Multiple phases of human occupation in Southeast Arabia between 210,000 and 120,000 years ago.

Changing climatic conditions are thought to be a major control of human presence in Arabia during the Paleolithic. Whilst the Pleistocene archaeological record shows that periods of increased monsoon rainfall attracted human occupation and led to increased population densities, the impact of arid conditions on human populations in Arabia remains largely speculative. Here, we present data from Jebel Faya in Southeast (SE) Arabia, which document four periods of human occupation between c. 210,000 and 120,000 years ago. The Jebel Faya record indicates that human occupation of SE Arabia was more regular and not exclusively linked to major humid periods. Our data show that brief phases of increased rainfall additionally enabled human settlement in the Faya region. These results imply that the mosaic environments in SE Arabia have likely formed a population refugia at the end of the Middle and the beginning of the Late Pleistocene.

Social inclusion, intersectionality, and profiles of vulnerable groups of young people seeking mental health support.

BACKGROUND: headspace centres provide enhanced primary mental healthcare for young people. A priority is to provide services for all young people irrespective of a range of social disadvantages or social exclusion. The aims of this study were to: (i) delineate extent of social inclusion across domains of housing, studying/employment, functioning, alcohol, and other drug use; and (ii) map profiles of young people deemed vulnerable to experiencing additional barriers to accessing services based on their social inclusion domains (e.g., those living in unstable housing, not in employment/education, and/or experiencing intersecting or multiple forms of disadvantage or difficulties), including detailing their clinical characteristics. METHODS: Young people were recruited from five headspace centres. Data relevant to social inclusion were examined. Multivariate logistic regression models were used to determine overlap between vulnerable groups, functional, social, clinical, and behavioural factors. RESULTS: 1107 young people participated, aged 12-25 years (M = 18.1 years, SD = 3.3), most living in stable housing (96.5%) and engaged in studying/employment (84.8%). Specific vulnerabilities were evident in young people with NEET status (15.2%); in unstable accommodation (3.5%); of culturally diverse backgrounds (CALD) (12.2%); living in regional areas (36.1%); and identifying as lesbian, gay, bisexual, transgender, intersex, queer/questioning, and asexual plus (LGBTIQA+; 28.2%). Higher levels of distress, substance use, functional impairment, and lower social support were reported by those who were NEET and/or in unstable housing. LGBTIQA+ status was associated with high distress, depressive symptoms, and suicidal ideation. CONCLUSIONS: Most participants reported good social support, stable housing, and engagement in work or education. Those deemed vulnerable were likely to experience social exclusion across multiple domains and reported more mental health problems. The co-occurrence of mental ill-health and social exclusion highlights the importance of integrated mental healthcare.

Boosting the sterile insect technique with pyriproxyfen increases tsetse flies Glossina palpalis gambiensis sterilization in controlled conditions.

Tsetse flies (Diptera: Glossinidae) are the main vectors of animal and human trypanosomoses in Africa. The Sterile Insect Technique (SIT) has proven effective in controlling tsetse flies when applied to isolated populations but necessitates the production of large numbers of sterile males. A new approach, called boosted SIT, combining SIT with the contamination of wild females by sterile males coated with biocides has been proposed for large-scale control of vector populations. The aim of the study was to evaluate this new approach using pyriproxyfen on the riverine species Glossina palpalis gambiensis (Vanderplank, 1949) in the laboratory. The contamination dose and persistence of pyriproxyfen on sterile males, the impact of pyriproxyfen on male survival, and the dynamics of pyriproxyfen transfer from a sterile male to a female during mating, as well as the impact of pyriproxyfen on pupal production and adult emergence, were evaluated in the laboratory. For this purpose, a method of treatment by impregnating sterile males with a powder containing 40% pyriproxyfen has been developed. The results showed that the pyriproxyfen has no impact on the survival of sterile males. Pyriproxyfen persisted on sterile males for up to 10 days at a dose of 100 ng per fly. In addition, the horizontal transfer of pyriproxyfen from a treated sterile male to a female during mating could be measured with an average of 50 ng of pyriproxyfen transferred. After contacts without mating, the average quantity transferred was more than 10 ng. Finally, the pyriproxyfen powder was very effective on G. p. gambiensis leading to 0% emergence of the pupae produced by contaminated females. These promising results must be confirmed in the field. A large-scale assessment of this boosted pyriproxyfen-based SIT approach will be carried out against tsetse flies in Senegal (West Africa).

