A regulatory role for NBS1 in strand-specific mutagenesis during somatic hypermutation

Activation-induced cytidine deaminase (AID) is believed to initiate somatic hypermutation (SHM) by deamination of deoxycytidines to deoxyuridines within the immunoglobulin variable regions genes. The deaminated bases can subsequently be replicated over, processed by base excision repair or mismatch repair, leading to introduction of different types of point mutations (G/C transitions, G/C transversions and A/T mutations). It is evident that the base excision repair pathway is largely dependent on uracil-DNA glycosylase (UNG) through its uracil excision activity. It is not known, however, which endonuclease acts in the step immediately downstream of UNG, i.e. that cleaves at the abasic sites generated by the latter. Two candidates have been proposed, an apurinic/apyrimidinic endonuclease (APE) and the Mre11-Rad50-NBS1 complex. The latter is intriguing as this might explain how the mutagenic pathway is primed during SHM. We have investigated the latter possibility by studying the in vivo SHM pattern in B cells from ataxia-telangiectasia-like disorder (Mre11 deficient) and Nijmegen breakage syndrome (NBS1 deficient) patients. Our results show that, although the pattern of mutations in the variable heavy chain (V(H)) genes was altered in NBS1 deficient patients, with a significantly increased number of G (but not C) transversions occurring in the SHM and/or AID targeting hotspots, the general pattern of mutations in the V(H) genes in Mre11 deficient patients was only slightly altered, with an increased frequency of A to C transversions. The Mre11-Rad50-NBS1 complex is thus unlikely to be the major nuclease involved in cleavage of the abasic sites during SHM, whereas NBS1 might have a specific role in regulating the strand-biased repair during phase Ib mutagenesis.     

Early endosome motility spatially organizes polysome distribution

Early endosomes (EEs) mediate protein sorting, and their cytoskeleton-dependent motility supports long-distance signaling in neurons. Here, we report an unexpected role of EE motility in distributing the translation machinery in a fungal model system. We visualize ribosomal subunit proteins and show that the large subunits diffused slowly throughout the cytoplasm (D_c,60S = 0.311 µm²/s), whereas entire polysomes underwent long-range motility along microtubules. This movement was mediated by “hitchhiking” on kinesin-3 and dynein-driven EEs, where the polysomes appeared to translate EE-associated mRNA into proteins. Modeling indicates that this motor-driven transport is required for even cellular distribution of newly formed ribosomes. Indeed, impaired EE motility in motor mutants, or their inability to bind EEs in mutants lacking the RNA-binding protein Rrm4, reduced ribosome transport and induced ribosome aggregation near the nucleus. As a consequence, cell growth was severely restricted. Collectively, our results indicate that polysomes associate with moving EEs and that “off- and reloading” distributes the protein translation machinery.     

Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy

CONTEXT:

Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders.

AIM:

To study the utility of MLPA in diagnosis and carrier detection for DMD.

MATERIALS AND METHODS:

Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared.

RESULTS AND CONCLUSIONS:

We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.     

Nephrotoxicity after radionuclide therapies

Radioligand therapies have opened new treatment avenues for cancer patients. They offer precise tumor targeting with a favorable efficacy-to-toxicity profile. Specifically, the kidneys, once regarded as the critical organ for radiation toxicity, also show excellent tolerance to radiation doses as high as 50–60 Gy in selected cases. However, the number of nephrons that form the structural and functional units of the kidney is determined before birth and is fixed. Thus, loss of nephrons secondary to any injury may lead to an irreversible decline in renal function over time. Our primary understanding of radiation-induced nephropathy is derived from the effects of external beam radiation on the renal tissue. With the growing adoption of radionuclide therapies, considerable evidence has been gained with regard to the occurrence of renal toxicity and its associated risk factors. In this review, we discuss the radionuclide therapies associated with the risk of nephrotoxicity, the present understanding of the factors and mechanisms that contribute to renal injury, and the current and potential methods for preventing, identifying, and managing nephrotoxicity, specifically acute onset nephropathies.     

