Identification of functional cis-regulatory elements by sequential enrichment from a randomized synthetic DNA library

BACKGROUND: The identification of endogenous cis-regulatory DNA elements (CREs) responsive to endogenous and environmental cues is important for studying gene regulation and for biotechnological applications but is labor and time intensive. Alternatively, by taking a synthetic biology approach small specific DNA binding sites tailored to the needs of the scientist can be generated and rapidly identified. RESULTS: Here we report a novel approach to identify stimulus-responsive synthetic CREs (SynCREs) from an unbiased random synthetic element (SynE) library. Functional SynCREs were isolated by screening the SynE libray for elements mediating transcriptional activity in plant protoplasts. Responsive elements were chromatin immunoprecipitated by targeting the active Ser-5 phosphorylated RNA polymerase II CTD (Pol II ChIP). Using sequential enrichment, deep sequencing and a bioinformatics pipeline, candidate responsive SynCREs were identified within a pool of constitutively active DNA elements and further validated. These included bonafide biotic/abiotic stress-responsive motifs along with novel SynCREs. We tested several SynCREs in Arabidopsis and confirmed their response to biotic stimuli. CONCLUSIONS: Successful isolation of synthetic stress-responsive elements from our screen illustrates the power of the described methodology. This approach can be applied to any transfectable eukaryotic system since it exploits a universal feature of the eukaryotic Pol II

Loss of CLN7 results in depletion of soluble lysosomal proteins and impaired mTOR reactivation

Defects in the MFSD8 gene encoding the lysosomal membrane protein CLN7 lead to CLN7 disease, a neurodegenerative lysosomal storage disorder belonging to the group of neuronal ceroid lipofuscinoses (NCLs). Here we have performed a SILAC-based quantitative analysis of the lysosomal proteome using Cln7-deficient mouse embryonic fibroblasts (MEFs) from a Cln7 knockout (ko) mouse model. From 3335 different proteins identified, we detected 56 soluble lysosomal proteins and 29 highly abundant lysosomal membrane proteins. Quantification revealed that the amounts of 12 different soluble lysosomal proteins were significantly reduced in Cln7 ko MEFs compared with wild type controls. One of the most significantly depleted lysosomal proteins was Cln5 protein that underlies another distinct NCL disorder. Expression analyses showed that the mRNA expression, biosynthesis, intracellular sorting and proteolytic processing of Cln5 were not affected, whereas the depletion of mature Cln5 protein was due to increased proteolytic degradation by cysteine proteases in Cln7 ko lysosomes. Considering the similar phenotypes of CLN5 and CLN7 patients, our data suggest that depletion of CLN5 may play an important part in the pathogenesis of CLN7 disease. In addition, we found a defect in the ability of Cln7 ko MEFs to adapt to starvation conditions as shown by impaired mammalian target of rapamycin complex 1 reactivation, reduced autolysosome tubulation and increased perinuclear accumulation of autolysosomes compared to controls. In summary, depletion of multiple soluble lysosomal proteins suggest a critical role of CLN7 for lysosomal function, which may contribute to the pathogenesis and progression of CLN7 disease

Evaluation of suspicious appearing microcalcification groups on mammogram: Comparing BI-RADS 5th edition descriptors with the BI-RADS 4th edition and pathologic association

Background: Groups of microcalcifications are the most frequent recognized features of ductal carcinoma on mammograms. However, heterogeneity (in size, morphology and density) and number of microcalcification groups as well as presence of accompanied soft-tissue density are not included in breast imaging reporting and data system (BI-RADS) descriptors. Objectives: The study purposes to determine the malignancy risk of microcalcification groups regarding these characteristics and also compare the 4th and 5th versions of BI-RADS. Patients and Methods: In a cross sectional study, 88 patients with microcalcification groups (age range, 26-80 years; mean, 53.4 years) who had undergone mammographically guided biopsy between March 2013 and March 2015 were evaluated. The overall number of microcalcification groups in each patient, number of deposits within each group, group location and heterogeneity in size, density and morphology were assessed and subsequently BI-RADS descriptors for 4th and 5th editions were recorded separately. Finally, correlation with histopathology was performed. Results: Overall, positive predictive value (PPV) of suspicious microcalcifications was 22.4. PPVs of morphology descriptors were as follows: amorphous, 7.9; coarse heterogeneous, 17.8; fine pleomorphic, 63.2; fine linear/fine linear branching, 100; (P < 0.001). Heterogenicity in size existed in 81 cases (92), in density in 69 cases (86.4) and in morphology in 86 cases (97.7). Additionally, microcalcification groups that were accompanied with soft-tissue density had a higher percentage of malignancy (67.5 vs. 54.5) but with no significant difference (P = 0.2). According to BI-RADS 4th edition, the risk of malignancy was 49.1, 66.7 and 88.1 in 4b, 4c and 5, respectively. These figures were 30, 82.9, and 100 for BI-RADS 5th version, respectively. The area under the receiver (AUC) of 4th and 5th versions of BI-RADS was 0.76 and 0.74 (both P values < 0.001, 95 confidence intervals = 0.66-0.87 and 0.63-0.85 respectively). P value for comparison was insignificant. Conclusion: The risk of malignancy increased with the heterogeneity of microcalcifications, especially in the groups with heterogeneity in density, however with no statistically significant difference. BI-RADS 5th edition could predict the likelihood of malignancy as well as 4th version. © 2018, Iranian Journal of Radiology

