Structure and repair of replication-coupled DNA breaks.

Using CRISPR/Cas9 nicking enzymes, we examine the interaction between the replication machinery and single strand breaks, one of the most common forms of endogenous DNA damage. We show that replication fork collapse at leading strand nicks generates resected single-ended double-strand breaks (seDSBs) that are repaired by homologous recombination (HR). If these seDSBs are not promptly repaired, arrival of adjacent forks creates double ended DSBs (deDSBs), which could drive genomic scarring in HR-deficient cancers. deDSBs can also be generated directly when the replication fork bypasses lagging strand nicks. Unlike deDSBs produced independently of replication, end-resection at nick-induced se/deDSBs is BRCA1-independent. Nevertheless, BRCA1 antagonizes 53BP1 suppression of RAD51 filament formation. These results highlight unique mechanisms that maintain replication fork stability.

Discovery of optimal cell type classification marker genes from single cell RNA sequencing data.

The use of single cell/nucleus RNA sequencing (scRNA-seq) technologies that quantitively describe cell transcriptional phenotypes is revolutionizing our understanding of cell biology, leading to new insights in cell type identification, disease mechanisms, and drug development. The tremendous growth in scRNA-seq data has posed new challenges in efficiently characterizing data-driven cell types and identifying quantifiable marker genes for cell type classification. The use of machine learning and explainable artificial intelligence has emerged as an effective approach to study large-scale scRNA-seq data. NS-Forest is a random forest machine learning-based algorithm that aims to provide a scalable data-driven solution to identify minimum combinations of necessary and sufficient marker genes that capture cell type identity with maximum classification accuracy. Here, we describe the latest version, NS-Forest version 4.0 and its companion Python package (https://github.com/JCVenterInstitute/NSForest), with several enhancements to select marker gene combinations that exhibit highly selective expression patterns among closely related cell types and more efficiently perform marker gene selection for large-scale scRNA-seq data atlases with millions of cells. By modularizing the final decision tree step, NS-Forest v4.0 can be used to compare the performance of user-defined marker genes with the NS-Forest computationally-derived marker genes based on the decision tree classifiers. To quantify how well the identified markers exhibit the desired pattern of being exclusively expressed at high levels within their target cell types, we introduce the On-Target Fraction metric that ranges from 0 to 1, with a metric of 1 assigned to markers that are only expressed within their target cell types and not in cells of any other cell types. NS-Forest v4.0 outperforms previous versions on its ability to identify markers with higher On-Target Fraction values for closely related cell types and outperforms other marker gene selection approaches at classification with significantly higher F-beta scores when applied to datasets from three human organs - brain, kidney, and lung.

Kuragel: A biomimetic hydrogel scaffold designed to promote corneal regeneration.

Cornea-related injuries are the most common cause of blindness worldwide. Transplantation remains the primary approach for addressing corneal blindness, though the demand for donor corneas outmatches the supply by millions. Tissue adhesives employed to seal corneal wounds have shown inefficient healing and incomplete vision restoration. We have developed a biodegradable hydrogel - Kuragel, with the ability to promote corneal regeneration. Functionalized gelatin and hyaluronic acid form photo-crosslinkable hydrogel with transparency and compressive modulus similar to healthy human cornea. Kuragel composition was tuned to achieve sufficient adhesive strength for sutureless integration to host tissue, with minimal swelling post-administration. Studies in the New Zealand rabbit mechanical injury model affecting corneal epithelium and stroma demonstrate that Kuragel efficiently promotes re-epithelialization within 1 month of administration, while stroma and sub-basal nerve plexus regenerate within 3 months. We propose Kuragel as a regenerative treatment for patients suffering from corneal defects including thinning, by restoration of transparency and thickness.

