Overcoming NMR line broadening of nitrogen containing compounds: A simple solution.

This study presents a straightforward solution to the challenge of elucidating the structures of nitrogen containing compounds undergoing isomerization. When spectral line broadening occurs related to isomerization, be it prototropic tautomerism or bond rotations, this poses a significant obstacle to structural elucidation. By adding acids, we demonstrate a simple approach to overcome this issue and effectively sharpen NMR signals for acid stable prototropic tautomers as well as the conformational isomers containing a morpholine or piperazine ring.

Hypoxemia in Young Children Undergoing One-lung Ventilation: A Retrospective Cohort Study.

BACKGROUND: One-lung ventilation in children remains a specialized practice with low case numbers even at tertiary centers, preventing an assessment of best practices. The authors hypothesized that certain case factors may be associated with a higher risk of intraprocedural hypoxemia in children undergoing thoracic surgery and one-lung ventilation. METHODS: The Multicenter Perioperative Outcomes database and a local quality improvement database were queried for documentation of one-lung ventilation in children 2 months to 3 yr of age inclusive between 2010 and 2020. Patients undergoing vascular or other cardiac procedures were excluded. All records were reviewed electronically for the presence of hypoxemia, oxygen saturation measured by pulse oximetry (Spo2) less than 90% for 3 min or more continuously, and severe hypoxemia, Spo2 less than 90% for 5 min or more continuously during one-lung ventilation. Records were also assessed for hypercarbia, end-tidal CO2 greater than 60 mmHg for 5 min or more or a Paco2 greater than 60 on arterial blood gas. Covariates assessed for association with these outcomes included age, weight, American Society of Anesthesiologists (Schaumburg, Illinois) Physical Status 3 or greater, duration of one-lung ventilation, preoperative Spo2 less than 98%, bronchial blocker versus endobronchial intubation, left operative side, video-assisted thoracoscopic surgery, lower tidal volume ventilation (tidal volume less than or equal to 6 ml/kg plus positive end expiratory pressure greater than or equal to 4 cm H2O for more than 80% of the duration of one-lung ventilation), and type of procedure. RESULTS: Three hundred six cases from 15 institutions were included for analysis. Hypoxemia and severe hypoxemia occurred in 81 of 306 (26%) patients and 56 of 306 (18%), respectively. Hypercarbia occurred in 153 of 306 (50%). Factors associated with lower risk of hypoxemia in multivariable analysis included left operative side (odds ratio, 0.45 [95% CI, 0.251 to 0.78]) and bronchial blocker use (odds ratio, 0.351 [95% CI, 0.177 to 0.67]). Additionally, use of a bronchial blocker was associated with a reduced risk of severe hypoxemia (odds ratio, 0.290 [95% CI, 0.125 to 0.62]). CONCLUSIONS: Use of a bronchial blocker was associated with a lower risk of hypoxemia in young children undergoing one-lung ventilation.

Preoperative Risk and the Association between Hypotension and Postoperative Acute Kidney Injury.

BACKGROUND: Despite the significant healthcare impact of acute kidney injury, little is known regarding prevention. Single-center data have implicated hypotension in developing postoperative acute kidney injury. The generalizability of this finding and the interaction between hypotension and baseline patient disease burden remain unknown. The authors sought to determine whether the association between intraoperative hypotension and acute kidney injury varies by preoperative risk. METHODS: Major noncardiac surgical procedures performed on adult patients across eight hospitals between 2008 and 2015 were reviewed. Derivation and validation cohorts were used, and cases were stratified into preoperative risk quartiles based upon comorbidities and surgical procedure. After preoperative risk stratification, associations between intraoperative hypotension and acute kidney injury were analyzed. Hypotension was defined as the lowest mean arterial pressure range achieved for more than 10 min; ranges were defined as absolute (mmHg) or relative (percentage of decrease from baseline). RESULTS: Among 138,021 cases reviewed, 12,431 (9.0%) developed postoperative acute kidney injury. Major risk factors included anemia, estimated glomerular filtration rate, surgery type, American Society of Anesthesiologists Physical Status, and expected anesthesia duration. Using such factors and others for risk stratification, patients with low baseline risk demonstrated no associations between intraoperative hypotension and acute kidney injury. Patients with medium risk demonstrated associations between severe-range intraoperative hypotension (mean arterial pressure less than 50 mmHg) and acute kidney injury (adjusted odds ratio, 2.62; 95% CI, 1.65 to 4.16 in validation cohort). In patients with the highest risk, mild hypotension ranges (mean arterial pressure 55 to 59 mmHg) were associated with acute kidney injury (adjusted odds ratio, 1.34; 95% CI, 1.16 to 1.56). Compared with absolute hypotension, relative hypotension demonstrated weak associations with acute kidney injury not replicable in the validation cohort. CONCLUSIONS: Adult patients undergoing noncardiac surgery demonstrate varying associations with distinct levels of hypotension when stratified by preoperative risk factors. Specific levels of absolute hypotension, but not relative hypotension, are an important independent risk factor for acute kidney injury.