Treating depression with physical activity in adolescents and young adults: a systematic review and meta-analysis of randomised controlled trials.

We aimed to establish the treatment effect of physical activity for depression in young people through meta-analysis. Four databases were searched to September 2016 for randomised controlled trials of physical activity interventions for adolescents and young adults, 12-25 years, experiencing a diagnosis or threshold symptoms of depression. Random-effects meta-analysis was used to estimate the standardised mean difference (SMD) between physical activity and control conditions. Subgroup analysis and meta-regression investigated potential treatment effect modifiers. Acceptability was estimated using dropout. Trials were assessed against risk of bias domains and overall quality of evidence was assessed using GRADE criteria. Seventeen trials were eligible and 16 provided data from 771 participants showing a large effect of physical activity on depression symptoms compared to controls (SMD = -0.82, 95% CI = -1.02 to -0.61, p < 0.05, I2 = 38%). The effect remained robust in trials with clinical samples (k = 5, SMD = -0.72, 95% CI = -1.15 to -0.30), and in trials using attention/activity placebo controls (k = 7, SMD = -0.82, 95% CI = -1.05 to -0.59). Dropout was 11% across physical activity arms and equivalent in controls (k = 12, RD = -0.01, 95% CI = -0.04 to 0.03, p = 0.70). However, the quality of RCT-level evidence contributing to the primary analysis was downgraded two levels to LOW (trial-level risk of bias, suspected publication bias), suggesting uncertainty in the size of effect and caution in its interpretation. While physical activity appears to be a promising and acceptable intervention for adolescents and young adults experiencing depression, robust clinical effectiveness trials that minimise risk of bias are required to increase confidence in the current finding. The specific intervention characteristics required to improve depression remain unclear, however best candidates given current evidence may include, but are not limited to, supervised, aerobic-based activity of moderate-to-vigorous intensity, engaged in multiple times per week over eight or more weeks. Further research is needed. (Registration: PROSPERO-CRD 42015024388).

Challenging the Wigglesworthia, Sodalis, Wolbachia symbiosis dogma in tsetse flies: Spiroplasma is present in both laboratory and natural populations.

Profiling of wild and laboratory tsetse populations using 16S rRNA gene amplicon sequencing allowed us to examine whether the "Wigglesworthia-Sodalis-Wolbachia dogma" operates across species and populations. The most abundant taxa, in wild and laboratory populations, were Wigglesworthia (the primary endosymbiont), Sodalis and Wolbachia as previously characterized. The species richness of the microbiota was greater in wild than laboratory populations. Spiroplasma was identified as a new symbiont exclusively in Glossina fuscipes fuscipes and G. tachinoides, members of the palpalis sub-group, and the infection prevalence in several laboratory and natural populations was surveyed. Multi locus sequencing typing (MLST) analysis identified two strains of tsetse-associated Spiroplasma, present in G. f. fuscipes and G. tachinoides. Spiroplasma density in G. f. fuscipes larva guts was significantly higher than in guts from teneral and 15-day old male and female adults. In gonads of teneral and 15-day old insects, Spiroplasma density was higher in testes than ovaries, and was significantly higher density in live versus prematurely deceased females indicating a potentially mutualistic association. Higher Spiroplasma density in testes than in ovaries was also detected by fluorescent in situ hybridization in G. f. fuscipes.

X-ray-induced sterility in Aedes albopictus (Diptera: Culicidae) and male longevity following irradiation.

