Intermediate conformations of CD4-bound HIV-1 Env heterotrimers.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops1-4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops3-9. To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref. 11), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity.

DEER Spectroscopy Measurements Reveal Multiple Conformations of HIV-1 SOSIP Envelopes that Show Similarities with Envelopes on Native Virions.

HIV-1 Envelope (Env) mediates viral-host membrane fusion after binding host-receptor CD4 and coreceptor. Soluble envelopes (SOSIPs), designed to mimic prefusion conformational states of virion-bound envelopes, are proposed immunogens for eliciting neutralizing antibodies, yet only static structures are available. To evaluate conformational landscapes of ligand-free, CD4-bound, inhibitor-bound, and antibody-bound SOSIPs, we measured inter-subunit distances throughout spin-labeled SOSIPs using double electron-electron resonance (DEER) spectroscopy and compared results to soluble and virion-bound Env structures, and single-molecule fluorescence resonance energy transfer (smFRET)-derived dynamics of virion-bound Envs. Unliganded SOSIP measurements were consistent with closed, neutralizing antibody-bound structures and shielding of non-neutralizing epitopes, demonstrating homogeneity at Env apex, increased flexibility near Env base, and no evidence for the intra-subunit flexibility near Env apex suggested by smFRET. CD4 binding increased inter-subunit distances and heterogeneity, consistent with rearrangements required for coreceptor binding. Results suggest similarities between SOSIPs and virion-bound Envs and demonstrate DEER's relevance for immunogen design.

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies.

The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein1-5 show promise therapeutically and are being evaluated clinically6-8. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies5 in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to 'up' RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and 'down' RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs9. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2.

Antibody Recognition of CD4-Induced Open HIV-1 Env Trimers.

Human immunodeficiency virus type 1 (HIV-1) envelope (Env), a heterotrimer of gp120-gp41 subunits, mediates fusion of the viral and host cell membranes after interactions with the host receptor CD4 and a coreceptor. CD4 binding induces rearrangements in Env trimer, resulting in a CD4-induced (CD4i) open Env conformation. Structural studies of antibodies isolated from infected donors have defined antibody-Env interactions, with one class of antibodies specifically recognizing the CD4i open Env conformation. In this study, we characterized a group of monoclonal antibodies isolated from HIV-1 infected donors (V2i MAbs) that displayed characteristics of CD4i antibodies. Binding experiments demonstrated that the V2i MAbs preferentially recognize CD4-bound open Env trimers. Structural characterizations of V2i MAb-Env-CD4 trimer complexes using single-particle cryo-electron microscopy showed recognition by V2i MAbs using different angles of approach to the gp120 V1V2 domain and the ß2/ß3 strands on a CD4i open conformation Env with no direct interactions of the MAbs with CD4. We also characterized CG10, a CD4i antibody that was raised in mice immunized with a gp120-CD4 complex, bound to an Env trimer plus CD4. CG10 exhibited characteristics similar to those of the V2i antibodies, i.e., recognition of the open Env conformation, but showed direct contacts to both CD4 and gp120. Structural comparisons of these and previously characterized CD4i antibody interactions with Env provide a suggested mechanism for how these antibodies are elicited during HIV-1 infection. IMPORTANCE The RV144 HIV-1 clinical vaccination trial showed modest protection against viral infection. Antibody responses to the V1V2 region of HIV-1 Env gp120 were correlated inversely with the risk of infection, and data from three other clinical vaccine trials suggested a similar signal. In addition, antibodies targeting V1V2 have been correlated with protections from simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) infections in nonhuman primates. We structurally characterized V2i antibodies directed against V1V2 isolated from HIV-1 infected humans in complex with open Env trimers bound to the host receptor CD4. We also characterized a CD4i antibody that interacts with CD4 as well as the gp120 subunit of an open Env trimer. Our study suggests how V2i and CD4i antibodies were elicited during HIV-1 infection.

Role of the kringle-like domain in glycoprotein NMB for its tumorigenic potential.

