Venom-inspired somatostatin receptor 4 (SSTR4) agonists as new drug leads for peripheral pain conditions.

Persistent pain affects one in five people worldwide, often with severely debilitating consequences. Current treatment options, which can be effective for mild or acute pain, are ill-suited for moderate-to-severe persistent pain, resulting in an urgent need for new therapeutics. In recent years, the somatostatin receptor 4 (SSTR 4 ), which is expressed in sensory neurons of the peripheral nervous system, has emerged as a promising target for pain relief. However, the presence of several closely related receptors with similar ligand-binding surfaces complicates the design of receptor-specific agonists. In this study, we report the discovery of a potent and selective SSTR 4 peptide, consomatin Fj1, derived from extensive venom gene datasets from marine cone snails. Consomatin Fj1 is a mimetic of the endogenous hormone somatostatin and contains a minimized binding motif that provides stability and drives peptide selectivity. Peripheral administration of synthetic consomatin Fj1 provided analgesia in mouse models of postoperative and neuropathic pain. Using structure-activity studies, we designed and functionally evaluated several Fj1 analogs, resulting in compounds with improved potency and selectivity. Our findings present a novel avenue for addressing persistent pain through the design of venom-inspired SSTR 4 -selective pain therapeutics. One Sentence Summary: Venom peptides from predatory marine mollusks provide new leads for treating peripheral pain conditions through a non-opioid target.

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.

Child-Directed Speech in Noise: Testing Signal- and Code-Based Phonetic Enhancement.

PURPOSE: Talkers adapt their speech according to the demands of their listeners and the communicative context, enhancing the properties of the signal (pitch, intensity) and/or properties of the code (enhancement of phonemic contrasts). This study asked how mothers adapt their child-directed speech (CDS) in ways that might serve the immediate goals of increasing intelligibility, as well as long-term goals of supporting speech and language development in their children. METHOD: Mothers (N = 28) participated in a real-time interactive speech production/perception paradigm, in which mothers instructed their young (3- to 5-year-old) children, or an adult listener, to select the picture corresponding to a target word. The task was performed at low and high levels (56 vs. 75 dB SPL) of background noise to examine the Lombard effects of decreased audibility on speech production. RESULTS: Acoustic-phonetic analyses of CDS and adult-directed speech (ADS) productions of target words and carrier phrase (e.g., "Find pig") revealed that mothers significantly enhanced the mean pitch, pitch variability, and intensity of target words in CDS, particularly at higher background noise levels and for younger children. Mothers produce CDS with a higher signal-to-noise ratio than ADS. However, limited evidence was found for phonetic enhancement of the segmental properties of speech. Although increased category separation was found in the voice onset time of stop consonants, decreased vowel category separation (an anti-enhancement effect) was observed in CDS. CONCLUSIONS: Mothers readily enhance the suprasegmental signal properties of their speech in CDS, but not the acoustic-phonetic properties of phonemes. This study fails to provide evidence of phonetic enhancement in preschool children in a dyadic communication task under noisy listening conditions. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.24645423.

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.

Asymptomatic sensitization to a cow's milk protein induces sustained neuroinflammation and behavioral changes with chronic allergen exposure.

Mouse models of food allergy have contributed to our understanding of various aspects of the disease, including susceptibilities, symptom spectra, cellular mechanisms, and therapeutic approaches. Previously, we used a mouse model of non-anaphylactic cow's milk allergy (CMA) and investigated sex- and strain-dependent differences in immunological, neurological, and behavioral sequelae. We showed that male C57BL/6J mice sensitized to a bovine whey protein, ß-lactoglobulin (BLG; Bos d 5), exhibited anxiety- and depression-like behavior upon acute allergen challenge. Systemic levels of BLG-specific immunoglobulins, cytokines and chemokines were also elevated in the sensitized mice. Furthermore, neuroinflammation and intestinal dysbiosis were evident as the possible causes of the altered behavior. To assess whether frequent allergen exposure influences CMA-associated pathologies over an extended period in this subclinical model, we placed BLG-sensitized mice on a whey protein (WP)-containing or whey-free control (CTL) diet for 3 months. As expected, allergen-specific IgE was significantly elevated in the plasma after completing the 5-week sensitization phase. However, the IgE levels declined in both diet groups after 3 months. In contrast, allergen-specific IgG1 stayed elevated in sensitized mice with the CTL diet, and the WP diet to a lesser extent. Interestingly, BLG-sensitized mice on the WP diet exhibited anxiety-like behavior and a trend toward spatial memory decline compared to the sham or the sensitized mice on the CTL diet. Moreover, increased immunoreactivities for GFAP and Iba1 and elevated levels of CXCL13 and CCL12, the chemokines involved in central leukocyte recruitment and other neurological diseases, were also observed in the brain. We demonstrated that sensitization to the whey protein, particularly with continuous allergen exposure, resulted in persistent neuroinflammation and associated behavioral changes despite lowered allergen-specific immunoglobulin levels. These results suggested that continuous consumption of the offending allergen may lead to adverse consequences in the brain even after desensitization.

