Double Negative Control Research Articles

Using Double Negative Controls to Adjust for Healthy User Bias in a Recombinant Zoster Vaccine Safety Study.

Unmeasured confounding is a major concern in many epidemiologic studies that are not randomized. Negative control methods can detect and reduce confounding by leveraging the proxies of the unmeasured confounders, including negative control outcomes (NCO) and exposures (NCE). An NCO is presumably unaffected by the exposure of interest but would be associated with unmeasured confounders; an NCE presumably does not affect the outcome of interest but would be associated with unmeasured confounders. A recently proposed double negative control method leverages both NCO and NCE for unmeasured confounding bias. To demonstrate this relatively new methodology in pharmacoepidemiologic studies, we re-analyzed data from a prior safety study of Recombinant Zoster Vaccine (RZV). The prior study compared risk of safety outcomes of RZV versus unvaccinated comparators, using logistic regression with propensity score adjustment. We identified NCOs and NCEs that could be used to adjust for unmeasured confounding bias that could arise if RZV recipients are incomparable to the comparators due to unmeasured factors. The double negative control analysis yielded relative risk estimates slightly closer to 1.0 than those reported previously, providing additional evidence of RZV safety that is less vulnerable to unmeasured confounding.

Air pollution below US regulatory standards and cardiovascular diseases using a double negative control approach

Growing evidence suggests that long-term air pollution exposure is a risk factor for cardiovascular mortality and morbidity. However, few studies have investigated air pollution below current regulatory limits, and causal evidence is limited. We use a double negative control approach to examine the association between long-term exposure to air pollution at low concentration and cardiovascular hospitalizations among US Medicare beneficiaries aged ≥65 years between 2000 and 2016. The expected values of the negative outcome control (preceding-year hospitalizations) regressed on exposure and negative exposure control (subsequent-year exposure) are treated as a surrogate for omitted confounders. With analyses separately restricted to low-pollution areas (PM2.5 < 9 μg/m³, NO2 < 75.2 µg/m3 [40 ppb], warm-season O3 < 88.2 μg/m3 [45 ppb]), we observed positive associations of the three pollutants with hospitalization rates of stroke, heart failure, and atrial fibrillation and flutter. The associations generally persisted in demographic subgroups. Stricter national air quality standards should be considered.

A confounding bridge approach for double negative control inference on causal effects

Unmeasured confounding is a key challenge for causal inference. In this paper, we establish a framework for unmeasured confounding adjustment with negative control variables. A negative control outcome is associated with the confounder but not causally affected by the exposure in view, and a negative control exposure is correlated with the primary exposure or the confounder but does not causally affect the outcome of interest. We introduce an outcome confounding bridge function that depicts the relationship between the confounding effects on the primary outcome and the negative control outcome, and we incorporate a negative control exposure to identify the bridge function and the average causal effect. We also consider the extension to the positive control setting by allowing for the nonzero causal effect of the primary exposure on the control outcome. We illustrate our approach with simulations and apply it to a study about the short-term effect of air pollution on mortality. Although a standard analysis shows a significant acute effect of PM2.5 on mortality, our analysis indicates that this effect may be confounded, and after double negative control adjustment, the effect is attenuated toward zero.

Interaction networks of Escherichia coli replication proteins under different bacterial growth conditions

In this work we analyzed protein-protein interactions (PPIs) formed by E. coli replication proteins under three disparate bacterial growth conditions. The chosen conditions corresponded to fast exponential growth, slow exponential growth and growth cessation at the stationary phase. We performed affinity purification coupled with mass spectrometry (AP-MS) of chromosomally expressed proteins (DnaA, DnaB, Hda, SeqA, DiaA, DnaG, HolD, NrdB), tagged with sequential peptide affinity (SPA) tag. Composition of protein complexes was characterized using MaxQuant software. To filter out unspecific interactions, we employed double negative control system and we proposed qualitative and quantitative data analysis strategies that can facilitate hits identification in other AP-MS datasets. Our motivation to undertake this task was still insufficient understanding of molecular mechanisms coupling DNA replication to cellular growth. Previous works suggested that such control mechanisms could involve physical interactions of replication factors with metabolic or cell envelope proteins. However, the dynamic replication protein interaction network (PIN) obtained in this study can be used to characterize links between DNA replication and various cellular processes in other contexts.

