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Milan D Antonijević,1 Ovidiu Novac,1 Barry MG O’Hagan2 1Faculty of Engineering and Science, University of Greenwich at Medway, Chatham Maritime, ME4 4TB, UK; 2Faculty of Life and Health Sciences, School of Biomedical Sciences, Ulster University, Coleraine, BT52 1SA, UK Introduction: Emollient therapy is the mainstay for treating skin conditions such as atopic dermatitis and psoriasis. New emollients have been introduced recently and are assumed to be therapeutically interchangeable with the innovator products because, superficially, they appear to have similar compositions. This study compares a) the ex vivo human skin occlusion performance and b) the visual and microscopic properties of Isomol gel (IMG) and Doublebase gel (DBG).Materials and Methods: Occlusion was measured gravimetrically by reduction in cumulative 48-hour evaporative weight loss from ex vivo human skin samples following single applications of the two test emollients and Vaseline®. Skin samples from a single donor were mounted in Franz diffusion cells and then the emollients were spread over the skin surface with an applied dose of approximately 2 mg/cm2. The assemblies (four replicates per treatment) were then accurately weighed at baseline (T0) and again after 5-, 24-, and 48-hour postapplication. The quality of the two emollient gel formulations was compared by visual examination of their film-forming characteristics and by microstructural examination using environmental scanning electron microscopy (ESEM). Results: Occlusivity of the DBG emollient gel formulation was comparable with Vaseline and substantially better than IMG, with the DBG-treated skin samples losing less than half as much weight as the IMG-treated skin samples over 48 hours and at a much slower rate during the first 5 hours. The film-forming characteristics and microstructure of the gels were also very different. Whereas DBG maintained a smooth, uniform film over 24 hours, the IMG formulation phase-separated. ESEM results showed that the DBG emulsion has a stable structural matrix with uniform oil droplets, whereas for IMG the emulsion system is inhomogeneous with the oil phase coalescing into larger irregular shaped rafts. Conclusions: We have demonstrated substantial performance differences between two prescribed emollient gels. Keywords: gel, emollient, occlusion, microscopy, comparison, performance

The design and development of new drug delivery systems with the aim of enchancing the efficacy of existing commercialized formulations represent an ongoing process in the pharmaceutical research. Captopril, an angiotensin converting enzyme inhibitor, was successfully loaded into unfunctionalized and functionalized mesoporous silica matrices. Pharmacokinetic evaluations were performed and UV-Vis and HPLC analysis were used in order to determine the plasma concentration of the released captopril. The following pharmacokinetic parameters were evaluated: time to the maximum concentration (Tmax), maximum value of plasma concentration, Cmax (µg/mL), half life (T1/2, h), area under curve AUC0-72 (µg h/mL). Pharmacokinetics experiments evidenced that temperature conditions for drug loading and chemical nature of silica surface are determining factors in the bioavailability of the loaded substance and directly influence the overall pharmacokinetics. Additionally, the toxicity of the obtained systems was evaluated using Kärber method. The tested silica-captopril formulations may be considered attractive controlled release systems, potentially offering better bioavailability and other benefits, compared to the commercial formulation. [ABSTRACT FROM AUTHOR]

Foreign Accent Syndrome (FAS) is a rare and complex speech disorder characterized by the sudden emergence of a foreign accent, typically following neurological events such as strokes, traumatic brain injuries, or neurodegenerative diseases. This comprehensive review explores the pathophysiology, neuroimaging findings, and prevalence of FAS. Neuroimaging studies, including MRI and fMRI, reveal significant brain reorganization predominantly in the left hemisphere, involving regions such as the superior temporal gyrus and medial frontal structures. Functional connectivity disruptions and the phenomenon of dynamic diaschisis are discussed as potential mechanisms. Additionally, the review addresses the psychogenic aspects of FAS, highlighting the role of psychological factors and functional neurological disorders. The high comorbidity of FAS with other conditions underscores the need for a multidisciplinary diagnostic and therapeutic approach. This paper aims to enhance the understanding of FAS and contribute to the development of effective treatment strategies for this unique speech disorder., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s) under exclusive licence to Belgian Neurological Society.)

