cDNA Levels Research Articles

Biallelic PIGM Coding Variant Causes Intractable Epilepsy and Intellectual Disability Without Thrombotic Events.

During the past two decades, an emerging group of genes coding for proteins involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis are being implicated in early-infantile epileptic encephalopathy. Amongst these, a hypomorphic promoter mutation in the mannosyltransferase-encoding PIGM gene was described in seven patients to date, exhibiting intractable absence epilepsy, portal and cerebral vein thrombosis and intellectual disability (ID). We describe here three siblings exhibiting intractable epilepsy and ID, found to harbor a homozygous c.224G>A p.(Arg75His) missense variant in PIGM, which segregated with the disease in the family. The variant is evolutionary conserved, extremely rare in general population databases and predicted to be deleterious. Structural modeling of the PIGM protein and the p.(Arg75His) variant indicates that it is located in a short luminal region of the protein, predicted to be hydrophilic. Functional prediction suggests that the entire local region is sensitive to mutations, with the p.(Arg75His) variant in particular. This is the first report of a PIGM coding variant, and the second variant altogether to be described affecting this gene. This phenotype differs from that of patients with the shared PIGM promoter mutation by lack of thrombotic events and no decrease in PIGM cDNA levels or CD59 expression on red blood cells.

Time-dependent effects of tumor necrosis factor α on Ca2+-dependent secretion in murine small intestinal organoids.

Intestinal organoids are stem cell-derived, 3D "mini-guts" with similar functions as the native intestinal epithelium such as electrolyte transport or establishment of an epithelial barrier. During intestinal inflammation, epithelial functions are dysregulated by proinflammatory cytokines like tumor necrosis factor α (TNFα) and other messengers from the immune system resulting in a loss of electrolytes and water due to an impaired epithelial barrier and higher net secretion. A murine small intestinal organoid model was established to study (long-term) effects of TNFα on the intestinal epithelium in vitro using live imaging, immunohistochemical staining and qPCR. TNFα induced apoptosis in intestinal organoids as indicated by an increased number of cells with immunoreactivity for cleaved caspase 3. Furthermore, TNFα exposure led to swelling of the organoids which was inhibited by bumetanide and was concomitant with an upregulation of the bumetanide-sensitive Na+-K+-2Cl- symporter 1 (NKCC1) as shown by qPCR. Fura-2 imaging experiments revealed time-dependent changes in Ca2+ signaling consisting of a rise in the basal cytosolic Ca2+ concentration at day 1 and an increase of the carbachol-induced Ca2+ response after 3days TNFα exposure. This was prevented by preincubation with La3+, an inhibitor of non-selective cation channels, or by using a Ca2+-free buffer indicating an enhancement of the Ca2+ influx from the extracellular side by the cytokine. No significant changes in cDNA levels of epithelial barrier proteins could be observed in the presence of TNFα. Intestinal organoids are a useful tool to study the mechanism underlying the TNFα-induced secretion on enterocytes such as the regulation of NKCC1 expression or the modulation of cellular Ca2+ signaling.

Abstract 253: Investigating key molecular players in putative stem cell subpopulations of prostate cancer

