Genome-wide Identification of Structure-Forming Repeats as Principal Sites of Fork Collapse upon ATR Inhibition.

DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods: replication protein A (RPA)-chromatin immunoprecipitation (ChIP) and breaks identified by TdT labeling (BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these distinct categories of repeats differed in the structures they formed and their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA-structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.

Overexpression of peroxisome proliferator-activated receptor γ co-activator-1⍺ (PGC-1⍺) in Chinese hamster ovary cells increases oxidative metabolism and IgG productivity.

Chinese hamster ovary (CHO) cells are used extensively to produce protein therapeutics, such as monoclonal antibodies (mAbs), in the biopharmaceutical industry. MAbs are large proteins that are energetically demanding to synthesize and secrete; therefore, high-producing CHO cell lines that are engineered for maximum metabolic efficiency are needed to meet increasing demands for mAb production. Previous studies have identified that high-producing cell lines possess a distinct metabolic phenotype when compared to low-producing cell lines. In particular, it was found that high mAb production is correlated to lactate consumption and elevated TCA cycle flux. We hypothesized that enhancing flux through the mitochondrial TCA cycle and oxidative phosphorylation would lead to increased mAb productivities and final titers. To test this hypothesis, we overexpressed peroxisome proliferator-activated receptor γ co-activator-1⍺ (PGC-1⍺), a gene that promotes mitochondrial metabolism, in an IgG-producing parental CHO cell line. Stable cell pools overexpressing PGC-1⍺ exhibited increased oxygen consumption, indicating increased mitochondrial metabolism, as well as increased mAb specific productivity compared to the parental line. We also performed 13C metabolic flux analysis (MFA) to quantify how PGC-1⍺ overexpression alters intracellular metabolic fluxes, revealing not only increased TCA cycle flux, but global upregulation of cellular metabolic activity. This study demonstrates the potential of rationally engineering the metabolism of industrial cell lines to improve overall mAb productivity and to increase the abundance of high-producing clones in stable cell pools.

Attenuation of glutamine synthetase selection marker improves product titer and reduces glutamine overflow in Chinese hamster ovary cells.

The glutamine synthetase (GS) expression system is commonly used to ensure stable transgene integration and amplification in Chinese hamster ovary (CHO) host lines. Transfected cell populations are typically grown in the presence of the GS inhibitor, methionine sulfoximine (MSX), to further select for increased transgene copy number. However, high levels of GS activity produce excess glutamine. We hypothesized that attenuating the GS promoter while keeping the strong IgG promoter on the GS-IgG expression vector would result in a more efficient cellular metabolic phenotype. Herein, we characterized CHO cell lines expressing GS from either an attenuated promoter or an SV40 promoter and selected with/without MSX. CHO cells with the attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to cells with SV40-driven GS expression. Selection with MSX increased both specific productivity and glutamine production, regardless of GS promoter strength. 13 C metabolic flux analysis (MFA) was performed to further assess metabolic differences between these cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate and glutamine varied depending on promoter strength and selection conditions. This study highlights the ability to optimize the GS expression system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central metabolism. Additionally, a detailed supplementary analysis of two "lactate runaway" reactors provides insight into the poorly understood phenomenon of excess lactate production by some CHO cell cultures.

Application of 13C flux analysis to identify high-productivity CHO metabolic phenotypes.

Industrial bioprocesses place high demands on the energy metabolism of host cells to meet biosynthetic requirements for maximal protein expression. Identifying metabolic phenotypes that promote high expression is therefore a major goal of the biotech industry. We conducted a series of 13C flux analysis studies to examine the metabolic response to IgG expression during early stationary phase of CHO cell cultures grown in 3L fed-batch bioreactors. We examined eight clones expressing four different IgGs and compared with three non-expressing host-cell controls. Some clones were genetically manipulated to be apoptosis-resistant by expressing Bcl-2Δ, which correlated with increased IgG production and elevated glucose metabolism. The metabolic phenotypes of the non-expressing, IgG-expressing, and Bcl-2Δ/IgG-expressing clones were fully segregated by hierarchical clustering analysis. Lactate consumption and citric acid cycle fluxes were most strongly associated with specific IgG productivity. These studies indicate that enhanced oxidative metabolism is a characteristic of high-producing CHO cell lines.

The impact of anti-apoptotic gene Bcl-2∆ expression on CHO central metabolism.

