Guidelines for establishing a cytometry laboratory.

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management.

Clinical correlation but no elevation of striatal dopamine synthesis capacity in two independent cohorts of medication-free individuals with schizophrenia.

Dysregulation of dopamine systems has been considered a foundational driver of pathophysiological processes in schizophrenia, an illness characterized by diverse domains of symptomatology. Prior work observing elevated presynaptic dopamine synthesis capacity in some patient groups has not always identified consistent symptom correlates, and studies of affected individuals in medication-free states have been challenging to obtain. Here we report on two separate cohorts of individuals with schizophrenia spectrum illness who underwent blinded medication withdrawal and medication-free neuroimaging with [18F]-FDOPA PET to assess striatal dopamine synthesis capacity. Consistently in both cohorts, we found no significant differences between patient and matched, healthy comparison groups; however, we did identify and replicate robust inverse relationships between negative symptom severity and tracer-specific uptake widely throughout the striatum: [18F]-FDOPA specific uptake was lower in patients with a greater preponderance of negative symptoms. Complementary voxel-wise and region of interest analyses, both with and without partial volume correction, yielded consistent results. These data suggest that for some individuals, striatal hyperdopaminergia may not be a defining or enduring feature of primary psychotic illness. However, clinical differences across individuals may be significantly linked to variability in striatal dopaminergic tone. These findings call for further experimentation aimed at parsing the heterogeneity of dopaminergic systems function in schizophrenia.

Importance of Asparagine 202 in Manipulating Active Site Structure and Substrate Preference for Human CYP17A1.

The multifunctional cytochrome P450 17A1 (CYP17A1) plays a crucial role in human steroid hormone synthesis (UniProtKB─P05093). It first carries out standard monooxygenase chemistry, converting pregnenolone (PREG) and progesterone (PROG) into 17OH-PREG and 17OH-PROG, utilizing a "Compound I" to initiate hydrogen abstraction and radical recombination in the classic "oxygen rebound" mechanism. Additionally, these hydroxylated products also serve as substrates in a second oxidative cycle which cleaves the 17-20 carbon-carbon bond to form dehydroepiandrosterone and androstenedione, which are key precursors in the generation of powerful androgens and estrogens. Interestingly, in humans, with 17OH-PREG, this so-called lyase reaction is more efficient than with 17OH-PROG, based on Kcat/Km values. In the present work, the asparagine residue at 202 position was replaced by serine, an alteration which can affect substrate orientation and control substrate preference for the lyase reaction. First, we report studies of solvent isotope effects for the N202S CYP17A1 mutant in the presence of 17OH-PREG and 17OH-PROG, which suggest that the ferric peroxo species is the predominant catalytically active intermediate in the lyase step. This conclusion is further supported by employing a combination of cryoradiolysis and resonance Raman techniques to successfully trap and structurally characterize the key reaction intermediates, including the peroxo, the hydroperoxo, and the crucial peroxo-hemiketal intermediate. Collectively, these studies show that the mutation causes active site structural changes that alter the H-bonding interactions with the key Fe-O-O fragment and the degree of protonation of the reactive ferric peroxo intermediate, thereby impacting lyase efficiency.

Considerations for user consultation in a flow cytometry shared resource laboratory.

User consultation is an essential first step in assuring high-quality flow cytometric data. A central challenge to shared resource laboratory (SRL) staff is how to best guide new and current users to meet each projects' needs. One solution to this challenge is to follow a standard user consultation platform addressing all critical steps between the conception of the experiment and the actual acquisition of samples. Here we describe considerations to help an SRL understand the researcher's goals and how best the SRL staff can provide expert advice in a structured manner. User consultation has an educational nature, informing users about current best practices in cytometry that apply to their specific utilization. A consultation report also improves communication between the SRL, principal investigator, and lab members of the collaborating researcher. Development of best SRL practices is spearheaded by the ISAC SRL committee and this communication sets the foundation to initiate such report for user consultation. Implementation of best practices during user consultation will improve rigor and reproducibility in cytometry.

Comparison of Transcranial Sonography and [18 F]-Fluorodopa PET Imaging in GBA1 Mutation Carriers.

