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Somatic hypermutation (SHM) of immunoglobulin ( ) 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 genes. The mutation rate of this programmed mutagenesis is ~10 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 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.

The prevalence of intermittent water supply in South Africa was investigated in this research study. Data on intermittent water supply in South Africa was collated by considering the following four sources: a targeted water services provider survey; published databases and related reports; open-access publications, such as online media articles; and field visits by the project team to selected areas. The data were spatially and temporally analysed to determine the prevalence of intermittent water supply. The population afected by intermittent water supply increased by ~26% between 2008 and 2017, which exceeds the population increase of ~12% over the same period. Moreover, 22 million people in South Africa were afected by intermittent water supply in 2017. Results from this study confirm an increased prevalence of intermittent water supply over time and show that 65 of the 231 municipalities in South Africa supplied water intermittently, 32 had continuous water supply and 134 had no data. Fundamentally, the outcomes highlight the widespread occurrence of intermittent water supply in South Africa, which is in line with the poor state of water services delivery in South Africa, as portrayed in the 2018 National Water and Sanitation Master Plan by the national Department of Water and Sanitation.

Water temperature is often monitored at water sources and treatment works; however, there is limited monitoring of the water temperature in the drinking water distribution system (DWDS), despite a known impact on physical, chemical and microbial reactions which impact water quality. A key parameter influencing drinking water temperature is soil temperature, which is influenced by the urban heat island effects. This paper provides critique and comprehensive summary of the current knowledge, policies and challenges regarding drinking water temperature research and presents the findings from a survey of international stakeholders. Knowledge gaps as well as challenges and opportunities for monitoring and research are identified. The conclusion of the study is that temperature in the DWDS is an emerging concern in various countries regardless of the water source and treatment, climate conditions, or network characteristics such as topology, pipe material or diameter. More research is needed, especially to determine (i) the effect of higher temperatures, (ii) a legislative limit on temperature and (iii) measures to comply with this limit.

As the world increasingly faces water shortages, it is critical to develop realistic estimates of water consumption based on water-use activities rather than top-down imposed limits. This study quantifies the absolute basic consumption (ABC) of an individual, irrespective of water-use location or water source, with a focus on personal water-use activities rather than measured household consumption. A theoretical model to stochastically describe 21 personal water-use activities was developed, corresponding to lifestyle levels based upon Maslow's hierarchy of physiological needs. From this model, the expected value of ABC was 92 L/capita/day (LPCD) and includes restrictions on several everyday activities and thus would be better as a minimum target during stringent water restrictions. A more realistic expected value for non-wasteful, indoor-only water use was determined to be 175 LPCD. The results of this study serve as a useful benchmark to inform policy choices and efforts to conserve water without affecting health and wellbeing.

DNA damage tolerance (DDT) enables bypassing of DNA lesions during replication, thereby preventing fork stalling, replication stress, and secondary DNA damage related to fork stalling. Three modes of DDT have been documented: translesion synthesis (TLS), template switching (TS), and repriming. TLS and TS depend on site-specific PCNA K164 monoubiquitination and polyubiquitination, respectively. To investigate the role of DDT in maintaining hematopoietic stem cells (HSCs) and progenitors, we used PcnaK164R/K164R mice as a unique DDT-defective mouse model. Analysis of the composition of HSCs and HSC-derived multipotent progenitors (MPPs) revealed a significantly reduced number of HSCs, likely owing to increased differentiation of HSCs toward myeloid/erythroid-associated MPP2s. This skewing came at the expense of the number of lymphoid-primed MPP4s, which appeared to be compensated for by increased MPP4 proliferation. Furthermore, defective DDT decreased the numbers of MPP-derived common lymphoid progenitor (CLP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), and granulocyte-macrophage progenitor (GMP) cells, accompanied by increased cell cycle arrest in CMPs. The HSC and MPP phenotypes are reminiscent of premature aging and stressed hematopoiesis, and indeed progressed with age and were exacerbated on cisplatin exposure. Bone marrow transplantations revealed a strong cell intrinsic defect of DDT-deficient HSCs in reconstituting lethally irradiated mice and a strong competitive disadvantage when cotransplanted with wild-type HSCs. These findings indicate a critical role of DDT in maintaining HSCs and progenitor cells, and in preventing premature aging.

