ELG1 gene of yeast lead to genomic instability, manifested in high levels of genetic recombination, chromosome loss, and gross chromosomal rearrangements. Elg1 shows similarity to the large subunit of the Replication Factor C clamp loader, and forms a RFC-like (RLC) complex in conjunction with the 4 small RFC subunits. Two additional RLCs exist in yeast: in one of them the large subunit is Ctf18, and in the other, Rad24. Ctf18 has been characterized as the RLC that functions in sister chromatid cohesion. Here we present evidence that the Elg1 RLC (but not Rad24) also plays an important role in this process. A genetic screen identified the cohesin subunit Mcd1/Scc1 and its loader Scc2 as suppressors of the synthetic lethality between elg1 and ctf4. We describe genetic interactions between ELG1 and genes encoding cohesin subunits and their accessory proteins. We also show that defects in Elg1 lead to higher precocious sister chromatid separation, and that Ctf18 and Elg1 affect cohesion via a joint pathway. Finally, we localize both Ctf18 and Elg1 to chromatin and show that Elg1 plays a role in the recruitment of Ctf18. Our results suggest that Elg1, Ctf4, and Ctf18 may coordinate the relative movement of the replication fork with respect to the cohesin ring.Mutations in the  Saccharomyces cerevisiae, are extremely useful for studying the basic principles of genome stability and maintenance.Genomic instability is a hallmark of cancer cells. Most human cancer cells show signs of genome instability, ranging from elevated mutation rates, to gross chromosomal rearrangements including deletions and translocations. Several surveillance and repair mechanisms operate in eukaryotic cells to ensure the stability of the genome. The current view is that most spontaneous chromosomal rearrangements arise during DNA replication. The activity of the DNA polymerases may be impaired by the presence of secondary structures, bound proteins or DNA lesions; this may lead to stalling or even collapse of replication forks. In response, cellular mechanisms are activated that arrest cell cycle progression, induce DNA repair, and restore replication ELG1 gene was identified in yeast as a mutant that causes enhanced levels of genomic instability ELG1 in yeast leads to increased recombination levels elg1 mutants also exhibit elongated telomeres RAD24 and CTF18. Elg1, Rad24 and Ctf18 form three alternative RFC-like (RLC) protein complexes, by interacting with the four small subunits of RFC S. pombe and hRad17 humans) S. cerevisiae, Rad9/Rad1/Hus1 in S. pombe and humans] The elg1 mutants have synthetic growth defects when combined with genes that are involved in homologous recombination, DNA repair, replication fork restart, checkpoint response and sister chromatid cohesion ctf4: double mutant spores elg1 ctf4 germinate, but fail to undergo cell divisions ctf4 and Δelg1 mutant cells show partially overlapping phenotypes. Both mutants have high levels of recombination, chromosome loss, as well as synthetic growth defects with rad52, mec1Genetic screens in yeast have shown that CTF4 was found, together with CTF18, in two independent screens for mutants that affect chromosome transmission fidelity ctf4 and ctf18 exhibit elevated levels of recombination ctf screen for increased chromosome loss ctf7 mutants, allowing them to grow at a higher temperature before arresting A clear linkage exists between replication progression and sister chromatid cohesion. First, Ctf7/Eco1, encoded by another gene that was found in the ELG1 has tight genetic interactions with CTF4 and CTF18, it is not known how it affects genomic stability. Here we performed a genetic screen in order to find high copy number suppressors of the SL interaction between elg1 and ctf4. We found a cohesin subunit and a cohesin loader, pointing to the fact that the synergistic genetic interaction between elg1 and ctf4 is due to enhanced defects in sister chromatid cohesion. Consistent with this possibility we also show that elg1 mutants exhibit defects in cohesion. Our results show that both the Elg1 and the Ctf18 clamp loaders affect sister chromatin cohesion and establish a link between Elg1 activity and cohesin loading.Although ELG1 and CTF4 are synthetic lethal (SL), namely, despite the fact that each single-mutant grows well, the double mutant is inviable ELG1 centromeric plasmid enabling the strain to stay alive. This plasmid has two marker genes: LEU2 and ADE3. In the appropriate genetic background, the presence of the ADE3 gene causes accumulation of a red pigment. Thus, elg1 ctf4 strains carrying the LEU2/ADE3/ELG1 plasmid grow well, and form uniformly red colonies, as they are unable to lose the ELG1-containing plasmid. These cells were then transformed with a yeast genomic library cloned in a high copy number plasmid (containing the URA3 marker). Cells that received a plasmid with a gene that, when over-expressed, can suppress the SL phenotype of elg1 ctf4 are now able to lose the ELG1-ADE3-LEU2 plasmid, and therefore show white/red sectored colonies  protein family, and two non-SMC subunits, Scc1, which is a member of the kleisin family, and Scc3. Smc1, Smc3 and Scc1 form a ring large enough to contain two dsDNA molecules SCC2, encodes a protein that forms, together with Scc4, a complex that helps loading cohesin on the DNA After transformation with the high copy number library, we screened about 30,000 colonies; 62 of them showed some degree of sectoring and were further analyzed. After re-introduction of the plasmid to naïve yeast strains, only 29 clones gave a positive result. Twenty six carried the SCC1 are related to its function as a structural component of cohesin. This raises the question of whether Scc1 is the only subunit of the complex able to suppress the synthetic lethal phenotype of elg1 ctf4 strains. The fact that CTF4 was isolated numerous times but ELG1 was not, suggest that the screen may not have been saturated enough. Alternatively, only Scc1 and Scc2 may be the limiting factors that determine the amount of cohesin complexes loaded onto the DNA. Scc1 may be the limiting component in cohesin formation, and Scc2 availability may be limiting the amount of Scc1 loaded. According to this possibility ctf4 elg1 strain is inviable due to a low level of the cohesin ring, for example, at the centromeres.All the known phenotypes of mutants in SCC1, SCC2, SMC1 or SMC3 from LEU2-marked plasmids in a ctf4 elg1 strain carrying a ELG1/URA3 plasmid. If overexpression of any of these genes can suppress the SL phenotype, the ELG1/URA3 plasmid can be lost, allowing the cells to grow on 5-FOA medium (which selects for Ura- cells). SCC1and SCC2, (and of course ELG1), but not SMC1 or SMC3, can rescue the SL phenotype of the ctf4 elg1 double mutant strain. Thus, in agreement with the second hypothesis expressed above, Scc1 and Scc2 are the only limiting components able to suppress the lethality when overexpressed. Scc1 seems to be a slightly better suppressor than Scc2  were detected in these experiments (data not shown).To investigate whether the suppression by Scc1 was due to bypass of the elg1 and mutants defective in each of the components of cohesin. elg1/ELG1 SCC1/scc1-73 and elg1/ELG1 SMC1/smc1-259 double heterozygous diploids. The double mutants elg1 scc1 and elg1 smc1 were very sick being either completely unable to form colonies or forming tiny, sick colonies that failed to develop.In order to determine the genetic relationship between Elg1 and the Cohesin complex, we attempted to create double mutants between elg1 and mutations in other components of the cohesin complex such as Scc3 and Smc3, and additional genes that interact with cohesin, such as the loaders Scc2 and Scc4 elg1 exhibited synthetic fitness phenotypes already at the permissive temperature when combined with any member of the cohesin complex or with the Scc2-Scc4 loaders, but not with mutations in the separin Esp1, in the securin Pds1 or in Pds5. These synthetic genetic interactions show that Elg1 activity is important in the early stages of cohesin establishment and not in the maintenance or the cleavage of cohesin, strengthening the possibility that both Elg1 and Ctf4 play an important role in the coordination between replication fork progression and cohesin loading.We used the above method to examine the genetic relationship between TetO array is integrated approximately 40 kb from the centromere of chromosome V. These strains constitutively express a GFP-tagged TetR protein, which binds the arrays and can be visualized as a single GFP “dot”. In G2-arrested cells, lack of chromatid cohesion is seen as a “double dot” elg1 mutants showed this phenotype in 18.5% of the cells. Mutations in CTF18 exhibited a stronger phenotype (33%). Interestingly, double mutants elg1 ctf18 exhibit the same phenotype as the ctf18 single, indicating epistatic interactions. In contrast, mutations in the gene encoding the third RFC-like protein, Rad24, had only a modest effect, if at all, in sister chromatid cohesion, and no aggravating phenotype was observed when the rad24 mutation was combined with the elg1 or ctf18 mutations, or with the double mutant  interaction ctf4 exhibits defects in sister chromatin cohesion elg1 mutant such as its sensitivity to MMS . Moreover, we show that both proteins localize to chromatin, and that Elg1 is required in order to direct Ctf18 to the chromatin fraction  the elg1 ctf18 double mutant showed a higher sensitivity compared to the single mutants, suggesting that these proteins work in different pathways in the presence of DNA damage elg1 and ctf18 mutants of S. pombeelg1 mutants exhibit elongated telomeres, whereas ctf18 mutants have short telomeres ELG1 or CTF18 have the same effect in suppressing the hst3 hst4 mutant that contains hyperacetylated histone H3 at position K56 CTF4, consistent again with a model in which Elg1, Ctf18 and Ctf4 carry out related functions.In previous studies, different interactions between ELG1 can partially suppress the temperature sensitivity and the cohesion defects of an ctf7/eco1-1 mutant, whereas Elg1 overexpression exacerbates its conditional growth defect. Ctf7 is a pivotal sister chromatid cohesion factor that was found to be important for cohesion establishment ELG1 and ctf7 strengthen our characterization of Elg1 as a cohesion factor. We further extend these conclusions by analyzing the interactions between elg1 and all known cohesion factors. In addition, we show that Elg1 plays a possible role in recruiting the Ctf18 RLC to the chromatin, thus providing a mechanistic explanation for the epistatic genetic interactions between elg1 and ctf18 in sister chromatid cohesion. Consistent with our results  and pSBA419 (LEU2-ELG1-ADE3) have been described URA3-marked and LEU2-marked plasmids pSCC1, pSCC2, pSMC1, pSMC3 and pELG1 were created by cloning the respective genes in YEplac181 (LEU2) or YEplac195 (URA3).A list of all strains used in this paper is presented as elg1 ctf4 ura3 ade2 ade3 leu2/pSBA419) was transformed with a high copy number URA3 library as described Strain MK1001 (elg1 and ctf4 mutants carrying various plasmids was measured as described The rate of recombination of wild type, Logarithmically growing cultures were arrested in G1 with α factor, at the beginning of S-phase with hydroxyurea or in G2 with nocodazole and incubated for one hour before being cropped by centrifugation This experiment was carried out as described


Salmonella Enteritidis phage type 6 (PT6) increased dramatically in the United Kingdom during 1997. The sharp rise suggests that PT6 contamination has spread rapidly throughout a basic food commodity; however, the source and food vehicle remain unknown. We present evidence from three outbreaks suggesting a possible link between PT6 and eggs. Poor documentation of the egg supply network continues to pose problems for public health investigators. Thorough investigation of all future PT6 outbreaks and case-control studies of sporadic infections are needed to confirm the etiology of PT6 infection.


