Optimization of physicochemical properties of pyrrolidine GPR40 AgoPAMs results in a differentiated profile with improved pharmacokinetics and reduced off-target activities.

GPR40 AgoPAMs are highly effective antidiabetic agents that have a dual mechanism of action, stimulating both glucose-dependent insulin and GLP-1 secretion. The early lipophilic, aromatic pyrrolidine and dihydropyrazole GPR40 AgoPAMs from our laboratory were highly efficacious in lowering plasma glucose levels in rodents but possessed off-target activities and triggered rebound hyperglycemia in rats at high doses. A focus on increasing molecular complexity through saturation and chirality in combination with reducing polarity for the pyrrolidine AgoPAM chemotype resulted in the discovery of compound 46, which shows significantly reduced off-target activities as well as improved aqueous solubility, rapid absorption, and linear PK. In vivo, compound 46 significantly lowers plasma glucose levels in rats during an oral glucose challenge yet does not demonstrate the reactive hyperglycemia effect at high doses that was observed with earlier GPR40 AgoPAMs.

Changes in Prefrontal Cortex-Thalamic Circuitry after Acoustic Trauma.

In the adult auditory system, loss of input resulting from peripheral deafferentation is well known to lead to plasticity in the central nervous system, manifested as reorganization of cortical maps and altered activity throughout the central auditory pathways. The auditory system also has strong afferent and efferent connections with cortico-limbic circuitry including the prefrontal cortex and the question arises whether this circuitry is also affected by loss of peripheral input. Recent studies in our laboratory showed that PFC activation can modulate activity of the auditory thalamus or medial geniculate nucleus (MGN) in normal hearing rats. In addition, we have shown in rats that cochlear trauma resulted in altered spontaneous burst firing in MGN. However, whether the PFC influence on MGN is changed after cochlear trauma is unknown. We investigated the effects of electrical stimulation of PFC on single neuron activity in the MGN in anaesthetized Wistar rats 2 weeks after acoustic trauma or sham surgery. Electrical stimulation of PFC showed a variety of effects in MGN neurons both in sham and acoustic trauma groups but inhibitory responses were significantly larger in the acoustic trauma animals. These results suggest an alteration in functional connectivity between PFC and MGN after cochlear trauma. This change may be a compensatory mechanism increasing sensory gating after the development of altered spontaneous activity in MGN, to prevent altered activity reaching the cortex and conscious perception.

BIOLUX P-III Passeo-18 Lux All-Comers Registry: 24-Month Results in Below-the-Knee Arteries.

PURPOSE: The BIOLUX P-III registry was initiated to further assess the safety and efficacy of the Passeo-18 Lux drug-coated balloon (DCB) in infrainguinal lesions in a real-world environment and in prespecified risk groups. MATERIALS AND METHODS: BIOLUX P-III is a prospective, global, all-comers registry with patients treated under real-world conditions. We herein report 24-month results of the prespecified subgroup of 151 patients with 185 below-the-knee (BTK) lesions. The primary safety and efficacy endpoints were freedom from major adverse events (a composite of freedom from device and procedure mortality through 30 days, major target limb amputation and clinically driven target lesion revascularization) at 6 months and freedom from clinically driven target lesion revascularization (FfTLR) at 12 months. RESULTS: At baseline, 76.0% of patients had critical limb ischemia and 48.9% of lesions were TASC C or D lesions. Technical success was achieved in 97.8%, and bailout stenting was required in 1.1%. Freedom from major adverse events was 86.2% [95% CI 79.4; 90.8] at 6 months, and FfTLR was 90.9% [95% CI 85.2; 94.4] at 12 months. At 24 months, FfTLR was 90.9% [95% CI 85.2; 94.4], freedom from major amputation was 90.1% [95% CI 83.9, 94.0], and overall survival was 79.2% [70.7, 85.5]. There was a significant clinical improvement (mean Rutherford class improvement of - 2.9 ± 1.9, p < 0.0001) and an improvement in pain (mean improvement on Wong-Baker Faces Pain Scale of - 2.7 ± 2.9, p < 0.0001). CONCLUSIONS: In this real-world DCB registry, 24-month outcomes of Passeo-18 Lux demonstrated safety and efficacy in BTK lesions with high patency rates and sustained clinical improvements at 24 months. TRIAL REGISTRATION: NCT02276313.

Targets of olivocochlear collaterals in cochlear nucleus of rat and guinea pig.