Differential Expression of SRY-Related HMG-Box Transcription Factor 2, Oligodendrocyte Lineage Transcription Factor 2, and Zinc Finger E-Box Binding Homeobox 1 in Serum-Derived Extracellular Vesicles: Implications for Mithramycin Sensitivity and Targeted Therapy in High-Grade Glioma.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and is often resistant to traditional therapies. Evidence suggests that glioma stem cells (GSCs) contribute to this resistance. Mithramycin (Mit-A) targets GSCs and exhibits antitumor activity in GBM by affecting transcriptional targets such as SRY-related HMG-box transcription factor 2 (SOX2), oligodendrocyte lineage transcription factor 2 (OLIG2), and zinc finger E-box binding homeobox 1 (ZEB1). However, its clinical use has been limited by toxicity. This study explored the diagnostic potential of serum extracellular vesicles (EVs) to identify Mit-A responders. Serum EVs were isolated from 70 glioma patients, and targeted gene expression was analyzed using qRT-PCR. Using chemosensitivity assay, we identified 8 Mit-A responders and 17 nonresponders among 25 glioma patients. The M-score showed a significant correlation (p = 0.045) with isocitrate dehydrogenase 1 mutation but not other clinical variables. The genes SOX2 (p = 0.005), OLIG2 (p = 0.003), and ZEB1 (p = 0.0281) were found to be upregulated in the responder EVs. SOX2 had the highest diagnostic potential (AUC = 0.875), followed by OLIG2 (AUC = 0.772) and ZEB1 (AUC = 0.632).The combined gene panel showed significant diagnostic efficacy (AUC = 0.956) through logistic regression analysis. The gene panel was further validated in the serum EVs of 45 glioma patients. These findings highlight the potential of Mit-A as a targeted therapy for high-grade glioma based on differential gene expression in serum EVs. The gene panel could serve as a diagnostic tool to predict Mit-A sensitivity, offering a promising approach for personalized treatment strategies and emphasizing the role of GSCs in therapeutic resistance.

Heterozygosity alters Msh5 binding to meiotic chromosomes in the baker's yeast.

Meiotic crossovers are initiated from programmed DNA double-strand breaks. The Msh4-Msh5 heterodimer is an evolutionarily conserved mismatch repair-related protein complex that promotes meiotic crossovers by stabilizing strand invasion intermediates and joint molecule structures such as Holliday junctions. In vivo studies using homozygous strains of the baker's yeast Saccharomyces cerevisiae (SK1) show that the Msh4-Msh5 complex associates with double-strand break hotspots, chromosome axes, and centromeres. Many organisms have heterozygous genomes that can affect the stability of strand invasion intermediates through heteroduplex rejection of mismatch-containing sequences. To examine Msh4-Msh5 function in a heterozygous context, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) analysis in a rapidly sporulating hybrid S. cerevisiae strain (S288c-sp/YJM789, containing sporulation-enhancing QTLs from SK1), using SNP information to distinguish reads from homologous chromosomes. Overall, Msh5 localization in this hybrid strain was similar to that determined in the homozygous strain (SK1). However, relative Msh5 levels were reduced in regions of high heterozygosity, suggesting that high mismatch densities reduce levels of recombination intermediates to which Msh4-Msh5 binds. Msh5 peaks were also wider in the hybrid background compared to the homozygous strain (SK1). We determined regions containing heteroduplex DNA by detecting chimeric sequence reads with SNPs from both parents. Msh5-bound double-strand break hotspots overlap with regions that have chimeric DNA, consistent with Msh5 binding to heteroduplex-containing recombination intermediates.

Comprehensive mapping of cell fates in microsatellite unstable cancer cells supports dual targeting of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.

Comprehensive mapping of cell fates in microsatellite unstable cancer cells support dual targe6ng of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN, knowledge that would be helpful for informing clinical development of WRN-targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system wherein the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We find that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we find no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provided the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggested a potential therapeutical rationale for dual targeting of WRN and ATR.

Reliability of Friedman Staging System and Modified Mallampati Scoring as clinical assessment methods for Obstructive Sleep Apnea - A cross sectional study.

OBJECTIVE: To assess the utility of oropharyngeal crowding indicators as predictors of Obstructive Sleep Apnea (OSA). METHODS: STOP-BANG Questionnaire was administered to 145 adults. Of the 95 with high OSA risk, 42 underwent polysomnography. Intraoral space was assessed using modified Mallampati (MMP) and Friedman Scoring (FS) System. RESULTS: Twenty-four were found to have OSA by polysomnography. Most with low risk (86%) had only Class I MMP. Whereas Class III and IV were seen in 37.9% and 17.9% patients with high risk (p < .001) and 50% and 20.8% patients with OSA (p < .831), respectively. All with low risk had Stage 1 FS. Stages 3 and 4 were observed in 27.4% and 1.1% in the high-risk group (p < .001) and in 29.2% and 4.2% with OSA (p < .092), respectively. CONCLUSION: In limited settings, either MMP or FS scores along with the STOP-BANG questionnaire can be used to diagnose OSA.