The I1307K allele was equally distributed among women with sporadic  and inherited  breast and/or ovarian cancer irrespective of their being diagnosed before or after 42 years of age and among asymptomatic  and cancer manifesting BRCA1/2 carriers . Taken together, the prevalence of the I1307K allele was significantly higher in BRCA1/2 carriers compared to non-BRCA1/2 carriers . The high prevalence of the I1307K allele among BRCA1/2 carriers is not associated with increased cancer risk but seems to be genetically connected because of Jewish ancestry. © 2000 Cancer Research CampaignThe frequency of the APC I1307K mutation and its association with disease pattern was examined in 996 Ashkenazi women consisting of individuals with either sporadic (


BRCA1 and BRCA2 are the two most important genes associated with familial breast and ovarian cancer susceptibility. In addition, PALB2 has recently been identified as a breast cancer susceptibility gene in several populations. Here we have evaluated whether large genomic rearrangement in these genes could explain some of Finnish breast and/or ovarian cancer families.BRCA1, BRCA2 and PALB2. The families have been comprehensively screened for germline mutation in these genes by conventional methods of mutation analysis and were found negative.Altogether 61 index patients of Northern Finnish breast and/or ovarian cancer families were analyzed by Multiplex ligation-dependent probe amplification (MLPA) method in order to identify exon deletions and duplications in BRCA1, deleting the most part of the gene (exon 1A-13) in one family with family history of ovarian cancer. No large genomic rearrangements were identified in either BRCA2 or PALB2.We identified one large deletion in BRCA1 or BRCA2 mutation screening, when found negative, could benefit from screening for large genomic rearrangements at least in BRCA1. On the contrary, the genomic rearrangements in PALB2 seem not to contribute to the hereditary breast cancer susceptibility.In Finland, women eligible for  BRCA1 and BRCA2 are the two major susceptibility genes, accounting for varying fraction of familial breast and ovarian cancer cases in different populations. In Finland, mutations in these genes explain approximately 20% of breast and ovarian cancer families [BRCA1 and BRCA2 are point mutations and small insertions/deletions, but increasing number of large genomic rearrangements in both genes have been identified in different populations [BRCA1 and BRCA2 mutations due to genomic rearrangements is not expected to vary markedly in different populations, although there might be accumulation of certain mutations due to a founder effect.Breast cancer is the most frequently occurring malignancy in women. ulations -6. Rearrulations ,6. The pBRCA1 or BRCA2 [BRCA1, BRCA2, ATM and RAD50 mutations [th century, and it was not until the 17th century that the vast inland regions were gradually inhabited by a relatively small number of individuals, resulting in several regionally occurring founder mutations [BRCA1 and BRCA2 might still be at least partly responsible for the hereditary predisposition to breast and ovarian cancer in Finland.Previous studies performed in the Finnish population have not observed large genomic rearrangements in or BRCA2 -9. Howevor BRCA2 , the stuor BRCA2 . In Finlutations -12, whicPALB2 was recently identified as a breast cancer susceptibility gene [PALB2 encodes a protein that binds to BRCA2 and this interaction is crucial for certain BRCA2 DNA damage response and tumor suppression functions [PALB2 are expected to be deleterious, and all result in protein truncations. The risk estimates for PALB2 mutations have ranged from two- to fourfold, although some PALB2 mutations have been suggested to have higher penetrance [PALB2 could also explain some of the Finnish breast and/or ovarian cancer families.ity gene ,14 and mity gene -17. PALBunctions . The brenetrance -15. HereBRCA1, BRCA2 and PALB2 genes. MLPA has been proven to be very useful in detecting copy number changes in genomic sequences [In the current study we have used Multiplex ligation-dependent probe amplification (MLPA) in order to identify exon deletions and duplications in the equences . The famequences  and wereBRCA1, BRCA2 and PALB2 and were found negative [Altogether 61 index patients of Northern Finnish breast and/or ovarian cancer families were selected for the study. These families have been screened for germline mutations in negative . 41 of tBRCA1 (primary screening kit P002B and confirmation kit P087), BRCA2 (P045B) and PALB2 (P057) kits  were used according to the manufacturer's instructions. After PCR amplification with IRD800 labeled primers the samples were analyzed with Li-Cor IR2 4200-S DNA Analysis system  and Gene Profiler 4.05 analysis program . For BRCA1 analysis the used deletion control had deletions in exons 1A-2, for BRCA2 analysis the control sample showed constant 50% reduction in band intensity resulting from a SNP locating three base pairs from the ligation site, and for PALB2 analysis the used control had heterozygous deletions in exons 1–3 and 5–10 in addition to homozygous deletion of exon 4.The SALSA MLPA kits for The information regarding the integrated density of each band received from GeneProfiler was analyzed by MLPA spreadsheets  in Excel Software according to the instructions. Dosage quotients 0.35–0.65 were considered deleted and dosage quotients 1.35–1.65 duplicated, and samples with quality value (standard deviation of the control ligation products) exceeding 0.1 were rejected. For the DNA sample positive for a genomic rearrangement, analysis was repeated using an independent sample in an independent assay.BRCA1, BRCA2 and PALB2 genes by MLPA. In BRCA2 and PALB2 no deletions or duplications were observed. We did, however, observe one large deletion in BRCA1 (exon 1A-13)  of the identified Northern Finnish BRCA1 positive families. Even though the deletion allele was observed only in one out 61 currently analyzed families, our results suggests that women eligible for BRCA1 or BRCA2 mutation screening, when found negative, could benefit from screening for large genomic rearrangements, at least in BRCA1. Additional work is still needed in order to determine the prevalence of BRCA2 rearrangements in Finland, as the current study was based only on relatively small number of families.To date, only three different utations , accountBRCA1, our results support the previous conclusions that the genomic rearrangements in BRCA1 and BRCA2 are not a major cause for increased breast cancer susceptibility in Finland, and that the previously reported Finnish founder mutations represent the majority of BRCA1 and BRCA2 positive families [PALB2 gene were identified in the studied index cases of 61 families. This suggests that genomic rearrangements in PALB2 are very rare, which has also been indicated by a previous study [PALB2 appears to be the previously reported founder truncation mutation [Despite the identification of one large genomic deletion in families -9,27. Nous study . At leasmutation .BRCA1 or BRCA2 mutation screening, when found negative, could benefit from screening for large genomic rearrangements at least in BRCA1. In contrast, the genomic rearrangements in PALB2 seem not to contribute to the hereditary breast cancer susceptibility in Finland.In Finland, women eligible for The authors declare that they have no competing interests.KP carried out the MLPA and data analysis, and drafted the manuscript. HE, JN and SS helped to draft the manuscript. RW participated in study design and in drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The Ad5CMV-αVEGF vector may be useful in targeting the tumour vasculature in the treatment of breast cancer. © 2001 Cancer Research Campaign http://www.bjcancer.comIncreased expression of VEGF in several types of tumours has been shown to correlate with poor prognosis. We used a replication-deficient adenoviral vector containing antisense VEGF cDNA (Ad5CMV-αVEGF) to down-regulate VEGF expression and increase the efficiency of delivery of the antisense sequence in the human breast cancer cell line MDA231-MB. Transfection of these cells with Ad5CMV-αVEGF in vitro reduced secreted levels of VEGF protein without affecting cell growth. Moreover, injection of the Ad5CMV-αVEGF vector into intramammary xenografts of these cells established in nude mice inhibited tumour growth and reduced the amount of VEGF protein and the density of microvessels in those tumours relative to tumours treated with the control vector Ad5(dl312). Our results showed that antisense VEGF


The amount of NO3 in warm seasons is twofold that of cold seasons , and there was a significant difference between cold and warm seasons (Table 1). Annual wet deposition of ammonia (NH3) was 10 kg/ha/year in the Chitgar Parkland[1].An investigation of air pollution in the Tehran metropolitan area between 1992–2000 indicated that there are significant amounts of nitrate ion (NO


Also in Table 1, a row of data for men was omitted and should have indicated 36 men (45.6%) with low trust and 43 (54.4%) with high trust. This article has been corrected.1In the Original Investigation titled “Characteristics of Health Care Organizations Associated With Clinician Trust: Results From the Healthy Work Place Study,”


