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Supplementary Data from Protein Expression Profiling in Esophageal Adenocarcinoma Patients Indicates Association of Heat-Shock Protein 27 Expression and Chemotherapy Response

Purpose: To identify pretherapeutic predictive biomarkers in tumor biopsies of patients with locally advanced esophageal adenocarcinomas treated with neoadjuvant chemotherapy, we used an explorative proteomic approach to correlate pretherapeutic protein expression profiles with tumor response to neoadjuvant chemotherapy.Experimental Design: Thirty-four patients with locally advanced esophageal adenocarcinomas who received neoadjuvant platin/5-fluorouracil–based chemotherapy before surgical resection were enrolled in this study. Response to chemotherapy was determined (a) by the amount of decline of [18F]fluorodeoxyglucose tumor uptake 2 weeks after the start of chemotherapy measured by positron emission tomography and (b) by histopathologic evaluation of tumor regression after surgical resection. Explorative quantitative and qualitative protein expression analysis was done through a quantitative differential protein expression analysis that used dual-isotope radioactive labeling of protein extracts. Selected identified biomarkers were validated by immunohistochemistry and quantitative real time reverse transcription-PCR.Results: Proteomic analysis revealed four cellular stress response–associated proteins [heat-shock protein (HSP) 27, HSP60, glucose-regulated protein (GRP) 94, GRP78] and a number of cytoskeletal proteins whose pretherapeutic abundance was significantly different (P < 0.001) between responders and nonresponders. Immunohistochemistry and gene expression analysis confirmed these data, showing a significant association between low HSP27 expression and nonresponse to neoadjuvant chemotherapy (P = 0.049 and P = 0.032, respectively).Conclusions: Albeit preliminary, our encouraging data suggest that protein expression profiling may distinguish cancers with a different response to chemotherapy. Our results suggest that response to chemotherapy may be related to a different activation of stress response and inflammatory biology in general. Moreover, the potential of HSPs and GRPs as biomarkers of chemotherapy response warrants further validation.

Sigma-2 receptor (S2R) is a S2R ligand-binding site historically associated with reportedly 21.5 kDa proteins that have been linked to several diseases, such as cancer, Alzheimer’s disease, and schizophrenia. The S2R is highly expressed in various tumors, where it correlates with the proliferative status of the malignant cells. Recently, S2R was reported to be the transmembrane protein TMEM97. Prior to that, we had been investigating the translocator protein (TSPO) as a potential 21.5 kDa S2R candidate protein with reported heme and sterol associations. Here, we investigate the contributions of TMEM97 and TSPO to S2R activity in MCF7 breast adenocarcinoma and MIA PaCa-2 (MP) pancreatic carcinoma cells. Additionally, the role of the reported S2R-interacting partner PGRMC1 was also elucidated. Proximity ligation assays and co-immunoprecipitation show a functional association between S2R and TSPO. Moreover, a close physical colocalization of TMEM97 and TSPO was found in MP cells. In MCF7 cells, co-immunoprecipitation only occurred with TMEM97 but not with PGRMC1, which was further confirmed by confocal microscopy experiments. Treatment with the TMEM97 ligand 20-(S)-hydroxycholesterol reduced co-immunoprecipitation of both TMEM97 and PGRMC1 in immune pellets of immunoprecipitated TSPO in MP cells. To the best of our knowledge, this is the first suggestion of a (functional) interaction between TSPO and TMEM97 that can be affected by S2R ligands.

BackgroundUtilization of sacubitril/valsartan is increasing as a component of guideline-directed medical therapy in patients with heart failure with reduced ejection fraction (HFrEF). Common adverse effects associated with the medication such as hypotension and hyperkalaemia have been described; however, hyponatraemia is very rarely reported to have a potential association with use of the medication. In this report, we describe what we believe to be the first reported case of acute hyponatraemia likely attributable to inpatient initiation of sacubitril/valsartan.Case SummaryA 71-year-old female presented with 2 weeks of progressively worsening dyspnoea and orthopnoea. Bedside echocardiography identified a dilated cardiomyopathy with an estimated left ventricular ejection fraction DiscussionAngiotensin receptor–neprilysin inhibitors are an integral component of guideline-directed medical therapy with proven benefits for patients with heart failure with reduced ejection fraction. Although the association between use of these medications and hyponatraemia appears to be exceedingly rare, clinicians should maintain awareness of this potential adverse effect.

