Proteosomes based on milk phospholipids and proteins to enhance the stability and bioaccessibility of ß-carotene.

Proteosomes (P) based on milk fat globule membrane's phospholipids (MPs), whey protein isolate (WPI) and sodium caseinate (CasNa) were developed by ultrasonication to encapsulate ß-carotene. Entirely milk-ingredients based proteosomes (WPI-MPs-P and CasNa-MPs-P) revealed homogenous distribution with size diameters < 250 nm. WPI-MPs-P depicted positive ζ-potential values (+15.7 ± 0.5 mV), while CasNa-MPs-P demonstrated negative (-32.5 ± 3.4 mV) values of surface charge, respectively and hydrophilic nature of proteosomes was observed by measuring contact-angle (θ). AFM and SEM exhibited spherical to oval and slightly irregular morphology of nanocarriers. For various concentrations of ß-carotene, the highest encapsulation efficiency of ß-carotene was 90 ± 0.2% and 92 ± 0.8% in WPI-MPs-P and CasNa-MPs-P respectively. FTIR analyses confirmed the hydrophobic and electrostatic interactions-based encapsulation of ß-carotene. Beneficial antioxidant-potential of ß-carotene was retained after its encapsulation in the proteosomes. Proteosomes increased the digestive-stability (>50%) and bioaccessibility (>85%) of ß-carotene. Thus, milk-ingredients based proteosomes offer a novel-strategy to develop functional dairy products to overcome widespread vitamin-A-deficiency.

Surface engineering of chitosan nanosystems and the impact of functionalized groups on the permeability of model drug across intestinal tissue.

Surface attributes of nanocarriers are crucial to determine their fate in the gastrointestinal (GI) tract. Herein, we have functionalized chitosan with biochemical moieties including rhamnolipid (RL), curcumin (Cur) and mannose (M). FTIR spectra of functionalized chitosan nanocarriers (FCNCs) demonstrated successful conjugation of M, Cur and RL. The functional moieties influenced the entrapment of model drug i.e., coumarin-6 (C6) in FCNCs with payload-hosting and non-leaching behavior i.e., >91 ± 2.5 % with negligible cumulative release of <2 % for 5 h in KREB, which was further verified in the simulated gastric and intestinal fluids. Consequently, substantial difference in the size and zeta potential was observed for FCNCs with different biochemical moieties. Scanning electron microscopy and atomic force microscopy of FCNCs displayed well-dispersed and spherical morphology. In addition, in vitro cytotoxicity results of FCNCs confirmed their hemocompatibility. In the ex-vivo rat intestinal models, FCNCs displayed a time-dependent-phenomenon in cellular-uptake and adherence. However, apparent-permeability-coefficient and flux values were in the order of C6-RL-FCNCs > C6-M-FCNCs > C6-Cur-FCNCs = C6-CNCs > Free-C6. Furthermore, the transepithelial electrical resistance revealed the FCNCs mediated recovery of membrane-integrity with reversible tight junctions opening. Thus, FCNCs have the potential to overcome the poor solubility and/or permeability issues of active pharmaceutical ingredients and transform the impact of functionalized-nanomedicines in the biomedical industry.

Epithelial Cell Adhesion Molecule (EpCAM) Expression Can Be Modulated via NFκB.

The epithelial cell adhesion molecule (EpCAM) is considered an essential proliferation signature in cancer. In the current research study, qPCR induced expression of EpCAM was noted in acute lymphoblastic leukemia (ALL) cases. Costunolide, a sesquiterpene lactone found in crepe ginger and lettuce, is a medicinal herb with anticancer properties. Expression of EpCAM and its downstream target genes (Myc and TERT) wasdownregulated upon treatment with costunolide in Jurkat cells. A significant change in the telomere length of Jurkat cells was not noted at 72 h of costunolide treatment. An in silico study revealed hydrophobic interactions between EpCAM extracellular domain and Myc bHLH with costunolide. Reduced expression of NFκB, a transcription factor of EpCAM, Myc, and TERT in costunolide-treated Jurkat cells, suggested that costunolide inhibits gene expression by targeting NFκB and its downstream targets. Overall, the study proposes that costunolide could be a promising therapeutic biomolecule for leukemia.

