Outpatient Virtual Visits and the "Right" Amount of Telehealth Going Forward.

Background: An exponential increase in outpatient telehealth visits occurred early in the pandemic period that has been followed by volumes that, although lower than peak numbers, are substantially greater than the pre-pandemic period. This provided an opportunity to assess provider perceptions regarding the right prevalence going forward and key obstacles to achieving it. Methods: A 10-question survey was distributed to all outpatient providers within the Dartmouth-Hitchcock Health System. Domains included practice location, specialty, professional degree, experience with telehealth, satisfaction, perception of the amount of telehealth that could be adequately delivered going forward, role of audio-only, and obstacles. Results: Three hundred thirty-six providers completed the survey representing 51 specialties. The most common response regarding the proportion of outpatient visits that could be delivered by video going forward was 21-50% (n = 104) followed by 6-20% (n = 99) and >50% (n = 71). A minority of respondents chose ≤5% (n = 17). In terms of the fraction of video visits for which phone was equally effective, a similar percentage of respondents felt that it was 1/10 (22%), 1/4 (20%), or 1/2 (26%) of visits. Fewer felt that all (7%) or 3/4 (15%) of visits were equally effective, and 10% felt that it was none. Common obstacles identified were the need for a physical exam, unique aspects of providers' patients, patient preference, and issues regarding technology and internet speed/connectivity. Conclusions: After a period of exponential growth in virtual visits due to the pandemic, outpatient providers within an academic health system felt that a substantial portion of future visits could be delivered by this modality.

United Kingdom recommendations for obstetric venous thromboembolism prophylaxis: Evidence and rationale.

Venous thromboembolism (VTE) is a leading cause of maternal death in the United Kingdom. To address this problem guidance from the Royal College of Obstetricians and Gynaecologists (RCOG) has been developed that recommends the assessment of a woman's risk of thrombosis at specific time-points during pregnancy and postnatally at the time of delivery. The RCOG guidelines provide clinicians with a framework to inform decision-making on the use of thromboprophylaxis and are based on the premise that the higher risk a woman has for VTE, the more likely she is to benefit from prophylaxis - determining her level of risk is based on the number and characteristics of the risk factors that she has. This article will address the pathophysiology of VTE in pregnancy, evidence behind the risk factors for VTE and the use of thromboprophylactic agents. Further, it will reflect on the rationale behind the RCOG guidance.

Clonidine restores vascular endothelial growth factor expression and improves tissue repair following severe trauma.

BACKGROUND: We hypothesized that clonidine and propranolol would increase VEGF and VEGF-receptor expression and promote lung healing following severe trauma and chronic stress. METHODS: Sprague-Dawley rats were subjected to lung contusion (LC), lung contusion/hemorrhagic shock (LCHS), or lung contusion/hemorrhagic shock/daily restraint stress (LCHS/CS). Clonidine and propranolol were administered daily. On day seven, lung VEGF, VEGFR-1, VEGFR-2, and HMGB1 were assessed by PCR. Lung injury was assessed by light microscopy (*p < 0.05). RESULTS: Clonidine increased VEGF expression following LCHS (43%*) and LCHS/CS (46%*). Clonidine increased VEGFR-1 and R-2 expression following LCHS/CS (203%* and 47%*, respectively). Clonidine decreased HMGB1 and TNF-alpha expression following LCHS/CS (22%* and 58%*, respectively.) Clonidine decreased inflammatory cell infiltration and total Lung Injury Score following LCHS/CS. Propranolol minimally affected VEGF and did not improve lung healing. CONCLUSIONS: Clonidine increased VEGF and VEGF-receptor expression, decreased HMGB1 expression, decreased lung inflammation, and improved lung tissue repair.