Statistical methods of unmeasured confounder control based on negative control theory

Controlling unmeasured confounders in non-randomized controlled studies is challenging. Negative control theory is based on the theoretical concept that the test result of negative controls must be negative. Setting appropriate negative control incorporates the specificity of association into population studies for the identification and control of unmeasured confounders. This paper explains the principles to control unmeasured confounders using negative control theory from a statistical perspective. A detailed introduction of derived methods based on negative control theory is also introduced, including adjusted standardized mortality ratio method, calibrating P-value method, generalized difference-in-difference model and double negative control method. The reasonable application of those derived methods is also comprehensively summarized based on representative case studies. Negative control is an important statistical design to identify, revise and control unmeasured confounders and a valuable method for comparative effectiveness research based on real-world data.

299-OR: A Combinatorial Approach with Monoclonal Antibodies to SerpinB13 and CD3 Is Therapeutically Superior to Single Antibody Use in Autoimmune Diabetes

A successful approach to autoimmune diabetes therapy may require simultaneous insulin-producing cell regeneration and T-cell mediated autoimmunity resolution in pancreatic islets. While CD3 antibody has been proved to reduce islet inflammation, the monoclonal antibody (mAb) to serpinB13 protease inhibitor, developed in our laboratory, was shown to cause regenerative changes in beta cells. Whether a combination of both antibodies is superior to single antibody use in the treatment of autoimmune diabetes is unclear. To address this non-obese diabetic (NOD) female mice at late pre-diabetic stage were injected with serpinB13 mAb (clone B29, 10, 40 or 200 micrograms/animal) and CD3 mAb (clone 145-2C11; at a suboptimal dose of 10 micrograms). All experimental injections were controlled with nonspecific hamster IgG and mouse IgG (clone HB-158). After the last antibody injection, random blood glucose levels were assessed for 20 weeks, and mice were declared diabetic after two measurements of glucose exceeding 300 mg/dL. We found that the combination of 40 micrograms of serpinB13 mAb with CD3 mAb offered a significant level of protection from diabetes (p=0.0027, Kaplan-Meyer with log rank test), while the combination with 10 micrograms of serpinB13 mAb had no effect. The higher dose of 200 micrograms of serpinB13 mAb showed a trend in favor of diabetes inhibition. Interestingly, at 6 to 7 weeks after completing the treatment with 40 micrograms of serpinB13 mAb alone, NOD mice showed a strong tendency to remain healthy, and separated from the double-negative control, which continued to progress to diabetes. Our results indicate that a combinatorial approach with mAbs to serpinB13 and CD3 is beneficial in autoimmune diabetes. The observations also suggest that an intermediate dose of serpinB13 mAb, and a sufficient time following administration of this mAb, is critical for its positive impact on diabetes prevention. Disclosure B.Szepietowska: None. S.V.Avdulov: None. Y.Kryvalap: None. J.Czyzyk: None. Funding JDRF (2-SRA-2022-1215-S-B)

Short term exposure to air pollution and mortality in the US: a double negative control analysis

RationaleStudies examining the association of short-term air pollution exposure and daily deaths have typically been limited to cities and used citywide average exposures, with few using causal models.ObjectivesTo estimate the associations between short-term exposures to fine particulate matter (PM2.5), ozone (O3), and nitrogen dioxide (NO2) and all-cause and cause-specific mortality in multiple US states using census tract or address exposure and including rural areas, using a double negative control analysis.MethodsWe conducted a time-stratified case-crossover study examining the entire population of seven US states from 2000–2015, with over 3 million non-accidental deaths. Daily predictions of PM2.5, O3, and NO2 at 1x1 km grid cells were linked to mortality based on census track or residential address. For each pollutant, we used conditional logistic regression to quantify the association between exposure and the relative risk of mortality conditioning on meteorological variables, other pollutants, and using double negative controls.ResultsA 10 μg/m3 increase in PM2.5 exposure at the moving average of lag 0–2 day was significantly associated with a 0.67% (95%CI: 0.34–1.01%) increase in all-cause mortality. 10 ppb increases in NO2 or O3 exposure at lag 0–2 day were marginally associated with and 0.19% (95%CI: −0.01-0.38%) and 0.20 (95% CI-0.01, 0.40), respectively. The adverse effects of PM2.5 persisted when pollution levels were restricted to below the current global air pollution standards. Negative control models indicated little likelihood of omitted confounders for PM2.5, and mixed results for the gases. PM2.5 was also significantly associated with respiratory mortality and cardiovascular mortality.ConclusionsShort-term exposure to PM2.5 and possibly O3 and NO2 are associated with increased risks for all-cause mortality. Our findings delivered evidence that risks of death persisted at levels below currently permissible.