Excessive Daytime Sleepiness as a Risk Factor for Obstructive Sleep Apnoea among Public Transport Drivers: A Cross-Sectional Study.

Context: Excessive daytime sleepiness (EDS) due to obstructive sleep apnoea (OSA) is reported to be a major contributor to many road traffic accidents. Lack of awareness and diagnosis of OSA among public transport workers remains a threat to the society. Aims: The primary aim of this study was to assess the risk of OSA among transport drivers of south Kerala using modified Berlin questionnaire. The secondary objective included craniofacial assessment of the high-risk patients identified through the questionnaire using lateral cephalogram. Settings and Design: A cross-sectional study was conducted among 180 transport drivers of south Kerala. Methods and Material: Modified Berlin questionnaire and limited physical examination [body mass index (kg/m2), neck circumference (cm), waist circumference (cm), hip circumference and waist to hip ratio, blood pressure (mm Hg)] were recorded. The screened subjects were categorized as high-risk snorers and low-risk snorers based on modified Berlin questionnaire. Craniofacial morphological variations of high-risk group were assessed by lateral cephalograms. Statistical Analysis Used: The descriptive statistics were represented as mean and standard deviation and percentage. Inter-group comparison was performed with independent sample t test. Results: The study demonstrated 64.4% of subjects were non-snorers and 35.6% were snorers. Furthermore, among the snorers, 46.9% were identified as high-risk snorers, whereas the remaining 53.1% represented low-risk snorers. Conclusions: The study revealed the concealed risk of OSA among transport drivers could be screened through the questionnaires and demographics assessment. The application of the proposed screening protocol would triage and enhance safety of OSA affected transport drivers.

Evaluation of Strain and Insertion Torque of Mini-implants at 90° and 45° Angulations on a Bone Model using Three-Dimensional Finite Element Analysis.

Background: Temporary anchorage devices or mini-implants have gained great attraction due to its capability to provide absolute anchorage, low cost, versatility, and can be loaded immediately after placement. Aims and Objectives: The aim of this study is to use FEA analysis to assess the strain and insertion torque of mini-implants on a bone model at two distinct angulations of 45° and 90°. Materials and Methods: A computer-aided three-dimensional (3-D) model representing alveolar bone and mini-implants were developed using ANSYS software. Computed tomography scan images of the implant and the alveolar bone were taken and exported in DICOM format for 3-D image processing. The thickness of the bone model is 1 mm. Ti6Al4V orthodontic single and double threaded mini-implants (L = 7 mm, D = 1.5 mm) were inclined at 45° and 90° on to the bone surface to measure the insertion torque and strain produced. Results: Maximum insertion torque (MIT) for single-threaded mini implant at 45° and 90° angulations are 20.001 Nmm and 19.977 Nmm, respectively. MIT for double-threaded mini-implants obtained is 19.977 Nmm at 90° and 19.991 Nmm at 45° angulation. The strain of the bone at 90° angulation for single-threaded mini-implant is 0.00893 mm and for single-threaded mini implant at 45° angulation is 0.01257 mm. The strain in double-threaded mini-implant at 90° angulation is 0.0125 mm and that of 45° angulation is 0.01773 mm. Conclusion: For maximum stability single-threaded mini-implant with perpendicular insertion, angle is preferred.

Exo1 protects DNA nicks from ligation to promote crossover formation during meiosis.