Descending auditory pathways can modify afferent auditory input en route to cortex. One component of these pathways is the olivocochlear system which originates in brainstem and terminates in cochlea. Medial olivocochlear (MOC) neurons also project collaterals to cochlear nucleus and make synaptic contacts with dendrites of multipolar neurons. Two broadly distinct populations of multipolar cells exist: T-stellate and D-stellate neurons, thought to project to inferior colliculus and contralateral cochlear nucleus, respectively. It is unclear which of these neurons receive direct MOC collateral input due to conflicting results between in vivo and in vitro studies. This study used anatomical techniques to identify which multipolar cell population receives synaptic innervation from MOC collaterals. The retrograde tracer Fluorogold was injected into inferior colliculus or cochlear nucleus to label T-stellate and D-stellate neurons, respectively. Axonal branches of MOC neurons were labeled by biocytin injections at the floor of the fourth ventricle. Fluorogold injections resulted in labeled cochlear nucleus multipolar neurons. Biocytin abundantly labeled MOC collaterals which entered cochlear nucleus. Microscopic analysis revealed that MOC collaterals made some putative synaptic contacts with the retrogradely labeled neurons but many more putative contacts were observed on unidentified neural targets. This suggest that both T- and D-stellate neurons receive synaptic innervation from the MOC collaterals on their somata and proximal dendrites. The prevalence of these contacts cannot be stated with certainty because of technical limitations, but the possibility exists that the collaterals may also make contacts with neurons not projecting to inferior colliculus or the contralateral cochlear nucleus.

Cholinergic responses of acoustically-characterized cochlear nucleus neurons: An in vivo iontophoretic study in Guinea pig.

The responses of guinea pig cochlear nucleus neurons to in vivo iontophoretic application of various neurotransmitter agonists were recorded with extracellular multi-barrelled electrodes. Where possible, neurons were physiologically identified using strict criteria. Emphasis was placed on the action of cholinergic agonists in relation to the possible action of olivocochlear collateral innervation. Excitatory responses (increase in action potential firing) to glutamate were confirmed in a number of neuronal response types. Application of acetylcholine (ACh) or the broad spectrum cholinergic agonist carbachol produced reliable excitatory responses in about 47% of neurons (n = 29 out of 61 neurons). The remaining neurons were unresponsive to cholinergic agonists and no inhibitory responses were observed. Cholinergic responses were more common in dorsal cochlear nucleus (DCN) (73% of 30 neurons tested) than in ventral cochlear nucleus (VCN) (23% of 31 neurons). Of the total neuron sample in which cholinergic responses were investigated, 41 neurons were able to be categorized according to established acoustic response features. Excitatory responses to cholinergic agonists were seen in "Pauser-buildup" (Pb) and "Transient chopper" (Ct) response types. Primary-like neurons (PL and Pn) as well as "Onset chopper" (Oc) neurons (n = 6) were unresponsive to either ACh or carbachol. Oc neurons also did not show any effect on their acoustic responses. Robust cholinergic responses were also seen in several VCN and DCN neurons that were either unresponsive to sound, or had acoustic response properties that did not fit standard classification. The results suggest a relatively more robust cholinergic innervation of DCN compared to VCN. The excitatory cholinergic responses of some Ct neurons and the lack of effect on Oc neurons are consistent with previous results in mouse brain slice studies, but are in conflict with reports of medial olivocochlear collateral excitatory responses in onset-type neurons in vivo. The results also indicate that a number of neurons of unknown identity may also receive cholinergic input.

Persistent hair cell malfunction contributes to hidden hearing loss.

Noise exposures that result in fully reversible changes in cochlear neural threshold can cause a reduced neural output at supra-threshold sound intensity. This so-called "hidden hearing loss" has been shown to be associated with selective degeneration of high threshold afferent nerve fiber-inner hair cell (IHC) synapses. However, the electrophysiological function of the IHCs themselves in hidden hearing loss has not been directly investigated. We have made round window (RW) measurements of cochlear action potentials (CAP) and summating potentials (SP) after two levels of a 10 kHz acoustic trauma. The more intense acoustic trauma lead to notch-like permanent threshold changes and both CAP and SP showed reductions in supra-threshold amplitudes at frequencies with altered thresholds as well as from fully recovered regions. However, the interpretation of the results in normal threshold regions was complicated by the likelihood of reduced contributions from adjacent regions with elevated thresholds. The milder trauma showed full recovery of all neural thresholds, but there was a persistent depression of the amplitudes of both CAP and SP in response to supra-threshold sounds. The effect on SP amplitude in particular shows that occult damage to hair cell transduction mechanisms can contribute to hidden hearing loss. Such damage could potentially affect the supra-threshold output properties of surviving primary afferent neurons.