Social Factors Influencing Russian Male Alcohol Use over the Life Course: A Qualitative Study Investigating Age Based Social Norms,Masculinity, and Workplace Context

The massive fluctuations occurring in Russian alcohol-related mortality since the mid-1980s cannot be seen outside of the context of great social and economic change. There is a dearth of qualitative studies about Russian male drinking and especially needed are those that address social processes and individual changes in drinking. Conducted as part of a longitudinal study on men’s alcohol consumption in Izhevsk, this qualitative study uses 25 semi-structured biographical interviews with men aged 33–60 years to explore life course variation in drinking. The dominant pattern was decreasing binge and frequent drinking as men reached middle age which was precipitated by family building, reductions in drinking with work colleagues, and health concerns. A minority of men described chaotic drinking histories with periods of abstinence and heavy drinking. The results highlight the importance of the blue-collar work environment for conditioning male heavy drinking in young adulthood through a variety of social, normative and structural mechanisms. Post-Soviet changes had a structural influence on the propensity for workplace drinking but the important social function of male drinking sessions remained. Bonding with workmates through heavy drinking was seen as an unavoidable and essential part of young men’s social life. With age peer pressure to drink decreased and the need to perform the role of responsible breadwinner put different behavioural demands on men. For some resisting social pressure to drink became an important site of self-determination and a mark of masculine maturity. Over the lifetime the place where masculine identity was asserted shifted from the workplace to the home, which commonly resulted in a reduction in drinking. We contribute to existing theories of Russian male drinking by showing that the performance of age-related social roles influences Russian men’s drinking patterns, drinking contexts and their attitudes. Further research should be conducted investigating drinking trajectories in Russian men.     

Breakpoint mapping of a novel de novo translocation t(X;20)(q11.1;p13) by positional cloning and long read sequencing

Disease associated chromosomal rearrangements often have break points located within disease causing genes or in their vicinity. The purpose of this study is to characterize a balanced reciprocal translocation in a girl with intellectual disability and seizures by positional cloning and whole genome sequencing. The translocation was identification by G- banding and confirmed by WCP FISH. Fine mapping using BAC clones and whole genome sequencing using Oxford nanopore long read sequencing technology for a 1.46 X coverage of the genome was done. The positional cloning showed split signals with BAC RP11-943 J20. Long read sequencing analysis of chimeric reads carrying parts of chromosomes X and 20 helped to identify the breakpoints to be in intron 2 of ARHGEF9 gene on Xp11.1 and on 20p13 between RASSF2 and SLC23A2 genes. This is the first report of translocation which successfully delineated to single base resolution using Nanopore sequencing. The genotype-phenotype correlation is discussed.     

Chitosan in Surface Modification for Bone Tissue Engineering Applications

Traditional methods of bone defect repair include autografts, allografts, surgical reconstruction, and metal implants that have several disadvantages such as donor site morbidity, rejection, risk of disease transmission, and repetitive surgery. Biomaterial‐based bone reconstructions can, therefore, be an efficient alternative due to the inherent properties of the materials. Chitosan (CS), the deacetylated form of chitin, is a biopolymer having a wide array of applicability in regenerative tissue applications owing to its biocompatible, in vitro degradative and bioresorbable nature. Extensive studies are being carried out using CS to augment the properties of the already existing methods and to also improve the applicability of CS‐based biocomposites in bone tissue repair. In this review, the suitability of CS as a surface modifier has been discussed in detail for the already existing implants, surface modifications of CS‐based natural biocomposites for bone tissue regeneration, and the wide range of techniques that can introduce these modifications. CS, being a natural polymer, possesses advantageous properties including surface modifier that makes it a suitable candidate for bone regeneration, and further research to investigate its osteogenic potential in vivo along with the molecular and signaling mechanisms involved in bone regeneration can aid in expanding its applicability in clinical trials.     

Normative Database of Retinal Oximetry in Asian Indian Eyes

Objective

To study the oxygen saturation profile in normal Asian Indian eyes.

Design

A cross sectional prospective study.

Subjects

Ninety eight consecutive patients presenting to our hospital with best corrected distance visual acuity (BCVA) of 20/20 and a normal ophthalmic examination were included in the study. Patients having any ocular or systemic disease were excluded from the study.

Materials and Methods

Oximetry was performed on all subjects with the Oxymap T1 retinal oximeter (Oxymap hf, Reykjavik, Iceland).

Main Outcome Measures

The images were analysed for oxygen saturation and diameter.

Results

The mean age was 33 years (Range: 18-63; SD: 12.4). The average arteriolar saturation was 90.3 ± 6.6% and the venous saturation was 56.9% ± 6.3. The average A-V (arterio-venous) difference was 33.2% ± 5.2. There was an increase in arteriolar (R² = 0.264; p=0.001) and venous saturation (R² = 0.151; p=0.001) with age. There was no significant change in the arterio-venous saturation difference (AVSD). The inferotemporal quadrant had the lowest saturations. Age correlated positively with ocular perfusion pressure (OPP)(R² = 0.07; p=0.007). OPP correlated positively with global arteriolar saturation (R²=0.057, p=0.018).

Conclusion

This study provides the normative database for an Indian population and is comparable to previous studies. Age, vessel diameter and OPP were the significant factors that influenced the saturation. Arteriolar and venous saturations increased with age while the AVSD did not change significantly.