Whole-Cell MALDI-ToF MS Coupled with Untargeted Metabolomics Facilitates Investigations of Microbial Chemical Interactions.

The emergence of drug-resistant pathogens necessitates the development of new countermeasures. In this regard, the introduction of probiotics to directly attack or competitively exclude pathogens presents a useful strategy. Application of this approach requires an understanding of how a probiotic and its target pathogen interact. A key means of probiotic-pathogen interaction involves the production of small molecules called natural products (NPs). Here, we report the use of whole-cell matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry to characterize NP production by candidate probiotics (mouse airway microbiome isolates) when co-cultured with the respiratory pathogen Burkholderia. We found that a Bacillus velezensis strain inhibits growth of and elicits NP production by Burkholderia thailandensis. Dereplication of known NPs detected in the metabolome of this B. velezensis strain suggests that a previously unannotated bioactive compound is involved. Thus, we present the use of whole-cell MALDI as a broadly applicable method for screening the NP composition of microbial co-cultures; this can be combined with other -omics methods to characterize probiotic-pathogen and other microbe-microbe interactions.

Understanding Perceptions of Nonsuicidal Self-Injury: Descriptive and Injunctive Norms.

Nonsuicidal self-injury (NSSI) is a major public health concern. Web-based personalized feedback interventions (PFIs) may be a cost-effective and efficient way to treat NSSI. In order to develop a PFI, it is imperative to assess descriptive and injunctive norms. The current study examines descriptive and injunctive norms of NSSI within college students and adults in the community, comparing how perceived norms may differ for those who do or do not engage in NSSI. Study 1 calculated percentages of NSSI behavior within the student sample. Study 2 then examined perceived descriptive and injunctive norms between those with and without history of NSSI in both samples. Study 1 indicated that 19% of undergraduate students had histories of NSSI. Additionally, there was a general tendency to overestimate the percentage of people who engage in NSSI and the number of times a typical person engages in NSSI. Finally, those who engaged in NSSI believed that most people do not understand why individuals engage in NSSI; comparatively, the majority of people without history of NSSI still indicated that they understand why others would engage in NSSI. These research findings may be utilized in a PFI to reduce shame and NSSI behavior.

Development of a cyclin-dependent kinase inhibitor devoid of ABC transporter-dependent drug resistance.

BACKGROUND: Cyclin-dependent kinases (CDKs) control cell cycle progression, RNA transcription and apoptosis, making them attractive targets for anticancer drug development. Unfortunately, CDK inhibitors developed to date have demonstrated variable efficacy. METHODS: We generated drug-resistant cells by continuous low-dose exposure to a model pyrazolo[1,5-a]pyrimidine CDK inhibitor and investigated potential structural alterations for optimal efficacy. RESULTS: We identified induction of the ATP-binding cassette (ABC) transporters, ABCB1 and ABCG2, in resistant cells. Assessment of features involved in the ABC transporter substrate specificity from a compound library revealed high polar surface area (>100 Å(2)) as a key determinant of transporter interaction. We developed ICEC-0782 that preferentially inhibited CDK2, CDK7 and CDK9 in the nanomolar range. The compound inhibited phosphorylation of CDK substrates and downregulated the short-lived proteins, Mcl-1 and cyclin D1. ICEC-0782 induced G2/M arrest and apoptosis. The permeability and cytotoxicity of ICEC-0782 were unaffected by ABC transporter expression. Following daily oral dosing, the compound inhibited growth of human colon HCT-116 and human breast MCF7 tumour xenografts in vivo by 84% and 94%, respectively. CONCLUSION: We identified a promising pyrazolo[1,5-a]pyrimidine compound devoid of ABC transporter interaction, highly suitable for further preclinical and clinical evaluation for the treatment of cancer.

Cystic fibrosis transmembrane conductance regulator splice variants are not conserved and fail to produce chloride channels.

In the human CFTR only the rare exon 4- splice variant is conserved in mice. We have discovered two novel murine variants, exon 5- and exon 11b+. The exon 5- variant represents up to 40% of mRNA in all CFTR-expressing tissues and leaves the reading frame intact. The exon 11b+ variant inserts a novel exon between exons 11 and 12 with expression restricted to the testis. Two variants of 11b have been found and both introduce premature stop codons. When we expressed human CFTR variants lacking either exon 5 or exon 9 in HeLa cells, they failed to generate cAMP-mediated chloride transport, due to defective intracellular processing. The lack of conservation of splice variants between species and the inability of the more abundant splice variants to generate protein that is correctly processed argue against a physiological role and may simply represent aberrant splicing that is tolerated by the cell and organism.

