Dietary supplements, guideline alignment and biochemical nutrient status in pregnancy: Findings from the Queensland Family Cohort pilot study.

In high-income nations, multiple micronutrient (MMN) supplementation during pregnancy is a common practice. We aimed to describe maternal characteristics associated with supplement use and daily dose of supplemental nutrients consumed in pregnancy, and whether guideline alignment and nutrient status are related to supplement use. The Queensland Family Cohort is a prospective, Australian observational longitudinal study. Maternal characteristics, nutrient intake from food and supplements, and biochemical nutrient status were assessed in the second trimester (n = 127). Supplement use was reported by 89% of participants, of whom 91% reported taking an MMN supplement. Participants who received private obstetric care, had private health insurance and had greater alignment to meat/vegetarian alternatives recommendations were more likely to report MMN supplement use. Private obstetric care and general practitioner shared care were associated with higher daily dose of supplemental nutrients consumed compared with midwifery group practice. There was high reliance on supplements to meet nutrient reference values for folate, iodine and iron, but only plasma folate concentrations were higher in MMN supplement versus nonsupplement users. Exceeding the upper level of intake for folic acid and iron was more likely among combined MMN and individual supplement/s users, and associated with higher plasma concentrations of the respective nutrients. Given the low alignment with food group recommendations and potential risks associated with high MMN supplement use, whole food diets should be emphasized. This study confirms the need to define effective strategies for optimizing nutrient intake in pregnancy, especially among those most vulnerable where MMN supplement use may be appropriate.

Prenatal alcohol consumption and placental outcomes: a systematic review and meta-analysis of clinical studies.

OBJECTIVE: A systematic review was conducted to determine placental outcomes following prenatal alcohol exposure in women. DATA SOURCES: The search terms "maternal OR prenatal OR pregnant OR periconception" AND "placenta" AND "alcohol OR ethanol" were used across 5 databases (PubMed, Embase, Cochrane Library, Web of Science, and CINAHL) from inception until November 2020. STUDY ELIGIBILITY CRITERIA: Articles were included if they reported placental outcomes in an alcohol exposure group compared with a control group. Studies were excluded if placentas were from elective termination before 20 weeks' gestation, animal studies, in vitro studies, case studies, or coexposure studies. METHODS: Study quality was assessed by 2 reviewers using the Newcastle-Ottawa Quality Assessment Scale. Title and abstract screening was conducted by 2 reviewers to remove duplicates and irrelevant studies. Remaining full text articles were screened by 2 reviewers against inclusion and exclusion criteria. Placental outcome data were extracted and tabulated separately for studies of placentation, placental weight, placental morphology, and placental molecular studies. Meta-analyses were conducted for outcomes reported by >3 studies. RESULTS: Database searching retrieved 640 unique records. Screening against inclusion and exclusion criteria resulted in 33 included studies. The quality assessment identified that 61% of studies were high quality, 30% were average quality, and 9% were low quality. Meta-analyses indicated that prenatal alcohol exposure increased the likelihood of placental abruption (odds ratio, 1.48; 95% confidence interval, 1.37-1.60) but not placenta previa (odds ratio, 1.14; 95% confidence interval, 0.84-1.34) and resulted in a reduction in placental weight of 51 g (95% confidence interval, -82.8 to -19.3). Reports of altered placental vasculature, placental DNA methylation, and gene expression following prenatal alcohol exposure were identified. A single study examined placentas from male and female infants separately and found sex-specific placental outcomes. CONCLUSION: Prenatal alcohol exposure increases the likelihood of placental abruption and is associated with decreased placental weight, altered placental vasculature, DNA methylation, and molecular pathways. Given the critical role of the placenta in determining pregnancy outcomes, further studies investigating the molecular mechanisms underlying alcohol-induced placental dysfunction are required. Sex-specific placental adaptations to adverse conditions in utero have been well documented; thus, future studies should examine prenatal alcohol exposure-associated placental outcomes separately by sex.

Phase I trial of intravesical recombinant adenovirus mediated interferon-α2b formulated in Syn3 for Bacillus Calmette-Guérin failures in nonmuscle invasive bladder cancer.

