Proteomic analysis of sensitive and multi drug resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains

Multidrug-resistant tuberculosis (MDR-TB) is caused by bacteria that are resistant to the most effective anti TB drugs (Isoniazid and Rifampicin) with or without resistance to other drugs. Novel intervention strategies to eliminate this disease based on finding proteins can be used for designing new drugs or new and reliable kits for diagnosis. The aim of this study was to compare the protein profile of MDR-TB with sensitive isolates. Two-dimensional gel electrophoresis (2DE) along with mass spectrometry is a powerful and effective tool to identification and characterization of Mycobacterium tuberculosis. Two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for diagnosis and comparison of proteins. We identified 14 protein spots in MDR-TB isolates that 2DE analysis showed these spots absent in M. tuberculosis sensitive isolates (Rv1876, Rv0379, Rv0147, Rv2031c, Rv3597c, Rv1886c, MT0493, Rv0440, Rv3614c, Rv1626, Rv0443, Rv0475, Rv3057 and unknown protein. The results showed 22 protein spots which were up regulated (or expressed) by the MDR-TB isolates, (Rv1240, Rv3028c, Rv2971, Rv2114c, Rv3311, Rv3699, Rv1023, Rv1308, Rv3774, Rv0831c, Rv2890c, Rv1392, Rv0719, Rv0054, Rv3418c, Rv0462, Rv2215, Rv2986c, Rv3248c and Rv1908c)). Two up regulated protein spots were identified in sensitive isolate (Rv1133c and Rv0685). These data will provide valuable clues in further investigation for suitable TB rapid tests or drug targets against drug resistant and sensitive of M. tuberculosis.     

Genotype and Phenotype of Echinococcus granulosus Derived from Wild Sheep (Ovis orientalis) in Iran

The aim of the present study is to determine the characteristics of genotype and phenotype of Echinococcus granulosus derived from wild sheep and to compare them with the strains of E. granulosus sensu stricto (sheep-dog) and E. granulosus camel strain (camel-dog) in Iran. In Khojir National Park, near Tehran, Iran, a fertile hydatid cyst was recently found in the liver of a dead wild sheep (Ovis orientalis). The number of protoscolices (n=6,000) proved enough for an experimental infection in a dog. The characteristics of large and small hooks of metacestode were statistically determined as the sensu stricto strain but not the camel strain (P=0.5). To determine E. granulosus genotype, 20 adult worms of this type were collected from the infected dog. The second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) and cytochrome c oxidase 1 subunit (COX1) of the mitochondrial DNA were amplified from individual adult worm by PCR. Subsequently, the PCR product was sequenced by Sanger method. The lengths of ITS2 and COX1 sequences were 378 and 857 bp, respectively, for all the sequenced samples. The amplified DNA sequences from both ribosomal and mitochondrial genes were highly similar (99% and 98%, respectively) to that of the ovine strain in the GenBank database. The results of the present study indicate that the morpho-molecular features and characteristics of E. granulosus in the Iranian wild sheep are the same as those of the sheep-dog E. granulosus sensu stricto strain.     

Mechanical and Chemical Characterization of a TiC/C System Synthesized Using a Focus Plasma Arc

Titanium carbide-graphite (TiC/C) composite was successfully synthesized from Ti and C starting elemental powders using self-propagating high-temperature synthesis technique in an ultra-high plasma inert medium in a single stage. The TiC was exposed to a high-temperature inert medium to allow recrystallization. The product was then characterized using field emission scanning electron microscopy (FESEM) coupled with energy dispersive X-ray analysis (EDX), X-ray diffraction (XRD), Rietveld refinement, nanoindentation, and micro-hardness to determine the product’s properties. The recorded micro-hardness of the product was 3660 HV, which is a 14% enhancement and makes is comparable to TiC materials.     

Type 2 IDI performs better than type 1 for improving lycopene production in metabolically engineered E. coli strains