The as-prepared CsPbBr3 PNCs and Cs4PbBr6 PNCs exhibit different optical properties that depend on their morphology, size, and structure. The photoluminescence (PL) emission and quantum yield (QY) of the CsPbBr3 PNCs can be tuned by changing the ultrasound power, radiation time, and the height of the vibrating spear. The optimized CsPbBr3 PNCs show a good stability and high PL QY of up to 85%. In addition, the phase transformation between CsPbBr3 PNCs and Cs4PbBr6 PNCs can be obtained through varying the amount of oleylamine (OAm) and water. The mechanism of this transformation between the CsPbBr3 PNCs and Cs4PbBr6 PNCs and their morphology change are studied, involving ions equilibrium, anisotropic growth kinetics, and CsBr-stripping process.We demonstrate an ultrasonication-assisted synthesis without polar solvent of CsPbBr A weak 4PbBr6 PNCs, typically a green emission arises either from defects or from impurities or from a combination of both [4PbBr6 PNCs did not demonstrate PL emission over the whole visible spectrum due to their wide bandgap (Eg(Cs4PbBr6) = 3.94 eV), while the observed weak PL emission results from a small portion of CsPbBr3 impurities in the Cs4PbBr6 PNCs  emerge, which have been confirmed to result from the formation of Cs4PbBr6 PNCs [3 PNCs. Based on this process it can be concluded that the excess amount of OAm triggers the transformation between CsPbBr3 PNCs and Cs4PbBr6 PNCs.Furthermore, the effect of the amount of OAm on the phase transformation was investigated. As shown in Br6 PNCs . When ad3 PNCs to Cs4PbBr6 PNCs was further confirmed by using TEM. When the amount of OAm is between 0.5 and 1.5 mL, the morphology of PNCs gradually becomes irregular and some hexagonal shapes emerge , suggesting the formation of CsPbBr3 PNCs. Moreover, the PL QY of as-prepared CsPbBr3 PNCs was calculated to be ca. 70%. Interestingly, the CsPbBr3 PNCs show a high stability in ambient environment, as shown in 3 PNCs to Cs4PbBr6 PNCs was achieved  solubility of hexane in water [3 PNCs have a higher stability than Cs4PbBr6 PNCs against water.The addition of led to the decomposition of CsBr3 PNCs  and the Br3 PNCs . This isin water . The abo3 PNCs [3 PNCs could be easily tuned. More importantly, with lower the immersion heights of the vibrating spear higher PL QY of CsPbBr3 PNCs were achieved. The as-prepared CsPbBr3 PNCs show a high PL QY of up to 85% and a considerable photostability and chemical stability. The Cs4PbBr6 PNCs are obtained via direct ultrasonication of precursors or after adding OAm in the pre-synthesized CsPbBr3 PNCs solution. The phase transformation of orthorhombic CsPbBr3 NCs to rhombohedral Cs4PbBr6 NCs is promoted by the capacity of organic ligands to dissolve PbBr2, and by the formation of lead oleate and the dissociation of CsPbBr3 PNCs. Morphology changes are mainly ascribed to the anisotropic growth of the crystals. In addition, a reverse transformation from Cs4PbBr6 PNCs to CsPbBr3 PNCs can be achieved by adding water to pre-synthesized Cs4PbBr6 PNCs. The developed ultrasonication assistance results in the successful control over the phase transformation of PNCs, which can find widespread application in photoelectronic devices. We anticipate that this work can be extended to prepare other halide perovskites.In summary, we demonstrate the effect of small changes in the environment of capping ligands and water on the crystal structure and stoichiometry of PNCs. This study expanded our recent work of synthesizing differently shaped CsPbBr3 PNCs . Similar2CO3, 99%), lead bromide , liquid paraffin (90%), oleic acid , oleylamine , and anhydrous toluene (99.8%) were purchased from Shanghai Aladdin Biochemical Technology Co. The chemicals used in the present work were of analytical grade and used without further purifications.Cesium carbonate (CsCsPbBr3 PNCs: The PNCs were prepared via modifying the procedures reported by Tong and Rao and co-workers [2CO3 (0.15 mmol) and PbBr2 (0.30 mmol) powders were added to a mixture of 10 mL liquid paraffin (LP), 0.50 mL OA and 0.50 mL OAm. Then the reaction medium was processed by tip-sonication at a power of 120 W for 40 min. During the sonication, the colorless reaction medium gradually transformed into a yellow and then an orange-yellow solution, which suggests the formation of PNCs and demonstrates strong fluorescence emission under 365 nm UV light excitation. After completion of the reaction, unreacted precursors and excess ligands were removed by centrifugation at a speed of 3000 rpm for 10 min and then the precipitate was redispersed in 5.0 mL of toluene. Then, the colloidal solution was centrifuged at a speed of 12000 rpm for 5 min and the sediment was redispersed in toluene for further characterization.-workers –30. In aCs4PbBr6 PNCs: Cs2CO3 (0.15 mmol) and PbBr2 (0.30 mmol) powders were added to a mixture of 10 mL liquid paraffin, 0.50 mL OA and 3.0 mL OAm, while keeping other synthesis conditions as the same as that of CsPbBr3 PNCs.4PbBr6 PNCs solution and shaken slightly, which is a modification of the work reported by Wu and co-workers [50–250 μL of water was added to 5.0 mL of the pre-synthesized Cs-workers .4PbBr6 PNCs was characterized by using a field-emission scanning electron microscope . The UV–vis absorption spectra of the samples were measured using a UV–vis spectrometer  over the wavelength range from 300 to 700 nm, at 1 nm intervals. The PL spectra of the PNCs were recorded using a fluorescence spectrophotometer  using a Xe lamp as an excitation source.The crystal surface morphology of the PNCs was characterized by transmission electron microscopy  with an accelerating voltage of 100 kV. High-resolution TEM (HRTEM) was carried out on a JEOL JEM-2100F instrument operating at 200 kV. The crystal phases of the products were measured using an X-ray diffractometer  with a Cu Kα radiation source (λ = 0.15418 nm) at a counting rate of 2° per minute in the scanning angle (2θ) range from 5° to 50°. The surface morphology of CsFile 1Additional PL spectra, SEM image, and UV–vis absorption spectra.File 24PbBr6 to CsPbBr3 PNCs after addition of water.Video showing the transformation from Cs


World Arthritis DayA report in this issue found that adults with arthritis had higher prevalences of symptoms of anxiety (22.5%) and depression (12.1%) compared with adults without arthritis (


SLC2A3) is present in the human population at variable copy number. An overt disease phenotype of SLC2A3 copy number variants has not been reported; however, deletion of SLC2A3 has been previously reported to protect carriers from rheumatoid arthritis, implicating GLUT3 as a therapeutic target in rheumatoid arthritis. Here we aim to perform functional analysis of GLUT3 copy number variants in immune cells, and test the reported protective association of the GLUT3 copy number variants for rheumatoid arthritis in a genetic replication study.The gene encoding glucose transporter 3 (GLUT3, SLC2A3/GLUT3 expression and glycolysis capacity. We genotyped the SLC2A3 copy number variant in four independent cohorts of rheumatoid arthritis and controls and one cohort of multiple sclerosis and controls.Cells from genotyped healthy controls were analyzed for SLC2A3 correlates directly with expression levels of GLUT3 and influences glycolysis rates in the human immune system. The frequency of the SLC2A3 copy number variant is not different between rheumatoid arthritis, multiple sclerosis and control groups.Heterozygous deletion of SLC2A3 gene copy number dependent phenotype, our study of large groups of rheumatoid arthritis cases and controls provides no evidence for rheumatoid arthritis disease protection in deletion carriers. These data emphasize the importance of well powered replication studies to confirm or refute genetic associations, particularly for relatively rare variants.Despite a robust  •SLC2A3/GLUT3 correlates to SLC2A3 gene copy number in a dose dependent manner.T cell and macrophage expression of •SLC2A3Glycolysis rates are reduced in individuals harboring a deletion of the GLUT3 gene •SLC2A3 is not associated with protection from rheumatoid arthritisDeletion of •SLC2A3 is not associated with risk for multiple sclerosisDeletion of •SLC2A3 deletion.GLUT3 is not a viable therapeutic target for RA as previously proposed based on a protective association of  We also did not observe evidence of an association with multiple sclerosis, despite initial encouraging results in multiplex families with autoimmunity.Prompted by a previous study of protection from RA, we explored the biological effects of SLC2A3 deletion. Foremost, we note that our current data reveal that the number of PennCNV false calls can be significant (tableS4). By their nature, random sampling of rare events can lead to false differences more often than common events [There are a number of possible explanations of why this study did not replicate the previously reported association of protection from RA with the n events . We analSLC2A3 CNV with RA [SLC2A3 CNV.Our result is consistent with two previous genome-wide CNV association studies that also found no association of the  with RA ,16, howeSLC2A3 CNVs in the human population at fairly constant frequencies between ethnically diverse groups . K.R.S is supported by the NIH CDA K01AR071502 (USA). PKG is supported by NIH grant UH2AR067694 (USA).This work was supported by the


Nature Communications 10.1038/ncomms6404, published online 20 November 2014Correction to: This Article contains an error in Equation 2 in that the denominator is inverted. The correct form of Equation 2 is:


Saccharomyces cerevisiae as well as contributing to the integration of the nuclear export of mature mRNA with preceding steps in the nuclear phase of the gene expression pathway. Nab2 is constructed from an N‐terminal PWI‐fold domain, followed by QQQP and RGG motifs and then seven CCCH Zn fingers. The nuclear pore‐associated proteins Gfd1 and Mlp1 bind to opposite sides of the Nab2 N‐terminal domain and function in the nuclear export of mRNA, whereas the Zn fingers, especially fingers 5–7, bind to A‐rich regions of mature transcripts and function to regulate poly(A) tail length as well as mRNA compaction prior to nuclear export. Nab2 Zn fingers 5–7 have a defined spatial arrangement, with fingers 5 and 7 arranged on one side of the cluster and finger 6 on the other side. This spatial arrangement facilitates the dimerization of Nab2 when bound to adenine‐rich RNAs and regulates both the termination of 3′ polyadenylation and transcript compaction. Nab2 also functions to coordinate steps in the nuclear phase of the gene expression pathway, such as splicing and polyadenylation, with the generation of mature mRNA and its nuclear export. Nab2 orthologues in higher Eukaryotes have similar domain structures and play roles associated with the regulation of splicing and polyadenylation. Importantly, mutations in the gene encoding the human Nab2 orthologue ZC3H14 and cause intellectual disability.The poly(A) RNA binding Zn finger ribonucleoprotein Nab2 functions to control the length of 3′ poly(A) tails in  Saccharomyces cerevisiae Nab2 (ScNab2) accompanies mature transcripts as they move through nuclear pores to the cytoplasm, after which ScNab2 is thought to be removed from the mRNA by the DEAD‐box RNA helicase, Dbp5ScNab2 is then recycled back to the nucleus through nuclear pores using the transport factor, karyopherin β2 .ScNab2 shows genetic interactions with the splicing machineryIn eukaryotes, the separation by the nuclear envelope of transcription from translation enables mRNAs to be modified by capping, splicing, and polyadenylation. These processing steps are mediated by a large number of different proteins that interact with transcripts as they pass through the nuclear phase of the gene expression pathway before they are finally exported to the cytoplasm through nuclear pores.Drosophila orthologue, DmNab2 are also required for proper poly(A) tail length control,ZC3H14 gene have been linked to a nonsyndromic form of autosomal recessive intellectual disability,DmNab2 mutant flies exhibit impaired short‐term memory and defects in neuronal patterning in the learning and memory center (mushroom body) of the fly brain.DmNab2 mutant flies rescues function, indicating that ZC3H14 is a functional orthologue of DmNab2.In higher eukaryotes, the human Nab2 orthologue, ZC3H14, and ScNab2 in S. cerevisiae. ScNab2 contains an N‐terminal domain that has a Proline‐Tryptophan‐Isoleucine (PWI)‐like fold,ScNab2 Zn fingers (ZnFs) are arranged into three groups: ZnF12; ZnF34; and ZnF567. The Nab2 orthologues in other species—S. pombe Nab2 (SpNab2); C. thermophilum Nab2 (CtNab2); D. melanogaster Nab2 (DmNab2); C. elegans SUT‐2 (CeSUT‐2); H. sapiens ZC3H14 (HsZC3H14)—have a similar overall domain architecture  RNA using CtNab2 and ScNab2.ScNab2 Zn fingers 5, 6, and 7 (ZnF567) are critical for function, and studies demonstrate that a nab2 mutant lacking ZnF567 is not functional in budding yeast.ScNab2.Binding to polyadenosine RNA, a primary function of the Nab2 protein family, is mediated by the Zn finger motifs.ScNab2 N‐terminal domain (Nab2‐N)nab2 mutant in which the N‐terminal domain has been deleted (nab2‐ΔN) exhibits severely impaired growth and nuclear accumulation of poly(A) RNA.nab2‐ΔN cells, indicating that the N‐terminal domain might contribute to the control of poly(A) tail length.ScNab2 N‐terminal domain interacts physically with both Mlp1, a component of the nuclear basket that is located on the nuclear face of nuclear pores,ScNab2 Phe73, which is located on a hydrophobic surface patch on the Nab2 N‐terminal domain [Fig. ScNab2 F73A and F73D variants show impaired binding to Mlp1 in yeast lysates and in vitro.The crystal and solution structures of the ScNab2 N‐terminal domain also interacts with Gfd1,dbp5(rat8‐2) RNA helicase mutantgle1‐8 RNA export factor mutant.ScNab2 N‐terminal domain and identified a key contribution made by ScNab2 Tyr34, which is located on the opposite side of the N‐terminal domain to ScNab2 Phe73, which is important for Mlp1 bindingnab2‐Y34A dbp5(rat8‐2) double mutant shows a synthetic slow growth phenotype.in vivo.The ScNab2‐N interacts with the nuclear pore associated proteins, Mlp1 and Gfd1, suggests that Nab2‐N PWI domain could also function as a protein–protein interaction module in higher eukaryotes, although no partners have currently been identified.Although the N‐terminal domain is conserved in Nab2 orthologues [Fig. ScNab2 ZnF567CtNab2ScNab2 ZnF1 and ZnF2 interact with one another as do ScNab2 ZnF3 and ZnF4 to form defined structural units.The structures of several Nab2 Zn finger clusters have been established using both X‐ray crystallography and NMR.ScNab2 ZnF567, NMR chemical shift perturbations associated with binding either AMP or A3 identified a series of basic and aromatic residues associated with RNA bindingScNab2 ZnF567 residues are strongly conserved between Nab2 orthologues [Fig. ScNab2 variants in which these ZnF residues were substituted showed reduced affinity for A9 RNA.nab2 ZnF567 mutants containing substitutions of these basic and aromatic residues had growth rates similar to wild‐type cells, the nab2 ZnF567 mutants generated longer poly(A) tails in vivo and also showed genetic interactions with both Dbp5 and Yra1, consistent with their also influencing the generation of mature mRNPs.ScNab2, structural coherence between ZnF567 was lost in the ScNab2 RNA‐binding mutant, nab2‐C437S, in which a Ser was substituted for the first Zn‐coordinated Cys in ZnF6 [Fig. nab2‐C437S yeast mutant exhibited cold‐sensitive growth and hyperadenylation of bulk poly(A) tails.nab2‐C437S mutant with the dbp5(rat8‐2) RNA helicase mutant suppressed the growth defect of the dbp5(rat8‐2) mutant.nab2 ZnF mutants in combination with the dbp5(rat8‐2) mutant indicated that dbp5(rat8‐2) suppression by nab2 ZnF mutants was more closely linked to hyperadenylation and suppression of mutant alleles of the nuclear RNA export adaptor, Yra1, than to the affinity of the mutant Nab2 for poly(A) RNA.ScNab2 has an unanticipated function associated with generating export‐competent mRNPs, and that changes within ZnF567 lead to suboptimal assembly of mRNP export complexes that are more easily disassembled by Dbp5 upon reaching the cytoplasm.In ScNab2 ZnF567CtNab2 ZnF34511G and A8 RNA, respectively, indicated the basis for selective binding of Nab2 Zn fingers to adenine and identified the importance of H‐bonds formed by adenine N6  RNA in vitro and generated longer bulk poly(A) tails in vivo.The crystal structures of ScNab2 Zn fingers 567 and CtNab2 Zn fingers 345 complexed with poly(A) RNA, the spatial arrangement of the fingers precludes them from all binding to the same RNA chain, so that the first and third Zn fingers of the module were bound to one RNA chain, whereas the middle Zn finger was bound to a second RNA chain. Moreover, the crystal structure of ScNab2 ZnF567 complexed with A11GIn vitro binding studiesScNab2 ZnF567 and either A12 or A11G, indicating that it was not a crystallization artifact and also that the 3′ terminal G was not necessary for its formation. Unusually, the dimerization of the ScNab2 protein chains was mediated almost entirely by each RNA chain binding to both protein chains and not by specific interactions between residues on the protein chains themselves, precluding the engineering of ScNab2 variants in which the Nab2‐Nab2 interaction was impaired. However, ScNab2 dimerization was impaired in Nab2 variants in which RNA binding was disturbed, with substitution of Phe450 in ZnF6, that is important for the interaction with the two adenines that bind ZnF6  tail length in vivo.ScNab2 F450A variant exhibited some of the strongest genetic interactions with Yra1 and Dbp5.In the structures of both ScNab2 ZnF567‐A11G heterotetramer, not all the adenines interact directly with the Nab2 protein chains, suggesting that Nab2 could also form analogous dimers with RNA sequences that only contain adenines in key positions [Fig. ScNab2 binds along the coding region of transcripts as well as to the poly(A) tail,ScNab2 ZnF567 in mediating the compaction of GAL1 transcripts in vitro [Fig. ScNab2 ZnF567, which were able to form dimers, resulted in much more compact complexes than those formed by the ScNab2 F450A variant, in which dimerization was impaired.In the S. cerevisiae, the 3′‐end processing machinery comprises cleavage factors IA and 1B and the cleavage and polyadenylation factor (CPF), a complex including the riboendonuclease, Ysh1/Brr5, the poly(A) polymerase, Pap1, and the Pap1 regulation factor, Fip1.S. cerevisiae, the CPF component, Fip1, binds Pap1 directly and tethers it to CPF to stimulate/regulate Pap1 activity.S. cerevisiae poly(A) tails have a length of ~60–80 nucleotides  RNA as it loops between Nab2 chains. Consequently, it is not clear whether generating a single Nab2 dimer is sufficient to terminate polyadenylation or whether instead it is necessary to form two dimers. Further work will be required to address this question.Polyadenylation is the final processing step in the nuclear phase of the gene expression pathway. In ScNab2 and SpNab2 can protect poly(A) RNA from degradation by the RNA exosome in vitro.In addition to terminating polyadenylation by dissociating Pap1 from the CPF, Nab2 may also contribute to regulating poly(A) tail length through interactions with the 3′‐5′ riboexonuclease exosome complex,ScNab2 Zn finger variants has indicated that the ScNab2 ZnF567 interaction with polyadenosine RNA plays a central role in the regulation of poly(A) tail length and mRNA compaction in S. cerevisiae. In this context, the observation that the loss of DmNab2 in Drosophila and ZC3H14 in mice and humans results in longer poly(A) tailsDmNab2 ZnF123 and ZC3H14 ZnF123, which are most similar to ScNab2 ZnF567 [Fig. ScNab2 to contribute to the control of poly(A) tail length. In the future, it will be informative to assess the functional consequences of specific DmNab2/ZC3H14 Zn finger variants, such as DmNab2 C879S and HsZC3H14 C622S in ZnF2 that are equivalent to ScNab2 C437S in ZnF6 [Fig. Overall, extensive analyses of S. cerevisiae, Nab2 also shows genetic interactions with components of the mRNA nuclear export machinery, such as Yra1, Sub2, and Mex67.ScNab2 may also interact directly with Mex67, one possible mechanism for signaling the completion of polyadenylation could be mediated through Pcf11 and the THO complex as a result of the dissociation of poly(A) polymerase Pap1 from the CPF complex.In addition to its function in controlling poly(A) tail length in S. cerevisiae, direct evidence for such a dimer containing full‐length Nab2 or the way in which the protein chains are arranged in the Nab2 dimer has not yet been obtained either in vitro or in vivo. It is also unclear whether one or two Nab2 dimers are needed to terminate polyadenylation. Although CPF components, such as Pcf11, appear to participate in signaling the termination of polyadenylation to the mRNA export machinery (TREX/Yra1/Sub2/Mex67:Mtr2) that generates an export‐competent mRNP, precise details of the signaling pathway and how polyadenylation termination is transmitted to Pcf11 remain to be established. Similarly, although there is clearly crosstalk between Nab2/ZC3H14 and the splicing machinery, most notably involving genetic interactions between ScNab2 and the splicosome component, Mud2,Although Nab2 dimerization following binding to the growing poly(A) tail could provide a mechanism by which Nab2 terminates polyadenylation in S. cerevisiae. Nab2 orthologues also contribute to these functions in higher Eukaryotes, however, the greater complexity of these systems has made establishing precise molecular mechanisms and signaling pathways more difficult. The spatial arrangement of Nab2 Zn fingers facilitates dimerization when bound to adenine‐rich RNAs that is important for the termination of 3′ polyadenylation and transcript compaction, albeit the precise structure of the Nab2 dimers generated in vivo remains to be established. Overall, the wealth of information that has been generated about the structure of Nab2 and its interactions with other components of the nuclear gene expression machinery has laid the foundation for beginning to define the precise ways in which these pathways are coordinated and also provides insight into the contribution made by Nab2 orthologues to these processes in higher Eukaryotes.In summary, a combination of functional and structural studies has provided a wealth of insight into the way in which Nab2 regulates mRNA poly(A) tail length, contributes to mRNA compaction and the integration of nuclear steps in the gene expression pathway with nuclear export in


A small amount of Al doping into the HfO2 film can stabilize the tetragonal phase of the HfO2, which helps to achieve a higher dielectric constant (k) and lower leakage current density, as well as a higher breakdown voltage than HfO2 film on its own. Moreover, assimilation of Al2O3 into HfO2 can reduce the hysteresis width and frequency dispersion. These are indications of border trap reduction, which was also verified by the border trap extraction mechanism. X-ray photoelectron spectroscopy (XPS) analysis also verified the HfAlO microstructural properties for various Al compositions. In addition, higher amounts of Al2O3 in HfAlO resulted in better interface and dielectric behavior through trap minimization, although the equivalent-oxide-thickness (EOT) values show the opposite trend.This study presents the characteristics of HfAlO films for a series of Al incorporation ratios into a HfO Inte2 film-based MOSCAPs along with Al2O3 and HfO2 ones. The C–V hysteresis was measured by performing the sweep from inversion to accumulation and (without any delay) again sweeping back to inversion at a frequency of 1 MHz to exclude the response of interface traps. It is obvious from the figure that Al incorporation reduces the hysteresis of HfO2 due to the charges trapped at the oxide/semiconductor interface, as well as inside the dielectric. This reduction can be explained as follows, Al2O3 exhibits micromolecular properties, while TMA has high reactivity, which allows the pinholes of HfO2 to be filled with Al ions and promotes formation of a more condensed film during ALD deposition  and sample H [3:3] showed higher breakdown voltages as they have higher thicknesses and greater Al content based on deposition cycle design.2O3 and HfO2 stacks. Lower Al incorporation increased the dielectric constant of HfO2 film though phase transition, while relative permittivity decreased with an increasing Al2O3 amount. Furthermore, leakage current decreased with breakdown voltage augmentation by Al2O3 assimilation since it provides better trap minimization and a bandgap increase for the pure HfO2 film. Both the border trap and interface traps were better minimized with an increased amount of Al2O3 in the HfAlO alloy, and a similar trend was observed in hysteresis and frequency dispersion analyses. In almost all cases, the HfAlO alloy showed behavior within the range set by single Al2O3 and HfO2 films. The behavior of the HfAlO alloy with respect to the Al: Hf ratio was verified, along with that of the single Al