Sleep apnea (SA) during pregnancy is detrimental to the health of the pregnancy and neonate, but little is known regarding long-lasting consequences of maternal SA during pregnancy on adult offspring. SA is characterized by repeated cessations in breathing during sleep, resulting in intermittent hypoxia (IH). We show that gestational IH (GIH) in rats reprograms the male fetal neuroimmune system toward enhanced inflammation in a region- and sex-specific manner, which persists into adulthood. Male GIH offspring also had deficits in the neural control of breathing, specifically in the ability to mount compensatory responses to central apnea, an effect that was rescued by a localized anti-inflammatory or microglial depletion. Female GIH offspring appeared unaffected. These results indicate that SA during pregnancy sex- and region-dependently skews offspring microglia toward a pro-inflammatory phenotype, which leads to long-lasting deficits in the capacity to elicit important forms of respiratory neuroplasticity in response to breathing instability. These studies contribute to the growing body of recent evidence indicating that SA during pregnancy may lead to sex-specific neurological deficits in offspring that persist into adulthood.

Right heart failure (RHF) is a common, yet difficult to manage, complication of severe acute respiratory distress syndrome requiring extracorporeal membrane oxygenation (ECMO) that is associated with increased mortality. Reports of the use of percutaneous mechanical circulatory support devices for concurrent right heart and respiratory failure are limited. This series describes the percutaneous cannulation of the pulmonary artery for conversion from veno-venous to veno-pulmonary artery return ECMO in 21 patients who developed secondary RHF. All patients cannulated between May 2019 and September 2021 were included. Either a 19 or 21 French venous cannula was placed percutaneously into the pulmonary artery via the internal jugular or subclavian vein, providing a total of 821 days of support (median 23 [4-71] days per patient) with flows up to 6 L/min. Five patients underwent cannulation at the bedside, with the remainder performed in the cardiac catheterization laboratory. Pulmonary artery cannulation occurred after 12 [8.5-23.5] days of ECMO support. Vasoactive infusion requirements decreased significantly within 24 hours of pulmonary artery cannula placement (p = 0.0004). Nonetheless, 75% of these patients expired after a median of 12 [4-63] days of support, with three patients found to have had significant pericardial effusions peri-arrest. This cannulation technique may be an effective alternative to veno-arterial ECMO cannulation or the placement of a dual-lumen cannula for the treatment of RHF.

Previous studies have found that students' concept-building approaches, identified a priori with a cognitive psychology laboratory task, are associated with student exam performances in chemistry classes. Abstraction learners (those who extract the principles underlying related examples) performed better than exemplar learners (those who focus on memorizing the training exemplars and responses) on transfer exam questions but not retention questions, after accounting for general ability. We extended these findings to introductory biology courses in which active-learning techniques were used to try to foster deep conceptual learning. Exams were constructed to contain both transfer and retention questions. Abstraction learners demonstrated better performance than exemplar learners on the transfer questions but not on the retention questions. These results were not moderated by indices of crystallized or fluid intelligence. Our central interpretation is that students identified as abstraction learners appear to construct a deep understanding of the concepts (presumably based on abstract underpinnings), thereby enabling them to apply and generalize the concepts to scenarios and instantiations not seen during instruction (transfer questions). By contrast, other students appear to base their representations on memorized instructed examples, leading to good performance on retention questions but not transfer questions.