Gastroschisis ultrasound bowel characteristics demonstrate minimal impact on perinatal outcomes.

BACKGROUND: Bowel dilation and bowel wall thickness are common prenatal ultrasound measurements for fetuses with gastroschisis. Data regarding antenatal sonographic bowel findings and postnatal outcomes are conflicting. Our objective was to evaluate the impact of in utero bowel measurements on perinatal outcomes in gastroschisis pregnancies. METHODS: Retrospective cohort study of 116 pregnancies complicated by gastroschisis between 2011 and 2020. We reviewed ultrasounds documenting fetal bowel measurements. To evaluate the association of these measurements with antepartum and delivery outcomes, we ran logistic and linear models using generalized estimating equations. RESULTS: Eleven perinatal outcomes reached statistical significance, although with minimal clinical impact given small magnitude of effect. Intra-abdominal bowel dilation was associated with a 0.5 week decrease in delivery gestational age (GA) (95% CI -0.07, -0.03) and a 6.93 g increase in birth weight (95% CI 1.54, 28.73). Intra-abdominal bowel wall thickness was associated with later GA of non-stress test (NST) start of 0.22 weeks (95% CI 0.07, 0.37), increased delivery GA of 0.08 weeks (95% CI 0.02, 0.15), 0.006 decrease in umbilical artery (UA) pH (95% CI -0.009, -0.003), 0.26 increase in UA base deficit (95% CI 0.09, 0.43), and decreased odds of cesarean delivery (OR = 0.83, 95% CI 0.70, 0.99). Extra-abdominal bowel wall thickness was associated with a 0.1 increase in UA base deficit (95% CI 0.02, 0.19) and a 0.05 increase in 5-min APGAR score (95% CI 0.01, 0.09). Stomach cross-section was associated with a 0.01 week decrease in delivery GA (95% CI -0.02, -0.001) and increased odds of receiving betamethasone (OR = 1.02, 95% CI 1.01, 1.04). CONCLUSIONS: In utero bowel characteristics reached statistical significance for several outcomes, but with minimal meaningful clinical differences in outcomes.

Short-term mating orientation as a predictor of alcohol use and risky sexual behavior.

OBJECTIVES: Sexual Strategies Theory suggests people fall on a continuum between having short-term mating orientation (STMO) and long-term mating orientation. One way STMO individuals signal mating goals is via risky drinking. The current study therefore aims to investigate drinks per week (DPW) as a mediator between STMO and risky sexual behavior (RSB), with gender as a moderator between STMO and DPW. PARTICIPANTS: Undergraduate students (N = 300) from a Midwestern university during Fall 2019. METHOD: Participants completed questionnaires assessing STMO, DPW, and RSB frequency. RESULTS: A moderated-mediation model indicated DPW significantly mediated the relationship between STMO and RSB. Positive associations were found among all three variables. Gender was not a moderator between STMO and DPW. CONCLUSIONS: Mating orientation was a correlate of alcohol use and RSB for women and men, contributing to the literature identifying STMO as an indicator of those in need of substance use and RSB intervention.

Displacement imposition scale assesses reactions of cigarette and e-cigarette users impacted by a campus-wide smoking ban.

OBJECTIVE: The present study developed a measure assessing the emotional responses, "Displacement Imposition," of cigarette and e-cigarette users on a college campus with a smoking/vaping ban. It also examined the relationship between Displacement Imposition and readiness to quit smoking/vaping, and how this relationship differed between cigarette and e-cigarette users. PARTICIPANTS: Participants (N = 297) were from a large, Midwestern university. METHODS: Participants completed online questionnaires assessing demographics, cigarette and e-cigarette use, Displacement Imposition, and readiness to quit. RESULTS: All six Displacement Imposition items loaded onto a single factor. A significant interaction emerged between Displacement Imposition and product use in predicting readiness to quit. At high levels of Displacement Imposition, cigarette users were less ready to quit than e-cigarette users. CONCLUSIONS: Findings suggest restrictions imposed on cigarette and e-cigarette users were associated with reduced readiness to quit. Findings inform tobacco control policies as tobacco denormalization may increase the burden placed on tobacco users.

The expendables: Bioarchaeological evidence for pauper apprentices in 19th century England and the health consequences of child labour.