BACKGROUND: Mutations in GBA1 are a common genetic risk factor for parkinsonism; however, penetrance is incomplete, and biomarkers of future progression to parkinsonism are needed. Both nigral sonography and striatal [18 F]-FDOPA PET assay dopamine system health, but their utility and coherence in this context are unclear. OBJECTIVE: The aim of this study is to evaluate the utility and coherence of these modalities in GBA1-associated parkinsonism. METHODS: A total of 34 patients with GBA1 mutations (7 with parkinsonism) underwent both transcranial studies for substantia nigra echogenicity and [18 F]-FDOPA PET to determine striatal tracer-specific uptake (Ki ). RESULTS: Larger nigral echogenic areas and reduced striatal Ki were exclusively observed in parkinsonian patients. Sonographic and PET measurements showed strong inverse correlations but only in individuals with clinical parkinsonism. CONCLUSIONS: Close correspondence between nigral echogenicity and striatal presynaptic dopamine synthesis capacity observed only in GBA1 carriers with parkinsonism provides validation that these two modalities may conjointly capture aspects of the biology underlying clinical parkinsonism but raises questions about their utility as predictive tools in at-risk subjects. © 2022 International Parkinson and Movement Disorder Society.

Mechanism of the Clinically Relevant E305G Mutation in Human P450 CYP17A1.

Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C17-C20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.

Beyond linearity in neuroimaging: Capturing nonlinear relationships with application to longitudinal studies.

The ubiquitous adoption of linearity for quantitative predictors in statistical modeling is likely attributable to its advantages of straightforward interpretation and computational feasibility. The linearity assumption may be a reasonable approximation especially when the variable is confined within a narrow range, but it can be problematic when the variable's effect is non-monotonic or complex. Furthermore, visualization and model assessment of a linear fit are usually omitted because of challenges at the whole brain level in neuroimaging. By adopting a principle of learning from the data in the presence of uncertainty to resolve the problematic aspects of conventional polynomial fitting, we introduce a flexible and adaptive approach of multilevel smoothing splines (MSS) to capture any nonlinearity of a quantitative predictor for population-level neuroimaging data analysis. With no prior knowledge regarding the underlying relationship other than a parsimonious assumption about the extent of smoothness (e.g., no sharp corners), we express the unknown relationship with a sufficient number of smoothing splines and use the data to adaptively determine the specifics of the nonlinearity. In addition to introducing the theoretical framework of MSS as an efficient approach with a counterbalance between flexibility and stability, we strive to (a) lay out the specific schemes for population-level nonlinear analyses that may involve task (e.g., contrasting conditions) and subject-grouping (e.g., patients vs controls) factors; (b) provide modeling accommodations to adaptively reveal, estimate and compare any nonlinear effects of a predictor across the brain, or to more accurately account for the effects (including nonlinear effects) of a quantitative confound; (c) offer the associated program 3dMSS to the neuroimaging community for whole-brain voxel-wise analysis as part of the AFNI suite; and (d) demonstrate the modeling approach and visualization processes with a longitudinal dataset of structural MRI scans.

The NIMH Intramural Longitudinal Study of the Endocrine and Neurobiological Events Accompanying Puberty: Protocol and rationale for methods and measures.