Succinylcholine Use and Dantrolene Availability for Malignant Hyperthermia Treatment: Database Analyses and Systematic Review.

BACKGROUND: Although dantrolene effectively treats malignant hyperthermia (MH), discrepant recommendations exist concerning dantrolene availability. Whereas Malignant Hyperthermia Association of the United States guidelines state dantrolene must be available within 10 min of the decision to treat MH wherever volatile anesthetics or succinylcholine are administered, a Society for Ambulatory Anesthesia protocol permits Class B ambulatory facilities to stock succinylcholine for airway rescue without dantrolene. The authors investigated (1) succinylcholine use rates, including for airway rescue, in anesthetizing/sedating locations; (2) whether succinylcholine without volatile anesthetics triggers MH warranting dantrolene; and (3) the relationship between dantrolene administration and MH morbidity/mortality. METHODS: The authors performed focused analyses of the Multicenter Perioperative Outcomes Group (2005 through 2016), North American MH Registry (2013 through 2016), and Anesthesia Closed Claims Project (1970 through 2014) databases, as well as a systematic literature review (1987 through 2017). The authors used difficult mask ventilation (grades III and IV) as a surrogate for airway rescue. MH experts judged dantrolene treatment. For MH morbidity/mortality analyses, the authors included U.S. and Canadian cases that were fulminant or scored 20 or higher on the clinical grading scale and in which volatile anesthetics or succinylcholine were given. RESULTS: Among 6,368,356 queried outcomes cases, 246,904 (3.9%) received succinylcholine without volatile agents. Succinylcholine was used in 46% (n = 710) of grade IV mask ventilation cases (median dose, 100 mg, 1.2 mg/kg). Succinylcholine without volatile anesthetics triggered 24 MH cases, 13 requiring dantrolene. Among 310 anesthetic-triggered MH cases, morbidity was 20 to 37%. Treatment delay increased complications every 10 min, reaching 100% with a 50-min delay. Overall mortality was 1 to 10%; 15 U.S. patients died, including 4 after anesthetics in freestanding facilities. CONCLUSIONS: Providers use succinylcholine commonly, including during difficult mask ventilation. Succinylcholine administered without volatile anesthetics may trigger MH events requiring dantrolene. Delayed dantrolene treatment increases the likelihood of MH complications. The data reported herein support stocking dantrolene wherever succinylcholine or volatile anesthetics may be used.

Deuterated active pharmaceutical ingredients: A science-based proposal for synthesis, analysis, and control. Part 1: Framing the problem.

The International Consortium for Innovation & Quality (IQ) in Pharmaceutical Development recently established a working group focused on the development of a guidance to address Deuterated Active Pharmaceutical Ingredients. Deuteration of an Active Pharmaceutical Ingredient (API) in some cases can retard and/or alter API metabolism by exploiting the primary kinetic isotope effect. Several deuterated APIs have entered into the clinic, and one has recently been approved. In most cases, it is very difficult to nearly impossible to synthesize a 100% isotopically pure compound. This raises synthetic, analytical, and regulatory questions that warrant a science-based assessment and recommendations for synthetic methods, analytical methods, and specifications. A cross functional team of scientists with expertise in isotope chemistry, process chemistry, analytical chemistry, and drug metabolism and pharmacokinetics have been meeting under the auspices of IQ to define and address these questions. This paper strives to frame chemistry, manufacturing, and controls challenges.

Improving Mass Spectral Quality of Monoclonal Antibody Middle-Up LC-MS Analysis by Shifting the Protein Charge State Distribution.

