diff --git "a/deduped/dedup_0794.jsonl" "b/deduped/dedup_0794.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0794.jsonl" @@ -0,0 +1,40 @@ +{"text": "The administration of granulocyte colony-stimulating factor (G-CSF) to peripheral blood progenitor cell (PBPC) donors causes spleen length to increase, but the duration of enlargement is not known. Eighteen healthy subjects were given 10 \u03bcg/kg of G-CSF for 5 days and a PBSC concentrate was collected by apheresis. Ultrasound scans were used to assess craniocaudal spleen length before and after G-CSF administration. Mean spleen length increased from a baseline length of 10.7 \u00b1 1.3 cm to 12.1 \u00b1 1.2 cm on the apheresis day (p < 0.001). Ten days after apheresis, spleen length fell to 10.5 \u00b1 1.2 cm and did not differ from baseline levels (p = 0.57), but in 3 subjects remained 0.5 cm greater than baseline length. Increases in spleen length in PBPC donors are transient and reversible. Peripheral blood progenitor cell (PBPC) concentrates donors are routinely given granulocyte colony-stimulating factor (G-CSF) to increase the concentration of circulating PBPCs and hence the number of progenitors that can be collected by apheresis. Typically 10 to 16 \u03bcg/kg of G-CSF are given subcutaneously daily for 4 to 6 days prior to the collection . The admWhile spontaneous rupture of the spleen in PBSC donors given G-CSF is rare, the administration of G-CSF for five days causes spleen length to increase in almost all healthy donors ,9. The iSince allogeneic PBPC donors may be at risk for splenic rupture while the spleen is enlarged, it is important to determine when spleen size returns to baseline levels. The purpose of this study was to determine if spleen length returns to baseline 10 days after G-CSF-mobilized PBPC concentrates are collected by apheresis from healthy subjects.All of the subjects were in good health and were donating G-CSF-mobilized PBPC concentrates for laboratory investigations. The donors were given 10 \u03bcg/kg of G-CSF daily for 5 days, and a PBSC concentrate was collected approximately 2 hours after the last G-CSF dose was given. PBPC concentrates were collected with a CS3000 blood cell separator . Spleen length was evaluated by ultrasound examination three times: prior to the administration of the first dose of G-CSF, on the day of apheresis, and 10 or 11 days after apheresis. This study was approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.Craniocaudal spleen length was assessed using ultrasound with a sector transducer . The intra-observer error for measuring spleen length using ultrasound is 4.9 mm when healthy subjects are evaluated at separate settings .Complete blood counts were performed with an automated cell counter . CD34+ cell counts were performed using a flow cytometer .Spleen lengths measured before and after the G-CSF course were compared using 2-tailed paired t-tests. Spleen length changes were also compared among males and females and Caucasians and non-Caucasians using 2-tailed t-tests. The percent change in spleen length was compared with blood counts, CD34+ cell counts, and donor age using linear regression.The median age of the 18 healthy subjects was 34 years old and ranged from 22 to 55 years of age. Eight of the subjects were male, 13 of the donors were Caucasian, 3 were African American, and 2 Asian. Apheresis day spleen length increased above baseline length in 17 of 18 donors . There was no difference between the 10-day post-apheresis and pre-G-CSF spleen length (p = 0.57). The spleen length 10 days after apheresis was less than the apheresis day length in all 17 donors whose spleen length increased. However, the spleen length 10 days after apheresis remained more than 0.5 cm greater than baseline spleen length in 3 subjects or between Caucasians and non-Caucasians . Spleen length increase was not related to donor age (r = 0.13). In addition, spleen length increase was not related to preapheresis CD34+ (r = 0.04), WBC (r = 0.05), neutrophil (r = 0.07), lymphocyte (r = -0.14), monocyte (r = -0.04), and platelet counts (r = 0.19) or hemoglobin level (r = -0.04).Healthy PBPC concentrate donors given G-CSF should be warned that their spleens will be enlarged for a brief time and that they may be at risk of splenic rupture. Most donors are likely at risk for splenic rupture only during the time of G-CSF administration and for about 10 days after the completion of the G-CSF course. Since splenic enlargement may persist for longer periods in some donor, until more data are available it may be worthwhile to counsel PBPC donors to avoid activities that could lead to abdominal and splenic trauma for 2 to 3 weeks after the last dose of G-CSF."} +{"text": "TGFBI) gene in patients from three unrelated Chilean families with lattice corneal dystrophy type I (LCDI).To describe clinical data and to characterize mutations in the transforming growth factor beta-induced was screened using PCR-RFLP for the seven patients and four healthy relatives. Exons 11, 12, 13, and 14 were sequenced in one patient not carrying the mutation in codon 124. Comparison of phenotype to genotype was performed.The seven patients studied exhibited LCDI in both eyes, most of which were symmetric. Affected individuals demonstrated progression from central subepithelial needlelike deposits and polymorphic anterior stromal opacities. The age at onset of symptoms varied between six to 15 years old in Family One; the patient in Family Two was five years old and the patient in Family Three was 21 years old. Visual acuity varied from 1.0 to 0.05. Two patients, aged 50 and 45 years, underwent penetrating keratoplasty in both eyes, and two patients, aged 47 and 24 years, underwent penetrating keratoplasty in one eye. The only patient in Family Three exhibited a somewhat distinct phenotype, with yellowish discoloration in the anterior stroma and fewer, but thicker lattice lines than the patients in Families One and Two. Screening for the mutation C>T at the nucleotide position 417 (R124C) in exon 4 in the three families revealed the heterozygous R124C mutation in Families One and Two. In Family Two, the mutation was a de novo mutation, as neither parent was a carrier. Screening by sequencing analysis for mutation in exons 11, 12, 13, and 14 in the affected patient in Family Three revealed a heterozygous A1762G mutation (H572R) in exon 13.This is the second report of the 417C>T mutation and the first report of 1762 A>G mutation (H572R) in Chilean patients. The H572R mutation identified is associated with a distinct lattice corneal dystrophy type I phenotype. Lattice corneal dystrophy type I is an early onset autosomal dominant dystrophy with variable clinical expression [Lattice corneal dystrophy (LCD) is one of the most common inherited corneal diseases, characterized by the accumulation of amyloid throughout the middle and anterior stroma, forming a network of branching refractile lines. It develops with recurrent corneal erosion and keratoplasty is frequently required . Four diTGFBI) gene as causative of LCD type I. The mutation list includes R124C, V505D, L518P, V539D, A546D, P551Q, L569R, H572R, and V625D [Thus far, few mutations have been described in the transforming growth factor beta-induced and one carries a 1762A>G (H572R) mutation in All examinations were performed according to the tenets of the Declaration of Helsinki and the present study was approved by the ethics committee of the Clinical Hospital of the University of Chile. All patients were informed about the study and gave signed consent. Three index cases were identified during ophthalmic examination at the Clinical Hospital of the University of Chile. After obtaining informed consent, seven affected and four unaffected members from three Chilean families with lattice corneal dystrophy were enrolled. Families One and Two were not related and the last names and family histories suggest Spanish origin. The proband of Family Three was adopted and we do not have any information about his biological family.All participants received a detailed clinical examination that included best-corrected visual acuity (BCVA) according to the best line of Snellen acuity, slit lamp biomicroscopy, color cornea photography, pneumatic tonometry , and dilated fundus examination. Autorefractometry measurement and keratometry were performed (model RM-A7000). We considered high myopia when the refractive error was greater than -6.0 diopters.The LCD type 1 diagnosis was based on clinical examination. The corneal phenotype of all index patients was assessed by slit lamp examination and the review of biomicroscopic photographs by an investigator who did not know the genetic status.The lesions were considered to be synchronic if the patients perceived the first symptoms in the second eye within a month of perceiving them in the first. All individuals with corneal commitment were considered clinically affected. Patients were classified in degrees of