Abstract Background: Androgen deprivation therapy is the standard treatment for prostate cancer (PCa). Nevertheless, despite initial effectiveness, pre-existing cancer stem cell (CSC) populations invariably lead to incurable castration-resistant prostate cancer. CSCs are a subset of cancer cells possessing self-renewal properties, driving tumor progression and regrowth. CD44+ PCa cells exhibit more stemness features and are enriched in tumorigenic and metastatic progenitor cells. Here, we aim to explore different subpopulations in tumors based on levels of CD44 expression and investigate whether they have distinct molecular properties and functional characteristics. Material and Method: The number of CD44+ cells was evaluated in a tissue microarray from primary prostate cancer patient samples (EMPACT cohort) with clinical follow-up. Flow cytometry sorted the CRPC model LAPC9 tumor into CD44 high (CD44-H) and CD44 low (CD44-L) cells. RNA sequencing was performed on these subpopulations to explore their transcriptomic profiles. CD44 expression was validated at the cDNA and protein levels using qPCR and Western blot (WB) analysis. Sorted cells were maintained as organoids in vitro. To trace the dynamics of CD44-H and CD44-L subpopulations in vivo, LAPC9 tumor was labeled by fluorescent and luciferent markers. The CD44 status was determined by flow cytometry when tumor growth. Results: Patients with elevated expression of CD44 (higher number of CD44+ cells) at the time of surgery exhibited a greater propensity for clinic progression (5-year progression-free survival: 75% vs 95%, P=0.03). Furthermore, CD44-H cell ratio increased in the LAPC9 after castration, indicating that CD44-H cells can survive after treatment. Subsequent WB analysis confirmed that CD44 was highly expressed in sorted CD44-H cells, validating the flow cytometry results. Analysis of the CD44 RNA-seq data showed CD44 transcript variants CD44v10 (CD44-201) and CD44v7-10 (CD44-209) were notably upregulated in CD44-H cells. In organoid culture, sorted CD44-H cells displayed enhanced formation, indicating they possessed higher proliferative capacity and clonogenicity than CD44-L cells. Furthermore, when GFP-labeled CD44-H cells and RFP-labeled CD44-L cells were recombined in vivo, CD44-H cells exhibited robust proliferation, while CD44-L cells transitioned to a CD44hi state to facilitate tumor growth. Moreover, those GFP and RFP subpopulations displayed the same CD44hi cell ratio at the endpoint. Conclusion: Elevated CD44 expression is related to an increased risk in primary PCa. In addition, CD44-H cells exhibit high tumorigenicity in vitro and in vivo and can convert to each other. Moreover, the ratio of CD44-H cells in tumor at endpoint is consistent despite the starting CD44 expression status. Furthermore, the upregulation of CD44v10 and v7-10 in the CD44-H subpopulation may promote tumor formation and metastasis. Citation Format: Wanli Cheng, Marta De Menna, Francesco Bonollo, Panagiotis Chouvardas, George N. Thalmann, Sofia Karkampouna, Marianna Kruithof Julio. Investigating key molecular players in putative stem cell subpopulations of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 253.

Impact of dissolved oxygen levels on N2O emissions and metabolically active bacterial community in biological nitrogen removal

Biological nutrient removal (BNR) is an effective process for removing nitrogen from wastewater using microorganisms under different environmental conditions. However, these processes can result in nitrous oxide (N2O) emission. Dissolved oxygen (DO) levels shape bacterial communities and affect N2O emissions in BNR. Ammonia-oxidizing bacteria (AOB), the main N2O producers in the BNR process, are significantly affected by DO. The bacterial communities responding to DO at the DNA level in BNR have been elucidated. However, the metabolically active microbes are truly participant in the biodegradation process. Thus, it is worth exploring how metabolically active bulk and AOB microbial communities and their N2O production respond to the DO levels. In this study, N2O emission were measured in lab-scale sequencing batch reactors at varying DO levels. High-throughput sequencing of the metabolically active bacterial 16S rRNA and amoA genes was performed. The results indicate that increasing DO levels positively influence metabolically active bacterial biodiversity and AOB relative abundance without increasing N2O emissions. The carbon source significantly affected denitrification and nitrification in activated sludge. Optimal DO levels and adequate carbon sources can effectively reduce N2O accumulation. However, excessive aeration reduces the abundance of Zoogloea, leading to compromised sludge settling. This study revealed the effect of DO on N2O producing microorganisms based on cDNA levels, which provided an important basis for mitigating N2O emission in BNR.

Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses.