Assisted suicide and euthanasia are long debated topics in amyotrophic lateral sclerosis (ALS) patients care. We conducted a meta-analysis to evaluate the attitudes of ALS patients and their caregivers toward physician-assisted suicide (PAS) and euthanasia. Also, we were interested to identify the factors associated with the positive or negative attitude of patients and caregivers towards PAS/euthanasia. A thorough search of the online databases (PubMed, Cochrane Library, and Web of Science) was conducted and eligibility criteria according to the PRISMA guidelines were used to include the studies in the current meta-analysis. The assessment of the quality of the selected studies was carried out using a pre-specified set of criteria by Cochrane. The studies that were selected for this meta-analysis suggested that the expression of the wish to die is more likely correlated with depression, anxiety, hopelessness, and lack of optimism. The overall prevalence of considering PAS/euthanasia significantly varies in a dependent manner over the cultural, legal, and societal factors. In this context, we found that the opinion on this topic can be deeply personal and may vary widely among individuals and communities. Lower quality of life and lower religiosity were associated with a positive attitude toward PAS/euthanasia. On the other hand, patients who are more religious are less likely to choose PAS/euthanasia. Gender does not appear to play a significant role in determining attitudes towards PAS/euthanasia in ALS patients. Other factors, such as education and psychological state, could also be important. In conclusion, end-of-life decisions in ALS patients are complex and require careful consideration of individual values, beliefs, and preferences. Understanding the factors that influence a patient's attitude towards PAS/euthanasia can help healthcare providers to offer appropriate care and support for these patients and their families., Competing Interests: Declarations. Conflict of interest: The authors have no competing interests to declare that are relevant to the content of this article., (© 2024. The Author(s) under exclusive licence to Belgian Neurological Society.)

The COVID-19 pandemic has introduced new challenges in managing neurological conditions, particularly among athletes. This paper explores the intersection of post-COVID-19 neurological syndrome (PCNS/PASC) and post-concussion syndrome (PCS), focusing on their implications in sports medicine. Our analysis covers the symptomatology, pathophysiology, and management strategies for PCNS/PASC and PPCS, with special attention paid to the unique challenges faced by athletes recovering from these conditions, including the risk of symptom exacerbation and prolonged recovery. Key findings reveal that both PCNS/PASC and PPCS present with overlapping symptoms such as cognitive difficulties, exercise intolerance, and mental health issues, but differ in specific manifestations like anosmia and ageusia, unique to COVID-19. Pathophysiological analysis reveals similarities in blood-brain barrier disruption (BBB) but differences in the extent of immune activation. Management strategies emphasize a gradual increase in physical activity, close symptom monitoring, and psychological support, with a tailored approach for athletes. Specific interventions include progressive aerobic exercises, resistance training, and cognitive rehabilitation. Furthermore, our study highlights the importance of integrating neurology, psychiatry, physical therapy, and sports medicine to develop comprehensive care strategies. Our findings underscore the dual challenge of COVID-19 and concussion in athletes, necessitating a nuanced, interdisciplinary approach to effective management. Future research should focus on the long-term neurological effects of both conditions and optimizing treatment protocols to improve patient outcomes. This comprehensive understanding is crucial for advancing the management of athletes affected by these overlapping conditions and ensuring their safe return to sports.