Anti-apoptosis engineering is an established technique to prolong the viability of mammalian cell cultures used for industrial production of recombinant proteins. However, the effect of overexpressing anti-apoptotic proteins on central carbon metabolism has not been systematically studied. We transfected CHO-S cells to express Bcl-2∆, an engineered anti-apoptotic gene, and selected clones that differed in their Bcl-2∆ expression and caspase activity. (13)C metabolic flux analysis (MFA) was then applied to elucidate the metabolic alterations induced by Bcl-2∆. Expression of Bcl-2Δ reduced lactate accumulation by redirecting the fate of intracellular pyruvate toward mitochondrial oxidation during the lactate-producing phase, and it significantly increased lactate re-uptake during the lactate-consuming phase. This flux redistribution was associated with significant increases in biomass yield, peak viable cell density (VCD), and integrated VCD. Additionally, Bcl-2∆ expression was associated with significant increases in isocitrate dehydrogenase and NADH oxidase activities, both rate-controlling mitochondrial enzymes. This is the first comprehensive (13)C MFA study to demonstrate that expression of anti-apoptotic genes has a significant impact on intracellular metabolic fluxes, especially in controlling the fate of pyruvate carbon, which has important biotechnology applications for reducing lactate accumulation and enhancing productivity in mammalian cell cultures.

Toward Alleviating Clinician Documentation Burden: A Scoping Review of Burden Reduction Efforts.

BACKGROUND: Studies have shown that documentation burden experienced by clinicians may lead to less direct patient care, increased errors, and job dissatisfaction. Implementing effective strategies within health care systems to mitigate documentation burden can result in improved clinician satisfaction and more time spent with patients. However, there is a gap in the literature regarding evidence-based interventions to reduce documentation burden. OBJECTIVES: The objective of this review was to identify and comprehensively summarize the state of the science related to documentation burden reduction efforts. METHODS: Following Joanna Briggs Institute Manual for Evidence Synthesis and Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines, we conducted a comprehensive search of multiple databases, including PubMed, Medline, Embase, CINAHL Complete, Scopus, and Web of Science. Additionally, we searched gray literature and used Google Scholar to ensure a thorough review. Two reviewers independently screened titles and abstracts, followed by full-text review, with a third reviewer resolving any discrepancies. Data extraction was performed and a table of evidence was created. RESULTS: A total of 34 articles were included in the review, published between 2016 and 2022, with a majority focusing on the United States. The efforts described can be categorized into medical scribes, workflow improvements, educational interventions, user-driven approaches, technology-based solutions, combination approaches, and other strategies. The outcomes of these efforts often resulted in improvements in documentation time, workflow efficiency, provider satisfaction, and patient interactions. CONCLUSION: This scoping review provides a comprehensive summary of health system documentation burden reduction efforts. The positive outcomes reported in the literature emphasize the potential effectiveness of these efforts. However, more research is needed to identify universally applicable best practices, and considerations should be given to the transfer of burden among members of the health care team, quality of education, clinician involvement, and evaluation methods.

A Common Longitudinal Intensive Care Unit data Format (CLIF) to enable multi-institutional federated critical illness research.

Background: Critical illness, or acute organ failure requiring life support, threatens over five million American lives annually. Electronic health record (EHR) data are a source of granular information that could generate crucial insights into the nature and optimal treatment of critical illness. However, data management, security, and standardization are barriers to large-scale critical illness EHR studies. Methods: A consortium of critical care physicians and data scientists from eight US healthcare systems developed the Common Longitudinal Intensive Care Unit (ICU) data Format (CLIF), an open-source database format that harmonizes a minimum set of ICU Data Elements for use in critical illness research. We created a pipeline to process adult ICU EHR data at each site. After development and iteration, we conducted two proof-of-concept studies with a federated research architecture: 1) an external validation of an in-hospital mortality prediction model for critically ill patients and 2) an assessment of 72-hour temperature trajectories and their association with mechanical ventilation and in-hospital mortality using group-based trajectory models. Results: We converted longitudinal data from 94,356 critically ill patients treated in 2020-2021 (mean age 60.6 years [standard deviation 17.2], 30% Black, 7% Hispanic, 45% female) across 8 health systems and 33 hospitals into the CLIF format, The in-hospital mortality prediction model performed well in the health system where it was derived (0.81 AUC, 0.06 Brier score). Performance across CLIF consortium sites varied (AUCs: 0.74-0.83, Brier scores: 0.06-0.01), and demonstrated some degradation in predictive capability. Temperature trajectories were similar across health systems. Hypothermic and hyperthermic-slow-resolver patients consistently had the highest mortality. Conclusions: CLIF facilitates efficient, rigorous, and reproducible critical care research. Our federated case studies showcase CLIF's potential for disease sub-phenotyping and clinical decision-support evaluation. Future applications include pragmatic EHR-based trials, target trial emulations, foundational multi-modal AI models of critical illness, and real-time critical care quality dashboards.

