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The African grasscutter (AGC) (Thryonomys swinderianus) is the second largest rodent in sub-Saharan Africa. It is bred for its organoleptic and culinary properties but also serves as a research model. The aim of this study was to investigate the relationship between age-related changes in brain weight, brain volume, and spatial and nonspatial memory performance in the AGC. A total of forty-two (42) captive-derived AGCs were divided into seven age groups: 6 neonates (6 days), 6 juveniles (1 month), 6 pubertals (3 months), 6 subadults (8 months), 6 young adults (2 years), 6 middle adults (4 years), and 6 old adults (5 years). The subjects were given a series of tests to assess their spatial memory (location test) and their nonspatial memory (object recognition test). Brain samples were then collected using basic neuroanatomical techniques. The weight and volume of the brain samples were determined and their encephalization quotient (EQ) was also calculated. The results showed that spatial and nonspatial memory in AGC develops into adulthood and then declines with age. Mean brain weight increased from neonates to mid-adulthood (5.20 ± 0.31 g–11.76 ± 0.23 g) and decreased in older AGC (11.75 ± 0.22 g). In contrast, the mean weight of the olfactory bulb (OB) increased from neonates to young adults (0.28 ± 0.02 g–0.80 ± 0.03 g) and the cerebellum increased from neonates to older (0.59 ± 0 0.01 g–1.86 ± 0.06 g). Finally, the EQ decreased with age (0.73 ± 0.05–0.29 ± 0.01). Mean brain volume increased with age from neonates to young adult (5 mL–11.25 mL). Conversely, the mean volume of the OB and cerebellum increases from neonates to older age (0.33 ± 0.03 mL–0.95 ± 0.04 mL). This study shows that spatial and nonspatial memory declines during the aging process in AGC. Neonates and juveniles have higher cognitive abilities than adults and older AGC. The weight of the brain, OB, and cerebellum increases from neonates to adult and decreases slightly from middle to old adults. However, the volume of the OB and cerebellum increases with age.

Purpose: To assess the added value of artificial intelligence (AI) for radiologists and emergency physicians in the radiographic detection of pelvic fractures., Materials & Methods: In this retrospective study, one junior radiologist reviewed 940 X-rays of patients admitted to emergency for a fall with suspicion of pelvic fracture between March 2020 and June 2021. The radiologist analyzed the X-rays alone and then using an AI system (BoneView). In a random sample of 100 exams, the same procedure was repeated alongside five other readers (three radiologists and two emergency physicians with 3-30 years of experience). The reference diagnosis was based on the patient's full set of medical imaging exams and medical records in the months following emergency admission., Results: A total of 633 confirmed pelvic fractures (64.8% from hip and 35.2% from pelvic ring) in 940 patients and 68 pelvic fractures (60% from hip and 40% from pelvic ring) in the 100-patient sample were included. In the whole dataset, the junior radiologist achieved a significant sensitivity improvement with AI assistance (Se -PELVIC  = 77.25% to 83.73%; p < 0.001, Se -HIP 93.24 to 96.49%; p < 0.001 and Se -PELVIC RING 54.60% to 64.50%; p < 0.001). However, there was a significant decrease in specificity with AI assistance (Spe -PELVIC  = 95.24% to 93.25%; p = 0.005 and Spe -HIP  = 98.30% to 96.90%; p = 0.005). In the 100-patient sample, the two emergency physicians obtained an improvement in fracture detection sensitivity across the pelvic area + 14.70% (p = 0.0011) and + 10.29% (p < 0.007) respectively without a significant decrease in specificity. For hip fractures, E1's sensitivity increased from 59.46% to 70.27% (p = 0.04), and E2's sensitivity increased from 78.38% to 86.49% (p = 0.08). For pelvic ring fractures, E1's sensitivity increased from 12.90% to 32.26% (p = 0.012), and E2's sensitivity increased from 19.35% to 32.26% (p = 0.043)., Conclusion: AI improved the diagnostic performance for emergency physicians and radiologists with limited experience in pelvic fracture screening., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no known competing financial or personal relationships that could be viewed as influencing the work reported in this paper. Ethical approval: The appropriate institutional ethics committee approved: IRB ID: 202000393. The authors declare that this article does not contain any personal information that could lead to the identification of the patients., (© 2025. The Author(s) under exclusive licence to Japan Radiological Society.)

