Nano-Magnesium Oxide: A Novel Bactericide Against Copper-Tolerant Xanthomonas perforans Causing Tomato Bacterial Spot.

Bacterial spot caused by Xanthomonas perforans causes significant damage on tomato in Florida. Due to the presence of copper (Cu)-tolerant X. perforans strains, Cu bactericides are not effective in disease management. Hence, there is a critical need to find alternatives for Cu. Antibacterial activity of magnesium oxide (Nano-MgO), and other metal oxide nanoparticles, were evaluated against a Cu-tolerant and -sensitive X. perforans strain. In vitro experiments demonstrated high antibacterial activity of Nano-MgO against both strains compared with the commercial Cu. The minimum inhibitory concentration of Nano-MgO is 25 µg/ml and the minimum bactericidal concentration is 100 µg/ml against a Cu-tolerant X. perforans strain after 4 h of exposure. Structural changes in the bacterial membrane following exposure to Nano-MgO treatments compared with the controls were observed using transmission electron microscopy. In two greenhouse experiments with a Cu-tolerant strain, bacterial spot severity was significantly reduced by Nano-MgO at 200 µg/ml compared with Cu-ethylene bis-dithiocarbamate (grower standard), and the untreated control (P = 0.05). In three field experiments, Nano-MgO at 200 µg/ml significantly reduced disease severity with no negative impact on yield compared with the untreated control. Inductively coupled plasma mass spectrometric analysis of the fruit confirmed that Nano-MgO application did not lead to the accumulation of Mg, Cu, Ca, K, Mn, P, and S. This study is the first to demonstrate the potential of Nano-MgO against bacterial spot of tomato.

Human papillomavirus genotypes in Pacific Islander cervical cancer patients.

OBJECTIVE: The role of human papillomavirus (HPV) in the development of invasive cervical cancers is widely known. Few HPV studies have targeted geographically isolated regions. The objective of this study was to determine the HPV genotypes in cervical cancer patients from the Pacific Islands referred to Tripler Army Medical Center (TAMC). METHODS: All cases of invasive cervical cancer treated at TAMC through the Pacific Island Health Care Project between January 2004 and October 2014 were identified through a review of pathology specimens. DNA was extracted from paraffin-embedded tissue blocks. PCR was performed using PLEX-ID plates to isolate and amplify HPV-specific DNA. Mass spectrometry was subsequently performed to identify specific HPV genotypes. RESULTS: Thirty-five patients had their pathology specimens analyzed. Ten patients had localized disease (Stage 1); 21 had regional disease (Stages 2 and 3); and 4 had distant disease (Stage 4). Thirty-three squamous cell carcinomas and 3 adenocarcinomas were identified. The most common HPV subtypes found were 16 (6, 24%), 45 (6, 24%), and 52 (6, 24%). Other HPV subtypes isolated included 18 (1, 4%), 33 (3, 12%), 39 (2, 8%), 54 (1, 4%), and 67 (1, 4%). In 10 samples, HPV was not isolated. CONCLUSION: Pacific Islanders referred to TAMC present with a disproportionally higher rate of regional and advanced disease. Significantly, only 28% of invasive cervical cancers in the Pacific Island population sampled could have been potentially be prevented using the available quadrivalent vaccine targeting HPV 16/18; however, 88% could be covered by the recently licensed nonavalent vaccine.

Advanced Copper Composites Against Copper-Tolerant Xanthomonas perforans and Tomato Bacterial Spot.

Bacterial spot, caused by Xanthomonas spp., is a widespread and damaging bacterial disease of tomato (Solanum lycopersicum). For disease management, growers rely on copper bactericides, which are often ineffective due to the presence of copper-tolerant Xanthomonas strains. This study evaluated the antibacterial activity of the new copper composites core-shell copper (CS-Cu), multivalent copper (MV-Cu), and fixed quaternary ammonium copper (FQ-Cu) as potential alternatives to commercially available micron-sized copper bactericides for controlling copper-tolerant Xanthomonas perforans. In vitro, metallic copper from CS-Cu and FQ-Cu at 100 µg/ml killed the copper-tolerant X. perforans strain within 1 h of exposure. In contrast, none of the micron-sized copper rates (100 to 1,000 µg/ml) from Kocide 3000 significantly reduced copper-tolerant X. perforans populations after 48 h of exposure compared with the water control (P < 0.05). All copper-based treatments killed the copper-sensitive X. perforans strain within 1 h. Greenhouse studies demonstrated that all copper composites significantly reduced bacterial spot disease severity when compared with copper-mancozeb and water controls (P < 0.05). Although there was no significant impact on yield, copper composites significantly reduced disease severity when compared with water controls, using 80% less metallic copper in comparison with copper-mancozeb in field studies (P < 0.05). This study highlights the discovery that copper composites have the potential to manage copper-tolerant X. perforans and tomato bacterial spot.