"The Terrors of Kingly Power": The Unusual Dental Pathology of King Pyrrhus of Epirus.

Pyrrhus of Epirus, widely respected and feared by his contemporaries, was a legendary figure in the ancient world. In this paper, we investigate Plutarch's description of the king's unique dental pathology. There are several possibilities to explain the ancient king's presentation, including several different types of developmental dysplasia. However, our conclusion is that it was likely due to a significant dental calculus overgrowth, often seen in the ancient Greek diet of the time. Whatever the underlying cause, Pyrrhus' intimidating visage helped secure the king a legacy that lasts to this day.

The analysis of glycosylation: a continued need for high pH anion exchange chromatography.

An appreciation of the structures of the oligosaccharide chains which become attached to biomolecules (the process known as glycosylation), and their relevance to the biological function of the molecule concerned, has progressed rapidly in recent years with developments in site-selective protein glycosylation, oligosaccharide synthesis and in vivo targeting of oligosaccharides. These developments have necessitated the parallel development of effective analytical tools for the determination of the structures of glycosylation. The conclusion of studies in the 1980s and 1990s was that high pH anion exchange chromatography (HPAEC) was the most effective HPLC mode for the analysis of glycosylation. It allowed the fractionation of complex mixtures of monosaccharides or oligosaccharides, the latter in terms of charge, size, composition, anomerity and intra-chain linkages. This review reinvestigates whether HPAEC still appears to offer the most effective means of analysing glycosylation.

Predicting favorable landing pads for targeted integrations in Chinese hamster ovary cell lines by learning stability characteristics from random transgene integrations.

Chinese Hamster Ovary (CHO) cell lines are considered to be the preferred platform for the production of biotherapeutics, but issues related to expression instability remain unresolved. In this study, we investigated potential causes for an unstable phenotype by comparing cell lines that express stably to such that undergo loss in titer across 10 passages. Factors related to transgene integrity and copy number as well as the genomic profile around the integration sites were analyzed. Horizon Discovery CHO-K1 (HD-BIOP3) derived production cell lines selected for phenotypes with low, medium or high copy number, each with stable and unstable transgene expression, were sequenced to capture changes at genomic and transcriptomic levels. The exact sites of the random integration events in each cell line were also identified, followed by profiling of the genomic, transcriptomic and epigenetic patterns around them. Based on the information deduced from these random integration events, genomic loci that potentially favor reliable and stable transgene expression were reported for use as targeted transgene integration sites. By comparing stable vs unstable phenotypes across these parameters, we could establish that expression stability may be controlled at three levels: 1) Good choice of integration site, 2) Ensuring integrity of transgene and observing concatemerization pattern after integration, and 3) Checking for potential stress related cellular processes. Genome wide favorable and unfavorable genomic loci for targeted transgene integration can be browsed at https://www.borthlabchoresources.boku.ac.at/.

Laparoscopic ureteral reimplantation: technique and outcomes.

PURPOSE: We describe our experience with laparoscopic ureteral reimplantation in 45 adults, and report success rates and complications at intermediate term followup. MATERIALS AND METHODS: We performed a retrospective chart review of 45 patients who underwent laparoscopic ureteral reimplantation between 1997 and 2007. Demographics, clinicopathological parameters, perioperative course, complications and followup studies were analyzed. RESULTS: Elective laparoscopic ureteral reimplantation was performed in 35 female and 10 male patients with a mean followup of 24.1 months (range 1 to 76). All patients presented with distal ureteral stricture with a mean stricture length of 3 cm and a mean +/- SD preoperative serum creatinine of 0.91 +/- 0.04 mg/dl. Mean patient age was 47.8 +/- 2.2 years (range 17 to 87). Mean American Society of Anesthesiologists score was 2 (range 1 to 3). Median estimated blood loss was 150 ml. The overall success rate, defined as radiographic evidence of no residual obstruction, symptoms, renal deterioration or need for subsequent procedures, was 96%. Two patients had recurrent strictures and 1 underwent nephrectomy for flank pain and preexisting chronic pyelonephritis. CONCLUSIONS: According to intermediate followup data laparoscopic ureteral reimplantation can be performed with an excellent success rate and low morbidity. Our data substantiate this technique as an effective method for managing distal ureteral stricture.