Delineating the relationship between human neurodevelopment and the maturation of the hypothalamic-pituitary-gonadal (HPG) axis during puberty is critical for investigating the increase in vulnerability to neuropsychiatric disorders that is well documented during this period. Preclinical research demonstrates a clear association between gonadal production of sex steroids and neurodevelopment; however, identifying similar associations in humans has been complicated by confounding variables (such as age) and the coactivation of two additional endocrine systems (the adrenal androgenic system and the somatotropic growth axis) and requires further elucidation. In this paper, we present the design of, and preliminary observations from, the ongoing NIMH Intramural Longitudinal Study of the Endocrine and Neurobiological Events Accompanying Puberty. The aim of this study is to directly examine how the increase in sex steroid hormone production following activation of the HPG-axis (i.e., gonadarche) impacts neurodevelopment, and, additionally, to determine how gonadal development and maturation is associated with longitudinal changes in brain structure and function in boys and girls. To disentangle the effects of sex steroids from those of age and other endocrine events on brain development, our study design includes 1) selection criteria that establish a well-characterized baseline cohort of healthy 8-year-old children prior to the onset of puberty (e.g., prior to puberty-related sex steroid hormone production); 2) temporally dense longitudinal, repeated-measures sampling of typically developing children at 8-10 month intervals over a 10-year period between the ages of eight and 18; 3) contemporaneous collection of endocrine and other measures of gonadal, adrenal, and growth axis function at each timepoint; and 4) collection of multimodal neuroimaging measures at these same timepoints, including brain structure (gray and white matter volume, cortical thickness and area, white matter integrity, myelination) and function (reward processing, emotional processing, inhibition/impulsivity, working memory, resting-state network connectivity, regional cerebral blood flow). This report of our ongoing longitudinal study 1) provides a comprehensive review of the endocrine events of puberty; 2) details our overall study design; 3) presents our selection criteria for study entry (e.g., well-characterized prepubertal baseline) along with the endocrinological considerations and guiding principles that underlie these criteria; 4) describes our longitudinal outcome measures and how they specifically relate to investigating the effects of gonadal development on brain development; and 5) documents patterns of fMRI activation and resting-state networks from an early, representative subsample of our cohort of prepubertal 8-year-old children.

Longitudinal Positron Emission Tomography of Dopamine Synthesis in Subjects with GBA1 Mutations.

Mutations in GBA1, the gene mutated in Gaucher disease, are a common genetic risk factor for Parkinson disease, although the penetrance is low. We performed [18 F]-fluorodopa positron emission tomography studies of 57 homozygous and heterozygous GBA1 mutation carriers (15 with parkinsonism) and 98 controls looking for early indications of dopamine loss using voxelwise analyses to identify group differences in striatal [18 F]-fluorodopa uptake (Ki ). Forty-eight subjects were followed longitudinally. Cross-sectional and longitudinal comparisons of Ki and Ki change found significant effects of Parkinson disease. However, at baseline and over time, striatal [18 F]-fluorodopa uptake in mutation carriers without parkinsonism did not significantly differ from controls. ANN NEUROL 2020;87:652-657.

Risk factors for malaria in high incidence areas of Viet Nam: a case-control study.

BACKGROUND: A key step to advancing the goal of malaria elimination in Viet Nam by 2030 is focusing limited resources for treatment and prevention to groups most at risk for malaria transmission. METHODS: To better understand risk factors for malaria transmission in central Viet Nam, a survey of 1000 malaria positive cases and 1000 malaria negative controls was conducted. Cases and controls were matched for age and gender and self-presented at commune health stations (CHS) in Binh Phuoc, Dak Nong and Dak Lak Provinces. Diagnoses were confirmed with microscopy, rapid diagnostic test and PCR. Participants were interviewed about 50 potential risk factors for malaria, which included information about occupation, forest visitation, travel, healthcare-seeking behaviour and prior use of anti-malaria interventions. Participants were enrolled by trained government health workers and the samples were analysed in Vietnamese government laboratories. Data were analysed by univariable, block-wise and multivariable logistic regression. RESULTS: Among cases, 61.8% had Plasmodium falciparum, 35.2% Plasmodium vivax and 3% mixed species infections. Median (IQR) age was 27 (21-36) years and 91.2% were male. Twenty-five risk factors were associated with being a case and eleven with being a control. Multivariable analysis found that malaria cases correlated with forest workers, recent forest visitation, longer duration of illness, having a recorded fever, number of malaria infections in the past year, having had prior malaria treatment and having previously visited a clinic. CONCLUSIONS: This study demonstrates the benefits of increased statistical power from matched controls in malaria surveillance studies, which allows identification of additional independent risk factors. It also illustrates an example of research partnership between academia and government to collect high quality data relevant to planning malaria elimination activities. Modifiable risk factors and implications of the findings for malaria elimination strategy are presented.

Neanderthal-derived genetic variation in living humans relates to schizophrenia diagnosis, to psychotic symptom severity, and to dopamine synthesis.