Purpose To investigate the injury rate in National Football League (NFL) athletes during the first 4 weeks of the 2020 NFL season. Methods This study was a retrospective review of all NFL players who were placed on the injury report during the preseason and the first 4 weeks of the regular season from the 2016-2017 through the 2020-2021 NFL regular seasons. Players’ dates of injury were cross-referenced with an absence of statistics from the respective games for which they were ruled out so as to ensure accuracy. Injury rates were calculated per 1,000 athletic exposures. Relative risk with 95% confidence intervals compared injury rates between the 2 cohorts. Results Over the course of the study period of 4 NFL seasons, 3,025 injuries were reported. Of the 3,025 injuries reported, 582 (19%) occurred during weeks 1-4 of the 2020-2021 regular season, whereas 1,292 (53%) occurred during preseason weeks 1-4, and 1,151 (38%) occurred during regular-season weeks 1-4 of NFL seasons 2016-2017, 2018-2019, and 2019-2020. There was a significant increase in the injury rate during weeks 1-4 of the 2020-2021 regular season for all comparisons with the injury rate both during the preseasons and the regular seasons of 3 recent past NFL seasons. Conclusions The rate of injury in NFL players during weeks 1-4 of the 2020-2021 regular seasons was significantly higher than during 3 recent past NFL preseasons and regular seasons. Level of Evidence Level IV, diagnostic case series.

We recently reported that the membrane-associated progesterone receptor (MAPR) protein family (mammalian members: PGRMC1, PGRMC2, NEUFC and NENF) originated from a new class of prokaryotic cytochrome b

The neural molecular and biochemical response to stress is a distinct physiological process, and multiple lines of evidence indicate that the prefrontal cortex (PFC) is particularly sensitive to, and afflicted by, exposure to stress. Largely through this PFC dysfunction, stress has a characterized role in facilitating cognitive impairment, which is often dissociable from its effects on non-cognitive behaviors. The Rap1 small GTPase pathway has emerged as a commonly disrupted intracellular target in neuropsychiatric conditions, whether it be via alterations in Rap1 expression or through alterations in the expression of direct and specific upstream Rap1 activators and inhibitors. Here we demonstrate that escalating, intermittent stress increases Rap1 in mouse PFC synapses, results in cognitive impairments, and reduces the preponderance of mature dendritic spines in PFC neurons. Using viral-mediated gene transfer, we reveal that the hyper-induction of Rap1 in the PFC is sufficient to drive stress-relevant cognitive and synaptic phenotypes. These findings point to Rap1 as a critical mediator of stress-driven neuronal and behavioral pathology and highlight a previously unrecognized involvement for Rap1 in novelty-driven PFC engagement.

11 pag, 6 figs. Coordinates for the model of SrrB DHp-CA region solved by X-ray crystallography have been deposited in the Protein Data Bank, https://www.rcsb.org/ (ID code 6PAJ). This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921307117/-/DCSupplemental., Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis., This work was supported by funding from the National Institutes of Health (NIH) and National Institute of Allergy and Infectious Diseases (NIAID) to E.J.F. and P.M.S. (NIAID Grant AI135305). J.M.B. was funded by the NIH (NIAID Grant AI139100-01) and US Department of Agriculture Multistate Reseach Fund (Project NE−1028). W.S.-P. was supported by NIH (NIAID Grant AI134692-03). The J.K.M. lab was supported by the Canadian Institutes of Health Research (Grant PJT-166050). A.M. was supported by grant BIO2016-78571-P from the Ministerio de Economia y Competitividad (Spain).

Background Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in many cancer cells, where it is associated with detrimental patient outcomes. It contains phosphorylated tyrosines which evolutionarily preceded deuterostome gastrulation and tissue differentiation mechanisms. Results We demonstrate that manipulating PGRMC1 phosphorylation status in MIA PaCa-2 (MP) cells imposes broad pleiotropic effects. Relative to parental cells over-expressing hemagglutinin-tagged wild-type (WT) PGRMC1-HA, cells expressing a PGRMC1-HA-S57A/S181A double mutant (DM) exhibited reduced levels of proteins involved in energy metabolism and mitochondrial function, and altered glucose metabolism suggesting modulation of the Warburg effect. This was associated with increased PI3K/AKT activity, altered cell shape, actin cytoskeleton, motility, and mitochondrial properties. An S57A/Y180F/S181A triple mutant (TM) indicated the involvement of Y180 in PI3K/AKT activation. Mutation of Y180F strongly attenuated subcutaneous xenograft tumor growth in NOD-SCID gamma mice. Elsewhere we demonstrate altered metabolism, mutation incidence, and epigenetic status in these cells. Conclusions Altogether, these results indicate that mutational manipulation of PGRMC1 phosphorylation status exerts broad pleiotropic effects relevant to cancer and other cell biology.