Transcriptomic Analysis of Conserved Telomere Maintenance Component 1 (CTC1) and Its Association with Leukemia.

Telomere length (TEL) regulation is important for genome stability and is governed by the coordinated role of shelterin proteins, telomerase (TERT), and CST (CTC1/OBFC1/TEN1) complex. Previous studies have shown the association of telomerase expression with the risk of acute lymphoblastic leukemia (ALL). However, no data are available for CST association with the ALL. The current pilot study was designed to evaluate the CST expression levels in ALL. In total, 350 subjects were recruited, including 250 ALL cases and 100 controls. The subjects were stratified by age and categorized into pediatrics (1-18 years) and adults (19-54 years). TEL and expression patterns of CTC1, OBFC1, and TERT genes were determined by qPCR. The univariable logistic regression analysis was performed to determine the association of gene expression with ALL, and the results were adjusted for age and sex in multivariable analyses. Pediatric and adult cases did not reflect any change in telomere lengths relative to controls. However, expression of CTC1, OBFC1, and TERT genes were induced among ALL cases. Multivariable logistic regression analyses showed association of CTC1 with ALL in pediatric [ß estimate (standard error (SE)= -0.013 (0.007), p = 0.049, and adults [0.053 (0.023), p = 0.025]. The association of CTC1 remained significant when taken together with OBFC1 and TERT in a multivariable model. Furthermore, CTC1 showed significant association with B-cell ALL [-0.057(0.017), p = 0.002) and T-cell ALL [-0.050 (0.018), p = 0.008] in pediatric group while no such association was noted in adults. Together, our findings demonstrated that telomere modulating genes, particularly CTC1, are strongly associated with ALL. Therefore, CTC1 can potentially be used as a risk biomarker for the identification of ALL in both pediatrics and adults.

Integration of RT-LAMP and Microfluidic Technology for Detection of SARS-CoV-2 in Wastewater as an Advanced Point-of-Care Platform.

Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of seven wastewater samples from COVID-19 hotspots. RT­LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best target gene for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be the best target gene for SARS-CoV-2 detection. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except microchannels which subjected to RT-LAMP for targeting N region after RNA extraction (yellow color) in 6 out of 7 samples. This study shows that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips. This study brings the novelty involving the use of wastewater samples for detection of SARS-CoV-2 without previous virus concentration and with/without RNA extraction.

Expression of HK2, PKM2, and PFKM Is Associated with Metastasis and Late Disease Onset in Breast Cancer Patients.

The reprogramming of energy metabolism is one of the hallmarks of cancer and is crucial for tumor progression. Altered aerobic glycolysis is a well-known characteristic of cancer cell metabolism. In the present study, the expression profiles of key metabolic genes (HK2, PFKM, and PKM2) were assessed in the breast cancer cohort of Pakistan using quantitative polymerase chain reaction (qPCR) and IHC. Expression patterns were correlated with molecular subtypes and clinical parameters in the patients. A significant upregulation of key glycolytic genes was observed in tumor samples in comparison to their adjacent controls (p < 0.0001). The expression of the studied glycolytic genes was significantly increased in late clinical stages, positive nodal involvement, and distant metastasis (p < 0.05). HK2 and PKM2 were found to be upregulated in luminal B, whereas PFKM was overexpressed in the luminal A subtype of breast cancer. The genes were positively correlated with the proliferation marker Ki67 (p < 0.001). Moreover, moderate positive linear correlations between HK2 and PKM2 (r = 0.476), HK2 and PFKM (r = 0.473), and PKM2 and PFKM (r = 0.501) were also observed (p < 0.01). These findings validate that the key regulatory genes in glycolysis can serve as potential biomarkers and/or molecular targets for breast cancer management. However, the clinical significance of these molecules needs to be further validated through in vitro and in vivo experiments.

