FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.

Patellar tilt, congruence angle, and tibial tubercle-trochlear groove distance are correlated with positive J-sign in adolescents.

PURPOSE: The J-sign is a clinical evaluation tool that assesses for patellar maltracking and is considered positive if lateral translation of the patella in extension, in the pattern of an inverted J is observed. This study aims to determine the association of clinical J-sign with imaging features noted on dynamic kinematic computed tomography (DKCT). METHODS: A retrospective review was conducted by reviewing the clinical records of all patients aged 18 years or younger who had a CT patellar tracking scan done between 1 January 2005 to 31 December 2016 in a single institution. Patients who had the presence or absence of a 'J-sign' evaluated clinically were included. Radiographic parameters evaluated using the axial cuts include the patellar tilt angle, congruence angle, Dejour's classification, femoral sulcus angle, trochlear groove depth, and Wiberg's classification. Patients were then divided into two groups based on the presence or absence of J-sign on clinical examination. The radiographic measurements were then analyzed for association with the presence or absence of J-sign on clinical examination. RESULTS: Patients with a positive J-sign had an increased patellar tilt of 23.3° ± 14.2° and an increased congruence angle of 47.1° ± 28.5° when measured in extension as compared to a patellar tilt of 18.3° ± 10.8° and a congruence angle of 32.1° ± 20.8° in patients with a negative J-sign (p = 0.024 and 0.004, respectively). Comparisons of the change in congruence angles with the knee in full extension and at 20° flexion also yielded significantly higher change of 28.0° ± 20.4° in patients with a positive J-sign as compared to 11.9° ± 17.5° in patients with a negative J-sign. Patients with a positive J-sign also had an increased TT-TG distance of 17.6 ± 5.6 mm as compared to a TT-TG distance of 14.7 ± 6.9 mm in patients with a negative J-sign (p = 0.01). CONCLUSION: Patients with a positive J-sign had an increased patellar tilt and an increased congruence angle when measured in extension. Increased TT-TG distance was also significantly associated with positive J-sign. Patients with a positive J-sign also had a greater change in their congruence angle when measured with the knee in full extension and at 20° of flexion.

An In Crystallo Reaction with an Engineered Cytochrome P450 Peroxygenase.

The cytochrome P450 monooxygenases (CYPs) are a class of heme-thiolate enzymes that insert oxygen into unactivated C-H bonds. These enzymes can be converted into peroxygenases via protein engineering, which enables their activity to occur using hydrogen peroxide (H2 O2 ) without the requirement for additional nicotinamide co-factors or partner proteins. Here, we demonstrate that soaking crystals of an engineered P450 peroxygenase with H2 O2 enables the enzymatic reaction to occur within the crystal. Crystals of the designed P450 peroxygenase, the T252E mutant of CYP199A4, in complex with 4-methoxybenzoic acid were soaked with different concentrations of H2 O2 for varying times to initiate the in crystallo O-demethylation reaction. Crystal structures of T252E-CYP199A4 showed a distinct loss of electron density that was consistent with the O-demethylated metabolite, 4-hydroxybenzoic acid. A new X-ray crystal structure of this enzyme with the 4-hydroxybenzoic acid product was obtained to enable comparison alongside the existing substrate-bound structure. The visualisation of enzymatic catalysis in action is challenging in structural biology and the ability to initiate the reactions of P450 enzymes, in crystallo by simply soaking crystals with H2 O2 will enable new structural biology methods and techniques to be applied to study their mechanism of action.

A2AR eGFP reporter mouse enables elucidation of A2AR expression dynamics during anti-tumor immune responses.

There is significant clinical interest in targeting adenosine-mediated immunosuppression, with several small molecule inhibitors having been developed for targeting the A2AR receptor. Understanding of the mechanism by which A2AR is regulated has been hindered by difficulty in identifying the cell types that express A2AR due to a lack of robust antibodies for these receptors. To overcome this limitation, here an A2AR eGFP reporter mouse is developed, enabling the expression of A2AR during ongoing anti-tumor immune responses to be assessed. This reveals that A2AR is highly expressed on all tumor-infiltrating lymphocyte subsets including Natural Killer (NK) cells, NKT cells, γδ T cells, conventional CD4+ and CD8+ T lymphocytes and on a MHCIIhiCD86hi subset of type 2 conventional dendritic cells. In response to PD-L1 blockade, the emergence of PD-1+A2AR- cells correlates with successful therapeutic responses, whilst IL-18 is identified as a cytokine that potently upregulates A2AR and synergizes with A2AR deficiency to improve anti-tumor immunity. These studies provide insight into the biology of A2AR in the context of anti-tumor immunity and reveals potential combination immunotherapy approaches.