Monoclonal antibodies (mAbs) are experiencing accelerated development in the pharmaceutical industry. Utilization of middle-up LC-MS methodology can provide detailed characterization of mAbs via reduction and/or enzymatic cleavage of the mAb into smaller protein fragments. However, under typical LC-MS conditions, these fragments, especially the more heterogeneous heavy chain, can present charge state distributions (CSD) featuring a severe interference in the low mass-to-charge (m/z) region in the mass spectrum, adversely impacting spectral quality of these proteins and ultimately the deconvoluted mass spectrum. Here, we introduce a novel method to characterize protein fragments by partially reducing mAbs and using acidic mobile phases (MPs) with a trace amount of base additive. Gas-phase charge stripping occurs with the basic MP additive, causing the CSD to shift to a higher m/z region resulting in high-quality mass spectra with enhanced resolution of protein charge states. Subsequently, high-quality deconvoluted spectra and accurate mass measurement of the fragments are achieved. This method has been applied to the intact mass measurement of mAbs and antibody drug conjugates.

State-based Medicaid costs for pediatric asthma emergency department visits.

INTRODUCTION: The prevalence of childhood asthma in the United States increased from 8.7% in 2001 to 9.5% in 2011. This increased prevalence adds to the costs incurred by state Medicaid programs. We provide state-based cost estimates of pediatric asthma emergency department (ED) visits and highlight an opportunity for states to reduce these costs through a recently changed Centers for Medicare and Medicaid Services (CMS) regulation. METHODS: We used a cross-sectional design across multiple data sets to produce state-based cost estimates for asthma-related ED visits among children younger than 18, where Medicaid/CHIP (Children's Health Insurance Program) was the primary payer. RESULTS: There were approximately 629,000 ED visits for pediatric asthma for Medicaid/CHIP enrollees, which cost $272 million in 2010. The average cost per visit was $433. Costs ranged from $282,000 in Alaska to more than $25 million in California. CONCLUSIONS: Costs to states for pediatric asthma ED visits vary widely. Effective January 1, 2014, the CMS rule expanded which type of providers can be reimbursed for providing preventive services to Medicaid/CHIP beneficiaries. This rule change, in combination with existing flexibility for states to define practice setting, allows state Medicaid programs to reimburse for asthma interventions that use nontraditional providers (such as community health workers or certified asthma educators) in a nonclinical setting, as long as the service was initially recommended by a physician or other licensed practitioner. The rule change may help states reduce Medicaid costs of asthma treatment and the severity of pediatric asthma.

A programmatic update on COVID-19 vaccination in rural communities in the United States.

When public health experts think of rural barriers to vaccines, they often initially focus on access, which makes sense with a new vaccine during a pandemic. This commentary highlights that there can be more complexity to vaccine uptake in rural communities. What follows are some examples of CDC's efforts to better understand rural health and learnings to inform ongoing vaccination efforts in rural communities.

Teaching, Learning, and Climate Change: Anticipated Impacts and Mitigation Strategies for Educators.

The impacts of climate change present numerous risks to the present and future state of teaching and learning. Natural disasters such as hurricanes, heat waves, flooding, blizzards, wildfires, sea level rise, and droughts threaten our ability to produce the learning outcomes promised to our pupils. Taking action to adapt to imminent climate-related challenges and mitigating measures that provoke and prolong ecological challenges is critical to the survival of these cultural institutions. Paradoxically, centers of teaching and learning can be seen as both victims of climate change as well as an instrumental part of the solution. Providing an efficient and effective education to the world's youth is a catalyst for the innovations that future generations of skilled professionals will use to combat climate change. Educational settings are also crucial venues for raising social awareness about anthropogenic climate change to undermine the complacency and denialism that have stagnated the global response to this crisis thus far. This paper incorporates suggestions from climate scientists and learning scientists about how to change how we teach, where we teach, and what we teach to ensure teaching enterprises survive and thrive in the face of a changing climate.

NMR spectroscopy as a characterization tool enabling biologics formulation development.