Glycoprotein NMB (GPNMB) is highly expressed in many types of malignant tumors and thought to be a poor prognostic factor in those cancers, including breast cancer. Glycoprotein NMB is a type IA transmembrane protein that has a long extracellular domain (ECD) and a short intracellular domain (ICD). In general, the ECD of a protein is involved in protein-protein or protein-carbohydrate interactions, whereas the ICD is important for intracellular signaling. We previously reported that GPNMB contributes to the initiation and malignant progression of breast cancer through the hemi-immunoreceptor tyrosine-based activation motif (hemITAM) in its ICD. Furthermore, we showed that the tyrosine residue in hemITAM is involved in induction of the stem-like properties of breast cancer cells. However, the contribution of the ECD to its tumorigenic function has yet to be fully elucidated. In this study, we focused on the region, the so-called kringle-like domain (KLD), that is conserved among species, and made a deletion mutant, GPNMB(ΔKLD). Enhanced expression of WT GPNMB induced sphere and tumor formation in breast epithelial cells; in contrast, GPNMB(ΔKLD) lacked these activities without affecting its molecular properties, such as subcellular localization, Src-induced tyrosine phosphorylation at least in overexpression experiments, and homo-oligomerization. Additionally, GPNMB(ΔKLD) lost its cell migration promoting activity, even though it reduced E-cadherin expression. Although the interaction partner binding to KLD has not yet been identified, we found that the KLD of GPNMB plays an important role in its tumorigenic potential.

Providers' Perceptions and Training Needs for Counseling Adolescents Undergoing Voluntary Medical Male Circumcision.

Background: The majority of individuals who seek voluntary medical male circumcision (VMMC) services in sub-Saharan Africa are adolescents (ages 10-19 years). However, adolescents who obtain VMMC services report receiving little information on human immunodeficiency virus (HIV) prevention and care. In this study, we assessed the perceptions of VMMC facility managers and providers about current training content and their perspectives on age-appropriate adolescent counseling. Methods: Semistructured in-depth interviews were conducted with 33 VMMC providers in Tanzania (n = 12), South Africa (n = 9), and Zimbabwe (n = 12) and with 4 key informant facility managers in each country (total 12). Two coders independently coded the data thematically using a 2-step process and Atlas.ti qualitative coding software. Results: Providers and facility managers discussed limitations with current VMMC training, noting the need for adolescent-specific guidelines and counseling skills. Providers expressed hesitation in communicating complete sexual health information-including HIV testing, HIV prevention, proper condom usage, the importance of knowing a partner's HIV status, and abstinence from sex or masturbation during wound healing-with younger males (aged <15 years) and/or those assumed to be sexually inexperienced. Many providers revealed that they did not assess adolescent clients' sexual experience and deemed sexual topics to be irrelevant or inappropriate. Providers preferred counseling younger adolescents with their parents or guardians present, typically focusing primarily on wound care and procedural information. Conclusions: Lack of training for working with adolescents influences the type of information communicated. Preconceptions hinder counseling that supports comprehensive HIV preventive behaviors and complete wound care information, particularly for younger adolescents.

Females' Peer Influence and Support for Adolescent Males Receiving Voluntary Medical Male Circumcision Services.

Background: While female involvement in voluntary medical male circumcision (VMMC) has been studied among adults, little is known about the influence of adolescent females on their male counterparts. This study explored adolescent females' involvement in VMMC decision making and the postoperative wound healing process in South Africa, Tanzania, and Zimbabwe. Methods: Across 3 countries, 12 focus group discussions were conducted with a total of 90 adolescent females (aged 16-19 years). Individual in-depth interviews were conducted 6-10 weeks post-VMMC with 92 adolescent males (aged 10-19 years). Transcribed and translated qualitative data were coded into categories and subcategories by 2 independent coders. Results: Adolescent female participants reported being supportive of male peers' decisions to seek VMMC, with the caveat that some thought VMMC gives males a chance to be promiscuous. Regardless, females from all countries expressed preference for circumcised over uncircumcised sexual partners. Adolescent females believed VMMC to be beneficial for the sexual health of both partners, viewed males with a circumcised penis as more attractive than uncircumcised males, used their romantic relationships with males or the potential for sex as leveraging points to convince males to become circumcised, and demonstrated supportive attitudes in the wound-healing period. Interviews with males confirmed that encouragement from females was a motivating factor in seeking VMMC. Conclusions: Adolescent female participants played a role in convincing young males to seek VMMC and remained supportive of the decision postprocedure. Programs aiming to increase uptake of VMMC and other health-related initiatives for adolescent males should consider the perspective and influence of adolescent females.

Age Differences in Perceptions of and Motivations for Voluntary Medical Male Circumcision Among Adolescents in South Africa, Tanzania, and Zimbabwe.