Effects of trauma, hemorrhagic shock, and chronic stress on lung vascular endothelial growth factor.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2) regulate vascular permeability and endothelial cell survival. We hypothesized that hemorrhagic shock (HS) and chronic stress (CS) would increase expression of lung VEGF and its receptors, potentiating pulmonary edema in lung tissue. MATERIALS AND METHODS: Male Sprague-Dawley rats aged 8-9 wk were randomized: naïve control, lung contusion (LC), LC followed by HS (LCHS), and LCHS with CS in a restraint cylinder for 2 h/d (LCHS/CS). Animals were sacrificed on days 1 and 7. Expressions of lung VEGF, VEGFR-1, and VEGFR-2 were determined by polymerase chain reaction. Lung Injury Score (LIS) was graded on light microscopy by inflammatory cell counts, interstitial edema, pulmonary edema, and alveolar integrity (range: 0 = normal; 8 = severe injury). RESULTS: Seven days after LC, lung VEGF and VEGFR-1 were increased, and lung tissue healed (LIS: 0.8 ± 0.8). However, 7 d after LCHS and LCHS/CS, lung VEGF and VEGFR-1 expressions were decreased. VEGFR-2 was also decreased after LCHS/CS. LIS was elevated 7 d after LCHS and LCHS/CS (6.5 ± 1.0 and 8.2 ± 0.8). Increased LIS after LCHS and LCHS/CS was because of higher inflammatory cell counts, increased interstitial edema, and loss of alveolar integrity, whereas pulmonary edema was unchanged. CONCLUSIONS: Elevation of lung VEGF and VEGFR-1 expressions after LC alone was associated with healing of injured lung tissue. Expressions of VEGF, VEGFR-1, and VEGFR-2 were reduced after LCHS and LCHS/CS, and injured lung tissue did not heal. Persistent lung injury after severe trauma was because of inflammation rather than pulmonary edema.

Mass spectrometric identification of intermediates in the O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion in FNR.

The iron-sulfur cluster containing protein Fumarate and Nitrate Reduction (FNR) is the master regulator for the switch between anaerobic and aerobic respiration in Escherichia coli and many other bacteria. The [4Fe-4S] cluster functions as the sensory module, undergoing reaction with O2 that leads to conversion to a [2Fe-2S] form with loss of high-affinity DNA binding. Here, we report studies of the FNR cluster conversion reaction using time-resolved electrospray ionization mass spectrometry. The data provide insight into the reaction, permitting the detection of cluster conversion intermediates and products, including a [3Fe-3S] cluster and persulfide-coordinated [2Fe-2S] clusters [[2Fe-2S](S) n , where n = 1 or 2]. Analysis of kinetic data revealed a branched mechanism in which cluster sulfide oxidation occurs in parallel with cluster conversion and not as a subsequent, secondary reaction to generate [2Fe-2S](S) n species. This methodology shows great potential for broad application to studies of protein cofactor-small molecule interactions.

A combined EPR and MD simulation study of a nitroxyl spin label with restricted internal mobility sensitive to protein dynamics.

EPR studies combined with fully atomistic Molecular Dynamics (MD) simulations and an MD-EPR simulation method provide evidence for intrinsic low rotameric mobility of a nitroxyl spin label, Rn, compared to the more widely employed label MTSL (R1). Both experimental and modelling results using two structurally different sites of attachment to Myoglobin show that the EPR spectra of Rn are more sensitive to the local protein environment than that of MTSL. This study reveals the potential of using the Rn spin label as a reporter of protein motions.

Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes.

The reaction of protein-bound iron-sulfur (Fe-S) clusters with nitric oxide (NO) plays key roles in NO-mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe-S clusters has been hampered by a lack of information about the nature of the iron-nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe-4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe2 (NO)4 (Cys)2 ]) and Roussin's Black Salt (RBS, [Fe4 (NO)7 S3 ]. In the latter case, the absence of 32 S/34 S shifts in the Fe-S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.

Differentiated, Promoter-specific Response of [4Fe-4S] NsrR DNA Binding to Reaction with Nitric Oxide.