Pakistan is among leading countries of world in prevalence of chronic hepatitis C Daclatasvir plus sofosbuvir is recommended for treatment of CHC. The purpose of study was to determine the sustained virological response in patients with chronic viral hepatitis C genotype 3a irrespective of previous treatment experience or presence of liver cirrhosis.Open label observational study was conducted at ABSTH Gujrat from January 2017 to April 2018 using non-probability purposive sampling. Patients chronically infected with hepatitis C virus having genotype 3a irrespective of presence of cirrhosis or previous treatment experience were included. Treatment naive patients without cirrhosis were given 12 weeks regimen of daily daclatasvir 60mg along with daily sofosbuvir 400mg. Patients with either compensated cirrhosis or treatment experienced were given 24 weeks regimen of daily daclatasvir 60mg along with daily sofosbuvir 400mg with weight based ribavirin. Data analysis was done using SPSS 20.024 was available for 101(80.8%) patients out of which 48 were having cirrhosis and 53 were without cirrhosis. SVR24 was achieved by 96 patients (95%). Virological response was better in treatment naive patients and without cirrhosis compared to treatment experienced and those with cirrhosis.Total 125 patients were included in study out of which 42 (33.6%) were male and 83 (66.4%) were female. Early virological response and end treatment response was achieved by 124 (99.2%) patients. Twenty four patients were lost to further follow-up and SVRDaclatasvir plus sofosbuvir is an effective combination in patients with chronic hepatitis C genotype 3a infection. Chronic hepatitis C affects 71 million people globally according to WHO estimates.5Decreased prices of DAAs and advent of generics has boosted up the efforts in treatment of chronic hepatitis C in Pakistan. Pakistan along with Egypt had half of people starting DAAs for treatment of chronic hepatitis C in the world during 2016Twelve week regimen of once daily daclatasvir plus sofosbuvir has showen good results in patients with HCV genotype 3 with sustained virological response of 91%.9Combination of daclatasvir and sofosbuvir is recommended for genotype 3 patients according to AASLD 2017 guidelines having a strong evidence. Recommended duration is 12 weeks in patients without cirrhosis and 24 weeks in patients with cirrhosis when weight based ribavirin is added to the regimen irrespective of previous peglated interferon plus ribavirin treatment.11There are no studies previously published in Pakistan regarding efficacy of daclatasvir plus sofosbuvir in patients with hepatitis C genotype 3a up to best of our knowledge. As this is an effective regimen its efficacy should be evaluated in a population having high incidence of infection. Thus the purpose of study was to determine the sustained virological response in patients with chronic viral hepatitis C genotype 3a irrespective of previous treatment experience or presence of liver cirrhosis.This open label observational study was conducted at Aziz Bhatti Shaheed Teaching Hospital Gujrat from January 2017 to April 2018. Patients were included in study after informed consent and approval of ethical committee of hospital. Patients chronically infected with hepatitis C virus having genotype 3a irrespective of presence of cirrhosis or previous treatment experience with interferon plus ribavirin were included. Patients were given this regimen due to free availability of DAAs in government setups and those who could not afford the first line regimen i.e combination of Velpatasvir plus Sofosbuvir.Presence of chronic hepatitis C was confirmed with baseline quantitative PCR testing and a value of > 15 ng/ml was considered positive. Genotype testing was done for every patient by University of Gujrat Laboratory free of cost and those with genotype 3a were selected using non-probability purposive sampling. Patients having liver cirrhosis were confirmed by abdominal ultrasound done by consultant radiologist and presence of coarse echotexture of liver was considered as liver cirrhosis. Although Shear Wave Elastography and Fibroscan are recommended but non-availability of these modalities led us to rely on ultrasound for presence of cirrhosis. Severity of liver disease was assessed using Child Pugh Score and patients with score 5-6 were defined as Child Class A, 7-9 as Child Class B and 10-15 as Child Class C. Patients with Child class A and B were included while those having Child class C were excluded from study.Treatment experienced patients were further divided in two groups. Patients who did not respond to 24 weeks interferon plus ribavirin were classified as non-responders while those who had a positive PCR after achieving ETR with 24 weeks interferon plus ribavirin treatment were classified as relapsers.24) 24 weeks after completion of treatment.Treatment naive patients without cirrhosis were given 12 weeks regimen of daily daclatasvir 60mg along with daily sofosbuvir 400mg. Patients with either compensated cirrhosis (Child Class A & B) or treatment experienced were given 24 weeks regimen of daily daclatasvir 60mg along with daily sofosbuvir 400mg with weight based ribavirin. PCR was done to assess the treatment response at four weeks of treatment , at end of 12 or 24 weeks treatment (End Treatment Response or ETR). Primary end point of study was to determine sustained virological response  were male and 83 (66.4%) were female. Mean age was 47.06 + 10.8 years. 102 (81.6%) patients were treatment naive, 7 (5.6%) treatment non-responders and 16 (12.8%) were relapsers. Fifty eight (46.4%) patients had cirrhosis including 43 treatment naive, 4 non-responders and 11 relapsers patients. Fifty patients with cirrhosis had Child Class A while 8 patients had Child Class B.24 was available for 101(80.8%) patients out of which 48 were having cirrhosis and 53 were without cirrhosis. SVR24 was achieved by 96(95%) patients out of 101. Patients who did not achieve SVR24 included both treatment naive and experienced patients and patients with and without cirrhosis. SVR24 in patients with cirrhosis were 91.66% while in those without cirrhosis were 98.11%. 97.65% treatment naive patients achieved SVR24 however only 90.9% relapsers and 60% non-responders achieved SVR24. Results are depicted in Early virological response (EVR) and end treatment response (ETR) was achieved by 123(98.4%) and 124 (99.2%) patients respectively. Taking in account treatment history; treatment naive, relapsers and non-responders achieved EVR of 99%, 100% and 85.7% and ETR was achieved by 99%, 100% and 100% respectively. One fifth of patients (24) were lost to further follow-up who were alive and did not report back despite multiple reminders. SVR24 rates were more in patients without cirrhosis than as compared to cirrhosis and treatment naive patients as compared to treatment experienced patients. However no statistical significance of cirrhosis and treatment history were found.This study shows that combination of daily daclatasvir plus daily sofosbuvir with or without weight based ribavirin is highly effective in Pakistani population with HCV genotype 3a and independent of presence of cirrhosis or previous treatment with interferon plus weight based ribavirin. SVRResults of this study are comparable to ALLY 3 phase III study by Nelson et al.Patients with compensated cirrhosis has good treatment response in this study. 98.3% patients with compensated cirrhosis achieved EVR and ETR. ALLY 3+ study also determined the role of daclatasvir and sofosbuvir in patients with compensated cirrhosis and resulting in 83.3% patients achieving treatment response at week 4 and 100% at end of treatment.24 rates as compared to treatment naive and without cirrhosis. Alonso et al. also found a high SVR rates (94%) among patients with HCV genotype 3 infection treated with sofosbuvir plus daclatasvir24 = 95%).Welzel et al. conducted a study about efficacy of daclatasvir plus sofosbuvir with or without ribavirin in HCV patients. HCV RNA was undetectable in 73% patients at week 4, 97% at week 12 and >99% at week 24. However they included all genotypes of HCV. In genotype 3, 92% patients achieved SVR which was minimally less in treatment experienced patients having decompensated cirrhosis.Mehta et al. conducted a study in HCV genotype 3 patients in India using combination of sofosbuvir plus daclatasvir and 97.3% patients achieved SVR showing it to be highly effective regimen in genotype 3Study by Ferrieria et al. showed lower SVR rates (84.7%) in patients with genotype 3 taking sofosbuvir plus daclatasvir regimen which do not augment results of this study. However they found out no significant association of presence of cirrhosis or treatment experience with achieving SVR which are consistent with results of this study.15This is perhaps first study in Pakistan determining the role sofosbuvir plus daclatasvir in treatment of chronic hepatitis C genotype 3a infection. Although SVR at 12 week is considered the end point in many studies, SVR at 24 weeks was focused in this study. However 19.2% patients lost to follow up which shows a need for a free of cost screening, treatment and follow up program for treatment of hepatitis C at national level owing to high prevalence of chronic hepatitis C Pakistan.Study showed good outcomes in treatment of hepatitis C genotype 3a which is prevalent genotype in Pakistan.Daclatasvir plus sofosbuvir is a highly effective combination in patients with chronic hepatitis C genotype 3a infection in Pakistani population and independent of treatment history and presence of cirrhosis.ZB: Conceived the study, collected data and did final review and approval of manuscript.SMAS: Planned the study, did statistical analysis and wrote manuscript.


