Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture

Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 ± 0.1) × 10⁶ cells mL^?1 was obtained, with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.     

Aryloxy cyclohexyl imidazoles: A novel class of antileishmanial agents

Thirteen novel aryloxy cyclohexane-based mono and bis imidazoles were synthesized and evaluated in vitro as antileishmanials against Leishmania donovani and cytotoxicity assessed. These compounds were better than the existing drugs, sodium stibogluconate and pentamidine in respect to IC₅₀ and SI values. Promising compounds were tested further in vivo. Among all, the bis methylimidazole with 2-fluoro, 4-nitro aryloxy group (9) exhibited significant in vivo inhibition of 77.9%, thus providing new structural lead for antileishmanials.     

A Histidine Switch in Hemagglutinin-Neuraminidase Triggers Paramyxovirus-Cell Membrane Fusion

Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine “switch” in HN that triggers fusion.     

Quantitative determination of phyllanthin and hypophyllanthin in Phyllanthus species by high-performance thin layer chromatography

A simple, precise and rapid high-performance thin-layer chromatographic method has been developed for the estimation of phyllanthin (1) and hypophyllanthin (2), the important lignans of Phyllanthus species, especially Phyllanthus amarus. Separation of 1 and 2 was carried out on silica gel 60 F254 layers eluted with hexane:acetone:ethyl acetate (74:12:8), and the analytes were visualised through colour development with vanillin in concentrated sulphuric acid and ethanol. Scanning and quantification of spots was performed at 580 nm. Recoveries of 1 and 2 were 98.7 and 97.3%, respectively. The method was validated and the peak purities and limits of detection and quantification were determined.     

Aberrant epigenetic and genetic marks are seen in myelodysplastic leukocytes and reveal Dock4 as a candidate pathogenic gene on chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.     

Prenatal music stimulation facilitates the postnatal functional development of the auditory as well as visual system in chicks (Gallus domesticus)

Rhythmic sound or music is known to improve cognition in animals and humans. We wanted to evaluate the effects of prenatal repetitive music stimulation on the remodelling of the auditory cortex and visual Wulst in chicks. Fertilized eggs (0 day) of white leghorn chicken (Gallus domesticus) during incubation were exposed either to music or no sound from embryonic day 10 until hatching. Auditory and visual perceptual learning and synaptic plasticity, as evident by synaptophysin and PSD-95 expression, were done at posthatch days (PH) 1, 2 and 3. The number of responders was significantly higher in the music stimulated group as compared to controls at PH1 in both auditory and visual preference tests. The stimulated chicks took significantly lesser time to enter and spent more time in the maternal area in both preference tests. A significantly higher expression of synaptophysin and PSD-95 was observed in the stimulated group in comparison to control at PH1-3 both in the auditory cortex and visual Wulst. A significant inter-hemispheric and gender-based difference in expression was also found in all groups. These results suggest facilitation of postnatal perceptual behaviour and synaptic plasticity in both auditory and visual systems following prenatal stimulation with complex rhythmic music.     

Synthesis and SAR of novel oxazolidinones: discovery of ranbezolid

Novel oxazolidinones were synthesized containing a number of substituted five-membered heterocycles attached to the 'piperazinyl-phenyl-oxazolidinone' core of eperezolid. Further, the piperazine ring of the core was replaced by other diamino-heterocycles. These modifications led to several compounds with potent activity against a spectrum of resistant and susceptible gram-positive organisms, along with the identification of ranbezolid (RBx 7644) as a clinical candidate.     

The mechanism of phosphatidylcholine‐induced interference of PAP (248‐286) aggregation

Seminal amyloids are well known for their role in enhancing HIV infection. Among all the amyloidogenic peptides identified in human semen, PAP_248‐286 was found to be the most active and was termed as semen‐derived enhancer of viral infection (SEVI). Although amyloidogenic nature of the peptide is mainly linked with enhancement of the viral infection, the most active physiological conformation of the aggregated peptide remains inconclusive. Lipids are known to modulate aggregation pathway of a variety of proteins and peptides and constitute one of the most abundant biomolecules in human semen. PAP_248‐286 significantly differs from the other known amyloidogenic peptides, including Aβ and IAPP, in terms of critical concentration, surface charge, fibril morphology, and structural transition during aggregation. Hence, in the present study, we aimed to assess the effect of a lipid, 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC), on PAP_248‐286 aggregation and the consequent conformational outcomes. Our initial observation suggested that the presence of the lipid considerably influenced the aggregation of PAP_248‐286. Further, ZDOCK and MD simulation studies of peptide multimerization have suggested that the hydrophobic residues at C‐terminus are crucial for PAP_248‐286 aggregation and are anticipated to be major DOPC‐interacting partners. Therefore, we further assessed the aggregation behaviour of C‐terminal (PAP_273‐286) fragment of PAP_248‐286 and observed that DOPC possesses the ability to interfere with the aggregation behaviour of both the peptides used in the current study. Mechanistically, we propose that the presence of DOPC causes considerable inhibition of the peptide aggregation by interfering with the peptide's disordered state to β‐sheet transition.