PURPOSE: A phase I trial of intravesical recombinant adenovirus mediated interferon-α2b gene therapy (rAd-IFNα) formulated with the excipient SCH Syn3 was conducted in patients with nonmuscle invasive bladder cancer who had disease recurrence after treatment with bacillus Calmette-Guérin. The primary objective was to determine the safety of rAd-IFNα/Syn3. Secondary end points were demonstrated effective rAd-IFNα gene expression and preliminary evidence of clinical activity at 3 months. MATERIALS AND METHODS: A total of 17 patients with recurrent nonmuscle invasive bladder cancer after bacillus Calmette-Guérin treatment were enrolled in the study. A single treatment of rAd-IFNα (3 × 10(9) to 3 × 10(11) particles per ml) formulated with the excipient Syn3 was administered. Patient safety was evaluated for 12 or more weeks. Efficacy of gene transfer was determined by urine IFNα protein concentrations. Preliminary drug efficacy was determined at 3 months. RESULTS: Intravesical rAd-IFNα/Syn3 was well tolerated as no dose limiting toxicity was encountered. Urgency was the most common adverse event and all cases were grade 1 or 2. rAd-IFNα DNA was not detected in the blood. However, transient low serum IFNα and Syn3 levels were measured. High and prolonged dose related urine IFNα levels were achieved with the initial treatment. Of the 14 patients treated at doses of 10(10) or more particles per ml with detectable urine IFNα, 6 (43%) experienced a complete response at 3 months and 2 remained disease-free at 29.0 and 39.2 months, respectively. CONCLUSIONS: Intravesical rAd-IFNα/Syn3 was well tolerated with no dose limiting toxicity encountered. Dose dependent urinary IFNα concentrations confirmed efficient gene transfer and expression. Intravesical rAd-IFNα/Syn3 demonstrated clinical activity in nonmuscle invasive bladder cancer recurring after bacillus Calmette-Guérin.

Freely Available Training Videos for Suicide Prevention: Scoping Review.

BACKGROUND: Freely available and asynchronous implementation supports can reduce the resource burden of evidence-based practice training to facilitate uptake. Freely available web-based training videos have proliferated, yet there have been no efforts to quantify their breadth, depth, and content for suicide prevention. OBJECTIVE: This study presents results from a scoping review of freely available training videos for suicide prevention and describes a methodological framework for reviewing such videos. METHODS: A scoping review of freely available training videos (≥2 minutes) for suicide prevention practices was conducted using 4 large video-sharing platforms: YouTube, Vimeo, Bing Video, and Google Video. Identified suicide prevention training videos (N=506) were reviewed and coded. RESULTS: Most content was targeted toward gatekeepers or other lay providers (n=370) versus clinical providers (n=136). Videos most commonly provided content related to suicidal thoughts or behaviors (n=420). Many videos (n=274, 54.2%) included content designed for certain communities or organizations. Less than half (n=232, 45.8%) of training videos included formal clinical content pertaining to assessment or intervention for suicide prevention. CONCLUSIONS: Results suggested an abundance of videos providing broad informational content (eg, "signs and symptoms of someone at risk for suicide") and a limited portion of videos with instructional content aimed at clinical providers delivering formal evidence-based assessments or interventions for suicide prevention. Development of resources to address identified gaps may be needed. Future work may leverage machine learning techniques to expedite the review process.

Exploring resource implications and models of care for assessment and diagnosis of fetal alcohol spectrum disorder: A scoping review.

Previous reviews have examined annual mean costs of care for individuals with fetal alcohol spectrum disorder (FASD), costs of the health burden, costs to the justice system, productivity losses for caregivers, and both the monetary and nonmonetary costs of reduced quality of life. However, because there have been no published reviews focused on understanding the resource implications and specific service features for the assessment and diagnostic process for FASD, the current scoping review investigated the available evidence on these topics. Eligible studies were identified through a systematic search of six databases and included if they contained information on the potential costs or models of care associated with undertaking an assessment for FASD. Data were charted, underwent content analysis, and were reported according to the PRISMA extension for scoping reviews. Eleven studies were included in the final qualitative synthesis. The primary patient costs were attributed to the lengthy time required for diagnosis (up to 47 h). The primary service costs were attributed to costs of clinicians and support personnel and the involvement of multidisciplinary teams in the assessment process. Estimates of the specific dollar values of diagnostic costs were limited and varied between studies. Several models of care were explored, primarily in Canadian clinics, which aimed to capitalize on available services to improve accessibility and patient care and reduce service costs. This study provides important preliminary insights into the resource implications and models of care involved in the diagnostic assessment of FASD. However, the low number of available studies and variability in available data highlight the need for formal costing studies and detailed information gathering on available models of care to inform future clinical practice and policy development.