Chitosan-curcumin complexation to develop functionalized nanosystems with enhanced antimicrobial activity against hetero-resistant gastric pathogen.

With the apparent stagnation in the antibiotic discovery and the propagation of multidrug resistance, Helicobacter pylori associated gastric infections are hard to eradicate. In pursuance of alternative medicines, in this study, covalent modification of chitosan (CS) polymer with curcumin (Cur) was accomplished. Proton Nuclear Magnetic Resonance and Fourier Transform Infrared spectroscopy elucidated the covalent interaction between Cur and CS with characteristic peak of imine functional group (C=N). Scanning Electron Microscopy provided visual proof for surface topology, while size and zeta potential values further affirmed the development of curcumin functionalized chitosan nanosystems (Cur-FCNS). The complexation efficiency of CS with Cur was found as 70 ± 3% at an optimal ratio of 5:1 for CS and Cur, respectively. Cur-FCNS developed with ionic gelation and ultrasonication method demonstrated synergistic anti-H. pylori activity in growth-kinetics and anti-biofilm assays, which was superior to free Cur and even chitosan nanosystems. Under simulated gastric conditions, Cur-FCNS revealed cumulative-release of only 16 ± 0.8% till 40 h, which indicated its improved stability to interact with H. pylori. In silico findings affirmed high binding affinity of Cur-FCNS with multiple bacterial virulence factors. Thus, our results affirmed the exceptional potential of Cur-FCNS as next-generation alternative-medicine to treat resistant H. pylori.

Upregulation of CD271 transcriptome in breast cancer promotes cell survival via NFκB pathway.

BACKGROUND: Biological treatment of many cancers currently targets membrane bound receptors located on a cell surface. We are in a great to need identify novel membrane proteins associated with migration and metastasis of breast cancer cells. CD271, a single transmembrane protein belongs to tumor necrosis factor receptor family acts and play its role in proliferation of cancer cell. The purpose of this study is to investigate the role of CD271 in breast cancer. METHODS AND RESULTS: In this study we analyzed the mRNA expression of CD271 in breast tumor tissue, breast cancer cell line MCF7 and isolated cancer stem cells (MCF7-CSCs) by RT-qPCR. We also measured the protein levels through western blotting in MCF-7 cell line. CD271 was upregulated in breast cancer patients among all age groups. Within the promoter region of CD271, there is a binding site for NF-κB1 which overlaps a putative quadraplex forming sequence. While CD271 also activates NF-κB pathway, down regulation of CD271 through quadraplex targeting resulted in inhibition of NF-κB and its downstream targets Nanog and Sox2. CONCLUSION: In conclusion, our data shows that CD271 and NF-κB are regulated in interdependent manner. Upon CD271 inhibition, the NF-κB expression also reduces which in turn affects the cell proliferation and migration. These results suggest that CD271 is playing a crucial rule in cancer progression by regulating NF-κB and is a good candidate for the therapeutic targeting.

Incidence and Molecular Characterization of Carbapenemase Genes in Association with Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa from Tertiary Healthcare Facilities in Southwest Nigeria.