This review highlights recent advancements in using high resolution nuclear magnetic resonance (NMR) spectroscopy as a characterization tool to expedite biologics formulation development, meeting a current need in the biopharmaceutical industry. Conformational changes of protein therapeutics during formulation development can result in various protein-protein and protein-excipient interactions, which can lead to physical aggregation and/or chemical degradation. Innovative analytical techniques that allow studying protein integrity with high specificity during formulation development are urgently needed in order to assess protein formulation stability and mitigate product quality risks. Solution NMR spectroscopy is emerging as a powerful analytical tool for biophysical characterization of protein therapeutics. For instance, one-dimensional (1D) NMR has been employed in high sensitivity monitoring of monoclonal antibody (mAb) structural changes and protein-excipient interactions in parenteral formulations, demonstrating it as a potential tool for formulation screening. 2D NMR, such as ALSOFAST-[1H-13C]-HMQC experiments, on the other hand, offer superior capability to detect higher order structural (HOS) changes of mAbs in formulated solutions and their interactions with excipients. These determinations need to be achieved in actual formulations, where proteins of natural abundance are typically at low concentrations depending on the actual dose regimen. Studying proteins with natural abundance in the presence of hundredfold more concentrated excipients makes NMR studies of proteins in formulations extremely difficult considering the sample matrix interferences. Thus, successfully suppressing buffer signals while enhancing the protein signals of interest by optimizing the instrument specific parameters is critically important. Given the large size of typical mAbs, with a molecular weight (MW) ranging from 100 to 240 kDa, coupled with low protein concentrations, data collection becomes a demanding task in terms of NMR instrument time. As such, the biopharmaceutical industry is facing the common challenge of developing innovative NMR approaches to enhance signal detection (sensitivity and selectivity) and reduce experimental/instrument time. XL-ALSOFAST -[1H-13C]-HMQC was recently developed for tackling high MW proteins (up to 240 kDa) with much improved sensitivity and selectivity. We at BMS have implemented the XL-ALSOFAST experiment utilizing its high sensitivity and superior artifact suppression to successfully analyze formulations of several investigational proteins. In this manuscript we will discuss the general utility of this superior tool for studying therapeutic proteins across a range of molecular sizes and buffers. We envisage that this manuscript will serve as a primer to expand the role of NMR spectroscopy as a characterization tool supporting biologics formulation development.

Investigation of Unusual N-(Triphenyl-λ5-phosphanylidene) Amide Fragmentation Observed upon MS/MS Collision-Induced Dissociation.

A mechanism of unusual tandem (MS/MS) fragmentation of protonated species of N-(triphenyl-λ5-phosphanylidene) derivatives, [M + H]+ to generate triphenylphosphine oxide (TPPO) within the mass spectrometer has been investigated and reported. Collision-induced dissociation of these molecules resulted in the generation of TPPO as a signature fragment. This fragment suggested the presence of a P-O bond in the structure which was contrary to the structure of the compound identified by nuclear magnetic resonance spectrometry (NMR) and single-crystal X-ray diffractometry (SXRD) techniques with a P═N bond rather than a P-O bond. In order to confirm the generation of the TPPO fragment within the mass spectrometer, 14 different N-(triphenyl-λ5-phosphanylidene) derivatives containing amide, 18O-labeled amide, thiamide, and nonacyl phosphazene derivatives were synthesized and their MS/MS behavior was studied by liquid chromatography-high-resolution mass spectrometry. Fragmentation of these amide derivatives generated TPPO/TPPS or their 18O-labeled analogues as the major fragment in almost all cases under similar MS conditions. Based on the outcome of these experiments, a plausible mechanism for such fragmentation, involving the intramolecular shifting of oxygen from carbon to phosphorus, has been proposed. DFT calculations for the protonated species at B3LYP-D3/6-31+G(d,p) further supported the proposed mechanism involving a four-membered ring, P-O-C-N, as the transition state. Details of this work are presented here.

Procedures for determining and then modifying the extinction component of multiple schedules for destructive behavior.

As a component of reinforcer schedule thinning following functional communication training, multiple schedules of reinforcement produce desirable rates and patterns of communication responses as an alternative response to destructive behavior. However, reinforcement schedule thinning is a gradual process that can take many sessions to obtain therapeutic goals. The desired outcome is that manding occurs only during signaled intervals of reinforcement with a sufficiently lean terminal schedule of reinforcement availability and low rates of destructive behavior. The purposes of this study were to (a) evaluate an assessment for informing the initial duration of extinction for alternative responding, (b) evaluate the utility of competing stimuli during extinction for alternative responding, and (c) assess a method for fading the availability of competing stimuli. With these procedures, all 4 participants experienced terminal schedules of reinforcement with rapid, robust reductions in destructive behavior soon after baseline. We discuss the implications and directions for future research.

Addressing Racial Disparities in Pregnancy-Related Deaths: An Analysis of Maternal Mortality-Related Federal Legislation, 2017-2021.