severity according to best-corrected vision, the number of lesions, and corneal commitment .TGFBI was analyzed in all patients and controls using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP), as previously described [Peripheral blood (5\u00a0ml) was collected from seven patients and four unaffected family members and genomic DNA was isolated . The 417escribed . BrieflyTGFBI were amplified by PCR using the primers and conditions described previously [Exons 11, 12, 13, and 14 of eviously . The priPaternity was confirmed by microsatellite typing of 15 short tandem repeat (STR) loci and Amelogenin using the AmpF/STR Identifiler PCR amplification kit under the recommended conditions.Three unrelated index cases were identified during ophthalmic examination at the Clinical Hospital of the University of Chile, Santiago, Chile. The pedigrees of their families were delineated, revealing there were eleven living cases of affected patients in Family One and only one affected member in each of Families Two and Three . We examThe pedigree of Family One is shown in The proband, a 27-year-old woman, began with episodes of acute ocular pain, redness, and photophobia at six years of age. The frequency and severity of these episodes increased coincident with a gradual deterioration of vision in both eyes. Slit lamp examination showed the presence of large, typical fine branching lattice lines in the anterior stroma of both eyes . A clearThe proband\u2019s maternal grandmother, an 81-year-old woman, began having symptoms at 14 years of age. She underwent penetrating keratoplasty in the right eye at age 47. Slit lamp examination revealed clinical signs of the recurrence of LCD type 1 in the corneal grafts of the right eye; the corneal graft exhibited a network of linear opacities associated with other smaller opaque spots and refractive lattice lines and diffuse anterior stromal opacity (not shown). Her left eye revealed irregularity of the epithelial surface with subepithelial and anterior stromal scarring, resulting in diffuse clouding of the central cornea . PeripheThe proband\u2019s maternal uncle, a 53-year-old man, began with episodes of recurrent corneal erosions at 15 years of age. Slit lamp examination showed the presence of a network of linear opacities associated with other smaller opaque spots and refractile lattice lines in both eyes OD, . No vascThe proband\u2019s mother, a 50-year-old woman, began having episodes of acute ocular pain, redness, and photophobia at 10 years of age. The frequency and severity of these episodes increased coincident with a gradual deterioration of vision in both eyes. At 32 years of age, she was diagnosed with lattice corneal dystrophy. She underwent keratoplasty in her left eye at age 45 and in her right eye at age 47. When she was initially examined at the Clinical Hospital of the University of Chile, slit lamp examination revealed clinical signs of the recurrence of LCD type 1 in the corneal grafts of both eyes. She had bilateral blurred vision; both corneal grafts showed a network of linear opacities associated with other smaller opaque spots and refractive lattice lines and diffuse anterior stromal opacity . No vascThe proband\u2019s maternal first cousin, a 22-year-old woman, began with episodes of recurrent corneal erosions at 15 years of age. However, an ophthalmic examination revealed no lesions characteristic of lattice corneal dystrophy, i.e., no typical fine branching lattice lines or vascularization of the cornea were observed. Best-corrected vision was 1.0 in both eyes.This was a two-generation family with one affected individual in the second generation. The pedigree of this family is shown in The proband, a 25-year-old women, began with episodes of recurrent corneal erosions at five years of age. She also had high myopia in both eyes. Slit lamp examination showed the presence of elevated subepithelial opacities, fine lattice lines, diffuse \u201cground glass\u201d haze in the anterior stroma, and corneal grafts . Best-coThe proband\u2019s parents, who had no history of recurrent corneal erosions, had a completely normal ophthalmic examination. Best-corrected vision was 1.0 in both eyes for each parent. We also examined three other family members . All were phenotypically normal.This was a two-generation family with one affected individual in the first generation. The pedigree of this family is shown in The proband, a 52-year-old man, was adopted and had no knowledge of his parents' ocular status. He began with episodes of recurrent corneal erosions at 21 years of age. He underwent penetrating keratoplasty in the left eye at age 50 and in the right eye at age 51. Slit lamp examination performed before surgery showed the presence of elevated subepithelial opacities, stromal thick lattice lines, diffuse \u201cground glass\u201d haze, and a yellowish discoloration in the anterior stroma of both eyes . Best-coDilated fundus examination and tonometry were normal in all members of the three families.TGFB1 in individuals III-1, IV-10, IV-13, V-15, V-17, V-19, and V-21 of Family One, I-1, I-2, and II-1 of Family Two, and I-1 of Family Three were analyzed using PCR-RFLP and were digested using the PstI restriction enzyme, as previously described [TGFBI. This mutation was not present in healthy controls. The same mutation was detected in individual II-1 of Family Two, but was absent in both parents . This mutation was observed in both direct in the first FAS domain. Families One and Two were not related, indicating that the mutations at the codon 124 hotspot arose separately and therefore they do not represent a founder effect. Moreover, the parents of the proband of Family Two were both healthy and paternity was proved, demonstrating that the mutation in this family is a de novo mutation and also reaffirming that the 417C>T mutation in Families One and Two did not result from a founder effect. The age at onset of symptoms in individuals of Families One and Two carrying the R124C mutation varied from six to 15 years old. Family One, a relatively extended family, did not exhibit anticipation as we described in a previous report .TGFBI; it has been reported in several ethnic groups throughout the world, including Chile [TGFBI. In addition, three other different mutations have been described at the same codon, substituting arginine for three different amino acids and generating different phenotypes. These mutations are a transversion 417C>A causing the substitution for serine (R124S) observed in late onset granular corneal dystrophy type I [The 417C>T mutation is the most frequent one reported in ng Chile . Consequy type I , a transy type I , and a ty type I . The cauy type I ,31. In fy type I .On the other hand, the DNA sequencing of the affected subject of Family Three revealed a heterozygous mutation in exon 13 (A1762G). This mutation changes histidine to arginine at codon 572 (H572R) located in the fourth FAS domain; this has only been reported once, in a Thai family . The ageTGFBI mutations may modify the secondary and tertiary structures of TGFBIp. This is supported by the observations that TGFBIp may form dimers and tetramers, a characteristic of many proteins capable of amyloid production [TGFBIp interacts with several extracellular matrix (ECM) proteins, including fibronectin, biglycan, decorin, and several types of collagen . Most ofoduction . The queoduction . The abiIn summary, we report two new families carrying the 417C>T (R124C) mutation and one family with the mutation A1762G (H572R) associated with LCDI. The latter mutation is associated with a distinct phenotype. Further studies are necessary to understand the normal function of TGFBIp and the molecular mechanisms underlying the variegation of phenotypes caused by different mutations."