In most sexually reproducing organisms crossing over between chromosome homologs during meiosis is essential to produce haploid gametes. Most crossovers that form in meiosis in budding yeast result from the biased resolution of double Holliday junction (dHJ) intermediates. This dHJ resolution step involves the actions of Rad2/XPG family nuclease Exo1 and the Mlh1-Mlh3 mismatch repair endonuclease. Here, we provide genetic evidence in baker's yeast that Exo1 promotes meiotic crossing over by protecting DNA nicks from ligation. We found that structural elements in Exo1 that interact with DNA, such as those required for the bending of DNA during nick/flap recognition, are critical for its role in crossing over. Consistent with these observations, meiotic expression of the Rad2/XPG family member Rad27 partially rescued the crossover defect in exo1 null mutants, and meiotic overexpression of Cdc9 ligase reduced the crossover levels of exo1 DNA-binding mutants to levels that approached the exo1 null. In addition, our work identified a role for Exo1 in crossover interference. Together, these studies provide experimental evidence for Exo1-protected nicks being critical for the formation of meiotic crossovers and their distribution.

Translational Diffusion of a Fluorescent Tracer Molecule in Nanoconfined Water.

Diffusion of tracer dye molecules in water confined to the nanoscale is an important subject with a direct bearing on many technological applications. It is not yet clear, however, if the dynamics of water in hydrophilic as well as hydrophobic nanochannels remains bulk-like. Here, we present diffusion measurement of a fluorescent dye molecule in water confined to the nanoscale between two hydrophilic surfaces whose separation can be controlled with a precision of less than a nm. We observe that the fluorescence intensities correlate over fast (∼30 µs) and slow (∼1000 µs) time components. The slow time scale is due to adsorption of fluorophores to the confining walls, and it disappears in the presence of 1 M salt. The fast component is attributed to diffusion of dye molecules in the gap. It is found to be bulk-like for sub-10 nm separations and indicates that the viscosity of water under confinement remains unaltered up to a confinement gap as small as ∼5 nm. Our findings contradict some of the recent measurements of diffusion under nanoconfinement; however, they are consistent with many estimates of self-diffusion using molecular dynamics simulations and measurements using neutron scattering experiments.

Regulation of Msh4-Msh5 association with meiotic chromosomes in budding yeast.

In the baker's yeast Saccharomyces cerevisiae, most of the meiotic crossovers are generated through a pathway involving the highly conserved mismatch repair related Msh4-Msh5 complex. To understand the role of Msh4-Msh5 in meiotic crossing over, we determined its genome wide in vivo binding sites in meiotic cells. We show that Msh5 specifically associates with DSB hotspots, chromosome axes, and centromeres on chromosomes. A basal level of Msh5 association with these chromosomal features is observed even in the absence of DSB formation (spo11Δ mutant) at the early stages of meiosis. But efficient binding to DSB hotspots and chromosome axes requires DSB formation and resection and is enhanced by double Holliday junction structures. Msh5 binding is also correlated to DSB frequency and enhanced on small chromosomes with higher DSB and crossover density. The axis protein Red1 is required for Msh5 association with the chromosome axes and DSB hotspots but not centromeres. Although binding sites of Msh5 and other pro-crossover factors like Zip3 show extensive overlap, Msh5 associates with centromeres independent of Zip3. These results on Msh5 localization in wild type and meiotic mutants have implications for how Msh4-Msh5 works with other pro-crossover factors to ensure crossover formation.

Validity of point-mass model in off-resonance dynamic atomic force microscopy.

The quantitative measurement of viscoelasticity of nano-scale entities is an important goal of nanotechnology research and there is considerable progress with advent of dynamic atomic force microscopy. The hydrodynamics of cantilever, the force sensor in AFM measurements, plays a pivotal role in quantitative estimates of nano-scale viscoelasticity. The point-mass (PM) model, wherein the AFM cantilever is approximated as a point-mass with mass-less spring is widely used in dynamic AFM analysis and its validity, particularly in liquid environments, is debated. It is suggested that the cantilever must be treated as a continuous rectangular beam to obtain accurate estimates of nano-scale viscoelasticity of materials it is probing. Here, we derived equations, which relate stiffness and damping coefficient of the material under investigation to measured parameters, by approximating cantilever as a point-mass and also considering the full geometric details. These equations are derived for both tip-excited as well as base-excited cantilevers. We have performed off-resonance dynamic atomic force spectroscopy on a single protein molecule to investigate the validity of widely used PM model. We performed measurements with AFMs equipped with different cantilever excitation methods as well as detection schemes to measure cantilever response. The data was analyzed using both, continuous beam model and the PM model. We found that both models yield same results when the experiments are performed in truly off-resonance regime with small amplitudes and the cantilever stiffness is much higher than the interaction stiffness. Our findings suggest that a simple PM approximation based model is adequate to describe the dynamics, provided care is taken while performing experiments so that the approximations used in these models are valid.