Child labour is the most common form of child abuse in the world today, with almost half of child workers employed in hazardous industries. The large-scale employment of children during the rapid industrialisation of the late 18th and early 19th centuries in England is well documented. During this period, the removal of pauper children from workhouses in cities to work as apprentices in rural mills in the North of England was commonplace. Whilst the experiences of some of these children have been recorded historically, this study provides the first direct evidence of their lives through bioarchaeological analysis. The excavation of a rural churchyard cemetery in the village of Fewston, North Yorkshire, yielded the skeletal remains of 154 individuals, including an unusually large proportion of children aged between 8 to 20 years. A multi-method approach was undertaken, including osteological and palaeopathological examination, stable isotope and amelogenin peptide analysis. The bioarchaeological results were integrated with historical data regarding a local textile mill in operation during the 18th-19th centuries. The results for the children were compared to those obtained from contemporaneous individuals of known identity (from coffin plates) of comparable date. Most of the children exhibited distinctive 'non-local' isotope signatures and a diet low in animal protein when compared to the named local individuals. These children also showed severe growth delays and pathological lesions indicative of early life adversity, as well as respiratory disease, which is a known occupational hazard of mill work. This study has provided unique insights into the harrowing lives of these children; born into poverty and forced to work long hours in dangerous conditions. This analysis provides a stark testimony of the impacts of industrial labour on the health, growth and mortality risk of children, with implications for the present as well as our understanding of the past.

Intentions to Reduce Alcohol Use Following Brief Alcohol-Related Health Messages Among College Students.

OBJECTIVE: Health care providers using brief alcohol-related health messaging is an effective manner of reducing risky drinking; however, research is needed to guide the content of such messages. The present study compared current drinkers' and nondrinkers' perspectives on the value of four different alcohol-related messages and the hypothetical impact of the messages on intentions to reduce drinking. METHOD: Undergraduates (n = 286 current drinkers, n = 101 nondrinkers) from a large, public, Southern Plains university identified primarily as White (82.9%) and female (70%), with a mean age of 19.98 years. They viewed four video recordings containing different alcohol-related messages in random order and were asked to rate how likely they were to change their drinking behaviors after watching each video. RESULTS: All participants generally had a comparably positive appraisal of all four messages. Among current drinkers, one-way analyses of variance revealed significant differences across messages for intention to change drinking frequency, F(3, 260) = 5.69, p = .001, ηp2 = .06, and quantity, F(3, 263) = 4.95, p = .002, ηp2 = .05. Post hoc tests showed that the condition warning students of severe consequences resulted in higher intentions to reduce drinking compared with other conditions describing less severe consequences, drinking norms, or protective behavioral strategies. No significant differences emerged among nondrinkers. CONCLUSIONS: Despite mixed research regarding the effectiveness of fear-based warning messages in reducing high-risk drinking, the message containing severe alcohol-related consequences evoked the greatest hypothetical intentions to reduce quantity and frequency of drinking. Future studies should track actual drinking behaviors longitudinally following each message.

Differences in reasons for little cigar/cigarillo use across white and black/African American young adult users.

INTRODUCTION: The prevalence of little cigar and cigarillo (LCC) use among young adults is high. Research shows there are racial/ethnic differences in this prevalence, with Black/African American users more likely to report current LCC use. Given these discrepancies in LCC use, the present study aimed to assess potential differences in reasons for LCC use between White and Black/African American young adult ever and past 30-day users. METHODS: Participants were White (n = 2150), and Black/African American (n = 308) young adults (aged 18 to 24) recruited from Amazon Mechanical Turk who completed an online survey of tobacco use (December 2018-January 2019). LCC users were asked eight reasons for using LCCs. RESULTS: Flavoring was cited as the most popular reason for LCC use among White ever users while affordability was the most popular among Black/African American ever users. Adjusted logistic regressions among ever users revealed that Black/African American respondents (vs White) were more likely to use LCCs because of their affordability, the perception that LCCs are less harmful than cigarettes, and because of LCC advertising appeal. Among past 30-day users, adjusted logistic regression models showed that Black/African American respondents (vs White) were more likely to use LCCs because of their affordability. CONCLUSIONS: Findings align with previous studies showing that LLCs are more heavily marketed in predominantly Black/African American communities. Prevention efforts should account for racial differences in reasons for use in message development.

Dynamic regulation of spinal pro-inflammatory cytokine release in the rat in vivo following peripheral nerve injury.

