A portable immunomagnetic cell capture system to accelerate culture diagnosis of bacterial infections.

Bacterial infections continue to be a major cause of deaths globally, particularly in resource-poor settings. In the absence of rapid and affordable diagnostic solutions, patients are mostly administered broad spectrum antibiotics leading to antibiotics resistance and poor recovery. Culture diagnosis continues to be a gold standard for diagnosis of bacterial infection, despite its long turnaround time of 24 to 48 h. We have developed a portable immunomagnetic cell capture (iMC(2)) system that allows rapid culture diagnosis of bacterial pathogens. Our approach involves the culture growth of the blood samples in broth media for 6 to 8 h, followed by immunomagnetic enrichment of the target cells using the iMC(2) device. The device comprises a disposable capture chip that has two chambers of 5 ml and 50 µl volume connected through a channel with a manual valve. Bacterial cells bound to antibody coated magnetic nanoparticles are swept from the 5 ml sample chamber into the 50 µl recovery chamber by moving an external magnetic field with respect to the capture chip using a linear positioner. This enables specific isolation and up to 100× enrichment of the target cells. The presence of bacteria in the recovered sample is confirmed visually using a lateral flow immunoassay. The system is demonstrated in buffer and blood samples spiked with S. typhi. The method has high sensitivity (10 CFU ml(-1)), specificity and a rapid turnaround time of less than 7 h, a significant improvement over conventional methods.

Depletion of CpG Dinucleotides in Papillomaviruses and Polyomaviruses: A Role for Divergent Evolutionary Pressures.

BACKGROUND: Papillomaviruses and polyomaviruses are small ds-DNA viruses infecting a wide-range of vertebrate hosts. Evidence supporting co-evolution of the virus with the host does not fully explain the evolutionary path of papillomaviruses and polyomaviruses. Studies analyzing CpG dinucleotide frequencies in virus genomes have provided interesting insights on virus evolution. CpG dinucleotide depletion has not been extensively studied among papillomaviruses and polyomaviruses. We sought to analyze the relative abundance of dinucleotides and the relative roles of evolutionary pressures in papillomaviruses and polyomaviruses. METHODS: We studied 127 full-length sequences from papillomaviruses and 56 full-length sequences from polyomaviruses. We analyzed the relative abundance of dinucleotides, effective codon number (ENC), differences in synonymous codon usage. We examined the association, if any, between the extent of CpG dinucleotide depletion and the evolutionary lineage of the infected host. We also investigated the contribution of mutational pressure and translational selection to the evolution of papillomaviruses and polyomaviruses. RESULTS: All papillomaviruses and polyomaviruses are CpG depleted. Interestingly, the evolutionary lineage of the infected host determines the extent of CpG depletion among papillomaviruses and polyomaviruses. CpG dinucleotide depletion was more pronounced among papillomaviruses and polyomaviruses infecting human and other mammals as compared to those infecting birds. Our findings demonstrate that CpG depletion among papillomaviruses is linked to mutational pressure; while CpG depletion among polyomaviruses is linked to translational selection. We also present evidence that suggests methylation of CpG dinucleotides may explain, at least in part, the depletion of CpG dinucleotides among papillomaviruses but not polyomaviruses. CONCLUSIONS: The extent of CpG depletion among papillomaviruses and polyomaviruses is linked to the evolutionary lineage of the infected host. Our results highlight the existence of divergent evolutionary pressures leading to CpG dinucleotide depletion among small ds-DNA viruses infecting vertebrate hosts.

Novel Insights on Hantavirus Evolution: The Dichotomy in Evolutionary Pressures Acting on Different Hantavirus Segments.

BACKGROUND: Hantaviruses are important emerging zoonotic pathogens. The current understanding of hantavirus evolution is complicated by the lack of consensus on co-divergence of hantaviruses with their animal hosts. In addition, hantaviruses have long-term associations with their reservoir hosts. Analyzing the relative abundance of dinucleotides may shed new light on hantavirus evolution. We studied the relative abundance of dinucleotides and the evolutionary pressures shaping different hantavirus segments. METHODS: A total of 118 sequences were analyzed; this includes 51 sequences of the S segment, 43 sequences of the M segment and 23 sequences of the L segment. The relative abundance of dinucleotides, effective codon number (ENC), codon usage biases were analyzed. Standard methods were used to investigate the relative roles of mutational pressure and translational selection on the three hantavirus segments. RESULTS: All three segments of hantaviruses are CpG depleted. Mutational pressure is the predominant evolutionary force leading to CpG depletion among hantaviruses. Interestingly, the S segment of hantaviruses is GpU depleted and in contrast to CpG depletion, the depletion of GpU dinucleotides from the S segment is driven by translational selection. Our findings also suggest that mutational pressure is the primary evolutionary pressure acting on the S and the M segments of hantaviruses. While translational selection plays a key role in shaping the evolution of the L segment. Our findings highlight how different evolutionary pressures may contribute disproportionally to the evolution of the three hantavirus segments. These findings provide new insights on the current understanding of hantavirus evolution. CONCLUSIONS: There is a dichotomy among evolutionary pressures shaping a) the relative abundance of different dinucleotides in hantavirus genomes b) the evolution of the three hantavirus segments.

