An EPR/HYSCORE, Mössbauer, and resonance Raman study of the hydrogenase maturation enzyme HydF: a model for N-coordination to [4Fe-4S] clusters.

The biosynthesis of the organometallic H cluster of [Fe-Fe] hydrogenase requires three accessory proteins, two of which (HydE and HydG) belong to the radical S-adenosylmethionine enzyme superfamily. The third, HydF, is an Fe-S protein with GTPase activity. The [4Fe-4S] cluster of HydF is bound to the polypeptide chain through only the three, conserved, cysteine residues present in the binding sequence motif CysXHisX(46-53)HisCysXXCys. However, the involvement of the two highly conserved histidines as a fourth ligand for the cluster coordination is controversial. In this study, we set out to characterize further the [4Fe-4S] cluster of HydF using Mössbauer, EPR, hyperfine sublevel correlation (HYSCORE), and resonance Raman spectroscopy in order to investigate the influence of nitrogen ligands on the spectroscopic properties of [4Fe-4S](2+/+) clusters. Our results show that Mössbauer, resonance Raman, and EPR spectroscopy are not able to readily discriminate between the imidazole-coordinated [4Fe-4S] cluster and the non-imidazole-bound [4Fe-4S] cluster with an exchangeable fourth ligand that is present in wild-type HydF. HYSCORE spectroscopy, on the other hand, detects the presence of an imidazole/histidine ligand on the cluster on the basis of the appearance of a specific spectral pattern in the strongly coupled region, with a coupling constant of approximately 6 MHz. We also discovered that a His-tagged version of HydF, with a hexahistidine tag at the N-terminus, has a [4Fe-4S] cluster coordinated by one histidine from the tag. This observation strongly indicates that care has to be taken in the analysis of data obtained on tagged forms of metalloproteins.

Get it together, together: Creating whole community continuity through cross-sector collaboration in Texas.

Continuity of operations for government is an evolving philosophy, much like exercises and after-action reports. Continuity continues to identify areas for growth and improvement as more people become involved in the conversation. This paper briefly describes the evolution of continuity in the USA and its application in the State of Texas. Moving forward, it discusses the application of the concept of 'whole community continuity' as the driving force of the Continuity Council in Texas, which focuses on preparedness at all levels, from individuals to private industry, to all levels of government.

Insecticide Resistance in Aedes aegypti Varies Seasonally and Geographically in Texas/Mexico Border Cities.

Insecticide use is the primary method of attempting to reduce or control the spread of mosquito-borne diseases. Insecticide resistance is a major concern as resistance will limit the efficacy of vector-control efforts. The lower Rio Grande Valley region of South Texas has had autochthonous transmission of multiple mosquito-borne diseases including those caused by dengue virus, chikungunya virus, and Zika virus. However, the current status of mosquito resistance to commonly used pesticides in this region is unknown. In this study, we collected field samples from multiple municipalities in South Texas and assessed resistance using the Centers for Disease Control and Prevention bottle bioassay. All populations exhibited characteristics of resistance, and permethrin was the most effective insecticide with an average mortality rate of 44.78%. Deltamethrin and sumethrin had significantly lower mortality rates of 20.31% and 32.16%, respectively, although neither of these insecticides are commonly used for vector-control activities in this region. Depending on which insecticide was used, there was little significance between each of the 7 cities. Seasonal variation in resistance was observed among the collection sites. Both deltamethrin and sumethrin exhibited an increase in susceptibility over the course of 10 months, while permethrin exhibited a decrease in susceptibility. These data highlight the need for further studies to determine if variations in resistance observed are repeated. The data and future findings may be useful in determining the most effective strategies for pesticide use and rotation.

Validation of SARS-CoV-2 pooled testing for surveillance using the Panther Fusion® system: Impact of pool size, automation, and assay chemistry.