The mosquito Aedes albopictus (Skuse, 1895) is a potent vector of several arboviral diseases, most notably chikungunya and dengue fever. In the context of the sterile insect technique (SIT), the sterilization of the male mosquitoes before their release can be achieved by gamma-ray irradiation. As gamma-ray irradiators are becoming increasingly problematic to purchase and transport, the suitability of an X-ray irradiator as an alternative for the sterilization of Ae. albopictus males was studied. The sterilization of up to 200,000 pupae at one time can be achieved with relative ease, and the sterility results obtained were comparable with those achieved by gamma irradiation, where 99% sterility is induced with a dose of 40 Gy. A significant reduction of longevity was observed in the latter stages of the males' life after irradiation treatments, especially at doses > 40 Gy, which is consistent with the negative effects on longevity induced by similar radiation doses using gamma rays. Females irradiated at 40 Gy were not only 100% sterile, but also failed to oviposit entirely, i.e., all of the females laid 0 eggs. Overall, it was found that the X-ray irradiator is generally suitable for the sterilization process for sterile insect technique programs, as it showed a high processing capacity, practicality, high effectiveness, and reproducibility.

Prevalence and genetic variation of salivary gland hypertrophy virus in wild populations of the tsetse fly Glossina pallidipes from southern and eastern Africa.

The Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is a rod-shaped, non-occluded double-stranded DNA virus that causes salivary gland hypertrophy (SGH) and reduced fecundity in the tsetse fly G. pallidipes. High GpSGHV prevalence (up to 80%) makes it impossible to mass-rear G. pallidipes colonies for the sterile insect technique (SIT). To evaluate the feasibility of molecular-based GpSGHV management strategies, we investigated the prevalence and genetic diversity of GpSGHV in wild populations of G. pallidipes collected from ten geographical locations in eastern and southern Africa. Virus diversity was examined using a total sequence of 1497 nucleotides (≈ 1% of the GpSGHV genome) from five putative conserved ORFs, p74, pif1, pif2, pif3 and dnapol. Overall, 34.08% of the analyzed flies (n=1972) tested positive by nested PCR. GpSGHV prevalence varied from 2% to 100% from one location to another but phylogenetic and gene genealogy analyses using concatenated sequences of the five putative ORFs revealed low virus diversity. Although no correlation of the virus diversity to geographical locations was detected, the GpSGHV haplotypes could be assigned to one of two distinct clades. The reference (Tororo) haplotype was the most widely distributed, and was shared by 47 individuals in seven of the 11 locations. The Ethiopian haplotypes were restricted to one clade, and showed the highest divergence (with 14-16 single nucleotide mutation steps) from the reference haplotype. The current study suggests that the proposed molecular-based virus management strategies have a good prospect of working throughout eastern and southern Africa due to the low diversity of the GpSGHV strains.

Risk factors for relapse following treatment for first episode psychosis: a systematic review and meta-analysis of longitudinal studies.

BACKGROUND: Preventing relapse is an essential element of early intervention in psychosis, but relevant risk factors and precise relapse rates remain to be clarified. The aim of this study was to systematically compile and analyse risk factors for and rates of relapse in the early course of psychosis. METHODS: Systematic review and meta-analysis of English and non-English language, peer-reviewed, longitudinal studies, with a minimum 12-month follow-up and at least 80% of participants diagnosed with a first episode of psychosis (FEP) that reported risk factors for relapse. RESULTS: Of 153 potentially relevant articles, 29 were included in the study. Pooled prevalence of relapse of positive symptoms was 28% (range=12-47%), 43% (35-54%), 54% (40-63%) at 1, 1.5-2, and 3 years follow-up, in that order. A total of 109 predictors were analysed, with 24 being assessed in at least 3 studies. Of those, 20 predictors could be extracted for meta-analysis. Medication non-adherence, persistent substance use disorder, carers' critical comments (but not overall expressed emotion) and poorer premorbid adjustment, increased the risk for relapse 4-fold, 3-fold, 2.3-fold and 2.2-fold, respectively. CONCLUSIONS: Clinical variables and general demographic variables have little impact on relapse rates. Conversely, non-adherence with medication, persistent substance use disorder, carers' criticism and poorer premorbid adjustment significantly increase the risk for relapse in FEP. Future studies need to address the methodological limitations of the extant research (e.g. definition of relapse), focus on the identification of protective factors and evaluate theoretically derived models of relapse.

Analysis of the antiviral drugs acyclovir and valacyclovir-hydrochloride in tsetse flies (Glossina pallidipes) using LC-MSMS.