Altered attentional filters in subjects with graded levels of sensorineural hearing loss.

Near-threshold tones (targets) in noise that are preceded by cues of the same frequency or occur with a high probability are detected better than tones of other frequencies that may occur with a lower probability (probes); the better detection of targets than probes defines the attentional filter. We measured attentional filters using a cued probe-signal procedure with a two-interval forced-choice (2IFC) method in normal-hearing subjects (N = 15) and subjects with sensorineural hearing loss (SNHL; N = 14) with a range of hearing levels. Attentional filters were altered in SNHL subjects, who detected low-frequency probes as well as targets at all hearing levels and who detected high-frequency probes increasingly well with increasing hearing level. These effects were present in both intervals of the 2IFC procedure. As auditory filters measured psychophysically are typically asymmetric in subjects with SNHL, these results suggest that the signal frequencies affected by the attentional filter are governed by the shapes of the auditory filters at and around the cue frequency. The normal-hearing subjects showed the expected attentional filters in the first interval and shallower filters in the second interval, suggesting that the cue-evoked attentional process is transient. In the first interval, both low- and high-frequency probes were detected better as hearing level increased over a narrow range (from -5 to 10 dB at the target frequency), with a resultant loss of attentional filtering. This finding adds to observations of variable auditory function in individuals with clinically normal hearing thresholds established by audiometry.

Metabolomic profiling distinction of human nonalcoholic fatty liver disease progression from a common rat model.

OBJECTIVE: Characteristic pathological changes define the progression of steatosis to nonalcoholic steatohepatitis (NASH) and are correlated to metabolic pathways. A common rodent model of NASH is the methionine and choline deficient (MCD) diet. The objective of this study was to perform full metabolomic analyses on liver samples to determine which pathways are altered most pronouncedly in this condition in humans, and to compare these changes to rodent models of nonalcoholic fatty liver disease (NAFLD). METHODS: A principal component analysis for all 91 metabolites measured indicated that metabolome perturbation is greater and less varied for humans than for rodents. RESULTS: Metabolome changes in human and rat NAFLD were greatest for the amino acid and bile acid metabolite families (e.g., asparagine, citrulline, gamma-aminobutyric acid, lysine); although, in many cases, the trends were reversed when compared between species (cholic acid, betaine). CONCLUSIONS: Overall, these results indicate that metabolites of specific pathways may be useful biomarkers for NASH progression, although these markers may not correspond to rodent NASH models. The MCD model may be useful when studying certain end points of NASH; however, the metabolomics results indicate important differences between humans and rodents in the biochemical pathogenesis of the disease.

Effects of Neonatal Dexamethasone Exposure on Adult Neuropsychiatric Traits in Rats.

The effects of early life stress in utero or in neonates has long-term consequences on hypothalamic-pituitary-adrenal (HPA) stress axis function and neurodevelopment. These effects extend into adulthood and may underpin a variety of mental illnesses and be related to various developmental and cognitive changes. We examined the potential role of neonatal HPA axis activation on adult psychopathology and dopamine sensitivity in the mature rat using neonatal exposure to the synthetic glucocorticoid receptor agonist and stress hormone, dexamethasone. We utilized a comprehensive battery of assessments for behaviour, brain function and gene expression to determine if elevated early life HPA activation is associated with adult-onset neuropsychiatric traits. Dexamethasone exposure increased startle reactivity under all conditions tested, but decreased sensitivity of sensorimotor gating to dopaminergic disruption-contrasting with what is observed in several neuropsychiatric diseases. Under certain conditions there also appeared to be mild long-term changes in stress and anxiety-related behaviours with neonatal dexamethasone exposure. Electrophysiology revealed that there were no consistent neuropsychiatric abnormalities in auditory processing or resting state brain function with dexamethasone exposure. However, neonatal dexamethasone altered auditory cortex glucocorticoid activation, and auditory cortex synchronization. Our results indicate that neonatal HPA axis activation by dexamethasone alters several aspects of adult brain function and behaviour and may induce long-term changes in emotional stress-reactivity. However, neonatal dexamethasone exposure is not specifically related to any particular neuropsychiatric disease.