NsrR is an iron-sulfur cluster protein that regulates the nitric oxide (NO) stress response of many bacteria. NsrR from Streptomyces coelicolor regulates its own expression and that of only two other genes, hmpA1 and hmpA2, which encode HmpA enzymes predicted to detoxify NO. NsrR binds promoter DNA with high affinity only when coordinating a [4Fe-4S] cluster. Here we show that reaction of [4Fe-4S] NsrR with NO affects DNA binding differently depending on the gene promoter. Binding to the hmpA2 promoter was abolished at ∼2 NO per cluster, although for the hmpA1 and nsrR promoters, ∼4 and ∼8 NO molecules, respectively, were required to abolish DNA binding. Spectroscopic and kinetic studies of the NO reaction revealed a rapid, multi-phase, non-concerted process involving up to 8-10 NO molecules per cluster, leading to the formation of several iron-nitrosyl species. A distinct intermediate was observed at ∼2 NO per cluster, along with two further intermediates at ∼4 and ∼6 NO. The NsrR nitrosylation reaction was not significantly affected by DNA binding. These results show that NsrR regulates different promoters in response to different concentrations of NO. Spectroscopic evidence indicates that this is achieved by different NO-FeS complexes.

Biochemical properties of Paracoccus denitrificans FnrP: reactions with molecular oxygen and nitric oxide.

In Paracoccus denitrificans, three CRP/FNR family regulatory proteins, NarR, NnrR and FnrP, control the switch between aerobic and anaerobic (denitrification) respiration. FnrP is a [4Fe-4S] cluster-containing homologue of the archetypal O2 sensor FNR from E. coli and accordingly regulates genes encoding aerobic and anaerobic respiratory enzymes in response to O2, and also NO, availability. Here we show that FnrP undergoes O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion that involves up to 2 O2 per cluster, with significant oxidation of released cluster sulfide to sulfane observed at higher O2 concentrations. The rate of the cluster reaction was found to be ~sixfold lower than that of E. coli FNR, suggesting that FnrP can remain transcriptionally active under microaerobic conditions. This is consistent with a role for FnrP in activating expression of the high O2 affinity cytochrome c oxidase under microaerobic conditions. Cluster conversion resulted in dissociation of the transcriptionally active FnrP dimer into monomers. Therefore, along with E. coli FNR, FnrP belongs to the subset of FNR proteins in which cluster type is correlated with association state. Interestingly, two key charged residues, Arg140 and Asp154, that have been shown to play key roles in the monomer-dimer equilibrium in E. coli FNR are not conserved in FnrP, indicating that different protomer interactions are important for this equilibrium. Finally, the FnrP [4Fe-4S] cluster is shown to undergo reaction with multiple NO molecules, resulting in iron nitrosyl species and dissociation into monomers.

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2.

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2.

NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function.

The Rrf2 family transcription factor NsrR controls expression of genes in a wide range of bacteria in response to nitric oxide (NO). The precise form of the NO-sensing module of NsrR is the subject of controversy because NsrR proteins containing either [2Fe-2S] or [4Fe-4S] clusters have been observed previously. Optical, Mössbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that Streptomyces coelicolor NsrR (ScNsrR), previously reported to contain a [2Fe-2S] cluster, can be isolated containing a [4Fe-4S] cluster. ChIP-seq experiments indicated that the ScNsrR regulon is small, consisting of only hmpA1, hmpA2, and nsrR itself. The hmpA genes encode NO-detoxifying flavohemoglobins, indicating that ScNsrR has a specialized regulatory function focused on NO detoxification and is not a global regulator like some NsrR orthologues. EMSAs and DNase I footprinting showed that the [4Fe-4S] form of ScNsrR binds specifically and tightly to an 11-bp inverted repeat sequence in the promoter regions of the identified target genes and that DNA binding is abolished following reaction with NO. Resonance Raman data were consistent with cluster coordination by three Cys residues and one oxygen-containing residue, and analysis of ScNsrR variants suggested that highly conserved Glu-85 may be the fourth ligand. Finally, we demonstrate that some low molecular weight thiols, but importantly not physiologically relevant thiols, such as cysteine and an analogue of mycothiol, bind weakly to the [4Fe-4S] cluster, and exposure of this bound form to O2 results in cluster conversion to the [2Fe-2S] form, which does not bind to DNA. These data help to account for the observation of [2Fe-2S] forms of NsrR.