Schizophrenia has been hypothesized to be a human-specific condition, but experimental approaches to testing this idea have been limited. Because Neanderthals, our closest evolutionary relatives, interbred with modern humans prior to their disappearance from the fossil record, leaving a residual echo that survives in our DNA today, we leveraged new discoveries about ancient hominid DNA to explore this hypothesis in living people in three converging ways. First, in four independent case-control datasets totaling 9,362 individuals, individuals with schizophrenia had less Neanderthal-derived genetic variation than controls (p = .044). Second, in 49 unmedicated inpatients with schizophrenia, having more Neanderthal admixture predicted less severe positive symptoms (p = .046). Finally, using 18 F-fluorodopa PET scanning in 172 healthy individuals, having greater Neanderthal introgression was significantly associated with lower dopamine synthesis capacity in the striatum and pons (p's < 2 × 10-5 ), which is fundamentally important in the pathophysiology and treatment of psychosis. These results may help to elucidate the evolutionary history of a devastating neuropsychiatric disease by supporting the notion of schizophrenia as a human-specific condition. Additionally, the relationship between Neanderthal admixture and dopamine function suggests a potential mechanism whereby Neanderthal admixture may have affected our gene pool to alter schizophrenia risk and/or course.

Evaluation of the Immunogenicity and Protective Efficacy of an Enterotoxigenic Escherichia coli CFA/I Adhesin-Heat-Labile Toxin Chimera.

Recent efforts to develop an enterotoxigenic Escherichia coli (ETEC) vaccine have focused on the antigenically conserved tip adhesins of colonization factors. We showed previously that intranasal immunization with dsc19CfaE, a soluble variant of the in cis donor strand-complemented tip adhesin of a colonization factor of the class 5 family (CFA/I) fimbria, is highly immunogenic and protects against oral challenge with CFA/I-positive (CFA/I+) ETEC strain H10407 in the Aotus nancymaae nonhuman primate. We also reported a cholera toxin (CT)-like chimera (called dsc19CfaE-CTA2/CTB) in which the CTA1 domain of CT was replaced by dsc19CfaE that was strongly immunogenic when administered intranasally or orogastrically in mice. Here, we evaluate the immunogenicity and protective efficacy (PE) of a refined and more stable chimera comprised of a pentameric B subunit of ETEC heat-labile toxin (LTB) in lieu of the CTB pentamer and a donor strand truncation (dsc14) of CfaE. The refined chimera, dsc14CfaE-sCTA2/LTB, was highly immunogenic in mice when administered intranasally or intradermally, eliciting serum and fecal antibody responses against CfaE and LTB, as well as strong hemagglutination inhibition titers, a surrogate for neutralization of intestinal adhesion mediated by CfaE. Moreover, the chimera was safe and highly immunogenic when administered intradermally to guinea pigs. In A. nancymaae, intradermal (i.d.) immunization with chimera plus single-mutant heat-labile toxin [LT(R192G)] elicited strong serum anti-CfaE and anti-LTB antibody responses and conferred significant reduction of diarrhea compared to phosphate-buffered saline (PBS) controls (PE = 84.1%; P < 0.02). These data support the further evaluation of dsc14CfaE-sCTA2/LTB as an ETEC vaccine in humans.

Williams syndrome hemideletion and LIMK1 variation both affect dorsal stream functional connectivity.