A new simple, sensitive and precise liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of valacyclovir-HCl and acyclovir in tsetse flies (Glossina pallipides). Tsetse flies were extracted by ultrasonication with acidified methanol/acetonitrile, centrifuged and cleaned up by solid phase dispersion using MgSO(4) and MSPD C(18) material. Samples were analysed using a Waters Alliance 2695 series HPLC with a C(18) Gemini analytical column (150 mm x 4.6 mm x 5 microm) and a guard cartridge column connected to a Waters Quattro-Micro triple-quadrupole mass spectrometer. The isocratic mobile phase consisted of methanol:acetonitrile:water (60:30:10, v/v/v) plus formic acid (0.1%) at a flow rate of 0.25 ml/min. The precursor>product ion transition for valacyclovir (m/z 325.1>152) and acyclovir (m/z 226.1>151.9) were monitored in positive electrospray multiple reaction monitoring mode. The method was validated at fortification levels of 0.5, 1 and 2 microg/g. The range of calibration for both drugs was 0.45-4.5 microg/g. The overall accuracy of the method was 92% for valacyclovir and 95% for acyclovir with corresponding within-laboratory reproducibilities of 4.4 and 3.4%, respectively. Mean recoveries were above 80% for both drugs and repeatability ranged from 0.7 to 6.1%. For both drugs the limits of detection and quantification were 0.0625 and 0.2 microg/g, respectively. The method was applied in experiments on the mass rearing of tsetse flies for sterile insect technique (SIT) applications, in which the flies were fed with blood meals containing acyclovir or valcyclovir-HCl prior to analysis to assess effects on Glossina pallidipes Salivary Gland Hypertrophy syndrome.

A new generation of X ray irradiators for insect sterilization.

Recent fears of terrorism have provoked an increase in delays and denials of transboundary shipments of radioisotopes. This represents a serious constraint to sterile insect technique (SIT) programs around the world as they rely on the use of ionizing radiation from radioisotopes for insect sterilization. To validate a novel X ray irradiator, a series of studies on Ceratitis capitata (Wiedemann) and Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) were carried out, comparing the relative biological effectiveness (RBE) between X rays and traditional gamma radiation from 60Co. Male C. capitata pupae and pupae of both sexes of A. fraterculus, both 24-48 h before adult emergence, were irradiated with doses ranging from 15 to 120 Gy and 10-70 Gy, respectively. Estimated mean doses of 91.2 Gy of X and 124.9 Gy of gamma radiation induced 99% sterility in C. capitata males. Irradiated A. fraterculus were 99% sterile at approximately 40-60 Gy for both radiation treatments. Standard quality control parameters and mating indices were not significantly affected by the two types of radiation. The RBE did not differ significantly between the tested X and gamma radiation, and X rays are as biologically effective for SIT purposes as gamma rays are. This work confirms the suitability of this new generation of X ray irradiators for pest control programs that integrate the SIT.

Who needs antipsychotic medication in the earliest stages of psychosis? A reconsideration of benefits, risks, neurobiology and ethics in the era of early intervention.

In recent years, early intervention services have attempted to identify people with a first episode of psychosis as early as possible, reducing the duration of untreated psychosis and changing the timing of delivery of interventions. The logic of early intervention is based partly on accessing people in a more treatment responsive stage of illness in which psychosocial damage is less extensive, and partly on remediating a putatively active process of neuroprogression that leads to pathophysiological, symptomatic and structural changes, hence improving symptomatic and functional outcomes. However, as in other areas of health care, earlier identification of new patients may mean that different treatment approaches are indicated. The corollary of early detection is that the sequence and complexion of treatment strategies for first episode psychosis has been revaluated. Examples include the minimal effective dosage of antipsychotic medication and the content of psychosocial interventions. With the substantial reductions of DUP now seen in many early psychosis services, based on clinical staging and stepped care principles, it is even possible that the immediate introduction of antipsychotic medication may not be necessary for all first episode psychosis cases, but that potentially safer interventions, which may be more acceptable to many patients, such as comprehensive psychosocial intervention, may constitute effective treatment at least for a subgroup of patients. In this paper, we review this theoretical background and describe a randomised controlled trial currently underway at the Early Psychosis Prevention and Intervention Centre (EPPIC) in Melbourne designed to test outcomes for first episode psychosis patients in response to two different treatments: intensive psychosocial intervention plus antipsychotic medication versus intensive psychosocial intervention plus placebo. This is a theoretically and pragmatically novel study in that it will provide evidence as to whether intensive psychosocial intervention alone is sufficient for a subgroup of first episode psychosis patients in a specialised early intervention service, and provide a test of the heuristic clinical staging model. By experimentally manipulating duration of untreated psychosis, the study will also provide a methodologically strong test of the effect of delaying the introduction of antipsychotic medication, as well as helping to disentangle the effects of antipsychotic medications and the putative neurobiological processes associated with brain changes and symptom profiles in the early phase of psychotic disorders. The study has been carefully crafted to satisfy critical ethical demands in this challenging research domain.

