Increased risk of non-fatal overdose associated with non-prescribed benzodiazepine use in Scotland, UK.

BACKGROUND: Drug-related deaths (DRDs) in Scotland increased for seven years in a row between 2014 and 2020, consolidating Scotland's place at the top of the United Kingdom and European drug-related mortality charts. One of the defining features of this recent and rapid rise has been the role of benzodiazepines, which are now involved in the majority of all DRDs. These deaths are linked to use of non-prescribed, benzodiazepine-type novel psychoactive substances (NPS) which have been identified by the United Nations as a global threat to public health. This study aimed to estimate the prevalence and determinants of non-prescribed benzodiazepine use and its association with recent non-fatal overdose among a national sample of people who inject drugs (PWID). METHODS: Data from the 2019-20 Needle Exchange Surveillance Initiative (NESI) was analysed using logistic regression. NESI is a voluntary, anonymous, biennial, cross-sectional, bio-behavioural survey of PWID attending community-based services providing injecting equipment in mainland Scotland. RESULTS: Prevalence of non-prescribed benzodiazepine use in the past six months was 52% and significantly associated with age (aOR 0.97, 0.96-0.98), frequent incarceration (aOR 1.29, 1.07-1.57), recent public injecting (aOR 3.25, 2.33-4.55), a recent methadone prescription (aOR 1.87, 1.51-2.33), and a history of benzodiazepine prescription (aOR 1.92, 1.47-2.52). In addition, non-prescribed benzodiazepine use was significantly associated with non-fatal overdose in the past year among PWID (aOR 2.47, 1.90-3.21). CONCLUSION: This study found a high prevalence of non-prescribed benzodiazepine use among a national sample of PWID in Scotland. Prevalence was highest among populations known to be at increased risk of drug-related death and use was strongly associated with overdose. These novel findings highlight the scale of the non-prescribed benzodiazepine issue Scotland faces, and the urgency required to expand its harm reduction infrastructure to address this unique element of its overdose crisis.

Economic analysis of open versus laparoscopic versus robot-assisted versus transanal total mesorectal excision in rectal cancer patients: A systematic review.

OBJECTIVES: Minimally invasive total mesorectal excision is increasingly being used as an alternative to open surgery in the treatment of patients with rectal cancer. This systematic review aimed to compare the total, operative and hospitalization costs of open, laparoscopic, robot-assisted and transanal total mesorectal excision. METHODS: This systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement (PRISMA) (S1 File) A literature review was conducted (end-of-search date: January 1, 2023) and quality assessment performed using the Consensus Health Economic Criteria. RESULTS: 12 studies were included, reporting on 2542 patients (226 open, 1192 laparoscopic, 998 robot-assisted and 126 transanal total mesorectal excision). Total costs of minimally invasive total mesorectal excision were higher compared to the open technique in the majority of included studies. For robot-assisted total mesorectal excision, higher operative costs and lower hospitalization costs were reported compared to the open and laparoscopic technique. A meta-analysis could not be performed due to low study quality and a high level of heterogeneity. Heterogeneity was caused by differences in the learning curve and statistical methods used. CONCLUSION: Literature regarding costs of total mesorectal excision techniques is limited in quality and number. Available evidence suggests minimally invasive techniques may be more expensive compared to open total mesorectal excision. High-quality economical evaluations, accounting for the learning curve, are needed to properly assess costs of the different techniques.

Cost analysis and cost-effectiveness of open versus laparoscopic versus robot-assisted versus transanal total mesorectal excision in patients with rectal cancer: a protocol for a systematic review.

INTRODUCTION: Nowadays, most rectal tumours are treated open or minimally invasive, using laparoscopic, robot-assisted or transanal total mesorectal excision. However, insight into the total costs of these techniques is limited. Since all three techniques are currently being performed, including cost considerations in the choice of treatment technique may significantly impact future healthcare costs. Therefore, this systematic review aims to provide an overview of evidence regarding costs in patients with rectal cancer following open, laparoscopic, robot-assisted and transanal total mesorectal excision. METHODS AND ANALYSIS: A systematic search will be conducted for papers between January 2000 and March 2022. Databases PubMed/MEDLINE, EMBASE, Scopus, Web of Science and Cochrane Library databases will be searched. Study selection, data extraction and quality assessment will be performed independently by four reviewers and discrepancies will be resolved through discussion. The Consensus Health Economic Criteria list will be used for assessing risk of bias. Total costs of the different techniques, consisting of but not limited to, theatre, in-hospital and postoperative costs, will be the primary outcome. ETHICS AND DISSEMINATION: No ethical approval is required, as there is no collection of patient data at an individual level. Findings will be disseminated widely, through peer-reviewed publication and presentation at relevant national and international conferences. TRIAL REGISTRATION NUMBER: CRD42021261125.