Background In Romania, as in other parts of the world, the family doctor is the first to make contact with a healthy patient and is also the first to notice even the smallest pathological changes. In the context of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the patient's communication with the family doctor became even closer and some behavioral changes could be easily noticed. Objective To assess the symptoms of anxiety and depression in the Romanian population using social media platforms in the context of the COVID-19 pandemic. Methods We conducted an anonymous, web-based cross-sectional survey consisting of 31 questions related to general characteristics (age, gender, education, inhabitancy, residence, smoking status, and alcohol consumption) and adapted GAD-7 (7-item General Anxiety Disorders questionnaire) and PHQ-9 (9-item Patient Health Questionnaire). This questionnaire was sent to volunteers in an electronic format through a social network (Facebook, Twitter). The data collected were statistically processed using IBM SPSS v25.0 (IBM Corp., Armonk, NY). The inclusion criteria were age over 18 years and no history of chronic disease. The exclusion criteria consisted of the absence of a mental illness diagnosis. Results From the 1254 respondents, 1232 cases were selected for statistical analysis after applying the exclusion criteria. The mean age was 35.94 (SD = 11.4, 95%CI=10.9-11.9) with a minimum of 18 years and a maximum of 97 years. Eighty-four point nine percent (84.9%; N=1046) of all study participants are female and 79.13% (N= 975) live in the urban area. A total of 188 (15.25%) were diagnosed with COVID-19 of which 31 (16.66%) were male and 157 (15%) were female. N=170 (13.8%) reported moderate symptoms of anxiety during the last two weeks before the survey while N=96 (7.8%) had severe anxiety. Twenty-two point two percent (22.2%; N=274) of the participants reported moderate symptoms of depression while 10.1% (N=125) had moderately severe symptoms and 6.6% (N=81) could be diagnosed with severe depression. A greater likelihood of screening for depression diagnosis was associated with ages between 25 and 34 years (OR=0.90, 95%CI=0.86-0.94, P<. 001), 35 and 44 years (OR=0.88, 95%CI=0.84-0.93, P<. 001), and 45 and 54 years (OR=0.87, 95%CI=0.82-0.92, P<. 001). Also, a tendency was observed for women to be more prone to high levels of anxiety (OR=1.21, 95%CI=1.08-1.35, P< . 001) and depression (OR=2.16, 95%CI=1.51-308, P< . 005). Conclusions Regarding the high prevalence of depression and anxiety, especially in women, appropriate measures for the risk categories should be applied. In the new social context created by the COVID-19 pandemic, screening for psychiatric and psychological disorders should be performed by telemedicine., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Popa et al.)

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is often not detected until drugs have reached clinical stages, risking patients' safety and leading to substantial losses for the pharma industry. Taking into account that standard 2D models have limitations in detecting DILI it is essential to develop in vitro models that are more predictive to improve data translatability. To understand causality and mechanistic aspects of DILI in detail, a human liver MPS consisting of human primary liver parenchymal and non-parenchymal cells (NPCs) and cultured in 3D microtissues on an engineered scaffold under perfusion has been developed. Cryopreserved primary human hepatocytes (PHHs) and Kupffer cells (HKCs) were cocultured as microtissues in the MPS platform for up to two weeks, and each compound of interest was repeatably dosed onto liver microtissues at seven test concentrations for up to four days. Functional liver-specific endpoints were analyzed (including clinical biomarkers such as alanine aminotransferase, ALT) to evaluate liver function. Acute and chronic exposure to compounds of various DILI severities can be assessed by comparing responses to single and multi-dosed microtissues. The methodology has been validated with a broad set of severe and mildly hepatotoxic compounds. Here we show the data for pioglitazone and troglitazone, well-known hepatotoxic compounds withdrawn from the market for causing liver failures. Overall, it has been shown that the liver MPS model can be a useful tool to assess DILI and its association with changes in hepatic function. The model can additionally be used to assess how novel compounds behave in distinct patient subsets and how toxicity profiles may be affected by liver disease states (e.g., viral hepatitis, fatty liver disease).