Iron-sulfur clusters as biological sensors: the chemistry of reactions with molecular oxygen and nitric oxide.

Iron-sulfur cluster proteins exhibit a range of physicochemical properties that underpin their functional diversity in biology, which includes roles in electron transfer, catalysis, and gene regulation. Transcriptional regulators that utilize iron-sulfur clusters are a growing group that exploit the redox and coordination properties of the clusters to act as sensors of environmental conditions including O2, oxidative and nitrosative stress, and metabolic nutritional status. To understand the mechanism by which a cluster detects such analytes and then generates modulation of DNA-binding affinity, we have undertaken a combined strategy of in vivo and in vitro studies of a range of regulators. In vitro studies of iron-sulfur cluster proteins are particularly challenging because of the inherent reactivity and fragility of the cluster, often necessitating strict anaerobic conditions for all manipulations. Nevertheless, and as discussed in this Account, significant progress has been made over the past decade in studies of O2-sensing by the fumarate and nitrate reduction (FNR) regulator and, more recently, nitric oxide (NO)-sensing by WhiB-like (Wbl) and FNR proteins. Escherichia coli FNR binds a [4Fe-4S] cluster under anaerobic conditions leading to a DNA-binding dimeric form. Exposure to O2 converts the cluster to a [2Fe-2S] form, leading to protein monomerization and hence loss of DNA binding ability. Spectroscopic and kinetic studies have shown that the conversion proceeds via at least two steps and involves a [3Fe-4S](1+) intermediate. The second step involves the release of two bridging sulfide ions from the cluster that, unusually, are not released into solution but rather undergo oxidation to sulfane (S(0)) subsequently forming cysteine persulfides that then coordinate the [2Fe-2S] cluster. Studies of other [4Fe-4S] cluster proteins that undergo oxidative cluster conversion indicate that persulfide formation and coordination may be more common than previously recognized. This remarkable feature suggested that the original [4Fe-4S] cluster can be restored using persulfide as the source of sulfide ion. We have demonstrated that only iron and a source of electrons are required to promote efficient conversion back from the [2Fe-2S] to the [4Fe-4S] form. We propose this as a novel in vivo repair mechanism that does not require the intervention of an iron-sulfur cluster biogenesis pathway. A number of iron-sulfur regulators have evolved to function as sensors of NO. Although it has long been known that the iron-sulfur clusters of many phylogenetically unrelated proteins are vulnerable to attack by NO, our recent studies of Wbl proteins and FNR have provided new insights into the mechanism of cluster nitrosylation, which overturn the commonly accepted view that the product is solely a mononuclear iron dinitrosyl complex (known as a DNIC). The major reaction is a rapid, multiphase process involving stepwise addition of up to eight NO molecules per [4Fe-4S] cluster. The major iron nitrosyl product is EPR silent and has optical characteristics similar to Roussin's red ester, [Fe2(NO)4(RS)2] (RRE), although a species similar to Roussin's black salt, [Fe4(NO)7(S)3](-) (RBS) cannot be ruled out. A major future challenge will be to clarify the nature of these species.

Influence of association state and DNA binding on the O2-reactivity of [4Fe-4S] fumarate and nitrate reduction (FNR) regulator.

The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2.

The central cavity of ABCB1 undergoes alternating access during ATP hydrolysis.

