DNA Damage, Genome Stability, and Adaptation: A Question of Chance or Necessity?

DNA damage causes the mutations that are the principal source of genetic variation. DNA damage detection and repair mechanisms therefore play a determining role in generating the genetic diversity on which natural selection acts. Speciation, it is commonly assumed, occurs at a rate set by the level of standing allelic diversity in a population. The process of speciation is driven by a combination of two evolutionary forces: genetic drift and ecological selection. Genetic drift takes place under the conditions of relaxed selection, and results in a balance between the rates of mutation and the rates of genetic substitution. These two processes, drift and selection, are necessarily mediated by a variety of mechanisms guaranteeing genome stability in any given species. One of the outstanding questions in evolutionary biology concerns the origin of the widely varying phylogenetic distribution of biodiversity across the Tree of Life and how the forces of drift and selection contribute to shaping that distribution. The following examines some of the molecular mechanisms underlying genome stability and the adaptive radiations that are associated with biodiversity and the widely varying species richness and evenness in the different eukaryotic lineages.

Delayed Onset of Symptoms After a Rattlesnake Bite in a Renal Transplant Patient: A Case Report.

Introduction: The United States is home to two major families of venomous snakes, Crotalids and Elapids. The Crotalid family, also known as pit vipers, is well known for being among the most frequent causes of snakebites reported. Crotalid envenomation can present with local findings, hematologic toxicity, and systemic toxicity. Identification of envenomated patients is key to determining who needs antivenom. Most sources recommend an observation period of six to eight hours after the snakebite to determine whether the bite was "dry" or the patient was exposed to venom. Case Report: We present the case of a 33-year-old patient with a history of renal transplantation who had delayed onset of symptoms of envenomation 18 hours after an initial emergency department observation. The patient did well after a course of antivenom and was discharged on hospital day three. Conclusion: The patient's immunosuppressive regimen may have delayed the onset of clinical symptoms, thus delaying treatment. To the best of our knowledge, this is the first case reported of a patient presenting with a delayed onset of initial snakebite envenomation symptoms.

Open Questions about the Roles of DnaA, Related Proteins, and Hyperstructure Dynamics in the Cell Cycle.

The DnaA protein has long been considered to play the key role in the initiation of chromosome replication in modern bacteria. Many questions about this role, however, remain unanswered. Here, we raise these questions within a framework based on the dynamics of hyperstructures, alias large assemblies of molecules and macromolecules that perform a function. In these dynamics, hyperstructures can (1) emit and receive signals or (2) fuse and separate from one another. We ask whether the DnaA-based initiation hyperstructure acts as a logic gate receiving information from the membrane, the chromosome, and metabolism to trigger replication; we try to phrase some of these questions in terms of DNA supercoiling, strand opening, glycolytic enzymes, SeqA, ribonucleotide reductase, the macromolecular synthesis operon, post-translational modifications, and metabolic pools. Finally, we ask whether, underpinning the regulation of the cell cycle, there is a physico-chemical clock inherited from the first protocells, and whether this clock emits a single signal that triggers both chromosome replication and cell division.

Kimura's Theory of Non-Adaptive Radiation and Peto's Paradox: A Missing Link?

Karyotype diversity reflects genome integrity and stability. A strong correlation between karyotype diversity and species richness, meaning the number of species in a phylogenetic clade, was first reported in mammals over forty years ago: in mammalian phylogenetic clades, the standard deviation of karyotype diversity (KD) closely corresponded to species richness (SR) at the order level. These initial studies, however, did not control for phylogenetic signal, raising the possibility that the correlation was due to phylogenetic relatedness among species in a clade. Accordingly, karyotype diversity trivially reflects species richness simply as a passive consequence of adaptive radiation. A more recent study in mammals controlled for phylogenetic signals and established the correlation as phylogenetically independent, suggesting that species richness cannot, in itself, explain the observed corresponding karyotype diversity. The correlation is, therefore, remarkable because the molecular mechanisms contributing to karyotype diversity are evolutionarily independent of the ecological mechanisms contributing to species richness. Recently, it was shown in salamanders that the two processes generating genome size diversity and species richness were indeed independent and operate in parallel, suggesting a potential non-adaptive, non-causal but biologically meaningful relationship. KD depends on mutational input generating genetic diversity and reflects genome stability, whereas species richness depends on ecological factors and reflects natural selection acting on phenotypic diversity. As mutation and selection operate independently and involve separate and unrelated evolutionary mechanisms-there is no reason a priori to expect such a strong, let alone any, correlation between KD and SR. That such a correlation exists is more consistent with Kimura's theory of non-adaptive radiation than with ecologically based adaptive theories of macro-evolution, which are not excluded in Kimura's non-adaptive theory. The following reviews recent evidence in support of Kimura's proposal, and other findings that contribute to a wider understanding of the molecular mechanisms underlying the process of non-adaptive radiation.