Aim: This paper is focused on the investigation the effects of two polymeric matrices for indomethacin in cutaneous pain models in mice. There were used two different co-polymers polyhydroxyethyl methacrylate and undecan, polymerization reactions being conducted under nitrogen, at 80 degrees C., Material and Methods: The experiments were carried out on white Swiss mice (20-25 g), divided into 6 groups of 7 animals each, treated orally. Biocompatibility properties of indomethacin-loaded copolymeric matrices ware evaluated by assessing the effects on the blood parameters, the serum biochemical tests of animals treated. The nociceptive somatic testing was performed using hot plate assay and tail immersion test. The latency (second) response to paw, respectively tail thermal noxious stimulation, was measured before the experiment and 15, 30, 60, 90, 120 minutes, 4, 6, 8, 10, 12 hours after the substances administration., Results: Laboratory analysis did not show significant differences of blood parameters, serum biochemical tests between control mice group (IND) and groups treated with 1 M, 1 IND, 3 M, 3 IND. In our experimental conditions IND determined a significant increasing of the latency period response, in hot plate and also in tail immersion tests. Using two different co-polymers for indomethacin incorporation we obtained an increasing of the latency time pain reaction in hot plate assay, respectively in tail immersion test, statistically significant (*p < 0.05) compared with the simple copolymers administration., Conclusions: We demonstrated that indomethacin co-polymeric matrices 1 IND and 3 IND determined similar immune responses with indomethacin and simple co-polymers after oral administration in mice, indicative of good in vivo biocompatibility. Oral administration of both 1 IND and 3 IND resulted in prolonged antinociceptive effects in hot plate assay and also in tail immersion test in mice.

Material and Method: The study has been conducted on a period of ten years and it included 1570 children with congenital abnormalities (CA), of which 371 (24%) were cardiac abnormalities, 312 (20%) were skeletal abnormalities, 55 (3%) were Down Syndrome and 832 (53%) were other pathologies., Results: 48% of the 371 children that were diagnosed with cardiac CA were males, while 52% were females; 52% of the children were from the city, while 48% were from the country-side; 42% of the children have been born prematurely, while 58% of them have been born at normal term. 38% of the children had an APGAR score lower than 7 and 62% of them had an APGAR score higher than 7. Of the total number of births, 72% were caesarian births and 28% were natural births. The different types of Cardiac CA that have been encountered in the study were atrioventricular canal (56%), transposition of the great vessels (18%), common arterial trunk (10%), atrial septal defect (8%), ventricular septal defect (5%) and tetralogy of Fallot (3%). 66% of the total number of deaths were represented by those with cardiac pathology, 21% were caused by hydrocephalus, 7% were caused by diaphragmatic hernia, 4% had renal CA, while 2% were caused by other pathologies.

Unlabelled: Obesity is a complex, pluricausal disease that develops through the interaction of social,'behavioural, cultural, physiological, metabolic and genetical causes., Material and Methods: The study was conducted on 1764 school age children from Iaşi city. Data was collected by measuring their weight, height and arterial pressure. 283 children were diagnosed with weight excess and have been questioned about their nutritional habits and life quality., Results: It has been concluded that 50.17% of the children were breastfed in the first months of life. The weight excess was frequently correlated with: irregular meal schedule (57.59%), roast meal cooking (69.61%), eating white bread (77.38%), predilection for fat meat (66.78%), processed cheese and butter (69.25%), french fries (68.19%), everyday dessert (62.89%), highly concentrated sweets (cookies, chocolate) (63.95%), biscuits, snacks, chips (73.85%) and sweet juices (66.42%). Inactivity was also an important factor, often associated with weight excess, considering that 60.42% of the children spend their spare time watching TV or playing computer games and only 32.86% of the children practice sport during their spare time., Conclusions: Obesity and overweight are pathologies with increasing incidence in our country and they are caused by a combination between genetical factors and, more important, nutritional habits and the amount of time dedicated to physical exercise.