Epidemiology and management of bacterial leaf spot on watermelon caused by Pseudomonas syringae.

Bacterial leaf spot of watermelon caused by Pseudomonas syringae has been an emerging disease in the southeastern United States in recent years. Disease outbreaks in Florida were widespread from 2013 to 2014 and resulted in foliar blighting at the early stages of the crop and transplant losses. We conducted a series of field trials at two locations over the course of two years to examine the chemical control options that may be effective in management of this disease, and to investigate the environmental conditions conducive for bacterial leaf spot development. Weekly applications of acibenzolar-S-methyl (ASM) foliar, ASM drip, or copper hydroxide mixed with ethylene bis-dithiocarbamate were effective in reducing the standardized area under the disease progress curve (P < 0.05). Pearson's correlation test demonstrated a negative relationship between the average weekly temperature and disease severity (-0.77, P = 0.0002). When incorporated into a multiple regression model with the square root transformed average weekly rainfall, these two variables accounted for 71% of the variability observed in the weekly disease severity (P < 0.0001). This information should be considered when choosing the planting date for watermelon seedlings as the cool conditions often encountered early in the spring season are conducive for bacterial leaf spot development.

Lesions in the central nucleus of the amygdala: discriminative avoidance learning, discriminative approach learning, and cingulothalamic training-induced neuronal activity.

The amygdala is critically involved in discriminative avoidance learning. Large lesions of the amygdala block discriminative avoidance learning and abolish cingulothalamic training-induced neuronal activity. These results indicated that amygdalar processing is critical for cingulothalamic plasticity. The larger lesions did not allow differentiation of the specific functioning of various amygdalar nuclei. Anatomical analysis showed that damage in the central (CE) nucleus of the amygdala was correlated with the severity of the behavioral deficit. The present study was carried out to determine whether smaller lesions, centered in the CE nucleus, would impair discriminative avoidance learning and block cingulothalamic plasticity. In addition, the possible role of the CE nucleus in appetitively motivated discriminative approach learning was examined for the first time. New Zealand White rabbits with CE nuclear lesions were first trained in the discriminative approach task. After attaining asymptotic performance, discriminative avoidance training sessions were alternated with continuing approach training sessions, one session each day. The rabbits with lesions were severely impaired in avoidance learning but showed no impairment of approach learning. Surprisingly, the attenuating effects of the lesions on cingulothalamic training-induced neuronal activity were more prevalent during approach learning than during avoidance learning. These results indicated that avoidance learning can be impaired by lesions centered in the CE nucleus that leave cingulothalamic plasticity largely intact and that the CE nucleus is involved in extra-cingulothalamic learning processes.

Ontogenetic changes in the neural mechanisms of eyeblink conditioning.

The rodent eyeblink conditioning paradigm is an ideal model system for examining the relationship between neural maturation and the ontogeny of associative learning. Elucidation of the neural mechanisms underlying the ontogeny of learning is tractable using eyeblink conditioning because the necessary neural circuitry (cerebellum and interconnected brainstem nuclei) underlying the acquisition and retention of the conditioned response (CR) has been identified in adult organisms. Moreover, the cerebellum exhibits substantial postnatal anatomical and physiological maturation in rats. The eyeblink CR emerges developmentally between postnatal day (PND) 17 and 24 in rats. A series of experiments found that the ontogenetic emergence of eyeblink conditioning is related to the development of associative learning and not related to changes in performance. More recent studies have examined the relationship between the development of eyeblink conditioning and the physiological maturation of the cerebellum, a brain structure that is necessary for eyeblink conditioning in adult organisms. Disrupting cerebellar development with lesions or antimitotic treatments impairs the ontogeny of eyeblink conditioning. Studies of the development of physiological processes within the cerebellum have revealed striking ontogenetic changes in stimulus-elicited and learning-related neuronal activity. Neurons in the interpositus nucleus and Purkinje cells in the cortex exhibit developmental increases in neuronal discharges following the unconditioned stimulus (US) and in neuronal discharges that model the amplitude and time-course of the eyeblink CR. The developmental changes in CR-related neuronal activity in the cerebellum suggest that the ontogeny of eyeblink conditioning depends on the development of mechanisms that establish cerebellar plasticity. Learning and the induction of neural plasticity depend on the magnitude of the US input to the cerebellum. The role of developmental changes in the efficacy of the US pathway has been investigated by monitoring neuronal activity in the inferior olive and with stimulation techniques. The results of these experiments indicate that the development of the conditioned eyeblink response may depend on dynamic interactions between multiple developmental processes within the eyeblink neural circuitry.

