Ethanolic extract of Euphorbia royleana Boiss. reduces metastasis of breast cancer cells and inhibits tumor progression in vivo.

Metastasis is the most devastating attribute of breast cancer (BC) that leads to high mortality. It is a complex process of tumor cell migration, invasion, and angiogenesis. In this study, we evaluated the effect of ERA on BC metastasis and BC progression in vivo. The transwell invasion/migration and wound healing assays showed that ERA treatment significantly reduced the invasion and migration of BC cell lines. The expression of mesenchymal (E-cadherin and N-cadherin), matrix metalloproteinases (MMP2, MMP9), and stemness markers (Oct3) were down-regulated by ERA. Furthermore, ERA down-regulated angiogenic chemokines (CXCL1/2/3, CXCL5, and CXCL12) expression in the highly metastatic MDA-MB-231 cell line. The clonogenic survival of BC cells was also reduced by ERA treatment. Strikingly, ERA prevented DMBA-induced tumor growth in Swiss albino mice as depicted by a high animal survival rate (84%) in the ERA group and histopathological analysis. Conclusively, this study revealed that ERA possesses anti-metastatic potential and also reduces the growth of BC in vivo. Moreover, the GC-MS data revealed the presence of biologically active compounds (Lupeol, Phytol, phytosterol) and some rare (9, 19-Cyclolanost) phyto metabolites in ERA extract. However, further studies are suggestive to identify and isolate the therapeutic agents from ERA to combat BC and metastasis.

SHISA3, an antagonist of the Wnt/ß-catenin signaling, is epigenetically silenced and its ectopic expression suppresses growth in breast cancer.

Breast cancer (BC) is the foremost cause of cancer related deaths in women globally. Currently there is a scarcity of reliable biomarkers for its early stage diagnosis and theranostics monitoring. Altered DNA methylation patterns leading to the silencing of tumor suppressor genes are considered as an important mechanism underlying tumor development and progression in various cancer types, including BC. Very recently, epigenetic silencing of SHISA3, an antagonist of ß-catenin, has been reported in various types of tumor. However, the role of SHISA3 in BC has not been investigated yet. Therefore, we aimed at evaluating the contribution of SHISA3 in BC causation by analyzing its expression and methylation levels in BC cell lines (MDA-MB231, MCF-7 and BT-474) and in 103 paired BC tissue samples. The SHISA3 expression and methylation status was determined by qPCR and methylation specific PCR (MSP) respectively. The role of SHISA3 in BC tumorigenesis was evaluated by proliferation and migration assays after ectopic expression of SHISA3. The association between SHISA3 hypermethylation and clinicopathological parameters of BC patients was also studied. The downregulation of SHISA3 expression was found in three BC cell lines used and in all BC tissue samples. However, SHISA3 promoter region was hypermethylated in 61% (63/103) tumorous tissues in comparison to the 18% of their matched normal tissues. The 5-aza-2'-deoxycytidine treatment restored SHISA3 expression by reversing promoter hypermethylation in both MDA-MB231 and MCF-7 cells. Furthermore, ectopic expression of SHISA3 significantly reduced the proliferation and migration ability of these cells. Taken together, our findings for the first time reveal epigenetic silencing and tumor suppressing role of SHISA3 in BC. Henceforth, this study has identified SHISA3 as potentially powerful target for the development of new therapies against BC, as well as novel diagnostic and therapy response monitoring approaches.

Human BK and JC polyomaviruses: Molecular insights and prevalence in Asia.