In vitro control release, cytotoxicity assessment and cellular uptake of methotrexate loaded liquid-crystalline folate nanocarrier.

Folate molecules self-assemble in the form of stacks to form liquid-crystalline solutions. Nanocarriers from self-assembled folates are composed of highly ordered structures, which offer high encapsulation of drug (95-98%), controlled drug release rates, active cellular uptake and biocompatibility. Recently, we have shown that the release rates of methotrexate can be controlled by varying the size of nanoparticles, cross-linking cation and cross-linking concentration. The present study reports the in vitro cytotoxic behavior of methotrexate loaded liquid-crystalline folate nanoparticles on cultured HeLa cells. Changing drug release rates can influence cytotoxicity of cancer cells. Therefore, to study the correlation of release rate and cytotoxic behavior, the effect of release controlling parameters on HeLa cells was studied through MTT assay. It is reported that by controlling the methotrexate release, the survival rates of HeLa cells can be controlled. Released methotrexate kills HeLa cells as effectively as free methotrexate solution. The co-culture based in vitro cellular uptake study through fluorescence microscopy on folate receptor positive and negative cancer cells shows that the present nanocarrier has the potential to distinguish cancer cells from normal cells. Overall, the present study reports the in vitro performance of self-assembled liquid-crystalline folate nanoparticles, which will be a platform for further in vivo studies and clinical trials.

Systematic CpT (ApG) depletion and CpG excess are unique genomic signatures of large DNA viruses infecting invertebrates.

Differences in the relative abundance of dinucleotides, if any may provide important clues on host-driven evolution of viruses. We studied dinucleotide frequencies of large DNA viruses infecting vertebrates (n = 105; viruses infecting mammals = 99; viruses infecting aves = 6; viruses infecting reptiles = 1) and invertebrates (n = 88; viruses infecting insects = 84; viruses infecting crustaceans = 4). We have identified systematic depletion of CpT(ApG) dinucleotides and over-representation of CpG dinucleotides as the unique genomic signature of large DNA viruses infecting invertebrates. Detailed investigation of this unique genomic signature suggests the existence of invertebrate host-induced pressures specifically targeting CpT(ApG) and CpG dinucleotides. The depletion of CpT dinucleotides among large DNA viruses infecting invertebrates is at least in part, explained by non-canonical DNA methylation by the infected host. Our findings highlight the role of invertebrate host-related factors in shaping virus evolution and they also provide the necessary framework for future studies on evolution, epigenetics and molecular biology of viruses infecting this group of hosts.

CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution.

Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.

The Warburg effect: insights from the past decade.

Several decades ago, Otto Warburg discovered that cancer cells produce energy predominantly by glycolysis; a phenomenon now termed "Warburg effect". Warburg linked mitochondrial respiratory defects in cancer cells to aerobic glycolysis; this theory of his gradually lost its importance with the lack of conclusive evidence confirming the presence of mitochondrial defects in cancer cells. Scientists began to believe that this altered mechanism of energy production in cancer cells was more of an effect than the cause. More than 50 years later, the clinical use of FDG-PET imaging in the diagnosis and monitoring of cancers rekindled the interest of the scientific community in Warburg's hypothesis. In the last ten years considerable progress in the field has advanced our understanding of the Warburg effect. However, it still remains unclear if the Warburg effect plays a causal role in cancers or it is an epiphenomenon in tumorigenesis. In this review we aim to discuss the molecular mechanisms associated with the Warburg effect with emphasis on recent advances in the field including the role of epigenetic changes, miRNAs and post-translational modification of proteins. In addition, we also discuss emerging therapeutic strategies that target the dependence of cancer cells on altered energy processing through aerobic glycolysis.