A Selective Review of Negative Control Methods in Epidemiology.

Negative controls are a powerful tool to detect and adjust for bias in epidemiological research. This paper introduces negative controls to a broader audience and provides guidance on principled design and causal analysis based on a formal negative control framework. We review and summarize causal and statistical assumptions, practical strategies, and validation criteria that can be combined with subject-matter knowledge to perform negative control analyses. We also review existing statistical methodologies for the detection, reduction, and correction of confounding bias, and briefly discuss recent advances towards nonparametric identification of causal effects in a double-negative control design. There is great potential for valid and accurate causal inference leveraging contemporary healthcare data in which negative controls are routinely available. Design and analysis of observational data leveraging negative controls is an area of growing interest in health and social sciences. Despite these developments, further effort is needed to disseminate these novel methods to ensure they are adopted by practicing epidemiologists.

Multiply robust causal inference with double-negative control adjustment for categorical unmeasured confounding.

Unmeasured confounding is a threat to causal inference in observational studies. In recent years, the use of negative controls to mitigate unmeasured confounding has gained increasing recognition and popularity. Negative controls have a long-standing tradition in laboratory sciences and epidemiology to rule out non-causal explanations, although they have been used primarily for bias detection. Recently, Miao and colleagues have described sufficient conditions under which a pair of negative control exposure and outcome variables can be used to identify non-parametrically the average treatment effect (ATE) from observational data subject to uncontrolled confounding. We establish non-parametric identification of the ATE under weaker conditions in the case of categorical unmeasured confounding and negative control variables. We also provide a general semiparametric framework for obtaining inferences about the ATE while leveraging information about a possibly large number of measured covariates. In particular, we derive the semiparametric efficiency bound in the non-parametric model, and we propose multiply robust and locally efficient estimators when non-parametric estimation may not be feasible. We assess the finite sample performance of our methods in extensive simulation studies. Finally, we illustrate our methods with an application to the post-licensure surveillance of vaccine safety among children.

Clove (Syzygium aromaticum) and honey extracts significantly reduce inflammatory cytokines and liver function enzymes in experimental rats fed on carbon tetrachloride (CCl4)

Liver is the key organ (detoxifier) that gets injured by reactive oxygen species (ROS) which deplete the body's stores of anti-oxidants. ROS is present in all chronic liver diseases leading to oxidative stress. Consumption of foods high in anti-oxidants replenishes and supplies the body with important anti-oxidants which break down ROS lowering inflammatory responses and preventing liver damage. In this work, clove and honey, which are known to be rich in anti-oxidants extracts were evaluated for their potential to breakdown ROS, Reactive nitrogen species (RNS) and lower oxidative stress in rats. Purified extracts were tested in vitro for ROS scavenging by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and in vivo using CCl4-induced liver injury model. A total of 50 experimental rats were assigned to 5 groups each consisting of 10 animals and treated as follows; 1. CCl4 group: fed on CCl4 in olive oil 2. honey group: fed CCl4 then honey extract, 3. clove group: CCl4 then clove extract, 4. double negative control group: rats fed on standard diet and 5 Negative control: fed olive oil, At 10 weeks, liver histopathology was done and sera collected for liver and kidney function tests. CCl4 -only treated rats showed fibroblastic cells proliferation and inflammatory cells infiltration which was absent in the control animals. Clove extract did not exhibit fibroblastic cells proliferation at all. Both clove and honey significantly reduced cytokines (TGF-β, TNF-α and EGF) and liver function enzymes'(ALT, AST and GGT) activity (58.5,56; 119,121 and 6.1; 6.3) compared to the CCl4 group (97; 191 and 10.1). Clove highly inhibited DPPH (77.2%) compared to honey (6.5%) and conferred better liver protection against CCl4. These findings indicate that clove and honey contain compounds that have anti-oxidant activity with the ability to reverse liver fibrosis. Frequent consumption of these foods may lower liver damage and prevent development of some liver diseases. Public health education on nutritional benefits of these foods is strongly encouraged.