A Bayesian approach for diagnostic accuracy of malignant peripheral nerve sheath tumors: a systematic review and meta-analysis.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST) carry a dismal prognosis and require early detection and complete resection. However, MPNSTs are prone to sampling errors and biopsies or resections are cumbersome and possibly damaging in benign peripheral nerve sheath tumor (BPNST). This study aimed to systematically review and quantify the diagnostic accuracy of noninvasive tests for distinguishing MPNST from BPNST. METHODS: Studies on accuracy of MRI, FDG-PET (fluorodeoxyglucose positron emission tomography), and liquid biopsies were identified in PubMed and Embase from 2000 to 2019. Pooled accuracies were calculated using Bayesian bivariate meta-analyses. Individual level-patient data were analyzed for ideal maximum standardized uptake value (SUVmax) threshold on FDG-PET. RESULTS: Forty-three studies were selected for qualitative synthesis including data on 1875 patients and 2939 lesions. Thirty-five studies were included for meta-analyses. For MRI, the absence of target sign showed highest sensitivity (0.99, 95% CI: 0.94-1.00); ill-defined margins (0.94, 95% CI: 0.88-0.98); and perilesional edema (0.95, 95% CI: 0.83-1.00) showed highest specificity. For FDG-PET, SUVmax and tumor-to-liver ratio show similar accuracy; sensitivity 0.94, 95% CI: 0.91-0.97 and 0.93, 95% CI: 0.87-0.97, respectively, specificity 0.81, 95% CI: 0.76-0.87 and 0.79, 95% CI: 0.70-0.86, respectively. SUVmax ≥3.5 yielded the best accuracy with a sensitivity of 0.99 (95% CI: 0.93-1.00) and specificity of 0.75 (95% CI: 0.56-0.90). CONCLUSIONS: Biopsies may be omitted in the presence of a target sign and the absence of ill-defined margins or perilesional edema. Because of diverse radiological characteristics of MPNST, biopsies may still commonly be required. In neurofibromatosis type 1, FDG-PET scans may further reduce biopsies. Ideal SUVmax threshold is ≥3.5.

Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer nanodiscs.

Self-assembled phospholipid bilayer Nanodiscs have become an important and versatile tool among model membrane systems to functionally reconstitute membrane proteins. Nanodiscs consist of lipid domains encased within an engineered derivative of apolipoprotein A-1 scaffold proteins, which can be tailored to yield homogeneous preparations of disks with different diameters, and with epitope tags for exploitation in various purification strategies. A critical aspect of the self-assembly of target membranes into Nanodiscs lies in the optimization of the lipid:protein ratio. Here we describe strategies for performing this optimization and provide examples for reconstituting bacteriorhodopsin as a trimer, rhodopsin, and functionally active P-glycoprotein. Together, these demonstrate the versatility of Nanodisc technology for preparing monodisperse samples of membrane proteins of wide-ranging structure.

Harmful drinking in military veterans with post-traumatic stress disorder: association with the D2 dopamine receptor A1 allele.

AIMS: The frequency of the Taq I A alleles (A1 and A2) of the D2 dopamine receptor (DRD2) gene was examined in Caucasian post-traumatic stress disorder (PTSD) patients and controls. RESULTS: In 91 PTSD patients, the frequency of the A1 allele was higher (P = 6.12 x 10(-3)) than in the 51 controls. In the 38 PTSD harmful drinkers (>or=60 g alcohol/day), A1 allelic frequency was higher (P = 3.91 x 10(-2)) than in the 53 non-harmful drinkers (<60 g alcohol/day), the former being also higher (P = 3.76 x 10(-4)) than in controls. However, there was no difference between non-harmful drinkers and controls. Based on DRD2 allelic association, the 35 PTSD patients with the A1(+) (A1A1, A1A2) allele consumed more than twice the daily amount of alcohol than the 56 patients with the A1(-) (A2A2) allele (P = 1.94 x 10(-3)). When the hourly rate of alcohol consumed was compared, A1(+) allelic patients consumed twice the rate of the A1(-) allelic patients (P < 10(-7)). CONCLUSION: The DRD2 A1 allele was associated with PTSD. However, this association was found only in the harmful drinkers. PTSD patients with the A1(+) allele consumed more alcohol than patients with the A1(-) allele. The importance of determining alcohol consumption in DRD2 association studies with PTSD is suggested.