There has been increasing national attention to the issue of racial disparities in pregnancy-related deaths. Federal legislation can support approaches at multiple levels of intervention to improve maternal health. As part of the CDC Policy Academy, a team of CDC staff completed a policy analysis to determine the approaches addressed in federal legislation to reduce racial disparities in pregnancy-related deaths. We analyzed federal maternal mortality legislation introduced January 2017 through December 2021. Common approaches addressed by the legislation were categorized into themes and reviewed for their alignment with approaches identified in clinical and public health literature to reduce pregnancy-related deaths, with an emphasis on social determinants of health (SDOH) approaches and reducing racial disparities. Thirty-seven unduplicated bills addressed pregnancy-related deaths, including 27 House or Senate bills that were introduced but not passed, 6 resolutions highlighting the maternal health crisis, 2 bills that passed the House only, and 2 bills enacted into law (Preventing Maternal Deaths Act of 2018 and Protecting Moms Who Served Act). The most common themes mentioned in federal legislation were improving maternal health care, addressing health inequities and SDOH, enhancing data, and promoting women's health. Legislation focused on health inequities and SDOH emphasized implicit bias training and improving SDOH, including racism and other social factors. The reviewed federal legislation reflected common clinical and public health approaches to prevent pregnancy-related deaths, including a significant focus on reducing bias and improving SDOH to address racial disparities.

Circumventing glass vial and diluent effects on solution stability of small molecule analytes during analytical method development and validation.

Solution stability of analytes plays an important part in qualitative analysis, especially in conducting accurate, quantitative analyses. Sample diluents and glass vials as sample containers for HPLC analyses can play a critical role and should be evaluated during chromatographic method development. We have encountered several instances during pharmaceutical development where the glass vial/diluent combination has negatively impacted method performance. One case encompasses adsorption of piperazine, a secondary amine, to non-silanized glass vials, resulting in recovery failures during analytical method transfer. Two further cases describe the propensity for peracetylated C-aryl glucosides being subject chemical transformations relating to sample diluent. The first reports transesterification with methanol-based diluents and the second describes hydrolysis with acetonitrile/water diluents mediated by the mild alkalinity of certain brands of Type I borosilicate vials. A final case explores development of a related substance method, it was found that an impurity was prone to hydrolysis and another impurity with a primary amine tended to be adsorbed on glass vials. Diluents of appropriate pH and buffer strength were strategically selected to neutralize the mild alkalinity of the glass vials as well as to mitigate adsorption of the amine analyte on glass vials. As a result, excellent sample stability and reproducibility were achieved, regardless the quality and brand of Type I glass vials used. Here we present four case studies that demonstrate how the negative impact of Type I glass vials on those susceptible analytes can be effectively eliminated by using appropriate sample diluents, which is essential to ensure accurate analytical data and provide for a smooth method validation and transfer.

Multifunctional nanoparticles as simulants for a gravimetric immunoassay.

Immunoassays are important tools for the rapid detection and identification of pathogens, both clinically and in the research laboratory. An immunoassay with the potential for the detection of influenza was developed and tested using hemagglutinin (HA), a commonly studied glycoprotein found on the surface of influenza virions. Gold nanoparticles were synthesized, which present multiple peptide epitopes, including the HA epitope, in order to increase the gravimetric response achieved with the use of a QCM immunosensor for influenza. Specifically, epitopes associated with HA and FLAG peptides were affixed to gold nanoparticles by a six-mer PEG spacer between the epitope and the terminal cysteine. The PEG spacer was shown to enhance the probability for interaction with antibodies by increasing the distance the epitope extends from the gold surface. These nanoparticles were characterized using thermogravimetric analysis, transmission electron microscopy, matrix-assisted laser desorption/ionization-time of flight, and (1)H nuclear magnetic resonance analysis. Anti-FLAG and anti-HA antibodies were adhered to the surface of a QCM, and the response of each antibody upon exposure to HA, FLAG, and dual functionalized nanoparticles was compared with binding of Au-tiopronin nanoparticles and H5 HA proteins from influenza virus (H5N1). Results demonstrate that the immunoassay was capable of differentiating between nanoparticles presenting orthogonal epitopes in real-time with minimal nonspecific binding. The detection of H5 HA protein demonstrates the logical extension of using these nanoparticle mimics as a safe positive control in the detection of influenza, making this a vital step in improving influenza detection methodology.

Characterizing and understanding the formation of cysteine conjugates and other by-products in a random, lysine-linked antibody drug conjugate.