The utility of stable isotope labeled (SIL) analogues in the bioanalysis of endogenous compounds by LC-MS applied to the study of bile acids in a metabolomics assay.

The growing field of biomarker bioanalysis by liquid chromatography mass spectrometry (LC-MS) is challenged with the selection of suitable matrices to construct relevant and valid calibration curves resulting in not only precise but also accurate data. Because surrogate matrices are often employed with the associated concerns about the accuracy of the obtained data, here we present an assay using surrogate analytes in naive biological matrices. This approach is illustrated with the analysis of endogenous bile acids (e-BAs) in serum and plasma using stable isotope-labeled (SIL) analogues as calibration standards to address the matrix concerns. Several deuterated BAs (d-BAs) were used as standards representing respectively grouped e-BAs with structural similarity allowing for the simultaneous bioanalysis of 16 e-BA. The utility of this LC-MS assay employing d-BAs is demonstrated with the analysis of samples resultant of a controlled metabolomics study where a cohort of rats was fed/fasted to investigate the change of e-BAs dependent on food consumption and fasting time.

Influence of the paraflocculus on normal and abnormal spontaneous firing rates in the inferior colliculus.

Spontaneous firing rates of neurons in the central auditory pathway, such as in the inferior colliculus, are known to be increased after cochlear trauma. This so-called hyperactivity is thought to be involved in the generation of tinnitus, a phantom auditory perception. Recent research in an animal model suggests behavioural signs of tinnitus can be significantly reduced by silencing or removal of the paraflocculus (PF) of the cerebellum. The current study investigated the effects of acute PF removal on spontaneous firing rates recorded from single neurons in the right inferior colliculus of guinea pigs with normal hearing (which did not receive acoustic trauma) or with hearing loss caused by acoustic trauma. Spontaneous firing rates were obtained at either 2 or 13 weeks after initial surgery on the left side. In half of the animals in each group the left PF was removed immediately prior to the spontaneous firing rates recordings. In the acoustic trauma groups, spontaneous firing rates in the inferior colliculus were higher when the PF was removed compared to animals with an intact PF. This effect of PF removal was not observed in animals that did not receive acoustic trauma. These results suggest that the PF has a tonic inhibitory effect on hyperactivity in the inferior colliculus in animals with hearing loss, but not on normal spontaneous firing rates in normal hearing animals.

Selective attentional processes in cochlear implant recipients: Measurements of the attentional filter.

In normal hearing subjects, detection of near-threshold tones in noise is influenced by signal certainty. Thus, tones that are presented more frequently than others, and/or are preceded by a clearly audible cue tone of the same frequency (target tones) are detected better than other tones (probe tones). This auditory attentional filter was examined in six cochlear implant (CI) recipients, using acoustic stimuli and direct programmed electrode stimulation. Three of the subjects showed no evidence of an attentional filter. Three subjects showed a relatively higher detection rate of the target frequency or electrode stimulated during the attentional task, and in two of these subjects the target benefit was influenced by stimulus certainty. The absence of an attentional filter in some CI recipients is consistent with suggestions that the attentional filter may be generated by efferent modulation of outer hair cells, which would presumably be absent in CI recipients, however, the presence of some frequency-selective attentional effects and a near-normal attentional filter in two CI subjects imply that central processes can modulate signal detection in CI recipients according to stimulus certainty. Such central processes might serve as a neural substrate to improve signal detection in CI recipients.

Quality assurance of metabolomics.

Metabolomics promises a holistic phenotypic characterization of biological responses to toxicants. This technology is based on advanced chemical analytical tools with reasonable throughput, including mass-spectroscopy and NMR. Quality assurance, however - from experimental design, sample preparation, metabolite identification, to bioinformatics data-mining - is urgently needed to assure both quality of metabolomics data and reproducibility of biological models. In contrast to microarray-based transcriptomics, where consensus on quality assurance and reporting standards has been fostered over the last two decades, quality assurance of metabolomics is only now emerging. Regulatory use in safety sciences, and even proper scientific use of these technologies, demand quality assurance. In an effort to promote this discussion, an expert workshop discussed the quality assurance needs of metabolomics. The goals for this workshop were 1) to consider the challenges associated with metabolomics as an emerging science, with an emphasis on its application in toxicology and 2) to identify the key issues to be addressed in order to establish and implement quality assurance procedures in metabolomics-based toxicology. Consensus has still to be achieved regarding best practices to make sure sound, useful, and relevant information is derived from these new tools.