HLA-II Alleles Influence Physical and Behavioral Responses to a Whey Allergen in a Transgenic Mouse Model of Cow's Milk Allergy.

The symptoms of food allergies vary significantly between individuals, likely due to genetic determinants. In humans, allergy development is initiated by antigen-presenting cells via class II human leukocyte antigen (HLA-II). The HLA-II gene is highly polymorphic, and its allelic variance is thought to influence the susceptibility of individuals to a particular allergen. However, whether antigen presentation by different HLA-II variants contributes to symptom variation is not clear. We hypothesized that HLA-II allelic variance affects symptom phenotypes, including immediate physical reactions and delayed behavioral changes, in individuals with food hypersensitivity. To test our hypothesis, male and female mice of three transgenic strains expressing an HLA-II variant, DR3, DR15, or DQ8, were used to establish a cow's milk allergy model. Mice were sensitized to a bovine whey allergen, ß-lactoglobulin (BLG; Bos d 5), weekly for 5 weeks, followed by an acute oral allergen challenge. At 30 min post-challenge, BLG-sensitized DR3 mice showed moderate to severe anaphylaxis resulting in perioral redness, swelling, and death. In contrast, DQ8 and DR15 mice were generally asymptomatic. The production of allergen-specific immunoglobulins was also HLA- and sex-dependent. Both male and female DR3 and female DR15 mice significantly increased BLG-specific IgE production, while robust elevation in BLG-specific IgG1 was observed in sensitized DQ8 mice of both sexes and, to a lesser extent, in DR15 males. Furthermore, BLG-sensitized DR15 mice showed sex-specific behavior changes, with males exhibiting mobility changes and anxiety-like behavior and females showing spatial memory impairment. When splenocytes from transgenic mice were stimulated in vitro with BLG, phenotypes of immune cells were HLA- and sex-specific, further underscoring the influence of HLA-II on immune responses. Our results support that HLA-II alleles influence behavioral responses in addition to immune and physical reactions of food allergy, suggesting that certain HLA-II variants may predispose individuals to food-allergy-associated behavioral changes.

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.

Structure of the BAK-activating antibody 7D10 bound to BAK reveals an unexpected role for the α1-α2 loop in BAK activation.

Pro-apoptotic BAK and BAX are activated by BH3-only proteins to permeabilise the outer mitochondrial membrane. The antibody 7D10 also activates BAK on mitochondria and its epitope has previously been mapped to BAK residues in the loop connecting helices α1 and α2 of BAK. A crystal structure of the complex between the Fv fragment of 7D10 and the BAK mutant L100A suggests a possible mechanism of activation involving the α1-α2 loop residue M60. M60 mutants of BAK have reduced stability and elevated sensitivity to activation by BID, illustrating that M60, through its contacts with residues in helices α1, α5 and α6, is a linchpin stabilising the inert, monomeric structure of BAK. Our data demonstrate that BAK's α1-α2 loop is not a passive covalent connector between secondary structure elements, but a direct restraint on BAK's activation.

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.

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.

Salvage Enbloc Thulium Fiber Laser Enucleation of Prostate for Giant Prostatomegaly After Prostatic Urethral Lift.

OBJECTIVE: To demonstrate technique of salvage thylium fiber laser enucleation of prostate in men with history of prior prostatic urethral lift (PUL) implant. PUL is an accepted treatment modality for benign prostatic hyperplasia (BPH) and is currently recommended for surgical management of prostates <80 g in size.1 However, in reality some patients with prostate >80 g also receive PUL.2 A substantial number of these patients may requirement re-treatment for recurrent or persistent lower urinary tract symptoms after PUL.3-5 Patients with larger prostates who fail PUL might be better managed with endoscopic enucleation which is a size-independent modality for treatment of BPH.1,6 Endoscopic enucleation can be accomplished via a variety of energy sources. Thulium fiber laser is a new addition to urologist armamentarium for endoscopic enucleation of prostate. We hereby present a video demonstrating salvage thulium fiber laser enucleation of 198 cc prostate in a men with history of prior PUL. METHODS: A 66-year-old male presented with a history of recurrent urine retention after prior PUL done 2 years prior at an outside institution. Pre-operative international prostate symptom score was 13, maximum uroflow rate was 6.1 ml/sec, residual urine was 26 ml despite maximal medical management. MRI demonstrated a 198 g prostate and PSA was 13.4 ng/dl with negative prostate biopsy. After a detailed discussion of options, he elected endoscopic enucleation. We employed a 550-micron Soltive superpulsed laser fiber set at 2 J and 30 Hz to perform en-bloc enucleation of prostate, and morcellation was performed with the VersaCut Morcellator. We edited the video to demonstrate the technical nuances of this procedure. RESULTS: Surgery was uneventful with enucleation time of 70 minutes and morcellation time of 142 minutes. Implants encountered during enucleation were easily separated with the thulium fiber laser. No attempt was made to remove implants segments extending beyond the plane of enucleation. Morcellation was challenging, with evidence of damage to morcellation probes requiring replacement of 3 morcellator probes. The patient was discharged on post-operative day one after successful voiding trial. At six-week follow up, his international prostate symptom score of 3 with maximum urinary flow rate of 50 mL/sec. He reported no urinary incontinence and his pathology demonstrated BPH. CONCLUSION: This video demonstrates the feasibility of thulium fiber laser enucleation of prostate after PUL, however the findings need to be reproduced in cohort studies. It also demonstrates difficulties encountered during morcellation. For smaller prostates, vaporization may be preferable, thereby avoiding difficulties associated with morcellation.