In this study a comparison was made between type 1 and type 2 isopentenyl diphosphate isomerases (IDI) in improving lycopene production in Escherichia coli. The corresponding genes of Bacillus licheniformis and the host (i _Bl and i _Ec , respectively) were expressed in lycopene producing E. coli strains by pTlyci_Bl and pTlyci_Ec plasmids, under the control of tac promoter. The results showed that the overexpression of i _Ec improved the lycopene production from 33 ± 1 in E. coli Tlyc to 68 ± 3 mg/gDCW in E. coli Tlyci_Ec. In contrast, the expression of i _Bl increased the lycopene production more efficiently up to 80 ± 9 mg/gDCW in E. coli Tlyci_Bl. The introduction of a heterologous mevalonate pathway to elevate the IPP abundance resulted in a lycopene production up to 132 ± 5 mg/gDCW with i _Ec in E. coli Tlyci_Ec-mev and 181 ± 9 mg/gDCW with i _Bl in E. coli Tlyci_Bl-mev, that is, 4 and 5.6 times respectively. When fructose, mannose, arabinose, and acetate were each used as an auxiliary substrate with glycerol, lycopene production was inhibited by different extents. Among auxiliary substrates tested, only citrate was an improving one for lycopene production in all strains with a maximum of 198 ± 3 mg/gDCW in E. coli Tlyci_Bl-mev. It may be concluded that the type 2 IDI performs better than the type 1 in metabolic engineering attempts for isoprenoid production in E. coli. In addition, the metabolic engineering of citrate pathway seems a promising approach to have more isoprenoid accumulation in E. coli.     

Portable,Battery-Operated,Low-Cost,Bright Field and Fluorescence Microscope

This study describes the design and evaluation of a portable bright-field and fluorescence microscope that can be manufactured for $240 USD. The microscope uses a battery-operated LED-based flashlight as the light source and achieves a resolution of 0.8 µm at 1000× magnification in fluorescence mode. We tested the diagnostic capability of this new instrument to identify infections caused by the human pathogen, Mycobacterium tuberculosis. Sixty-four direct, decontaminated, and serially diluted smears were prepared from sputa obtained from 19 patients suspected to have M. tuberculosis infection. Slides were stained with auramine orange and evaluated as being positive or negative for M. tuberculosis with both the new portable fluorescence microscope and a laboratory grade fluorescence microscope. Concordant results were obtained in 98.4% of cases. This highly portable, low cost, fluorescence microscope may be a useful diagnostic tool to expand the availability of M. tuberculosis testing at the point-of-care in low resource settings.     

Hypermethylated RASSF1 and SLC5A8 promoters alongside BRAFV600E mutation as biomarkers for papillary thyroid carcinoma

Circulating cell-free DNA (cfDNA) has been considered as a diagnostic source to track genetic and epigenetic alterations in cancer. We aimed to study mutation in addition to the methylation status in the promoter regions of RASSF1 and SLC5A8 genes in tissues and circulating free DNA samples of patients affected with papillary thyroid carcinoma (PTC) and thyroid nodules as controls. BRAF^V600E mutation was studied by ARMS-scorpion real-time polymerase chain reaction method in 57 PTC and 45 thyroid nodule cases. Methylation status of RASSF1 and SLC5A8 promoter regions was analyzed by methylation-specific high-resolution melting curve analysis. BRAF^V600E mutation was found in 39 (68.4%) out of 57 PTC tissue samples, while in 33 (49.1%) cases of cfDNA, this mutation was detected. The frequency of BRAF^V600E mutation in cfDNA was significantly different between metastatic and nonmetastatic PTC cases (22 of 33 PTC cases vs. 5 of 34 thyroid nodule samples). Methylation levels of three promoter regions of SLC5A8 and proximal promoter region of RASSF1 was significantly different between PTC and thyroid nodule cases in both cfDNA and tissue DNA. In addition, the methylation status of these two genes in tissue DNA was reflected in methylation status observed in cfDNA. This study confirmed that BRAF^V600E mutation is better for discrimination between papillary thyroid carcinoma and thyroid nodules. On the other hand, hypermethylation in the more proximal promoter regions to RASSF1 and SLC5A8 genes showed higher sensitivity and more acceptable specificity for this discrimination.     

Physiological responses to NaCl stress in three wild species of potato in vitro

In this study, responses of wild species of potato to NaCl stress were investigated in vitro. In S. stoloniferum and S. bulbosum, length of the shoot, fresh and dry weight, photosynthetic pigments, K⁺ concentration, K⁺/Na⁺ ratio, ascorbate pool, anthocyanin, and phenolic and flavonoid compounds were decreased in response to salinity. In these species, salinity increased the level of Na⁺, lipid peroxidation, proline and ion leakage percentage. In S. acaule, the length of the shoot, and fresh and dry weight were not affected by salinity. Photosynthetic pigments, Na⁺ concentration, proline, flavonoid and phenolic compounds quantities were increased and K⁺/Na⁺ ratio were decreased. K⁺ concentration, lipid peroxidation, ascorbate pool, anthocyanin and ion leakage were not changed by NaCl stress. Superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase and catalase activities were increased in all species. The results suggest that the non-enzymatic antioxidant capacity in S. acaule (salt tolerant) is more important than the enzymatic antioxidant capacity in comparison with the other species.     

