Key residues in the VDAC2-BAK complex can be targeted to modulate apoptosis.

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial channel protein VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy.

Histopathological assessment of depth of coagulation necrosis with Holmium, Moses, and Thulium fiber lasers in human prostate tissue.

PURPOSE: To evaluate coagulation necrosis depth (CND) of Holmium (HL), Moses (ML), and Thulium fiber laser (TFL) in ex vivo human prostate tissue at various energy settings. METHODS: After endoscopic HL enucleation, small prostate tissue fragments were removed from the bladder with graspers and used for study. Immediately after surgery, a single incision was made on the surface of the tissue kept under normal saline at room temperature using a hand-held 550-µm laser fiber. Variable energy settings were tested for all three lasers. Two pathologists measured the CND with light microscopy using ocular micrometer. Impact of various laser settings on CND was analyzed. The differences in CND of all three lasers at similar laser power were compared. RESULTS: Mean CND was 0.56 ± 0.53 mm for long-pulse HL, 0.54 ± 0.53 mm for ML, 0.67 ± 0.67 mm for low-pulse TFL, and 0.81 ± 0.78 mm for high-pulse TFL. There was no significant difference between mean CND of HL and ML at various laser settings ranging from 10 to 120 W and CND with long- and short-pulse settings of TFL at settings from 10 to 60 W. There was a trend of increasing CND in HL and ML with increasing laser power; however, it was not statistically significant. TFL had similar tissue effects as HL and ML. CONCLUSION: There is no significant difference in CND of HL, ML, and TFL in ex vivo human prostate tissue. Other factors besides laser type and settings need to be studied to explain clinical differences among various lasers used for prostate enucleation.

Single-chain insulin analogs threaded by the insulin receptor αCT domain.

Insulin is a mainstay of therapy for diabetes mellitus, yet its thermal stability complicates global transportation and storage. Cold-chain transport, coupled with optimized formulation and materials, prevents to some degree nucleation of amyloid and hence inactivation of hormonal activity. These issues hence motivate the design of analogs with increased stability, with a promising approach being single-chain insulins (SCIs), whose C domains (foreshortened relative to proinsulin) resemble those of the single-chain growth factors (IGFs). We have previously demonstrated that optimized SCIs can exhibit native-like hormonal activity with enhanced thermal stability and marked resistance to fibrillation. Here, we describe the crystal structure of an ultrastable SCI (C-domain length 6; sequence EEGPRR) bound to modules of the insulin receptor (IR) ectodomain (N-terminal α-subunit domains L1-CR and C-terminal αCT peptide; "microreceptor" [µIR]). The structure of the SCI-µIR complex, stabilized by an Fv module, was determined using diffraction data to a resolution of 2.6 Å. Remarkably, the αCT peptide (IR-A isoform) "threads" through a gap between the flexible C domain and the insulin core. To explore such threading, we undertook molecular dynamics simulations to 1) compare threaded with unthreaded binding modes and 2) evaluate effects of C-domain length on these alternate modes. The simulations (employing both conventional and enhanced sampling simulations) provide evidence that very short linkers (C-domain length of -1) would limit gap opening in the SCI and so impair threading. We envisage that analogous threading occurs in the intact SCI-IR complex-rationalizing why minimal C-domain lengths block complete activity-and might be exploited to design novel receptor-isoform-specific analogs.

The Bak core dimer focuses triacylglycerides in the membrane.

Apoptosis, the intrinsic programmed cell death process, is mediated by the Bcl-2 family members Bak and Bax. Activation via formation of symmetric core dimers and oligomerization on the mitochondrial outer membrane (MOM) leads to permeabilization and cell death. Although this process is linked to the MOM, the role of the membrane in facilitating such pores is poorly understood. We recently described Bak core domain dimers, revealing lipid binding sites and an initial role of lipids in oligomerization. Here we describe simulations that identified localized clustering and interaction of triacylglycerides (TAGs) with a minimized Bak dimer construct. Coalescence of TAGs occurred beneath this Bak dimer, mitigating dimer-induced local membrane thinning and curvature in representative coarse-grain MOM and model membrane systems. Furthermore, the effects observed as a result of coarse-grain TAG cluster formation was concentration dependent, scaling from low physiological MOM concentrations to those found in other organelles. We find that increasing the TAG concentration in liposomes mimicking the MOM decreased the ability of activated Bak to permeabilize these liposomes. These results suggest that the presence of TAGs within a Bak-lipid membrane preserves membrane integrity and is associated with reduced membrane stress, suggesting a possible role of TAGs in Bak-mediated apoptosis.