Histone deacetylases (HDACs) counterbalance acetylation of lysine residues, a protein modification involved in numerous biological processes. Here, Hdac1 and Hdac2 conditional knock‐out alleles were used to study the function of class I Hdac1 and Hdac2 in cell cycle progression and haematopoietic differentiation. Combined deletion of Hdac1 and Hdac2, or inactivation of their deacetylase activity in primary or oncogenic‐transformed fibroblasts, results in a senescence‐like G 1 cell cycle arrest, accompanied by up‐regulation of the cyclin‐dependent kinase inhibitor p21 Cip . Notably, concomitant genetic inactivation of p53 or p21 Cip indicates that Hdac1 and Hdac2 regulate p53–p21 Cip ‐independent pathways critical for maintaining cell cycle progression. In vivo , we show that Hdac1 and Hdac2 are not essential for liver homeostasis. In contrast, total levels of Hdac1 and Hdac2 in the haematopoietic system are critical for erythrocyte‐megakaryocyte differentiation. Dual inactivation of Hdac1 and Hdac2 results in apoptosis of megakaryocytes and thrombocytopenia. Together, these data indicate that Hdac1 and Hdac2 have overlapping functions in cell cycle regulation and haematopoiesis. In addition, this work provides insights into mechanism‐based toxicities observed in patients treated with HDAC inhibitors.

Background DNA damage tolerance (DDT) enables replication to continue in the presence of fork stalling lesions. In mammalian cells, DDT is regulated by two independent pathways, controlled by the polymerase REV1 and ubiquitinated PCNA, respectively. Results To determine the molecular and genomic impact of a global DDT defect, we studied Pcna K164R/− ;Rev1 −/− compound mutants in mouse cells. Double-mutant cells display increased replication stress, hypersensitivity to genotoxic agents, replication speed, and repriming. A whole-genome CRISPR-Cas9 screen revealed a strict reliance of double-mutant cells on the CST complex, where CST promotes fork stability. Whole-genome sequencing indicated that this double-mutant DDT defect favors the generation of large, replication-stress inducible deletions of 0.4–4.0 kbp, defined as type 3 deletions. Junction break sites of these deletions reveal microhomology preferences of 1–2 base pairs, differing from the smaller type 1 and type 2 deletions. These differential characteristics suggest the existence of molecularly distinct deletion pathways. Type 3 deletions are abundant in human tumors, can dominate the deletion landscape, and are associated with DNA damage response status and treatment modality. Conclusions Our data highlight the essential contribution of the DDT system to genome maintenance and type 3 deletions as mutational signature of replication stress. The unique characteristics of type 3 deletions implicate the existence of a novel deletion pathway in mice and humans that is counteracted by DDT. Graphical Abstract

Ageing is associated with changes in the cellular composition of the immune system. During ageing, hematopoietic stem and progenitor cells (HSPCs) that produce immune cells are thought to decline in their regenerative capacity. However, HSPC function has been mostly assessed using transplantation assays, and it remains unclear how HSPCs age in the native bone marrow niche. To address this issue, we present an in situ single cell lineage tracing technology to quantify the clonal composition and cell production of single cells in their native niche. Our results demonstrate that a pool of HSPCs with unequal output maintains myelopoiesis through overlapping waves of cell production throughout adult life. During ageing, the increased frequency of myeloid cells is explained by greater numbers of HSPCs contributing to myelopoiesis rather than the increased myeloid output of individual HSPCs. Strikingly, the myeloid output of HSPCs remains constant over time despite accumulating significant transcriptomic changes throughout adulthood. Together, these results show that, unlike emergency myelopoiesis post-transplantation, aged HSPCs in their native microenvironment do not functionally decline in their regenerative capacity. Using in situ single cell lineage tracing technology DRAG, we show that, unlike emergency myelopoiesis post-transplantation, aged HSPCs do not functionally decline in the number of myeloid cells that they produce.

This paper addresses on-site supplementary household water sources with a focus on groundwater abstraction, rainwater harvesting and greywater reuse as available non-potable water sources to residential consumers. An end-use model is presented and used to assess the theoretical impact of household water sources on potable water demand in formal residential areas. Reliable potable municipal supply to urban consumers via the water distribution system is typically linked to relatively low uptake of household water sources. However, stringent water restrictions in some large South African cities that prohibit outdoor use, and reports of intermittent water supply, have led to increased uptake of household sources in South Africa. This paper describes the legal position regarding such sources in South Africa, and describes an end-use model to assess the theoretical impact on water demand in formal residential areas. The model provides valuable strategic direction and indicates a significant theoretical reduction in potable municipal water demand of between 55% and 69% for relatively large properties when household sources are maximally utilised (when compared to exclusive unrestricted municipal use as a baseline). This load reduction on piped reticulation systems could be an advantage in order to augment municipal supply, but water service planning and demand management are complicated by the introduction, and possible future decommissioning, of any household water source. The extent of both positive and negative impacts of household water sources requires further research.

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 [m.sup.2] 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 area