Early identification and intervention in depressive disorders: towards a clinical staging model.

BACKGROUND: Depressive disorders are common and associated risks include the onset of secondary disorders, substance use disorders, impairment in social and occupational functioning, and an increase in suicidality. As the onset often occurs in youth, there is a clear imperative for early identification and intervention to ameliorate, if not prevent, associated distress. METHODS: An extensive search of relevant databases and an ancestry search was undertaken. RESULTS: There is a limited but growing body of literature on this topic that is discussed in relation to a clinical staging model, which may prove to be a useful framework for identifying where an individual lies along the continuum of the course of a depressive illness thus allowing interventions to be matched for that stage. The identification of a subsyndromal and prodromal stage of depressive disorders provides early intervention opportunities. CONCLUSIONS: It is argued that a clinical staging heuristic may increase the number of those treated early, which may in turn delay or prevent onset, reduce severity, or prevent progression in the course of depressive disorders.

The prevention of schizophrenia.

Preventive strategies can be divided into universal, selective and indicated prevention and early intervention. Universal interventions are directed to the general population. Selective approaches are targeted at people who have risk factors for an illness, but who do not show any current signs. Indicated approaches target high risk individuals with minimal signs or symptoms foreshadowing mental disorder, but who do not meet diagnostic levels at the current time. Early intervention involves treating those with already diagnosable disorder in a timely and optimal manner aiming to decrease the severity of the illness, and reduce secondary morbidity. Although universal and selective interventions are not yet viable strategies, indicated prevention and early intervention are now realistic possibilities in schizophrenia. Development of methods to identify those at risk of psychosis continues to evolve. Promising results in the prevention and delay of transition to psychotic disorder from high risk state have been found. Early intervention in schizophrenia, including promotion of early help-seeking, has been shown to reduce the duration of untreated psychosis, which is known to be associated with poor outcome in schizophrenia. Early intervention programmes which optimise the care of the first episode have been shown to produce better outcomes than routine management.

Sex separation of tsetse fly pupae using near-infrared spectroscopy.

Implementation of the sterile insect technique for tsetse (Glossina spp.) requires that only sterile male insects be released; thus, at some stage of the fly production process the females have to be removed. A further constraint in the use of the sterile insect technique for tsetse is that the females are needed for colony production and hence, a non-destructive method of sex separation is required. In most tsetse sterile insect technique programmes thus far, females have been eliminated from the released material by hand-separation of chilled adults. Using near-infrared (NIR) spectroscopy, significant differences have been found between the spectra for the pupae of male and female G. pallidipes Austen. Significantly, the differences appear to be maximized 4-5 days before emergence of the adults. Tsetse fly pupae up to five days before emergence can be sexed with accuracies that generally range from 80 to 100%. This system, when refined, will enable effective separation of male and female pupae to be carried out, with emerged females being returned to the colony and males being irradiated and released. If separation can be achieved five days before emergence, this will also enable irradiated male pupae to be shipped to other destinations as required. Other Diptera were evaluated using this system but had lower classification accuracies of 50-74%. This may be due to the difference in reproductive physiology between these different fly groups.

The effect of age on the mating competitiveness of male Glossina fuscipes fuscipes and G. palpalis palpalis.