Unhoused and unhireable? Examining employment biases in service contexts related to perceived warmth and competence of people experiencing houselessness.

Lack of safe and stable housing is a pernicious and growing social concern, and stereotypes about individuals experiencing houselessness are generally quite negative. Little scholarly work has examined housing insecurity and its associated stereotypes in employment contexts. The purpose of the current research was to examine, in the context of the hospitality industry, whether housing status influences hiring managers' perceptions of hireability (Study 1) and customers' evaluations of an organization and its employees (Study 2) using the stereotype content model. Across two experimental studies, we assessed participant attitudes toward individuals experiencing houselessness. In Study 1, we instructed 148 hotel managers to listen to a hypothetical job interview with either an unhoused or housed job applicant, and then complete measures of hireability. In Study 2, we instructed 139 hotel customers to observe a hypothetical interaction with either an unhoused or housed employee, and then evaluate the employee and the organization. Study 1's findings suggested an indirect effect of housing status on perceived hireability through warmth, and this indirect relationship was moderated by gender. Men who were houseless were rated lower in warmth, and thus lower in hireability, than non-houseless men or women regardless of their housing status. However, houseless men were perceived by customers as warmer than non-houseless men as employees, driving higher evaluations of the organization and the employee (Study 2). Hiring initiatives targeted at providing short-term housing for unhoused employees will benefit employees, employers, and the larger communities they encompass.

"Register-shift" insulin analogs uncover constraints of proteotoxicity in protein evolution.

Globular protein sequences encode not only functional structures (the native state) but also protein foldability, i.e. a conformational search that is both efficient and robustly minimizes misfolding. Studies of mutations associated with toxic misfolding have yielded insights into molecular determinants of protein foldability. Of particular interest are residues that are conserved yet dispensable in the native state. Here, we exploited the mutant proinsulin syndrome (a major cause of permanent neonatal-onset diabetes mellitus) to investigate whether toxic misfolding poses an evolutionary constraint. Our experiments focused on an invariant aromatic motif (PheB24-PheB25-TyrB26) with complementary roles in native self-assembly and receptor binding. A novel class of mutations provided evidence that insulin can bind to the insulin receptor (IR) in two different modes, distinguished by a "register shift" in this motif, as visualized by molecular dynamics (MD) simulations. Register-shift variants are active but defective in cellular foldability and exquisitely susceptible to fibrillation in vitro Indeed, expression of the corresponding proinsulin variant induced endoplasmic reticulum stress, a general feature of the mutant proinsulin syndrome. Although not present among vertebrate insulin and insulin-like sequences, a prototypical variant ([GlyB24]insulin) was as potent as WT insulin in a rat model of diabetes. Although in MD simulations the shifted register of receptor engagement is compatible with the structure and allosteric reorganization of the IR-signaling complex, our results suggest that this binding mode is associated with toxic misfolding and so is disallowed in evolution. The implicit threat of proteotoxicity limits sequence variation among vertebrate insulins and insulin-like growth factors.

CD21 (Complement Receptor 2) Is the Receptor for Epstein-Barr Virus Entry into T Cells.