Cytotoxicity of the CD3×CD20 bispecific antibody epcoritamab in CLL is increased by concurrent BTK or BCL-2 targeting.

Chronic lymphocytic leukemia (CLL) is an immunosuppressive disease characterized by increased infectious morbidity and inferior antitumor activity of immunotherapies. Targeted therapy with Bruton's tyrosine kinase inhibitors (BTKis) or the Bcl-2 inhibitor venetoclax has profoundly improved treatment outcomes in CLL. To overcome or prevent drug resistance and extend the duration of response after a time-limited therapy, combination regimens are tested. Anti-CD20 antibodies that recruit cell- and complement-mediated effector functions are commonly used. Epcoritamab (GEN3013), an anti-CD3×CD20 bispecific antibody that recruits T-cell effector functions, has demonstrated potent clinical activity in patients with relapsed CD20+ B-cell non-Hodgkin lymphoma. Development of CLL therapy is ongoing. To characterize epcoritamab-mediated cytotoxicity against primary CLL cells, peripheral blood mononuclear cells from treatment-naive and BTKi-treated patients, including patients progressing on therapy, were cultured with epcoritamab alone or in combination with venetoclax. Ongoing treatment with BTKi and high effector-to-target ratios were associated with superior in vitro cytotoxicity. Cytotoxic activity was independent of CD20 expression on CLL cells and observed in samples from patients whose condition progressed while receiving BTKi. Epcoritamab induced significant T-cell expansion, activation, and differentiation into Th1 and effector memory cells in all patient samples. In patient-derived xenografts, epcoritamab reduced the blood and spleen disease burden compared with that in mice receiving a nontargeting control. In vitro, the combination of venetoclax with epcoritamab induced superior killing of CLL cells than either agent alone. These data support the investigation of epcoritamab in combination with BTKis or venetoclax to consolidate responses and target emergent drug-resistant subclones.

Patient-derived Siglec-6-targeting antibodies engineered for T-cell recruitment have potential therapeutic utility in chronic lymphocytic leukemia.

BACKGROUND: Despite numerous therapeutic options, safe and curative therapy is unavailable for most patients with chronic lymphocytic leukemia (CLL). A drawback of current therapies such as the anti-CD20 monoclonal antibody (mAb) rituximab is the elimination of all healthy B cells, resulting in impaired humoral immunity. We previously reported the identification of a patient-derived, CLL-binding mAb, JML-1, and identified sialic acid-binding immunoglobulin-like lectin-6 (Siglec-6) as the target of JML-1. Although little is known about Siglec-6, it appears to be an attractive target for cancer immunotherapy due to its absence on most healthy cells and tissues. METHODS: We used a target-specific approach to mine for additional patient-derived anti-Siglec-6 mAbs. To assess the therapeutic utility of targeting Siglec-6 in the context of CLL, T cell-recruiting bispecific antibodies (T-biAbs) that bind to Siglec-6 and CD3 were engineered into single-chain variable fragment-Fc and dual-affinity retargeting (DART)-Fc constructs. T-biAbs were evaluated for their activity in vitro, ex vivo, and in vivo. RESULTS: We discovered the anti-Siglec-6 mAbs RC-1 and RC-2, which bind with higher affinity than JML-1 yet maintain similar specificity. Both JML-1 and RC-1 T-biAbs were effective at activating T cells and killing Siglec-6+ target cells. The RC-1 clone in the DART-Fc format was the most potent T-biAb tested and was the only anti-Siglec-6 T-biAb that eliminated Siglec-6+ primary CLL cells via autologous T cells at pathological T-to-CLL cell ratios. Tested at healthy T-to-B cell ratios, it also eliminated a Siglec-6+ fraction of primary B cells from healthy donors. The subpicomolar potency of the DART-Fc format was attributed to the reduction in the length and flexibility of the cytolytic synapse. Furthermore, the RC-1 T-biAb was effective at clearing MEC1 CLL cells in vivo and demonstrated a circulatory half-life of over 7 days. CONCLUSION: Siglec-6-targeting T-biAbs are highly potent and specific for eliminating Siglec-6+ leukemic and healthy B cells while sparing Siglec-6- healthy B cells, suggesting a unique treatment strategy for CLL with diminished suppression of humoral immunity. Our data corroborate reports that T-biAb efficacy is dependent on synapse geometry and reveal that synapse architecture can be tuned via antibody engineering. Our fully human anti-Siglec-6 antibodies and T-biAbs have potential for cancer immunotherapy. TRIAL REGISTRATION NUMBER: NCT00923507.