Polyomaviridae family consists of small circular dsDNA viruses. Out of the 14 human polyomaviruses described so far, BKPyV and JCPyV have been studied extensively since their discovery in 1971. Reportedly, both BKPyV and JCPyV are widely distributed across the globe with the frequency of 80-90 % in different populations. The primary infection of these viruses is usually asymptomatic and latent which is activated as a consequence of immunosuppression. Activated BKPyV and JCPyV viruses lead to the development of BK Virus Associated Nephropathy and Progressive Multifocal Leukoencephalopathy, respectively. Immense progress has been made during the last few decades regarding the molecular understanding of polyomaviruses. Epidemiology of polyomaviruses has also been studied extensively. However, most of the epidemiological studies have focused on European and American populations. Therefore, limited data is available regarding the geographical distribution of these potentially oncogenic viruses in Asian countries. In this article, we have presented a compendium of latest advances in the molecular understanding of polyomaviruses and their pathobiology. We also present a comprehensive review of published literature regarding the epidemiology and prevalence of BKPyV and JCPyV in Asian regions. For this purpose, a thorough search of available online resources was performed. As a result, we retrieved 24 studies for BKPyV and 22 studies for JCPyV, that describe their prevalence in Asia. These studies unanimously report high occurrence of both BKPyV and JCPyV in Asian populations. The available data from these studies was categorized into two groups: on the basis of prevalence (low, medium and high) and disease development (healthy and diseased). Altogether, Korean population hasbeen evidenced to possess highest frequency of BKPyV (66.7 %), while JCPyV was found to be most prevalent in Taiwan (88 %). Due to high and ubiquitous distribution of these viruses, frequent studies are required to develop a better understanding regarding the epidemiology and pathobiology of these viruses in Asia.

Genetic diversity of Paramecium species on the basis of multiple loci analysis and ITS secondary structure models.

Among ciliates, Paramecium has become a privileged model for the study of "species problem" particularly in the case of the "Paramecium aurelia complex" that has been intensely investigated. Despite extensive studies, the taxonomy of Paramecium is still challenging. The major problem is an uneven sampling of Paramecium with relatively few representatives of each species. To investigate species from the less discovered region (Pakistan), 10 isolates of Paramecium species including a standing-alone FT8 strain previously isolated by some of us were subjected to molecular characterization. Fragments of 18S recombinant DNA (rDNA), ITS1-5.8S-ITS2-5'LSU rDNA, cytochrome c oxidase subunit II, and hsp70 genes were used as molecular markers for phylogenetic analysis of particular isolates. The nucleotide sequences of polymerase chain reaction products of all markers were compared with the available sequences of relevant markers of other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all the nine strains had a very close relationship with Paramecium primaurelia except for the FT8 strain. FT8 consistently showed its unique position in comparison to all other species in the phylogenetic trees. Available sequences of internal transcribed spacer 1 (ITS1) and ITS2 and some other ciliate sequences from GenBank were used for the construction of secondary models. Two highly conserved helices supported by compensatory base changes among all ciliates of ITS2 secondary structures were found similar to other eukaryotes. Therefore, the most conserved 120 to 180 base pairs regions were identified for their comparative studies. We found that out of the three helices in ITS1 structure, helix B was more conserved in Paramecium species. Despite various substitutions in the primary sequence, it was observed that secondary structures of ITS1 and ITS2 could be helpful in interpreting the phylogenetic relationships both at species as well as at generic level.

Merkel cell polyomavirus DNA sequences in the blood of healthy population of Pakistan.

Aim: This study aimed at detecting and quantifying Merkel cell polyomavirus (MCPyV) viral loads in the peripheral blood of healthy Pakistani individuals. Patients & methods: A total of 221 whole blood samples obtained from healthy individuals were examined by qPCR. Results & conclusion: MCPyV was detected in the peripheral blood of 31.2% healthy individuals. The rate of MCPyV positivity decreased from young (36%), to middle (33.7%) and elder (25.3%) age groups. Our data revealed higher prevalence of MCPyV in women (43.93%) than men (25.80%). The MCPyV viral load was calculated in the range of 0.06 -11 copies/ng of isolated DNA. The MCPyV viral load increased from young (median = 3.35) to elder (median = 5.66) age groups. The MCPyV circulate at a higher frequency by residing dormant in certain blood cells, which might act as potential vehicles for the spread of MCPyV infection among general population.

The implication of CRISPR/Cas9 genome editing technology in combating human oncoviruses.