Does an association exist between the sponsorship and conduct of phase 3 randomized clinical trials for cancer drugs despite negative or absent phase 2 trials for the drug, and does an association exist for overall patient survival and such phase 3 trials?This analysis of 67 studies found that both industry and academia conducted negative phase 3 trials of cancer drugs. No association was found between trial sponsorship and lack of a positive phase 2 trial; there was no association with patient overall survival, although the survival hazard ratio was greater than 1 for 37% of such trials.Phase 3 trials conducted without supporting phase 2 trial evidence risks loss of resources owing to trial failure and may be associated with decreased patient survival. Only 3.4% of cancer drugs evaluated in phase 1 trials are approved by the US Food and Drug Administration, with most failing in phase 3 trials.To investigate whether an association exists between the sponsorship and conduct of a negative phase 3 randomized clinical trial (RCT) investigating a cancer drug that lacked supporting phase 2 trial evidence for that drug, and to evaluate the association with overall survival among patients randomized to the experimental arm of such phase 3 trials.Lancet, Lancet Oncology, JAMA, JAMA Oncology, and Journal of Clinical Oncology published between January 2016 and June 2018 were searched.Articles in the Phase 3 RCTs of cancer drugs that failed to improve the primary end point were selected and any prior phase 2 trial of the same drug that supported the phase 3 trial was selected without any date or journal restrictions.Percentages of negative phase 3 RCTs of cancer drugs that lacked any phase 2 evidence, had a negative phase 2 trial, or had a positive phase 2 study were extracted. Associations were assessed using the Fisher exact test. Pooled hazard ratios and 95% CIs for the overall survival of patients enrolled in these negative phase 3 RCTs were estimated using a random-effects model.Negative phase 3 RCTs with a lack of a phase 2 trial or the presence of a negative phase 2 trial and overall survival of enrolled patients in the phase 3 RCTs.In this meta-epidemiological study, 67 negative phase 3 RCTs on cancer drugs, which included 64 600 patients, met the criteria of being sponsored by industry or academic groups, of which 42 RCTs (63%) were industry sponsored and the remaining 25 RCTs (37%) were academic. A phase 2 trial was not available for 28 of these trials (42%). Of 29 trials (43%) with a phase 2 trial available, 8 trials (28%) failed to meet their primary end points and 5 of those were industry sponsored. There was no association with overall survival for patients participating in these negative phase 3 RCTs . When the pooled analysis was limited to the 27 RCTs with a hazard ratio above 1.00, the overall pooled hazard ratio for overall survival was 1.11 . No association between having a negative or undefined phase 2 trial and trial sponsorship was found using the Fisher exact test.More than 40% of the negative phase 3 RCTs in oncology published in these 5 journals were conducted without a supporting phase 2 trial and were sponsored by both academia and industry. Running such trials not only may risk loss of resources owing to a failed trial but also may be associated with decreased patient survival. Further research and regulations in this area appear warranted. This study assesses whether an association exists between the sponsorship and conduct of a phase 3 randomized clinical trial investigating a cancer drug and the lack of supporting phase 2 trial evidence for that drug. Phase 3 RCTs usually involve more patients and resources than earlier phases and are costly undertakings. According to a 2014 report, the cost of a cancer drug trial was $22.1 million, $11.2 million, and $4.5 million, for a phase 3, phase 2, and phase 1 trial, respectively.2 Thus, a drug failing the phase 3 trial  is an undesirable outcome, even from an economic point of view.Phase 3 randomized clinical trials (RCTs) are the final barrier in establishing the efficacy of cancer drugs. One study estimates that only 3.4% of cancer drugs being evaluated in phase 1 trials are approved by the US Food and Drug Administration, with most (35.5%) failing in the phase 3 stage when all indications are considered.3 Second, a trial involves significant investment of time and logistics from health care professionals and patients. Third, a failed phase 3 trial also means unmet patient expectations. Thus, although negative RCTs can provide us with important knowledge for future research and patient care, precautions can be taken to minimize the frequency of negative RCTs, especially if they can be avoided. The US Food and Drug Administration resource “Information for Consumers (Drugs)” states that “if the phase 2 trials indicate that the drug may be effective—and the risks are considered acceptable, given the observed efficacy and the severity of the disease—the drug moves to phase 3.”4 Another document on the clinical review of investigational new drug applications also states that a phase 2 trial review is conducted on completion before initiation of phase 3 trials, during which questions such as whether sufficient data are available to plan a phase 3 program are asked.5 However, in a previous study, our group discovered that less than 20% of the negative phase 3 RCTs on cancer had a supportive phase 2 trial.6 One hypothesis for such a practice is that it is financially lucrative for the industry to continue to test compounds that have low probability of success in a phase 3 trial because even a chance finding of a positive outcome will offset all associated costs.7 However, associations of sponsors of such negative phase 3 RCTs with phase 2 trial outcomes or the association of such negative phase 3 RCTs on patient survival has not been studied systematically to our knowledge.However, the larger concern is the effect on human resources. First, patient resources for trial enrollment are scarce given that only 3% of patients with cancer in the United States participate in trials and that nearly 60% of phase 3 trials fail to achieve minimum patient enrollment.In the present study, using a larger data set of negative phase 3 RCTs, we investigated whether an association existed between the sponsorship of negative phase 3 RCTs and the lack of supportive prior phase 2 trial evidence. We also studied what proportion of the industry-sponsored negative phase 3 RCTs had a history of the experimental drug being acquired or licensed from a small pharmaceutical company . We hypothesized that such an arrangement might motivate the sponsor to pursue a phase 3 RCT to recoup the costs of the investment. Finally, we investigated whether there was an association with overall survival (OS) among patients who were enrolled in these negative phase 3 RCTs.PRISMA) reporting guidelines for meta-epidemiological studies.8 We searched the top oncology journals based on their impact factor  and their parent journals (Lancet and JAMA) between January 2016 and June 2018 for any negative phase 3 RCTs of cancer drugs. We excluded the RCTs that did not involve a cancer drug in any arm, such as RCTs of radiotherapies or surgical procedures in both the arms. We extracted the hazard ratio (HR) and 95% CI for OS for each of the included trials, where available. We also categorized the sponsors of these negative phase 3 RCTs into academic (cooperative groups) or industry based on the posted ClinicalTrials.gov registration information. We then searched the literature and meeting abstracts for any prior phase 2 trial of the same drug that supported the phase 3 trial. We categorized these findings into yes, no, and not applicable (NA). Not applicable was applied to drugs that had already been approved for later lines of therapy or in an advanced setting but the phase 3 RCT tested the drug in an earlier or curative setting. For those trials categorized as yes for any prior phase 2 trial, we categorized the outcomes of the phase 2 trials into positive, negative, or inconclusive based on whether the primary end point of the phase 2 trial was met, unmet, or unstated, respectively. Finally, for industry-sponsored trials, to find out if the drug was purchased or licensed from a small pharmaceutical company , we used search terms in Google  and then reviewed the news article or press release for details, if any, regarding a transaction or business arrangement or deal with another pharmaceutical company or entity. All study and data extractions as well as categorizations were made by 2 of us (A.A. and G.W.) and rechecked by the third author (B.G.). Any discrepancies were resolved by consensus among the 3 authors.This study was conducted based on modified Preferred Reporting Items for Systematic Reviews and Meta-analyses , and a 2-sided 75 which included 64 600 patients, that met our criteria for inclusion, of which 42 RCTs (63%) were industry sponsored and the remaining 25 RCTs (37%) were academic  of these negative phase 3 RCTs, it was available for 29 (43%) of them, and it was not applicable for 10 (15%) of them. Excluding the 10 nonapplicable trials, the availability of a phase 2 trial was not associated with negative phase 3 trial sponsorship . Of the 29 trials in which a phase 2 trial was available, 8 trials  failed to meet the primary end point, whereas the phase 2 trial was inconclusive in 5 cases (17%). The phase 2 trial met its primary end point and was considered positive in 16 trials . There was no association between having a negative or undefined phase 2 trial and phase 3 trial sponsorship .We found 67 negative phase 3 RCTs,9 The other was the RCT of lapatinib in bladder cancer, which was also tested in a biomarker-selected population (ERBB1/2 [formerly HER1/2] positive) in phase 3, despite a negative phase 2 trial in an unselected population.34 Of the 5 industry-sponsored trials that were conducted despite a negative phase 2 trial, 4 trials were associated with purchase or licensure of the molecule from a small pharmaceutical company.Of the 8 RCTs that were conducted despite a negative phase 2 trial, 5 were industry sponsored. Two of the 3 academic phase 3 RCTs were conducted despite a negative phase 2 trial because the phase 2 trial showed beneficial effects in a subgroup of biomarker-positive patients. One was the Alliance CALGB 30801 trial of celecoxib in non–small cell lung cancer with cyclooxygenase-2 overexpression that was conducted despite a negative phase 2 trial because cyclooxygenase-2 overexpression was supposed to be a predictive biomarker.36 the overall pooled HR for overall survival was 1.11   with no subgroup differences by trial sponsorship or by phase 2 trial results. However, the HR was greater than 1.00 in 27 RCTs (46%). When the pooled analysis was limited to these 27 RCTs,06-1.16) .Of the negative phase 3 RCTs of cancer drugs, 42% did not have phase 2 trial evidence and only 28% had a positive phase 2 trial. Nearly 14% of negative phase 3 RCTs were conducted despite a negative phase 2 trial. The availability of a phase 2 trial or whether or not the phase 2 trial was positive was not associated with trial sponsorship, but of the 5 industry-sponsored negative phase 3 RCTs, 4 were associated with drug purchase or licensure from a small pharmaceutical company. Patients who were enrolled in the negative phase 3 RCTs were not associated with poorer OS although when limited to 46% of negative phase 3 RCTs reporting an HR greater than 1.00, the pooled HR showed a significant increase in mortality among the participants randomized to the experimental arm of the trials.There are several hypotheses as to why a cancer drug may be tested in a phase 3 RCT despite having no or even negative phase 2 trial evidence. Among industry-sponsored trials, we found that acquisition of the investigational drug from small pharmaceutical companies could be one factor. Financial milestones among the small pharmaceutical companies to move a compound from phase 2 to phase 3 trials can influence decisions. However, in the case of academia-sponsored trials, a belief in the biomarker-based subgroup analysis of phase 2 trials seemed to be a motivation for conducting a larger phase 3 trial in the biomarker-enriched subgroup population.76Negative phase 3 RCTs are not futile or meaningless; they are important to advance our understanding of disease and drugs. Nevertheless, because of the financial, human, logistic, and time resources needed to conduct a phase 3 RCT and the patients’ expectation of therapeutic benefit, phase 3 RCTs should be conducted only when there is some prior evidence to suggest a potential therapeutic benefit. If a cancer drug has failed to meet its primary end point in a phase 2 trial, there is neither therapeutic equipoise nor rationale to test it in a phase 3 RCT, and we question if it is against the ethical norms of participating in a phase 3 RCT. Institutional review boards and ethical committees should take a proactive step in discouraging the conduct of such phase 3 RCTs of cancer drugs that have already failed in phase 2 trials. Indeed, in a recent editorial, the US Food and Drug Administration questions the practice of conducting phase 3 RCTs of cancer drugs known to have poor activity in phase 2 trials.We included only those negative phase 3 RCTs published in journals with high impact factors; thus, our findings should be considered conservative. Indeed, our results reflected the best-case scenarios of the negative phase 3 RCTs, and the results would likely be worse for the negative phase 3 RCTs published elsewhere or not published at all. However, our search for phase 2 trials was conducted comprehensively without any journal or time restrictions. Another limitation of our study was the relative difficulty in accessing information on purchase or licensure of a drug from small pharmaceutical companies. Lack of a control group of positive phase 3 RCTs was another limitation; however, our objectives were to assess the association of negative phase 3 RCTs with sponsorship as well as to assess the association of negative phase 3 RCTs with patient survival, and therefore for our purpose, the cohort of negative trials alone sufficed. Furthermore, even though the trials were categorized into groups based on sponsorship, most trials will involve both industry and academia collaboration. We also acknowledge that in some instances of extraordinary responses in phase 1, it may be more efficient to take a drug directly to a phase 3 RCT; however, the activity and criteria for success (taking to a phase 3 RCT) must be prospectively defined in such early-phase trials.In this study of trials published in 2016 through 2018, approximately 40% of negative phase 3 RCTs in oncology were conducted without supporting phase 2 trials, and such phase 3 trials were sponsored by both academia and industry. On the basis of our results, proactive steps from regulators and ethical committees should be contemplated to encourage greater consideration before allowing conduct of phase 3 RCTs despite negative results from phase 2 trials in the interest of protecting patients and trial resources.