We recently reported that the membrane associated progesterone receptor (MAPR) protein family (mammalian members: PGRMC1, PGRMC2, NEUFC and NENF) originated from a new class of prokaryotic cytochrome b5 (cytb5) domain proteins, called cytb5M (MAPR-like). Relative to classical cytb5 proteins, MAPR and ctyb5M proteins shared unique sequence elements and a distinct heme binding orientation at an approximately 90⁰ rotation relative to classical cytb5, as demonstrated in the archetypal crystal structure of a cytb5M protein (PDB accession number 6NZX). Here, we present the second crystal structure of an archaeal cytb5M domain (Methanococcoides burtonii WP_011499504.1, PDB:6VZ6). It exhibits similar heme-binding to the 6NZX cytb5M, supporting the deduction that MAPR-like heme orientation was inherited from the prokaryotic ancestor of the original eukaryotic MAPR gene.

The multiple functions of PGRMC1, the archetypal heme-binding eukaryotic MAPR family member, include steroidogenic regulation, membrane trafficking, and steroid responsiveness. The interrelationships between these functions are currently poorly understood. Previous work has shown that different MAPR subclasses were present early in eukaryotic evolution, and that tyrosine phosphorylated residues appeared in the eumetazoan ancestor, coincident with a gastrulation organizer. Here we show that MAPR proteins are related to a newly recognized class of prokaryotic cytochrome-b5 domain proteins. Our first solved structure of this new class exhibits shared MAPR-like folded architecture and heme-binding orientation. We also report that a protein subgroup from Candidate Phyla Radiation bacteria shares MAPR-like heme-interacting tyrosines. Our results support bacterial origins for both PGRMC1 and CYP51A, that catalyze the meiosis-associated 14-demethylation of the first sterol lanosterol from yeast to humans. We propose that eukaryotic acquisition of a membrane-trafficking function related to sterol metabolism was associated with the appearance of MAPR genes early in eukaryotic evolution.

Hard ceramic and glass material with tough mechanical properties and complex geometries can present a significant challenge in manufacturing. Slower processing times, excessive tool wear, and increased setup times are common with these materials and geometries. To overcome these challenges OptiPro has developed OptiSonic, an ultrasonic assisted machining technology. OptiSonic utilizes a custom tool holder that is designed to produce oscillations, in microns of amplitude, when loaded into a rotating spindle. A software package, IntelliSonic is used for setting up and controlling the ultrasonic motion during operation. Initially, the software is used to characterize each tool to find its optimal resonant peak to operate. Then, while grinding, IntelliSonic continuously adjusts the frequency to maintain optimal grinding efficiency during the operation. OptiSonic has shown, through testing, a reduction of force while machining. The combination of OptiSonic, kinematics, and RTCP (rotating tool center point) allows for full 5-axis simultaneous motion for grinding which allows operators to keep a more consistent wear band on the tool and aid in setup, operation, and part quality. After machine kinematic alignment is completed, the RTCP in the CNC control can make compensations without having to generate new tool paths. Also, the same tool paths can be shared between machines that have kinematics enabled. This can lead to a significant time reduction in setting up and adjusting tool paths.