D(2) dopamine receptor (DRD2) polymorphism is associated with severity of alcohol dependence.

The A(1) allele of the D(2) dopamine receptor (DRD2) gene has been associated with alcohol dependence. However, the expression of this allele risk on the severity of drinking behavior in patients with alcohol dependence has not been systematically explored. The present study examines the association between DRD2 A(1)(+) (A(1)/A(1) and A(1)/A(2) genotypes) and A(1)- (A(2)/A(2) genotype) allele status and key drinking parameters in alcohol-dependent patients. A sample of Caucasian adults was recruited from an alcohol detoxification unit. A clinical interview and the Alcohol Dependence Scale (ADS) questionnaire provided data on consumption, dependence, chronology of drinking and prior detoxification. A(1)(+) allele compared to A(1)- allele patients consumed higher quantities of alcohol, commenced problem drinking at an earlier age, experienced a shorter latency between first introduction to alcohol to the onset of problem drinking and had higher ADS scores. Moreover, A(1)(+) allele patients had more detoxification attempts than their A(1)- allele counterparts. In sum, alcohol-dependent patients with the DRD2 A(1) allele compared to patients without this allele are characterized by greater severity of their disorder across a range of problem drinking indices. The implications of these findings are discussed.

GABA(A) receptor beta 3 subunit gene and psychiatric morbidity in a post-traumatic stress disorder population.

GABAergic systems have been implicated in the pathogenesis of anxiety, depression and insomnia. These symptoms are part of the core and comorbid psychiatric disturbances in post-traumatic stress disorder (PTSD). In a sample of Caucasian male PTSD patients, dinucleotide repeat polymorphisms of the GABA(A) receptor beta 3 subunit gene were compared to scores on the General Health Questionnaire-28 (GHQ). As the major allele at this gene locus (GABRB3) was G1, the alleles were divided into G1 and non-G1 groups. On the total score of the GHQ, which comprises the somatic symptoms, anxiety/insomnia, social dysfunction and depression subscales, patients with the G1 non-G1 genotype had a significantly higher score when compared to either the G1G1 genotype (alpha=0.01) or the non-G1 non-G1 genotype (alpha=0.05). No significant difference was found between the G1G1 and non-G1 non-G1 genotypes. When the G1 non-G1 heterozygotes were compared to the combined G1G1 and non-G1 non-G1 homozygotes, a significantly higher total GHQ score was found in the heterozygotes (P=0.002). These observations suggest a heterosis effect. Further analysis of GHQ subscale scores showed that heterozygotes compared to the combined homozygotes had higher scores on the somatic symptoms (P=0.006), anxiety/insomnia (P=0.003), social dysfunction (P=0.054) and depression (P=0.004) subscales. In conclusion, the present study indicates that in a population of PTSD patients, heterozygosity of the GABRB3 major (G1) allele confers higher levels of somatic symptoms, anxiety/insomnia, social dysfunction and depression than found in homozygosity.

GPx-1 gene delivery modulates NFkappaB activation following diverse environmental injuries through a specific subunit of the IKK complex.

Numerous environmental stimuli alter cell functions by the induction of intracellular reactive oxygen species, such as superoxide and hydrogen peroxide (H2O2). These redox alterations can change the activity of kinases and phosphatases responsible for controlling intracellular signal transduction cascades important in determining how cells react to their environment. One such well known pathway includes nuclear factor-kappaB (NFkappaB); however, the exact redox-sensitive factors important in controlling H2O2-mediated activation of NFkappaB remain unclear. In the present study, we have investigated how intracellular clearance of H2O2, using a recombinant adenovirus expressing glutathione peroxidase-1 (GPx-1), modulates NFkappaB activation following UV irradiation, tumor necrosis factor-alpha, or H2O2 treatment of MCF-7 cells. Findings from these studies demonstrate that GPx-1 overexpression can down-regulate NFkappaB DNA binding, and transcriptional activation of an NFkappaB-dependent luciferase reporter, to varying extents following these environmental stimuli. Studies using dominant negative adenoviral vectors expressing IKKalpha(KM) and IKKbeta(KA) suggest that GPx-1-mediated H2O2 clearance appears to preferentially inhibit the activity of IKKalpha, but not IKKbeta. These studies demonstrate for the first time that redox regulation of NFkappaB activation by intracellular H2O2 may be specific for a unique subunit in the IKK complex. Such findings suggest that IKK kinases or IKK phosphatases may have unique redox-regulated components. These studies have shed mechanistic insight into the potential application of redox-modulating gene therapies aimed at altering NFkappaB activation following environmental injury.

Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappa B.

Reactive oxygen species (ROS) are important second messengers generated in response to many types of environmental stress. In this setting, changes in intracellular ROS can activate signal transduction pathways that influence how cells react to their environment. In sepsis, a dynamic proinflammatory cellular response to bacterial toxins (e.g. lipopolysaccharide or LPS) leads to widespread organ damage and death. The present study demonstrates for the first time that the activation of Rac1 (a GTP-binding protein), and the subsequent production of ROS, constitutes a major pathway involved in NFkappaB-mediated tumor necrosis factor-alpha (TNFalpha) secretion following LPS challenge in macrophages. Expression of a dominant negative mutant of Rac1 (N17Rac1) reduced Rac1 activation, ROS formation, NFkappaB activation, and TNFalpha secretion following LPS stimulation. In contrast, expression of a dominant active form of Rac1 (V12Rac1) mimicked these effects in the absence of LPS stimulation. IKKalpha and IKKbeta were both required downstream modulators of LPS-activated Rac1, since the expression of either of the IKK dominant mutants (IKKalphaKM or IKKbetaKA) drastically reduced NFkappaB-dependent TNFalpha secretion. Moreover, studies using CD14 blocking antibodies suggest that Rac1 induces TNFalpha secretion through a pathway independent of CD14. However, a maximum therapeutic inhibition of LPS-induced TNFalpha secretion occurred when both CD14 and Rac1 pathways were inhibited. Our results suggest that targeting both Rac1- and CD14-dependent pathways could be a useful therapeutic strategy for attenuating the proinflammatory cytokine response during the course of sepsis.

A computer-based simulator for diagnostic peritoneal lavage.

Diagnostic peritoneal lavage (DPL) is an emergency diagnostic procedure performed when intra-abdominal bleeding secondary to trauma is suspected. This procedure is part of the surgical skills section of the Advanced Trauma Life Support course. DPL is traditionally taught using anesthetized animals or cadavers. For reasons described below, these alternatives are not ideal. We have developed a computer-based diagnostic peritoneal lavage simulator. Our system addresses the shortcomings of the traditional method. We have used our system to teach ATLS. Preliminary results suggests that our system is effective.

Developmental expression of catenins and associated proteins during submucosal gland morphogenesis in the airway.

Although lymphoid enhancer binding factor-1 (Lef-1) plays an obligatory role in airway submucosal gland (SMG) development, its expression alone is not an adequate signal for initiating gland morphogenesis. Because Lef-1 forms a bipartite transcription factor with beta-catenin to mediate wnt pathway signaling, we investigated the expression of beta-catenin and associated proteins during SMG development with both in situ hybridization and immunocytochemistry. Unexpectedly, high levels of E-cadherin mRNA were expressed by cells in developing gland buds from the earliest stages through subsequent differentiation into mature glands. In contrast, a decreased level of E-cadherin immunoreactivity in stage I gland bud cells suggested that post-translational modulation of E-cadherin protein levels may play a critical role in early stages of gland morphogenesis. Adenomatous polyposis coli (APC) mRNA was expressed relatively weakly in the developing ferret trachea, but higher levels of protein staining were observed throughout the cytoplasm of gland buds and surface epithelial cells. B-Catenin mRNA was abundantly expressed throughout the tracheal epithelium and at the highest levels in primordial gland buds. B-Catenin protein localized to the basolateral membranes of all airway epithelial cell types. However, no detectable increases in nuclear or cytoplasmic staining were associated with gland buds, as would be expected if beta-catenin served as a transcriptional cofactor for Lef-1 in gland morphogenesis. Additional studies demonstrated the gamma-catenin distribution to be remarkably similar to that of beta-catenin, whereas alpha-catenin staining was more diffuse in the cytoplasm of airway epithelial and gland bud cells. These descriptive results do not rule out a role for wnt signaling in SMG development , but provide no evidence that beta-catenin, or gamma-catenin, is a cofactor in Lef-1 regulation of SMG development.