A novel method for selectively labelling olivocochlear collaterals in the rat.

Axons of olivocochlear neurons originate from the brainstem and project to the cochlea. A subpopulation, medial olivocochlear (MOC) neurons, also projects collateral branches to the cochlear nucleus. The precise targets of these collaterals are as yet unknown. Previous methods for labelling these collaterals include firstly, cochlear injections of retrograde tracers, but this is technically demanding and can also label afferent projections or secondly, labelling by injecting tracers into the nuclei of origin of MOC neurons. However, this latter method is non-specific because it also labels non-MOC projections. A technique was used to specifically label MOC collaterals, which involved injections of the tracer biocytin at the floor of the fourth ventricle and fixation 3 hours later. Biocytin injections resulted in labelled neurons in the ventral nucleus of the trapezoid body and rostral periolivary nucleus, confirming MOC axonal labelling. Labelled neurons in dorsal cochlear nucleus indicated labelling of the dorsal acoustic stria and these injections were discarded. After selective MOC labelling, collateral branches were found to innervate granule cell regions, medial edge and core of the ventral cochlear nucleus, as well as the dorsal cochlear nucleus, in agreement with previous data. Therefore we conclude that injections at the floor of the fourth ventricle provide a simple, rapid and specific technique for labelling the majority of MOC axons and their collaterals and this technique may assist in defining the precise neuronal targets of olivocochlear collaterals in cochlear nucleus.

Branched chain amino acid metabolism profiles in progressive human nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a globally widespread disease of increasing clinical significance. The pathological progression of the disease from simple steatosis to nonalcoholic steatohepatitis (NASH) has been well defined, however, the contribution of altered branched chain amino acid metabolomic profiles to the progression of NAFLD is not known. The three BCAAs: leucine, isoleucine and valine are known to mediate activation of several important hepatic metabolic signaling pathways ranging from insulin signaling to glucose regulation. The purpose of this study is to profile changes in hepatic BCAA metabolite levels with transcriptomic changes in the progression of human NAFLD to discover novel mechanisms of disease progression. Metabolomic and transcriptomic data sets representing the spectrum of human NAFLD (normal, steatosis, NASH fatty, and NASH not fatty livers) were utilized for this study. During the transition from steatosis to NASH, increases in the levels of leucine (127% of normal), isoleucine (139%), and valine (147%) were observed. Carnitine metabolites also exhibited significantly elevated profiles in NASH fatty and NASH not fatty samples and included propionyl, hexanoyl, lauryl, acetyl and butyryl carnitine. Amino acid and BCAA metabolism gene sets were significantly enriched among downregulated genes during NASH. These cumulative alterations in BCAA metabolite and amino acid metabolism gene profiles represent adaptive physiological responses to disease-induced hepatic stress in NASH patients.

Modulation of gene expression in guinea pig paraflocculus after induction of hearing loss.

Hearing loss often results in plastic changes in the central auditory pathways, which may be involved in the generation of tinnitus, a phantom auditory sensation. However, although animal studies have consistently shown increased neural activity in auditory structures after hearing loss, tinnitus does not always develop. It has therefore been suggested that non-auditory structures perform a gating or regulatory role that determines whether the increased activity in auditory structures leads to conscious perception. Recent evidence points to the paraflocculus of the cerebellum as having such a role. Therefore, we investigated the early effects of hearing loss on gene expression in guinea pig paraflocculus. Gene expression was investigated after two weeks recovery from either acoustic or mechanical cochlear trauma. The genes investigated in our study were associated with inhibitory neurotransmission (GABA-A receptor subunit alpha 1; glutamate decarboxylase 1), excitatory neurotransmission (glutamate receptor NMDA subunit 1), and regulation of transmitter release (member of RAB family of small GTPase). Our results show increased mRNA levels of glutamate decarboxylase 1 in ipsilateral paraflocculus with no difference between the different methods of cochlear trauma. Early modulation of gene expression in the paraflocculus suggests that an early effect of hearing loss may affect the influence of this structure on auditory processing.

Effects of chronic furosemide on central neural hyperactivity and cochlear thresholds after cochlear trauma in Guinea pig.