Spinal release of cytokines may play a critical role in the maladapted nociceptive signaling underlying chronic pain states. In order to investigate this biology, we have developed a novel 'high flux' intrathecal microdialysis approach in combination with multiplex bead-based immunoassay technology to concurrently monitor the spinal release of interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF)alpha in rats with unilateral sciatic nerve chronic constriction injury (CCI). Intrathecal microdialysis was performed under isoflurane/N(2)O anaesthesia in rats with confirmed mechanical hypersensitivity. In a first study, C-fiber strength electrical stimulation of the operated nerve in neuropathic rats was found to evoke a dramatic increase in IL-1beta efflux ( approximately 15-fold) that was significantly greater than that observed in the sham-operated group. Spinal IL-6 efflux was also responsive to primary afferent stimulation, whereas TNFalpha was not. In a second study, treatment with the glial inhibitor propentofylline for 7days normalized CCI-induced mechanical hypersensitivity. In the same animals, this treatment also significantly reduced intrathecal IL-1beta, IL-6 and TNFalpha and prevented afferent stimulation-evoked cytokine release of both IL-1beta and IL-6. These results provide support for glia as the source of the majority of intrathecal IL-1beta, IL-6 and TNFalpha that accompanies mechanical hypersensitivity in the CCI rat. Moreover, our studies demonstrate the ability of a neurone-glia signaling mechanism to dynamically modulate this release and support a role of spinal IL-1beta in the phasic transmission of abnormal pain signals.

Analysis of the aetiology of epilepsy in 3,216 adult patients attending a tertiary referral center enabled by an electronic patient record.

PURPOSE: The aim of this study was to review the causes of the epilepsies in our institution, an adult tertiary referral center for neurology and neurosurgery in Dublin, Ireland. Data was obtained from a bespoke epilepsy electronic patient record (EPR). METHODS: Predetermined search parameters of well-established broad categories of epilepsy aetiology were used to identify patients with a diagnosis of epilepsy attending Beaumont Hospital, Dublin. There were 3216 patients that met the inclusion criteria for this study. We included living patients with epilepsy attending our institution. We then excluded patients with a diagnosis of pure non-epileptic attack disorder and patients found to have idiopathic generalised epilepsy (IGE) (n = 382) from our final cohort. We excluded IGE due to the complex polygenic basis underlying this patient group. RESULTS: An aetiology was identified in 54.3 % (n = 1747) of the total number of patients studied. Of the symptomatic epilepsies, 41.08 % (n = 1321) were acquired and 13.3 % (n = 426) were predominantly of genetic or developmental aetiology. The most common causes of the acquired epilepsies were hippocampal sclerosis (n = 380; 28.75 %), cerebral tumor (n = 279; 21.06 %), traumatic brain injury (n = 248; 18.77 %), stroke and cerebrovascular disease (n = 151; 11.43 %) and perinatal causes (n = 138; 10.45 %). The leading causes in the genetic / developmental category included cavernous haemangiomas (n = 62, 22.22 %), arteriovenous malformations (n = 59; 21.15 %) and cortical dysplasia (n = 55; 19.71 %). The aetiology of a patient's epilepsy was undetermined in 45.68 % (n = 1469) of individuals. CONCLUSION: This study emphasizes the clinical utility of the ILAE's 2017 revised classification of the epilepsies and highlights the evolving dynamic nature of attributing causality in epilepsy. This is the largest single centre analysis of the aetiology of the epilepsies described in the literature. It is also the first large scale study examining aetiology utilising a bespoke electronic patient record in epilepsy.

Correlation of gene and mediator expression with clinical endpoints in an acute interleukin-1beta-driven model of joint pathology.