Pseudomonas aeruginosa, resistant to multiple antibacterial agents including carbapenems, is of great global public health concern. There is limited data available regarding incidence of Metallo-Beta Lactamase producing P. aeruginosa, their molecular basis of resistance in particular carbapenem resistance and any genetic relatedness among circulating clinical isolates in Southwest Nigeria. Four hundred and thirty P. aeruginosa isolates were collected from seven tertiary care hospitals (predominantly from wound, ear, and urinary tract infections) and verified by PCR targeting oprI and oprL. Antibiotic susceptibility using 16 selected antibiotics and MBL screening was performed. The integrons (class 1, 2 and 3) and carbapenemase genes- blaGES, blaNMC-A, blaBIC-1, blaSME, blaIMP, blaVIM, blaSPM, blaNDM, blaAIM, blaDIM, blaSIM, blaGIM, blaOXA-48, blaOXA-58 were detected by PCR and were sequenced. Quantitative real-time polymerase chain reaction was used to quantify expression levels of eight efflux pump genes, ampC cephalosporinase and outer membrane porin, oprD. The isolates were genotyped using Enterobacterial Repetitive Intergenic Consensus sequence Polymerase Chain Reaction (ERIC-PCR). Four hundred and thirty P. aeruginosa isolates were subjected to antibiotic susceptibility testing, revealing that 109 (25.4%) isolates were multidrug-resistant, 47 (10.9%) were extensively drug-resistant and 25 (5.8%) were pandrug-resistant. MBL was seen in 17.0% (73/430) isolates. MBL-encoding genes; blaVIM-5 and blaNDM-1 were detected in 86.3% (63/73) isolates, with blaVIM-5 and blaNDM-1 in 35.6% (26/73) and 38.4% (28/73), respectively, whereas co-occurrence of blaVIM-5 and blaNDM-1 was found in 12.3% (9/73). Forty-one (56.2%) carbapenem-resistant P. aeruginosa strains carried class 1 integrons, while co-occurrence of class 1 and 2 integrons was seen in 12.3%. qPCR results indicated that MexXY-OprM was highly expressed pump in 58.9%, ampC upregulated in 26.0%, while oprD porin was downregulated in 65.8% isolates. ERIC-PCR results suggest that carbapenem-resistant strains exhibit genetic heterogeneity. The high incidence of MBL-encoding genes and integrons in diversified clinical P. aeruginosa from southwestern Nigeria is of great concern. The co-occurrence of blaVIM-5 and blaNDM-1 as well as resistance in general manifesting a gradient based on genotypic variation suggests that there is a strong need for efficient surveillance programs and antibiotic stewardship.

Could PCSK9 be a new therapeutic target of Eugenol? In vitro and in silico evaluation of hypothesis.

PCSK9 (Proprotein convertase Subtilisin/Kexin Type 9), an important regulator of lipid metabolism, has been shown to play a role in hepatocellular carcinoma by promoting metastasis. PCSK9 interferes with LDL metabolism and causes dyslipidemias in hematological malignancies particularly acute lymphoblastic leukemia. Nutraceuticals like berberine, curcumin and polydatin have been found effective in modulating PCSK9 expression by lowering LDL levels. Eugenol, a nutraceutical has shown a promising role in cancer due to its antioxidant and antihypercholesterolemic effects. In the present study, PCSK9 expression was measured in acute lymphoblastic leukemia (ALL) patients and was found to be significantly induced. Based on the results of expression analysis, a plausible hypothesis was made. Eugenol being an antioxidant will prevent oxidation of LDL. In the absence of ox-LDL, LOX1 scavenger receptor, which regulates PCSK9 expression, will not be activated. As the circulating LDL is reduced, it will no longer be able to support leukemia cell growth. The hypothesis was validated by an in silico and in vitro study. Molecular docking revealed hydrophobic interactions between ligand eugenol and macromolecules PCSK9 and LOX1. Expression of both PCSK9 and LOX1 were significantly reduced by eugenol in Jurkat cells. To conclude, PCSK9 could therapeutically be targeted by eugenol in leukemia cells.

Oligosaccharyltransferase PglB of Campylobacter jejuni is a glycoprotein.

