Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery.

Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.

An embryonic stem cell-specific heterochromatin state promotes core histone exchange in the absence of DNA accessibility.

Nucleosome turnover concomitant with incorporation of the replication-independent histone variant H3.3 is a hallmark of regulatory regions in the animal genome. Nucleosome turnover is known to be universally linked to DNA accessibility and histone acetylation. In mouse embryonic stem cells, H3.3 is also highly enriched at interstitial heterochromatin, most prominently at intracisternal A-particle endogenous retroviral elements. Interstitial heterochromatin is established over confined domains by the TRIM28-KAP1/SETDB1 corepressor complex and has stereotypical features of repressive chromatin, such as H3K9me3 and recruitment of all HP1 isoforms. Here, we demonstrate that fast histone turnover and H3.3 incorporation is compatible with these hallmarks of heterochromatin. Further, we find that Smarcad1 chromatin remodeler evicts nucleosomes generating accessible DNA. Free DNA is repackaged via DAXX-mediated nucleosome assembly with histone variant H3.3 in this dynamic heterochromatin state. Loss of H3.3 in mouse embryonic stem cells elicits a highly specific opening of interstitial heterochromatin with minimal effects on other silent or active regions of the genome.

NEIL1 is a candidate gene associated with common variable immunodeficiency in a patient with a chromosome 15q24 deletion.

We report the first patient with an interstitial deletion of chromosome 15q24.1-q24.3 associated with common variable immunodeficiency (CVID). The 18-year old female patient's clinical and immunological phenotype was compared with 8 additional previously published patients with chr15q24 deletions. A CGH analysis estimated the deletion to be 3.767Mb in size (chr15: 74,410,916-78,178,418) and the result was confirmed using qRT-PCR. We defined an immune-related commonly deleted region (ICDR) within the chromosomal band 15q24.2, deleted in all four patients with different forms of antibody deficiencies. Mutations in the 14 genes within this ICDR were not identified in the remaining allele in our patient by WES and gene expression analyses showed haploinsufficiency of all the genes. Among these genes, we consider Nei Like DNA Glycosylase 1 (NEIL1) as a likely candidate gene due to its crucial role in B-cell activation and terminal differentiation.

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency.

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)-related diseases. Three patients presented with EBV-associated Hodgkin's lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro-generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70-CD27 interactions therefore play a nonredundant role in T and B cell-mediated immunity, especially for protection against EBV and humoral immunity.

HIV-1 mother-to-child transmission, post-test counselling, and antiretroviral prophylaxis in Northern Viet Nam: a prospective observational study.

BACKGROUND: Pregnant women fear being identified as HIV-1-infected and this has hampered prevention programmes and the calculation of transmission rates in Viet Nam. We introduced post-test counselling, antiretroviral prophylaxis, and formula feeding, and determined the vertical transmission rate in parts of Northern Viet Nam. METHODS: HIV infection was identified in 234 pregnant women; 182 (77.8%) accepted follow-up of their children. Counselling was given on 3-7 occasions for altogether approximately 6 h on antiretroviral prophylaxis and formula feeding to avoid transmission, and on the importance of surveillance of the child. All children were formula-fed. A polymerase chain reaction (PCR) was used for the diagnosis of HIV-1 in the children. One hundred and thirty-five of the 182 mothers allowed ≥ 3 blood samples to be taken from birth to ≥ 1 y of age, 32/182 provided a birth sample only, and 15/182 provided a sample later only. Nevirapine was given at delivery to 93/135 (69%) women, and to 128/135 (95%) children. Additionally, combination therapy was given to 15/135 (11%) who entered the study before delivery, and azidothymidine to their children for 1 week. RESULTS: Nine of 135 (6.7%) children became infected and 2/15 of the others, giving altogether 11/150 infected (7.3%). Intrauterine transmission was identified in 7/167 (4.2%) children by a positive PCR test at birth. PCR was negative at birth but positive at 1 month in 2/135 (1.5%), pointing to delivery-associated transmission. Thus, intrauterine transmission accounted for 78% (7/9). None of the uninfected children died, but 3/11 (p =0.004) of the HIV-1-infected died (in AIDS). CONCLUSION: Post-test confidential counselling, formula feeding, and antiretroviral prophylaxis resulted in low rates of delivery-associated and late HIV-1 transmissions.

Short communication: phylogenetic characterization of HIV type 1 CRF01_AE V3 envelope sequences in pregnant women in Northern Vietnam.

Characterization of HIV-1 strains is important for surveillance of the HIV-1 epidemic. In Vietnam HIV-1-infected pregnant women often fail to receive the care they are entitled to. Here, we analyzed phylogenetically HIV-1 env sequences from 37 HIV-1-infected pregnant women from Ha Noi (n=22) and Hai Phong (n=15), where they delivered in 2005-2007. All carried CRF01_AE in the gp120 V3 region. In 21 women CRF01_AE was also found in the reverse transcriptase gene. We compared their env gp120 V3 sequences phylogenetically in a maximum likelihood tree to those of 198 other CRF01_AE sequences in Vietnam and 229 from neighboring countries, predominantly Thailand, from the HIV-1 database. Altogether 464 sequences were analyzed. All but one of the maternal sequences colocalized with sequences from northern Vietnam. The maternal sequences had evolved the least when compared to sequences collected in Ha Noi in 2002, as shown by analysis of synonymous and nonsynonymous changes, than to other Vietnamese sequences collected earlier and/or elsewhere. Since the HIV-1 epidemic in women in Vietnam may still be underestimated, characterization of HIV-1 in pregnant women is important to observe how HIV-1 has evolved and follow its molecular epidemiology.

Apolipoprotein E gene polymorphism in psoriasis.

Polymorphism in the apolipoprotein E gene has been associated with several neurological, cardiological and ophthalmological diseases. Apolipoprotein E is involved in psoriasis by modifying mitogen-activated T lymphocyte proliferation and by assuring protection against some infections. We evaluated the apolipoprotein E gene polymorphism in patients suffering from psoriasis (compared to matched controls) in Thrace, Northern Greece. One hundred and forty patients suffering from psoriasis vulgaris and 155 matched controls were included in this study and genotyped by semi-nested polymerase chain reaction RFLPs; the results were evaluated by conditional logistic regression. In psoriasis vulgaris patients, the e2 allele showed higher frequency (6.1%, P = 0.021) versus matched controls (2.3%). The above data were ascertained particularly enforced in psoriasis vulgaris male patients (P = 0.031) as well as in late onset psoriasis vulgaris (P = 0.029). Implication of apolipoprotein E in the pathogenesis of psoriasis has been indicated. Allele e2 is more frequent in psoriatic patients and its presence is more evident in late onset psoriasis vulgaris.