Epstein-Barr virus (EBV) is associated with a number of T-cell diseases, including some peripheral T-cell lymphomas, hemophagocytic lymphohistiocytosis, and chronic active EBV disease. The tropism of EBV for B cells and epithelial cell infection has been well characterized, but infection of T cells has been minimally explored. We have recently shown that the EBV type 2 (EBV-2) strain has the unique ability to infect mature T cells. Utilizing an ex vivo infection model, we sought to understand the viral glycoprotein and cellular receptor required for EBV-2 infection of T cells. Here, using a neutralizing-antibody assay, we found that viral gp350 and complement receptor 2 (CD21) are required for CD3+ T-cell infection. Using the HB5 anti-CD21 antibody clone but not the Bly-4 anti-CD21 antibody clone, we detected expression of CD21 on both CD4+ and CD8+ T cells, with the highest expression on naive CD4 and CD8+ T-cell subsets. Using CRISPR to knock out CD21, we demonstrated that CD21 is necessary for EBV entry into the Jurkat T-cell line. Together, these results indicate that EBV uses the same viral glycoprotein and cellular receptor for both T- and B-cell infection.IMPORTANCE Epstein-Barr virus (EBV) has a well-described tropism for B cells and epithelial cells. Recently, we described the ability of a second strain of EBV, EBV type 2, to infect mature peripheral T cells. Using a neutralizing antibody assay, we determined that EBV uses the viral glycoprotein gp350 and the cellular protein CD21 to gain entry into mature peripheral T cells. CRISPR-Cas9 deletion of CD21 on the Jurkat T-cell line confirmed that CD21 is required for EBV infection. This study has broad implications, as we have defined a function for CD21 on mature peripheral T cells, i.e., as a receptor for EBV. In addition, the requirement for gp350 for T-cell entry has implications for EBV vaccine studies currently targeting the gp350 glycoprotein to prevent EBV-associated diseases.

Human Cytomegalovirus-Induced Autophagy Prevents Necroptosis of Infected Monocytes.

Key to the viral dissemination strategy of human cytomegalovirus (HCMV) is the induction of monocyte survival, where monocytes are normally short-lived cells. Autophagy is a cellular process that preserves cellular homeostasis and promotes cellular survival during times of stress. We found that HCMV rapidly induced autophagy within infected monocytes. The early induction of autophagy during HCMV infection was distinctly required for the survival of HCMV-infected monocytes, as repression of autophagosome formation led to cellular death of infected cells but had no effect on the viability of uninfected monocytes. The inhibition of caspases was insufficient to rescue cell viability of autophagy-repressed infected monocytes, suggesting that autophagy was not protecting cells from apoptosis. Accordingly, we found that HCMV blocked the activation of caspase 8, which was maintained in the presence of autophagy inhibitors. Necroptosis is an alternative form of cell death triggered when apoptosis is impeded and is dependent on RIPK3 phosphorylation of MLKL. Although we found that HCMV activated RIP3K upon infection, MLKL was not activated. However, inhibition of autophagy removed the block in RIPK3 phosphorylation of MLKL, suggesting that autophagy was protecting infected monocytes from undergoing necroptosis. Indeed, survival of autophagy-inhibited HCMV-infected monocytes was rescued when MLKL and RIPK3 were suppressed. Taken together, these data indicate that HCMV induces autophagy to prevent necroptotic cell death in order to ensure the survival of infected monocytes and thus facilitate viral dissemination within the host.IMPORTANCE Human cytomegalovirus (HCMV) infection is endemic throughout the world, with a seroprevalence of 40 to 100% depending on geographic location. HCMV infection is generally asymptomatic, but can cause severe inflammatory organ diseases in immunocompromised individuals. The broad array of organ diseases caused by HCMV is directly linked to the systematic spread of the virus mediated by monocytes. Monocytes are naturally programmed to undergo apoptosis, which is rapidly blocked by HCMV to ensure the survival and dissemination of infected monocytes to different organ sites. In this work, we demonstrate infected monocytes also initiate necroptosis as a "trap door" death pathway in response to HCMV subversion of apoptosis. HCMV then activates cellular autophagy as a countermeasure to prevent the execution of necroptosis, thereby promoting the continued survival of infected monocytes. Elucidating the mechanisms by which HCMV stimulates monocyte survival is an important step to the development of novel anti-HCMV drugs that prevent the spread of infected monocytes.

A Novel Human Skin Tissue Model To Study Varicella-Zoster Virus and Human Cytomegalovirus.