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.

Optimization of Benzothiazole and Thiazole Hydrazones as Inhibitors of Schistosome BCL-2.

Limited therapeutic options are available for the treatment of human schistosomiasis caused by the parasitic Schistosoma flatworm. The B cell lymphoma-2 (BCL-2)-regulated apoptotic cell death pathway in schistosomes was recently characterized and shown to share similarities with the intrinsic apoptosis pathway in humans. Here, we exploit structural differences in the human and schistosome BCL-2 (sBCL-2) pro-survival proteins toward a novel treatment strategy for schistosomiasis. The benzothiazole hydrazone scaffold previously employed to target human BCL-XL was repurposed as a starting point to target sBCL-2. We utilized X-ray structural data to inform optimization and then applied a scaffold-hop strategy to identify the 5-carboxamide thiazole hydrazone scaffold (43) with potent sBCL-2 activity (IC50 30 nM). Human BCL-XL potency (IC50 13 nM) was inadvertently preserved during the optimization process. The lead analogues from this study exhibit on-target activity in model fibroblast cell lines dependent on either sBCL-2 or human BCL-XL for survival. Further optimization of the thiazole hydrazone class is required to exhibit activity in schistosomes and enhance the potential of this strategy for treating schistosomiasis.

Induction of Hypersensitivity with Purified Beta-Lactoglobulin as a Mouse Model of Cow's Milk Allergy.

Cow's milk allergy is one of the most prevalent food allergies in both children and adults. As dairy products are common dietary ingredients and the prevalence of chronic conditions is on the rise, milk allergy is a growing public health concern. To elucidate underlying mechanisms and develop therapeutic strategies, reliable animal models are essential research tools. Sensitization to a milk protein is the principal procedure for establishing animal models of cow's milk allergy. However, the methods of sensitization vary from laboratory to laboratory, using different milk proteins with different amounts, routes, and durations of allergen exposure during sensitization of varying sex and strains of mice, likely resulting in diverse immunological and physical responses. Furthermore, the sources and potential impurities of milk protein may also produce variable responses. Thus, standardization of sensitization protocol is important, particularly when results are compared across studies. Here, we describe a method to generate a mouse model of cow's milk allergy using purified ß-lactoglobulin as the milk allergen with cholera toxin as an adjuvant in a 5-week oral sensitization protocol.

BAK core dimers bind lipids and can be bridged by them.

BAK and BAX are essential mediators of apoptosis that oligomerize in response to death cues, thereby causing permeabilization of the mitochondrial outer membrane. Their transition from quiescent monomers to pore-forming oligomers involves a well-characterized symmetric dimer intermediate. However, no essential secondary interface that can be disrupted by mutagenesis has been identified. Here we describe crystal structures of human BAK core domain (α2-α5) dimers that reveal preferred binding sites for membrane lipids and detergents. The phospholipid headgroup and one acyl chain (sn2) associate with one core dimer while the other acyl chain (sn1) associates with a neighboring core dimer, suggesting a mechanism by which lipids contribute to the oligomerization of BAK. Our data support a model in which, unlike for other pore-forming proteins whose monomers assemble into oligomers primarily through protein-protein interfaces, the membrane itself plays a role in BAK and BAX oligomerization.

Structure of the Plasmodium falciparum PfSERA5 pseudo-zymogen.

PfSERA5, a significantly abundant protein present within the parasitophorous vacuole (PV) and essential for normal growth during the blood-stage life cycle of the malaria parasite Plasmodium falciparum, displays structural similarity to many other cysteine proteases. However, PfSERA5 does not exhibit any detectable protease activity and therefore the role of the PfSERA5 papain-like domain (PfSERA5E), thought to remain bound to its cognate prodomain, remains unknown. In this study, we present a revised structure of the central PfSERA5E domain at a resolution of 1.2 Å, and the first structure of the "zymogen" of this papain-like domain including its cognate prodomain (PfSERA5PE) to 2.2 Å resolution. PfSERA5PE is somewhat structurally similar to that of other known proenzymes, retaining the conserved overall folding and orientation of the prodomain through, and occluding, the archetypal papain-like catalytic triad "active-site" cleft, in the same reverse direction as conventional prodomains. Our findings are congruent with previously identified structures of PfSERA5E and of similar "zymogens" and provide a foundation for further investigation into the function of PfSERA5.

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.