Whole-genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), a viral disease of economic significance to poultry producers. The current strategies and procedures used to obtain the complete genome sequences of ARV isolates are not cost-effective because most of the genetic material data resulting from next-generation sequencing belong to the host and cannot be used to assemble the viral genome. The purpose of this study was to develop a workflow to enrich the ARV genomic content in a sample before subjecting it to next-generation sequencing (NGS). Herein, we compare four different ARV purification and enrichment approaches at the virion, RNA and cDNA levels to determine which treatment or treatment combination would provide a higher proportion of ARV-specific reads after WGS. Seven ARV isolates were subjected to different combinations of virion purification via ultracentrifugation in sucrose density gradient or Capto Core 700 resin with or without a subsequent Benzonase treatment, followed by a chicken rRNA depletion step after RNA extraction and a final ARV cDNA amplification step using a single-primer amplification assay. Our results show that the combination of Capto Core 700 resin, Chicken rRNA depletion and cDNA amplification is the most cost-effective strategy to obtain ARV whole genomes after short-read sequencing.

A novel heterozygous PKD1 variant causing alternative splicing in a Chinese family with autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by pathogenic variants of PKD1 and PKD2. Compared to PKD2-related patients, patients with PKD1 pathogenic variants have more severe symptoms, present a gradual decline in renal function, and finally progress to end-stage kidney disease with an earlier mean onset age. In this study, trio exome sequencing (ES) was performed to reveal the genetic etiology in a Chinese family clinically diagnosed with polycystic kidney, followed by validation through Sanger sequencing on both genomic DNA and cDNA levels. Subsequently, targeted preimplantation genetic testing was provided for the family. A novel heterozygous PKD1 variant (c.1717_1722+11del) was detected in the proband and other clinically-affected relatives. Interestingly, cDNA sequencing demonstrated that the variant, despite being annotated as non-frameshift within exon 8, impacted the splicing of PKD1. Two abnormal transcription products were formed: one induced frameshift, while the other caused 133 amino acids to be inserted between exon 8 and exon 9. Our study revealed a novel PKD1 variant using ES as the cause of ADPKD in a Chinese family with multiple affected members. The variant at the exon-intron boundary would induce alternative splicing, which should not be excluded from genetic analysis. Validated on the cDNA level could provide more comprehensive genetic information for disease stratification. And the novel variant expands the spectrum of PKD1 variants in ADPKD. The recurrent risk could be blocked accordingly for the families' offspring.

Prognostic importance of PD-L1 expression in bladder cancer patients.

PD-L1/PD-1 molecules are known as important mediators in immune-escape mechanisms of tumors. PD-L1 is highly expressed in various malignancies, including bladder cancer. However, the prognostic value of PD-L1 in bladder cancer patients remains controversial. To investigate the prognostic significance of PD-L1 expression in tumor tissues of bladder cancer patients. RNA was isolated from FFPE tumor tissues of 48 bladder cancer patients using the monophasic phenol and guanidine isothiocyanate method. Total RNA was converted to cDNA and gene expression levels were analyzed by qRT-PCR. The differential expression levels of the PD-L1 gene between tumor grade and cancer stage groups were analyzed by independent student's t-test and one-way ANOVA. Statistically significantly increased PD-L1 expression was observed in the high-grade tumor group (p < 0.05). No significant difference in PD-L1 expression was found among pTa, pT1, and pT2 groups. In addition, the difference in overall survival was not significantly different between groups. The results showed that high PD-L1 expression in bladder cancer was associated with tumor aggressiveness and grade. Despite the inability of the qRT-PCR to show the PD-L1 expression at different locations of tumor tissue, evaluation of PD-L1 mRNA expression by qRT-PCR, which is a highly sensitive and specific assay, appears to be a robust approach. Furthermore, these findings may contribute to a rationale for recommending anti-PD-L1 immunotherapy as an alternative to standard therapy for bladder cancer patients who are most likely to benefit from it.

Efficiency and Mechanism Evaluation of Magnolia officinalis Water Extract in Preventing Gastric Ulcer.

