroblasts appeared unaltered in the mutants, as were lesion size, neuronal survival and inflammation marker expression. Thus, genetic deletion of EphA4 does not significantly alter the astroglial response or the formation of the astroglial-fibrotic scar following a dorsal hemisection SCI in mice. In contrast to what has been proposed, these data do not support a major role for EphA4 in reactive astrogliosis following SCI.

Generation of an EphA4 conditional allele in mice.

Ephrins and Eph receptor tyrosine kinases are cell-surface molecules that serve a multitude of functions in cell-cell communication in development, physiology, and disease. EphA4 is a promiscuous member of the EphA subclass of Eph receptors and can bind to both EphrinAs and EphrinBs. In addition to its well-established roles in guiding the development of neuronal connectivity, EphA4 has been implicated for a role in synaptic plasticity, vascular formation, axon regeneration, and central nervous system repair following injury. However, the study of its role in the adult stage has been hampered by confounding developmental defects in EphA4 germline mutants. Here, we report the generation and molecular characterization of an EphA4 conditional allele along with a novel null allele with a knockin fluorescent reporter gene (mCFP). The conditional allele will be useful in ascertaining postdevelopmental and/or cell type-specific function of EphA4 in physiology, injury, and disease.

Structural insights into regioselectivity in the enzymatic chlorination of tryptophan.

The regioselectively controlled introduction of chlorine into organic molecules is an important biological and chemical process. This importance derives from the observation that many pharmaceutically active natural products contain a chlorine atom. Flavin-dependent halogenases are one of the principal enzyme families responsible for regioselective halogenation of natural products. Structural studies of two flavin-dependent tryptophan 7-halogenases (PrnA and RebH) have generated important insights into the chemical mechanism of halogenation by this enzyme family. These proteins comprise two modules: a flavin adenine dinucleotide (FAD)-binding module and a tryptophan-binding module. Although the 7-halogenase studies advance a hypothesis for regioselectivity, this has never been experimentally demonstrated. PyrH is a tryptophan 5-halogenase that catalyzes halogenation on tryptophan C5 position. We report the crystal structure of a tryptophan 5-halogenase (PyrH) bound to tryptophan and FAD. The FAD-binding module is essentially unchanged relative to PrnA (and RebH), and PyrH would appear to generate the same reactive species from Cl(-), O(2), and 1,5-dihydroflavin adenine dinucleotide. We report additional mutagenesis data that extend our mechanistic understanding of this process, in particular highlighting a strap region that regulates FAD binding, and may allow communication between the two modules. PyrH has a significantly different tryptophan-binding module. The data show that PyrH binds tryptophan and presents the C5 atom to the reactive chlorinating species, shielding other potential reactive sites. We have mutated residues identified by structural analysis as recognizing the tryptophan in order to confirm their role. This work establishes the method by which flavin-dependent tryptophan halogenases regioselectively control chlorine addition to tryptophan. This method would seem to be general across the superfamily.