Williams syndrome is a rare genetic disorder caused by hemizygous deletion of ∼1.6 Mb affecting 26 genes on chromosome 7 (7q11.23) and is clinically typified by two cognitive/behavioural hallmarks: marked visuospatial deficits relative to verbal and non-verbal reasoning abilities and hypersocial personality. Clear knowledge of the circumscribed set of genes that are affected in Williams syndrome, along with the well-characterized neurobehavioural phenotype, offers the potential to elucidate neurogenetic principles that may apply in genetically and clinically more complex settings. The intraparietal sulcus, in the dorsal visual processing stream, has been shown to be structurally and functionally altered in Williams syndrome, providing a target for investigating resting-state functional connectivity and effects of specific genes hemideleted in Williams syndrome. Here, we tested for effects of the LIMK1 gene, deleted in Williams syndrome and important for neuronal maturation and migration, on intraparietal sulcus functional connectivity. We first defined a target brain phenotype by comparing intraparietal sulcus resting functional connectivity in individuals with Williams syndrome, in whom LIMK1 is hemideleted, with typically developing children. Then in two separate cohorts from the general population, we asked whether intraparietal sulcus functional connectivity patterns similar to those found in Williams syndrome were associated with sequence variation of the LIMK1 gene. Four independent between-group comparisons of resting-state functional MRI data (total n = 510) were performed: (i) 20 children with Williams syndrome compared to 20 age- and sex-matched typically developing children; (ii) a discovery cohort of 99 healthy adults stratified by LIMK1 haplotype; (iii) a replication cohort of 32 healthy adults also stratified by LIMK1 haplotype; and (iv) 339 healthy adolescent children stratified by LIMK1 haplotype. For between-group analyses, differences in intraparietal sulcus resting-state functional connectivity were calculated comparing children with Williams syndrome to matched typically developing children and comparing LIMK1 haplotype groups in each of the three general population cohorts separately. Consistent with the visuospatial construction impairment and hypersocial personality that typify Williams syndrome, the Williams syndrome cohort exhibited opposite patterns of intraparietal sulcus functional connectivity with visual processing regions and social processing regions: decreased circuit function in the former and increased circuit function in the latter. All three general population groups also showed LIMK1 haplotype-related differences in intraparietal sulcus functional connectivity localized to the fusiform gyrus, a visual processing region also identified in the Williams syndrome-typically developing comparison. These results suggest a neurogenetic mechanism, in part involving LIMK1, that may bias neural circuit function in both the general population and individuals with Williams syndrome.

Sequence Variation Associated with SLC12A5 Gene Expression Is Linked to Brain Structure and Function in Healthy Adults.

A single-nucleotide polymorphism in the promoter region of the Matrix Metalloproteinase-9 (MMP9) gene, rs3918242, has been shown to affect MMP9 expression in macrophages and was associated with schizophrenia by two independent groups. However, rs3918242's effects on MMP9 expression were not replicable in cell lines or brain tissue. Additionally, publically available data indicate that rs3918242 genotype is related not to MMP9 expression, but rather to expression of SLC12A5, a nearby gene coding for a K+/Cl- cotransporter, whose expression has also been related to schizophrenia. Here, we studied brain structure and function in healthy participants stratified by rs3918242 genotype using structural MRI (N = 298), functional MRI during an N-back working memory task (N = 554), and magnetoencephalography (MEG) during the same task (N = 190). We found rs3918242 was associated with gray matter volume (GMV) in the insula and dorsolateral prefrontal cortex bilaterally, closely replicated in discovery and replication samples; and with inferior parietal lobule (IPL) GMV when the samples were meta-analytically combined. Additionally, using both fMRI and MEG, rs3918242 was associated with right IPL working memory-related activation, replicated in two cohorts and across imaging modalities. These convergent results provide further impetus for examinations of the relationship of SLC12A5 with brain structure and function in neuropsychiatric disease.

A Survey of Antimicrobial Resistance Determinants in Category A Select Agents, Exempt Strains, and Near-Neighbor Species.

A dramatic increase in global antimicrobial resistance (AMR) has been well documented. Of particular concern is the dearth of information regarding the spectrum and prevalence of AMR within Category A Select Agents. Here, we performed a survey of horizontally and vertically transferred AMR determinants among Category A agents and their near neighbors. Microarrays provided broad spectrum screening of 127 Francisella spp., Yersinia spp., and Bacillus spp. strains for the presence/absence of 500+ AMR genes (or families of genes). Detecting a broad variety of AMR genes in each genus, microarray analysis also picked up the presence of an engineered plasmid in a Y. pestis strain. High resolution melt analysis (HRMA) was also used to assess the presence of quinolone resistance-associated mutations in 100 of these strains. Though HRMA was able to detect resistance-causing point mutations in B. anthracis strains, it was not capable of discriminating these point mutations from other nucleotide substitutions (e.g., arising from sequence differences in near neighbors). Though these technologies are well-established, to our knowledge, this is the largest survey of Category A agents and their near-neighbor species for genes covering multiple mechanisms of AMR.

Tracking Antimicrobial Resistance Determinants in Diarrheal Pathogens: A Cross-Institutional Pilot Study.