In this study we aimed at demonstrating the ability of Magnolia officinalis water extract to ameliorate gastric ulcers in in vitro and in vivo experiments. The gastric mucosa epithelial cell line, RGM 1, was pretreated with Magnolia officinalis water extract (0, 0.1, 1, 2, 5, or 10 mg/ml) and cultured in DMEM/F12 medium (pH 7.4) for 2 h and then in DMEM/F12 medium (pH 4.0) for 10 min. Magnolia officinalis water extract protected the cell viability and decreased reactive oxygen species formation by the acidic medium. In the in vivo experiment, Magnolia officinalis water extract (100 mg/kg) was administrated daily for 28 days in ICR mice via oral gavage, and then Shay's ulcer surgical method was performed to induce gastric ulcers. We analyzed the pH value of stomach acid and the pathological section, inflammation, and cannabinoid receptor type 2 (CB2) cDNA levels of the stomach. Magnolia officinalis water extract not only enhanced the pH value of stomach acid but also ameliorated the ulcer index and inflammation and increased CB2 expression effectively. These results suggest that Magnolia officinalis water extract might be used to decrease the incidence of gastric ulcer.

Development and validation of SSR markers related to flower color based on full-length transcriptome sequencing in Chrysanthemum

Chrysanthemum (Chrysanthemum moriforlium Ramat.) is one of the most popular flowers worldwide, with very high ornamental and economic values. However, the limitations of available DNA molecular markers and the lack of full genomic sequences hinder the study of genetic diversity and the molecular breeding of chrysanthemum. Here, we developed simple sequence repeat (SSR) from the full-length transcriptome sequences of chrysanthemum cultivar ‘Hechengxinghuo’. A total of 11,699 SSRs with mono-, di-, tri-, tetra-, penta- and hexanucleotide repeats were identified, of which eight out of eighteen SSR loci identified based on sixteen transcripts participated in carotenoid metabolism or anthocyanin synthesis were validated as polymorphic SSR markers. These SSRs were used to classify 117 chrysanthemum accessions with different flower colors at the DNA and cDNA levels. The results showed that four SSR markers of carotenoid metabolic pathway divided 117 chrysanthemum accessions into five groups at cDNA level and all purple chrysanthemum accessions were in the group III. Furthermore, the SSR marker CHS-3, LCYE-1 and 3MaT may be related to green color and the PSY-1b marker may be related to yellow color. Overall, our work may be provide a novel method for mining SSR markers associated with specific traits.

Cis-Allosteric Regulation of HIV-1 Reverse Transcriptase by Integrase.

Reverse transcriptase (RT) and integrase (IN) are encoded tandemly in the pol genes of retroviruses. We reported recently that HIV-1 RT and IN need to be supplied as the pol precursor intermediates, in which RT and IN are in fusion form (RTIN) to exert efficient reverse transcription in the context of HIV-1 replication. The mechanism underlying RTIN's effect, however, remains to be elucidated. In this study, we examined the effect of IN fusion on RT during reverse transcription by an in vitro cell-free assay, using recombinant HIV-1 RTIN (rRTIN). We found that, compared to recombinant RT (rRT), rRTIN generated significantly higher cDNAs under physiological concentrations of dNTPs (less than 10 μM), suggesting increased affinity of RTIN to dNTPs. Importantly, the cleavage of RTIN with HIV-1 protease reduced cDNA levels at a low dose of dNTPs. Similarly, sensitivities against RT inhibitors were significantly altered in RTIN form. Finally, analysis of molecular dynamics simulations of RT and RTIN suggested that IN can influence the structural dynamics of the RT active center and the inhibitor binding pockets in cis. Thus, we demonstrated, for the first time, the cis-allosteric regulatory roles of IN in RT structure and enzymatic activity.