It is evidenced that 20% of all tumors in humans are caused by oncoviruses, including human papilloma viruses, Epstein-Barr virus, Kaposi sarcoma virus, human polyomaviruses, human T-lymphotrophic virus-1, and hepatitis B and C viruses. Human immunodeficiency virus is also involved in carcinogenesis, although not directly, but by facilitating the infection of many oncoviruses through compromising the immune system. Being intracellular parasites with the property of establishing latency and integrating into the host genome, these viruses are a therapeutic challenge for biomedical researchers. Therefore, strategies able to target nucleotide sequences within episomal or integrated viral genomes are of prime importance in antiviral or anticancerous armamentarium. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a powerful genome editing tool. Standing out as a precise and efficient oncoviruses method, it has been extensively applied in recent experimental ventures in the field of molecular medicine, particularly in combating infections including tumor inducing viruses. This review is aimed at collating the experimental and clinical advances in CRISPR/Cas9 technology in terms of its applications against oncoviruses. Primarily, it will focus on the application of CRISPR/Cas9 in combating tumor viruses, types of mechanisms targeted, and the significant outcomes till date. The technical pitfalls of the CRISPR/Cas9 and the comparative approaches in evaluating this technique with respect to other available alternatives are also described briefly. Furthermore, the review also discussed the clinical aspects and the ethical, legal, and social issues associated with the use of CRISPR/Cas9.

Specific and quantitative detection of Human polyomaviruses BKPyV and JCPyV in the healthy Pakistani population.

BACKGROUND: The BK Polyomavirus (BKPyV) and JC polyomavirus (JCPyV) infections are widespread in human population and have been associated with severe kidney and brain disorders, respectively. The viruses remain latent primarily in reno-urinary tract, reactivating only in case of a compromised immune system. The seroepidemiology and molecular prevalence of BKPyV and JCPyV have been widely studied both in healthy and immunocompromised patients worldwide. However, data regarding the prevalence of these viruses in the immunocompetent or apparently healthy Pakistani population is lacking. Herein, we present the first ever report on quantitative prevalence of BKPyV and JCPyV in the peripheral blood of a randomly selected cohort of healthy Pakistani population. METHODS: A total of 266 whole blood samples were examined. The subjects were divided into three age groups: ≤ 25 years (young), 26-50 years (middle) and ≥ 51 years (elder). Absolute real time PCR assay was designed to quantify the BKPyV and JCPyV viral copy numbers in the range of 106 to 100 copies/mL. RESULTS: Overall, BKPyV was detected in 27.1% (72/266) individuals while JCPyV in 11.6% (31/266) indicating significant difference (p < 0.005) in the distribution of these two viruses. The prevalence of BKPyV significantly decreased from 51% (49/96) in young age group to 8.2% (7/85) in eldest age group. Whereas, JCPyV positivity rate slightly increased from 8.3% (8/96) in young age group to 11.8% (10/85) in elder age group. The median viral load was calculated as 6.2 log and 3.38 log copies/mL of blood for BKPyV and JCPyV, respectively. Notably, no significant difference in viral load of either of the subtypes was found between different age groups. CONCLUSION: The current study provides an important baseline data on the prevalence and viral load of circulating BKPyV and JCPyV in Pakistani population. The prevalence and viral load of BKPyV was comparatively higher than JCPyV. The prevalence of BKPyV significantly decreased with increase in age while JCPyV positivity rate slightly increased with increasing age. Viral load of both BKPyV and JCPyV was not correlated with the individual ages.

Variability in secondary structure of 18S ribosomal RNA as topological marker for identification of Paramecium species.

Besides cytological and molecular applications, Paramecium is being used in water quality assessment and for determination of saprobic levels. An unambiguous identification of these unicellular eukaryotes is not only essential, but its ecological diversity must also be explored in the local environment. 18SrRNA genes of all the strains of Paramecium species isolated from waste water were amplified, cloned and sequenced. Phylogenetic comparison of the nucleotide sequences of these strains with 23 closely related Paramecium species from GenBank Database enabled identification of Paramecium multimicronucleatum and Paramecium jenningsi. Some isolates did not show significant close association with other Paramecium species, and because of their unique position in the phylogenetic tree, they were considered new to the field. In the present report, these isolates are being designated as Paramecium caudatum pakistanicus. In this article, secondary structure of 18SrRNA has also been analyzed as an additional and perhaps more reliable topological marker for species discrimination and for determining possible phylogenetic relationship between the ciliate species. On the basis of comparison of secondary structure of 18SrRNA of various isolated Paramacium strains, and among Paramecium caudatum pakistanicus, Tetrahymena thermophila, Drosophila melanogaster, and Homo sapiens, it can be deduced that variable regions are more helpful in differentiating the species at interspecific level rather than at intraspecific level. It was concluded that V3 was the least variable region in all the organisms, V2 and V7 were the longest expansion segments of D. melanogaster and there was continuous mutational bias towards G.C base pairing in H. sapiens.