Infectious diarrhea affects over four billion individuals annually and causes over a million deaths each year. Though not typically prescribed for treatment of uncomplicated diarrheal disease, antimicrobials serve as a critical part of the armamentarium used to treat severe or persistent cases. Due to widespread over- and misuse of antimicrobials, there has been an alarming increase in global resistance, for which a standardized methodology for geographic surveillance would be highly beneficial. To demonstrate that a standardized methodology could be used to provide molecular surveillance of antimicrobial resistance (AMR) genes, we initiated a pilot study to test 130 diarrheal pathogens (Campylobacter spp., Escherichia coli, Salmonella, and Shigella spp.) from the USA, Peru, Egypt, Cambodia, and Kenya for the presence/absence of over 200 AMR determinants. We detected a total of 55 different determinants conferring resistance to ten different categories of antimicrobials: genes detected in ≥ 25 samples included blaTEM, tet(A), tet(B), mac(A), mac(B), aadA1/A2, strA, strB, sul1, sul2, qacEΔ1, cmr, and dfrA1. The number of determinants per strain ranged from none (several Campylobacter spp. strains) to sixteen, with isolates from Egypt harboring a wider variety and greater number of genes per isolate than other sites. Two samples harbored carbapenemase genes, blaOXA-48 or blaNDM. Genes conferring resistance to azithromycin (ere(A), mph(A)/mph(K), erm(B)), a first-line therapeutic for severe diarrhea, were detected in over 10% of all Enterobacteriaceae tested: these included >25% of the Enterobacteriaceae from Egypt and Kenya. Forty-six percent of the Egyptian Enterobacteriaceae harbored genes encoding CTX-M-1 or CTX-M-9 families of extended-spectrum ß-lactamases. Overall, the data provide cross-comparable resistome information to establish regional trends in support of international surveillance activities and potentially guide geospatially informed medical care.

Influence of Transmembrane Helix Mutations on Cytochrome P450-Membrane Interactions and Function.

Human cytochrome P450 (CYP) enzymes play an important role in the metabolism of drugs, steroids, fatty acids, and xenobiotics. Microsomal CYPs are anchored in the endoplasmic reticulum membrane by an N-terminal transmembrane (TM) helix that is connected to the globular catalytic domain by a flexible linker sequence. However, the structural and functional importance of the TM-helix is unclear because it has been shown that CYPs can still associate with the membrane and have enzymatic activity in reconstituted systems after truncation or modification of the N-terminal sequence. Here, we investigated the effect of mutations in the N-terminal TM-helix residues of two human steroidogenic enzymes, CYP 17A1 and CYP 19A1, that are major drug targets for cancer therapy. These mutations were originally introduced to increase the expression of the proteins in Escherichia coli. To investigate the effect of the mutations on protein-membrane interactions and function, we carried out coarse-grained and all-atom molecular dynamics simulations of the CYPs in a phospholipid bilayer. We confirmed the orientations of the globular domain in the membrane observed in the simulations by linear dichroism measurements in a Nanodisc. Whereas the behavior of CYP 19A1 was rather insensitive to truncation of the TM-helix, mutations in the TM-helix of CYP 17A1, especially W2A and E3L, led to a gradual drifting of the TM-helix out of the hydrophobic core of the membrane. This instability of the TM-helix could affect interactions with the allosteric redox partner, cytochrome b5, required for CYP 17A1's lyase activity. Furthermore, the simulations showed that the mutant TM-helix influenced the membrane interactions of the CYP 17A1 globular domain. In some simulations, the mutated TM-helix obstructed the substrate access tunnel from the membrane to the CYP active site, indicating a possible effect on enzyme function.

Soluble Intercellular Adhesion Molecule-1 (sICAM-1) as a Biomarker of Vascular Cognitive Impairment in Older Adults.