Effects of various straw incorporation strategies on soil phosphorus fractions and transformations

AbstractReturning straw to the field is an effective method for optimizing the soil phosphorus (P) availability, in which bacteria play an important role. However, the effects of various straw incorporation strategies on P transformation between different soil P pools remain unclear. In this study, variations in soil P fractions, phosphatase activities and the abundance of phosphatase genes (phoD, phoX and phoC) as well as a P‐solubilizing gene (pqqC) at DNA (total) and cDNA (transcribed) levels were analysed in three straw incorporation treatments, including chopped straw (StrawD), straw compost (Compost) and straw‐derived biochar (Biochar), and control (no straw, CK). Compared with the CK, the moderately labile inorganic P (NaOH I‐Pi) content significantly decreased and the non‐available P (Residual P) content significantly increased in the StrawD treatment. At the same time, phosphodiesterase (PD) activity and the transcribed phoC and phoX genes as well as total pqqC gene abundance significantly increased in the StrawD treatment, suggesting that the input of chopped straw stimulated P transformations from both organic and inorganic P pools. In addition, the stable Pi (NaOH II‐Pi) content and total pqqC gene abundance in the Biochar treatment were significantly higher than that in the CK, indicating that the input of biochar increased the NaOH II‐Pi that could release available P by Pi‐solubilizing bacteria. In comparison to the CK, the Compost input significantly decreased one labile Pi (resin‐Pi) only. However, its P fractions were significantly different from that of CK, Biochar and StrawD treatments, suggesting that the effects of compost input on P should not be ignored. In conclusion, chopped straw input increased soil P transformation but not available P, biochar input may promote inorganic P transformation, and compost input has a latent effect on P transformation. The study provided a comprehensive understanding of straw incorporation strategies for regulating soil P availability.

Development and validation of a minimally invasive and image-guided tape stripping method to sample atopic skin in children.

Molecular skin profiling techniques, typically performed on skin samples taken by punch biopsy, have enhanced the understanding of the pathophysiology of atopic dermatitis (AD), thereby enabling the development of novel targeted therapeutics. However, punch biopsies are not always feasible or desirable, and novel minimally invasive methods such as skin tape stripping have been developed. To develop, optimize and validate a novel tape stripping method guided by noninvasive in vivo skin imaging to sample atopic skin in children. Skin tape stripping-based procedures were compared and optimized using data from 30 healthy controls (HCs: 5 adults, 25 children) and 39 atopic children. Evaluations were guided by high-resolution photography, reflectance confocal microscopy, optical coherence tomography and transepidermal water loss measurements. We assessed and compared adverse events (AEs), the time needed to perform the sampling and the cDNA levels obtained from the tapes. Tape stripping methods based on previously described protocols resulted in erosions in all participants and required a median time of 65 min to perform (range 60-70 min), but provided good cDNA yield. Shorter durations appeared less invasive but provided lower cDNA yield. The final optimized tape stripping protocol, using 11 tapes of 22 mm in diameter, each applied twice for 5 s with 90° rotation, did not produce significant AEs, was completed within a median time of 7 min (range 5-15 min) and provided good cDNA yield both in HCs and atopic children. Our minimally invasive method is safe and reliable, and provides reproducible acquisition of cDNA in atopic children. In addition, it enables rapid sample collection, a crucial factor in clinical practice.

Distribution and Oxidation Rates of Ammonia-Oxidizing Archaea Influenced by the Coastal Upwelling off Eastern Hainan Island.

Coastal upwelling causes variations in temperature, salinity and inorganic nutrients in the water column, consequently leading to the shift of microbial populations and their metabolic activities. Impacts of the eastern Hainan upwelling (EHU) on the ammonia-oxidizing archaea (AOA) were investigated based on the amoA gene using pyrosequencing and quantitative PCR at both DNA and cDNA levels, together with the determination of the ammonia oxidation (AO) rate measured with 15N-labelled ammonium. By comparing stations with and without upwelling influence, we found that coastal upwelling correlated with an increase in amoA gene abundance, the dominance of distinct clades for AOA communities at the respective gene and transcript levels, and a large increase in the proportion of the SCM1-like (Nitrosopumilus maritimus-like) cluster as well. The AO rates were generally higher in the deeper water (~25 m), which was in significant positive correlation with the proportion of cluster Water Column A (WCA) at the transcript level, indicating the potential contribution of this cluster to in situ ammonia oxidization. Our study demonstrated that coastal upwelling had a significant impact on the AOA community and ammonia oxidization rate; therefore, this physical forcing should be considered in the future assessment of the global nitrogen budgets and biogeochemical nitrogen cycles.

