DNA Double Strand Breaks Occur Independent of AID in Hypermutating Ig Genes

Somatic hypermutation (SHM) and class switch recombination (CSR) take place in B cells of the germinal center (GC) and are associated with DNA double-strand breaks (DNA-DSBs). Transcription favors the generation of DNA-DSBs in the V-regions and switch regions of Ig genes. Both SHM and CSR are controlled by the Activation Induced Cytidine Deaminase (AID), an enzyme exclusively expressed in B cells of the GC. Because AID is capable of deaminating deoxy-cytidine (dC) to deoxy-uracil (dU), it might directly induce nicks (single strand DNA breaks) and also DNA-DSBs via a U-DNA glycosylase mediated base excision repair pathway ('DNA-substrate model'). Alternatively, AID could function like its closest homologue Apobec-1 as a catalytic subunit of a RNA editing holoenzyme ('RNA-substrate model'). To determine whether AID lies upstream or downstream of the DNA lesions found in hypermutating Ig genes, we have analysed the Vλ locus of AID proficient and AID deficient GC B cells for the presence of DNA-DSBs. Although rearranged Vλ genes are preferred targets of SHM we find that AID-proficient and -deficient Vλ1/2-expressing GC B cells display a similar frequency, distribution and sequence preference of DNA-DSBs in rearranged and germline Vλ genes, favoring the idea that AID acts downstream of the DNA lesions to mediate error prone processing

A conceptual index for benchmarking intermittent water supply in a water distribution system zone

Various challenges, such as limited freshwater resources, climate change impacts, rapid population growth, urbanisation and underinvestment in water supply infrastructure, have led to intermittent water supply (IWS) in potable water distribution systems. Earlier research has confirmed that IWS negatively impacts the consumers, the infrastructure and the water supply authorities. Water supply authorities need tools to help understand IWS and the associated implications. A new indexing framework involving the causes and impacts associated with IWS is presented in this paper. In addition, a novel approach allows for quantification of the severity of IWS based on knowledge of a few readily available inputs. The severity quantification is based on two ratios: the intermittency ratio is a temporal measurement, accounting for supply duration; the connection ratio describes spatial aspects, using the number of service connections affected. The indexing framework and quantification tool could lead to improved understanding of IWS and could assist water supply authorities faced with IWS to make informed decisions. Improved planning of remedial actions to mitigate or avoid risks associated with IWS is aided. The tools presented in this paper could be used as basis for future development of a key performance indicator.Keywords: intermittent water supply, index, water scarcit

A conceptual theoretical framework to integrally assess the possible impacts of climate change on domestic irrigation water use

Southern Africa is likely to experience higher evapotranspiration and altered rainfall characteristics due to global warming and climate change. Climate-driven water use may potentially stress water supply  facilities due to increased demand and reduced surface water yield. This paper presents a conceptual  theoretical framework for assessing impacts of climate change on domestic irrigation water use. The  prediction of climatic conditions that may potentially influence future water use is reviewed together with regional capacity for downscaling global climate projections. The impact assessment of water use is based on the modification and adaptation of an existing end-use model for water demand to include  parameters for climate change. The Penman-Monteith equation and the soil water balance equation are incorporated for the estimation of daily water needs of vegetated areas in residential properties. The paper also discusses data requirements and a calibration procedure to improve model fit to the observed domestic irrigation water use. The proposed approach could form a basis for constructing a detailed model for planning various adaption measures relating to climate-driven domestic irrigation water use.Keywords: climate change, outdoor water use, end-use model, irrigation water us

Mutating for Good: DNA Damage Responses During Somatic Hypermutation

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes plays a key role in antibody mediated immunity. SHM in B cells provides the molecular basis for affinity maturation of antibodies. In this way SHM is key in optimizing antibody dependent immune responses. SHM is initiated by targeting the Activation-Induced Cytidine Deaminase (AID) to rearranged V(D)J and switch regions of Ig genes. The mutation rate of this programmed mutagenesis is ~10−3 base pairs per generation, a million-fold higher than the non-AID targeted genome of B cells. AID is a processive enzyme that binds single-stranded DNA and deaminates cytosines in DNA. Cytosine deamination generates highly mutagenic deoxy-uracil (U) in the DNA of both strands of the Ig loci. Mutagenic processing of the U by the DNA damage response generates the entire spectrum of base substitutions characterizing SHM at and around the initial U lesion. Starting from the U as a primary lesion, currently five mutagenic DNA damage response pathways have been identified in generating a well-defined SHM spectrum of C/G transitions, C/G transversions, and A/T mutations around this initial lesion. These pathways include (1) replication opposite template U generates transitions at C/G, (2) UNG2-dependent translesion synthesis (TLS) generates transversions at C/G, (3) a hybrid pathway comprising non-canonical mismatch repair (ncMMR) and UNG2-dependent TLS generates transversions at C/G, (4) ncMMR generates mutations at A/T, and (5) UNG2- and PCNA Ubiquitination (PCNA-Ub)-dependent mutations at A/T. Furthermore, specific strand-biases of SHM spectra arise as a consequence of a biased AID targeting, ncMMR, and anti-mutagenic repriming. Here, we review mammalian SHM with special focus on the mutagenic DNA damage response pathways involved in processing AID induced Us, the origin of characteristic strand biases, and relevance of the cell cycle