Background: The World Health Organization (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS) have set a Fast-Track goal to achieve 90% coverage of voluntary medical male circumcision (VMMC) among boys and men aged 10-29 years in priority settings by 2021. We aimed to identify age-specific facilitators of VMMC uptake among adolescents. Methods: Younger (aged 10-14 years; n = 967) and older (aged 15-19 years; n = 559) male adolescents completed structured interviews about perceptions of and motivations for VMMC before receiving VMMC counseling at 14 service provision sites across South Africa, Tanzania, and Zimbabwe. Adjusted prevalence ratios (aPRs) were estimated using multivariable modified Poisson regression models with generalized estimating equations and robust standard errors. Results: The majority of adolescents reported a strong desire for VMMC. Compared with older adolescents, younger adolescents were less likely to cite protection against human immunodeficiency virus (HIV) or other sexually transmitted infections (aPR, 0.77; 95% confidence interval [CI], .66-.91) and hygienic reasons (aPR, 0.55; 95% CI, .39-.77) as their motivation to undergo VMMC but were more likely to report being motivated by advice from others (aPR, 1.88; 95% CI, 1.54-2.29). Although most adolescents believed that undergoing VMMC was a normative behavior, younger adolescents were less likely to perceive higher descriptive norms (aPR, 0.79; .71-.89), injunctive norms (aPR, 0.86; 95% CI, .73-1.00), or anticipated stigma for being uncircumcised (aPR, 0.79; 95% CI, .68-.90). Younger adolescents were also less likely than older adolescents to correctly cite that VMMC offers men and boys partial HIV protection (aPR, 0.73; 95% CI, .65-.82). Irrespective of age, adolescents' main concern about undergoing VMMC was pain (aPR, 0.95; 95% CI, .87-1.04). Among younger adolescents, fear of pain was negatively associated with desire for VMMC (aPR, 0.89; 95% CI, .83-.96). Conclusions: Age-specific strategies are important to consider to generate sustainable demand for VMMC. Programmatic efforts should consider building on the social norms surrounding VMMC and aim to alleviate fears about pain.

Counseling Received by Adolescents Undergoing Voluntary Medical Male Circumcision: Moving Toward Age-Equitable Comprehensive Human Immunodeficiency Virus Prevention Measures.

Background: The minimum package of voluntary medical male circumcision (VMMC) services, as defined by the World Health Organization, includes human immunodeficiency virus (HIV) testing, HIV prevention counseling, screening/treatment for sexually transmitted infections, condom promotion, and the VMMC procedure. The current study aimed to assess whether adolescents received these key elements. Methods: Quantitative surveys were conducted among male adolescents aged 10-19 years (n = 1293) seeking VMMC in South Africa, Tanzania, and Zimbabwe. We used a summative index score of 8 self-reported binary items to measure receipt of important elements of the World Health Organization-recommended HIV minimum package and the US President's Emergency Plan for AIDS Relief VMMC recommendations. Counseling sessions were observed for a subset of adolescents (n = 44). To evaluate factors associated with counseling content, we used Poisson regression models with generalized estimating equations and robust variance estimation. Results: Although counseling included VMMC benefits, little attention was paid to risks, including how to identify complications, what to do if they arise, and why avoiding sex and masturbation could prevent complications. Overall, older adolescents (aged 15-19 years) reported receiving more items in the recommended minimum package than younger adolescents (aged 10-14 years; adjusted ß, 0.17; 95% confidence interval [CI], .12-.21; P < .001). Older adolescents were also more likely to report receiving HIV test education and promotion (42.7% vs 29.5%; adjusted prevalence ratio [aPR], 1.53; 95% CI, 1.16-2.02) and a condom demonstration with condoms to take home (16.8% vs 4.4%; aPR, 2.44; 95% CI, 1.30-4.58). No significant age differences appeared in reports of explanations of VMMC risks and benefits or uptake of HIV testing. These self-reported findings were confirmed during counseling observations. Conclusions: Moving toward age-equitable HIV prevention services during adolescent VMMC likely requires standardizing counseling content, as there are significant age differences in HIV prevention content received by adolescents.

Adolescent Wound-Care Self-Efficacy and Practices After Voluntary Medical Male Circumcision-A Multicountry Assessment.