L3 Gene: Exploring the Presence in Syrian Strain of Leishmania Tropica Genome

Cutaneous leishmaniasis represents a serious health problem in Syria, this problem has become noticeably aggravated after the civil war in the country. Leishmania tropica parasite is the main cause for the cutaneous leishmaniasis in Syria. In order to control the disease, we need an effective vaccine against leishmania parasite. DNA vaccination remains one of the favorable approaches that have been used to face cutaneous leishmaniasis. Ribosomal protein L3 is responsible for important roles in Leishmania parasite life. DNA vaccine based on L3 gene has been used against infections by many species of Leishmania parasite but leishmania tropica parasite, so this gene represents a good candidate for DNA vaccine construction. But the presence of this gene has not yet been demonstrated in the genome of Leishmania tropica. So, this study aims to explore the presence and the expression of this gene in Syrian strain of Leishmania tropica promastigotes. The DNA and RNA were extracted from Syrian strain of Leishmania tropica parasites, the cDNA was made, the primers for genes’ amplification were designed manually, the conditions of the PCR were optimized and cDNA was used as a template. Full-length double-stranded sequence analysis was done. The Extracted genomic DNA and total RNA were with high degree of integrity and purity. L3 PCR products were shown by gel electrophoresis, on the level of DNA and cDNA, only one band goes to L3 gene, and their sizes were approximately 1260 bp. The sequence of the cDNA-L3 gene was determined and published in GenBank, according to the sequence the exact size of the gene was 1260 bp. Expression was also proven at the level of cDNA. Ribosomal protein L3 gene is a part of Syrian strain of L. tropica, the expression of this gene has been proven. The sequence of the cDNA of ribosomal protein L3 gene has been defined and submitted to the Genbank with accession number NO. MN495878.1.

Spatio-Temporal Variation of Synechococcus Assemblages at DNA and cDNA Levels in the Tropical Estuarine and Coastal Waters.

Synechococcus is a major contributor to global marine primary production. Here, its spatio-temporal variations in abundance and phylogenetic structure were studied at three stations of the South China Sea at both DNA and cDNA levels. Synechococcus cell abundance was lowest in March, but highest in October at two coastal stations. Its abundance was higher at the estuarine station, which reached a peak value of 1.36 × 105 cells/ml in April, owing to the nitrogen nutrients discharged from the Sanya River. Gene and gene transcript abundances of four Synechococcus lineages, clades II, III, VIII, and S5.3, were studied by quantitative PCR, which showed that clade II was the most abundant lineage at both DNA and cDNA levels. High-throughput sequencing revealed that, at the DNA level, Synechococcus assemblage was dominated by clade SY4 (a novel clade defined in this study), S5.2, and clade II in the coastal waters and was dominated by freshwater/S5.2 Synechococcus, reaching a value up to 88.61% in June, in estuarine waters. Changes in salinity and nutrient concentration caused by seasonal monsoonal forcing and river discharge were the key determinants of the spatio-temporal variation in Synechococcus assemblages at the DNA level. In comparison, high dissimilation among samples at the same stations and in the same seasons leads to the imperceptible spatio-temporal variation pattern of Synechococcus assemblages at the cDNA level. Furthermore, co-occurrence networks disclosed that Synechococcus community had closer and more complex internal interactions at the cDNA level. These discrepancies highlighted the necessity to study Synechococcus assemblages at both DNA and cDNA levels.