This study describes the characterization of conjugation sites for a random, lysine conjugated 2-iminothiolane (2-IT) based antibody-drug-conjugate synthesized from an IgG1 antibody and a duocarmycin analog-based payload-linker. Of the 80 putative lysine sites, 78 were found to be conjugated via tryptic peptide mapping and LC-HRMS. Surprisingly, seven cysteine-linked conjugated peptides were also detected resulting from the conjugation of cysteine residues derived from the four inter-chain disulfide bonds during the reaction. This unexpected finding could be attributed to the free thiols of the 2-IT thiolated antibody intermediates and/or the 4-mercaptobutanamide by-product resulting from the hydrolysis of 2-IT. These free thiols could cause the four inter-chain disulfide bonds of the antibody to scramble via intra- or inter-molecular attack. The presence of only pair of non-reactive (unconjugated) lysine residues, along with the four intact intra-chain disulfide bonds, is attributed to their poor accessibility, which is consistent with solvent accessibility modeling analysis. We also discovered a major by-product derived from the hydrolysis of the amidine moiety of the N-terminus conjugate. In contrast, the amidine moiety in lysine-linked conjugates appeared stable. Based on our results, we propose plausible formation mechanisms of cysteine-linked conjugates and the hydrolysis of the N-terminus conjugate, which provide scientific insights that are beneficial to process development and drug quality control.

Evading metal adduct formation during desorption-ionization mass spectrometry.

An investigation into the propensity of metal adduct formation in the recently developed Desorption Ionization by Charge Exchange (DICE) mass spectrometric technique has demonstrated that this method could be utilized to minimize spectral complications caused by metal adducts. For example, peaks for sodium and other metal adducts were not observed in the mass spectra acquired by the ambient-pressure DICE technique from samples deposited on a solid surface, even after the salt content of samples was deliberately increased. A mass spectrum recorded from a urine sample by this technique showed peaks only for the proton adducts of urea and creatinine. This technique employs a nebulized spray of charged toluene droplets for analyte desorption. Because of the non-polar nature of the spray reagent, it neither contains any appreciable amount of cations nor provides any favored 'pickup' of metal cations from the sample matrix. Consequently, peaks for metal adducts that are commonly observed with other desorption techniques are minimal or absent in the spectra recorded by the DICE method.

Investigation of zone migration in a current rectifying nanofluidic/microfluidic analyte concentrator.

A simple microfluidic device that uses a nanocapillary membrane (NCM) to connect a microfluidic channel and solution reservoir is capable of rectifying ionic current and enrichment of ionic species. Application of a potential induces concentration polarization (CP), which creates ion-depleted and ion-enriched zones on opposite sides of the permselective NCM. A force balanced (FB) enriched zone forms at the interface of the bulk buffer solution and depleted CP zone in the off state or the low-current case. After polarity reversal, the migration of a FB enriched zone of anionic tracer is imaged. By decreasing the solution volume at the microchannel and NCM interface, the response time of the current rectifier is decreased and elution of the zone of anionic tracer is achieved. The decrease in response time is most dramatic for the on to off state transition. For this transition, the response time decreases from approximately 50 to approximately 1 s. The decrease in response time for the off to on state is not as dramatic and is characterized by the time from polarity reversal to current peak, which decreased from 84 to 21 s. The features in the I-t plots can be accounted for with schematics of the zone migration that show the migration of depleted CP and enriched CP zones. Together, the fluorescent images and the I-t plots provide the foundation for schematics that describe the zone elution following polarity reversal. These results provide an improved understanding of the zone migration and current rectification in nanofluidic-microfluidic interfaces with symmetric nanochannels.

Children's understanding of second-order mental states.

The most popular topic in theory-of-mind research has been first-order false belief: the realization that it is possible to hold false beliefs about events in the world. A more advanced development is second-order false belief: the realization that it is possible to hold a false belief about someone else's belief. This article reviews research directed to second-order false belief and other forms of higher order, recursive mentalistic reasoning. Three general issues are considered. Research directed to developmental changes indicates that preschoolers typically fail second-order tasks and that success emerges at about age 5 or 6, although results vary some with method of assessment. Research directed to the consequences of second-order competence has revealed positive relations with a number of other aspects of children's development. Finally, measures of both language and executive function relate positively to performance on second-order tasks; the causal bases for the correlations, however, remain to be established. This article concludes with suggestions for future research.