The aim of this paper is theophylline (THP) inclusion into xanthan-chitosan polyionic complex (Xa-CS) and the study of its in vitro and in vivo kinetic release. Xa-CS hydrogel was obtained by ionic complexation between two oppositely charged polysaccharides. THP was loaded into the Xa-CS matrix by diffusion of the drug solution. The obtained samples were characterized by FTIR spectroscopy, SEM microscopy and study of the swelling behavior. THP in vitro release experiments were carried out in conditions mimicking the gastrointestinal environment. The chosen drug dose for in vivo study was 15 mg THP/Kg body weight of THP powder or an equivalent dose in complex form. THP serum concentrations were determined by an HPLC assay. The THP peak serum concentration (C(max)) was 7.18 microg/ml for free THP and AUC(0-48) was 25.76 microg h/ml, while in the case of Xa-CS-THP, C(max) was of 5.72 microg/ml and AUC(0-48) = 45.72 microg h/ml. The in vivo study regarding the behaviour of the obtained formulation, showed an increase bioavailability of THP compared to the raw drug, suggesting the possible application of the complex Xa-CS as an oral controlled drug delivery system in the management of chronic pulmonary obstructive disease.

Unlabelled: Due to the higher incidence of children obesity, in the last decade, the scientific community as well as mass media have increased their interest towards obesity and eating habits., Material and Method: The study has been made on a number of 108 pediatric patients with intercurrent affections that has been diagnosed with obesity or weight excess between 2005 and 2008. The evaluation of the pacients has been done with the use of anamnesis, full clinical examination and biochemical investigations., Results: The case repartition based on body mass index has revealed that 46.29% of the pacients have excess weight and 53.70% suffer from obesity. The biochemical alterations have shown the following: regarding the glucidic metabolism--21 cases with impaired basal glycaemia, 5 cases with low glucose tolerance and 2 cases with diabetes mellitus type 1; regarding the lipidic metabolism--39 cases with modifications of triglycerides, of cholesterol fractions or of both categories. Also, we found the X metabolic syndrome in 20 of the studied cases, hyperuricaemia in 13 cases, hepatic steatosis in 10 cases and arterial hypertension in 34 of the cases. Almost half of the interviewed subjects have accused different types of psycho-behaviour changes., Conclusions: From the investigated complications, the dislipidemic syndrome, arterial hypertension and glucidic metabolism disorders have had an important place and have completed the metabolic syndrome picture in an important number of cases.

Chitosan, a natural polycationic polysaccharide, was coupled with two polyanionic polymers: Na-alginate and carboxymethylcellulose (CMC) and with tannic acid (TA) obtaining three species of self-assembled complexes: chitosan/alginate/TA (sample 1), chitosan/TA (sample 2) and chitosan/CMC/TA (sample 3). The microparticle formation was achieved by dropwise addition of one solution into other by using a coaxial airflow sprayer. These systems were characterized with regard to particle size distribution, thermal stability, tannic acid entrapment efficiency. Sample 2 showed quite a different behavior compared to the other two samples; the particle diameter is located in the nanometric region, the quantity of incorporated tannic acid is higher than in the other two samples and the material shows better thermal stability. The release of tannic acid from these complexes was studied in water (pH = 5.89), phosphates buffer (pH = 7.04) and acetate buffer (pH = 4.11). These studies revealed two distinct periods in tannic acid delivery process: an initial period, varying between 4 and 10 h, characterized by a high release rate with a delivered tannic acid amount of approximately 80% of the incorporated polyphenol and a second period, which starts after 20 to 30 h of delivery and it ends after approximately 120 h, when the release process takes place with low and constant rate and the kinetic curve is linear--characteristic for a zero order kinetic.

The aim of the study is to evaluate the efficiency of the first-line antibiotic treatment of the community-acquired respiratory tract infections in a population of young adults from an urban setting and to establish the pattern of antibiotic resistance of the germs involved. The bacteria most frequently identified have been: S. pneumoniae, H. influenzae, M. catarrhalis, atypical agents also being suspected. Antibiotic treatment has been chosen accordingly to the recent guidelines, total clinical remission rate being of 91.08%, despite the increasing resistance for the commonly used antibiotics; a close monitoring of the phenomenon is mandatory.