Blood smear from a pregnant cat that died shortly after partial abortion.

A 1-year-old, 5- to 6-week pregnant cat was presented with a history of aborting 3 kittens the previous night. During the examination, the cat began to seizure and died. At necropsy, formalin-fixed tissues and blood smears prepared from an EDTA blood sample collected via cardiac puncture were submitted to a diagnostic laboratory. Examination of the blood smears revealed numerous large (15-75 microm) clumped macrophages containing dark blue intracytoplasmic organisms consistent with Cytauxzoon merozoites. Scattered erythrophagocytic macrophages were also observed. Within several erythrocytes, 1-2 small, round, ring-like piroplasms consistent with Cytauxzoon were observed. Histologic examination revealed numerous large, schizont-laden macrophages filling the blood vessels of multiple organs. The cytologic and histologic findings were diagnostic for Cytaxuzoon felis infection. This case was of particular interest because of the unusually large number of organism-laden macrophages in the cardiac blood sample, an uncommon finding in peripheral blood. Although the cat was presented for complications of abortion, it remains uncertain whether C felis organisms crossed the placenta and infected the fetuses or lead to the abortions.

A Conservatively Managed Anatomical Variant of the Flexor Digitorum Superficialis Muscle in the Hand.

Anomalous flexor digitorum superficialis muscles in the hand are an uncommon phenomenon, and therefore present challenges in diagnosis and management. We report a case of a 16-year-old girl presenting with a painful, slowly enlarging palmar swelling. The swelling was investigated with ultrasound and magnetic resonance imaging, and was found to be an anomalous muscle belly of the flexor digitorum superficialis muscle. After careful consideration, multidisciplinary discussion, and thorough imaging, the patient was treated successfully without surgical exploration or excision, in comparison to previously reported cases. The patient was pain free and had no concerns at 8-month follow-up, demonstrating the value of conservative management in these cases.

The glycosylation of AGP and its associations with the binding to methadone.

Methadone remains the most common form of pharmacological therapy for opioid dependence; however, there is a lack of explanation for the reports of its relatively low success rate in achieving complete abstinence. One hypothesis is that in vivo binding of methadone to the plasma glycoprotein alpha-1-acid glycoprotein (AGP), to a degree dependent on the molecular structure, may render the drug inactive. This study sought to determine whether alterations present in the glycosylation pattern of AGP in patients undergoing various stages of methadone therapy (titration < two weeks, harm reduction < one year, long-term > one and a half years) could affect the affinity of the glycoprotein to bind methadone. The composition of AGP glycosylation was determined using high pH anion exchange chromatography (HPAEC) and intrinsic fluorescence analysed to determine the extent of binding to methadone. The monosaccharides galactose and N-acetyl-glucosamine were elevated in all methadone treatment groups indicating alterations in AGP glycosylation. AGP from all patients receiving methadone therapy exhibited a greater degree of binding than the normal population. This suggests that analysing the glycosylation of AGP in patients receiving methadone may aid in determining whether the therapy is likely to be effective.

Combining ATR suppression with oncogenic Ras synergistically increases genomic instability, causing synthetic lethality or tumorigenesis in a dosage-dependent manner.

Previous studies indicate that oncogenic stress activates the ATR-Chk1 pathway. Here, we show that ATR-Chk1 pathway engagement is essential for limiting genomic instability following oncogenic Ras transformation. ATR pathway inhibition in combination with oncogenic Ras expression synergistically increased genomic instability, as quantified by chromatid breaks, sister chromatid exchanges, and H2AX phosphorylation. This level of instability was significantly greater than that observed following ATR suppression in untransformed control cells. In addition, consistent with a deficiency in long-term genome maintenance, hypomorphic ATR pathway reduction to 16% of normal levels was synthetic lethal with oncogenic Ras expression in cultured cells. Notably, elevated genomic instability and synthetic lethality following suppression of ATR were not due to accelerated cycling rates in Ras-transformed cells, indicating that these synergistic effects were generated on a per-cell-cycle basis. In contrast to the synthetic lethal effects of hypomorphic ATR suppression, subtle reduction of ATR expression (haploinsufficiency) in combination with endogenous levels of K-ras(G12D) expression elevated the incidence of lung adenocarcinoma, spindle cell sarcoma, and thymic lymphoma in p53 heterozygous mice. K-ras(G12D)-induced tumorigenesis in ATR(+/-)p53(+/-) mice was associated with intrachromosomal deletions and loss of wild-type p53. These findings indicate that synergistic increases in genomic instability following ATR reduction in oncogenic Ras-transformed cells can produce 2 distinct biological outcomes: synthetic lethality upon significant suppression of ATR expression and tumor promotion in the context of ATR haploinsufficiency. These results highlight the importance of the ATR pathway both as a barrier to malignant progression and as a potential target for cancer treatment.