Developmental changes in eye-blink conditioning and neuronal activity in the inferior olive.

Neuronal activity was recorded in the dorsal accessory inferior olive in infant rats during classical conditioning of the eye-blink response. The percentage and amplitude of eye-blink conditioned responses (CRs) increased as a function of age. The magnitude of the neuronal response to the unconditioned stimulus (US) decreased with age. There were also age-specific modifications of US-elicited inferior olive neuronal activity during paired trials in which a conditioned eye-blink response was performed. The results indicate that the development of the conditioned eye-blink response may depend on dynamic interactions between multiple developmental processes within the eye-blink circuitry. Differences in the functional maturity of olivo-cerebellar pathways may limit the induction of plasticity in the cerebellum and thereby limit the development of eye-blink conditioned responses.

Lesions of the perirhinal cortex impair sensory preconditioning in rats.

The effects of lesions of the perirhinal cortex on the development of associations between two conditioned stimuli (CSs) were examined with a sensory preconditioning procedure. Rats were given either bilateral electrolytic lesions of the perirhinal cortex or control surgery. They were then given either paired or unpaired presentations of a light CS and a tone CS. All of the rats were then given eyeblink conditioning procedures that involved paired presentations of either the light or tone and a periorbital shock unconditioned stimulus (US). The rats were finally given a test session that consisted of unpaired presentations of the tone and light CSs. Sensory preconditioning was established in the control group, but not in the lesion group. The findings are consistent with the view that the perirhinal cortex is involved in forming associations between neutral stimuli (even in the absence of reinforcement).

Developmental changes in eye-blink conditioning and neuronal activity in the cerebellar interpositus nucleus.

Neuronal activity was recorded in the cerebellar interpositus nucleus in infant rats during classical conditioning of the eye-blink response. The percentage and amplitude of eye-blink conditioned responses increased as a function of postnatal age. Learning-specific neuronal activity in the cerebellum emerged ontogenetically in parallel with the eye-blink conditioned response. There were also age-specific changes in neuronal activity after the onset of the conditioned and unconditioned stimuli. The results indicate that the development of the eye-blink conditioned response may depend on the development of stimulus-evoked neuronal responses and learning-specific plasticity in the cerebellum. Functional immaturity in the afferent neural pathways may limit the induction of neural plasticity in the cerebellum and thereby limit the development of the eye-blink conditioned response.

Changes of cingulothalamic topographic excitation patterns and avoidance response incubation over time following initial discriminative conditioning in rabbits.

Neurons in particular layers of cingulate cortex and in limbic thalamic nuclei exhibit peak firing rates in response to a positive conditional stimulus (CS+) in particular stages of discriminative learning. A given area is maximally activated by the CS+ in the initial, an intermediate, or a late stage of behavioral acquisition, and activation in all of the areas diminishes as training continues after the peak of activation occurs. Thus, the topographic distribution of activation in these areas depends on the stage of behavioral acquisition. The present study determined whether the acquisition-related changes of the topographic distributions of peak firing rates in CS-elicited activity are driven exclusively by the repetition of conditioning trials (i.e., practice) or may occur as well with the passage of time, similar to putative processes of memory consolidation. Multiunit activity was recorded in cingulate cortex and in the anterior dorsal (AD), anterior ventral (AV), and medial dorsal (MD) thalamic nuclei as rabbits learned to step in response to a warning tone (CS+) to prevent a scheduled foot-shock, and to ignore a different tone (CS-) not predictive of foot-shock. The rabbits received two training sessions, S1 and S2. S2 followed S1 immediately in one group of rabbits and after 48 h in a different group. Significant neuronal discharge increments occurred from S1 to S2 in the 48-h group but not in the 0-h group, for the areas (posterior cingulate cortex, AV thalamic nucleus) that previously showed only late-stage activation. Significant discharge increments occurred from S1 to S2 in the 0-h group but not in the 48-h group in areas (anterior cingulate cortex, the AD, and MD thalamic nuclei) that previously exhibited early stage activation. These results indicate that the trial-driven topographic distribution changes also occur with the passage of time after limited initial training. It is suggested that the trial-driven and time-related changes may have a common functional relevance concerning memory consolidation.