A potential role of Staphylococcus aureus in bullous pemphigoid was explored by examining the colonization rate in patients with new-onset disease compared with that in age- and sex-matched controls. S. aureus colonization was observed in 85% of bullous pemphigoid lesions, 3–6-fold higher than the nares or unaffected skin from the same patients (P ≤ 0.003) and 6-fold higher than the nares or skin of controls (P ≤ 0.0015). Furthermore, 96% of the lesional isolates produced the toxic shock syndrome toxin-1 superantigen, and most of these additionally exhibited homogeneous expression of the enterotoxin gene cluster toxins. Toxic shock syndrome toxin-1‒neutralizing antibodies were not protective against colonization. However, S. aureus colonization was not observed in patients who had recently received antibiotics, and the addition of antibiotics with staphylococcal coverage eliminated S. aureus and resulted in clinical improvement. This study shows that toxic shock syndrome toxin-1‒positive S. aureus is prevalent in bullous pemphigoid lesions and suggests that early implementation of antibiotics may be of benefit. Furthermore, our results suggest that S. aureus colonization could provide a source of infection in patients with bullous pemphigoid, particularly in the setting of high-dose immunosuppression.

Mounting epidemiologic and scientific evidence indicates that many psychiatric disorders originate from a complex interplay between genetics and early life experiences, particularly in the womb. Despite decades of research, our understanding of the precise prenatal and perinatal experiences that increase susceptibility to neurodevelopmental disorders remains incomplete. Sleep apnea (SA) is increasingly common during pregnancy and is characterized by recurrent partial or complete cessations in breathing during sleep. SA causes pathological drops in blood oxygen levels (intermittent hypoxia, IH), often hundreds of times each night. Although SA is known to cause adverse pregnancy and neonatal outcomes, the long-term consequences of maternal SA during pregnancy on brain-based behavioral outcomes and associated neuronal functioning in the offspring remain unknown. We developed a rat model of maternal SA during pregnancy by exposing dams to IH, a hallmark feature of SA, during gestational days 10 to 21 and investigated the consequences on the offspring’s forebrain synaptic structure, synaptic function, and behavioral phenotypes across multiples stages of development. Our findings represent a rare example of prenatal factors causing sexually dimorphic behavioral phenotypes associated with excessive (rather than reduced) synapse numbers and implicate hyperactivity of the mammalian target of rapamycin (mTOR) pathway in contributing to the behavioral aberrations. These findings have implications for neuropsychiatric disorders typified by superfluous synapse maintenance that are believed to result, at least in part, from largely unknown insults to the maternal environment., Correlative data in humans has hinted at an association between maternal sleep apnea during pregnancy and altered neuronal function in offspring. This study shows that in a rat model of sleep apnea, maternal gestational intermittent hypoxia leads to sex-specific changes in neuronal structure and function in offspring, accompanied by impaired behavioral phenotypes.

This is a preface by the guest editors of the special issue of Cancers featuring the biology of progesterone (P4) receptor membrane component (PGRMC) proteins as it relates to metabolism and cancer [...]