} +{"text": "To report the clinical, ophthalmic, and genetic characteristics for lattice corneal dystrophy type I (LCDI) in a Chilean family.transforming growth factor-induced gene (TGFBI) was screened for the most frequent mutation, R124C, in the proband by sequencing. We also designed a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to analyze the same mutation, amplifying exon 4 and digesting with PstI restriction enzyme. Using this strategy, we analyzed the mutation in six affected and three healthy family members.Six affected family members were examined clinically including visual acuity, color cornea photography, applanation tonography, and fundoscopy. Genomic DNA was extracted from peripheral leukocytes from six affected and three unaffected members of a family with lattice corneal dystrophy type I. Exon 4 of the TGFBI of the proband exhibits the heterozygous single-nucleotide mutation, C417T, leading to amino acid substitution (R124C) in the encoded TGF\u2013induced protein. Using PCR-RFLP, we confirmed the heterozygous mutation in six affected family members and excluded it in three healthy members.Three generations of family members were positively diagnosed with lattice corneal dystrophy. Six participants demonstrated LCD1 in both eyes, most of whom were symmetric. Age at onset of symptoms was variable (3\u201342 years old). Moreover, in this family, the age of onset of the disease decreased in succeeding generations, which could be interpreted as anticipation. Visual acuity varied from 1.0 to 0.13. Two patients, ages 69 and 44 years old, demonstrated a degree of severity \u201cBad\u201d according to best-corrected vision and corneal commitment. The exon 4 sequence of TGFBI cosegregated with LCD type I in the investigated family. This is the first report of a molecular analysis of LCD type I in Chilean patients. The early onset affected persons in the fourth generation raises the possibility of anticipation.The R124C mutation in The corneal dystrophies are a group of genetically determined diseases usually characterized by loss of corneal transparency, which may be caused by a progressive accumulation of abnormal material within the cornea. Lattice corneal dystrophy (LCD) is a distinct clinical entity characterized by the accumulation of amyloid throughout the middle and anterior stroma .122200), also known as Biber-Haab-Dimmer dystrophy, is inherited as an autosomal dominant trait with variable clinical expression and a high degree of penetration and 0.13 in the left eye [OS]) and corneal commitment. Slit-lamp examination revealed an irregular epithelial surface with subepithelial and anterior stromal scarring, resulting in diffuse clouding of the central cornea. She showed a network of linear opacities associated with other smaller opaque spots and refractive lattice lines . No vascThe proband\u2019s mother, at 40 years of age, initially presented a mild subepithelial scarring and opacification, which gradually progressed to include elevated subepithelial opacities, fine lattice lines, diffuse \u201cground glass\u201d haze in the anterior stroma, and corneal grafts . Best-coAlthough patients IV-3 and IV-4 did not have lesions at time of the exams, they had a history of recurrent corneal erosions in both eyes since the ages of three and four, respectively.The proband had a history of recurrent corneal erosions in both eyes, which began when he was four years old. Slit lamp examination showed the presence of large, typical fine branching lattice lines in the anterior stroma in OD . A clearThe average age at onset for affected family members was 18.9\u00b115.7 years old ) ; and copIn our case, genetic anticipation might be explained in at least three ways. One way is the fact that a previous ophthalmic history of the parents and grandparents probably prompted the family to look earlier for medical examination. It is possible that several mild cases of the syndrome, especially when the affected person does not feel impaired, will go undiagnosed until the occurrence of a more severely affected sibling or offspring. Another explanation of anticipation may be the result of the loss of a protective allele or another interacting gene from the affected parent. Thus the protective allele or interacting gene could ameliorate the effect of the mutated allele in the affected parent. Since such protective allele or protective gene could be absent in the offspring, it can not exhibit any protective effect in these subjects. The final possible way may be the gain of a susceptibility allele from the non-affected parent, which could worsen the effect of the mutated allele. If we consider that individuals III-4 and III-7 are not related, the probability of gaining a susceptibility allele simultaneously by individuals IV-3, IV-4, and IV-5 is low.TGFBI mutations in LCD type I patients will also contribute to improve their clinical classification, management, and eventual genetic counseling. Although the R124C mutation is one of the genetic causes of the disease, different genetic and environmental factors may govern the age of onset.In conclusion, since the mutation at the hot spot nucleotide 417 can be easily, rapidly, and cost-effectively evaluated by PCR sequencing or by PCR-RFLP, identification of this mutation will allow Chilean patients to benefit from a timely and accurate molecular diagnosis of LCD type I. Genetic testing of"} +{"text": "MCT8 (SLC16A2) mutations. Intrauterine growth restriction (IUGR), usually due to uteroplacental failure, is associated with milder neurodevelopmental deficits, which have been partly attributed to dysregulated TH action in utero secondary to reduced circulating fetal TH concentrations and decreased cerebral thyroid hormone receptor expression. We postulate that altered MCT8 expression is implicated in this pathophysiology; therefore, in this study, we sought to quantify changes in cortical MCT8 expression with IUGR. First, MCT8 immunohistochemistry was performed on occipital and parietal cerebral cortex sections obtained from appropriately grown for gestational age (AGA) human fetuses between 19 weeks of gestation and term. Secondly, MCT8 immunostaining in the occipital cortex of stillborn IUGR human fetuses at 24\u201328 weeks of gestation was objectively compared with that in the occipital cortex of gestationally matched AGA fetuses. Fetuses demonstrated widespread MCT8 expression in neurons within the cortical plate and subplate, in the ventricular and subventricular zones, in the epithelium of the choroid plexus and ependyma, and in microvessel wall. When complicated by IUGR, fetuses showed a significant fivefold reduction in the percentage area of cortical plate immunostained for MCT8 compared with AGA fetuses (P<0.05), but there was no significant difference in the proportion of subplate microvessels immunostained. Cortical MCT8 expression was negatively correlated with the severity of IUGR indicated by the brain:liver weight ratios at post-mortem. Our results support the hypothesis that a reduction in MCT8 expression in the IUGR fetal brain could further compromise TH-dependent brain development.The importance of the thyroid hormone (TH) transporter, monocarboxylate transporter 8 (MCT8), to human neurodevelopment is highlighted by findings of severe global neurological impairment in subjects with IUGR is often characterized by continued head and brain growth at the expense of other less vital organs resulting in an elevated brain:liver weight ratio postnatally . IUGR coMCT8 gene (SLC16A2) in subjects with a variety of X-linked mental retardation syndromes, characterized by severe psychomotor and cognitive impairment and accompanied by elevated serum free T3 concentrations but normal or low free T4 concentrations (Monocarboxylate transporter 8 (MCT8) is a highly specific plasma membrane TH transporter , and demonstrates pre-receptor regulation by DIO2 and deiodinase type 3 (DIO3) (which inactivates T4 and T3) . Gestational ages were determined by first-trimester ultrasound scan for crown\u2013rump length. Sections of formalin-fixed paraffin-embedded (FFPE) samples were then obtained from the hospital archive of histopathology blocks.n=3) and third trimesters from AGA fetuses with unexplained intrauterine deaths were examined. Sections of normal adult occipital cortex sampled at post-mortem and donated to the London Neurodegenerative Diseases Brain Bank were obtained for comparison.First, sections of the fetal cerebral cortex obtained during the second or AGA (n=5) . IUGR waGA (n=5) . AlthougFFPE sections (5\u200a\u03bcm) of cortical samples were immunostained for MCT8 using an avidin\u2013biotin peroxidase technique unless otherwise stated) as per the kit instructions as described previously from the MCT8-immunostained section and five corresponding images from the adjacent section processed with the omission of the primary antibody as a negative control were analyzed. An objective measure of the area containing brown pixels corresponding to immunoreactive staining for MCT8 was quantified using the software ImageJ as described previously . BrieflyMCT8 immunoreactivity in microvessels was assessed in the subplate zone, a layer deep to the cortical plate with a lower density of cells, where it was easily possible to identify all the microvessels in bright field based on morphology at 40\u00d7 magnification. For each fetus, 20 non-overlapping images of the subplate were taken. The number of immunostained microvessels was counted and calculated as a percentage of all the microvessels present. An average of 40.4\u00b11.9 microvessels was counted per fetus. Non-specific staining of intravascular erythrocytes was disregarded. The percentage of microvessels stained was then expressed relative to the mean of the AGA group, which was assigned an arbitrary value of 1.t-test to compare continuous variables and the Fisher's exact test to compare contingency tables. Quantitative data expressed as relative values were used for analysis using the two-way ANOVA followed by the Holm\u2013Sidak all pairwise multiple comparisons post hoc analysis. The quantitative datasets passed the normality and equal