Background: Adolescent boys (aged 10-19 years) constitute the majority of voluntary medical male circumcision (VMMC) clients in sub-Saharan Africa. They are at higher risk of postoperative infections compared to adults. We explored adolescents' wound-care knowledge, self-efficacy, and practices after VMMC to inform strategies for reducing the risks of infectious complications postoperatively. Methods: Quantitative and qualitative data were collected in South Africa, Tanzania, and Zimbabwe between June 2015 to September 2016. A postprocedure survey was conducted approximately 7-10 days after VMMC among male adolescents (n = 1293) who had completed a preprocedure survey; the postprocedure survey assessed knowledge of proper wound care and wound-care self-efficacy. We also conducted in-depth interviews (n = 92) with male adolescents 6-10 weeks after the VMMC procedure to further explore comprehension of providers' wound-care instructions as well as wound-care practices, and we held 24 focus group discussions with randomly selected parents/guardians of the adolescents. Results: Adolescent VMMC clients face multiple challenges with postcircumcision wound care owing to factors such as forgetting, misinterpreting, and disregarding provider instructions. Although younger adolescents stated that parental intervention helped them overcome potential hindrances to wound care, parents and guardians lacked crucial information on wound care because most had not attended counseling sessions. Some older adolescents reported ignoring symptoms of infection and not returning to the clinic for review when an adverse event had occurred. Conclusions: Increased involvement of parents/guardians in wound-care counseling for younger adolescents and in wound-care supervision, alongside the development of age-appropriate materials on wound care, are needed to minimize postoperative complications after VMMC.

Parental Communication, Engagement, and Support During the Adolescent Voluntary Medical Male Circumcision Experience.

Background: Voluntary medical male circumcision (VMMC) is one of few opportunities in sub-Saharan Africa to engage male adolescents in the healthcare system. Limited data are available on the level of parental communication, engagement, and support adolescents receive during the VMMC experience. Methods: We conducted 24 focus group discussions with parents/guardians of adolescents (N = 192) who agreed to be circumcised or were recently circumcised in South Africa, Tanzania, and Zimbabwe. In addition, male adolescents (N = 1293) in South Africa (n = 299), Tanzania (n = 498), and Zimbabwe (n = 496) were interviewed about their VMMC experience within 7-10 days postprocedure. We estimated adjusted prevalence ratios (aPRs) using multivariable Poisson regression with generalized estimating equations and robust standard errors. Results: Parents/guardians noted challenges and gaps in communicating with their sons about VMMC, especially when they did not accompany them to the clinic. Adolescents aged 10-14 years were significantly more likely than 15- to 19-year-olds to report that their parent accompanied them to a preprocedure counseling session (56.5% vs 12.5%; P < .001). Among adolescents, younger age (aPR, 0.86; 95% confidence interval [CI], .76-.99) and rural setting (aPR, 0.34; 95% CI, .13-.89) were less likely to be associated with parental-adolescent communication barriers, while lower socioeconomic status (aPR, 1.37; 95% CI, 1.00-1.87), being agnostic (or of a nondominant religion; aPR, 2.87; 95% CI, 2.21-3.72), and living in South Africa (aPR, 2.63; 95% CI, 1.29-4.73) were associated with greater perceived barriers to parental-adolescent communication about VMMC. Parents/guardians found it more difficult to be involved in wound care for older adolescents than for adolescents <15 years of age. Conclusions: Parents play a vital role in the VMMC experience, especially for younger male adolescents. Strategies are needed to inform parents completely throughout the VMMC adolescent experience, whether or not they accompany their sons to clinics.

Perceived Quality of In-Service Communication and Counseling Among Adolescents Undergoing Voluntary Medical Male Circumcision.