Cells respond to pathogen infection by activating a complex innate immune signaling pathway, which culminates in the activation of transcription factors and secretion of a family of functionally and genetically related cytokines. However, excessive immune activation may cause tissue damage and detrimental effects on the host., Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts HIV-1 replication by limiting the intracellular deoxynucleoside triphosphate (dNTP) pool. SAMHD1 also suppresses the activation of NF-κB in response to viral infections and inflammatory stimuli. However, the mechanisms by which SAMHD1 negatively regulates this pathway remain unclear. Here, we show that SAMHD1-mediated suppression of NF-κB activation is modulated by two key mediators of NF-κB signaling, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and transforming growth factor β-activated kinase 1 (TAK1). We compared NF-κB activation stimulated by interleukin (IL)-1β in monocytic THP-1 control and SAMHD1 knockout (KO) cells with and without partial TRAF6 knockdown (KD), or in cells treated with TAK1 inhibitors. Relative to control cells, IL-1β-treated SAMHD1 KO cells showed increased phosphorylation of the inhibitor of NF-κB (IκBα), an indication of pathway activation, and elevated levels of TNF-α mRNA. Moreover, SAMHD1 KO combined with TRAF6 KD or pharmacological TAK1 inhibition reduced IκBα phosphorylation and TNF-α mRNA to the level of control cells. SAMHD1 KO cells infected with single-cycle HIV-1 showed elevated infection and TNF-α mRNA levels compared to control cells, and the effects were significantly reduced by TRAF6 KD or TAK1 inhibition. We further demonstrated that overexpressed SAMHD1 inhibited TRAF6-stimulated NF-κB reporter activity in HEK293T cells in a dose-dependent manner. SAMHD1 contains a nuclear localization signal (NLS), but an NLS-defective SAMHD1 exhibited a suppressive effect similar to the wild-type protein. Our data suggest that the TRAF6-TAK1 axis contributes to SAMHD1-mediated suppression of NF-κB activation and HIV-1 infection. IMPORTANCE Cells respond to pathogen infection by activating a complex innate immune signaling pathway, which culminates in the activation of transcription factors and secretion of a family of functionally and genetically related cytokines. However, excessive immune activation may cause tissue damage and detrimental effects on the host. Therefore, in order to maintain host homeostasis, the innate immune response is tightly regulated during viral infection. We have reported SAMHD1 as a novel negative regulator of the innate immune response. Here, we provide new insights into SAMHD1-mediated negative regulation of the NF-κB pathway at the TRAF6-TAK1 checkpoint. We show that SAMHD1 inhibits TAK1 activation and TRAF6 signaling in response to proinflammatory stimuli. Interestingly, TRAF6 knockdown in SAMHD1-deficient cells significantly inhibited HIV-1 infection and activation of NF-κB induced by virus infection. Our research reveals a new negative regulatory mechanism by which SAMHD1 participates in the maintenance of cellular homeostasis during HIV-1 infection and inflammation.

The σ-2 receptor (S2R) complex has been implicated in CNS disorders ranging from anxiety and depression to neurodegenerative disorders such as Alzheimer’s disease (AD). The proteins comprising the S2R complex impact processes including autophagy, cholesterol synthesis, progesterone signaling, lipid membrane-bound protein trafficking, and receptor stabilization at the cell surface. While there has been much progress in understanding the role of S2R in cellular processes and its potential therapeutic value, a great deal remains unknown. The International Symposium on Sigma-2 Receptors is held in conjunction with the annual Society for Neuroscience (SfN) conference to promote collaboration and advance the field of S2R research. This review summarizes updates presented at the Fourth International Symposium on Sigma-2 Receptors: Role in Health and Disease, a Satellite Symposium held at the 2019 SfN conference. Interdisciplinary members of the S2R research community presented both previously published and preliminary results from ongoing studies of the role of S2R in cellular metabolism, the anatomic and cellular expression patterns of S2R, the relationship between S2R and amyloid β (Aβ) in AD, the role of S2R complex protein PGRMC1 in health and disease, and the efforts to design new S2R ligands for the purposes of research and drug development. The proceedings from this symposium are reported here as an update on the field of S2R research, as well as to highlight the value of the symposia that occur yearly in conjunction with the SfN conference.