Characterization of systemic genomic instability in budding yeast.

Conventional models of genome evolution are centered around the principle that mutations form independently of each other and build up slowly over time. We characterized the occurrence of bursts of genome-wide loss-of-heterozygosity (LOH) in Saccharomyces cerevisiae, providing support for an additional nonindependent and faster mode of mutation accumulation. We initially characterized a yeast clone isolated for carrying an LOH event at a specific chromosome site, and surprisingly found that it also carried multiple unselected rearrangements elsewhere in its genome. Whole-genome analysis of over 100 additional clones selected for carrying primary LOH tracts revealed that they too contained unselected structural alterations more often than control clones obtained without any selection. We also measured the rates of coincident LOH at two different chromosomes and found that double LOH formed at rates 14- to 150-fold higher than expected if the two underlying single LOH events occurred independently of each other. These results were consistent across different strain backgrounds and in mutants incapable of entering meiosis. Our results indicate that a subset of mitotic cells within a population can experience discrete episodes of systemic genomic instability, when the entire genome becomes vulnerable and multiple chromosomal alterations can form over a narrow time window. They are reminiscent of early reports from the classic yeast genetics literature, as well as recent studies in humans, both in cancer and genomic disorder contexts. The experimental model we describe provides a system to further dissect the fundamental biological processes responsible for punctuated bursts of structural genomic variation.

Loss of Heterozygosity and Base Mutation Rates Vary Among Saccharomyces cerevisiae Hybrid Strains.

A growing body of evidence suggests that mutation rates exhibit intra-species specific variation. We estimated genome-wide loss of heterozygosity (LOH), gross chromosomal changes, and single nucleotide mutation rates to determine intra-species specific differences in hybrid and homozygous strains of Saccharomyces cerevisiae The mutation accumulation lines of the S. cerevisiae hybrid backgrounds - S288c/YJM789 (S/Y) and S288c/RM11-1a (S/R) were analyzed along with the homozygous diploids RM11, S288c, and YJM145. LOH was extensive in both S/Y and S/R hybrid backgrounds. The S/Y background also showed longer LOH tracts, gross chromosomal changes, and aneuploidy. Short copy number aberrations were observed in the S/R background. LOH data from the S/Y and S/R hybrids were used to construct a LOH map for S288c to identify hotspots. Further, we observe up to a sixfold difference in single nucleotide mutation rates among the S. cerevisiae S/Y and S/R genetic backgrounds. Our results demonstrate LOH is common during mitotic divisions in S. cerevisiae hybrids and also highlight genome-wide differences in LOH patterns and rates of single nucleotide mutations between commonly used S. cerevisiae hybrid genetic backgrounds.

A new method for measurement and quantification of tracer diffusion in nanoconfined liquids.

We report development of a novel instrument to measure tracer diffusion in water under nanoscale confinement. A direct optical access to the confinement region, where water is confined between a tapered fiber and a flat substrate, is made possible by coating the probe with metal and opening a small aperture (0.1 µm-1 µm) at its end. A well-controlled cut using an ion beam ensures desired lateral confinement area as well as adequate illumination of the confinement gap. The probe is mounted on a tuning-fork based force sensor to control the separation between the probe and the substrate with nanometer precision. Fluctuations in fluorescence intensity due to diffusion of a dye molecule in water confined between the probe and the sample are recorded using a confocal arrangement with a single photon precision. A Monte Carlo method is developed to determine the diffusion coefficient from the measured autocorrelation of intensity fluctuations which accommodates the specific geometry of confinement and the illumination profile. The instrument allows for measurement of diffusion laws under confinement. We found that the diffusion of a tracer molecule is slowed down by more than 10 times for the probe-substrate separations of 5 nm and below.

Epidemiological study on prevalent risk factors and craniofacial skeletal patterns in obstructive sleep apnea among South Indian population.