The effect of age on male Glossina fuscipes fuscipes, Newstead, and Glossina palpalis palpalis, Austin (Diptera: Glossinidae) competiveness were investigated with a view to estimate optimal age for sterile male release. Sterile insect technique involves the mass production, sterilization and sequential release of males of the target species to out compete the wild male population. Mating between released sterile males and wild females produce inviable progeny and the population is reduced over several generations to unsustainable levels. It is vital that the released male are of high quality and are sexually competitive. Age is one parameter affecting the sexual competiveness of the male tsetse fly. The optimal release age was estimated by assessing sexual competitiveness of flies of different age categories, 1, 5, 8 and 13-days after adult eclosion. A walk-in field-cage was used in order to approximate as closely as possible the actual field scenario during sterile insect release programes. It was shown that 8 and 13-day old males mated significantly more frequently, i.e. were more competitive, in the presence of equal numbers of 1 and 5-day old males. The age of male tsetse flies significantly affected competitiveness in both species studied. The ability of G. f. fuscipes to inseminate was not age dependent, and insemination occurred in all females that mated regardless of male age. In G. p. palpalis, however, 1-day old males were least able to inseminate. Mating duration was not significantly affected by age in both species. Eight to thirteen day old males of the test species are here recommended as the optimal sterile male release age.

Mating age of Glossina austeni Newstead.

Before removal from the emergence cage, 12.8% of 141 newly emerged females of Glossina austeni Newstead less than 24 h old were found on dissection to have been inseminated. Likewise, dissection of a sample of sterilised females destined for release showed that 5.43% of 2487 females had already been inseminated while still in the emergence cages. It was decided therefore to put female and male flies together in production cages from the day of emergence at a ratio of 1 male to 5 females and leave them to mature and mate in the cages. The females produced viable pupae of acceptable mean weight and desired quality with the proportion of A-Class pupae less than 10%. The number of pupae per initial female (PPIF) did not differ from pupae produced by pre-aged parent flies. It is now clear that there is no need to age female and male flies of G. austeni before mating. Parent flies of less than 1 day old put together in production cages from the day of emergence have been used for mass rearing G. austeni in the Tsetse and Trypanosomosis Research Institute (TTRI) colony to produce males for the eradication programme in Zanzibar since December 1995. This has substantially reduced the labour of fly production by removing the need to age flies and the need to chill and separate flies after mating.

Regulation of mouse liver microsomal esterases by clofibrate and sexual hormones.

Carboxylesterase activity was measured using six different substrates in microsomal preparations from female and ovariectomized female mice in order to evaluate the effects of female sex hormones on esterase expression. With three of the substrates (alpha-naphthyl acetate and esters 2 and 3), esterase activity was the same in both groups; however, with the others (rho-nitrophenyl acetate and esters 1 and 4), there was a small increase in activity in ovariectomized females, compared with intact females. Castration of males followed by treatment with testosterone caused only transient increases in activity for four of the substrates (alpha-naphthyl acetate and esters 1, 2, and 3) and no change in activity for the other two (rho-nitrophenyl acetate and ester 4). Treatment of male and female mice with the peroxisome proliferator clofibrate, with or without testosterone, resulted in increased hydrolysis of alpha-naphthyl acetate and rho-nitrophenyl acetate, but little change for the other substrates. Clofibrate also induced alpha-naphthyl acetate and rho-nitrophenyl acetate hydrolysis in castrated males, but clofibrate and testosterone administrated together resulted in significant increases of activity with all substrates, which were greater than the additive effects of the two compounds administered separately. These results indicate that clofibrate causes significant alterations in the regulation of esterase activity, whereas sex hormones only cause small changes. However, it would seem that testosterone can synergize the effect of clofibrate in castrated males, resulting in higher levels of activity than with clofibrate alone. Finally, an overall increase in esterase activity might be due to a large increase in the activity of a few esterases or to a small increase in many esterases. Enzyme staining of native polyacrylamide gels reveals that the latter is true, with the majority of esterases present in mouse liver microsomes being induced to a small degree by clofibrate.

Differential regulation of soluble epoxide hydrolase by clofibrate and sexual hormones in the liver and kidneys of mice.