Characterisation of the heme aqua-ligand coordination environment in an engineered peroxygenase cytochrome P450 variant.

The cytochrome P450 enzymes (CYPs) are heme-thiolate monooxygenases that catalyse the insertion of an oxygen atom into the C-H bonds of organic molecules. In most CYPs, the activation of dioxygen by the heme is aided by an acid-alcohol pair of residues located in the I-helix of the enzyme. Mutation of the threonine residue of this acid-alcohol pair of CYP199A4, from the bacterium Rhodospeudomonas palustris HaA2, to a glutamate residue induces peroxygenase activity. In the X-ray crystal structures of this variant an interaction of the glutamate side chain and the distal aqua ligand of the heme was observed and this results in this ligand not being readily displaced in the peroxygenase mutant on the addition of substrate. Here we use a range of bulky hydrophobic and nitrogen donor containing ligands in an attempt to displace the distal aqua ligand of the T252E mutant of CYP199A4. Ligand binding was assessed by UV-visible absorbance spectroscopy, native mass spectrometry, electron paramagnetic resonance and X-ray crystallography. None of the ligands tested, even the nitrogen donor ligands which bind directly to the iron in the wild-type enzyme, resulted in displacement of the aqua ligand. Therefore, modification of the I-helix threonine residue to a glutamate residue results in a significant strengthening of the ferric distal aqua ligand. This ligand was not displaced using any of the ligands during this study and this provides a rationale as to why this mutant can shutdown the monooxygenase pathway of this enzyme and switch to peroxygenase activity.

Engineering C-C Bond Cleavage Activity into a P450 Monooxygenase Enzyme.

The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.

Selective carbon-hydrogen bond hydroxylation using an engineered cytochrome P450 peroxygenase.

The cytochrome P450 enzyme CYP102A1 (P450BM3) is a versatile monooxygenase enzyme which has been adapted and engineered for multiple applications in chemical synthesis. Mutation of threonine 268 to glutamate (Thr268Glu) converted the heme domain of this enzyme into a H2O2 utilizing peroxygenase. This variant displayed significantly increased peroxide driven hydroxylation activity towards the saturated linear fatty acids tested (undecanoic through to hexadecenoic acid) when compared to the wild-type heme domain. The product distributions arising from fatty acid oxidation using this peroxygenase variant were broadly similar to those obtained with the wild-type monooxygenase holoenzyme, with oxidation occurring predominantly at the ω-1 through to ω-3 positions. 10-Undecenoic acid was regioselectively hydroxylated at the allylic ω-2 carbon by the Thr268Glu peroxygenase. The effect of isotopic substitution were measured using [9,9,10,10-d4]-dodecanoic acid. The kinetic isotope effect for both the monooxygenase and peroxygenase systems ranged between 7.9 and 9.5, with that of the peroxygenase enzyme being marginally lower. This highlights that carbon­hydrogen bond abstraction is important in the mechanism of both the monooxygenase and peroxygenase systems. This would infer that the ferryl-oxo radical cation intermediate, compound I, is the likely reactive intermediate in both systems. The peroxygenase variant offers the possibility of simpler cytochrome P450 systems for selective oxidations. To demonstrate this we used this system to oxidize tetradecanoic acid using light driven generation of H2O2 by a flavin.

Exploring the Factors which Result in Cytochrome P450 Catalyzed Desaturation Versus Hydroxylation.

The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon-hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4-ethyl- and 4-isopropyl- benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4-cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4-n-propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cß carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4-n-butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4-isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cß position (41%). The oxidation of 4-n-propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cß and a significant increase in metabolites arising from desaturation (73%). The X-ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4-n-propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.

Synaptic memory survives molecular turnover.

Activation of Ca2+/calmodulin-dependent kinase II (CaMKII) plays a critical role in long-term potentiation (LTP), a long accepted cellular model for learning and memory. However, how LTP and memories survive the turnover of synaptic proteins, particularly CaMKII, remains a mystery. Here, we take advantage of the finding that constitutive Ca2+-independent CaMKII activity, acquired prior to slice preparation, provides a lasting memory trace at synapses. In slice culture, this persistent CaMKII activity, in the absence of Ca2+ stimulation, remains stable over a 2-wk period, well beyond the turnover of CaMKII protein. We propose that the nascent CaMKII protein present at 2 wk acquired its activity from preexisting active CaMKII molecules, which transferred their activity to newly synthesized CaMKII molecules and thus maintain the memory in the face of protein turnover.