The temporal multimodal influence of optical and auditory cues on the repellent behavior of ring-billed gulls (Larus delewarensis)

Context A generation of new animal repellents is based on the premise that threat stimuli are best interpreted through multiple sensory pathways. Ring-billed gulls (RBG; Larus delawarensis) offer a unique opportunity to assess the efficacy of multimodal repellents over time. This pest species is repelled by both auditory and optical cues and persists in stable populations, often remaining in the same colony for life. This distinctive attribute makes it possible to assess colonies independently over time and space. Aims We assessed the unimodal (single-cue treatment) and multimodal (paired-cue) response by RBG to auditory (conspecific distress call) and optical (green or red laser) cues, along with a double-negative control (flashlight aimed at ground, background noise). Methods All stimuli were investigated separately and together within a 3 × 2 factorial design randomised by treatment and site. We predicted that paired stimuli would generate more pronounced (number of gulls fleeing from a roost) and faster (flight initiation time) responses than stimuli presented alone with a control. Key results The distress call was more effective than either visual signal and almost nullified our ability to detect a multimodal response. However, the multimodal influence was detected on two levels. Gulls were more likely to flee from either paired treatment (optical + auditory) than from unimodal stimuli (laser light only; P &lt; 0.001) and gulls fled more quickly from multiple cues (P &lt; 0.001). A more subtle, but important, benefit was observed in that – over time – gulls were more likely to flee from either paired treatment (optical or auditory), but not from unimodal treatments (P &lt; 0.005). The latter response may have been due to a fear-conditioned generalisation. Conclusions We provide evidence and a causal mechanism to address why multimodal stimuli may be more efficacious as deterrents than single-mode treatments. This species may be more effectively managed, over longer periods of time, through the use of multimodal repellents. Implications A better understanding of how multimodal repellents function may help frame novel approaches to animal conservation and to assay better tools and repellents for wildlife management. Even modest multimodal benefits may justify their use, if they delay habituation over time.

Mutation analysis of the purine operon leader region in Bacillus subtilis

Expression of Bacillus subtilis purine (purE) operon is a subject of double negative control involving repressor protein PurR and a transcription terminator located in the operon leader region. We have performed site-directed mutagenesis of the specific motives, which are involved in formation of alternative hairpin structures, one of which produces transcription termination at the leader region ofpurEoperon. In vivo and in vitro analyses of the generated mutants have shown that purine bases, guanine and hypoxantine, serve as effector metabolites capable of increasing stability of terminating hairpin within the leader mRNA. Therefore, the leader RNA of purE operon serves as a sensor towards these metabolites and a riboswitch that provides premature termination of the operon transcription. The synergistic effect of the PurR repressor protein and a transcription terminator located at the leader region on the expression of purE operon was also revealed.

Presence of ATG triplets in 5′ untranslated regions of eukaryotic cDNAs correlates with a ‘weak’ context of the start codon

The context of the start codon (typically, AUG) and the features of the 5' Untranslated Regions (5' UTRs) are important for understanding translation regulation in eukaryotic mRNAs and for accurate prediction of the coding region in genomic and cDNA sequences. The presence of AUG triplets in 5' UTRs (upstream AUGs) might effect the initiation rate and, in the context of gene prediction, could reduce the accuracy of the identification of the authentic start. To reveal potential connections between the presence of upstream AUGs and other features of 5' UTRs, such as their length and the start codon context, we undertook a systematic analysis of the available eukaryotic 5' UTR sequences. We show that a large fraction of 5' UTRs in the available cDNA sequences, 15-53% depending on the organism, contain upstream ATGs. A negative correlation was observed between the information content of the translation start signal and the length of the 5' UTR. Similarly, a negative correlation exists between the 'strength' of the start context and the number of upstream ATGs. Typically, cDNAs containing long 5' UTRs with multiple upstream ATGs have a 'weak' start context, and in contrast, cDNAs containing short 5' UTRs without ATGs have 'strong' starts. These counter-intuitive results may be interpreted in terms of upstream AUGs having an important role in the regulation of translation efficiency by ensuring low basal translation level via double negative control and creating the potential for additional regulatory mechanisms. One of such mechanisms, supported by experimental studies of some mRNAs, includes removal of the AUG-containing portion of the 5' UTR by alternative splicing. An ATG_ EVALUATOR program is available upon request or at www.itba.mi.cnr.it/webgene. rogozin@ncbi.nlm.nih.gov, milanesi@itba.mi.cnr.it.