BRCA1/2 mutations to familial breast cancer in Bahrain has not been explored. The objective of this study was to investigate the spectrum of BRCA1/2 genetic variants and estimate their frequencies in familial breast cancer. We also aim to test the efficiency of the next‐generation sequencing (NGS) as a powerful tool for detecting genetic variation within BRCA1/2 genes.Breast cancer is the most common malignancy in women worldwide. About 5%–10% are due to hereditary predisposition. The contribution of BRCA1/2 variants. All targeted coding exons and exon–intron boundaries of BRCA1/2 genes were amplified with 167 pairs of primers by NGS.Twenty‐five unrelated female patients diagnosed with familial breast cancer were screened for BRCA1/2 variants in two patients, one in BRCA1 gene (c.4850C>A) and other in BRCA2 gene (c.67+2T>C). In addition to the deleterious variants, we identified 24 distinct missense variants of uncertain significance, 10 of them are seen to confer minor but cumulatively significant risk of breast cancer.We have identified two deleterious BRCA1/2 variants may contribute to the pathogenesis of familial breast cancer in Bahrain. It also shows that NGS is useful tool for screening BRCA1/2 genetic variants of probands and unaffected relatives.Our data suggest that  These tumor suppressor genes were reported and identified as breast cancer susceptibility genes for the first time in 1990 and 1995 and are located on chromosome 17q21 and 13q12‐13, respectively  database  and Kuwait (46.6)  and to test the efficiency of the NGS in the diagnosis and screening programs of breast cancer patients in the region. The overall objective was to better understand the genetic risk factors associated with the disease which could eventually lead to an earlier detection with better prognosis and survival rates.In Bahrain, the breast cancer screening by mammography was introduced in 2005 and essentially subjected to high‐risk suspected cases for women aged 40 and above. The screening program since introduced has contributed to the diagnosis of 12.7% of the cases .Genomic DNA was isolated from 200 µl peripheral blood anti‐coagulated with EDTA on the MagNa Pure LC instrument using MagNa Pure LC DNA Isolation Kit , according to the manufacturer's instructions. The concentration of DNA was determined by using the Nano Drop2.4BRCA1/2 genes were amplified with 167 pairs of primers in three primer pair pools. After the targeted amplification and construction of a library through Ion AmpliSeq™ Library Kit 2.0, the nucleotide sequences of the targeted regions are analyzed by Life Technologies Ion PGM platform on an Ion 316 Chip. Sequence variants are identified by Torrent Suite 4.2 . The clinical significance of each sequence variant is suggested on the basis of reference to ClinVar and BIC .The reference sequences used were NM_007294.3 for 33.1BRCA1/2 genetic variants, we screened BRCA1/2 variants in the 25 recruited patients with personal and family history of breast cancer. All women were diagnosed with unilateral breast cancer. The median age at diagnosis of breast cancer was 47 years (range 33–63). DNA was extracted from patient's peripheral blood samples. We amplified targeted coding regions and exon–intron boundaries of BRCA1/2 genes using multiplex PCR. A total of 167 primer pairs were used in three primer pools covering 16.25 KB of target genomic sequence. Targeted genomic sequencing was performed using the Ion Torrent PGM System from Life Technologies generating short sequence reads of approximately 200 bp. We have identified two deleterious BRCA1/2 variants in two patients, one in BRCA1 gene (c.4850C>A) and other in BRCA2 gene (c.67+2T>C). In addition to the deleterious variants, we have identified 24 missense variants of uncertain significance (VUS).To analyse the prevalence of breast cancer with 3.2BRCA1 variant c.4850C>A was diagnosed with right breast cancer at age 33 and had a positive family history of breast cancer . There was no family history of ovarian cancer. Patient presented with right breast lump noticed 2 days prior to presentation that radiological investigations did not reveal any breast lesions or distant metastasis; however, fine needle aspiration reported malignancy.The carrier of The tumor was classified as a stage 2 loco‐regional malignancy, and the histopathology results confirmed the diagnosis of an invasive ductal carcinoma grade 3, measuring 23 mm in maximum dimension. Two palpable mobile axillary nodes were observed during examination. The patient underwent wide local excision of right breast tumor and right axillary clearance.3.3This is a Bahraini woman, known case of hypertension and hyperlipidemia with history of abdominal hysterectomy and bilateral oophorectomy for endometrial cancer 9 years prior to her presentation. The patient has a very strong family history of breast cancer including her mother and maternal aunt (diagnosed before age 50). She has presented with right axillary swelling and right arm pain. At investigation, a deep right breast lump was observed occupying the upper outer quadrant. Two palpable metastatic axillary nodes were observed during examination. The tumor was classified as a stage 2 loco‐regional malignancy, and the histopathology results confirmed the diagnosis of an invasive papillary carcinoma (grade 2), measuring 4 cm in maximum dimension. The patient underwent right mastectomy and axillary clearance.3.4BRCA1/2 genes of the 25 patients revealed two deleterious variants in two unrelated patients with total frequency of 8% (2/25), one within BRCA1 and other within BRCA2   which uses sequence homology to predict whether an amino acid substitution will affect protein function and hence, potentially alter phenotype to the missense substitutions associated with the disease. SIFT predicted 42% of the detected 24 variants to be damaging. The SIFT algorithm and software have been described previously  is identical to the figures previously reported for the Arab population, a decade earlier than western countries  or missense alterations at critical residues in functional domains, we defined one BRCA1 c.4850C>A and one BRCA2 c.67+2T>C as deleterious variants in two patients. Interestingly, both patients showed a very strong family history of breast cancer suggestive of genetic predisposition to breast cancer. The two deleterious variants were observed in two unrelated patients out of the 25 subjects studied providing an overall prevalence rate of 8%. Some studies conducted in other Arab countries such as Tunis and Saudi Arabia have reported frequencies of 16%–25% . BRCA1 gene contains 22 exons spanning about 110 kb of DNA and encoding a 1863 amino acid protein with an N‐terminal RING finger domain and two BRCT— domains. The identified BRCA1 nonsense variant c.4850C>A was predicted to be causative because it creates a premature stop codon at amino acid 1617 leading to premature truncated protein. To the best of our knowledge, the BRCA1 variant c.4850C>A was not reported previously elsewhere except in a recent study conducted in Qatar where two patients were found to be positive carriers for the c.4850C>A variant  including distinct polymorphisms and unclassified sequence variants.Additionally, we have identified 24 sequence variants (11 BRCA1 SNPs (rs1799950 (4%), rs16942 (64%), rs4986850 (20%), rs2227945 (16%), and rs1799966 (64%)) and 5 BRCA2 SNPs (rs766173 (12%), rs144848 (60%), rs4987117 (4%), rs4987047 (8%), and rs11571833 (4%)) are found to confer minor but cumulatively significant risk of breast cancer  and other in BRCA2 gene (c.67+2T>C). The identified BRCA1 variant c.4850C>A has been reported in a Qatari study and in this report only suggesting that it might be novel to the gulf region. We also showed that NGS is a useful tool to explore mutations in BRCA1 and BRCA2 genes and can be used to screen mutations in probands and unaffected relatives from the same family.In conclusion, this is the first report on the breast cancer predisposition factors in the population of Bahrain and our data suggest that The authors declare that there is no conflict of interest regarding the publication of this article.FA conceived the study, participated in its design and coordination, carried out the molecular genetics studies, analyzed the data, and drafted the manuscript. MK arranged for the study funding, participated in the study coordination, and revised the manuscript. ST supervised the sequencing experiment and revised the manuscript. LA looked after patients' recruitment, gathering the clinical data, and revised the manuscript. All the authors read and approved the final manuscript.


We previously reported the identification of monocarboxylate transporter 4 (MCT4) and glypican-3 (GPC3) as prognostic factors for hepatocellular carcinoma (HCC), which are now considered significant poor prognostic factors for the disease. This study aimed to clarify the detailed interaction of these two factors in HCC to improve our understanding of aggressive HCC phenotypes. A total of 225 Japanese patients with HCC from our previous study were subjected to immunohistochemical analyses.The number of MCT4-positive (MCT4+) HCC cases was 47 (21%), and most MCT4+ HCC showed high GPC3 expression . In 44 MCT4+/GPC3+ HCC cases, intratumoral heterogeneity of GPC3 or MCT4 expression was further evaluated. We observed reciprocal (inverse), synergistic, mixed reciprocal and synergistic, or irrelevant interaction of MCT4 and GPC3 expression in 29 (66%), 5 (11%), 1 (2%), and 9 cases (21%), respectively. The cases exhibiting reciprocal expression of both markers tended to have cirrhosis without a history of neoadjuvant therapy. In summary, although MCT4+ HCC cases are mostly GPC3+, intratumoral expression patterns of MCT4 and GPC3 are frequently reciprocal each other, suggesting that dual targeting of MCT4 and GPC3 may achieve a better antitumor effect for MCT4+ HCC cases. Liver cancer is the leading cause of cancer death worldwide and is the second leading cause of cancer death in men . As hepaWe previously reported that MCT4+ HCC cases were mostly GPC3 positive [The eligible cases included 225 Japanese patients  with HCC who had underwent partial hepatectomy in the University of Miyazaki Hospital from February 1999 to October 2012. The patients included in this study were the same as in our previous report , as wereSerial sections, which were prepared from formalin-fixed, paraffin-embedded HCC blocks of 225 cases, were used for hematoxylin and eosin staining and MCT4 and GPC3 immunohistochemistry  as the pAs reported in our previous studies , 4, we dp < 0.05. Data were analyzed by StatView 5.0 .Fisher’s exact test or the Chi-square test was used for assessment of the relationship between variables. Statistical significance was assumed if MCT4+ HCC and GPC3+ HCC were immunohistochemically identified in 21% (47 cases) and 84% (190 cases) of the 225 cases, respectively. The mean positive area of MCT4 and GPC3 was 20%  and 72% , respectively. MCT4+/GPC3+, MCT4+/GPC3-negative (GPC3−), MCT4-negative (MCT4−)/GPC3+, and MCT4−/GPC3− HCCs were observed in 44, 3, 146, and 32 cases, respectively , we statistically analyzed the correlation between clinicopathological variables of the reciprocal HCC cases (29 cases) and those of the non-reciprocal ones   of MCT4+ HCC cases in our cohort showed GPC3 positivity, and nearly 80% of MCT4+ HCC cases exhibited reciprocal or synergistic expression pattern between MCT4 and GPC3. Thus, the expression of MCT4 in HCC cells might be influenced by GPC3 expression and vice versa. Of note, 68% of MCT4+/GPC3+ HCC cases demonstrated reciprocal interaction of both markers. These findings may provide a novel therapeutic approach for MCT4+ HCC; dual targeting of MCT4 and GPC3 may achieve a better antitumor effect for MCT4+ HCC.In this study, we used the custom-made anti-GPC3 antibody GC33, which is a mouse monoclonal antibody that recognizes human GPC3. Humanized GC33 (codrituzumab) may serve as a treatment option for HCC because it has a significant antitumor activity to HCC cells in vivo via antibody-dependent cellular cytotoxicity , 20. We GPC3 is silenced partly by promoter hypermethylation in some cancers [GPC3 transcription in HCC may be suppressed by transcription factor zinc fingers and homeoboxes 2 (ZHX2), a well-known repressor of the GPC3 gene [The mechanism of reciprocal interaction of MCT4 and GPC3 in HCCs remains unknown. In the tumor areas showing reciprocal interaction of MCT4 and GPC3, MCT4 was likely induced by the hypoxic tumor microenvironment because MCT4+ HCC cells were observed primarily in the central portions of tumor nests distant from the tumor vessels. In fact, we previously showed that MCT4+ HCC cells were present near necrotic portions, and those tumor cells tended to be positive for the hypoxia marker carbonic anhydrase IX [ cancers , 22, and cancers . AlternaPC3 gene , 25, in Although the reciprocal pattern was predominant, 11% of the cases showed a synergistic expression pattern of MCT4 and GPC3. The mechanism underlying the synergistic interaction of MCT4 and GPC3 in HCC also remains unclear. In the areas of tumors showing synergistic interaction of MCT4 and GPC3, concomitant cell surface immunoreactivities of MCT4 and GPC3 were observed as reported previously , suggestBased on statistical analysis, the reciprocal interaction of MCT4 and GPC3 tended to be observed in non-treated HCCs derived from cirrhosis. Thus, severe cell damage induced by adjuvant therapy in HCC may disturb the reciprocal relationship and may result in a synergistic or irrelevant interaction of MCT4 and GPC3; however, this hypothesis remains highly speculative.In conclusion, we immunohistochemically explored the intratumoral relationship between MCT4 and GPC3 expression in HCC. Although the mechanism underlying the interaction of these molecules in HCC is currently unknown, the observed phenomena may have implications in the development of therapeutic strategies targeting MCT4 and GPC3 in HCC.Expression of MCT4 and GPC3 in HCC cells was immunohistochemically evaluated using formalin-fixed, paraffin-embedded HCC tissue blocks from 225 cases. In each case, one tumor block was randomly selected from the maximal section of the tumor. The expression status of MCT4 and GPC3 was not evaluated in whole tumor sections.Prognostic differences between patients with “reciprocal” and “non-reciprocal” HCC could not be statistically evaluated owing to the small sample size.With respect to the expression regulation mechanism of MCT4 and GPC3 in HCC cells, we could not sufficiently explain the mechanism with this morphological study.Additional file 1. Clinicopathological data of patients with HCC.Additional file 2. Intratumoral expression patterns of MCT4 and GPC3 in 44 cases of MCT4+ GPC3+ HCC.


