OBJECTIVES. Despite epidemiologic evidence to the contrary, claims of an association between measles-mumps-rubella vaccination and the development of autism have persisted. Such claims are based primarily on the identification of measles virus nucleic acids in tissues and body fluids by polymerase chain reaction. We sought to determine whether measles virus nucleic acids persist in children with autism spectrum disorder compared with control children. PATIENTS AND METHODS. Peripheral blood mononuclear cells were isolated from 54 children with autism spectrum disorder and 34 developmentally normal children, and up to 4 real-time polymerase chain reaction assays and 2 nested polymerase chain reaction assays were performed. These assays targeted the nucleoprotein, fusion, and hemagglutinin genes of measles virus using previously published primer pairs with detection by SYBR green I. Our own real-time assay targeted the fusion gene using novel primers and an internal fluorescent probe. Positive reactions were evaluated rigorously, and amplicons were sequenced. Finally, anti-measles antibody titers were measured by enzyme immunoassay. RESULTS. The real-time assays based on previously published primers gave rise to a large number of positive reactions in both autism spectrum disorder and control samples. Almost all of the positive reactions in these assays were eliminated by evaluation of melting curves and amplicon band size. The amplicons for the remaining positive reactions were cloned and sequenced. No sample from either autism spectrum disorder or control groups was found to contain nucleic acids from any measles virus gene. In the nested polymerase chain reaction and in-house assays, none of the samples yielded positive results. Furthermore, there was no difference in anti-measles antibody titers between the autism and control groups. INTERPRETATION. There is no evidence of measles virus persistence in the peripheral blood mononuclear cells of children with autism spectrum disorder.

Salmonella enterica is a bacterial species that is a major cause of illness in humans and food-producing animals. S. enterica exhibits considerable inter-serovar diversity, as evidenced by the large number of host adapted serovars that have been identified. The development of methods to assess genome diversity in S. enterica will help to further define the limits of diversity in this foodborne pathogen. Thus, we evaluated a PCR assay, which targets prophage integrase genes, as a rapid method to investigate S. enterica genome diversity. To evaluate the PCR prophage integrase assay, 49 isolates of S. enterica were selected, including 19 clinical isolates from clonal serovars (Enteritidis and Heidelberg) that commonly cause human illness, and 30 isolates from food-associated Salmonella serovars that rarely cause human illness. The number of integrase genes identified by the PCR assay was compared to the number of integrase genes within intact prophages identified by whole genome sequencing and phage finding program PHASTER. The PCR assay identified a total of 147 prophage integrase genes within the 49 S. enterica genomes (79 integrase genes in the food-associated Salmonella isolates, 50 integrase genes in S. Enteritidis, and 18 integrase genes in S. Heidelberg). In comparison, whole genome sequencing and PHASTER identified a total of 75 prophage integrase genes within 102 intact prophages in the 49 S. enterica genomes (44 integrase genes in the food-associated Salmonella isolates, 21 integrase genes in S. Enteritidis, and 9 integrase genes in S. Heidelberg). Collectively, both the PCR assay and PHASTER identified the presence of a large diversity of prophage integrase genes in the food-associated isolates compared to the clinical isolates, thus indicating a high degree of diversity in the food-associated isolates, and confirming the clonal nature of S. Enteritidis and S. Heidelberg. Moreover, PHASTER revealed a diversity of 29 different types of prophages and 23 different integrase genes within the food-associated isolates, but only identified 4 different phages and integrase genes within clonal isolates of S. Enteritidis and S. Heidelberg. These results demonstrate the potential usefulness of PCR based detection of prophage integrase genes as a rapid indicator of genome diversity in S. enterica.