variance tests. Spearman's rank correlation test was used to determine significant correlations between the variables. Significance was taken as P<0.05.Data were analyzed using the SigmaStat Software, v3.1 . Demographic data were analyzed using the unpaired Student's The developing human fetal cerebral cortex in mid-gestation is formed by several layers; from superficial to deep, they are the marginal zone, cortical plate, subplate, intermediate zone, subventricular zone, and ventricular zone , but the raw brain weights were not significantly different between the two groups, with brain weights being well preserved for gestation even in the IUGR cohort (1.08 relative to the expected mean). However, the relative brain weights (ratio to the expected mean for gestation) in the IUGR group were still lower compared with those in the AGA group (P<0.05). The brain:liver weight ratios in the IUGR group were significantly higher compared with those in the AGA group (P<0.01). Atrophy of the thymus secondary to chronic stress in IUGR (P<0.01) and thymus weights relative to the expected mean for gestation (P<0.001). All these indicate that the IUGR cohort comprised cases at the severe end of the spectrum. Most of the IUGR cases demonstrated features of chronic uteroplacental failure on placental examination . However, post hoc tests indicated that the difference was significant only for cortical plate immunostaining in the IUGR group compared with 23.3\u00b18.1% (1\u00b10.3 relative to AGA) in the AGA group (P<0.05), which represents approximately a fivefold decrease in MCT8 expression with IUGR compared with that in the AGA samples when all the samples were analyzed together. The positive correlation remained significant within the IUGR group (correlation coefficient=0.75, r2=0.12; P<0.05; However, there was a significant positive correlation between the area of cortical plate MCT8 immunostaining and the proportion of microvessels stained in the subplate (correlation coefficient=0.71, r2=0.28; P<0.05; When all the samples were analyzed together, a negative correlation was also observed between the area of cortical plate MCT8 immunostaining and brain:liver weight ratios (correlation coefficient=\u22120.64, Changes in TH transporter expression have never been described in the growth-restricted state. This study is the first to demonstrate significantly reduced cortical MCT8 expression within the developing CNS of human fetuses stillborn with severe IUGR. Our results suggest that altered TH transporter activity in cerebral neurons could be a contributory factor to the pathophysiology of neurodevelopmental impairment associated with IUGR.Mct8-knockout mice lack the neurological phenotype observed in humans with MCT8 mutations. However, a limitation is the restriction of the availability of human fetal tissue samples of adequate quality for investigation, hence, the small number of samples in this study.The strength of this study is the use of human fetal tissue samples, thus eliminating species differences, particularly relevant, as The localization of MCT8 in developing neurons across the different cortical layers, microvessels, and choroid plexus reported herein is generally consistent with the findings of previously published studies of human fetuses (3-independent manner (in vitro (Neurogenesis takes place in the ventricular and subventricular zones with much being completed by 28 weeks of gestation (During normal human fetal cortical development, over 70% of neurons undergo programmed cell death after 32 weeks of gestation (3 (MCT8 promotes cell death in non-proliferative cytotrophoblast cells from human placenta independently of TWhether the reduction in cortical cell number in the third trimester is due to reduced neurogenesis, reduced neuronal migration, or increased cell death in IUGR is not known. In rats, abnormal neuronal migration in the fetal CNS in both IUGR (Current understanding of the physiological regulation of MCT8 expression is poor. TH status has been shown to influence MCT8 expression in some tissues (Future studies should investigate whether there are compensatory alterations in the expression of other TH transporters in neurons and microvessels. Studies could also extend to other regions of the CNS and at different gestational ages to obtain a more comprehensive picture of the effects of IUGR on TH transport and how this could correlate with observed neurological impairments in IUGR survivors.In conclusion, our results showing perturbed patterns of cortical MCT8 expression support the hypothesis that a reduction in MCT8 expression in the IUGR fetal CNS could be a contributory factor implicated in the long-term neurodevelopmental impairments associated with this condition."} +{"text": "Staphylococcus aureus (MRSA) infection in orthopaedic patients. However, little is known about the effectiveness of and compliance with such policies in practice.Ring fencing of joint replacement (JR) surgery units is recommended to prevent the high morbidity and mortality associated with methicillin resistant Over 10 weeks in 2010, 250 prospectively recruited admissions to a busy, ring fenced JR unit underwent admission screening for MRSA and demographic screening using a standardised questionnaire based on the unit\u2019s admission policy which is designed to exclude patients at high risk of MRSA colonisation. Subjects comprised of patients admitted for reasons other than JR surgery, as well as JR patients who stayed in any other hospital ward prior to admission to the unit.Despite nearly perfect compliance with unit\u2019s admission policy, 2.8% (7/248) of subjects complying with the admission policy were colonised with MRSA at the nares and/or groin. MRSA carriers were disparate in age, gender and inpatient hospital admission in the last 12 months. Non-JR patients and transfers from high dependency units represented a high risk for introducing MRSA into a ring fenced unit.Demographic screening undoubtedly excludes a proportion of MRSA carriers; however, it is inadequate to completely prevent the admission of MRSA to a ring fenced unit. Where it is unavoidable that ring fenced units host patients not admitted for JR surgery and not screened for MRSA preadmission, these patients should be cohorted away from JR patients as they represent a higher risk for MRSA despite rigorous demographic screening.None declared."} +{"text": "Aim. The aim of this study was to evaluate the role of the Lys751Gln (rs13181) ERCC2 gene polymorphism in clinical parameters and the risk for development of ovarian cancer. Material and Methods. The study consisted of 430 patients with ovarian cancer (mean age: 53.2 \u00b1 10.11) and 430 healthy subjects (mean age: 50.31 \u00b1 18.21). Analysis of the gene polymorphisms was performed using the PCR-based restriction fragment length polymorphism (PCR-RFLP). The odds ratios (ORs) and 95% confidence intervals (CIs) for each genotype and allele were calculated. Results. The results obtained indicate that the genotype Gln/Gln is associated with an increased risk of ovarian cancer . Association of Lys751Gln polymorphism with histological grading showed increased ERCC2 Gln/Gln genotype in grading 1 as well as Gln allele overrepresentation in G1 ovarian patients. Finally, with clinical FIGO staging under evaluation, an increase in ERCC2 Gln/Gln homozygote frequencies in staging I and Gln allele frequencies in SI were observed. Conclusion. On the basis of these results, we conclude that ERCC2 gene polymorphism Lys751Gln may be associated with an increased risk of ovarian carcinoma. The system of DNA repair takes part in maintaining the genomic integrity which undergoes changes under exo- and endogenous factors. There were more than 130 DNA repair genes identified, in which a series of single nucleotide polymorphisms (SNPs) were discovered [The repair process usually encompasses two stages: the excision of lesion and the repair synthesis. This is how the repair system acts via base-excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Totally converse is the repair system activity by direct lesion reversal, in which there is merely a single-stage process with maintained integrity of the DNA phosphodiester chain and the system of recombination repair (HR).A NER system removes short DNA oligonucleotides containing a damaged base . NER recXPD), also called excision repair cross-complimentary group 2 (ERCC2), is one of the most important low-penetrant genes which is located at chromosome 19q13.3 and involved in the nucleotide excision repair (NER) pathway and removes certain DNA cross-links, ultraviolet photolesions, and bulky chemical adducts.Xeroderma pigmentosum complementation group D that is associated with a DNA damage repair phenotype. ERCC2 polymorphisms in the risk of various cancers have been done and different association between Lys751Gln polymorphism and the risk of lung cancer [Many epidemiological studies with the aim of identifying the