Understanding the process that underlies multidrug recognition and efflux by P-glycoprotein (ABCB1) remains a key biological challenge. Structural data have recently become available for the murine and Caenorhabditis elegans homologues of ABCB1; however all structures were obtained in the absence of nucleotide. A feature of these structures was the presence of a central cavity that is inaccessible from the extracellular face of the protein. To determine the conformational dynamics of this region several residues in transmembrane helices TM6 (331, 343 and 354) and TM12 (980) were mutated to cysteine. Based upon structural predictions, these residues are proposed to line, or reside proximal to, the central cavity. The mutant isoforms were labelled with a paramagnetic probe enabling the application of EPR spectroscopic methods. Power saturation EPR spectra were recorded in the presence of hydrophobic (O2 ) or hydrophilic (NiEDDA) quenching agents to study the local environment of each residue. ABCB1 was trapped in both its nucleotide-bound and post-hydrolytic conformations and EPR spectra were again recorded in the presence and absence of quenching agents. The EPR line shapes provide information on the movements of these residues within TM6 and TM12 during ATP hydrolysis. Rationalization of the data with molecular dynamic simulations indicates that the cavity is converted to a configuration open to the aqueous phase following nucleotide binding, thereby suggesting alternating access to the cavity on opposite sides of the membrane during translocation.

Techniques for the production, isolation, and analysis of iron-sulfur proteins.

Iron-sulfur clusters constitute a group of cofactors found in many proteins that play key roles in an exceptionally wide range of metabolic processes. The chemical reactivity of iron-sulfur clusters means that they can be particularly prone to damage when removed from the protective environment of the cell. In general, the key to obtaining an intact, biologically active iron-sulfur cluster-containing protein is to maintain a strictly anaerobic environment throughout the entire process of protein purification and analysis. For many proteins, particularly those with more labile clusters, it is essential.

Mechanism of [4Fe-4S](Cys)4 cluster nitrosylation is conserved among NO-responsive regulators.

The Fumarate nitrate reduction (FNR) regulator from Escherichia coli controls expression of >300 genes in response to O2 through reaction with its [4Fe-4S] cluster cofactor. FNR is the master switch for the transition between anaerobic and aerobic respiration. In response to physiological concentrations of nitric oxide (NO), FNR also regulates genes, including the nitrate reductase (nar) operon, a major source of endogenous cellular NO, and hmp, which encodes an NO-detoxifying enzyme. Here we show that the [4Fe-4S] cluster of FNR reacts rapidly in a multiphasic reaction with eight NO molecules. Oxidation of cluster sulfide ions (S(2-)) to sulfane (S(0)) occurs, some of which remains associated with the protein as Cys persulfide. The nitrosylation products are similar to a pair of dinuclear dinitrosyl iron complexes, [Fe(I)2(NO)4(Cys)2](0), known as Roussin's red ester. A similar reactivity with NO was reported for the Wbl family of [4Fe-4S]-containing proteins found only in actinomycetes, such as Streptomyces and Mycobacteria. These results show that NO reacts via a common mechanism with [4Fe-4S] clusters in phylogenetically unrelated regulatory proteins that, although coordinated by four Cys residues, have different cluster environments. The reactivity of E. coli FNR toward NO, in addition to its sensitivity toward O2, is part of a hierarchal network that monitors, and responds to, NO, both endogenously generated and exogenously derived.

Unexpected weak magnetic exchange coupling between haem and non-haem iron in the catalytic site of nitric oxide reductase (NorBC) from Paracoccus denitrificans1.

Bacterial NOR (nitric oxide reductase) is a major source of the powerful greenhouse gas N2O. NorBC from Paracoccus denitrificans is a heterodimeric multi-haem transmembrane complex. The active site, in NorB, comprises high-spin haem b3 in close proximity with non-haem iron, FeB. In oxidized NorBC, the active site is EPR-silent owing to exchange coupling between FeIII haem b3 and FeBIII (both S=5/2). On the basis of resonance Raman studies [Moënne-Loccoz, Richter, Huang, Wasser, Ghiladi, Karlin and de Vries (2000) J. Am. Chem. Soc. 122, 9344-9345], it has been assumed that the coupling is mediated by an oxo-bridge and subsequent studies have been interpreted on the basis of this model. In the present study we report a VFVT (variable-field variable-temperature) MCD (magnetic circular dichroism) study that determines an isotropic value of J=-1.7 cm-1 for the coupling. This is two orders of magnitude smaller than that encountered for oxo-bridged diferric systems, thus ruling out this configuration. Instead, it is proposed that weak coupling is mediated by a conserved glutamate residue.