The herpesviruses varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) are endemic to humans. VZV causes varicella (chicken pox) and herpes zoster (shingles), while HCMV causes serious disease in immunocompromised patients and neonates. More effective, less toxic antivirals are needed, necessitating better models to study these viruses and evaluate antivirals. Previously, VZV and HCMV models used fetal tissue; here, we developed an adult human skin model to study VZV and HCMV in culture and in vivo While VZV is known to grow in skin, it was unknown whether skin could support an HCMV infection. We used TB40/E HCMV and POka VZV strains to evaluate virus tropism in skin organ culture (SOC) and skin xenograft mouse models. Adult human skin from reduction mammoplasties was prepared for culture on NetWells or mouse implantation. In SOC, VZV infected the epidermis and HCMV infected the dermis. Specifically, HCMV infected fibroblasts, endothelial cells, and hematopoietic cells, with some infected cells able to transfer infection. VZV and HCMV mouse models were developed by subcutaneous transplantation of skin into SCID/beige or athymic nude mice at 2 independent sites. Viruses were inoculated directly into one xenograft, and widespread infection was observed for VZV and HCMV. Notably, we detected VZV- and HCMV-infected cells in the contralateral, uninoculated xenografts, suggesting dissemination from infected xenografts occurred. For the first time, we showed HCMV successfully grows in adult human skin, as does VZV. Thus, this novel system may provide a much-needed preclinical small-animal model for HCMV and VZV and, potentially, other human-restricted viruses.IMPORTANCE Varicella-zoster virus and human cytomegalovirus infect a majority of the global population. While they often cause mild disease, serious illness and complications can arise. Unfortunately, there are few effective drugs to treat these viruses, and many are toxic. To complicate this, these viruses are restricted to replication in human cells and tissues, making them difficult to study in traditional animal models. Current models rely heavily on fetal tissues, can be prohibitively expensive, and are often complicated to generate. While fetal tissue models provide helpful insights, it is necessary to study human viruses in human tissue systems to fully understand these viruses and adequately evaluate novel antivirals. Adult human skin is an appropriate model for these viruses because many target cells are present, including basal keratinocytes, fibroblasts, dendritic cells, and lymphocytes. Skin models, in culture and xenografts in immunodeficient mice, have potential for research on viral pathogenesis, tissue tropism, dissemination, and therapy.

Anxiety-like behavior and intestinal microbiota changes as strain-and sex-dependent sequelae of mild food allergy in mouse models of cow's milk allergy.

A number of studies have reported comorbidity of food allergies with various neuropsychiatric disorders, such as anxiety, depression, attention-deficit hyperactivity disorder, and autism. However, inconsistent results across clinical studies have left the association between food allergy and behavioral disorders inconclusive. We postulated that the heterogeneities in genetic background among allergic cohorts affect symptom presentation and severity of food allergy, introducing bias in patient selection criteria toward individuals with overt physical reactions. To understand the influence of genetic background on food allergy symptoms and behavioral changes beyond anaphylaxis, we generated mouse models with mild cow's milk allergy by sensitizing male and female C57BL/6J and BALB/cJ mice to a bovine whey protein, ß-lactoglobulin (BLG; Bos d 5). We compared strain- and sex-dependent differences in their immediate physical reactions to BLG challenge as well as anxiety-like behavior one day after the challenge. While reactions to the allergen challenge were either absent or mild for all groups, a greater number of BLG-sensitized BALB/cJ mice presented visible symptoms and hypothermia compared to C57BL/6J mice. Interestingly, male mice of both strains displayed anxiety-like behavior on an elevated zero maze without exhibiting cognitive impairment with the cross maze test. Further characterization of plasma cytokines/chemokines and fecal microbiota also differentiated strain- and sex-dependent effects of BLG sensitization on immune-mediator levels and bacterial populations, respectively. These results demonstrated that the genetic variables in mouse models of milk allergy influenced immediate physical reactions to the allergen, manifestation of anxiety-like behavior, levels of immune responses, and population shift in gut microbiota. Thus, stratification of allergic cohorts by their symptom presentations and severity may strengthen the link between food allergy and behavioral disorders and identify a population(s) with specific genetic background that have increased susceptibility to allergy-associated behavioral disorders.

Modulation of host cell signaling during cytomegalovirus latency and reactivation.