role ofg cancer \u20135, gliomg cancer , colorecg cancer , 9, and g cancer has been ERCC2 gene in a group of patients with ovarian cancer and in a group of healthy people. The Lys751Gln polymorphism of ERCC2 gene was selected on the basis of literature data, which are highly suggestive of its correlations with ovarian cancer [ ERCC2 gene (Lys751Gln and Asp312Asn) positively affect the response to therapy with carboplatin/paclitaxel [In the reported study, the interest of the authors was focused onto the studies of the Lys751Gln polymorphism ofn cancer \u201313. In pn cancer , 12. In ERCC2 with an attempt to determine the impact this polymorphism exerts on ovarian cancer.The aim of this study was to analyze the frequency of alleles and genotypes of Lys751Gln (rs13181) inn = 430) with ovarian carcinoma, treated at the Department of Surgical Gynaecology and Gynaecologic Oncology, Institute of Polish Mothers Memorial Hospital, between 2000 and 2012. We enrolled only women born and living in central Poland (\u0141\u00f3d\u017a region). The distribution of sociodemographic features of the study participants is shown in n = 430) served as control . They were nonrelated women that have never been diagnosed with ovarian tumors, other tumors, or chronic disease and were randomly selected and frequency matched to the cases on age. The Local Ethical Committee approved the study and each patient gave a written consent for participation in the study.Formalin-fixed paraffin-embedded (FFPE) tumour tissue specimens were obtained from women according to the manufacturer instruction.The PCR-restriction fragment length polymorphism (PCR-RFLP) method was used to detect the genotypes of the Lys751Gln polymorphisms as described previously .\u03bcL PCR mixture contained about 100\u2009ng of DNA, 12.5\u2009pmol of each primer, 0.2\u2009mmol/L of dNTPs, 2\u2009mmol/L of MgCl2, and 1\u2009U of Taq DNA polymerase . PCR products were electrophoresed in a 2% agarose gel and visualised by ethidium bromide staining. All PCR was carried out in a DNA Thermal Cycler PTC-100 TM . After an initial denaturation at 95\u00b0C for 5\u2009min, 35 cycles of amplification with denaturation at 95\u00b0C for 30\u2009s, annealing at 62\u00b0C for 30\u2009s, and extension at 72\u00b0C for 30\u2009s were performed, followed by a final extension step of 7\u2009min at 72\u00b0C. The PCR product was digested overnight with 1\u2009U of PstI at 37\u00b0C. The cleavage with PstI produced fragments of 161, 161/120/41, and 120/41\u2009bp corresponding to the Lys/Lys, Lys/Gln, and Gln/Gln genotypes of the ERCC2 gene, respectively.Primers were applied to assess SNP Lys751Gln (rs13181). The 25\u2009 ERCC2 genotype were compared with those expected for a population in Hardy-Weinberg equilibrium by using the Chi-square (\u03c72) test. Genotype and allele frequencies in cases and controls were compared by \u03c72 test. Logistic regression analysis was used to compute odds ratio (OR) and associated 95% confidence interval (95% CI) relating Lys751Gln SNP as well as combinations of Lys751Gln SNP and other analysed factors presented in p values < 0.05 were considered significant. All the statistical analyses were performed, using the STATISTICA 6.0 software .The observed numbers of each ERCC2 gene in patients and controls are presented in p < 0.0001) was found between the Gln/Gln genotype of the Lys751Gln polymorphism of ERCC2 gene and ovarian cancer occurrence. Variant 751Gln allele of ERCC2 increased cancer risk .The distribution of genotypes and the frequency of alleles of ERCC2 Lys751Gln SNP in the controls were in agreement with Hardy-Weinberg equilibrium (p > 0.05), but the observed genotype frequencies of ERCC2 Lys751Gln SNP in patients were not in agreement with Hardy-Weinberg equilibrium (p < 0.05). It is caused by the very low abundance of the Lys/Lys genotype in the examined Polish population.The observed genotype frequencies of ERCC2 polymorphism. Histological grades were evaluated in all the cases (n = 430). Grades 2 and 3 were accounted for together for statistical analysis (see p < 0.0001) in grade 1 patients, according to FIGO criteria. Moreover, ovarian cancer patients in G1 had an overrepresentation of Gln allele .Histological grading was related toysis see . An incr ERCC2 Lys751Gln polymorphism (p < 0.0001) in stage I patients, according to FIGO classification. A tendency for an increased risk of ovarian cancer progression was observed with the occurrence of Gln allele of ERCC2 polymorphism.Clinical FIGO staging was also related tomorphism . An incr ERCC2 polymorphisms and the risk factors for ovarian cancer, such as BMI (body mass index), smoking status, alcohol consumption, family history of cancer, pregnancy, ascites, HRT, size of tumour, menarche, and the women with ovarian cancer (\u201cdata not shown\u201d).Our data did not demonstrate any statistically significant correlation betweenERCC2-Lys751Gln) was associated with the risk of ovarian cancer in Polish women. DNA is regularly damaged by endogenous and exogenous mutagens. The genes involved in DNA repair and in the maintenance of genome integrity play a crucial role in providing protection against mutations that may lead to cancer [ ERCC1 gene is important in repairing DNA damage and genomic instability and is involved in the nucleotide excision repair pathway. Single nucleotide polymorphism Lys751Gln (rs13181) is one of the most widely studied genetic markers in ERCC2 and its role in various cancers' development is evident [ ERCC2 gene can lead to a conformational change in the encoded protein at the domain of the interaction between ERCC2 and its helicase activator, p44, inside the TFIIH Complex [ ERCC2 Lys751Gln SNP is associated with suboptimal DNA repair capacity [ ERCC2 expression is associated with increased chemotherapeutic sensitivity and thus considered a predictive marker for patients with ovarian cancer receiving combination gemcitabine and cisplatin chemotherapy [ ERCC2 Lys751Gln polymorphism and lower DNA repair capacity. The time to ovarian cancer progression was significantly higher in gemcitabine/cisplatin-treated patients with the Lys751Gln genotype than in those with the Lys751Lys genotype [ ERCC1 and ERCC2 genotype is associated with risk of ovarian carcinoma [ ERCC2 Lys751Gln polymorphism [An attempt was undertaken in the presented study to determine whether single nucleotide polymorphism in the DNA repair pathway and staging (SI) of ovarian carcinoma were presented. Our study was performed on an ethnically homogenous population, which may improve our knowledge, regarding to what extent the genotype-phenotype relationship variations are population related.Our results indicate that the Lys751Gln polymorphism of ERCC2 Lys751Gln polymorphism in ovarian carcinoma occurrence. Similar to our observation, the recent reports demonstrate that ERCC2 Lys751Gln genotype seems to be associated with an elevated ovarian cancer risk [Our results are in line with the data from other reports, introducing an important role ofcer risk . ERCC2 Lys751Gln SNP may be involved in the susceptibility of ovarian cancer in the Polish population. Further research on SNP in ovarian carcinoma is warranted to obtain more conclusive outcomes.In conclusion, our results indicate that the"} +{"text": "Recent studies have revealed the positive antiproliferative and cytotoxic effects of antiviral agents in cancer treatment. The real effect of adjuvant antiviral therapy is still controversial due to the lack of studies in biochemical mechanisms. Here, we studied the effect of the antiviral agent acyclovir on morphometric and migratory features of the MCF7 breast cancer cell line. Molecular levels of various proteins have also been examined.To evaluate and assess the effect of antiviral treatment on morphometric, migratory and other cellular characteristics of MCF7 breast cancer cells, the following experiments were performed: (i) MTT assay to measure the viability of MCF7 cells; (ii) Colony formation ability by soft agar assay; (iii) Morphometric characterization by immunofluorescent analysis using confocal microscopy; (iv) wound healing and transwell membrane assays to evaluate migration and invasion capacity of the cells; (v) ELISA colorimetric assays to assess expression levels of caspase-3, E-cadherin and enzymatic activity of aldehyde dehydrogenase (ALDH).We demonstrate the suppressive effect of acyclovir on breast cancer cells. Acyclovir treatment decreases the growth and the proliferation rate of cells and correlates with the upregulated levels of apoptosis associated cytokine Caspase-3. Moreover, acyclovir inhibits colony formation ability and cell invasion capacity of the cancer cells while enhancing the expression of E-cadherin protein in MCF7 cells. Breast cancer cells are characterized by high ALDH activity and associated with upregulated proliferation and invasion. According to this study, acyclovir downregulates ALDH activity in MCF7 cells.These results