OBJECTIVE: Interleukin-1beta (IL-1beta) plays a key role in the pathogenesis of chronic joint diseases, including osteoarthritis (OA), and drives a cascade of inflammatory and destructive responses within the synovial joint. Animal models of arthritis support the role of IL-1beta in joint pathology, however, the molecular changes downstream of IL-1beta are poorly understood in vivo. This study aimed to evaluate the intra-articular (i.a.) injection of IL-1beta in the rat joint as an acute model of joint disease and associate gene and mediator expression with clinical endpoints and pathological changes. METHODS: The effects of i.a. administration of a pathologically relevant dose of IL-1beta on joint swelling, mechanical hyperalgesia, histopathology, gene expression and biochemical changes were measured from 2 to 24h. RESULTS: IL-1beta-induced joint swelling and mechanical hyperalgesia. Gene expression analysis of joint tissue and biochemical analysis of joint lavage fluid identified pro-inflammatory and destructive mediators induced by IL-1beta. Histopathology of joint tissues showed evidence of synovitis and connective tissue inflammation, but not cartilage destruction. However, biochemical analysis of glucosaminoglycan levels in joint lavage fluids indicated cartilage breakdown and might be a sensitive marker of cartilage pathology. CONCLUSIONS: Intra-articular injection of IL-1beta is a reproducible acute model of joint pathology that is potentially useful to evaluate IL-1 pathway inhibitors. The correlation of molecular events with clinical and pathological changes in this model has enhanced the understanding of the role of IL-1beta in joint disease. Methods developed for gene expression analysis using multi-gene microfluidics cards and for biochemical analysis of joint lavage fluid might have utility for characterisation of other arthritis models and corresponding human disease.

A physiologically relevant 3D collagen-based scaffold-neuroblastoma cell system exhibits chemosensitivity similar to orthotopic xenograft models.

3D scaffold-based in vitro cell culturing is a recent technological advancement in cancer research bridging the gap between conventional 2D culture and in vivo tumours. The main challenge in treating neuroblastoma, a paediatric cancer of the sympathetic nervous system, is to combat tumour metastasis and resistance to multiple chemotherapeutic drugs. The aim of this study was to establish a physiologically relevant 3D neuroblastoma tissue-engineered system and explore its therapeutic relevance. Two neuroblastoma cell lines, chemotherapeutic sensitive Kelly and chemotherapeutic resistant KellyCis83 were cultured in a 3D in vitro model on two collagen-based scaffolds containing either glycosaminoglycan (Coll-GAG) or nanohydroxyapatite (Coll-nHA) and compared to 2D cell culture and an orthotopic murine model. Both neuroblastoma cell lines actively infiltrated the scaffolds and proliferated displaying >100-fold increased resistance to cisplatin treatment when compared to 2D cultures, exhibiting chemosensitivity similar to orthotopic xenograft in vivo models. This model demonstrated its applicability to validate miRNA-based gene delivery. The efficacy of liposomes bearing miRNA mimics uptake and gene knockdown was similar in both 2D and 3D in vitro culturing models highlighting the proof-of-principle for the applicability of 3D collagen-based scaffolds cell system for validation of miRNA function. Collectively, this data shows the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. While neuroblastoma is the specific disease being focused upon, the platform may have multi-functionality beyond this tumour type. STATEMENT OF SIGNIFICANCE: Traditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in our understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results. Here, we demonstrated the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs.

SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis.

KRE6 encodes a predicted type II membrane protein which, when disrupted, results in a slowly growing, killer toxin-resistant mutant possessing half the normal level of a structurally wild-type cell wall (1-->6)-beta-glucan (T. Roemer and H. Bussey, Proc. Natl. Acad. Sci. USA 88:11295-11299, 1991). The mutant phenotype and structure of the KRE6 gene product, Kre6p, suggest that it may be a beta-glucan synthase component, implying that (1-->6)-beta-glucan synthesis in Saccharomyces cerevisiae is functionally redundant. To examine this possibility, we screened a multicopy genomic library for suppression of both the slow-growth and killer resistance phenotypes of a kre6 mutant and identified SKN1, which encodes a protein sharing 66% overall identity to Kre6p. SKN1 suppresses kre6 null alleles in a dose-dependent manner, though disruption of the SKN1 locus has no effect on killer sensitivity, growth, or (1-->6)-beta-glucan levels. skn1 kre6 double disruptants, however, showed a dramatic reduction in both (1-->6)-beta-glucan levels and growth rate compared with either single disruptant. Moreover, the residual (1-->6)-beta-glucan polymer in skn1 kre6 double mutants is smaller in size and altered in structure. Since single disruptions of these genes lead to structurally wild-type (1-->6)-beta-glucan polymers, Kre6p and Skn1p appear to function independently, possibly in parallel, in (1-->6)-beta-glucan biosynthesis.

Direct-to-consumer genetic testing: Perspectives on its value in healthcare.

The direct-to-consumer genetic testing debate reached a fever pitch in November 2013 when the US Food and Drug Administration (FDA) instructed 23andMe to discontinue marketing and sale of their Personal Genome Service. In 2015, 23andMe emerged with FDA approval to market a carrier test for Bloom syndrome only, and plans to release additional reports. The dust has settled and it is time to ask: What have we learned, and where do we go from here?