BACKGROUND: Human cytomegalovirus (HCMV) resides latently in cells of the myeloid compartment, including CD34+ hematopoietic progenitor cells and circulating monocytes. Healthy hosts maintain the virus latently, and this infection is, for the most part, asymptomatic. However, given the proper external cues, HCMV reactivates from latency, at which point the virus disseminates, causing disease. The viral and cellular factors dictating the balance between these phases of infection are incompletely understood, though a large body of literature support a role for viral-mediated manipulation of host cell signaling. MAIN BODY: To establish and maintain latency, HCMV has evolved various means by which it usurps host cell factors to alter the cellular environment to its own advantage, including altering host cell signaling cascades. As early as virus entry into myeloid cells, HCMV usurps cellular signaling to change the cellular milieu, and this regulation includes upregulation, as well as downregulation, of different signaling cascades. Indeed, given proper reactivation cues, this signaling is again altered to allow for transactivation of viral lytic genes. CONCLUSIONS: HCMV modulation of host cell signaling is not binary, and many of the cellular pathways altered are finely regulated, wherein the slightest modification imparts profound changes to the cellular milieu. It is also evident that viral-mediated cell signaling differs not only between these phases of infection, but also is myeloid cell type specific. Nonetheless, understanding the exact pathways and the means by which HCMV mediates them will undoubtedly provide novel targets for therapeutic intervention.

Safety and efficacy of holmium laser enucleation of prostate as salvage procedure for persistent or recurrent lower urinary tract symptoms secondary to bladder outlet obstruction after prior prostate artery embolization: a match analysis.

PURPOSE: To evaluate safety and efficacy of Holmium laser enucleation of Prostate (HoLEP) for management of persistent or recurrent lower urinary tract symptoms after prior prostate artery embolization (PAE). We also evaluated histopathological changes in prostate after PAE. METHODS: Ten patients who underwent HoLEP after prior PAE were matched according to age, weight of resected prostate tissue, and anticoagulation status in 1:2 ratio with patients who underwent HoLEP without prior PAE by a researcher who was blinded to patient's outcome at the time of matching. Histopathological examination of prostate tissue was performed to look for changes related to prior PAE. Patient's demographics, perioperative parameters, and follow-up data were retrospectively compared. RESULTS: The median interval between PAE and HoLEP was 25 months [IQR 14.5-37.5]. Patients demographic were comparable in both groups. Intra-operatively plane of enucleation were well-maintained in spite of prior PAE. The differences in duration of surgery, enucleation efficiency, hemoglobin drop, duration of catheterization and hospital stay, and complications were statistically insignificant. Incidental prostate cancer was identified in 10% specimens from both groups. Post-PAE prostate specimens demonstrated evidence of remote-healed infarction represented by dense hyalinized paucicellur connective tissue with surrounding squamous metaplasia. There were no statistically significant differences in AUA symptom scores, maximum urine flow rate, post-void residual urine volume, and PSA at 3- and 6-month follow-up between both groups. CONCLUSIONS: Plane of enucleation is well-maintained after prior PAE. Salvage HoLEP is safe and effective after previous PAE and provide outcome comparable with HoLEP as a primary procedure.

Vocal Turn-Taking Between Mothers and Their Children With Cochlear Implants.

OBJECTIVES: The primary objective of the study was to examine the occurrence and temporal structure of vocal turn-taking during spontaneous interactions between mothers and their children with cochlear implants (CI) over the first year after cochlear implantation as compared with interactions between mothers and children with normal hearing (NH). DESIGN: Mothers' unstructured play sessions with children with CI (n = 12) were recorded at 2 time points, 3 months (mean age 18.3 months) and 9 months (mean age 27.5 months) post-CI. A separate control group of mothers with age-matched hearing children (n = 12) was recorded at the same 2 time points. Five types of events were coded: mother and child vocalizations, vocalizations including speech overlap, and between- and within-speaker pauses. We analyzed the proportion of child and mother vocalizations involved in turn-taking, the temporal structure of turn-taking, and the temporal reciprocity of turn-taking using proportions of simultaneous speech and the duration of between- and within-speaker pauses. RESULTS: The CI group produced a significantly smaller proportion of vocalizations in turn-taking than the NH group at the first session; however, CI children's proportion of vocalizations in turn-taking increased over time. There was a significantly larger proportion of simultaneous speech in the CI compared with the NH group at the first session. The CI group produced longer between-speaker pauses as compared with those in the NH group at the first session with mothers decreasing the duration of between-speaker pauses over time. NH infants and mothers in both groups produced longer within- than between-speaker pauses but CI infants demonstrated the opposite pattern. In addition, the duration of mothers' between-speaker pauses (CI and NH) was predicted by the duration of the infants' between-speaker pauses. CONCLUSIONS: Vocal turn-taking and timing in both members of the dyad, the mother and infant, were sensitive to the experiential effects of child hearing loss and remediation with CI. Child hearing status affected dyad-specific coordination in the timing of responses between mothers and their children.