Genome size variation and species diversity in salamanders.

Salamanders (Urodela) have among the largest vertebrate genomes, ranging in size from 10 to 120 pg. Although changes in genome size often occur randomly and in the absence of selection pressure, nonrandom patterns of genome size variation are evident among specific vertebrate lineages. Several reports suggest a relationship between species richness and genome size, but the exact nature of that relationship remains unclear both within and across different taxonomic groups. Here, we report (a) a negative relationship between haploid genome size (C-value) and species richness at the family taxonomic level in salamander clades; (b) a correlation of C-value and species richness with clade crown age but not with diversification rates; (c) strong associations between C-value and both geographic area and climatic-niche rate. Finally, we report a relationship between C-value diversity and species diversity at both the family- and genus-level clades in urodeles.

Exclusion of Non-English Speakers in Published Emergency Medicine Research - A Comparison of 2004 and 2014.

BACKGROUND: Non-English speakers (NES) as a proportion of the United States population have steadily increased in recent years. There remains substantial risk of excluding NES from research. OBJECTIVE: To assess whether the percentage of emergency medicine (EM) studies that exclude Non-English speakers from participation has changed with time. METHODS: In a structured fashion, the lead investigator analyzed all original research articles in Academic Emergency Medicine and Annals of Emergency Medicine retrospectively for 2004 and prospectively for 2014. An independent investigator conducted a blind review of a sample of articles to assess for interobserver agreement. Demographic data were analyzed using descriptive statistics. Chi-square, t-tests, and linear regression models were utilized; alpha set at 0.05. Cohen's kappa calculated to assess interrater reliability. RESULTS: We included a total of 236 original research articles. Overall, 11% excluded NES from research (10% AEM, 12% Annals). Cohen's kappa (nonweighted) was 0.73. Comparing all articles in 2004 vs. 2014, research excluded NES 6% vs. 16% of the time respectively (P=0.02). This was not statistically significant when comparing year to year for AEM (7.3% vs. 14.5%; P=0.12) and Annals (6.7% vs. 19%; P=0.06) separately. Factors affecting NES exclusion included type of study design (P<0.001), geographic area (P=0.009) and hospital type (P=0.035). Interestingly, 42% of articles failed to mention language as an exclusion or inclusion criteria. CONCLUSION: We found that the percentage of articles excluding NES from EM research increased between 2004 and 20014. Further, many investigators do not report whether NES are excluded/included in their studies.

Impact of Starting an Emergency Medicine Residency Program on Overall Mortality Rate in a Regional Trauma Center.

BACKGROUND: CHRISTUS Spohn Hospital Corpus Christi - Memorial began an Emergency Medicine Residency Program in March 2007. During each of the three years of their residency, residents are required to complete a trauma surgery rotation. These emergency medicine residents are the only residents participating on this rotation as there is no surgical residency. The Department of Acute Care Surgery, Trauma and Surgical Critical Care analyzed the impact of the residents on trauma patient care outcomes with the hypothesis that there were no differences. METHODS: Data including length of stay in the hospital, length of stay in the intensive care unit, time spent in the emergency department (ED), morbidities and mortalities were compiled from the trauma registry for patients from the year before the residents began (March 1, 2006 to February 28, 2007) and compared with patients from the first year the residents began their trauma rotations (March 1, 2007 to February 29, 2008). T-tests and Mann-Whitney U tests were used to compare continuous variables and a Chi-square test was used to analyze the categorical variable (mortality). Linear and logistic regression analyses were also performed in order to adjust for potential confounding factors. RESULTS: Trauma patient admission rates were 1,316 before and 1,391 after the residents began. No statistically significant differences were found among all of the outcome variables during the two time periods except for time spent in the ED (P = 0.00), which increased during the year the residents began (236.83 ± 4.53 minutes in 2006 compared to 297.40 ± 5.55 minutes in 2007). Linear and logistic regression analyses confirmed these results with the exception of a statistically significant decrease in mortality with the residents on the trauma service (2.8% in 2006 and 2.1% in 2007, P = 0.00) after adjustment for multiple confounding factors. CONCLUSION: The addition of emergency medicine residents to the trauma care service did increase ED length of stay, but did not increase overall hospital or intensive care unit length of stay. There was a statistically significant decrease in adjusted morbidity and mortality, thus supporting our hypothesis that the residency program did not negatively impact the trauma service and its goals of high quality patient care.