Sequence conservation around the 5' ends of the larval serum protein 1 genes of Drosophila melanogaster.

We have determined the nucleotide sequence at the 5' ends of the genes for the alpha, beta and gamma polypeptides of larval serum protein 1 (LSP1) of Drosophila melanogaster. In their upstream regions, the three genes share homology around the TATA boxes. There is also a homologous region of about 20 nucleotides at positions 200, 216 and 377 upstream from the alpha, beta and gamma genes, respectively. Another 18-nucleotide homology occurs between a sequence 111 nucleotides upstream from the alpha gene and 130 nucleotides upstream of the beta gene. This contains a seven-nucleotide match with a sequence 180 nucleotides upstream from the gamma gene. The sequences corresponding to the 5' non-translated regions of the RNA show two regions of strong homology: one being within the first 20 nucleotides at the very 5' end of the RNA, and the other being between nucleotides 27 and 52 of the three transcripts. The first AUG codon to precede a long open reading frame is found at nucleotides 89, 86 and 83 downstream from the 5' end of the alpha, beta and gamma RNAs, respectively. An extremely conserved nucleotide sequence with an exact homology of 66 nucleotides between the alpha and beta genes, and sharing 27 nucleotides with the gamma gene, is contained within this long open reading frame in the first exon. Conceptual translation of the long open reading frame shows that the hydrophobic nature of the first 20 amino acids of the three polypeptides has been conserved whereas the exact sequence has not. This suggests that the N termini contain signal sequences required for secretion of the protein into the haemolymph. The three genes have intervening sequences ranging from 65 to 68 nucleotides in length at comparable locations close to the 5' end of the genes.

Expression of the prokaryotic gene for chloramphenicol acetyl transferase in Drosophila under the control of larval serum protein 1 gene promoters.

We have linked the protein coding region of the prokaryotic gene for chloramphenicol acetyl transferase (CAT) to the promoter region of the Drosophila genes for larval serum protein 1 (LSP1). These regions consist of 1.65 X 10(3) and 2.25 X 10(3) base long DNA segments upstream from the LSP1 alpha and beta genes, respectively. The hybrid genes have been inserted into a P-element transformation vector and the constructs introduced into cultured Drosophila Kc cells by calcium phosphate-mediated transfection, or into the germ-lines of flies by P-element-mediated transformation. CAT expression occurs in approximately 1% of the cultured cells following transfection and the accumulation of protein is maximal three to four days after transfection; and it is dependent upon the presence of the LSP1 sequences. We have also obtained several lines of transformed flies that carry the LSP1-CAT genes in their germ-line. The onset of synthesis of functional chloramphenicol acetyl transferase in these organisms occurs in the late third larval instar, rising to a maximum at puparium formation. We continue to detect CAT until the first few hours of adulthood. Assays of CAT activity in the homogenates of dissected tissues indicated that the genes are only expressed in the fat body, as are the endogenous LSP1 genes. We have confirmed this tissue-specific localization of CAT in indirect immunofluorescence using an anti-CAT monoclonal antibody. Northern blots indicate that CAT transcripts are found only in the fat body but their abundance is an order of magnitude lower than endogenous LSP1 transcripts. Primer extension experiments show that transcription of the hybrid genes is initiated at the same nucleotide as the endogenous LSP1 genes. Taken together these data indicate that the 1.65 X 10(3) and 2.25 X 10(3) base segments of DNA upstream from the LSP1 alpha and LSP1 beta genes contain cis-acting regulatory elements necessary for correct tissue and temporal specificity of LSP1 gene expression.

Lymphocyte beta-adrenoceptors in social phobia.

Beta-adrenergic receptor kinetics were measured in leukocytes from 17 drug-free, nondepressed patients with social phobia (generalized type) and 17 gender-matched and age-matched healthy controls. Binding was characterized using the highly specific beta-adrenergic ligand [125I]pindolol (125IPIN). Contrary to some studies in panic disorder and many studies in depression, no significant difference was found in Bmax or Kd values between social phobic patients and controls. Neither severity of social phobic symptoms nor the severity of certain symptoms of beta-adrenergic activation (i.e., tachycardia, tremor, blushing) influenced Bmax or Kd. To the extent that these peripheral indices can be considered reflective of central processes, these findings suggest that a simple defect in beta-adrenoceptor number of affinity is unlikely to explain the pathophysiology of generalized social phobia.