are encouraging and demonstrate the possibility of partial suppression of cancer cell proliferation using an antiviral agent. Acyclovir antiviral agents have a great potential as an adjuvant therapy in the cancer treatment. However, more research is necessary to identify relevant biochemical mechanisms by which acyclovir induces a potent anti-cancer effect.The online version of this article (doi:10.1186/s13027-017-0128-7) contains supplementary material, which is available to authorized users. Current cancer therapy includes the use of chemotherapeutic agents, surgery and radiation therapy. It is estimated that four types of viruses alone could cause 12% of cancer cases worldwide. These are human papillomavirus (HPV), hepatitis B (HBV), hepatitis C (HCV), and Epstein\u2013Barr virus (EBV) . ComplexAdjuvant antiviral therapy also has a reported antiproliferative effect in some types of cancer . TreatmeNamba et al. demonstrated the use of zidovudine, an antiviral drug, in combination with gemcitabine, a chemotherapeutic agent - in an attempt to overcome a gemcitabine resistance for the pancreatic cancer treatment. In this type of malignancy, the gemcitabine resistance is associated with a decreased level of human equilibrative nucleoside transporter 1 (hENT1) and acquisition of epithelial-to-mesenchymal transition (EMT) - like phenotype. The zidovudine adjunct therapy was shown to reverse both events in this study . FurtherAlthough there is a plethora of evidence suggesting the beneficial effect of the antiviral agents in cancer treatment, the therapeutic benefit of their use in cancer treatment remains a grey area due to the lack of studies of the biochemical mechanisms. Antiviral agents such as acyclovir and ribavirin have been reported to have a suppressive effect on the proliferation and ability to increase an apoptosis in various cancers , 8. AcycIn the present study, we propose to investigate how cancer cells respond to the antiviral agent as acyclovir in vitro and whether this treatment can affect the metastatic phenotype of cancer cells. We report results on the potential effect of acyclovir treatment on the cell proliferation, invasion capacity, cytotoxicity, and the expression of tumor suppressing genes.2. Cells were subcultured every three days using 0.25% trypsin-EDTA for detachment.Breast cancer cell line MCF7 and human breast epithelial primary cells were cultured in a complete media (CM) (Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) supplemented with 10% fetal bovine serum , 100 U/mL penicillin, 100\u00a0\u03bcg/mL streptomycin and 25 ug/mL Amphotericin B at 37\u00a0\u00b0C in 5% CO2 in the presence of 5 uM acyclovir solution and incubated for 72\u00a0h at 37\u00a0\u00b0C in 5% CO2. In a positive control experiment, cells were cultured in the absence of acyclovir. A control of acyclovir without cells was also conducted. All experiments were performed in triplicate.Antiviral agent \u2013 acyclovir (ACV) in powder form was purchased from Sigma-Aldrich . A 10\u00a0mM (stock) solution was prepared in phosphate buffered saline (PBS) and sterilized through filtering . Stock solution was stored at -20\u00a0\u00b0C. MCF7 cells were cultured in 12-well plate at 26,000 cells/cm2. Then 400\u00a0\u03bcl of media was removed and crystals of formazan were diluted with 500\u00a0\u03bcl of dimethyl sulfoxide (DMSO) . Absorbance of cells was measured at 570\u00a0nm .Cell viability after the acyclovir treatment was evaluated with MTT -2,5-diphenyltetrazolium bromide assay. Concentration of 5\u00a0mg/ml was achieved by reconstituting MTT in DI water. After removal of the supernatant from the wells, 500\u00a0\u03bcl of warm culture media and 50\u00a0\u03bcl of MTT solution were added in each well for two hours at 37\u00a0\u00b0C in 5% CO2 into 12-well plate and cultured in a medium with and without ACV. After overnight incubation cells were detached with trypsin and counted using automated cell counter at 24, 48, 72 and 96\u00a0h.Proliferation in MCF7 cells was determined by plating 6500 cells/cm2, Dynalon Labware, Rochester, NY, USA) was coated with 0.7% agar and 0.3% CM by adding 3\u00a0ml/dish at room temperature for 30\u00a0min. following this, the upper layer of 3\u00a0mL of agar solution with 0.3% agar and 0.7% cell suspension (3125 cells/cm2) was plated. The top agar layer was allowed to solidify and then incubated for 3\u00a0weeks at 37\u00a0\u00b0C in 5% CO2. CM was refreshed 2 times a week. In 21\u00a0days crystal violet was used as a staining for colonies and counting was performed using Leica DMI3000 B light microscope.The ability of cancer cells to form colonies was characterized using a soft agar assay. This assay required 21\u00a0days of growth on the soft agar medium. At the end of 3 weeks, a number of colonies formed per petri dish were counted using a crystal violet stain. Briefly, 1% sterile agar solution was warmed in a microwave and place to 37\u00a0\u00b0C water bath to cool down. 500\u00a0\u03bcg of agarose powder was dissolved in 50\u00a0mL distilled water. The bottom of the petri dish and incubated at 37\u00a0\u00b0C in 5% CO2 overnight before the treatment. After the 72\u00a0h incubation with acyclovir, coverslips with cells were rinsed briefly with PBS for thrice for 5\u00a0min each time. 4% paraformaldehyde was used as a fixative for 10\u00a0min at room temperature. The samples were blocked with 1% BSA and 0.3% Tween-20 in PBS and incubated at room temperature for 1\u00a0h. Following 1\u00a0h incubation, \u03b1-tubulin rabbit mAb Alexa Fluor\u00ae 488 conjugate diluted as 1:200 was used for staining and incubation at 4\u00a0\u00b0C overnight in the dark. After 24\u00a0h, the coverslips were washed 3 times for 5\u00a0min with PBS and then incubated with 0.1\u00a0\u03bcg/mL DAPI for 2\u00a0min. After rinsing again with PBS, aqueous mounting medium was used for mounting coverslips on microscope slides. Finally, coverslips were sealed with a clear nail polish. Images were acquired using EVOS\u00ae FLoid\u00ae Cell Imaging Station . Cellprofiler software was used to evaluate morphometric features of treated MCF7 cells . The equation used for the quantitative measurement of the shape of the cell is given below. This equation uses form factor, FF:Glass coverslips were cleaned for 2\u00a0h in 200\u00a0mL ethanol, 50\u00a0g NaOH, and 300\u00a0mL DI water and finally rinsed with PBS. Cells were seeded on glass coverslips at 10,500 cells/cmThe pipeline for this analysis included four modules: Identify Primary Objects, Identify Secondary Objects, Identify Tertiary Objects and Measure Object Size Shape . Area ra4% paraformaldehyde in PBS was used as a fixative for 10\u00a0min at room temperature and 0.1% Triton was used as a permeabilization solution for 10\u00a0min on ice. Following this, cells were washed 3 times with ice cold PBS and blocked with 1% BSA in PBST at room temperature for 1\u00a0h. Blocked cells were stained with 1\u00a0mg/ml of Ms mAb to E-cadherin in 1% BSA in PBST in a humidified chamber for 24\u00a0h at 4\u00a0\u00b0C. The stained samples were then washed 3x5 min with PBS and incubated overnight with a 2\u00a0mg/ml of secondary antibody goat pAb to Ms IgG Alexa fluor 488 in 1% BSA in PBST. Coverslips were washed 3x5 min with PBS in the dark and incubated with DAPI for 2\u00a0min, and mounted on the microscope slides using glycerol. Images were acquired using EVOS\u00ae FLoid\u00ae Cell Imaging Station .ELx800 plate reader. Measurements were recorded every 3\u00a0min until the value of the control sample exceeded the value of the most active standard (10 nmole/well). The following equation was used to calculate the activity of the enzyme:NAD-dependent ALDH activity was measured using colorimetric assay kit and performed as described in the manufacturer\u2019s instructions. The absorbance was measured at 450\u00a0nm on BioTek 2. After detachment with 0.05% trypsin- EDTA the cells were re-suspended in a serum-free medium. Upper insert was filled with 100\u00a0\u03bcl of the cell suspension (~9x104-1x106 cells per well) while reservoir chamber was filled with 600\u00a0\u03bcl of culture medium. Migration of cells was monitored at 3, 6, and 12\u00a0h at 37\u00a0\u00b0C in 5% CO2. Crystal violet was used as the staining solution to distinguish between migrated and non-migrated cells. A cotton swab was used to remove the cells that were left in the upper chamber of the membrane. Those cells that migrated through the insert were examined and counted with bright-field microscope .Cells were cultured for 72\u00a0h with and without acyclovir at 37\u00a0\u00b0C in 5% CO2, and then in serum-free medium for another 24\u00a0h at 37\u00a0\u00b0C in 5% CO2 in 12-well plates incubated overnight at 37\u00a0\u00b0C in 5% CO2. The medium was then substituted with CO2 free media with and without ACV. A scratch using sharpened toothpick was made on the surface of the well to simulate a wound in vitro. Four randomly areas in a well were