Enabling Aromatic Hydroxylation in a Cytochrome P450 Monooxygenase Enzyme through Protein Engineering.

The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C-H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4-cyclohexylbenzoic acid but not the aromatic oxidation of 4-phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4-benzyl-, 4-phenoxy- and 4-benzoyl-benzoic acid and methoxy-substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4-benzylbenzoic acid. The methoxy-substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4-(3'-methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4-phenylbenzoic acid. 4-Phenoxy- and 4-benzoyl-benzoic acid bound with the ether or ketone oxygen atom hydrogen-bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4-phenylbenzoic acid; however the F182L mutant was able to catalyse 4-phenylbenzoic acid oxidation generating 2'-hydroxy-, 3'-hydroxy- and 4'-hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild-type enzyme, the phenyl ring of 4-phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.

Selective Oxidations Using a Cytochrome P450 Enzyme Variant Driven with Surrogate Oxygen Donors and Light.

Cytochrome P450 monooxygenase enzymes are versatile catalysts, which have been adapted for multiple applications in chemical synthesis. Mutation of a highly conserved active site threonine to a glutamate can convert these enzymes into peroxygenases that utilise hydrogen peroxide (H2 O2 ). Here, we use the T252E-CYP199A4 variant to study peroxide-driven oxidation activity by using H2 O2 and urea-hydrogen peroxide (UHP). We demonstrate that the T252E variant has a higher stability to H2 O2 in the presence of substrate that can undergo carbon-hydrogen abstraction. This peroxygenase variant could efficiently catalyse O-demethylation and an enantioselective epoxidation reaction (94 % ee). Neither the monooxygenase nor peroxygenase pathways of the P450 demonstrated a significant kinetic isotope effect (KIE) for the oxidation of deuterated substrates. These new peroxygenase variants offer the possibility of simpler cytochrome P450 systems for selective oxidations. To demonstrate this, a light driven H2 O2 generating system was used to support efficient product formation with this peroxygenase enzyme.

Synaptic memory requires CaMKII.

Long-term potentiation (LTP) is arguably the most compelling cellular model for learning and memory. While the mechanisms underlying the induction of LTP ('learning') are well understood, the maintenance of LTP ('memory') has remained contentious over the last 20 years. Here, we find that Ca2+-calmodulin-dependent kinase II (CaMKII) contributes to synaptic transmission and is required LTP maintenance. Acute inhibition of CaMKII erases LTP and transient inhibition of CaMKII enhances subsequent LTP. These findings strongly support the role of CaMKII as a molecular storage device.

How the brain stores information is a question that has fascinated neuroscientists for well over a century. Two general ideas have emerged. The first is that groups of neurons hold information by staying active. The second is that they hold information by strengthening their connections to one another, making it easier for them to work together in the future. Scientists call this second idea 'long-term potentiation'. One of the molecules involved in long-term potentiation is a protein called calcium-calmodulin-dependent kinase II, or CaMKII for short. Blocking CaMKII, or deleting its gene, stops the connections between neurons from becoming stronger. This suggests neurons need CaMKII to learn, but it remains unclear whether neurons also use CaMKII to maintain neuronal memories after they have been created. If CaMKII does play a role in maintaining memories, blocking it after learning should reverse the learning process, but so far, experiments have not been able to show this. Tao et al. revisited these experiments to find out more. They examined slices of brain tissue from mice that had been treated with fast-acting CaMKII inhibitors. It took tens of minutes, but the inhibitors were able to reverse long-term potentiation, both for newly acquired neuronal memories and for older memories that had formed when the mice were alive. The choice of CaMKII inhibitor and the time lag could explain why scientists have not observed the effect before. Understanding long-term potentiation is a fundamental part of understanding learning and memory. It could also reveal more about the opposite phenomenon: long-term depression. This is a type of learning where the connections between neurons become weaker. Long-term depression also takes tens of minutes to occur, suggesting that future research into CaMKII might shed light on how it works.

The Stereoselective Oxidation of para-Substituted Benzenes by a Cytochrome P450 Biocatalyst.

The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60 nmol.(nmol-CYP)-1 .min-1 ) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.

Advanced Practice Providers in Plastic Surgery.