CONTEXT: Obstructive sleep apnea (OSA) is a condition affecting the upper airway among a vast number of people around the world. AIMS: To determine the prevalent risk factors of OSA and its association with craniofacial skeletal pattern. SETTINGS AND DESIGN: Cross-sectional, community-based study. MATERIALS AND METHODS: In the first stage, questionnaire and physical examination were done for 1000 subjects between 20 and 70 years of age. Subjects were categorized as snorers and non-snorers. Snorers were further grouped as high-risk and low-risk snorers. In the second stage, polysomnography (PSG) was done for randomly selected high-risk subjects. Craniofacial skeletal pattern of OSA-diagnosed subjects were compared with non-OSA subjects using lateral cephalograms. STATISTICAL ANALYSIS: Analysis was performed using IBM SPSS 20. Independent sample t-test was used. A P value < 0.05 was considered as statistically significant. RESULTS: The study population represented the following: high-risk snorers: 22.4%, low-risk snorers: 13.9%, and non-snorers: 63.7%. Excessive daytime sleepiness was present in 7.7%. Among high-risk, 80 underwent PSG, and 75 were diagnosed as OSA (94%) and 5 non-OSA subjects. Increased body mass index and neck circumference were statistically significant. Cephalometric evaluation showed difference in maxillomandibular relationship, narrowing of airway space, and inferiorly displaced hyoid. CONCLUSION: OSA is a major public health problem. Obesity is a strong predictor for OSA. Thus, high-risk subjects for sleep apnea could be identified using routine clinical examination, investigations, and anthropometric parameters.

The Neurospora crassa Standard Oak Ridge Background Exhibits Atypically Efficient Meiotic Silencing by Unpaired DNA.

Meiotic silencing by unpaired DNA (MSUD), an RNAi-mediated gene silencing process, is efficient in crosses made in the Neurospora crassa standard Oak Ridge (OR) genetic background. However, MSUD was decidedly less efficient when the OR-derived MSUD testers were crossed with many wild-isolated strains (W), suggesting that either sequence heterozygosity in tester x W crosses suppresses MSUD, or that OR represents the MSUD-conducive extreme in the range of genetic variation in MSUD efficiency. Our results support the latter model. MSUD was less efficient in near-isogenic crosses made in the novel N. crassa B/S1 genetic background, and in N. tetrasperma strain 85. Possibly, in B/S1 and 85, additional regulatory cues, absent from OR, calibrate the MSUD response. A locus in distal chromosome 1R appears to underlie the OR vs. B/S1 difference. Repeat-induced point mutation (RIP) destroys duplicated genes by G:C to A:T mutation of duplicated DNA sequences. Chromosome segment duplications (Dps) dominantly suppress RIP, possibly by titrating out the RIP machinery. In Dp x N crosses, the Dp-borne genes cannot pair properly, hence efficient MSUD, as in OR, silences them and renders the crosses barren. We speculate that the increased productivity engendered by inefficient MSUD enables small duplications to escape RIP.

An analysis of surveillance screening for SDHB-related disease in childhood and adolescence.

Objective Phaeochromocytomas (PCC) and paragangliomas (PGL) are rare in children. A large proportion of these are now understood to be due to underlying germline mutations. Here we focus on succinate dehydrogenase subunit B (SDHB) gene mutation carriers as these tumours carry a high risk of malignant transformation. There remains no current consensus with respect to optimal surveillance for asymptomatic carriers and those in whom the presenting tumour has been resected. Method We undertook a retrospective analysis of longitudinal clinical data of all children and adolescents with SDHB mutations followed up in a single UK tertiary referral centre. This included index cases that pre-dated the introduction of surveillance screening and asymptomatic carriers identified through cascade genetic testing. We also conducted a literature review to inform a suggested surveillance protocol for children and adolescents harbouring SDHB mutations. Results Clinical outcomes of a total of 38 children are presented: 8 index cases and 30 mutation-positive asymptomatic carriers with 175 patient years of follow-up data. Three of the eight index cases developed metachronous disease and two developed metastatic disease. Of the 30 asymptomatic carriers, 3 were found to have PGLs on surveillance screening. Conclusions Surveillance screening was well tolerated in our paediatric cohort and asymptomatic paediatric subjects. Screening can identify tumours before they become secretory and/or symptomatic, thereby facilitating surgical resection and reducing the chance of distant spread. We propose a regular screening protocol commencing at age 5 years in this at-risk cohort of patients.