Campylobacter jejuni is the one of the leading cause of bacterial food borne gastroenteritis. PglB, a glycosyltransferase, plays a crucial role of mediating glycosylation of numerous periplasmic proteins. It catalyzes N-glycosylation at the sequon D/E-X1-N-X2-S/T in its substrate proteins. Here we report that the PglB itself is a glycoprotein which self-glycosylates at N534 site in its DYNQS sequon by its own catalytic WWDYG motif. Site-directed mutagenesis, lectin Immunoblot, and mobility shift assays confirmed that the DYNQS is an N-glycosylation motif. PglB's N-glycosylation motif is structurally and functionally similar to its widely studied glycosylation substrate, the OMPH1. Its DYNQS motif forms a solvent-exposed crest. This motif is close to a cluster of polar and hydrophilic residues, which form a loop flanked by two α helices. This arrangement extremely apposite for auto-glycosylation at N534. This self-glycosylation ability of PglB could mediate C. jejuni's ability to colonize the intestinal epithelium. Further this capability may also bear significance for the development of novel conjugated vaccines and diagnostic tests.

Mutagenic players in ALL progression and their associated signaling pathways.

An alarming increase in acute lymphoblastic leukemia among children and males has drawn attention of investigators to delve into the genetic causes of ALL and to discover new therapeutic strategies with better prognosis. Although the survival rate in children is much higher than adults, but there's a need to find new potential molecular targets with better treatment outcome. Genomic profiling has made it possible to identify various genetic defects important for driving leukemogenesis. Study of the genetic lesions not only give a better understanding of genes function but also helps to target various signaling pathways involved in disease progression. The current review provides an overview of important genetic defects and dysregulation in their downstream signaling pathways in acute lymphoblastic leukemia.

Analysis of gut microbiota of obese individuals with type 2 diabetes and healthy individuals.

Type 2 diabetes mellitus (T2DM) accounts for 90% of diabetes cases worldwide. The majority of T2DM patients are obese. Dysbiosis in the gut microflora is strongly associated with the pathogenesis of obesity and T2DM; however, the microbiome of obese-T2DM individuals in the Pakistani population remains unexplored. The gut microbiota signature of 60 Pakistani adults was studied using 16S rRNA sequencing targeting V3-V4 hypervariable regions. The sequence analysis revealed that bacteria from Firmicutes were predominant along with those from Clostridia and Negativicutes, whereas bacteria from Verrucomicrobia, Bacteroidetes, Proteobacteria, and Elusimicrobia were less abundant among the obese T2DM patients. These data distinctively vary from those in reports on the Indian population. The difference in gut microbiota could presumably be related to the distinct lifestyle and eastern dietary habits (high carbohydrate and fat intake, low fiber intake) and unregulated antibiotic consumption. This is the first study carried out to understand the gut microbiome and its correlation with individual life style of obese T2DM patients in the Pakistani population.

Catalysts for RNA and DNA modification.

To study DNAs and RNAs it is often necessary to chemically modify them. Nature's strategy for nucleic acid modification is to use selective catalysts, and chemists have begun to emulate this conceptual approach. In this review we present a summary of catalytic approaches toward the construction of modified RNAs and DNAs and outline our opinions on where new research is needed.

The BCL-2 5' untranslated region contains an RNA G-quadruplex-forming motif that modulates protein expression.

The BCL-2 gene encodes a 25 kDa membrane protein that plays critical roles in the control of apoptosis. The regulation of BCL-2 gene expression is highly complex and occurs both transcriptionally and posttranscriptionally. In particular, the 5' upstream region of BCL-2 contains a number of elements that control its expression. We have identified a highly conserved 25-nucleotide G-rich sequence (BCL2Q), with potential to fold into a RNA G-quadruplex structure, located 42 nucleotides upstream of the translation start site of human BCL-2. In this study, we used a series of biophysical experiments to show that the BCL2Q sequence folds into a stable RNA G-quadruplex in vitro, and we conducted functional luciferase reporter-based assays, in a cell-free lysate and in three types of human cell lines, to demonstrate that the BCL2Q sequence modulates protein expression in the context of the 493-nucleotide native 5' untranslated region of BCL-2.