Figure 3C on page 7 of this paper erroneously presented two identical western-blot panels (GRP78 and HSPA8).GRP78 and HSPA8 western-blot panels as originally intended.The corrected The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


The pristine and modified Li4Ti5O12 electrodes are characterized at an extended voltage range of 3–0.01 V. The reversible capacity reaches a high level of 286 mAh g−1, which is a little less than its theoretical capacity (293 mAh g−1). Electrodes modified by ZnO thin films with various thickness show elevated rate capability and improved cycle performance.Oxide is widely used in modifying cathode and anode materials for lithium-ion batteries. In this work, a facile method of radio magnetron sputtering is introduced to deposit a thin film on Li The calculated data agree with the measured data very well when the inserted equivalent circuit (as shown in figure 5b) is employed to fit the data. Both the measured plots consist of two compressed semicircles and an inconspicuous oblique tail. The high-frequency semicircle is related to the resistance of SEI film formed on the electrode at a low voltage of 0.7 V. The intermediate frequency semicircle is attributed to the charge transfer resistance between the interface of active materials and electrolyte. The oblique line is associated with the Li-ion diffusion within the active materials. The fitted values of Rs of the pristine and ZnO-coated Li4Ti5O12 composite electrodes are less than 5 Ω. The fitted value of the SEI film resistance (Rf) of ZnO-coated Li4Ti5O12 (5 min) composite electrodes is 101 Ω, which is much smaller than 364 Ω for the pristine electrode. The fitted values of charge transfer resistances (Rct) are 224 and 1019 Ω for pristine and ZnO-coated Li4Ti5O12 (5 min) composite electrodes, respectively. The reduced SEI film resistance and charge transfer resistance ensured elevated rate performance and stable cycle performance.The measured and calculated Nyquist plots are shown in figure 54.4Ti5O12 composite electrodes were successfully coated with ZnO thin films via a radio frequency magnetron sputtering method. The thickness of ZnO films was tailored by altering depositing time. A mixture layer of Zn-Li alloy and Li2O produced by ZnO coating film during the discharge process was suggested to improve the interface between electrode and electrolyte. As a result, the rate performance and cycle stability were greatly improved by ZnO coating layer.In summary, Li


In this study, we aimed to investigate the impact of the new recommendations on HER2 FISH interpretation in invasive breast cancers with immunohistochemically (IHC) equivocal results. 1810 breast cancer cases with IHC equivocal results were enrolled in this study between January 2012 and May 2019. Concomitant IHC was performed on the same tissue blocks detected by FISH testing. According to the 2018 guidelines, all the cases in ISH group 2 were categorized as HER2 negative; three of four cases in ISH group 3 were considered as HER2 positive, while the one scored IHC 1+ was reclassified as HER2 negative; Fifty-three previously ISH equivocal cases were redistributed into ten HER2-positive cases and forty-three HER2-negative cases. In conclusion, the utility of 2018 ASCO/CAP guidelines resulted in a slight decrease in HER2 positive rate, due to the reclassification of cases in ISH group 2 and group 4. The implementation of the new guidelines can reduce reflex FISH test and make the diagnosis of HER2 gene status more definitive.The American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) recently issued updated guidelines on human epidermal growth factor receptor 2 (HER2) testing by fluorescence  Amplification and/or overexpression of human epidermal growth factor receptor 2 (HER2) is observed in about 15–20% of invasive breast cancer patients2. HER2 positivity is closely related to the poor prognosis of patients5. Clinical trials have demonstrated that breast cancer patients with HER2 overexpression can benefit from the targeted therapy, showing the progression-free survival and the overall survival improvement8. Therefore, accurate assessment of HER2 status is a prerequisite for identifying the subset of breast cancer patients who may benefit from the anti-HER2 targeted therapy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the two most frequently performed technologies to determine HER2 status.Breast cancer is the most common carcinoma and the second leading cause of cancer-related death among Chinese women9? The diagnostic approach incorporates concomitant IHC review into ISH interpretation in ISH groups 2 to 4 to reach the most definitive HER2 status classification.The American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) have periodically issued and updated the HER2 testing guidelines in breast cancers, with first version released in 2007, first revised in 2013 and focused updated in 2018. The new guidelines have addressed five clinical questions, including the following: (1) What is the most appropriate definition for IHC 2+ ? (2) Must HER2 testing be repeated on a surgical specimen if initially negative test on core biopsy? (3) Should invasive cancers with a HER2/chromosome enumeration probe (CEP17) ratio of ≥2.0 but an average HER2 copy number of <4.0 signals per cell be considered ISH positive (designated as ISH group 2)? (4) Should invasive cancers with an average HER2 copy number of ≥6.0 signals per cell but a HER2/CEP17 ratio of <2.0 be considered ISH positive (designated as ISH group 3)? (5) What is the appropriate diagnostic work –up for invasive cancers with an average HER2 copy number of ≥4.0 but <6.0 signals per cell and a HER2/CEP17 ratio of <2.0 and initially deemed to have an equivocal HER2 ISH test result (designated as ISH group 4)In this study, we retrospectively reviewed HER2 status in 1810 breast cancer patients with equivocal IHC results to assess the impact of these revised guidelines.HER2 FISH results were collected from 1810 invasive breast cancer cases between January 2012 and May 2019. All cases were equivocal for HER2 IHC. Specimens included core needle biopsies, surgical excisions, and biopsy samples from metastatic sites. This research was approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University. The Committee waived the written informed consents for the data were analyzed anonymously. All experiments were performed in accordance with relevant guidelines and regulations.Automated IHC for HER2  was performed on 4-um-thick tissue sections using an automated slide stainer, the Ventana Benchmark XT . The results were interpreted according to the 2018 guidelines.4-um-thick tissue sections were deparaffinized, rehydrated, and immersed in distilled water for 40 min at above 90 °C. The slides were incubated for 18 min in protease solution at 37 °C. After dehydration with alcohol, a total of 10 uL HER2/CEP17 mixture probe  was added to the slides. The slides were then transferred to a hybridization oven . The procedure was as follows: denature at 83 °C for 5 min, and hybridization overnight at 42 °C. The second day, the slides were washed in preheated post-hybridization buffer, air dried and then counterstained with 15 μl DAPI. HER2 FISH signals were interpreted by one technologist (BW) and one pathologist (KS). Thirty nuclei from two non-overlapping areas were counted. When there was a conflict between the scores, another pathologist (XLW) would review the slide and reach the final result. The new guidelines were applied for the interpretation of FISH testing results.10, 318 cases with a HER2/CEP17 ratio ≥2.0 and ≥4.0 HER2 signals per cell (ISH group 1) were diagnosed as HER2 positive. 1406 cases with a HER2/CEP17 ratio <2.0 and <4.0 HER2 signals per cell (ISH group 5) were negative for HER2. There were 29, 4 and 53 cases in ISH group 2, 3 and 4 respectively. The positive rate was 19.40% (351/1810). Clinicopathological characteristics of the patients in each ISH group were summarized in Table A total of 1810 cases were enrolled in our study. Most of the cases (n = 1478) were sampled from surgical excisions. Specimens from core needle biopsies (n = 139) and metastatic sites n = 72) were much less common (Table  were mucTwenty-nine 1.60%) cases fell into ISH group 2. Among them, twenty-five cases were sampled from surgical excisions, three from core needle biopsies, and one from lymph node metastasis (Table .60% caseThere were only four (0.22%) cases in ISH group 3, which were composed of two specimens from surgical excisions, one from lymph node metastasis, and one from core needle biopsy Table . Two casFifty-three 2.92%) of the 1810 cases fell into ISH group 4. Most of the cases n = 35) were sampled from surgical excisions, followed by 14 from core needle biopsies and 4 from metastatic sites  of the cases had a different final interpretation by 2018 guidelines compared with 2013 guidelines, including 30 previously considered HER2-positive cases being categorized as negative, 53 previously ISH equivocal cases being redistributed into 10 positive cases and 43 negative cases. In total, 331 of the 1810 cases were positive for HER2, with a slight decrease in positive rate from 19.4% to 18.3% 20. Another research showed that of 35 patients with this ISH pattern tested by IHC, only 3 cases were IHC 2+ and none of them were IHC 3+14. In parallel with these findings, our data also found that there was no case scored IHC 3+ in this group. So far as we know, only one case with HER2 IHC positive was reported in ISH group 221.The HER2 status in ISH group 2 is uncommon. Twenty-nine cases were included in this group, accounting for 1.60% of the present cohort , which was consistent with other reports22. A ratio of <2.0 can be attributed to the increase in both HER2 and control centromere signals. All the four cases in the present study had gain of chromosome 17 (CEP17 ≥ 3.0). A remarkable variability of IHC score for cases in this group was observed across different laboratories21. The positive rate of IHC ranged from 8.3% to 75%. Cases with IHC 0/1+ could also be identified. Similarly, our data showed that two cases were scored as IHC 2+, while the other two were interpreted as IHC 3+ and 1+. Owing to the limited number of cases enrolled in clinical trials, a definitive conclusion on whether the patients with this ISH pattern can benefit from HER2-targeted therapy cannot be reached. Considering the heterogeneity of HER2 IHC results, the 2018 guidelines recommend that cases with concurrent IHC score of 2+/3+ be categorized as HER2 positive.There were only four cases in ISH group 3, with a lower incidence than other reports25. Some laboratories relied on alternative chromosome 17 genes as surrogate to deal with this issue, leading to a change in HER2 status from equivocal to positive in about 50% of patients27. Due to the presence of false-positive HER2 results and the absence of evidence in the therapy efficacy, this approach has not been recommended in the 2018 guidelines. In our study, after concomitant IHC review, 29 cases were scored as IHC 0/1+, 15 were IHC 2+ and 9 were IHC 3+. Previous studies proved that HER2 genetic heterogeneity in breast cancers was most frequent in cases with IHC 2+ and equivocal HER2 amplification29. Therefore, another tissue block was selected for further FISH testing in the 15 cases with IHC 2+. Only one case was turned out to be HER2 positive with a HER2/CEP17 ratio of 2.19 and an average of 8.03 HER2 signals per cell. Finally, ten cases were converted to HER2 positive and forty-three cases were diagnosed as HER2 negative. The suggestion that repeat testing on the other tissue samples from the same patients in the new guidelines is appropriate in this setting.Fifty-three cases were assigned as ISH group 4. Before the introduction of the 2018 guidelines, the patients with equivocal ISH results posed a challenge to oncologists of whether to recommend HER2-targeted therapy. Moreover, implementation of the 2013 guidelines resulted in the detection of more equivocal casesIn conclusion, the utility of 2018 guidelines resulted in a mild decrease in HER2 positive rate, due to the reclassification of cases in ISH group 2 and group 4. The implementation of the new guidelines can reduce reflex FISH test and make the diagnosis of HER2 gene status more definitive.