Lived experiences of the diagnostic assessment process for fetal alcohol spectrum disorder: A systematic review of qualitative evidence.

Early assessment and diagnosis of FASD are crucial in providing therapeutic interventions that aim to enhance meaningful participation and quality of life for individuals and their families, while reducing psychosocial difficulties that may arise during adolescence and adulthood. Individuals with lived experience of FASD have expertise based on their own lives and family needs. Their insights into the assessment and diagnostic process are valuable for improving service delivery and informing the provision of meaningful, person- and family-centered care. To date, reviews have focused broadly on the experiences of living with FASD. The aim of this systematic review is to synthesize qualitative evidence on the lived experiences of the diagnostic assessment process for FASD. Six electronic databases, including PubMed, the Cochrane Library, CINAH, EMBASE, PsycINFO, and Web of Science Core Collection were searched from inception until February 2021, and updated in December 2022. A manual search of reference lists of included studies identified additional studies for inclusion. The quality of included studies was assessed using the Critical Appraisal Skills Program Checklist for Qualitative Studies. Data from included studies were synthesized using a thematic analysis approach. GRADE-CERQual was used to assess confidence in the review findings. Ten studies met the selection criteria for inclusion in the review. Thematic analysis identified 10 first-level themes relating to four over-arching topics: (1) pre-assessment concerns and challenges, (2) the diagnostic assessment process, (3) receipt of the diagnosis, and (4) post-assessment adaptations and needs. GRADE-CERQual confidence ratings for each of the review themes were moderate to high. The findings from this review have implications for referral pathways, client-centered assessment processes, and post-diagnostic recommendations and support.

No differences in the pharmacodynamics and pharmacokinetics of the thrombin receptor antagonist vorapaxar between healthy Japanese and Caucasian subjects.

BACKGROUND: Vorapaxar, a novel antiplatelet agent in advanced clinical development for the prevention and treatment of atherothrombotic disease, is a potent, orally bioavailable thrombin receptor antagonist selective for the protease-activated receptor 1 (PAR-1). METHODS: Since race/ethnicity may affect the safety, efficacy and dosage of drugs, this study was conducted to evaluate potential differences in the pharmacodynamics, pharmacokinetics and safety of vorapaxar after single (5, 10, 20, or 40 mg) or multiple (0.5, 1, or 2.5 mg once daily) doses in healthy Japanese and matched (gender, age, height, and weight) Caucasian volunteers. RESULTS: Vorapaxar was well tolerated in both Japanese and Caucasian subjects. Pharmacodynamic and pharmacokinetic profiles of vorapaxar in the two racial/ethnic groups were similar. In both racial groups, complete inhibition of platelet aggregation was achieved most rapidly with vorapaxar 40 mg and was consistently achieved and maintained with a 2.5 mg daily maintenance dose. CONCLUSION: There were no substantial differences in the safety, pharmacokinetics or pharmacodynamics of vorapaxar between Japanese and Caucasian subjects.

Altered bone and body composition in children and adolescents with confirmed prenatal alcohol exposure.

Prenatal alcohol exposure can contribute to long term adverse health outcomes. Development of the skeletal system begins at the early embryonic stage and continues into early adulthood but the effect of prenatal alcohol exposure on skeletal growth is relatively unexplored in a clinical population. Here, we performed dual X-ray absorptiometry to examine bone, fat, and muscle accrual in children and adolescents diagnosed with, or at risk of, fetal alcohol spectrum disorders (FASDs). Children (aged 4-9 years) with FASD or at risk of FASD (n = 10) had similar growth to age matched controls (n = 27). By adolescence (aged ≥10 years), those with FASDs (n = 13) were shorter and had lower areal bone mineral density and lean tissue mass than typically developing peers (n = 29). Overall, adolescents diagnosed with FASDs had greater odds of impairments to bone and body composition. These findings highlight the importance of early FASD diagnosis and appropriate post-diagnostic medical follow-up to enable timely, effective interventions to optimize bone and body composition during paediatric growth.