Soluble epoxide hydrolase (sEH) activity was measured in the liver and kidneys of male, female, and castrated male mice in order to evaluate sex- and tissue-specific differences in enzyme expression. sEH activity was found to be higher in liver than in kidneys. Activity increased with age in the liver of females, males and castrated males, but only in males did activity in the kidneys increase. There was greater activity in both the liver and kidneys of adult males than females. This sexual dimorphism was more pronounced in the kidneys (283% higher) than in the liver (55% higher). Castration of males led to a decrease in activity in both organs, but it had a greater effect on renal activity (67% decrease) than on hepatic activity (27% decrease). Treatment of castrated mice with testosterone led to an increase in sEH activity of 400% in kidneys and 49% in liver compared with surgical controls. These results suggest differential regulation of sEH by testosterone in kidneys and liver. Ovariectomized female mice had renal and hepatic activities approximately 30% greater than control females. Feeding mice with the hypolipidemic drug clofibrate produced stronger induction of sEH in liver than in kidneys. Testosterone treatment, however, caused greater induction in kidneys than in liver of females and castrated males and had no effect in either kidneys or liver in males. When given together, the effects of these two compounds appeared to be additive in both liver and kidneys. Results from western blot showed that the increase in sEH enzyme activity in kidneys is correlated with an increase in sEH protein. These results suggest that clofibrate and testosterone independently regulate sEH activity in vivo, and that kidneys and liver respond differently to clofibrate and testosterone.

Molecular and biochemical evidence for the involvement of the Asp-333-His-523 pair in the catalytic mechanism of soluble epoxide hydrolase.

In order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. Our results are consistent with the involvement of Asp-333 and His-523 in a catalytic mechanism similar to that of other alpha/beta hydrolase fold enzymes. Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. When His-332 was replaced by asparagine, 96.7% of the specific activity was lost and mutation of the conserved His-523 to glutamine led to a more dramatic loss of 99.9% of the specific activity. No activity was detectable after the replacement of Asp-333 by serine. However, more than 20% of the wild-type activity was retained in an Asp-333-->Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333-->Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the epoxide containing radiolabeled substrate juvenile hormone III (JH III). The purified Asp-333-->Asn mutant bound 6% of the substrate compared to the wild-type soluble epoxide hydrolase. The mutant also showed 8% of the specific activity of the wild-type. Preincubation of the purified Asp-333-->Asn mutant at 37 degrees C (pH 8), however, led to a complete recovery of activity and to a change of isoelectric point (pI), both of which are consistent with hydrolysis of Asn-333 to aspartic acid. This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant. Wild-type enzyme that had been radiolabeled with the substrate was digested with trypsin. Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity. These peptides were not radiolabeled if the enzyme was preincubated with a selective competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone oxide. This strongly suggested that these peptides contained a catalytic amino acid. Each peptide was characterized with N-terminal amino acid sequencing and electrospray mass spectrometry. All four radiolabeled peptides contained overlapping sequences. The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333. These peptides resulted from cleavage at different trypsin sites and the mass of each was consistent with the covalent linkage of Asp-333 to the substrate.

Selection of dieldrin-resistant strains of Lucilia cuprina (Diptera: Calliphoridae) after ethyl methanesulfonate mutagenesis of a susceptible strain.

After ethyl methanesulfonate (EMS) mutagenesis of a susceptible strain (SWT), selective screening of Lucilia cuprina (Wiedemann) resulted in four strains that were resistant to the insecticide dieldrin. Concentrations used for selection were greater than LC99 of susceptible phenotypes. No resistant variants were screened from the standard laboratory strain (SWT) not treated with EMS. The resistance phenotypes of the four resistant strains were similar to each other and to that of a field-selected resistant strain. The genetic basis of resistance is monogenic in all strains and the data are consistent with the same locus, Rdl, determining resistance status in each strain. The Rdl locus maps to chromosome V, approximately 3.5 map units distal to the Sut locus. Dieldrin resistance may be caused by less effective blocking of insect neuronal GABA receptors by the chemical in resistant strains. The data indicate that the evolution of resistance to an insecticide in the field may be constrained by a limited number of genetical and biochemical options if a monogenic response is selected for and that the spontaneous mutation rate to the Rdl allele is less than 1 in 10(6) in the laboratory.