Head multidetector computed tomography: emergency medicine physicians overestimate the pretest probability and legal risk of significant findings.

OBJECTIVES: This study focuses on clinically assigned prospective estimated pretest probability and pretest perception of legal risk as independent variables in the ordering of multidetector computed tomographic (MDCT) head scans. Our primary aim is to measure the association between pretest probability of a significant finding and pretest perception of legal risk. Secondarily, we measure the percentage of MDCT scans that physicians would not order if there was no legal risk. METHODS: This study is a prospective, cross-sectional, descriptive analysis of patients 18 years and older for whom emergency medicine physicians ordered a head MDCT. RESULTS: We collected a sample of 138 patients subjected to head MDCT scans. The prevalence of a significant finding in our population was 6%, yet the pretest probability expectation of a significant finding was 33%. The legal risk presumed was even more dramatic at 54%. These data support the hypothesis that physicians presume the legal risk to be significantly higher than the risk of a significant finding. A total of 21% or 15% patients (95% confidence interval, ±5.9%) would not have been subjected to MDCT if there was no legal risk. CONCLUSIONS: Physicians overestimated the probability that the computed tomographic scan would yield a significant result and indicated an even greater perceived medicolegal risk if the scan was not obtained. Physician test-ordering behavior is complex, and our study queries pertinent aspects of MDCT testing. The magnification of legal risk vs the pretest probability of a significant finding is demonstrated. Physicians significantly overestimated pretest probability of a significant finding on head MDCT scans and presumed legal risk.

Genetic variation and DNA replication timing, or why is there late replicating DNA?

Mutation rates vary significantly within the genome and across species. Recent studies revealed a long suspected replication-timing effect on mutation rate, but the mechanisms that regulate the increase in mutation rate as the genome is replicated remain unclear. Evidence is emerging, however, that DNA repair systems, in general, are less efficient in late replicating heterochromatic regions compared to early replicating euchromatic regions of the genome. At the same time, mutation rates in both vertebrates and invertebrates have been shown to vary with generation time (GT). GT is correlated with genome size, which suggests a possible nucleotypic effect on species-specific mutation rates. These and other observations all converge on a role for DNA replication checkpoints in modulating generation times and mutation rates during the DNA synthetic phase (S phase) of the cell cycle. The following will examine the potential role of the intra-S checkpoint in regulating cell cycle times (GT) and mutation rates in eukaryotes. This article was published online on August 5, 2011. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected October 4, 2011.

Defects and DNA replication.

We introduce a rate-equation formalism to study DNA replication kinetics in the presence of defects resulting from DNA damage and find a crossover between two regimes: a normal regime, where the influence of defects is local, and an initiation-limited regime. In the latter, defects have a global impact on replication, whose progress is set by the rate at which origins of replication are activated, or initiated. Normal, healthy cells have defect densities in the normal regime. Our model can explain an observed correlation between interorigin separation and rate of DNA replication.

DnaA-ATP acts as a molecular switch to control levels of ribonucleotide reductase expression in Escherichia coli.

Ribonucleotide reductase (RNR) is the bottleneck enzyme in the synthesis of dNTPs required for DNA replication. In order to avoid the mutagenic effects of imbalances in dNTPs the amount and activity of RNR enzyme in the cell is tightly regulated. RNR expression from the nrdAB operon is thus coupled to coincide with the initiation of DNA replication. However, the mechanism for the co-ordination of gene transcription and DNA replication remains to be elucidated. The timing and synchrony of DNA replication initiation in Escherichia coli is controlled in part by the binding of the DnaA protein to the origin of replication. DnaA is also a transcription factor of the nrdAB operon and could thus be the link between these two processes. Here we show that RNA polymerase can form a stable transcription initiation complex at the nrdAB promoter by direct interaction with the far upstream sites required for the timing of expression as a function of DNA replication. In addition, we show that the binding of DnaA on the promoter can either activate or repress transcription as a function of its concentration and its nucleotide-bound state. However, transcription regulation by DnaA does not significantly affect the timing of expression of RNR from the nrdAB operon.

The dynamic replicon: adapting to a changing cellular environment.

Eukaryotic cells are often exposed to fluctuations in growth conditions as well as endogenous and exogenous stress-related agents. During development, global patterns of gene transcription change substantially, and these changes are associated with altered patterns of DNA replication and larger distances between replication origins in somatic cells compared to embryos. Conversely, when cells experience difficulties while replicating DNA, the replication program is dramatically altered and distances between replication origins decrease. Recent evidence indicates that each unit of replication, or replicon, can correspond to one or more potential replication origins, but in the case of multiple potential origins, only one is selected to initiate replication of the replicon. How one origin is selected from multiple potential origins and how origin densities are regulated during genome duplication remains unclear. The following review addresses some of the mechanisms involved in regulating replication origins during both a normal and perturbed eukaryotic cell cycle.