The D(2) dopamine receptor A(1) allele and opioid dependence: association with heroin use and response to methadone treatment.

A total of 95 Caucasian opioid-dependent patients were followed over a one-year period in an outpatient methadone treatment program. The frequency of the TaqI A(1) allele of the D(2) dopamine receptor (DRD2) gene was 19.0% in these patients compared with 4.6% in controls free of past and current alcohol and other drug abuse and free of family history of alcohol and other drug abuse (p = 0.009). Twenty-two of these patients dropped out of the methadone program (Group A), 54 had a successful treatment (Group B), and 19 had a poor treatment (Group C) outcome. The frequency of the A(1) allele was highest in Group C (42.1%), followed by Group A (22.7%) and was lowest in Group B (9.3%). The more than fourfold higher frequency of the A(1) allele in the poor treatment outcome group compared with the successful treatment outcome group was significant (p = 0.00002). Moreover, the average use of heroin (grams/day) during the year prior to study entry was more than twice as great in patients with the A(1)(+) allele (A(1)/A(1) or A(1)/A(2) genotype) than those with the A(1)(-) allele (A(2)/A(2) genotype) (A(1)(+) allele = 0.55 +/- 0. 10, A(1)(-) allele = 0.25 +/- 0.05; p = 0.003). The results indicate that DRD2 variants are predictors of heroin use and subsequent methadone treatment outcome and suggest a pharmacogenetic approach to the treatment of opioid dependence.

Haplotypes at the DRD2 locus and severe alcoholism.

Association studies of the minor TaqI A allele of the D(2) dopamine receptor (DRD2) gene with alcoholism have produced conflicting findings. Failure to assess alcoholics for severity of their disorder and to screen controls for substance use have been proposed as causes for the discrepant results. In the present study, five diallelic sites spanning the DRD2 gene were determined in combined Caucasian (non-Hispanic) studies of more severe alcoholics (n = 92) and controls screened for substance use (n = 85). The frequency of the minor alleles at the 3'-untranslated site (TaqI A) and two intronic sites (TaqI B and intron 6) of the DRD2 gene were each strongly associated with alcoholism. Moreover, the alcoholics compared with the controls at these three sites had a significantly higher frequency of the minor/major allele heterozygote haplotype combination (A1/A2 B1/B2 T/G) than the major allele homozygote haplotype combination (A2/A2 B2/B2 G/G). However, exon 7 and promoter alleles were not associated with alcoholism. In neither the alcoholics nor in the controls were there departures from Hardy-Weinberg equilibrium at any of the five sites examined. The most significant diallelic composite genotypic disequilibria were found when comparisons were made between TaqI A and TaqI B, TaqI A and intron 6, and TaqI B and intron 6 sites. Weaker but still significant disequilibria were observed when TaqI A and exon 7, TaqI B and exon 7, intron 6 and exon 7, and promoter and exon 7 sites were compared. However, no significant disequilibria were noted when TaqI A and promoter, TaqI B and promoter, and intron 6 and promoter sites were compared. In sum, the study found significant evidence for association of the minor alleles in the untranslated sites of the DRD2 gene and their haplotypes with the more severe alcoholic phenotype.

Association of D2 dopamine receptor and alcohol dehydrogenase 2 genes with Polynesian alcoholics.

Alleles of the D2 dopamine receptor (DRD2) and the alcohol dehydrogenase 2 (ADH2) genes were determined in 69 French Polynesian alcoholic patients and 57 controls matched for racial origin. Three racial groups were studied: pure Polynesians (PP), Polynesians mixed with Caucasian (PCA) ancestry and Polynesians mixed with Chinese (PCH) ancestry. DRD2 A1 allele frequencies in the alcoholics compared to their controls in these groups were: PP,.26 vs.32 (P =. 69); PCA,.44 vs.35 (P =.46); PCH,.40 vs 0.39 (P =.88). ADH2 1 allele frequencies in alcoholics compared to their controls groups were: PP, .56 vs.62 (P =.66); PCA,.75 vs.56 (P =.09); PCH,.78 vs.32 (P =.009). In the PCA group, the combination of the DRD2 A1 genotypes and the ADH2 1 homozygotes was strongly associated with alcoholism (P =. 0027). This preliminary study shows the importance of ascertaining racial ancestry in molecular genetic association studies. Moreover, it suggests that a combination of genes are involved in susceptibility to the development of alcoholism.