Prevalence and Patterns of Prenatal Alcohol Exposure in Australian Cohort and Cross-Sectional Studies: A Systematic Review of Data Collection Approaches.

This study sought to determine data collection approaches in Australian cohort studies and explore the potential impact on reported prenatal alcohol exposure (PAE) prevalence and patterns. Inclusion criteria were that studies related to a general Australian antenatal population where PAE was assessed and reported. Studies were excluded if they were not peer reviewed, examined the prevalence of PAE in pregnancies complicated by alcohol-use disorders, or were published in a language other than English. A systematic search of five electronic databases (PubMed, Embase, CINAHL, Web of Science, and Scopus) was conducted. Risk of bias was assessed using the Effective Public Health Practice Project quality assessment tool. Results were synthesised using MetaXL. Data from 16 separate birth cohorts (n = 78 articles) were included. Included cohorts were either general cohorts that included alcohol as a variable or alcohol-focused cohorts that were designed with a primary focus on PAE. PAE prevalence was estimated as 48% (95% CI: 38 to 57%). When subgroup analysis was performed, estimates of PAE prevalence when self-administered surveys and interviews were used for data collection were 53% (95% CI: 41% to 64%) and 43% (95% CI: 28% to 59%), respectively. Use of trained assessors was an influencing factor of the prevalence estimates when data were collected via interview. Alcohol-focused studies reported higher prevalence of PAE, regardless of method of survey administration. Where interviewer training is not possible, self-administered questionnaires will likely provide the most reliable PAE estimates. No funding sources are relevant to mention. Review was registered with PROSPERO (CRD42020204853).

Is the link between elevated TSH and gestational diabetes mellitus dependant on diagnostic criteria and thyroid antibody status: a systematic review and meta-analysis.

PURPOSE: Clinical studies have investigated the prevalence of gestational diabetes mellitus (GDM) in women with subclinical hypothyroidism (SCH). While some studies demonstrate a clear association, others do not. It is possible this may be due to varied diagnostic criteria for SCH and the presence of thyroid antibodies (TA). We conducted a meta-analysis, separating patients diagnosed with SCH using a diagnostic cut-off <4.0 mIU/L from those diagnosed using a cut-off >4.0 mIU/L and determined the association with GDM and factored TA status into our analysis. METHODS: A computerised search of five databases including PubMed, Embase, Cochrane Library, Web of Science and CINAHL returned 787 records. Two independent reviewers assessed abstracts and full texts against pre-specified inclusion and exclusion criteria. Ten cohort studies were included in the final analysis. The diagnostic criteria for SCH and incidence of GDM were extracted from each study. Study quality and risk of bias was assessed by two reviewers. RESULTS: TSH levels <4.0 mIU/L for SCH diagnosis was not associated with GDM unless patients were TA positive. Studies that used a diagnostic cut-off >4.0 mIU/L saw a significant increase in the odds of GDM, regardless of TA status (OR = 1.60, 95% CI 1.33-1.93). CONCLUSIONS: Women with TSH levels >4.0 mIU/L have an increased odds of GDM regardless of TA status but at TSH levels <4.0 mIU/L, GDM is dependent on TA status. The use of TSH levels to identify pregnancies at risk of GDM is a novel concept that warrants exploration.

Adherence to Dietary and Physical Activity Guidelines in Australian Undergraduate Biomedical Students and Associations with Body Composition and Metabolic Health: A Cross-Sectional Study.

There is a paucity of data on whether Australian university students are meeting specific nutrient guidelines, and the relationship between diet and physical activity patterns with body composition and metabolic health. In this study, biomedical students from The University of Queensland were recruited (150 males and 211 females, 19-25 years), and nutritional intake (ASA24-Australia) and physical activity levels (Active Australia Survey) quantified. Body composition (height, waist circumference, body mass, BMI, and percentage body fat; BOD POD) and metabolic health (oral glucose tolerance test) were also measured. Median daily energy intake was 6760 kJ in females and 10,338 kJ in males, with more than 30% of total energy coming from energy-dense, nutrient-poor foods. Only 1 in 10 students met fruit or vegetable recommendations, with less than one third meeting recommendations for fibre, calcium, and potassium. Intakes of calcium and iron were particularly low among female students, with only 16% and 6% of students meeting the recommended dietary intake (RDI), respectively. The majority of males and almost half of all females exceeded the suggested dietary target (SDT) for sodium. Sufficient physical activity (≥150 min over ≥5 sessions per week) was met by more than 80% of students. Body composition and blood glucose concentrations were largely normal but an early sign of insulin resistance (HOMA-IR > 2.0), measured in a subset of students, was present in 21% of males and 17% of females. Modest reductions in blood glucose levels and percentage body fat were associated with increasing vigorous activity. Low intakes of fibre, calcium, and potassium could be corrected by increasing fruit, vegetable, and dairy intake, and, among females, health promotion messages focusing on iron-rich foods should be prioritised. While these nutrient deficiencies did not translate into immediate metabolic heath concerns, dietary behaviours can track into adulthood and have lasting effects on overall health.