Garden footprint area and water use of gated communities in South Africa

Gated community homes in South Africa are popular amongst property buyers in urban environments such as cities and metropoles due to the  increased security and lifestyle improvements offered. Garden design and layout requirements are prescribed in architectural guidelines compiled by the homeowners associations of these communities. Garden footprint area in gated community homes is of importance to researchers and planners, because of the influence on water use. This study used a quantitative approach to evaluate the spatial data of garden footprint area as a percentage of total plot area for 1 813 gated community homes in different regions of South Africa. The research reviewed how garden footprint area is prescribed and how it is applied in gated community homes. The impact of garden footprint area on water use was also analysed. The results were compared to relevant information lifted from specific architectural design guidelines developed for each gated community. Data from 11 gated communities were analysed and the average garden footprint area was found to be 36% of the total plot area. Gated community homes with a garden area smaller than 100 m2 were found to have limited influence on monthly water consumption, while the water use of gated community homes with a larger garden footprint area increased proportionally with garden footprint area. The seasonal fluctuation of water use is illustrative of garden irrigation and other outdoor water use. The results provided useful input for incorporation in outdoor water use modelling of gated  community homes Keywords: garden irrigation household water consumption plot are

Rapid and quantitative detection of homologous and non-homologous recombination events using three oligonucleotide MLPA

Embryonic stem (ES) cell technology allows modification of the mouse germline from large deletions and insertions to single nucleotide substitutions by homologous recombination. Identification of these rare events demands an accurate and fast detection method. Current methods for detection rely on Southern blotting and/or conventional PCR. Both the techniques have major drawbacks, Southern blotting is time-consuming and PCR can generate false positives. As an alternative, we here demonstrate a novel approach of Multiplex Ligation-dependent Probe Amplification (MLPA) as a quick, quantitative and reliable method for the detection of homologous, non-homologous and incomplete recombination events in ES cell clones. We have adapted MLPA to detect homologous recombinants in ES cell clones targeted with two different constructs: one introduces a single nucleotide change in the PCNA gene and the other allows for a conditional inactivation of the wild-type PCNA allele. By using MLPA probes consisting of three oligonucleotides we were able to simultaneously detect and quantify both wild-type and mutant alleles

Constitutive expression of the pre-TCR enables development of mature T cells

Expression and signalling through the pre-TCR and the TCRαβ resemble two critical checkpoints during T cell development. We investigated to which extent a pre-TCR can functionally replace mature TCRα chains during T cell development. For this purpose, transgenic mice were generated expressing the pre-TCRα (pTα) under the transcriptional control of TCRβ regulatory elements. We report here on the interesting finding that constitutive pTα expression allows complete T cell maturation. The pre-TCR complex permits a subset of β-selected thymocytes to mature in the absence of TCRα into peripheral T cells (βT cells) comprising up to 10% of all lymphocytes. Lymphopenia-driven proliferation of these βT cells is similar to that of conventional αβT cells. Furthermore, βT cells proliferated and acquired effector function upon stimulation with allogeneic MH

DNA Double-Strand Breaks: Prior to but not Sufficient in Targeting Hypermutation

The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin (Ig) genes, both of which are associated with DNA double-strand breaks (DSBs). As AID is capable of deaminating deoxy-cytidine (dC) to deoxy-uracil (dU), it might induce nicks (single strand DNA breaks) and also DNA DSBs via a U-DNA glycosylase-mediated base excision repair pathway (‘DNA-substrate model’). Alternatively, AID functions like its closest homologue Apobec1 as a catalytic subunit of a RNA editing holoenzyme (‘RNA-substrate model’). Although rearranged Vλ genes are preferred targets of SHM we found that germinal center (GC) B cells of AID-proficient and -deficient Vλ1-expressing GC B cells display a similar frequency, distribution, and sequence preference of DSBs in rearranged and also in germline Vλ1 genes. The possible roles of DSBs in relation to AID function and SHM are discussed