Background: Helicobacter pylori (H. pylori) infection is a significant risk factor for gastric cancer (GC) development. A growing body of evidence suggests a causal link between infection with H. pylori and increased DNA breakage in the host cells. While several mechanisms have been proposed for this damage, their relative impact on the overall bacterial genotoxicity is unknown. Moreover, the link between the formation of DNA damage following infection and the emergence of cancerous structural variants (SV) in the genome of infected cells remained unexplored., Methods: We constructed a high-resolution map of genomic H. pylori-induced recurrent break sites using the END-seq method on AGS human gastric cells before and after infection. We next applied END-seq to cycling and arrested cells to identify the role of DNA replication on break formation. Recurrent H. pylori-mediated break sites were further characterized by analyzing published RNA-seq, DRIP-seq, and GRO-seq data at these sites. γH2AX staining and comet assay were used for DNA breakage quantification. Liquid chromatography-mass spectrometry (LC-MS) assay was used to quantify cellular concentrations of dNTPs., Results: Our data indicated that sites of recurrent H. pylori-mediated DNA breaks are ubiquitous across cell types, localized at replication-related fragile sites, and their breakage is dependent on replication. Consistent with that, we found that H. pylori inflicts nucleotide depletion, and that rescuing the cellular nucleotide pool largely reduced H. pylori-induced DNA breaks. Intriguingly, we found that this genotoxic mechanism operates independently of H. pylori cag pathogenicity island (CagPAI) that encodes for the bacterial type 4 secretion system (T4SS), and its virulence factor, CagA, which was previously implicated in increasing DNA damage by downregulating the DNA damage response. Finally, we show that sites of recurrent H. pylori-mediated breaks coincide with chromosomal deletions observed in patients with intestinal-type GC and that this link potentially elucidates the persistent transcriptional alterations observed in cancer driver genes., Conclusions: Our findings indicate that dNTP depletion by H. pylori is a key component of its genotoxicity and suggest a link between H. pylori genotoxicity and its oncogenic potential., Competing Interests: Declarations. Ethics approval and consent to participate: Research involved human samples (immunohistochemistry) was approved by Helsinki Committee of the Baruch Padeh Medical Center, Poriya, (Approval no. POR 0007–20). Competing interests: The authors declare that they have no competing interests., (© 2025. The Author(s).)

Background: Vegetarianism is constantly increasing worldwide. However, the role of metabolic and bariatric surgery (MBS) in vegetarians/vegans is unclear as there is very limited data on this topic. The aim of this study was to evaluate MBS outcomes in vegetarians or vegans., Methods: Retrospective analysis of a prospectively maintained database of a single-bariatric surgeon was carried out. All patients with a vegetarian or vegan lifestyle undergoing MBS were included., Results: Eleven patients were included; none were lost to follow-up. Ten patients were women, the mean age and Body Mass Index (BMI) were 40.8±14 years and 43.5±4.9 kg/m 2 , respectively. Five patients (45%) were lacto-ovo-vegetarians, one (9%) was ovo-vegetarian, two (18%) were lacto-ovo-pesco-vegetarians, and three (27%) were vegans. Eight patients consumed vitamin supplements preoperatively, the mean albumin level was 4.1±0.2, 3/11 patients had vitamin D deficiency, 2/11 patients had vitamin-B12 deficiency, and 2/11 patients had iron deficiency. Eight patients (73%) underwent one anastomosis gastric bypass (OAGB), 2/11 patients (18%) underwent single anastomosis duodeno-ileal bypass with sleeve gastrectomy, and one patient (9%) underwent sleeve gastrectomy, the mean biliopancreatic limb length in OAGB was 225 cm. The median follow-up time was 17 months, the mean BMI and percentage of total weight loss during follow-up were 28.2±5 kg/m 2 and 35.3±10.7%, respectively. The mean albumin level was 3.82±0.27, 3/11 patients had vitamin D deficiency, and 3/11 patients had iron deficiency., Conclusions: This study reports preliminary data on MBS outcomes in vegetarian/vegan patients. It was shown to be safe, effective, and an acceptable rate of nutritional deficiencies during follow-up. Further large cohort studies are required to clarify this data.