Neuronal activity in the cerebellar interpositus and lateral pontine nuclei during inhibitory classical conditioning of the eyeblink response.

Single-unit neuronal activity was recorded from the cerebellar interpositus nucleus and lateral pontine nuclei during conditioned inhibition of the eyeblink response in rats. Conditioned inhibition training sessions included 100 trials/day for 12 days. During each training session, the rats were given 50 presentations of a tone conditioned stimulus (CS) that was paired with a brief periocular shock unconditioned stimulus (US). They were also given 50 presentations of a compound stimulus that included the tone-CS and a light-CS. The compound-CS was not paired with the US. The two types of trials were mixed throughout the session and presented in an irregular sequence. This training procedure resulted in significant inhibition of the eyeblink response during the compound-CS. Neurons in the interpositus and lateral pontine nuclei exhibited significantly less activity during the compound-CS relative to the tone-CS. The suppression of cerebellar and pontine learning-related neuronal activity during the inhibitory CS may be critical for inhibiting the conditioned eyeblink response.

Impairment of cingulothalamic learning-related neuronal coding in rabbits exposed to cocaine in utero: general and sex-specific effects.

Neuronal activity was recorded in the cingulate cortex and the limbic thalamus in Dutch-belted rabbits (Oryctolagus cuniculus) exposed to cocaine (8 mg/kg/day i.v.) or saline in utero during acquisition and reversal learning of a discriminative avoidance response. Anterior cingulate cortical excitatory training-induced activity (TIA) was attenuated in cocaine-exposed female rabbits during acquisition and reversal learning, but only during reversal learning in male rabbits. Posterior cingulate cortical excitatory TIA was lessened in cocaine-exposed rabbits during acquisition, whereas discrimination between the positive and negative cues was enhanced. Neuronal firing was attenuated in the anterior ventral thalamus in cocaine-exposed rabbits during acquisition and reversal learning. Behavioral learning was normal in cocaine-exposed rabbits. Other data suggest that rabbits exposed to cocaine in utero exhibit a learning deficit when trained with nonsalient cues.

Pairing-specific long-term depression prevented by blockade of PKC or intracellular Ca2+.

Long-term depression was established in cerebellar Purkinje cells using 20 pairings of a brief, high frequency train of parallel fiber stimulation with a subsequent 100 ms depolarizing current injection. Effects were assessed on the peak amplitude of Purkinje cell excitatory post synaptic potentials (EPSPs) elicited by single parallel fiber test pulses. Intradendritically recorded Purkinje cell EPSPs exhibited long-term (>20 min) reduction in peak amplitude following paired stimulation but not if pretreated with the protein kinase C inhibitor calphostin C or iontophoretically injected with the calcium chelator EGTA. The similarity of the stimulation protocol and behavioral conditioning paradigms suggests pairing-specific long-term synaptic depression is a valuable model for understanding the cellular mechanisms underlying cerebellar cortical contributions to learning.

Classical conditioning increases membrane-bound protein kinase C in rabbit cerebellum.

We examined membrane-bound protein kinase C (PKC) in the cerebellum of rabbits given paired presentations of a tone conditioned stimulus (CS) that co-terminated with a periocular electrical stimulation unconditioned stimulus (US) or unpaired presentations of the CS and US or restraint in the experimental context. PKC activation was measured by quantitative film autoradiography of [3H]phorbol 12,13-dibutyrate ([3H]PBt2) binding in the molecular and granule cells layers of lobule HVI, anterior vermis and Crus I, and in the dentate/interpositus nuclei. There was a statistically significant increase in [3H]PBt2 binding within the molecular layer of lobule HVI in rabbits given paired training relative to controls. The results indicate PKC activation in lobule HVI may be important in acquisition of conditioned eyeblink responses.