Military sexual trauma among women Veterans: The buffering effect of coworker support.

Prior research has demonstrated the impact of military sexual trauma (MST) on health and well-being. However, little empirical work has been published identifying protective factors for women who have experienced MST. We examined the impact of two different forms of MST, harassment-only and assault MST, on PTSD symptoms and social functional impairment in a sample of women Veterans employed in the civilian workforce. The effects of MST were examined at three different times over a period of 9 months. We found that MST that included both harassment and assault was associated with significantly higher levels of PTSD symptoms and social functional impairment across three different time points among women Veterans employed in civilian jobs. Further, the pattern of results suggested that coworker support can buffer against these negative outcomes experienced by women who reported assault MST. Overall, findings suggest that coworker support is one critical resource for women Veterans who experienced assault MST.

Differences in the Epstein-Barr Virus gp350 IgA Antibody Response Are Associated With Increased Risk for Coinfection With a Second Strain of Epstein-Barr Virus.

BACKGROUND: The Epstein-Barr virus (EBV) viral glycoprotein gp350 has been proposed as a candidate antigen for an EBV vaccine. However, the proposed formulations of these vaccines have not taken into account the presence of 2 unique EBV strains (EBV-1 and EBV-2) present in areas of high incidence of the EBV-associated cancer, Burkitt lymphoma. METHODS: In this study, we analyze the kinetics of EBV-1 and EBV-2 infection in an asymptomatic infant cohort from Kisumu, Kenya. We also analyzed the kinetics of the antibody response against 5 EBV antigens, gp350 (IgG and IgA), VCA (IgG), EBNA-1 (IgG), EAd (IgG), and Zta (IgG). RESULTS: We observed a high frequency of coinfection with both EBV types over time, with the only observable defect in the antibody response in infants coinfected being a significantly lower level of anti-gp350 IgA at peak response. Gp350 IgA levels were also significantly lower in coinfected infants 2.5 months postinfection and at the time of coinfection. CONCLUSIONS: These results suggest that anti-gp350 IgA antibodies may be important for sterilizing immunity against secondary infection. These findings have implications for the development of an efficacious EBV vaccine to prevent both EBV-1 and EBV-2 infection in a population at high risk for Burkitt lymphoma.

Solution structure of an ultra-stable single-chain insulin analog connects protein dynamics to a novel mechanism of receptor binding.

Domain-minimized insulin receptors (IRs) have enabled crystallographic analysis of insulin-bound "micro-receptors." In such structures, the C-terminal segment of the insulin B chain inserts between conserved IR domains, unmasking an invariant receptor-binding surface that spans both insulin A and B chains. This "open" conformation not only rationalizes the inactivity of single-chain insulin (SCI) analogs (in which the A and B chains are directly linked), but also suggests that connecting (C) domains of sufficient length will bind the IR. Here, we report the high-resolution solution structure and dynamics of such an active SCI. The hormone's closed-to-open transition is foreshadowed by segmental flexibility in the native state as probed by heteronuclear NMR spectroscopy and multiple conformer simulations of crystallographic protomers as described in the companion article. We propose a model of the SCI's IR-bound state based on molecular-dynamics simulations of a micro-receptor complex. In this model, a loop defined by the SCI's B and C domains encircles the C-terminal segment of the IR α-subunit. This binding mode predicts a conformational transition between an ultra-stable closed state (in the free hormone) and an active open state (on receptor binding). Optimization of this switch within an ultra-stable SCI promises to circumvent insulin's complex global cold chain. The analog's biphasic activity, which serendipitously resembles current premixed formulations of soluble insulin and microcrystalline suspension, may be of particular utility in the developing world.

Ablation of amyloid precursor protein increases insulin-degrading enzyme levels and activity in brain and peripheral tissues.