Background: Major abdominal wall defects remain a highly morbid complication. Occasionally a fascial defect is encountered, that despite all surgical efforts, is unable to completely approximate at the midline. Here we describe our method and outcomes of using a bridging mesh when the posterior fascia was unable to be approximated during the repair of large postoperative ventral hernias using the modified Rives-Stoppa technique., Methods: A retrospective review was conducted looking at all the open abdominal wall hernia repairs between 2014 and 2020. The cohort of patients who had a bridge placed in addition to the traditional open modified Rives-Stoppa repair were used for this study., Results: Nineteen patients had a mesh inlay bridge placed in addition to a modified Rives-Stoppa repair with a sublay (retrorectus) Ultrapro mesh. For the inlay mesh 13 Symbotex composite meshes were placed and 6 Vicryl meshes used. The average surface area of the defect was 358.1 cm^2. The average length of hospitalization was 8.8 days with a range of 3-24 days. During the immediate postoperative course there were 6 minor complications. During the follow-up period there were 2 recurrences., Discussion: The use of inlay mesh bridge as an adjuvant to a modified Rives-Stoppa repair with a sublay ultrapro mesh is an effective technique for difficult abdominal wall repairs where the posterior fascia is unable to be approximated without tension., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: The recommended treatment for resectable pancreatic cancer (PC) is resection followed by adjuvant FOLFIRINOX. We assessed the proportion of patients that managed to complete the 12 courses of adjuvant FOLFIRINOX and compared their outcome with that of patients with borderline resectable pancreatic cancer (BRPC) who underwent resection after neoadjuvant FOLFIRINOX., Methods: A retrospective analysis was performed on a prospectively maintained database of all PC patients who underwent resection with (2/2015-12/2021) or without (1/2018-12/2021) neoadjuvant therapy., Results: A total of 100 patients underwent upfront resection, and 51 patients with BRPC received neoadjuvant treatment. Only 46 resection patients started adjuvant FOLFIRINOX, and only 23 completed 12 courses. The main reasons for not starting/completing adjuvant therapy were poor tolerance and rapid recurrence. Significantly more patients in the neoadjuvant group received at least six FOLFIRINOX courses (80.4% vs. 31%, p < 0.001). Patients who completed at least 6 courses, either pre- or postoperatively, had better overall survival ( p = 0.025) than those who did not. In spite of having more advanced disease, the neoadjuvant group had comparable overall survival ( p = 0.062) regardless of the number of treatment courses., Conclusion: Only a minority of patients (23%) undergoing upfront pancreatic resection completed the planned 12 courses of FOLFIRINOX. Patients who received neoadjuvant treatment were significantly more likely to receive at least six treatment courses. Patients receiving at least six courses had better overall survival than those who received fewer than six courses, regardless of the timing of treatment relative to surgery. Potential ways to increase chemotherapy adherence, such as administering treatment before surgery, should be considered.