Secreted Protein Acidic and Rich in Cysteine (SPARC) and Chemokine’s mRNA expression in Endometrial Cancer versus Normal Endometrium: A Possible Pattern of Haematogenous and Lymphovascular Metastases

Objectives: Secreted Protein Acidic and Rich in Cysteine (SPARC) is a multifunctional glycoprotein, participating in tissue remodeling, morphogenesis and bone mineralization. Furthermore, SPARC controls important mechanisms involved in cancer progression, including angiogenesis regulation. However, in some studies SPARC was found to show tumor suppression while in other a protumorigenic and prometastatic action. In tumor microenvironment some chemokines and their receptors, thanks to their ability to modulate cancer cells migration and proliferation, are involved in the angiogenetic and metastatic process. In this study we compared, in human endometrial cancer tissue (EC) vs normal endometrium counterpart (NE), SPARC with CXCL12, CXCL11, CXCL8, and CXCR7 mRNA expression. Material and Methods: Fresh specimens from 15 patients with EC and corresponding NE were stored at –80°. One mcg of mRNA was reverse-transcribed in cDNA. A Real-Time PCR determined relative cDNA levels of targeted gene mRNA. Results: In EC vs NE, we observed down-regulation of SPARC mRNA in 91% (P&lt;.05), down regulation of CXCL12 mRNA in 91% (P&lt;.001), and down-regulation of CXCR7 mRNA in 91% (P&lt;.001). In EC, SPARC mRNA down-regulation was directly related in 100% of samples to CXCL12 and CXCR7 (P&lt;.001) and in 73% of samples to VEGF (P&lt;.03). Conclusion: In endometrial cancer, under expression of SPARC is directly related to CXCL12 and CXCR7 and this result might be consistent with a SPARC function on tumor progression and invasion mediated by CXCL12 and CXCR7 on blood and lymphatic spread, respectively.

Detecting the presence of GRP-78 gene in syrian strain of leishmania tropica genome and sequencing it

Background: Leishmania tropica is the main cause of cutaneous leishmaniasis which represents a common issue in Syria. After the start of the current war, cutaneous leishmaniasis has become yet more widespread, so an effective vaccine is needed to prevent a largescale spread of leishmania in the country. DNA vaccination is a promising approach for developing such needed vaccine against leishmaniasis. Glucose-regulated protein 78 plays important roles in Leishmania parasite. The gene that codes this protein had been used as DNA vaccine against infections by different species of Leishmania, so this gene represents a good candidate for DNA vaccine designing. But the existence of glucose-regulated protein 78 gene has not yet been proven in the genome of Leishmania tropica. So, this study aims to detect the existence and the expression of glucoseregulated protein 78 gene in Syrian strain of Leishmania tropica promastigotes. Materials &amp; methods: The DNA and RNA were isolated from Syrian strain of Leishmania tropica parasites, the cDNA was synthesized, necessary primers for genes’ amplification were designed manually, the PCR conditions were optimized. Full-length double-stranded sequence analysis was performed on PCR products from cDNA. Results: Extracted genomic DNA and total RNA showed high degree of purity and integrity. GRP78 PCR products showed by gel electrophoresis, on the level of DNA and cDNA, only one band belongs to the gene, and their sizes were approximately 1977 bp. The sequence of the cDNA-GRP78 gene was determined and published in GenBank, and the precise size of the gene was 1977 bp. Expression was also demonstrated at the level of cDNA. Conclusion: Glucose-regulated protein 78 gene is a part of Syrian strain of L. tropica, the expression of this gene has been demonstrated. The sequence of the cDNA of glucose-regulated protein 78 gene has defined and submitted to the Genbank with accession number NO. MN458469

A nuclear pore sub-complex restricts the propagation of Ty retrotransposons by limiting their transcription.