Background: Experience with providers shapes the quality of adolescent health services, including voluntary medical male circumcision (VMMC). This study examined the perceived quality of in-service communication and counseling during adolescent VMMC services. Methods: A postprocedure quantitative survey measuring overall satisfaction, comfort, perceived quality of in-service communication and counseling, and perceived quality of facility-level factors was administered across 14 VMMC sites in South Africa, Tanzania, and Zimbabwe. Participants were adolescent male clients aged 10-14 years (n = 836) and 15-19 years (n = 457) and completed the survey 7 to 10 days following VMMC. Adjusted prevalence ratios (aPRs) were estimated by multivariable modified Poisson regression with generalized estimating equations and robust variance estimation to account for site-level clustering. Results: Of 10- to 14-year-olds and 15- to 19-year-olds, 97.7% and 98.7%, respectively, reported they were either satisfied or very satisfied with their VMMC counseling experience. Most were also very likely or somewhat likely (93.6% of 10- to 14-year olds and 94.7% of 15- to 19-year olds) to recommend VMMC to their peers. On a 9-point scale, the median perceived quality of in-service (counselor) communication was 9 (interquartile range [IQR], 8-9) among 15- to 19-year-olds and 8 (IQR, 7-9) among 10- to 14-year-olds. The 10- to 14-year-olds were more likely than 15- to 19-year-olds to perceive a lower quality of in-service (counselor) communication (score <7; 21.5% vs. 8.2%; aPR, 1.61 [95% confidence interval, 1.33-1.95]). Most adolescents were more comfortable with a male rather than female counselor and provider. Adolescents of all ages wanted more discussion about pain, wound care, and healing time. Conclusions: Adolescents perceive the quality of in-service communication as high and recommend VMMC to their peers; however, many adolescents desire more discussion about key topics outlined in World Health Organization guidance.

Impact of Counseling Received by Adolescents Undergoing Voluntary Medical Male Circumcision on Knowledge and Sexual Intentions.

Background: Little is known regarding the impact of counseling delivered during voluntary medical male circumcision (VMMC) services on adolescents' human immunodeficiency virus (HIV) knowledge, VMMC knowledge, or post-VMMC preventive sexual intentions. This study assessed the effect of counseling on knowledge and intentions. Methods: Surveys were conducted with 1293 adolescent clients in 3 countries (South Africa, n = 299; Tanzania, n = 498; Zimbabwe, n = 496). Adolescents were assessed on HIV and VMMC knowledge-based items before receiving VMMC preprocedure counseling and at a follow-up survey approximately 10 days postprocedure. Sexually active adolescents were asked about their sexual intentions in the follow-up survey. Prevalence ratios (PRs) and 95% confidence intervals (CIs) were calculated by modified Poisson regression models with generalized estimating equations and robust variance estimators. Results: Regarding post-VMMC HIV prevention knowledge, older adolescents were significantly more likely than younger adolescents to know that a male should use condoms (age 10-14 years, 41.1%; 15-19 years, 84.2%; aPR, 1.38 [95% CI, 1.19-1.60]), have fewer sex partners (age 10-14 years, 8.1%; age 15-19 years, 24.5%; aPR, 2.10 [95% CI, 1.30-3.39]), and be faithful to one partner (age 10-14 years, 5.7%; age 15-19 years, 23.2%; aPR, 2.79 [95% CI, 1.97-3.97]) to further protect himself from HIV. Older adolescents demonstrated greater improvement in knowledge in most categories, differences that were significant for questions regarding number of sex partners (aPR, 2.01 [95% CI, 1.18-3.44]) and faithfulness to one partner post-VMMC (aPR, 3.28 [95% CI, 2.22-4.86]). However, prevention knowledge levels overall and HIV risk reduction sexual intentions among sexually active adolescents were notably low, especially given that adolescents had been counseled only 7-10 days prior. Conclusions: Adolescent VMMC counseling needs to be improved to increase knowledge and postprocedure preventive sexual intentions.

Measuring pediatric quality of care in rural clinics-a multi-country assessment-Cambodia, Guatemala, Zambia and Kenya.

OBJECTIVE: To assess the quality of care provided in rural pediatric facilities in Cambodia, Guatemala, Kenya and Zambia DESIGN: All public health facilities in four districts in each country were included in the assessment. Based on utilization patterns, five children under five were selected randomly from each facility to perform the Integrated Management of Childhood Illness (IMCI) assessments followed by exit interviews with their caretakers. SETTING: Seventy rural ambulatory pediatric care facilities. PARTICIPANTS: Three hundred and forty pediatric case management observations and exit interviews with child caretakers. MAIN OUTCOME MEASURE: IMCI index of observed quality of care for patient assessment and counseling RESULTS: Screening for danger signs, diarrhea and fever showed significant differences between countries (P < 0.001), with facilities in Cambodia and Guatemala performing better. More than 90% of the children were screened for fever in all three countries, but <75% were screened in Cambodia. The assessment of nutritional status, checking weight against growth chart and palmar pallor for anemia, was suboptimal in all countries. Mean consultation time ranged from 8.2 minutes in Zambia and 12.6 minutes in Guatemala. Child age, consultation time, health provider cadre and presenting symptoms were significantly associated with higher quality of assessment and counseling care as measured by the IMCI index. CONCLUSIONS: Achieving the goals of universal health coverage in these contexts must be complimented with accelerated efforts for capacity investments at the primary care level to ensure optimal quality of healthcare and favorable health outcomes for children, who still experience a high disease burden for these common IMCI conditions.