Background & Aims: Hepatocellular carcinoma (HCC) is a model of a diverse spectrum of cancers because it is induced by well-known etiologies, mainly hepatitis C virus (HCV) and hepatitis B virus. Here, we aimed to identify HCV-specific mutational signatures and explored the link between the HCV-related regional variation in mutations rates and HCV-induced alterations in genome-wide chromatin organization., Methods: To identify an HCV-specific mutational signature in HCC, we performed high-resolution targeted sequencing to detect passenger mutations on 64 HCC samples from 3 etiology groups: hepatitis B virus, HCV, or other. To explore the link between the genomic signature and genome-wide chromatin organization we performed chromatin immunoprecipitation sequencing for the transcriptionally permissive H3K4Me3, H3K9Ac, and suppressive H3K9Me3 modifications after HCV infection., Results: Regional variation in mutation rate analysis showed significant etiology-dependent regional mutation rates in 12 genes: LRP2, KRT84, TMEM132B, DOCK2, DMD, INADL, JAK2, DNAH6, MTMR9, ATM, SLX4, and ARSD. We found an enrichment of C->T transversion mutations in the HCV-associated HCC cases. Furthermore, these cases showed regional variation in mutation rates associated with genomic intervals in which HCV infection dictated epigenetic alterations. This signature may be related to the HCV-induced decreased expression of genes encoding key enzymes in the base excision repair pathway., Conclusions: We identified novel distinct HCV etiology-dependent mutation signatures in HCC associated with HCV-induced alterations in histone modification. This study presents a link between cancer-causing mutagenesis and the increased predisposition to liver cancer in chronic HCV-infected individuals, and unveils novel etiology-specific mechanisms leading to HCC and cancer in general., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent studies suggest the enhancement of liver injury in COVID-19 patients infected with Hepatitis C virus (HCV). Hepatocytes express low levels of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 entry receptor, raising the possibility of HCV-SARS-CoV-2 coinfection in the liver. This work aimed to explore whether HCV and SARS-CoV-2 coinfect hepatocytes and the interplay between these viruses. We demonstrate that SARS-CoV-2 coinfects HCV-infected Huh7.5 (Huh7.5 HCV ) cells. Both viruses replicated efficiently in the coinfected cells, with HCV replication enhanced in coinfected compared to HCV-mono-infected cells. Strikingly, Huh7.5 HCV cells were eight fold more susceptible to SARS-CoV-2 pseudoviruses than naive Huh7.5 cells, suggesting enhanced SARS-CoV-2 entry into HCV-preinfected hepatocytes. In addition, we observed increased binding of spike receptor-binding domain (RBD) protein to Huh7.5 HCV cells, as well as enhanced cell-to-cell fusion of Huh7.5 HCV cells with spike-expressing Huh7.5 cells. We explored the mechanism of enhanced SARS-CoV-2 entry and identified an increased ACE2 mRNA and protein levels in Huh7.5 HCV cells, primary hepatocytes, and in data from infected liver biopsies obtained from database. Importantly, higher expression of ACE2 increased HCV infection by enhancing its binding to the host cell, underscoring its role in the HCV life cycle as well. Transcriptome analysis revealed that shared host signaling pathways were induced in HCV-SARS-CoV-2 coinfection. This study revealed complex interactions between HCV and SARS-CoV-2 infections in hepatocytes, which may lead to the increased liver damage recently reported in HCV-positive COVID-19 patients. IMPORTANCE Here, we provide the first experimental evidence for the coexistence of SARS-CoV-2 infection with HCV, and the interplay between them. The study revealed a complex relationship of enhancement between the two viruses, where HCV infection increased the expression of the SARS-CoV-2 entry receptor ACE2, thus facilitating SARS-CoV-2 entry, and potentially, also HCV entry. Thereafter, SARS-CoV-2 infection enhanced HCV replication in hepatocytes. This study may explain the aggravation of liver damage that was recently reported in COVID-19 patients with HCV coinfection and suggests preinfection with HCV as a risk factor for severe COVID-19. Moreover, it highlights the possible importance of HCV treatment for coinfected patients. In a broader view, these findings emphasize the importance of identifying coinfecting pathogens that increase the risk of SARS-CoV-2 infection and that may accelerate COVID-19-related co-morbidities.

Background: The COVID-19 pandemic has transformed and affected every aspect of health care. Like any catastrophic event, the stress on hospitals to maintain a certain level of function is immense. Acute surgical pathologies cannot be prevented or curtailed; therefore, it is important to understand patterns and outcomes during catastrophes in order to optimize care and organize the health care system., Methods: In a single urban tertiary care center, a retrospective study examined the first complete lockdown period of Israel during the COVID-19 pandemic. This was compared to the same time period the previous year., Results: During the pandemic, time to hospitalization was significantly decreased. There was also an overall reduction in surgical admissions yet with a higher percentage being hospitalized for further treatment (69.2% vs 23.5%). The patients admitted during this time had a higher APACHE-II score and Charlson comorbidity index score. During the pandemic, time to surgery was decreased, there were less laparoscopic procedures, and more RBC units were used per patient. There were no differences in overall complications, except when sub-analyzed for major complications (9.7% vs 6.3%). There was no significant difference in overall in-house mortality or morbidity. Length of hospitalization was significantly decreased in the elderly population during the pandemic., Conclusion: During the COVID-19 pandemic, despite a significantly less number of patients presenting to the hospital, there was a higher percentage of those admitted needing surgical intervention, and they were overall sicker than the previous year.

Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis -acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9-KRAB (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by using microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated and isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty-one of the 64 hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-photoswitchable screening (AI-PS), offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale., (© 2021 Kanfer et al.)

Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm -/- spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1 Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1 Δ11 53BP1 S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1 D11 53BP1 S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51., (Copyright © 2019 Elsevier Inc. All rights reserved.)

During V(D)J recombination, RAG proteins introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) that contain either 12- or 23-nt spacer regions. Coordinated 12/23 cleavage predicts that DSBs at variable (V) gene segments should equal the level of breakage at joining (J) segments. Contrary to this, here we report abundant RAG-dependent DSBs at multiple Vκ gene segments independent of V-J rearrangement. We find that a large fraction of Vκ gene segments are flanked not only by a bone-fide 12 spacer but also an overlapping, 23-spacer flipped RSS. These compatible pairs of RSSs mediate recombination and deletion inside the Vκ cluster even in the complete absence of Jκ gene segments and support a V(D)J recombination center (RC) independent of the conventional Jκ-centered RC. We propose an improved model of Vκ-Jκ repertoire formation by incorporating these surprisingly frequent, evolutionarily conserved intra-Vκ cluster recombination events., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes but also causes mutations in other parts of the genome. We have used lentiviral SHM reporter vectors to identify regions of the genome that are susceptible ("hot") and resistant ("cold") to SHM, revealing that SHM susceptibility and resistance are often properties of entire topologically associated domains (TADs). Comparison of hot and cold TADs reveals that while levels of transcription are equivalent, hot TADs are enriched for the cohesin loader NIPBL, super-enhancers, markers of paused/stalled RNA polymerase 2, and multiple important B cell transcription factors. We demonstrate that at least some hot TADs contain enhancers that possess SHM targeting activity and that insertion of a strong Ig SHM-targeting element into a cold TAD renders it hot. Our findings lead to a model for SHM susceptibility involving the cooperative action of cis-acting SHM targeting elements and the dynamic and architectural properties of TADs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs., (Published by Elsevier Inc.)

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility., (Published by Elsevier Inc.)

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT., (Published by Elsevier Inc.)

The RAG1/RAG2 endonuclease initiates V(D)J recombination at antigen receptor loci but also binds to thousands of places outside of these loci. RAG2 localizes directly to lysine 4 trimethylated histone 3 (H3K4me3) through a plant homeodomain (PHD) finger. The relative contribution of RAG2-dependent and RAG1-intrinsic mechanisms in determining RAG1 binding patterns is not known. Through analysis of deep RAG1 ChIP-seq data, we provide a quantitative description of the forces underlying genome-wide targeting of RAG1. Surprisingly, sequence-specific DNA binding contributes minimally to RAG1 targeting outside of antigen receptor loci. Instead, RAG1 binding is driven by two distinct modes of interaction with chromatin: the first is driven by H3K4me3, promoter-focused and dependent on the RAG2 PHD, and the second is defined by H3K27Ac, enhancer-focused and dependent on 'non-core' portions of RAG1. Based on this and additional chromatin and genomic features, we formulated a predictive model of RAG1 targeting to the genome. RAG1 binding sites predicted by our model correlate well with observed patterns of RAG1-mediated breaks in human pro-B acute lymphoblastic leukemia. Overall, this study provides an integrative model for RAG1 genome-wide binding and off-target activity and reveals a novel role for the RAG1 non-core region in RAG1 targeting., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Incremental selection within a population, defined as limited fitness changes following mutation, is an important aspect of many evolutionary processes. Strongly advantageous or deleterious mutations are detected using the synonymous to non-synonymous mutations ratio. However, there are currently no precise methods to estimate incremental selection. We here provide for the first time such a detailed method and show its precision in multiple cases of micro-evolution. The proposed method is a novel mixed lineage tree/sequence based method to detect within population selection as defined by the effect of mutations on the average number of offspring. Specifically, we propose to measure the log of the ratio between the number of leaves in lineage trees branches following synonymous and non-synonymous mutations. The method requires a high enough number of sequences, and a large enough number of independent mutations. It assumes that all mutations are independent events. It does not require of a baseline model and is practically not affected by sampling biases. We show the method's wide applicability by testing it on multiple cases of micro-evolution. We show that it can detect genes and inter-genic regions using the selection rate and detect selection pressures in viral proteins and in the immune response to pathogens., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma of skin-homing T lymphocytes. We performed exome and whole-genome DNA sequencing and RNA sequencing on purified CTCL and matched normal cells. The results implicate mutations in 17 genes in CTCL pathogenesis, including genes involved in T cell activation and apoptosis, NF-κB signaling, chromatin remodeling and DNA damage response. CTCL is distinctive in that somatic copy number variants (SCNVs) comprise 92% of all driver mutations (mean of 11.8 pathogenic SCNVs versus 1.0 somatic single-nucleotide variant per CTCL). These findings have implications for new therapeutics.