Introduction to molecular combing: genomics, DNA replication, and cancer.

The sequencing of the human genome inaugurated a new era in both fundamental and applied genetics. At the same time, the emergence of new technologies for probing the genome has transformed the field of pharmaco-genetics and made personalized genomic profiling and high-throughput screening of new therapeutic agents all but a matter of routine. One of these technologies, molecular combing, has served to bridge the technical gap between the examination of gross chromosomal abnormalities and sequence-specific alterations. Molecular combing provides a new perspective on the structure and dynamics of the human genome at the whole genome and sub-chromosomal levels with a resolution ranging from a few kilobases up to a megabase and more. Originally developed to study genetic rearrangements and to map genes for positional cloning, recent advances have extended the spectrum of its applications to studying the real-time dynamics of the replication of the genome. Understanding how the genome is replicated is essential for elucidating the mechanisms that both maintain genome integrity and result in the instabilities leading to human genetic disease and cancer. In the following, we will examine recent discoveries and advances due to the application of molecular combing to new areas of research in the fields of molecular cytogenetics and cancer genomics.

Pseudoaneurysm of the radial artery diagnosed by bedside ultrasound.

A 42-year-old male presented to the emergency department with pain and swelling of his distal right wrist. Bedside ultrasound placed over the swelling revealed a pseudoaneurysm of the radial artery. The patient received percutaneous thrombin injection of the aneurysm sac followed by direct ultrasound compression therapy of the pseudoaneurysm neck, resulting in thrombosis of the sac. The use of bedside ultrasound by the emergency physician led to appropriate care and proper disposition for definitive management.

Global regulation of genome duplication in eukaryotes: an overview from the epifluorescence microscope.

In eukaryotes, DNA replication is initiated along each chromosome at multiple sites called replication origins. Locally, each replication origin is "licensed" or specified at the end of the M and the beginning of the G1 phases of the cell cycle. During the S phase when DNA synthesis takes place, origins are activated in stages corresponding to early and late-replicating domains. The staged and progressive activation of replication origins reflects the need to maintain a strict balance between the number of active replication forks and the rate at which DNA synthesis proceeds. This suggests that origin densities (frequency of initiation) and replication fork movement (rates of elongation) must be coregulated to guarantee the efficient and complete duplication of each subchromosomal domain. Emerging evidence supports this proposal and suggests that the ATM/ATR intra-S phase checkpoint plays an important role in the coregulation of initiation frequencies and rates of elongation. In this paper, we review recent results concerning the mechanisms governing the global regulation of DNA replication and discuss the roles these mechanisms play in maintaining genome stability during both a normal and perturbed S phase.

Acute ischemic stroke in a pediatric patient.

Acute ischemic stroke in a pediatric patient is a complex disease with a variety of etiologies that differ from adults. Though rare, they are a real phenomenon with potentially devastating consequences. Some treating institutions are using anti-thrombotic drug therapy with unclear benefits. Available literature, which is limited to case reports and retrospective reviews of databases, clouds this topic with both positive and negative outcomes. Emergency department management should focus on stabilization and resuscitation with immediate involvement of a pediatric neurologist and intensivist. The decision to use anti-thrombotic drug therapy, including anti-platelet drugs and thrombolytics, should be in consult with the specialists involved until randomized controlled trials determine their safety and efficacy in the pediatric population.

Replication fork velocities at adjacent replication origins are coordinately modified during DNA replication in human cells.

The spatial organization of replicons into clusters is believed to be of critical importance for genome duplication in higher eukaryotes, but its functional organization still remains to be fully clarified. The coordinated activation of origins is insufficient on its own to account for a timely completion of genome duplication when interorigin distances vary significantly and fork velocities are constant. Mechanisms coordinating origin distribution with fork progression are still poorly elucidated, because of technical difficulties of visualizing the process. Taking advantage of a single molecule approach, we delineated and compared the DNA replication kinetics at the genome level in human normal primary and malignant cells. Our results show that replication forks moving from one origin, as well as from neighboring origins, tend to exhibit the same velocity, although the plasticity of the replication program allows for their adaptation to variable interorigin distances. We also found that forks that emanated from closely spaced origins tended to move slower than those associated with long replicons. Taken together, our results indicate a functional role for origin clustering in the dynamic regulation of genome duplication.