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein widely studied for its role as the source of ß-amyloid peptide, accumulation of which is causal in at least some cases of Alzheimer's disease (AD). APP is expressed ubiquitously and is involved in diverse biological processes. Growing bodies of evidence indicate connections between AD and somatic metabolic disorders related to type 2 diabetes, and App-/- mice show alterations in glycemic regulation. We find that App-/- mice have higher levels of insulin-degrading enzyme (IDE) mRNA, protein, and activity compared with wild-type controls. This regulation of IDE by APP was widespread across numerous tissues, including liver, skeletal muscle, and brain as well as cell types within neural tissue, including neurons, astrocytes, and microglia. RNA interference-mediated knockdown of APP in the SIM-A9 microglia cell line elevated IDE levels. Fasting levels of blood insulin were lower in App-/- than App+/+ mice, but the former showed a larger increase in response to glucose. These low basal levels may enhance peripheral insulin sensitivity, as App-/- mice failed to develop impairment of glucose tolerance on a high-fat, high-sucrose ("Western") diet. Insulin levels and insulin signaling were also lower in the App-/- brain; synaptosomes prepared from App-/- hippocampus showed diminished insulin receptor phosphorylation compared with App+/+ mice when stimulated ex vivo. These findings represent a new molecular link connecting APP to metabolic homeostasis and demonstrate a novel role for APP as an upstream regulator of IDE in vivo.

Epstein-Barr Virus Type 2 Infects T Cells and Induces B Cell Lymphomagenesis in Humanized Mice.

Epstein-Barr virus (EBV) has been classified into two strains, EBV type 1 (EBV-1) and EBV type 2 (EBV-2) based on genetic variances and differences in transforming capacity. EBV-1 readily transforms B cells in culture while EBV-2 is poorly transforming. The differing abilities to immortalize B cells in vitro suggest that in vivo these viruses likely use alternative approaches to establish latency. Indeed, we recently reported that EBV-2 has a unique cell tropism for T cells, infecting T cells in culture and in healthy Kenyan infants, strongly suggesting that EBV-2 infection of T cells is a natural part of the EBV-2 life cycle. However, limitations of human studies hamper further investigation into how EBV-2 utilizes T cells. Therefore, BALB/c Rag2null IL2rγnull SIRPα humanized mice were utilized to develop an EBV-2 in vivo model. Infection of humanized mice with EBV-2 led to infection of both T and B cells, unlike infection with EBV-1, in which only B cells were infected. Gene expression analysis demonstrated that EBV-2 established a latency III infection with evidence of ongoing viral reactivation in both B and T cells. Importantly, EBV-2-infected mice developed tumors resembling diffuse large B cell lymphoma (DLBCL). These lymphomas had morphological features comparable to those of EBV-1-induced DLBCLs, developed at similar rates with equivalent frequencies, and expressed a latency III gene profile. Thus, despite the impaired ability of EBV-2 to immortalize B cells in vitro, EBV-2 efficiently induces lymphomagenesis in humanized mice. Further research utilizing this model will enhance our understanding of EBV-2 biology, the consequence of EBV infection of T cells, and the capacity of EBV-2 to drive lymphomagenesis.IMPORTANCE EBV is a well-established B cell-tropic virus. However, we have recently shown that the EBV type 2 (EBV-2) strain also infects primary T cells in culture and in healthy Kenyan children. This finding suggests that EBV-2, unlike the well-studied EBV-1 strain, utilizes the T cell compartment to persist. As EBV is human specific, studies to understand the role of T cells in EBV-2 persistence require an in vivo model. Thus, we developed an EBV-2 humanized mouse model, utilizing immunodeficient mice engrafted with human cord blood CD34+ stem cells. Characterization of the EBV-2-infected humanized mice established that both T cells and B cells are infected by EBV-2 and that the majority of infected mice develop a B cell lymphoma resembling diffuse large B cell lymphoma. This new in vivo model can be utilized for studies to enhance our understanding of how EBV-2 infection of T cells contributes to persistence and lymphomagenesis.

Oral sensitization to whey proteins induces age- and sex-dependent behavioral abnormality and neuroinflammatory responses in a mouse model of food allergy: a potential role of mast cells.