Sotagliflozin, a Dual SGLT1/2 Inhibitor, Improves Cardiac Outcomes in a Normoglycemic Mouse Model of Cardiac Pressure Overload.

Selective SGLT2 inhibition reduces the risk of worsening heart failure and cardiovascular death in patients with existing heart failure, irrespective of diabetic status. We aimed to investigate the effects of dual SGLT1/2 inhibition, using sotagliflozin, on cardiac outcomes in normal diet (ND) and high fat diet (HFD) mice with cardiac pressure overload. Five-week-old male C57BL/6J mice were randomized to receive a HFD (60% of calories from fat) or remain on ND for 12 weeks. One week later, transverse aortic constriction (TAC) was employed to induce cardiac pressure-overload (50% increase in right:left carotid pressure versus sham surgery), resulting in left ventricular hypertrophic remodeling and cardiac fibrosis, albeit preserved ejection fraction. At 4 weeks post-TAC, mice were treated for 7 weeks by oral gavage once daily with sotagliflozin (10 mg/kg body weight) or vehicle (0.1% tween 80). In ND mice, treatment with sotagliflozin attenuated cardiac hypertrophy and histological markers of cardiac fibrosis induced by TAC. These benefits were associated with profound diuresis and glucosuria, without shifts toward whole-body fatty acid utilization, increased circulating ketones, nor increased cardiac ketolysis. In HFD mice, sotagliflozin reduced the mildly elevated glucose and insulin levels but did not attenuate cardiac injury induced by TAC. HFD mice had vacuolation of proximal tubular cells, associated with less profound sotagliflozin-induced diuresis and glucosuria, which suggests dampened drug action. We demonstrate the utility of dual SGLT1/2 inhibition in treating cardiac injury induced by pressure overload in normoglycemic mice. Its efficacy in high fat-fed mice with mild hyperglycemia and compromised renal morphology requires further study.

The Impact of Isolation Measures Due to COVID-19 on Energy Intake and Physical Activity Levels in Australian University Students.

The coronavirus disease 2019 (COVID-19) pandemic resulted in physical isolation measures in many parts of the world. In Australia, nationwide restrictions included staying at home, unless seeking medical care, providing care, purchasing food, undertaking exercise, or attending work in an essential service. All undergraduate university classes transitioned to online, mostly home-based learning. We, therefore, examined the effect of isolation measures during the early phase of the COVID-19 pandemic in Australia (March/April) on diet (24-h recall) and physical activity (Active Australia Survey) patterns in third-year biomedical students. Findings were compared with students enrolled in the same course in the previous two years. In females, but not males, energy intake was ~20% greater during the pandemic, and snacking frequency and energy density of consumed snacks also increased compared with 2018 and 2019. Physical activity was impacted for both sexes during the pandemic with ~30% fewer students achieving "sufficient" levels of activity, defined by at least 150 min over at least five sessions, compared with the previous two years. In a follow-up study six to eight weeks later (14-18% response rate), during gradual easing of nationwide restrictions albeit continued gym closures and online learning, higher energy intake in females and reduced physical activity levels in both sexes persisted. These data demonstrate the health impacts of isolation measures, with the potential to affect long-term diet and activity behaviours.

Engaging student health organizations in reducing health disparities in underserved communities through volunteerism: developing a student health corps.