Tim-Tipin dysfunction creates an indispensible reliance on the ATR-Chk1 pathway for continued DNA synthesis.

The Tim (Timeless)-Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim-Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR-Chk1 pathway engagement. We now demonstrate that Tim-Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR-Chk1 pathway in Tim-Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim-Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim-Tipin dysfunction. Together, these experiments indicate that the Tim-Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR-Chk1 function and by facilitating phosphorylation of Chk1 by ATR.

Timeless Maintains Genomic Stability and Suppresses Sister Chromatid Exchange during Unperturbed DNA Replication.

Genome integrity is maintained during DNA replication by coordination of various replisome-regulated processes. Although it is known that Timeless (Tim) is a replisome component that participates in replication checkpoint responses to genotoxic stress, its importance for genome maintenance during normal DNA synthesis has not been reported. Here we demonstrate that Tim reduction leads to genomic instability during unperturbed DNA replication, culminating in increased chromatid breaks and translocations (triradials, quadriradials, and fusions). Tim deficiency led to increased H2AX phosphorylation and Rad51 and Rad52 foci formation selectively during DNA synthesis and caused a 3-4-fold increase in sister chromatid exchange. The sister chromatid exchange events stimulated by Tim reduction were largely mediated via a Brca2/Rad51-dependent mechanism and were additively increased by deletion of the Blm helicase. Therefore, Tim deficiency leads to an increased reliance on homologous recombination for proper continuation of DNA synthesis. Together, these results indicate a pivotal role for Tim in maintaining genome stability throughout normal DNA replication.

The efficacy of certain anti-tuberculosis drugs is affected by binding to alpha-1-acid glycoprotein.

One of the most ubiquitous plasma proteins, alpha-1-acid glycoprotein (AGP), has a high affinity, low capacity binding for basic drugs positively charged at physiological pH. Moreover, as an acute phase protein its level is increased in various disease states in a manner that is likely to influence the free plasma level of a drug, the ability to attain minimum effective concentration and overall in vivo effectiveness. AGP is a glycoprotein known to display disease specific changes in glycosylation and although this secondary modification is not directly involved in drug binding, it may influence the conformation of the binding site. Binding studies reveal that alpha-1-acid glycoprotein bind mainly to the tuberculosis drugs: rifampicin; isoniazid; pyrazinamide; p-aminosalicylic acid; capreomycin; ethionamide; levofloxacin and ofloxacin out with the therapeutic plasma range tested. These results are however still considered significant as not only are alpha-1-acid glycoprotein levels increased during the acute phase response but specific alpha-1-acid glycoprotein from tuberculosis samples are subject to glycosylation changes which can increase the binding affinity and cause binding to occur at the therapeutic concentration.

Analysis of the interaction between alpha-1-acid glycoprotein and tamoxifen and its metabolites.

Tamoxifen is administered for the treatment of breast cancer; however resistance to therapy is commonplace. Postulated mechanisms of resistance to tamoxifen include altered pharmacology of the drug, changes in the structure and function of the oestrogen receptor and expression of genes that function to support the growth of cells resistant to tamoxifen. However, binding of drugs to proteins found in the plasma is known to affect the efficacy of drugs and alter their distribution. It is already known that tamoxifen is bound 99% to albumin. We investigated the interaction between the plasma protein, alpha-1-acid glycoprotein (AGP), and tamoxifen, since if binding did occur then the free plasma concentration of the drug would be reduced, resulting in the minimum effective concentration of tamoxifen not being attained. Using a recently described intrinsic fluorescence technique for the study of drug-protein interactions, the extent of binding between tamoxifen citrate and AGP was determined. Furthermore, analysis of binding of the known active metabolites of tamoxifen (4-hydroxytamoxifen, N-desmethyltamoxifen, N-desdimethyltamoxifen, cis-alpha-hydroxytamoxifen and trans-alpha-hydroxytamoxifen) to AGP was conducted. Tamoxifen citrate and metabolites were shown to bind AGP, however the level of interaction was low and negligible at the concentration of the drug found in the plasma.