Combining diagnostic specimens into pools has been considered as a strategy to augment throughput, decrease turnaround time, and leverage resources. This study utilized a multi-parametric approach to assess optimum pool size, impact of automation, and effect of nucleic acid amplification chemistries on the detection of SARS-CoV-2 RNA in pooled samples for surveillance testing on the Hologic Panther Fusion® System. Dorfman pooled testing was conducted with previously tested SARS-CoV-2 nasopharyngeal samples using Hologic's Aptima® and Panther Fusion® SARS-CoV-2 Emergency Use Authorization assays. A manual workflow was used to generate pool sizes of 5:1 (five samples: one positive, four negative) and 10:1. An automated workflow was used to generate pool sizes of 3:1, 4:1, 5:1, 8:1 and 10:1. The impact of pool size, pooling method, and assay chemistry on sensitivity, specificity, and lower limit of detection (LLOD) was evaluated. Both the Hologic Aptima® and Panther Fusion® SARS-CoV-2 assays demonstrated >85% positive percent agreement between neat testing and pool sizes ≤5:1, satisfying FDA recommendation. Discordant results between neat and pooled testing were more frequent for positive samples with CT>35. Fusion® CT (cycle threshold) values for pooled samples increased as expected for pool sizes of 5:1 (CT increase of 1.92-2.41) and 10:1 (CT increase of 3.03-3.29). The Fusion® assay demonstrated lower LLOD than the Aptima® assay for pooled testing (956 vs 1503 cp/mL, pool size of 5:1). Lowering the cut-off threshold of the Aptima® assay from 560 kRLU (manufacturer's setting) to 350 kRLU improved the assay sensitivity to that of the Fusion® assay for pooled testing. Both Hologic's SARS-CoV-2 assays met the FDA recommended guidelines for percent positive agreement (>85%) for pool sizes ≤5:1. Automated pooling increased test throughput and enabled automated sample tracking while requiring less labor. The Fusion® SARS-CoV-2 assay, which demonstrated a lower LLOD, may be more appropriate for surveillance testing.

A Method for Repeated, Longitudinal Sampling of Individual Aedes aegypti for Transmission Potential of Arboviruses.

Mosquito-borne viruses are the cause of significant morbidity and mortality worldwide, especially in low- and middle-income countries. Assessing risk for viral transmission often involves characterization of the vector competence of vector-virus pairings. The most common determination of vector competence uses discreet, terminal time points, which cannot be used to investigate variation in transmission aspects, such as biting behavior, over time. Here, we present a novel method to longitudinally measure individual biting behavior and Zika virus (ZIKV) transmission. Individual mosquitoes were exposed to ZIKV, and from 9 to 24 days post-exposure, individuals were each offered a 180 µL bloodmeal every other day. Biting behavior was observed and characterized as either active probing, feeding, or no bite. The bloodmeal was then collected, spun down, serum collected, and tested for ZIKV RNA via qRT-PCR to determine individuals' vector competence over time. This included whether transmission to the bloodmeal was successful and the titer of expectorated virus. Additionally, serum was inoculated onto Vero cells in order to determine infectiousness of positive recovered sera. Results demonstrate heterogeneity in not only biting patterns but expectorated viral titers among individual mosquitoes over time. These findings demonstrate that the act of transmission is a complex process governed by mosquito behavior and mosquito-virus interaction, and herein we offer a method to investigate this phenomenon.

A SARS-CoV-2 spike ferritin nanoparticle vaccine protects against heterologous challenge with B.1.1.7 and B.1.351 virus variants in Syrian golden hamsters.

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the B.1.1.7 and B.1.351 VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 µg) or low (0.2 µg) immunogen dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose two vaccinations. SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

A SARS-CoV-2 spike ferritin nanoparticle vaccine protects hamsters against Alpha and Beta virus variant challenge.

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a SARS-CoV-2 spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the Alpha (B.1.1.7), and Beta (B.1.351) VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 µg) or low (0.2 µg) dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose vaccinations. Dose-dependent SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

ENDOR spectroscopy shows that guanine N1 binds to [4Fe-4S] cluster II of the S-adenosylmethionine-dependent enzyme MoaA: mechanistic implications.