Targeting LINC00152 activates cAMP/Ca2+/ferroptosis axis and overcomes tamoxifen resistance in ER+ breast cancer.

Tamoxifen has been the mainstay therapy to treat early, locally advanced, and metastatic estrogen receptor-positive (ER + ) breast cancer, constituting around 75% of all cases. However, the emergence of resistance is common, necessitating the identification of novel therapeutic targets. Here, we demonstrated that long-noncoding RNA LINC00152 confers tamoxifen resistance by blocking tamoxifen-induced ferroptosis, an iron-mediated cell death. Mechanistically, inhibiting LINC00152 reduces the mRNA stability of phosphodiesterase 4D (PDE4D), leading to activation of the cAMP/PKA/CREB axis and increased expression of the TRPC1 Ca2+ channel. This causes cytosolic Ca2+ overload and generation of reactive oxygen species (ROS) that is, on the one hand, accompanied by downregulation of FTH1, a member of the iron sequestration unit, thus increasing intracellular Fe2+ levels; and on the other hand, inhibition of the peroxidase activity upon reduced GPX4 and xCT levels, in part by cAMP/CREB. These ultimately restore tamoxifen-dependent lipid peroxidation and ferroptotic cell death which are reversed upon chelating Ca2+ or overexpressing GPX4 or xCT. Overexpressing PDE4D reverses LINC00152 inhibition-mediated tamoxifen sensitization by de-activating the cAMP/Ca2+/ferroptosis axis. Importantly, high LINC00152 expression is significantly correlated with high PDE4D/low ferroptosis and worse survival in multiple cohorts of tamoxifen- or tamoxifen-containing endocrine therapy-treated ER+ breast cancer patients. Overall, we identified LINC00152 inhibition as a novel mechanism of tamoxifen sensitization via restoring tamoxifen-dependent ferroptosis upon destabilizing PDE4D, increasing cAMP and Ca2+ levels, thus leading to ROS generation and lipid peroxidation. Our findings reveal LINC00152 and its effectors as actionable therapeutic targets to improve clinical outcome in refractory ER+ breast cancer.

Clinical recognition of mid-aortic syndrome in children.

Mid-aortic syndrome is characterized by narrowing of the abdominal aorta, usually with the involvement of renal arteries and other visceral branches. The combination of the presence of an abdominal bruit, diminished or absent pulsations of the lower extremities, and a blood pressure discrepancy between upper and lower extremities is the classic triad associated with mid-aortic syndrome. However, it has a wide variety of clinical symptoms, and awareness of the variable presentation can lead to early diagnosis of the vascular anomaly. We report three cases presenting at three different stages of this disease, such as hydrops fetalis, refractory hypertension, and intracerebral bleeding. In conclusion, these cases highlight the importance of blood pressure measurements in all patients and accurate physical examination for early recognition of a mid-aortic syndrome.

Structural characterization of HIV-1 Env heterotrimers bound to one or two CD4 receptors reveals intermediate Env conformations.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120/gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops, and in fully-saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops. To investigate changes resulting from sub-stoichiometric CD4 binding, we solved 3.4Å and 3.9Å single-particle cryo-EM structures of soluble, native-like Envs bound to one or two CD4 molecules. Env trimer bound to one CD4 adopted the closed, prefusion Env state. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state that included gp120 outward rotation but maintained the prefusion, three-stranded gp120 bridging sheet and showed only partial V1V2 displacement and V3 exposure. We conclude that engagement of one CD4 molecule was insufficient to stimulate CD4-induced conformational changes, while binding two CD4 molecules led to Env opening in CD4-bound protomers only. Together, these results illuminate HIV-1 Env intermediate conformations and illustrate the structural plasticity of HIV-1 Env.

Timeline of changes in spike conformational dynamics in emergent SARS-CoV-2 variants reveal progressive stabilization of trimer stalk with altered NTD dynamics.

SARS-CoV-2 emergent variants are characterized by increased viral fitness and each shows multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in subsequent variants. Stabilization preceded altered dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from S mutations detail the evolutionary trajectory of S in emerging variants. These carry major implications for SARS-CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.