Lesions of the entorhinal cortex disrupt behavioral and neuronal responses to context change during extinction of discriminative avoidance behavior.

Rabbits given either electrolytic lesions of the entorhinal cortex or sham-lesions were trained to prevent a foot-shock by stepping in an activity wheel after one tone, a positive conditioned stimulus (CS+), and to ignore a different tone, a negative conditioned stimulus (CS-). Neuronal activity was recorded simultaneously in the basolateral nucleus of the amygdala, the CA1 cell field of hippocampus, anterior cingulate cortical area 24b and posterior cingulate cortical area 29c/d. The activity of neurons in the entorhinal cortex was recorded in the controls. Acquisition of conditioned avoidance responses (CRs) was not affected by lesions of the entorhinal cortex. Discriminative neuronal activity (greater neuronal responses to the CS+ than to the CS-) during CR acquisition was significantly enhanced in hippocampal area CA1 and attenuated in the basolateral amygdala in rabbits with lesions. Following acquisition to a criterion, two counterbalanced extinction tests were administered, one in the original context and the other in the presence of novel contextual stimuli. CR frequency was significantly reduced in controls but not in rabbits with lesions, during extinction with novel contextual stimuli, relative to performance in the original context. The rabbits with lesions also showed fewer inter-trial responses than controls during extinction in the original context but intertrial response frequency in rabbits with lesions did not differ from the frequency in controls during extinction in the novel context. Neurons in the basolateral amygdala in controls showed discriminative activity during extinction in the original context but not in the novel context. Amygdalar neurons in the rabbits with lesions did not show discriminative activity during extinction in either context. Posterior cingulate cortical neurons in control rabbits did not show discriminative activity during extinction in the original context but these neurons exhibited robust discriminative activity in the novel context. Posterior cingulate cortical neurons in rabbits with lesions showed discriminative activity in both extinction sessions. The results indicated that the entorhinal cortex does not play a significant role in the acquisition of discriminative avoidance behavior, under the employed conditions of training. However, the interactions of neurons in the entorhinal cortex, amygdala and cingulate cortex are essential for contextual modulation of CRs during extinction.

Limbic thalamic, cingulate cortical and hippocampal neuronal correlates of discriminative approach learning in rabbits.

Previous research employing lesions and recording of neuronal activity has implicated cingulothalamic and hippocampal circuitry in the mediation of discriminative instrumental avoidance learning in rabbits. This study was directed at the question of whether the cingulothalamic circuitry is specialized for avoidance learning, or whether it is also involved in appetitively motivated learning. Multi-unit neuronal recordings in the aforementioned areas were obtained as adult New-Zealand white rabbits learned to approach and orally contact a drinking spout for water reward after a tone conditional stimulus (CS+), and to ignore the spout after a different, non-predictive tone conditional stimulus (CS-). As during avoidance learning, excitatory and discriminative training-induced neuronal activity (TIA) developed during the course of approach learning. Discriminative TIA refers to development of greater neuronal firing response to the CS+ than to the CS-. Excitatory TIA refers to increased neuronal discharge magnitude during training compared to the activity elicited before training, when CS presentations were unpaired with foot-shock presentations. As during avoidance learning, TIA in anterior cingulate cortical and interconnected mediodorsal (MD) thalamic neuronal records preceded TIA in posterior cingulate cortical and interconnected anterior ventral thalamic records. Delayed changes also occurred in area CA1 of the hippocampus in parallel with changes in the posterior cingulate cortex and the anterior thalamic nuclei. In contrast to the avoidance-related activity, the changes in the thalamic areas preceded or occurred concurrently with changes in the related cingulate cortical areas. This difference is hypothesized to be due to a reduced or absent contribution of amygdaloid efferents to the approach learning-related TIA. The overall magnitude of the elicited training-induced neuronal responses was reduced, relative to the discharges during avoidance conditioning. The discharge magnitude differences suggested a greater recruitment of limbic circuit functions during avoidance learning, possibly due to the aversiveness and high arousal associated with the avoidance task. In general, the results indicate that the circuitry formed by interconnected cingulate cortical, limbic thalamic and hippocampal neurons has fundamentally similar functions in both approach and avoidance learning.