Beyond their canonical function in nucleocytoplasmic exchanges, nuclear pore complexes (NPCs) regulate the expression of protein-coding genes. Here, we have implemented transcriptomic and molecular methods to specifically address the impact of the NPC on retroelements, which are present in multiple copies in genomes. We report a novel function for the Nup84 complex, a core NPC building block, in specifically restricting the transcription of LTR-retrotransposons in yeast. Nup84 complex-dependent repression impacts both Copia and Gypsy Ty LTR-retrotransposons, all over the S. cerevisiae genome. Mechanistically, the Nup84 complex restricts the transcription of Ty1, the most active yeast retrotransposon, through the tethering of the SUMO-deconjugating enzyme Ulp1 to NPCs. Strikingly, the modest accumulation of Ty1 RNAs caused by Nup84 complex loss-of-function is sufficient to trigger an important increase of Ty1 cDNA levels, resulting in massive Ty1 retrotransposition. Altogether, our study expands our understanding of the complex interactions between retrotransposons and the NPC, and highlights the importance for the cells to keep retrotransposons under tight transcriptional control.

Investigating the Existence of Ribosomal Protein L5 Gene in Syrian Strain of Leishmania tropica Genome: Sequencing It and Evaluating Its Immune Response as DNA Vaccine.

Cutaneous leishmaniasis in Syria is caused mainly by Leishmania tropica. It represents a serious health problem, which has aggravated further after the civil war in the country. Until now, there are no effective protective strategies, safe therapy, or efficacious vaccine to protect from this infection. DNA vaccines represent a promising approach for achieving protection against leishmaniasis. The L5 ribosomal protein plays fundamental roles in the assembly process of the ribosome subunits, so this study has chosen the ribosomal protein L5 gene to design a DNA vaccine against Leishmania tropica infection. After proving the existence of the ribosomal protein L5 gene in a Syrian strain of Leishmania tropica (LCED Syrian 01), it was sequenced and cloned into a pCI plasmid, and the designed DNA vaccine was administered to BALB/c mice. The protective response was evaluated by measuring lesion development in immunized BALB/c mice for 6 weeks after challenging mice with the parasite. RT-qPCR was used to quantify IL-12, IFN-γ, and IL-4 in draining lymph nodes (DLNs) of immunized mice. In the final week, the parasite burden was determined in footpad lesions and local draining lymph nodes (DLNs). This study demonstrated the presence and expression of the ribosomal protein L5 gene in the Syrian strain of Leishmania tropica promastigotes. The sequence of the ribosomal protein cDNA L5 gene was determined and published in Genbank. The gene size was 918 bp. Expression was also demonstrated at the level of cDNA. This study also demonstrated that vaccination with the ribosomal protein L5 gene induces TH1 response in immunized mice. This response prevents the partial development of a skin lesion of Leishmania.

Phenotypic Definition and Genotype-Phenotype Correlates in PMPCA-Related Disease

Background: Peptidase mitochondrial processing alpha (PMPCA) biallelic mutations cause a spectrum of disorders ranging from severe progressive multisystemic mitochondrial encephalopathy to a milder non-progressive cerebellar ataxia with or without intellectual disability. Recently, we and others described an intermediate phenotype in two unrelated patients. Methods: We report a second Italian patient carrying novel PMPCA variants (p.Trp278Leu; p.Arg362Gly). Molecular modeling, dynamics simulation, RT-qPCR, and Western blotting were performed to predict the pathogenic impact of variants in the two Italian patients and attempt genotype-phenotype correlates. Results: In line with the two patients with intermediate phenotypes, our case presented global psychomotor delay with regression, intellectual disability, spastic-ataxic gait, and hyperkinetic movements, with cerebellar atrophy and bilateral striatal hyperintensities. However, blood lactate, muscle biopsy, and MRI spectroscopy were normal. PMPCA protein levels were significantly higher than controls despite normal cDNA levels. Dynamics simulation of several PMPCA missense variants showed a variable impact on the flexibility of the glycine rich loop and, for some cases, on the overall protein stability, without clear genotype-phenotype correlates. Conclusion: We confirm the expansion of PMPCA phenotypic spectrum including an intermediate phenotype of progressive encephalopathy without systemic involvement. The association of cerebellar atrophy with “Leigh-like” striatal hyperintensities may represent a “red flag” for this condition.