BACKGROUND: The aims of this article are to examine the scope of practice differences between physician assistant and nurse practitioner providers, to identify financial cost and benefits, and to posit the impact of physician extenders on plastic surgery practices. METHODS: A review of the literature was performed using the PubMed database. Key words included "plastic surgery AND physician extender AND cost," "plastic surgery AND physician assistant AND cost," and "plastic surgery AND nurse practitioner AND cost." Secondarily, a search was performed for plastic surgery-related specialties of maxillofacial surgery, orthopedic surgery, and otolaryngology. Inclusion criteria consisted of any study design measuring the financial benefits associated with integrating physician extenders. RESULTS: The PubMed search yielded 91 articles. Eight articles were ultimately included, of which four (plastic, maxillofacial, and orthopedic) discussed the impact of physician assistants and four (orthopedic and otolaryngology) discussed the impact of nurse practitioners. All eight studies demonstrated that integration of physician assistants and nurse practitioners into practices was associated with a net financial gain even after taking into account their overall costs, along with other outcomes such as productivity or time involvement. CONCLUSIONS: As the number of physician extenders continues to grow, especially in subspecialties, plastic surgeons should be aware of their roles and the potentially positive impact of these providers, their respective training, and their quantifiable financial impact toward a plastic surgery practice. Both physician assistants and nurse practitioners appear to have a positive effect on costs in plastic surgery and plastic surgery-related practices.

The First Descriptions of Dorsal Preservation Rhinoplasty in the 19th and Early- to Mid-20th Centuries and Relevance Today.

INTRODUCTION: The basis of dorsal preservation rhinoplasty goes back to the late 19th and the early 20th centuries. In that era, pioneers such as Drs. Goodale, Lothrop, and Cottle were prominent surgeons who reported on this technique. Currently, there has been a renewed interest of this technique that stems from the nasal anatomy and an interest in less destructive techniques. In this review, we discuss examples of the contributions of those surgeons, which represent some of the earliest experiences in this field. METHODS: We reviewed several journals from the late 19th and early 20th centuries as detailed in the references section. We collected the related publications on closed reduction techniques performed by Drs. Goodale, Lothrop, and Cottle. RESULTS: The publications on closed reduction techniques by Drs. Goodale, Lothrop, and Cottle described similar thought processes and techniques comparable to current dorsal preservation rhinoplasty techniques. The thought processes of these 3 renowned rhinoplasty surgeons appear to be very much relevant today. CONCLUSIONS: Although there has been recent resurgence in dorsal preservation rhinoplasty techniques due to anatomical and functional aspects of the nose, the basis of dorsal preservation rhinoplasty goes far back to more than 100 years ago.

A preliminary study of resting brain metabolism in treatment-resistant depression before and after treatment with olanzapine-fluoxetine combination.

Treatment-resistant depression (TRD) occurs in many patients and causes high morbidity and mortality. Because TRD subjects are particularly difficult to study especially longitudinally, biological data remain very limited. In a preliminary study to judge feasibility and power, 25 TRD patients were referred from specialty psychiatric practices. All were severely and chronically depressed and mostly had comorbid psychiatric disorders as is typical in TRD. Nine patients were able to complete all required components of the protocol that included diagnostic interview; rating scales; clinical magnetic resonance imaging; medication washout; treatment with maximally tolerated olanzapine-fluoxetine combination for 8 weeks; and pre- and post-treatment fluorodeoxyglucose positron emission tomography. This drug combination is an accepted standard of treatment for TRD. Dropouts arose from worsening depression, insomnia, and anxiety. One patient remitted; three responded. A priori regions of interest included the amygdala and subgenual cingulate cortex (sgACC; Brodmann area BA25). Responders showed decreased metabolism with treatment in the right amygdala that correlated with clinical response; no significant changes in BA25; better response to treatment the higher the baseline BA25 metabolism; and decreased right ventromedial prefrontal metabolism (VMPFC; broader than BA25) with treatment which did not correlate with depression scores. The baseline metabolism of all individuals showed heterogeneous patterns when compared to a normative metabolic database. Although preliminary given the sample size, this study highlights several issues important for future work: marked dropout rate in this study design; need for large sample size for adequate power; baseline metabolic heterogeneity of TRD requiring careful subject characterization for future studies of interventions; relationship of amygdala activity decreases with response; and the relationship between baseline sgACC and VMPFC activity with response. Successful treatment of TRD with olanzapine-fluoxetine combination shows changes in cerebral metabolism like those seen in treatment-responsive major depression.

Aging-Related Hypometabolism in the Anterior Cingulate Cortex of Cognitively Intact, Amyloid-Negative Seniors at Rest Mediates the Relationship between Age and Executive Function but Not Memory.