The purpose of the study was to determine the prevalence and to describe the clinical presentations of Attention Deficit Hyperkinetic Syndrome in the pediatric patients of the Outpatient Specialty Clinic of the "Sf. Spiridon" University Hospital of Iaşi (a pediatric neuro-psychiatry practice and a general pediatric practice). The studied group consisted of 51 children and adolescents ages 2 years 6 month-18 years of age (urban environment 34 children--66%, rural environment 17 children--34% ; male gender 36 children--70%, female gender 15 children--30%). The pediatric patients represented a prevalence of 1.9% of the Attention Deficit Hyperkinetic Syndrome having the main features: motor and psychological instability, lack of attention, negativism, impulsivity, and social and school integration difficulties. The existence in 40% of cases of family problems (couple pathology, parents with psychiatric disorders, alcoholism, etc) proves the importance of the family patterns description in the apparition and evolution of Attention Deficit Hyperkinetic Syndrome. The study performed underlines the existence of the Attention Deficit Hyperkinetic Syndrome, the necessity of multi-disciplinary intervention with the purpose of precise and on-time diagnosis of this syndrome, to ensure a better integration of these children and teenagers in the social and school environment.

The Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (Delta/-) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated. p53 was inactivated during the derivation of the Orc2 hypomorphic cell lines, accounting for their survival despite active Chk2. These cells also show a defect in the G1 to S-phase transition. Cdk2 kinase activation in G1 is decreased due to decreased Cyclin E levels, preventing progression into S-phase. Molecular combing of bromodeoxyuridine-labeled DNA revealed that once the Orc2 hypomorphic cells enter S-phase, fork density and fork progression are approximately comparable with wild type cells. Therefore, the low level of Orc2 hinders normal cell cycle progression by delaying the activation of G1 cyclin-dependent kinases. The results suggest that hypomorphic mutations in initiation factor genes may be particularly deleterious in cancers with mutant p53 or increased activity of Cyclin E/Cdk2.

RNA helicases are the largest group of enzymes in eukaryotic RNA metabolism. The DEXD/H-box putative RNA helicases form the helicase superfamily II, whose members are defined by seven highly conserved amino acid motifs, making specific targeting of selected members a challenging pharmacological problem. The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase that works in conjunction with eIF4B and eIF4H and as a subunit of eIF4F to prepare the mRNA template for ribosome binding, possibly by unwinding the secondary structure proximal to the 5' m7GpppN cap structure. We report the identification and characterization of a small molecule inhibitor of eukaryotic translation initiation that acts in an unusual manner by stimulating eIF4A-associated activities. Our results suggest that proper control of eIF4A helicase activity is necessary for efficient ribosome binding and demonstrate the feasibility of selectively targeting DEAD-box RNA helicases with small molecules.

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

Ku antigen (Ku70/Ku80) is a regulatory subunit of DNA-dependent protein kinase, which participates in the regulation of DNA replication and gene transcription through specific DNA sequences. In this study, we have compared the mechanism of action of Ku from A3/4, a DNA sequence that appears in mammalian origins of DNA replication, and NRE1, a transcriptional regulatory element in the long terminal repeat of mouse mammary tumor virus through which Ku antigen and its associated kinase, DNA-dependent protein kinase (DNA-PK(cs)), act to repress steroid-induced transcription. Our results indicate that replication from a minimal replication origin of ors8 is independent of DNA-PK(cs) and that Ku interacts with A3/4-like sequences and NRE1 in fundamentally different ways. UV crosslinking experiments revealed differential interactions of the Ku subunits with A3/4, NRE1, and two other proposed Ku transcriptional regulatory elements. In vitro footprinting experiments showed direct contact of Ku on A3/4 and over the region of ors8 homologous to A3/4. In vitro replication assays using ors8 templates bearing mutations in the A3/4-like sequence suggested that Ku binding to this element was necessary for replication. By contrast, in vitro replication experiments revealed that NRE1 was not involved in DNA replication. Our results establish A3/4 as a new class of Ku DNA binding site. Classification of Ku DNA binding into eight categories of interaction based on recognition and DNA crosslinking experiments is discussed.