selected and imaged in 10\u00a0min intervals for 12\u00a0h using time-lapse microscopy system . The images obtained were processed using ImageJ (image processing software). The sequence of images was analyzed and the open wound area was measured for each image at every hour. A scatter plot of wound area measurements (units) vs. time (in hours) was generated as seen in Additional data 5. A line-of-best fit was used to calculate the slope, which corresponded to the rate of migrated cells.Cell culture was performed at a concentration of 260,000 cells/cmSecretion levels of E-cadherin in a cell culture were measured using a human E-cadherin ELISA colorimetric assay kit and performed as described in the manufacturer\u2019s instructions. Cellular levels of C-Myc, NF-kB p65 and caspase-3 in cell lysates were measured using a human C-Myc ELISA colorimetric assay kit ; a human NF-kB p65 Total ELISA colorimetric assay kit and a human caspase-3 ELISA Kit .Cell lysates were prepared using a 1\u00a0mM phenylmethylsulfonyl fluoride (PMSF) cell extraction buffer, a protease inhibitor cocktail and RIPA buffer . Lysis was performed by adding 500\u00a0\u03bcl extraction buffer to the cell pellet for 30\u00a0min on ice while vortexing every 10\u00a0min. Then the cells were placed in the microcentrifuge tubes at 13,000\u00a0rpm for 10\u00a0min at 4\u00a0\u00b0C. Lysates were stored at -80\u00a0\u00b0C. All experiments were performed in triplicate. Results were expressed as mean concentration\u2009\u00b1\u2009standard deviation and normalized to the number of live cells.6/ml in 1.5\u00a0ml Eppendorf tubes. Cells were centrifuged for 1\u00a0min at 3000\u00a0rpm, washed with ice cold PBS and centrifuged again. Cell pellets were re-suspended in 190 ul of 1x binding buffer with 10 ul Annexin V-FITC and 10 ul of 20ug/ml propidium iodide for 15\u00a0min at room temperature in the dark. Apoptosis of the cells was analyzed by flow cytometry .Programmed cell death was studied using annexin V-FITC apoptosis detection kit . Treated and control cells were harvested and re-suspended in CM to obtain a target concentration of 1\u00d710All reported results below are presented as mean values\u2009\u00b1\u2009standard error values. To calculate differences between means, one-way analysis of variance (ANOVA) was implemented, where a null hypothesis was accepted when all means were equal. Population differences were calculated only for a treatment and a control within the cell line. If at least one mean was different, a follow-up Tukey\u2019s HSD test to compare between groups and calculate p-values of each sample (\u03b1\u2009=\u20090.05).Viability of MCF7 cells and breast epithelial cells after the treatment with 5\u00a0\u03bcM ACV were measured and normalized to the untreated culture cells [Additional file p\u2009<\u20090.05) Fig.\u00a0. Annexinis) Fig.\u00a0.Fig. 1ThWhen examining normal cells and cancerous cells under the microscope, we observed distinctive external characteristic features. Results of the IF staining indicate that cancer cells underwent changes in their morphological characteristics in response to ACV treatment Fig.\u00a0. FF shapp\u2009<\u20090.05). Fig.\u00a0p\u2009<\u20090.05) [Additional file The effect of ACV treatment on the migratory and invasive capacities of the breast cancer cells was also tested. Various environmental factors can modulate the motility of cancer cells and affect invasion capacity of these cells. Teng et al. showed that antiviral drug ribavirin causes a considerable suppression of the migration of renal cell carcinoma cell lines . Boyden p\u2009<\u20090.05) as shown in Fig.\u00a0E-cadherin is secreted in most of the epithelial tissues and normal expression of E-cadherin has been reported to inhibit metastasis and invasion by suppressing epithelial-mesenchymal transition as well as stimulating cell-cell adhesions , 16. We Next, we evaluated the action of ACV on the ability of MCF7 cells to form colonies. This distinguishing feature of the cell transformation and its deregulated growth served as a marker to distinguish between cancer and normal cells, since normal cells do not have the ability to grow in semisolid matrices . ACV treThe concentration changes of C-Myc protein secreted by MCF7 cells were determined in response to ACV treatment. C-Myc oncogene regulates cellular growth and metabolic mechanisms as well as their interconnection . Our resALDH activity is one of the detectors of cancer progression . UpregulRecently, several antiviral agents have been found to possess the ability to decrease the rate of the cells\u2019 proliferation and to promote apoptosis in cancer cells. However, despite the compelling results supporting the clinical use of antiviral agents as an adjuvant therapy in cancer treatment, there is still a lack of studies of the biochemical mechanisms of their anticancer effects , 24. TheHerpesviridae family [in situ hybridization shows that the tumor cell populations were reduced in EBV-encoded RNAs [In this study, we used acyclovir (ACV) to examine the potential of the antiviral treatment on MCF7 breast cancer cell line. Acyclovir is an antiviral drug used in treating infections of e family . In sevee family , 27. Rece family . Moreoveded RNAs . In anotBased on our results, we conclude that ACV as an antiviral agent has a potential suppressive effect on MCF7 breast cancer cells. ACV does not affect viability of non-cancerous breast epithelial cells, while showing a decrease of the viability of MCF7 breast cancer cells. Observed morphological changes and apoptosis analysis demonstrated the ability of ACV to affect the process of programmed cell death of MCF7 cells. The mechanism of apoptosis requires a number of proteins that regulate a proper cell death. One of these proteins is caspase-3 which is included in a family of cysteine proteases . An upreACV was also able to decrease the rate of the growth, colony formation ability, and cell invasion capacity of MCF7 breast cancer cells. These observations correlate with an upregulated secretion of E-cadherin in ACV treated cells. As previously mentioned, E-cadherin is an essential marker in the building of cell-to-cell adhesion and downregulation of this protein leads to the stimulation of invasion and metastasis .Previous studies also report that antiviral agents can affect the secretion of the specific translation initiation factors, oncogenes, and angiogenic genes , 31, 32.Moreover, our study showed that acyclovir was able to influence ALDH activity in the breast cancer cells. The ALDH superfamily consists of 19 isoenzymes with various cellular localizations, tissue/organ distributions and functions. ALDH enzymes catalyze highly reactive aldehydes and some isoenzymes play structural roles related to the osmoregulation and possess antioxidant functions . FollowiA study by Curiel et al. reported that ACV had an inhibitory effect on one of the immune system components as T-regulatory cells (Treg) in glioblastomas through the suppression of indoleamine 2, 3-dioxygenase activity . AnotherThere are several limitations in this study. All experiments were performed in vitro only on one cell line. Future research should focus on an adjuvant strategy of different antiviral agents to determine whether a combinatorial effect exists, and if so, which pathways are affected during the mechanism. Additionally, an examination of epigenetic modifications might serve as a platform for understanding the molecular mechanism underlying the antiviral therapy.In summary, we present evidence that ACV has an anticancer effect on breast cancer cell line. Our study shows that ACV was able to inhibit cancer cells proliferation, colony formation ability and cell invasion capacity, while having no effect on the secretion of certain tumor suppressor genes. Treatment with ACV induced downregulation of ALDH activity, suggesting a decrease of the tumorigenic potential of the treated cancer cells. These results provide new insights on the effect of antiviral agents on the tumorigenesis and metastasis. However, more research is necessary to identify the primary target of ACV and maximize its potential.Additional file 1:p <0.05 as compared with other samples and for pairwise comparison. One way ANOVA followed by Tukey\u2019s test were used for statistical analysis. The data for each cell type were taken from same culture experiment. (DOCX 144\u00a0kb)Viability of MCF7 breast cancer and normal breast epithelial cells in response to acyclovir. Error bars represent 95% confidence interval based on the standard deviation. (*) indicates Additional file 2:p <0.05 as compared with other samples and for pairwise comparison. One way ANOVA followed by Tukey\u2019s test were used for statistical analysis. The data for each cell type were taken from same culture experiment. (DOCX 301\u00a0kb)Population doubling time (hours) of proliferation of MCF7 cells treated with ACV. Error bars represent 95% confidence interval based on the