The S-adenosylmethionine-dependent enzyme MoaA, in concert with MoaC, catalyzes the first step of molybdenum cofactor biosynthesis, the conversion of guanosine 5'-triphosphate (5'-GTP) into precursor Z. A published X-ray crystal structure of MoaA with the substrate 5'-GTP revealed that the substrate might be bound to the unique iron of one of two 4Fe-4S clusters through either or both the amino and N1 nitrogen nuclei. Use of 35 GHz continuous-wave ENDOR spectroscopy of MoaA with unlabeled and (15)N-labeled substrate and a reduced [4Fe-4S](+) cluster now demonstrates that only one nitrogen nucleus is bound to the cluster. Experiments with the substrate analogue inosine 5'-triphosphate further demonstrate that it is the N1 nitrogen that binds. Two of the more distant nitrogen nuclei have also been detected by 35 GHz pulsed ENDOR spectroscopy, allowing a rough approximation of their distances from the cluster to be calculated. Combining this information with the crystal structure, we propose that the guanine base adopts the enol tautomer as N1 binds to Fe4 and the O6-H hydroxyl group forms a hydrogen bond with S4 of the 4Fe-4S cluster, and that this binding-induced tautomerization may have important mechanistic ramifications.

Possible direct involvement of the active-site [4Fe-4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP.

The GcpE enzyme converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the penultimate step of the DOXP pathway for isoprene biosynthesis. Purification of the enzyme under exclusion of air leads to a preparation that contains solely [4Fe-4S] clusters. Kinetic studies showed that in the presence of the artificial reductant dithionite and MEcPP a new transient iron-sulfur-based signal is detected in electron paramagnetic resonance (EPR) spectroscopy. Similarity of this EPR signal to that detected in ferredoxin:thioredoxin reductase indicates that during the reaction an intermediate is directly bound to the active-site cluster.

Effects of chronic opioid exposure on guinea pig mu opioid receptor in Chinese hamster ovary cells: comparison with human and rat receptor.

Chronic opioid treatment leads to agonist-specific effects at the mu opioid receptor. The molecular mechanisms resulting from chronic opioid exposure include desensitization, internalization and down-regulation of membrane-bound mu opioid receptors (MOP). The purpose of this study was to compare the cellular regulation of guinea pig, human and rat MOP expressed in Chinese hamster ovary (CHO) cells, following exposure to two clinically important opioids, morphine and methadone. MOP expressing CHO cells were treated in culture with methadone or morphine for up to 48 h. Radioligand diprenorphine and [D-AIa(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO)-stimulated GTP gamma S binding assays were carried out using paired control and opioid-exposed CHO cells. Methadone induced downregulation of the mu opioid receptor, while morphine induced desensitization of the receptor for all three species. Furthermore, morphine predominantly decreased the potency of DAMGO to stimulate GTP gamma S binding, whereas methadone primarily reduced its efficacy. Changes in DAMGO potency and efficacy differed among species and depended on the opioid used to treat the cells. Our results showed similarities between guinea pig and human MOP for morphine-induced desensitization, but identified differences between the two for methadone-induced desensitization. In contrast, human and rat MOP differed in response to morphine treatment, but were not distinct in their response to methadone treatment. The guinea pig is an excellent and established animal model to study opioid effects, but its molecular opioid pharmacology has not been investigated thus far. These results can assist in understanding species differences in the effects of opioid ligands activating the mu opioid receptor.

MiaB, a bifunctional radical-S-adenosylmethionine enzyme involved in the thiolation and methylation of tRNA, contains two essential [4Fe-4S] clusters.

The radical-S-adenosylmethionine (radical-AdoMet) enzyme MiaB catalyzes the posttranscriptional methylthiolation of N-6-isopentenyladenosine in tRNAs. Spectroscopic and analytical studies of the reconstituted wild-type and C150/154/157A triple variant forms of Thermotoga maritima MiaB have revealed the presence of two distinct [4Fe-4S](2+,1+) clusters in the protein. One is coordinated by the three conserved cysteines in the radical-AdoMet motif (Cys150, Cys154, and Cys157) as previously reported, and the other, here observed for the first time, is proposed to be coordinated by the three N-terminal conserved cysteines (Cys10, Cys46, and Cys79). The two [4Fe-4S]2+ clusters have similar UV-visible absorption, resonance Raman, and Mössbauer properties but differ in terms of redox properties and the EPR properties of the reduced [4Fe-4S]1+ clusters. Reconstituted forms of MiaB containing two [4Fe-4S] clusters are more active than previously reported. Comparison of MiaB with other radical-AdoMet enzymes involved in thiolation reactions, such as biotin synthase and lipoate synthase, is discussed as well as a possible role of the second cluster as a sacrificial S-donor in the MiaB-catalyzed reaction.