Anti-Fas (CD95/Apo-I) autoantibodies and soluble Fas levels concur in T cell depletion in HIV type 1 infection.

Deregulation of the Fas/FasL pathway in activated T cells is suspected to contribute to the abnormal apoptosis that drives their progressive depletion during HIV-1 infection. However, the role of serum soluble Fas (sFas) is unclear. Here we investigated both sFas and anti-Fas IgG levels in a cohort of 227 HIV-1-infected patients with respect to their T cell apoptosis. By using optimized ELISAs, we found that serum titers of sFas and anti-Fas were linearly correlated in 17 severely lymphopenic subjects as compared with other patients grouped in relation to their single expression of anti-Fas and sFas, or with double-negative control patients. Cytofluorimetric measurement of the subdiploid DNA-containing cell population by both PI and TUNEL revealed an increased occurrence of cell death in vitro, in particular in patients with elevations of sFas. We also found that fresh CD4(+) cells from these patients showed high levels of both caspase 3 (CPP32) and its molecular targets, namely PARP and CK18. In addition, their in vitro proliferative rate was inhibited by sFas, in particular in patients with undetectable levels of the soluble receptor in vivo as well as in normal donors. In these subjects the Fas-related caspase 8 (FLICE) was significantly increased in cells treated with the recombinant Fas. These results support the contention that functionally exhausted T cells may undergo apoptosis in response to the persistent in vivo stimulation by sFas. This may elucidate the described occurrence of enhanced cell death in advanced HIV-1 infection in association with serum elevations of the soluble receptor.

Double negative and positive control of tsx expression in Escherichia coli.

The Escherichia coli tsx gene encodes an outer membrane protein that is involved in nucleoside uptake and serves as the receptor protein for colicin K and several bacteriophages. Regulation of its expression was studied by using tsx-lacZ protein and operon fusion strains carrying mutations in deoR, cytR, and crp. The cytR-encoded repressor had a stronger influence on tsx transcription than the DeoR repressor did, and the level of tsx expression in a deoR cytR double mutant was approximately the sum of those found in the single deoR and cytR strains. This double negative control of Tsx synthesis was superceded by a positive control mechanism mediated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex. Our results suggest that tsx expression is controlled at two separate and differently regulated promoters: the weaker promoter (P1) is repressible by DeoR, while the stronger promoter (P2) is subject to negative and positive control by the CytR repressor and the cAMP-CAP complex, respectively. A mutant was isolated that showed unaltered tsx regulation by DeoR and the cAMP-CAP complex but strongly reduced repression by CytR. This tsx operator mutant was used to obtain a suppressor mutation located on a plasmid carrying the cloned cytR gene that restored CytR control of tsx expression. The direction of tsx transcription was determined and found to be counterclockwise on the E. coli chromosome.

Regulation of the deo operon in Escherichia coli

The synthesis of the four enzymes of the deo operon in Escherichia coli is known from in vivo experiments to be subject to a double negative control, exerted by the products of the cytR and deoR genes. A DNA-directed in vitro protein synthesizing system makes the deo enzymes (exemplified by thymidine phosphorylase) in agreement with in vivo results. Enzyme synthesis is stimulated by cyclic AMP and repressed by the cytR and deoR gene products. Repression by the cytR repressor is reversed by cytidine or adenosine in the presence of cyclic AMP, while repression by the deoR repressor is reversed by deoxyribose-5-phosphate. Assays for the presence of the cytR and deoR repressors were established by use of S-30 extracts prepared from the regulatory mutants. Dissociation constants for repressor-operator binding as well as for repressor-inducer interactions have been estimated from the results.