Bradyzide, a potent non-peptide B(2) bradykinin receptor antagonist with long-lasting oral activity in animal models of inflammatory hyperalgesia.

Bradyzide is from a novel class of rodent-selective non-peptide B(2) bradykinin antagonists (1-(2-Nitrophenyl)thiosemicarbazides). Bradyzide has high affinity for the rodent B(2) receptor, displacing [(3)H]-bradykinin binding in NG108-15 cells and in Cos-7 cells expressing the rat receptor with K(I) values of 0.51+/-0.18 nM (n=3) and 0.89+/-0.27 nM (n=3), respectively. Bradyzide is a competitive antagonist, inhibiting B(2) receptor-induced (45)Ca efflux from NG108-15 cells with a pK(B) of 8.0+/-0.16 (n=5) and a Schild slope of 1.05. In the rat spinal cord and tail preparation, bradyzide inhibits bradykinin-induced ventral root depolarizations (IC(50) value; 1.6+/-0.05 nM (n=3)). Bradyzide is much less potent at the human than at the rodent B(2) receptor, displacing [(3)H]-bradykinin binding in human fibroblasts and in Cos-7 cells expressing the human B(2) receptor with K(I) values of 393+/-90 nM (n=3) and 772+/-144 nM (n=3), respectively. Bradyzide inhibits bradykinin-induced [(3)H]-inositol trisphosphate (IP(3)) formation with IC(50) values of 11.6+/-1.4 nM (n=3) at the rat and 2.4+/-0.3 microM (n=3) at the human receptor. Bradyzide does not interact with a range of other receptors, including human and rat B(1) bradykinin receptors. Bradyzide is orally available and blocks bradykinin-induced hypotension and plasma extravasation. Bradyzide shows long-lasting oral activity in rodent models of inflammatory hyperalgesia, reversing Freund's complete adjuvant (FCA)-induced mechanical hyperalgesia in the rat knee joint (ED(50), 0.84 micromol kg(-1); duration of action >4 h). It is equipotent with morphine and diclofenac, and 1000 times more potent than paracetamol, its maximal effect exceeding that of the non-steroidal anti-inflammatory drugs (NSAIDs). Bradyzide does not exhibit tolerance when administered over 6 days. In summary, bradyzide is a potent, orally active, antagonist of the B(2) bradykinin receptor, with selectivity for the rodent over the human receptor. British Journal of Pharmacology (2000) 129, 77 - 86

Concatamerization of adeno-associated virus circular genomes occurs through intermolecular recombination.

Long-term recombinant AAV (rAAV) transgene expression in muscle has been associated with the molecular conversion of single-stranded rAAV genomes to high-molecular-weight head-to-tail circular concatamers. However, the mechanisms by which these large multimeric concatamers form remain to be defined. To this end, we tested whether concatamerization of rAAV circular intermediates occurs through intra- or intermolecular mechanisms of amplification. Coinfection of the tibialis muscle of mice with rAAV alkaline phosphatase (Alkphos)- and green fluorescent protein (GFP)-encoding vectors was used to evaluate the frequency of circular concatamer formation by intermolecular recombination of independent viral genomes. The GFP shuttle vector also encoded ampicillin resistance and contained a bacterial origin of replication to allow for bacterial rescue of circular intermediates from Hirt DNA of infected muscle samples. The results demonstrated a time-dependent increase in the abundance of rescued plasmids encoding both GFP and Alkphos, which reached 33% of the total circular intermediates by 120 days postinfection. Furthermore, these large circular concatamers were capable of expressing both GFP- and Alkphos-encoding transgenes following transient transfection in cell lines. These findings demonstrate that concatamerization of AAV genomes in vivo occurs through intermolecular recombination of independent monomer circular viral genomes and suggest new viable strategies for delivering multiple DNA segments at a single locus. Such developments will expand the utility of rAAV for splicing large gene inserts or large promoter-gene combinations carried by two or more independent rAAV vectors.