MiaB protein from Thermotoga maritima. Characterization of an extremely thermophilic tRNA-methylthiotransferase.

In Escherichia coli, the MiaB protein catalyzes the methylthiolation of N-6-isopentenyl adenosine in tRNAs, the last reaction step during biosynthesis of 2-methylthio-N-6-isopentenyl adenosine (ms2i6A-37). For the first time the thermophilic bacterium Thermotoga maritima is shown here to contain such a MiaB tRNA-modifying enzyme, named MiaBTm, and to synthesize ms2i6A-37 as demonstrated by an analysis of modified nucleosides from tRNA hydrolysates. The corresponding gene (TM0653) was identified by sequence similarity to the miaB gene cloned and expressed in E. coli. MiaBTm was purified to homogeneity and thoroughly characterized by biochemical and spectroscopic methods. It is a monomer of 443 residues with a molecular mass of 50,710 kilodaltons. Its amino acid sequence shares the CysXXX-CysXXCys sequence with MiaB from E. coli as well as with biotin synthase and lipoate synthase. This sequence was shown to be essential for chelation of an iron-sulfur center and for activity in these enzymes. As isolated, MiaBTm contains both iron and sulfide and an apoprotein form can coordinate up to 4 iron and 4 sulfur atoms per polypeptide chain. UV-visible absorption, resonance Raman, variable temperature magnetic circular dichroism, and EPR spectroscopy of MiaBTm indicate the presence of a [4Fe-4S]+2/+1 cluster under reducing and anaerobic conditions, whereas [3Fe-4S]+1 and [2Fe-2S]+2 forms are generated under aerobic conditions. The redox potential of the [4Fe-4S]+2/+1 transition is -495 +/- 10 mV (versus the normal hydrogen electrode). Finally, the expression of MiaBTm from T. maritima in an E. coli mutant strain lacking functional miaB gene allowed production of ms2i6A-37. These results provide further information on the enzymes involved in methylthiolation of tRNAs.

Characterization of the cofactor composition of Escherichia coli biotin synthase.

The cofactor content of in vivo, as-isolated, and reconstituted forms of recombinant Escherichia coli biotin synthase (BioB) has been investigated using the combination of UV-visible absorption, resonance Raman, and Mössbauer spectroscopies along with parallel analytical and activity assays. In contrast to the recent report that E. coli BioB is a pyridoxal phosphate (PLP)-dependent enzyme with intrinsic cysteine desulfurase activity (Ollagnier-deChoudens, S., Mulliez, E., Hewitson, K. S., and Fontecave, M. (2002) Biochemistry 41, 9145-9152), no evidence for PLP binding or for PLP-induced cysteine desulfurase or biotin synthase activity was observed with any of the forms of BioB investigated in this work. The results confirm that BioB contains two distinct Fe-S cluster binding sites. One site accommodates a [2Fe-2S](2+) cluster with partial noncysteinyl ligation that can only be reconstituted in vitro in the presence of O(2). The other site accommodates a [4Fe-4S](2+,+) cluster that binds S-adenosylmethionine (SAM) at a unique Fe site of the [4Fe-4S](2+) cluster and undergoes O(2)-induced degradation via a distinct type of [2Fe-2S](2+) cluster intermediate. In vivo Mössbauer studies show that recombinant BioB in anaerobically grown cells is expressed exclusively in an inactive form containing only the as-isolated [2Fe-2S](2+) cluster and demonstrate that the [2Fe-2S](2+) cluster is not a consequence of overexpressing the recombinant enzyme under aerobic growth conditions. Overall the results clarify the confusion in the literature concerning the Fe-S cluster composition and the in vitro reconstitution and O(2)-induced cluster transformations that are possible for recombinant BioB. In addition, they provide a firm foundation for assessing cluster transformations that occur during turnover and the catalytic competence of the [2Fe-2S](2+) cluster as the immediate S-donor for biotin biosynthesis.

Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase.

Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom between C6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purified recombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe-2S](2+) cluster per monomer. It is inactive in vitro without the addition of exogenous Fe. Anaerobic reconstitution of the as-purified [2Fe-2S]-containing BioB with Fe(2+) and S(2)(-) produces a form of BioB that contains approximately one [2Fe-2S](2+) and one [4Fe-4S](2+) cluster per monomer ([2Fe-2S]/[4Fe-4S] BioB). In the absence of added Fe, the [2Fe-2S]/[4Fe-4S] BioB is active and can produce up to approximately 0.7 equiv of biotin per monomer. To better define the roles of the Fe-S clusters in the BioB reaction, Mössbauer and electron paramagnetic resonance (EPR) spectroscopy have been used to monitor the states of the Fe-S clusters during the conversion of dethiobiotin to biotin. The results show that the [4Fe-4S](2+) cluster is stable during the reaction and present in the SAM-bound form, supporting the current consensus that the functional role of the [4Fe-4S] cluster is to bind SAM and facilitate the reductive cleavage of SAM to generate the catalytically essential 5'-deoxyadenosyl radical. The results also demonstrate that approximately (2)/(3) of the [2Fe-2S] clusters are degraded by the end of the turnover experiment (24 h at 25 degrees C). A transient species with spectroscopic properties consistent with a [2Fe-2S](+) cluster is observed during turnover, suggesting that the degradation of the [2Fe-2S](2+) cluster is initiated by reduction of the cluster. This observed degradation of the [2Fe-2S] cluster during biotin formation is consistent with the proposed sacrificial S-donating function of the [2Fe-2S] cluster put forth by Jarrett and co-workers (Ugulava et al. (2001) Biochemistry 40, 8352-8358). Interestingly, degradation of the [2Fe-2S](2+) cluster was found not to parallel biotin formation. The initial decay rate of the [2Fe-2S](2+) cluster is about 1 order of magnitude faster than the initial formation rate of biotin, indicating that if the [2Fe-2S] cluster is the immediate S donor for biotin synthesis, insertion of S into dethiobiotin would not be the rate-limiting step. Alternatively, the [2Fe-2S] cluster may not be the immediate S donor. Instead, degradation of the [2Fe-2S] cluster may generate a protein-bound polysulfide or persulfide that serves as the immediate S donor for biotin production.

Characterization of MOCS1A, an oxygen-sensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis.

The human proteins MOCS1A and MOCS1B catalyze the conversion of a guanosine derivative to precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine (AdoMet)-dependent radical enzymes, which catalyze the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus. MOCS1A was heterologously expressed in Escherichia coli and purified under aerobic and anaerobic conditions. Individual mutations of the conserved cysteines to serine revealed that all are essential for synthesis of precursor Z in vivo. The type and properties of the iron-sulfur (FeS) clusters were investigated using a combination of UV-visible absorption, variable temperature magnetic circular dichroism, resonance Raman, Mössbauer, and EPR spectroscopies coupled with iron and acid-labile sulfide analyses. The results indicated that anaerobically purified MOCS1A is a monomeric protein containing two oxygen-sensitive FeS clusters, each coordinated by only three cysteine residues. A redox-active [4Fe-4S](2+,+) cluster is ligated by an N-terminal CX(3)CX(2)C motif as is the case with all other AdoMet-dependent radical enzymes investigated thus far. A C-terminal CX(2)CX(13)C motif that is unique to MOCS1A and its orthologs primarily ligates a [3Fe-4S](0) cluster. However, MOCS1A could be reconstituted in vitro under anaerobic conditions to yield a form containing two [4Fe-4S](2+) clusters. The N-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen via a semistable [2Fe-2S](2+) cluster intermediate, and the C-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen to yield a semistable [3Fe-4S](0) cluster intermediate.