BACKGROUND: Growing evidence has strengthened the association of food allergy with neuropsychiatric symptoms such as depression, anxiety, and autism. However, underlying mechanisms by which peripheral allergic responses lead to behavioral dysfunction are yet to be determined. Allergen-activated mast cells may serve as mediators by releasing histamine and other inflammatory factors that could adversely affect brain function. We hypothesized that eliciting food allergy in experimental animals would result in behavioral changes accompanied by mast cell accumulation in the brain. Our hypothesis was tested in a mouse model of milk allergy using bovine milk whey proteins (WP) as the allergen. METHODS: Male and female C57BL/6 mice at 4 weeks (young) and 10 months (old) of age underwent 5-week WP sensitization with weekly intragastric administration of 20 mg WP and 10 µg cholera toxin as an adjuvant. Age-matched sham animals were given the vehicle containing only the adjuvant. All animals were orally challenged with 50 mg WP in week 6 and their intrinsic digging behavior was assessed the next day. Animals were sacrificed 3 days after the challenge, and WP-specific serum IgE, intestinal and brain mast cells, glial activation, and epigenetic DNA modification in the brain were examined. RESULTS: WP-sensitized males showed significantly less digging activity than the sham males in both age groups while no apparent difference was observed in females. Mast cells and their activities were evident in the intestines in an age- and sex-dependent manner. Brain mast cells were predominantly located in the region between the lateral midbrain and medial hippocampus, and their number increased in the WP-sensitized young, but not old, male brains. Noticeable differences in for 5-hydroxymethylcytosine immunoreactivity were observed in WP mice of both age groups in the amygdala, suggesting epigenetic regulation. Increased microglial Iba1 immunoreactivity and perivascular astrocytes hypertrophy were also observed in the WP-sensitized old male mice. CONCLUSIONS: Our results demonstrated that food allergy induced behavioral abnormality, increases in the number of mast cells, epigenetic DNA modification in the brain, microgliosis, and astrocyte hypertrophy in a sex- and age-dependent manner, providing a potential mechanism by which peripheral allergic responses evoke behavioral dysfunction.

Looking Behavior and Audiovisual Speech Understanding in Children With Normal Hearing and Children With Mild Bilateral or Unilateral Hearing Loss.

OBJECTIVES: Visual information from talkers facilitates speech intelligibility for listeners when audibility is challenged by environmental noise and hearing loss. Less is known about how listeners actively process and attend to visual information from different talkers in complex multi-talker environments. This study tracked looking behavior in children with normal hearing (NH), mild bilateral hearing loss (MBHL), and unilateral hearing loss (UHL) in a complex multi-talker environment to examine the extent to which children look at talkers and whether looking patterns relate to performance on a speech-understanding task. It was hypothesized that performance would decrease as perceptual complexity increased and that children with hearing loss would perform more poorly than their peers with NH. Children with MBHL or UHL were expected to demonstrate greater attention to individual talkers during multi-talker exchanges, indicating that they were more likely to attempt to use visual information from talkers to assist in speech understanding in adverse acoustics. It also was of interest to examine whether MBHL, versus UHL, would differentially affect performance and looking behavior. DESIGN: Eighteen children with NH, eight children with MBHL, and 10 children with UHL participated (8-12 years). They followed audiovisual instructions for placing objects on a mat under three conditions: a single talker providing instructions via a video monitor, four possible talkers alternately providing instructions on separate monitors in front of the listener, and the same four talkers providing both target and nontarget information. Multi-talker background noise was presented at a 5 dB signal-to-noise ratio during testing. An eye tracker monitored looking behavior while children performed the experimental task. RESULTS: Behavioral task performance was higher for children with NH than for either group of children with hearing loss. There were no differences in performance between children with UHL and children with MBHL. Eye-tracker analysis revealed that children with NH looked more at the screens overall than did children with MBHL or UHL, though individual differences were greater in the groups with hearing loss. Listeners in all groups spent a small proportion of time looking at relevant screens as talkers spoke. Although looking was distributed across all screens, there was a bias toward the right side of the display. There was no relationship between overall looking behavior and performance on the task. CONCLUSIONS: The present study examined the processing of audiovisual speech in the context of a naturalistic task. Results demonstrated that children distributed their looking to a variety of sources during the task, but that children with NH were more likely to look at screens than were those with MBHL/UHL. However, all groups looked at the relevant talkers as they were speaking only a small proportion of the time. Despite variability in looking behavior, listeners were able to follow the audiovisual instructions and children with NH demonstrated better performance than children with MBHL/UHL. These results suggest that performance on some challenging multi-talker audiovisual tasks is not dependent on visual fixation to relevant talkers for children with NH or with MBHL/UHL.