BACKGROUND: Endothelial dysfunction and subsequent inflammation contribute to the development of vascular cognitive impairment (VCI). Soluble intercellular adhesion molecule-1 (sICAM-1) is upregulated in endothelial dysfunction and promotes an inflammatory response; however, the relationship between sICAM-1 and VCI remains equivocal. OBJECTIVE: To determine whether sICAM-1 contributes to the prediction of VCI. METHODS: Community-dwelling older adults (n = 172) from the "Cohort of Obesity, Sarcopenia and Frailty of Older Mexican Adults" (COSFOMA) study were identified as VCI or controls using standard neuropsychological evaluations and neuroimaging. sICAM-1 was quantified using ELISA, and multivariate logistic regression determined the association between sICAM-1 and VCI. RESULTS: A total of 31 VCI cases were identified. sICAM-1 was higher in VCI (VCI: 450.7 [241.6] ng/mL vs. controls: 296.9 [140.9] ng/mL). sICAM-1 concentrations above the 90th percentile (464.1 ng/mL) were associated with VCI group membership in all models (OR: 6.9, 95% CI: 1.1-42.2). The final saturated model explained 64% of the variance in VCI group membership. CONCLUSION: High concentrations of sICAM-1 are independently associated with VCI group membership. Efforts to further characterize the relationship between indices of endothelial dysfunction and pathological changes to the aging brain should be further pursued.

Human P450 CYP17A1: Control of Substrate Preference by Asparagine 202.

CYP17A1 is a key steroidogenic enzyme known to conduct several distinct chemical transformations on multiple substrates. In its hydroxylase activity, this enzyme adds a hydroxyl group at the 17α position of both pregnenolone and progesterone at approximately equal rates. However, the subsequent 17,20 carbon-carbon scission reaction displays variable substrate specificity in the numerous CYP17A1 isozymes operating in vertebrates, manifesting as different Kd and kcat values when presented with 17α-hydroxypregnenlone (OHPREG) versus 17α-hydroxyprogesterone (OHPROG). Here we show that the identity of the residue at position 202 in human CYP17A1, thought to form a hydrogen bond with the A-ring alcohol substituent on the pregnene- nucleus, is a key driver of this enzyme's native preference for OHPREG. Replacement of asparagine 202 with serine completely reverses the preference of CYP17A1, more than doubling the rate of turnover of the OHPROG to androstenedione reaction and substantially decreasing the rate of formation of dehydroepiandrosterone from OHPREG. In a series of resonance Raman experiments, it was observed that, in contrast with the case for the wild-type protein, in the mutant the 17α alcohol of OHPROG tends to form a H-bond with the proximal rather than terminal oxygen of the oxy-ferrous complex. When OHPREG was a substrate, the mutant enzyme was found to have a H-bonding interaction with the proximal oxygen that is substantially weaker than that of the wild type. These results demonstrate that a single-point mutation in the active site pocket of CYP17A1, even when far from the heme, has profound effects on steroidogenic selectivity in androgen biosynthesis.

Human Cytochrome CYP17A1: The Structural Basis for Compromised Lyase Activity with 17-Hydroxyprogesterone.

The multifunctional enzyme, cytochrome P450 (CYP17A1), plays a crucial role in the production of androgens, catalyzing two key reactions on pregnenolone (PREG) and progesterone (PROG), the first being a 17-hydroxylation to generate 17-OH PREG and 17-OH PROG, with roughly equal efficiencies. The second is a C-C bond scission or "lyase" reaction in which the C17-C20 bond is cleaved, leading to the eventual production of powerful androgens, whose involvement in the proliferation of prostate cancer has generated intense interest in developing inhibitors of CYP17A1. For humans, the significance of the C-C bond cleavage of 17-OH PROG is lessened, because it is about 50 times less efficient than for 17-OH PREG in terms of kcat/Km. Recognizing the need to clarify relevant reaction mechanisms involved with such transformations, we first report studies of solvent isotope effects, results of which are consistent with a Compound I mediated PROG hydroxylase activity, yet exclude this intermediate as a participant in the formation of androstenedione (AD) via the lyase reaction. This finding is also supported by a combination of cryoreduction and resonance Raman spectroscopy that traps and structurally characterizes the key hemiketal reaction intermediates. Adding to a previous study of PREG and 17-OH PREG metabolism, the current work provides definitive evidence for a more facile protonation of the initially formed ferric peroxo-intermediate for 17-OH PROG-bound CYP17A1, compared to the complex with 17-OH PREG. Importantly, Raman characterization also reveals an H-bonding interaction with the terminal oxygen of the peroxo fragment, rather than with the proximal oxygen, as is present for 17-OH PREG. These factors would favor a diminished lyase activity of the sample with 17-OH PROG relative to the complex with 17-OH PREG, thereby providing a convincing structural explanation for the dramatic differences in activity for these lyase substrates in humans.