The RAG1 endonuclease, together with its cofactor RAG2, is essential for V(D)J recombination but is a potent threat to genome stability. The sources of RAG1 mis-targeting and the mechanisms that have evolved to suppress it are poorly understood. Here, we report that RAG1 associates with chromatin at thousands of active promoters and enhancers in the genome of developing lymphocytes. The mouse and human genomes appear to have responded by reducing the abundance of "cryptic" recombination signals near RAG1 binding sites. This depletion operates specifically on the RSS heptamer, whereas nonamers are enriched at RAG1 binding sites. Reversing this RAG-driven depletion of cleavage sites by insertion of strong recombination signals creates an ectopic hub of RAG-mediated V(D)J recombination and chromosomal translocations. Our findings delineate rules governing RAG binding in the genome, identify areas at risk of RAG-mediated damage, and highlight the evolutionary struggle to accommodate programmed DNA damage in developing lymphocytes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Viral epitopes have a distinct codon usage that reflects their dual role in infection and immunity. On the one hand, epitopes are part of proteins important to viral function; on the other hand, they are targets of the immune response. Studies of selection are most commonly based on changes of amino acid and seen through the accumulation of non-synonymous mutations. An independent measure of selection is the codon usage and underlying changeability of the nucleotide sequences. We here use multiple tools and a large-scale analysis of viral genomes to demonstrate that viral epitopes have a distinct codon usage and that this codon usage reflects distinct short- and long-term types of selection during viral evolution. We show that CD8(+) T cell epitopes are encoded by codons more distant from stop codons and more changeable than codons outside epitopes. This biased codon usage reflects the viral population toggling back and forth from a wild-type sequence to an escape mode, which enable them to avoid immune detection when needed, and go back to the functionally favorable form when the threat is removed (i.e., in a new host).