Evaluation of suspicious appearing microcalcification groups on mammogram: Comparing BI-RADS 5th edition descriptors with the BI-RADS 4th edition and pathologic association

Background: Groups of microcalcifications are the most frequent recognized features of ductal carcinoma on mammograms. However, heterogeneity (in size, morphology and density) and number of microcalcification groups as well as presence of accompanied soft-tissue density are not included in breast imaging reporting and data system (BI-RADS) descriptors. Objectives: The study purposes to determine the malignancy risk of microcalcification groups regarding these characteristics and also compare the 4th and 5th versions of BI-RADS. Patients and Methods: In a cross sectional study, 88 patients with microcalcification groups (age range, 26-80 years; mean, 53.4 years) who had undergone mammographically guided biopsy between March 2013 and March 2015 were evaluated. The overall number of microcalcification groups in each patient, number of deposits within each group, group location and heterogeneity in size, density and morphology were assessed and subsequently BI-RADS descriptors for 4th and 5th editions were recorded separately. Finally, correlation with histopathology was performed. Results: Overall, positive predictive value (PPV) of suspicious microcalcifications was 22.4. PPVs of morphology descriptors were as follows: amorphous, 7.9; coarse heterogeneous, 17.8; fine pleomorphic, 63.2; fine linear/fine linear branching, 100; (P < 0.001). Heterogenicity in size existed in 81 cases (92), in density in 69 cases (86.4) and in morphology in 86 cases (97.7). Additionally, microcalcification groups that were accompanied with soft-tissue density had a higher percentage of malignancy (67.5 vs. 54.5) but with no significant difference (P = 0.2). According to BI-RADS 4th edition, the risk of malignancy was 49.1, 66.7 and 88.1 in 4b, 4c and 5, respectively. These figures were 30, 82.9, and 100 for BI-RADS 5th version, respectively. The area under the receiver (AUC) of 4th and 5th versions of BI-RADS was 0.76 and 0.74 (both P values < 0.001, 95 confidence intervals = 0.66-0.87 and 0.63-0.85 respectively). P value for comparison was insignificant. Conclusion: The risk of malignancy increased with the heterogeneity of microcalcifications, especially in the groups with heterogeneity in density, however with no statistically significant difference. BI-RADS 5th edition could predict the likelihood of malignancy as well as 4th version. © 2018, Iranian Journal of Radiology

Risk factors of developing critical conditions in Iranian patients with COVID-19

COVID-19 due to novel Coronavirus was first reported in Wuhan, China. Nowadays, the Islamic Republic of Iran stands among countries with high COVID-19 prevalence and high burden of disease. Since the medical resources are limited, we aimed to identify the risk factors for patients developing critical conditions. This can help to improve resource management and treatment outcomes. In this retrospective study, we included 12,677 patients who were from 26 hospitals, supervised by Tehran University of Medical Sciences with signs and symptoms of COVID-19, until April 12. University integrated IT system was adopted to collect the data. We performed Logistic regression to evaluate the association between death in COVID-19 positive patients and other variables. Cough, respiratory distress and fever were the most common symptoms in our patients, respectively. Cancer, chronic lung diseases and chronic neurologic diseases were the strongest risk factors for death in COVID-19 patients. © 202

Barley Ror1 encodes a class XI myosin required for mlo-based broad-spectrum resistance to the fungal powdery mildew pathogen

Loss-of-function alleles of plant MLO genes confer broad-spectrum resistance to powdery mildews in many eudicot and monocot species. Although barley (Hordeum vulgare) mlo mutants have been used in agriculture for more than 40 years, understanding of the molecular principles underlying this type of disease resistance remains fragmentary. Forward genetic screens in barley have revealed mutations in two Required for mlo resistance (Ror) genes that partially impair immunity conferred by mlo mutants. While Ror2 encodes a soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE), the identity of Ror1, located at the pericentromeric region of barley chromosome 1H, remained elusive. We report the identification of Ror1 based on combined barley genomic sequence information and transcriptomic data from ror1 mutant plants. Ror1 encodes the barley class XI myosin Myo11A (HORVU.MOREX.r3.1HG0046420). Single amino acid substitutions of this myosin, deduced from non-functional ror1 mutant alleles, map to the nucleotide-binding region and the interface between the relay-helix and the converter domain of the motor protein. Ror1 myosin accumulates transiently in the course of powdery mildew infection. Functional fluorophore-labeled Ror1 variants associate with mobile intracellular compartments that partially colocalize with peroxisomes. Single-cell expression of the Ror1 tail region causes a dominant-negative effect that phenocopies ror1 loss-of-function mutants. We define a myosin motor for the establishment of mlo-mediated resistance, suggesting that motor protein-driven intracellular transport processes are critical for extracellular immunity, possibly through the targeted transfer of antifungal and/or cell wall cargoes to pathogen contact sites