One underutilized method for reducing health disparities and training culturally competent health care workers is the engagement of undergraduate student health organizations in conducting health screenings, promotion, and health education outreach activities in in underserved racial/ethnic communities. We conducted a needs assessment of 14 predominantly racial/ethnic minority undergraduate student-run health organizations. The 14 organizations annually served approximately 12,425 people (67% Hispanic, 25% African American, 6.33% Asian Pacific Islander), predominantly at health fairs within Los Angeles County (averaging 138 attendees). Student organizations provided screenings on general health conditions and diseases, with less emphasis on behavioral risk factors (e.g., drinking, smoking). Organizations indicated a need for increased and affordable training in preventive health screenings and help in understanding target populations' needs. Universities are in an excellent position to train, supervise, and organize volunteer health corps in order to engage students in reducing health disparities and to train culturally competent health care providers.

The effect of multiple doses of ketoconazole or rifampin on the single- and multiple-dose pharmacokinetics of vorapaxar.

This randomized, open-label, parallel-group study evaluated the effects of multiple-dose ketoconazole or rifampin on the single- and multiple-dose pharmacokinetics of vorapaxar. Healthy subjects randomly received one of the following three treatments (N = 12/group): (1) ketoconazole 400 mg once daily (QD) for 28 days (Days 1-28) and single-dose vorapaxar 20 mg on Day 7 followed by vorapaxar 2.5 mg QD for 21 days (Days 8-28); (2) rifampin 600 mg QD for 28 days (Days 1-28) and single-dose vorapaxar 20 mg on Day 7 followed by vorapaxar 2.5 mg QD for 21 days (Days 8-28); and (3) placebo QD for 28 days (Days 1-28) and single-dose vorapaxar 20 mg on Day 7 followed by vorapaxar 2.5 mg QD for 21 days (Days 8-28). Ketoconazole increased the steady-state vorapaxar AUC(0-24 h) and C(max) by approximately twofold (GMR [90% CI]: 196% [173,222]; 193% [166,223], respectively), while rifampin decreased vorapaxar AUC(0-24 h) and C(max) by approximately 50% (GMR [90% CI]: 45.5% [40,52]; 61.4% [52,72], respectively) versus vorapaxar alone. Potent CYP3A4 inhibitors or inducers may cause moderate increases or decreases in vorapaxar exposure, respectively, which may have safety and/or efficacy implications; therefore, their concomitant use with vorapaxar is not recommended.

Polybasic KKR motif in the cytoplasmic tail of Nipah virus fusion protein modulates membrane fusion by inside-out signaling.

The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where approximately 20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r(2) = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.

Identification of the optimal DC-SIGN binding site on human immunodeficiency virus type 1 gp120.

Human immunodeficiency virus type 1 (HIV-1) envelope (gp120) binding to DC-SIGN, a C-type lectin that can facilitate HIV infection in cis and in trans, is largely dependent on high-mannose-content moieties. Here, we delineate the N-linked glycosylation (N-glycan) sites in gp120 that contribute to optimal DC-SIGN binding. Soluble DC-SIGN was able to block 2G12 binding to gp120, but not vice versa, suggesting that DC-SIGN binds to a more flexible combination of N-glycans than 2G12. Consistent with this observation, HIV strain JRCSF gp120 prebound to 2G12 was 10-fold more sensitive to mannan competition than gp120 that was not prebound in a DC-SIGN cell surface binding assay. The analysis of multiple mutant forms of the 2G12 epitope revealed one triple glycosylation mutant form, termed 134mut (carrying N293Q, N382Q, and N388Q mutations), that exhibited a significant increase in sensitivity to both mannan competition and endoglycosidase H digestion compared to that of the 124mut form (carrying N293Q, N328Q, and N388Q mutations) and wild-type gp120 in a DC-SIGN binding assay. Importantly, no such differences were observed when binding to Galanthus nivalis was assessed. The 134mut form of gp120 also exhibited decreased binding to DC-SIGN in the context of native envelope spikes on a virion, and virus bearing 134mut exhibited less efficient DC-SIGN-mediated infection in trans. Significantly, 124mut and 134mut differed by only one glycosylation site mutation in each construct, and both 124mut and 134mut viruses exhibited wild-type levels of infectivity when used in a direct infection assay. In summary, while DC-SIGN can bind to a flexible combination of N-glycans on gp120, its optimal binding site overlaps with specific N-glycans within the 2G12 epitope. Conformationally intact envelopes that are DC-SIGN binding deficient can be used to probe the in vivo biological functions of DC-SIGN.