The anterior cingulate cortex (ACC) shows the most aging-related brain metabolic dysfunction that correlates with decreasing executive processing in otherwise healthy, cognitively intact volunteers. Here, data from ADNI are used to elucidate potential pathophysiological mechanisms involved in cognitive aging, that is, age-related decline in cognitive performance in the absence of known neurodegenerative disease. Amyloid-negative volunteers showed statistically significant mediation of ACC metabolism in the relationship between age and verbal fluency. A nonlinguistic task of executive function, Trails B, showed also negative correlation between performance and age, albeit weaker, but was not significant in the mediation analysis. Recall of story items, minimizing attentional demands compared with learning of word lists, did not correlate with age. ADNI subjects selected for low vascular risks also showed correlation between age and declining ACC metabolism. In the whole-brain amyloid-negative subset, ACC amyloid was not correlated with age. As expected, the metabolism in an arbitrary region such as motor cortex that was not expected to decline with cognitive aging showed no correlation with age or ACC metabolism suggesting regional specificity. These findings motivate the search for the pathophysiology of aging-related ACC dysfunction to prevent, diagnose, and treat the decline in executive function associated with cognitive aging.

Empowerment and HIV Risk Behaviors in Couples: Modeling the Theory of Gender and Power in an African Context.

Background: Young women and girls in Eastern and Southern Africa are at elevated risk of acquiring human immunodeficiency virus (HIV) compared with men, largely due to power dynamics within heterosexual relationships that contribute to HIV risk behaviors. Few studies employ a comprehensive framework to examine divisions between men and women and HIV risk behaviors in an African context. Thus, we examined associations between levels of women's empowerment and HIV risk behaviors applying the Theory of Gender and Power. Methods: We used logistic regression (adjusted odds ratios or AORs) to assess associations between women's empowerment indicators and HIV risk behaviors (multiple sexual partners) and self-efficacy (ability to negotiate sex/sex refusal) with couples data (n = 12,670) from Malawi, Namibia, Zambia, and Zimbabwe. Results: Specifically, key drivers of high levels of empowerment among women were household decision-making involvement, female economic independence, and rejecting all reasons for wife-beating. Furthermore, higher levels of women's empowerment in coupled relationships was associated with safer sex negotiation in Malawi (AOR = 1.57, p < 0.05) and Zambia (AOR = 1.60, p < 0.0001) and sex refusal in Malawi (AOR = 1.62, p < 0.0001) and Zimbabwe (AOR = 1.29, p < 0.05). However, empowerment was not associated with the likelihood of the male partner having multiple sexual partners across all countries studied. Conclusions: These findings provide evidence that high levels of women's empowerment were associated with safer sex practices, although this varied by country. Policymakers should incorporate empowerment indicators to address women's empowerment and HIV prevention within African couples.

Immunotherapeutic Challenges for Pediatric Cancers.

Solid tumors contain a mixture of malignant cells and non-malignant infiltrating cells that often create a chronic inflammatory and immunosuppressive microenvironment that restricts immunotherapeutic approaches. Although childhood and adult cancers share some similarities related to microenvironmental changes, pediatric cancers are unique, and adult cancer practices may not be wholly applicable to our pediatric patients. This review highlights the differences in tumorigenesis, viral infection, and immunologic response between children and adults that need to be considered when trying to apply experiences from experimental therapies in adult cancer patients to pediatric cancers.

STING Restricts oHSV Replication and Spread in Resistant MPNSTs but Is Dispensable for Basal IFN-Stimulated Gene Upregulation.

Malignant peripheral nerve sheath tumors (MPNSTs) are an aggressive soft-tissue sarcoma amenable only to surgical resection. Oncolytic herpes simplex viruses (oHSVs) are a promising experimental therapy. We previously showed that basal interferon (IFN) and nuclear factor κB (NFκB) signaling upregulate IFN-stimulated gene (ISG) expression and restrict efficient viral infection and cell-to-cell spread in ∼50% of tested MPNSTs. Stimulator of Interferon Genes (STING) integrates DNA sensor activity and mediates downstream IFN signaling in infected cells. We sought to identify STING's role in oHSV resistance and contribution to basal ISG upregulation in MPNSTs. We show that the level of STING activity in human MPNST cell lines is predictive of oHSV sensitivity and that resistant cell lines have intact mechanisms for detection of cytosolic double-stranded DNA (dsDNA). Furthermore, we show that STING downregulation renders MPNSTs more permissive to oHSV infection and cell-to-cell spread. While next-generation viruses can exploit this loss of STING activity, first-generation viruses remain restricted. Finally, STING is not integral to the previously-observed basal ISG upregulation, indicating that other pathways contribute to basal IFN signaling in resistant MPNSTs. These data broaden our understanding of the intrinsic pathways in MPNSTs and their role in oHSV resistance and offer potential targets to potentiate oncolytic virus activity.