Phylogenetic Distribution of Intron Positions in Alpha-Amylase Genes of Bilateria Suggests Numerous Gains and Losses

Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that “resets” of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures

A Survey On Gastrointestinal And Hepatic Manifestations Of Brucellosis Imam Hospital (1995-2001)

Brucellosis is a zoonotic disease of worldwide distribution. Despite its control in many developing countries the disease remains endemic in Iran. The symptoms, signs and laboratory results are variable and nonspecific. This case series study was conducted to determine the liver complications of Brucellosis in Iran&quot;nMaterials and Methods: We studied 188 patients (108 males and 80 females) with Brucellosis, fulfilled the diagnostic criteria, aged 1-79 years (mean 34.8 years) were registered in Imam Khomeini Hospital, a referral center in Tehran, during the six years (1995-2001).&quot;nResults: Thirty-four of 188 cases (18.08 percent) had elevated liver enzyme (elevated SGOT only, 6 patients; elevated SGPT only 1 patient; elevation of both transaminases, 27 patients). The prominent symptoms included anorexia (74 cases), weight loss (62 cases), right upper quadrant pain (32 cases), epigastric pain (25 cases) and nausea and vomiting (23 cases). Among the gastrointestinal signs were found in these patients, hepatomegaly was seen in 28 patients. Jaundice and ascitis were present in only 7 and 3 patients, respectively. Other laboratory results showed elevated alkaline phosphatase in 28 cases and abnormal bilirubin in 10 cases. Fifty-seven patients had a focal illness, representing 30.32 percent of all patients. Osteoarticular complications were the most frequent focal forms, being present in 34 cases. Twelve male patients had genitourinary Brucellosis, representing 10.53 percent of focal forms. Also, 5 patients had neurologic complications.&quot;nConclusion: In conclusion liver involvement is frequent in Brucellosis, although the rate of this complication in our study was lower than other studies. So, in patients with evidence of overt clinical or laboratory findings compatible with liver disturbance etiologies other than brucellosis should be considered in Iran.&quot;n&quot;n&quot;n&amp;nbsp

Mutational Decay and Age of Chloroplast and Mitochondrial Genomes Transferred Recently to Angiosperm Nuclear Chromosomes

Transfers of organelle DNA to the nucleus established several thousand functional genes in eukaryotic chromosomes over evolutionary time. Recent transfers have also contributed nonfunctional plastid (pt)- and mitochondrion (mt)-derived DNA (termed nupts and numts, respectively) to plant nuclear genomes. The two largest transferred organelle genome copies are 131-kb nuptDNA in rice (Oryza sativa) and 262-kb numtDNA in Arabidopsis (Arabidopsis thaliana). These transferred copies were compared in detail with their bona fide organelle counterparts, to which they are 99.77% and 99.91% identical, respectively. No evidence for purifying selection was found in either nuclear integrant, indicating that they are nonfunctional. Mutations attributable to 5-methylcytosine hypermutation have occurred at a 6- to 10-fold higher rate than other point mutations in Arabidopsis numtDNA and rice nuptDNA, respectively, revealing this as a major mechanism of mutational decay for these transferred organelle sequences. Short indels occurred preferentially within homopolymeric stretches but were less frequent than point mutations. The 131-kb nuptDNA is absent in the O. sativa subsp. indica or Oryza rufipogon nuclear genome, suggesting that it was transferred within the O. sativa subsp. japonica lineage and, as revealed by sequence comparisons, after its divergence from the indica chloroplast lineage. The time of the transfer for the rice nupt was estimated as 148,000 (74,000–296,000) years ago and that for the Arabidopsis numtDNA as 88,000 (44,000–176,000) years ago. The results reveal transfer and integration of entire organelle genomes into the nucleus as an ongoing evolutionary process and uncover mutational mechanisms affecting organelle genomes recently transferred into a new mutational environment

Lrp1/LDL receptor play critical roles in mannose 6-phosphate-independent lysosomal enzyme targeting

Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P residues results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types suggesting the existence of M6P-independent transport routes. Here, we quantify the lysosomal proteome in M6P-deficient mouse fibroblasts (PT(ki) ) using Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based comparative mass spectrometry, and find unchanged amounts of 20 % of lysosomal enzymes, including cathepsin D and B (Ctsd, Ctsb). Examination of fibroblasts from a new mouse line lacking both M6P and sortilin, a candidate for M6P-independent transport of lysosomal enzymes, revealed that sortilin does not act as cargo receptor for Ctsb and Ctsd. Using fibroblast lines deficient for endocytic lipoprotein receptors we could demonstrate that both LDL receptor and Lrp1 mediate the internalization of non-phosphorylated Ctsb and Ctsd. Furthermore, the presence of Lrp1 inhibitor increased the secretion of Ctsd from PT(ki) cells. These findings establish Lrp1 and LDL receptors in M6P-independent secretion-recapture targeting mechanism for lysosomal enzymes