N6-methyladenosine (m6A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m6A modification is regulated by two groups of cellular proteins, writers and erasers that add or remove m6A, respectively. HIV-1 RNA contains m6A modifications that modulate viral infection and gene expression in CD4+ T cells. However, it remains unclear whether m6A modifications of HIV-1 RNA modulate innate immune responses in myeloid cells that are important for antiviral immunity. Here we show that m6A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in differentiated human monocytic cells and primary monocyte-derived macrophages. Transfection of differentiated monocytic U937 cells with HIV-1 RNA fragments containing a single m6A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered m6A levels of RNA by manipulating the expression of the m6A erasers (FTO and ALKBH5) or pharmacological inhibition of m6A addition in virus-producing cells, or by treating HIV-1 RNA with recombinant FTO in vitro. HIV-1 RNA transfection or viral infection of differentiated U937 cells and primary macrophages demonstrated that HIV-1 RNA with decreased m6A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m6A modifications had opposite effects. Our mechanistic studies indicated that m6A of HIV-1 RNA escaped retinoic acid-induced gene I (RIG-I)-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Together, these findings suggest that m6A modifications of HIV-1 RNA evade innate immune sensing in myeloid cells., Author summary HIV-1 is known as a weak inducer of antiviral cytokines including IFN-I, but it is unclear how HIV-1 evades innate immunity. Different types of RNA modifications including m6A within the HIV-1 genome modulate viral replication; however, the role of m6A modifications of HIV-1 RNA in regulating innate immune responses remains elusive. Myeloid cells including macrophages are HIV-1 target cells and critical for generating antiviral immunity. In this study, we aimed to investigate the role of m6A modifications of HIV-1 RNA in regulating innate immune responses in myeloid cells. We found that m6A-modified HIV-1 RNA suppresses IFN-I expression in differentiated monocytic cells and primary macrophages. Our data suggest that the cellular protein RIG-I contributes to innate sensing of m6A-defective HIV-1 RNA in differentiated monocytic cells. Our findings provide new insights into the functions and mechanisms of m6A modifications of HIV-1 RNA in regulating innate immune sensing and responses in myeloid cells.

The Akt family of kinases exert many of their cellular effects via the activation of the mammalian target of rapamycin (mTOR) kinase through a series of intermediary proteins. Multiple lines of evidence have identified the Akt family of kinases as candidate schizophrenia and bipolar disorder genes. Although dysfunction of the prefrontal cortex (PFC) is a key feature of both schizophrenia and bipolar disorder, no studies have comprehensively assessed potential alterations in Akt-mTOR pathway activity in the PFC of either disorder. Here, we examined the activity and expression profile of key proteins in the Akt-mTOR pathway in bipolar disorder and schizophrenia homogenates from two different PFC subregions. Our findings identify reduced Akt-mTOR PFC signaling in a subset of bipolar disorder subjects. Using a reverse-translational approach, we demonstrated that Akt hypofunction the PFC is sufficient to give rise to key cognitive phenotypes that are paralleled by alterations in synaptic connectivity and function.

BackgroundThe membrane-associated progesterone receptor (MAPR) family consists of heme-binding proteins containing a cytochrome b5(cytb5) domain characterized by the presence of a MAPR-specific interhelical insert region (MIHIR) between helices 3 and 4 of the canonical cytb5-domain fold. Animals possess three MAPR families (PGRMC-like, Neuferricin and Neudesin).ResultsHere we show that all animal MAPR families were already present in the common ancestor of the Opisthokonta (comprising animals and fungi as well as related protistan taxa). All three MAPR genes acquired extensions C-terminal to the cytb5domain, either before or with the evolution of animals. The archetypical MAPR protein, progesterone receptor membrane component 1 (PGRMC1), contains phosphorylated tyrosines Y139 and Y180. The combination of Y139/Y180 appeared in the common ancestor of Cnidaria and bilaterally symmetrical animals, along with an early embryological organizer and synapsed neurons, and is strongly conserved in all bilateral animals. A predicted protein interaction motif in the PGRMC1 MIHIR is potentially regulated by Y139 phosphorylation. A multilayered model of animal MAPR function acquisition includes some pre-metazoan functions (e.g., heme binding and cytochrome P450 interactions) and some acquired animal-specific functions that involve regulation of strongly conserved protein interaction motifs acquired by early-branching animals.ConclusionsThis study provides a conceptual framework for future studies, against which PGRMC1’s multiple functions can perhaps be stratified and functionally dissected. In accompanying papers we show that mutational perturbation of PGRMC1 phosphorylation status of the Y180 motif is associated with dramatic changes cell pasticity assayed by protein abundances, cell morphology, mitochondrial function, genomic stability, and epigenetic status, with pathways analysis associating Y180 mutation with processes related to organizer function. These combined works reveal previously unrecognized involvement of PGRMC1 in foundational animal processes of great relevance to disease.

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.