The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell line, is defective in DNA double-strand break repair and V(D)J recombination. The defective phenotypes of xrs-5 cells are complemented by the 86 kDa subunit of Ku antigen. OBA is a protein, previously purified from HeLa cells, that binds in a sequence-specific manner to mammalian origins of DNA replication. The DNA-binding subunit of OBA has been identified as Ku86. We tested the xrs-5 cell line for its ability to replicate a mammalian origin-containing plasmid, p186, in vivo and in vitro. In vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% when compared with the CHO K1 cells transfected with p186. In vitro, although total and cytoplasmic cell extracts from xrs-5 cells replicated the p186 with the same efficiency as the parental CHO K1 cell extracts, xrs-5 nuclear extracts did not possess any detectable replication activity. Addition of affinity-purified OBA/Ku restored replication in the xrs-5 nuclear extract reaction. Western blot analyses showed that the levels of other replication proteins (Orc2, PCNA, DNA polymerase epsilon and delta, Primase and Topoisomerase IIalpha) were comparable in both the xrs-5 mutant and CHO K1 wild-type cell lines. In addition, the in vivo association of Ku with the DHFR origin-containing sequence (oribeta) was examined in both the CHO K1 and xrs-5 cell lines by a chromatin immunoprecipitation (ChIP) assay. Anti-Ku antibodies did not immunoprecipitate a detectable amount of Ku from the xrs-5 cells in the origin-containing sequence, in contrast to the CHO K1 cells, wherein Ku was found to be associated with the oribeta origin. The data implicate Ku antigen in in vivo and in vitro DNA replication and suggest the existence of another protein with Ku-like functions in the xrs-5 cells.

We previously identified and purified from human (HeLa) cells a 66-kDa cruciform-binding protein, CBP, with binding specificity for cruciform DNA regardless of its sequence. DNA cruciforms have been implicated in the regulation of initiation of DNA replication. CBP is a member of the 14-3-3 family of proteins, which are conserved regulatory molecules expressed in all eukaryotes. Here, the in vivo association of CBP/14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey replication origins ors8 and ors12, as assayed by a chromatin immunoprecipitation assay and quantitative PCR analysis. The association of the 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with these origins was found to be approximately 9-fold higher, compared with other portions of the genome, in logarithmically growing cells. In addition, the association of these isoforms with ors8 and ors12 was also analyzed as a function of the cell cycle. Higher binding of 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with ors8 and ors12 was found at the G(1)/S border, by comparison with other stages of the cell cycle. The CBP/14-3-3 cruciform binding activity was also found to be maximal at the G(1)/S boundary. The involvement of 14-3-3 in mammalian DNA replication was analyzed by studying the effect of anti-14-3-3beta, -epsilon, -gamma, and -zeta antibodies in the in vitro replication of p186, a plasmid containing the minimal replication origin of ors8. Anti-14-3-3epsilon, -gamma, and -zeta antibodies alone or in combination inhibited p186 replication by approximately 50-80%, while anti-14-3-3beta antibodies had a lesser effect ( approximately 25-50%). All of the antibodies tested were also able to interfere with CBP binding to cruciform DNA. The results indicate that CBP/14-3-3 is an origin-binding protein, acting at the initiation step of DNA replication by binding to cruciform-containing molecules, and dissociates after origin firing.

A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding., (Copyright 2002 Wiley-Liss, Inc.)

Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association with ors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80(+/+) and Ku80(-/-) mouse embryonic fibroblasts. A decrease of approximately 70% in in vitro DNA replication was observed when the Ku80(-/-) extracts were used, compared with the Ku80(+/+) extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.