Increased neuronal spontaneous firing rates have been observed throughout the central auditory system after trauma to the cochlea and this hyperactivity is believed to be associated with the phantom perception of tinnitus. Previously, we have shown in an animal model of hearing loss, that an acute injection with furosemide can significantly decrease hyperactivity after cochlear trauma and eliminate behavioral evidence of tinnitus of early onset. However, furosemide also has the potential to affect cochlear thresholds. In this paper, we measured the effects of a chronic (daily injections for 7 days) furosemide treatment on the spontaneous firing rate of inferior colliculus neurons and on cochlear thresholds in order to establish whether a beneficial effect on hyperactivity can be obtained without causing additional hearing loss. Guinea pigs were exposed to a 10-kHz, 124 dB, 2 h acoustic trauma, and after 5 days of recovery, were given daily i.p. injections of 80 mg/kg furosemide or an equivalent amount of saline. The activity of single IC neurons was recorded 24 h following the last injection. The furosemide treatment had no effect on cochlear thresholds compared to saline injections but did result in significant reductions in spontaneous firing rates recorded in inferior colliculus. These results that suggest a long-term beneficial effect of furosemide on hyperactivity after cochlear trauma may be achievable without detrimental effects on hearing, which is important when considering therapeutic potential.

Effects of furosemide on cochlear neural activity, central hyperactivity and behavioural tinnitus after cochlear trauma in guinea pig.

Cochlear trauma causes increased spontaneous activity (hyperactivity) to develop in central auditory structures, and this has been suggested as a neural substrate for tinnitus. Using a guinea pig model we have previously demonstrated that for some time after cochlear trauma, central hyperactivity is dependent on peripheral afferent drive and only later becomes generated intrinsically within central structures. Furosemide, a loop diuretic, reduces spontaneous firing of auditory afferents. We investigated in our guinea pig model the efficacy of furosemide in reducing 1) spontaneous firing of auditory afferents, using the spectrum of neural noise (SNN) from round window recording, 2) hyperactivity in inferior colliculus, using extracellular single neuron recordings and 3) tinnitus at early time-points after cochlear trauma. Tinnitus was assessed using gap prepulse inhibition of acoustic startle (GPIAS). Intraperitoneal furosemide, but not saline, caused a marked decrease in both SNN and central hyperactivity. Intracochlear perfusion with furosemide similarly reversed central hyperactivity. In animals in which GPIAS measurements suggested the presence of tinnitus (reduced GPIAS), this could be reversed with an intraperitoneal injection with furosemide but not saline. The results are consistent with furosemide reducing central hyperactivity and behavioural signs of tinnitus by acting peripherally to decrease spontaneous firing of auditory afferents. The data support the notion that hyperactivity may be involved in the generation of tinnitus and further suggest that there may be a therapeutic window after cochlear trauma using drug treatments that target peripheral spontaneous activity.

Characterization of heat-stable (STa) toxoids of enterotoxigenic Escherichia coli fused to double mutant heat-labile toxin peptide in inducing neutralizing Anti-STa antibodies.

A long-standing challenge in developing vaccines against enterotoxigenic Escherichia coli (ETEC), the most common bacteria causing diarrhea in children of developing countries and travelers to these countries, is to protect against heat-stable toxin type Ib (STa or hSTa). STa and heat-labile toxin (LT) are virulence determinants in ETEC diarrhea. LT antigens are often used in vaccine development, but STa has not been included because of its poor immunogenicity and potent toxicity. Toxic STa is not safe for vaccines, but only STa possessing toxicity is believed to be able to induce neutralizing antibodies. However, recent studies demonstrated that nontoxic STa derivatives (toxoids), after being fused to an LT protein, induced neutralizing antibodies and suggested that different STa toxoids fused to an LT protein might exhibit different STa antigenic propensity. In this study, we selected 14 STa toxoids from a mini-STa toxoid library based on toxicity reduction and reactivity to anti-native STa antibodies, and genetically fused each toxoid to a monomeric double mutant LT (dmLT) peptide for 14 STa-toxoid-dmLT toxoid fusions. These toxoid fusions were used to immunize mice and were characterized for induction of anti-STa antibody response. The results showed that different STa toxoids (in fusions) varied greatly in anti-STa antigenicity. Among them, STaN12S, STaN12T, and STaA14H were the top toxoids in inducing anti-STa antibodies. In vitro neutralization assays indicated that antibodies induced by the 3×STaN12S-dmLT fusion antigen exhibited the greatest neutralizing activity against STa toxin. These results suggested 3×STaN12S-dmLT is a preferred fusion antigen to induce an anti-STa antibody response and provided long-awaited information for effective ETEC vaccine development.