Context-specific multi-site cingulate cortical, limbic thalamic, and hippocampal neuronal activity during concurrent discriminative approach and avoidance training in rabbits.

This study assessed the context specificity of learning-related neuronal activity: whether the same physical stimuli would elicit different neuronal responses depending on the learning situation. Neuronal activity was recorded simultaneously in six limbic areas as rabbits learned to approach a spout for water reinforcement after a tone (CS+) and to ignore the spout after a different tone (CS-). The rabbits then received avoidance training in which they learned to prevent a foot-shock by stepping in an activity wheel after one tone (CS+) and to ignore a different tone (CS-). Avoidance training sessions were alternated (1 session daily) with sessions in the well learned approach task. The tone assigned as the CS+ for approach training was the CS- for avoidance training and vice versa. The neuronal records of the anterior ventral and medial dorsal thalamic nuclei and the anterior and posterior cingulate cortices showed neuronal discrimination appropriate to the approach task during pretraining in the avoidance training apparatus with unpaired presentations of the tones and foot-shock. This finding demonstrated that the discriminative neuronal activity for approach learning was unaffected by a change in context in the pretraining session. However, context-appropriate discrimination occurred in both tasks thereafter, with the exception that medial dorsal thalamic neurons no longer showed discrimination during overtraining in the approach task. Hippocampal area CA1 neurons showed entirely context-appropriate discrimination in both tasks, with no carryover of the approach-relevant discrimination to the avoidance training apparatus. Avoidance training stage-specific peaks of training-induced excitation in different brain areas were not elicited by the same physical stimuli during concurrent approach training sessions. The results are consistent with an involvement of limbic-circuit neuronal activity in the use of context cues for mnemonic retrieval. Differential persistence of the approach-related neuronal discrimination in anterior and posterior cingulate cortex confirmed the previously hypothesized distinct mnemonic functions of these areas.

Disruption of cerebellar maturation by an antimitotic agent impairs the ontogeny of eyeblink conditioning in rats.

This study represents an attempt to establish a relationship between maturation of the cerebellum and the ontogeny of eyeblink conditioning in the rat. Experiments 1 and 2 examined the effects of disrupting cerebellar maturation by neonatal exposure to the antimitotic agent methylazoxymethanol (MAM) on the ontogeny of eyeblink conditioning in infant rats. Experiment 1 demonstrated that neonatal exposure to MAM on Postnatal Day 4 (PND4) and 7 severely disrupted cerebellar maturation. This effect appeared to be specific in that there was no overt dysmorphology in other brain regions. MAM treatment also severely disrupted associative eyeblink conditioning in rats given training on PND24 and 25. However, exposure to MAM had no effect on the unconditioned response, T-maze delayed alternation, or conditioned suppression of ongoing behavior. In Experiment 2, MAM was given on PND4 and 7 and pups were tested behaviorally on PND17-18, 20-21, or 31-32. Cerebellar hypoplasia was most dramatic shortly after exposure. The cerebellar cortex continued to mature after exposure to MAM, but development of morphological endpoints examined here were static from PND19 to 33. Eyeblink conditioning was impaired at all ages, indicating that there was no functional recovery following neonatal exposure to MAM over the age range tested. These experiments suggest that normal cerebellar maturation may be important for the ontogeny of eyeblink conditioning.

Early cerebellar lesions impair eyeblink conditioning in developing rats: differential effects of unilateral lesions on postnatal day 10 or 20.

Experiment 1 demonstrated that the ipsilateral cerebellar hemisphere is essential for the acquisition of eyeblink conditioning in infant rats and that cerebellar lesions given on Postnatal Day 10 (PND10) produced deficits in eyeblink conditioning when given to either hemisphere. For both hemispheres, lesions that were restricted to the cerebellar cortex produced less severe deficits than lesions that included the deep nuclei. Experiment 2 showed that the age at which the cerebellar lesions occurred determined whether damage to the contralateral cerebellar hemisphere impaired conditioning. Lesions of either the ipsilateral or contralateral hemisphere that included the deep nuclei disrupted eyeblink conditioning when given on PND10. In contrast, when lesions were given on PND20, ipsilateral lesions that included the deep nuclei abolished conditioning, while the same lesion given to the contralateral hemisphere had no effect.