standard deviation. (*) indicates Additional file 3:p\u2009>\u20090.05. P-value for early apoptosis\u2009=\u20091.31579; for late apoptosis\u2009=\u20090.91371. The data for each cell type were taken from same culture experiment. (DOCX 288\u00a0kb)Annexin V staining of apoptotic MCF7 cells after treatment with acyclovir. Left panel is early apoptosis, right panel is late apoptosis. Error bars represent 95% confidence interval based on the standard deviation. One way ANOVA followed by Tukey\u2019s test were used for statistical analysis. Means are not significant, Additional file 4:Quantitative analysis of nucleus and cytoplasm of MCF7 breast cancer cells without and with acyclovir treatment. (DOCX 10\u00a0kb)Additional file 5:A scatter plot of measurements where best fit line and a slope indicate rate of migrating cells. (DOCX 62\u00a0kb)Additional file 6:p <0.05 as compared with other samples and for pairwise comparison. One way ANOVA followed by Tukey\u2019s test were used for statistical analysis. The data for each cell type were taken from the same culture experiment. (DOCX 28\u00a0kb)NF-kB p65 (pg/10^3 cells) expression of MCF7 cells in response to ACV treatment. Error bars represent 95% confidence interval based on the standard deviation. (*) indicates"} +{"text": "One of the reasons for the popularity of meta\u2010analysis is the notion that these analyses will possess more power to detect effects than individual studies. This is inevitably the case under a fixed\u2010effect model. However, the inclusion of the between\u2010study variance in the random\u2010effects model, and the need to estimate this parameter, can have unfortunate implications for this power. We develop methods for assessing the power of random\u2010effects meta\u2010analyses, and the average power of the individual studies that contribute to meta\u2010analyses, so that these powers can be compared. In addition to deriving new analytical results and methods, we apply our methods to 1991 meta\u2010analyses taken from the Cochrane Database of Systematic Reviews to retrospectively calculate their powers. We find that, in practice, 5 or more studies are needed to reasonably consistently achieve powers from random\u2010effects meta\u2010analyses that are greater than the studies that contribute to them. Not only is statistical inference under the random\u2010effects model challenging when there are very few studies but also less worthwhile in such cases. The assumption that meta\u2010analysis will result in an increase in power is challenged by our findings. There are many motivations for including meta\u2010analyses in systematic reviews. The Cochrane HandbookThe power of a hypothesis test is the probability that the null hypothesis is rejected when it is false. Bayesian methods would be needed to instead calculate the probability that the null hypothesis is false, but here we focus on classical methods. Power analysis for meta\u2010analysis is a sufficiently important topic to warrant an entire chapter devoted to it in the introductory text by Borenstein et alOur other main focus is on the use of random\u2010effects meta\u2010analyses. The random\u2010effects modelOthers have previously discussed power calculations under the random\u2010effects model.We should however be clear from the outset that meta\u2010analysis and study specific hypothesis tests involve testing different types of hypotheses: in a meta\u2010analysis, we test whether or not the average effect is a particular value (for example zero), and for individual studies we test whether or not the true study specific treatment effect is a particular value. The distinction between these 2 types of hypotheses is especially clear when using the random\u2010effects model, where we assume that the true treatment effects differ across studies. Whilst recognising that the meta\u2010analysis and study specific hypothesis tests differ in this way, we will still compare the power of these two types of tests. We should also recognise that the study\u2010specific and fixed\u2010effect model hypothesis tests possess, under the assumptions that these methods make, the correct significance level. However, the conventional random\u2010effects model hypothesis test only retains the nominal significance level approximately. To more fairly compare the power of different hypothesis tests, we should use the same actual significance level throughout, and this is only approximately the case here. Some of the power of the conventional random\u2010effects model hypothesis test is therefore an artifact of the approximate nature of the methods used.One motivation for developing methods for power analysis under the random\u2010effects model is so that we can retrospectively determine the powers of the random\u2010effects meta\u2010analyses from a large sample of meta\u2010analyses from Cochrane. We therefore calculate what is sometimes referred to as the \u201cobserved power\u201d in this empirical investigation. Then, by comparing the power of these meta\u2010analyses to the average power of the studies that contribute to them, we can empirically investigate the validity of the notion that random\u2010effects meta\u2010analyses result in an increase in power. This empirical investigation of a large number of random\u2010effects meta\u2010analyses is one important contribution of this paper. The new Monte Carlo method that we develop for this purpose is another important contribution. Retrospective power calculations have some value in practice because they have the potential to explain why effects were not detected. For example, a systematic reviewer might be disappointed or surprised not to detect a particular effect, but this is likely to be mitigated or explained by a calculation that reveals that the power was in any case very low. Although low power also manifests itself as wide confidence intervals, a power calculation provides a much more direct statement about the difficulty in detecting effects than a confidence interval. However this should not be taken to suggest that the usual statistical inferences, made for example by using confidence intervals, are in any sense deficient because they do not involve power calculations. Readers may also be able to suggest other reasons why retrospective power calculations could be of interest.Despite this, we would not advocate the routine use of retrospective power calculations in meta\u2010analysis, rather they are likely to be useful in some instances to clearly convey the difficulty in detecting particular effects. In our empirical investigation, we retrospectively investigate the powers of random\u2010effects meta\u2010analyses so that these powers can be compared to study specific powers. Our retrospective power calculations are performed to answer the more general question of whether or not random\u2010effects meta\u2010analyses usually provide an increase in power, rather than to retrospectively investigate the power for any specific meta\u2010analysis. Those who might advocate the routine use of retrospective power calculations should examine the arguments made by Hoenig and HeiseyAnother motivation is to develop a method for power analysis that is most suitable for those who wish to perform such an analysis at the planning stage. As Borenstein et alThe rest of the paper is set out as follows. In section 2In this section, we derive the power of the individual studies that contribute to the meta\u2010analysis. At this stage, we make no use of meta\u2010analysis methodology, because we make no assumptions about how the true treatment effects for each study relate to each other.\u03bci denote the true effect in study i and let k denotes the number of studies. We let Yi denotes this study's estimate of \u03bci and let \u03c3i denotes the corresponding standard error. These standard errors are usually estimated in practice but treated as if fixed and known in analysis. We suppress the fact that the within\u2010study standard errors are estimated prior to performing the meta\u2010analysis, and so write \u03c3i instead of \u03c3^i. We also use normal within\u2010study approximations Yi\u223cN, as is conventional in meta\u2010analysis and common when analysing data from individual trials. We assume that 2\u2010tailed hypothesis tests are used throughout. We make no attempt to distinguish \u201caccepting the null hypothesis\u201d and \u201cnot rejecting the null hypothesis\u201d and other more subtle issues related to the interpretation of hypothesis and significance testing.We let H0:\u03bci=\u03bc0 versus H1:\u03bci\u2260\u03bc0 in the ith study is given by Zi=(Yi\u2212\u03bc0)/\u03c3i; typically we set \u03bc0=0 to test for no effect. Under the null hypothesis, H0:\u03bci=\u03bc0, Zi\u223cN. Under the alternative hypothesis Zi\u223cN, where \u03b4i=\u03bci\u2212\u03bc0. The null hypothesis is rejected using a 2\u2010tailed test by the ith study if |Zi|\u2a7eZa, and this hypothesis is accepted if |Zi|