The relation between the complexity of organisms and proteins and their evolution rates has been discussed in the context of multiple generic models. The main robust claim from most such models is the negative relation between complexity and the accumulation rate of mutations. Viruses accumulate escape mutations in their epitopes to avoid detection and destruction of their host cell by CD8+ T cells. The extreme regime of immune escape, namely, strong selection and high mutation rate, provide an opportunity to extend and validate the existing models of relation between complexity and evolution rate as proposed by Fisher and Kimura. Using epitope prediction algorithms to compute the epitopes presented on the most frequent human HLA alleles in over 100 fully sequenced human viruses, and over 900 non-human viruses, we here study the correlation between viruses/proteins complexity (as measured by the number of proteins in the virus and the length of each protein, respectively) and the rate of accumulation of escape mutation. The latter is evaluated by measuring the normalized epitope density of viral proteins. If the virus/protein complexity prevents the accumulation of escape mutations, the epitope density is expected to be positively correlated with both the number of proteins in the virus and the length of proteins. We show that such correlations are indeed observed for most human viruses. For non-human viruses the correlations were much less significant, indicating that the correlation is indeed induced by human HLA molecules., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Predictive tools for all levels of CD8+ T cell epitopes processing have reached a maturation level. Good prediction algorithms have been developed for proteasomal cleavage, TAP and MHC class I peptide binding. The same cannot be said of CD4+ T cell epitopes. While multiple algorithms of varying accuracy have been proposed for MHC class II peptide binding, the preprocessing of CD4+ T cell epitopes is still lacking a good prediction algorithm. CD4+ T cell epitopes generation includes several stages, not all which are well-defined. We here group these stages to produce a generic preprocessing stage predictor for the cleavage processes preceding the presentation of epitopes to CD4+ T cell. The predictor is learnt using a combination of in vitro cleavage experiments and observed naturally processed MHC class II binding peptides. The properties of the predictor highlight the effect of different factors on CD4+ T cell epitopes preprocessing. The most important factor emerging from the predictor is the secondary structure of the cleaved region in the protein. The effect of the secondary structure is expected since CD4+ T cell epitopes are not denatured before cleavage. A website developed based on this predictor is available at: http://peptibase.cs.biu.ac.il/PepCleave&#95;cd4/., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Rapidly mutating viruses such as the hepatitis C virus (HCV), the human immunodeficiency virus (HIV), or influenza viruses (Flu) call for highly effective universal peptide vaccines, i.e. vaccines that do not only yield broad population coverage but also broad coverage of various viral strains. The efficacy of such vaccines is determined by multiple properties of the epitopes they comprise. Beyond the specific properties of each epitope, properties of the corresponding source antigens are of great importance. If a response is mounted against viral proteins with a low copy number within the cell or against proteins expressed very late, this response may fail to induce lysis of the infected cells before budding can take place. We here propose a novel methodology to optimize the epitope composition and assembly in order to induce maximum protection. In order for a peptide vaccine to yield the best possible universal protection, several conditions should be met: (a) an optimal choice of target antigens, (b) an optimal choice of highly conserved epitopes, (c) maximum coverage of the target population, and (d) the proper ordering of the epitopes in the final vaccine to ensure favorable cleavage. We propose a mathematical formalism for epitope selection and ordering that balances the constraints imposed by these different conditions. Focusing on HCV, HIV, and Flu, we show that not all of the conditions can be satisfied for all viruses. Depending on the virus, different constraints are harder to fulfill: for Flu, the conservation constraint is violated first, while for HIV, it is difficult to focus the response at the optimal target antigens. The proposed methodology can be applied to any virus to assess the feasibility of optimally combining the above-mentioned constraints., (Copyright © 2011. Published by Elsevier Ltd.)

The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested.

Viruses employ various means to evade immune detection. Reduction of CD8(+) T cell epitopes is one of the common strategies used for this purpose. Hepatitis B virus (HBV), a member of the Hepadnaviridae family, has four open reading frames, with about 50% overlap between the genes they encode. We computed the CD8(+) T cell epitope density within HBV proteins and the mutations within the epitopes. Our results suggest that HBV accumulates escape mutations that reduce the number of epitopes. These mutations are not equally distributed among genes and reading frames. While the highly expressed core and X proteins are selected to have low epitope density, polymerase, which is expressed at low levels, does not undergo the same selection. In overlapping regions, mutations in one protein-coding sequence also affect the other protein-coding sequence. We show that mutations lead to the removal of epitopes in X and surface proteins even at the expense of the addition of epitopes in polymerase. The total escape mutation rate for overlapping regions is lower than that for nonoverlapping regions. The lower epitope replacement rate for overlapping regions slows the evolutionary escape rate of these regions but leads to the accumulation of mutations more robust in the transfer between hosts, such as mutations preventing proteasomal cleavage into epitopes.

Existing Clinical Decision Support Systems (CDSSs) typically rely on rule-based algorithms and focus on tasks like guidelines adherence and drug prescribing and monitoring. However, the increasing dominance of Electronic Health Record technologies and personalized medicine suggest great potential for prognostic data-driven CDSS. A major goal for such systems would be to accurately predict the outcome of patients' candidate treatments by statistical analysis of the clinical data stored at a Health Care Organization. We formally define the concepts involved in the development of such a system, highlight an inherent difficulty arising from bias in treatment allocation, and propose a general strategy to address this difficulty. Experiments over hypertension clinical data demonstrate the validity of our approach.

One of the challenges of healthcare data processing, analysis and warehousing is the integration of data gathered from disparate and diverse data sources. Promoting the adoption of worldwide accepted information standards along with common terminologies and the use of technologies derived from semantic web representation, is a suitable path to achieve that. To that end, the HL7 V3 Reference Information Model (RIM) [1] has been used as the underlying information model coupled with the Web Ontology Language (OWL) [2] as the semantic data integration technology. In this paper we depict a biomedical data integration process and demonstrate how it was used for integrating various data sources, containing clinical, environmental and genomic data, within Hypergenes, a European Commission funded project exploring the Essential Hypertension [3] disease model.