Zellweger spectrum disorder (ZSD) is a rare, debilitating genetic disorder of peroxisome biogenesis that affects multiple organ systems and presents with broad clinical heterogeneity. Although severe, intermediate, and mild forms of ZSD have been described, these designations are often arbitrary, presenting difficulty in understanding individual prognosis and treatment effectiveness. The purpose of this study is to conduct a scoping review and meta-analysis of existing literature and a medical chart review to determine if characterization of clinical findings can predict severity in ZSD. Our PubMed search for articles describing severity, clinical findings, and survival in ZSD resulted in 107 studies (representing 307 patients) that were included in the review and meta-analysis. We also collected and analyzed these same parameters from medical records of 136 ZSD individuals from our natural history study. Common clinical findings that were significantly different across severity categories included seizures, hypotonia, reduced mobility, feeding difficulties, renal cysts, adrenal insufficiency, hearing and vision loss, and a shortened lifespan. Our primary data analysis also revealed significant differences across severity categories in failure to thrive, gastroesophageal reflux, bone fractures, global developmental delay, verbal communication difficulties, and cardiac abnormalities. Univariable multinomial logistic modeling analysis of clinical findings and very long chain fatty acid (VLCFA) hexacosanoic acid (C26:0) levels showed that the number of clinical findings present among seizures, abnormal EEG, renal cysts, and cardiac abnormalities, as well as plasma C26:0 fatty acid levels could differentiate severity categories. We report the largest characterization of clinical findings in relation to overall disease severity in ZSD. This information will be useful in determining appropriate outcomes for specific subjects in clinical trials for ZSD.

In most human cancer cells, cellular immortalization relies on the activation and recruitment of telomerase to telomeres. The telomere binding protein, TPP1, and the TEN domain of the telomerase catalytic subunit, TERT, regulate telomerase recruitment. TERT contains a unique ‘insertion in fingers domain' (IFD) located within the conserved reverse transcriptase domain. We report the role of specific hTERT IFD residues in the regulation of telomerase activity and processivity, recruitment to telomeres and cell survival. One hTERT IFD variant, hTERT-L805A, with reduced activity and processivity, showed impaired telomere association, which could be partially rescued by overexpression of TPP1-POT1. Another previously reported hTERT IFD mutant enzyme with similarly low levels of activity and processivity, hTERT-V791Y, displayed defects in telomere binding and was insensitive to TPP1-POT1 overexpression. Our results provide the first evidence that the IFD can mediate enzyme processivity and telomerase recruitment to telomeres in a TPP1-dependent manner. Moreover, unlike hTERT-V791Y, hTERT-V763S, a variant with reduced activity but increased processivity, and hTERT-L805A, could both immortalize limited lifespan cells, but cells expressing these two mutant enzymes displayed growth defects, increased apoptosis, DNA damage at telomeres and short telomeres. Our results highlight the importance of the IFD in maintaining short telomeres, and cell survival.

Summary Telomerase is a ribonucleoprotein consisting of a catalytic subunit, the telomerase reverse transcriptase (TERT), and an integrally associated RNA that contains a template for the synthesis of short repetitive G-rich DNA sequences at the ends of telomeres. Telomerase can repetitively reverse transcribe its short RNA template, acting processively to add multiple telomeric repeats onto the same DNA substrate. The contribution of enzyme processivity to telomere length regulation in human cells is not well characterized. In cancer cells, under homeostatic telomere length-maintenance conditions, telomerase acts processively, whereas under nonequilibrium conditions, telomerase acts distributively on the shortest telomeres. To investigate the role of increased telomerase processivity on telomere length regulation in human cells with limited lifespan that are dependent on human TERT for lifespan extension and immortalization, we mutated the leucine at position 866 in the reverse transcriptase C motif of human TERT to a tyrosine (L866Y), which is the amino acid found at the equivalent position in HIV-1 reverse transcriptase. We report that, similar to the previously reported gain-of-function Tetrahymena telomerase mutant (L813Y), the human telomerase variant displays increased processivity. Human TERT-L866Y, like wild-type human TERT, can immortalize and extend the lifespan of limited-lifespan cells. Moreover, cells expressing human TERT-L866Y display heterogenous telomere lengths, telomere elongation, multiple telomeric signals indicative of fragile sites and replicative stress, and an increase in short telomeres, which is accompanied by telomere trimming events. Our results suggest that telomere length and homeostasis in human cells may be regulated by telomerase enzyme processivity.