Distinct functions of the Drosophila Nup153 and Nup214 FG domains in nuclear protein transport

The phenylanine-glycine (FG)-rich regions of several nucleoporins both bind to nuclear transport receptors and collectively provide a diffusion barrier to the nuclear pores. However, the in vivo roles of FG nucleoporins in transport remain unclear. We have inactivated 30 putative nucleoporins in cultured Drosophila melanogaster S2 cells by RNA interference and analyzed the phenotypes on importin alpha/beta-mediated import and CRM1-dependent protein export. The fly homologues of FG nucleoporins Nup358, Nup153, and Nup54 are selectively required for import. The FG repeats of Nup153 are necessary for its function in transport, whereas the remainder of the protein maintains pore integrity. Inactivation of the CRM1 cofactor RanBP3 decreased the nuclear accumulation of CRM1 and protein export. We report a surprisingly antagonistic relationship between RanBP3 and the Nup214 FG region in determining CRM1 localization and its function in protein export. Our data suggest that peripheral metazoan FG nucleoporins have distinct functions in nuclear protein transport events.     

Molecular detection and identification of phytoplasmas associated with Catharanthus roseus,Pinus eldarica,and Petunia hybrida in southeastern Iran's urban green spaces

Given the potential for urban green spaces to provide fresh and healthy environments for humans, exploring the issues that threaten plants in these places is crucial. Phytoplasma-related symptoms were encountered on some plants in urban green spaces in the province of Kerman, southeastern Iran, between 2017 and 2019. Affected periwinkles and petunias exhibited phytoplasma disease symptoms, including virescence, phyllody, and witches'-broom. However, ball or disc-like shoot proliferation symptoms were noticed on the trunks and branches of pine trees. PCR was performed with phytoplasma-detecting universal primers, targetting and amplifying the 16S rRNA gene, and determining whether phytoplasmas are implicated in the symptomatic plants. The infection of the symptomatic plants was confirmed using nested-PCR amplification of expected DNA sizes for phytoplasmas. No product, however, was amplified from sampled symptomless plants. The sequencing of nested-PCR products was performed to obtain sequences encasing the standard F2nR2 fragments. The resulted sequences were submitted to iPhyClassifier, the universal phytoplasma classification platform, for the taxonomic assignment of the found phytoplasmas compared with previously identified ‘Candidatus Phytoplasma’ species, groups, and subgroups. The results revealed that phytoplasma strains related to the species ‘Ca. P. trifolii’ (16SrVI-A subgroup) infect periwinkles and pines. However, strains from the species ‘Ca. P. aurantifolia’ (16SrII-D subgroup) and ‘Ca. P. phoenicium’ (16SrIX-C subgroup) were found in petunias and periwinkles, respectively. To the best of our knowledge, phytoplasmas from the 16SrVI-A and 16SrII-D subgroups are the first reported to infect these plants in Kerman province, while a related strain from the subgroup 16SrIX-C is the first recorded to infect periwinkles in Iran and the second in the world.     

TWIST1, MMP-21, and HLAG-1 co-overexpression is associated with ESCC aggressiveness

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive types of cancer, requiring reliable biomarkers for prognosis and therapeutic responsiveness. TWIST1, as an important factor responsible for metastasis of several cancers, is involved in tumor invasion and metastasis through indirectly regulation of MMP-21 expression. On the other hand, NF-ĸβ which is a regulator of HLAG-1 has direct interaction with TWIST1 protein. In this retrospective study we investigated the clinical significance of TWIST1, MMP-21, and HLAG-1 expression in ESCC, and the possible correlation between these genes and progression of the disease. The gene expression analyses of TWIST1, MMP-21, and HLA-G1 were performed by relative comparative real-time polymerase chain reaction in 58 ESCCs compared with corresponding margin-normal esophageal tissues. Significant overexpression of HLAG-1, TWIST1, and MMP-21 messenger RNA was observed in 22.4%, 41.4%, and 60.3% of tumor samples, respectively. Concomitant overexpression of TWIST1/MMP-21 and TWIST1/HLAG-1 were significantly correlated to each other in various clinicopathological features, including depth of tumor invasion, stage of tumor progression, lymphatic invasion, and grade of tumor cell differentiation ( P < 0.05). The current study is the first report of coexpression of TWIST1, MMP-21, and HLAG-1 in ESCC. Such findings suggest an oncogenic role for concomitant expression of these genes in ESCC invasion and metastasis.