Strategic engineering of alkyl spacer length for a pH-tolerant lysosome marker and dual organelle localization

Long-term visualization of lysosomal properties is extremely crucial to evaluate diseases related to their dysfunction. However, many of the reported lysotrackers are less conducive to imaging lysosomes precisely because they suffer from fluorescence quenching and other inherent drawbacks such as pH-sensitivity, polarity insensitivity, water insolubility, slow diffusibility, and poor photostability. To overcome these limitations, we have utilized an alkyl chain length engineering strategy and synthesized a series of lysosome targeting fluorescent derivatives namely NIMCs by attaching a morpholine moiety at the peri position of the 1,8-naphthalimide (NI) ring through varying alkyl spacers between morpholine and 1,8-naphthalimide. The structural and optical properties of the synthesized NIMCs were explored by ¹H-NMR, single-crystal X-ray diffraction, UV-Vis, and fluorescence spectroscopy. Afterward, optical spectroscopic measurements were carefully performed to identify a pH-tolerant, polarity sensitive, and highly photostable fluoroprobes for further live-cell imaging applications. NIMC6 displayed excellent pH-tolerant and polarity-sensitive properties. Consequently, all NIMCs were employed in kidney fibroblast cells (BHK-21) to investigate their applicability for lysosome targeting and probing lysosomal micropolarity. Interestingly, a switching of localization from lysosomes to the endoplasmic reticulum (ER) was also achieved by controlling the linker length and this phenomenon was subsequently applied in determining ER micropolarity. Additionally, the selected probe NIMC6 was also employed in BHK-21 cells for 3-D spheroid imaging and in Caenorhabditis elegans (C. elegans) for in vivo imaging, to evaluate its efficacy for imaging animal models.

A series naphthalimide-based fluorophores were designed by alkyl spacer length engineering to discover a pH-tolerant lysosomal marker. This approach also allows to probe lysosome-related organelles in C. elegans and communication between organelles.     

Identification of a set of genes showing regionally enriched expression in the mouse brain

Background

We have recorded responses from single neurons in murine visual cortex to determine the effectiveness of the input from the two murine cone photoreceptor mechanisms and whether there is any unique selectivity for cone inputs at this higher region of the visual system that would support the possibility of colour vision in mice. Each eye was stimulated by diffuse light, either 370 (strong stimulus for the ultra-violet (UV) cone opsin) or 505 nm (exclusively stimulating the middle wavelength sensitive (M) cone opsin), obtained from light emitting diodes (LEDs) in the presence of a strong adapting light that suppressed the responses of rods.

Results

Single cells responded to these diffuse stimuli in all areas of striate cortex. Two types of responsive cells were encountered. One type (135/323 – 42%) had little to no spontaneous activity and responded at either the on and/or the off phase of the light stimulus with a few impulses often of relatively large amplitude. A second type (166/323 – 51%) had spontaneous activity and responded tonically to light stimuli with impulses often of small amplitude. Most of the cells responded similarly to both spectral stimuli. A few (18/323 – 6%) responded strongly or exclusively to one or the other spectral stimulus and rarely in a spectrally opponent manner.

Conclusion

Most cells in murine striate cortex receive excitatory inputs from both UV- and M-cones. A small fraction shows either strong selectivity for one or the other cone mechanism and occasionally cone opponent responses. Cells that could underlie chromatic contrast detection are present but extremely rare in murine striate cortex.     

Investigation of Ti/CuO interface by X-ray photoelectron spectroscopy and atomic force microscopy

The Ti/CuO interface has been studied by the techniques of X-ray photoelectron spectroscopy and atomic force microscopy. Thin films of titanium were deposited on a CuO substrate at room temperature by the e-beam technique. The photoelectron spectra of titanium and copper were found to exhibit significant chemical interaction at the interface. The titanium overlayer was observed to get oxidized to TiO₂, while the CuO was observed to get reduced to elemental copper. This chemical interaction was observed to occur until a thickness of 7 nm of the titanium overlayer. For thicknesses greater than this value, the presence of unreacted titanium in the sample was detected. Barrier characteristics at the Ti/CuO interface were also carried out for substrate temperatures of 300°C, 400°C, 500°C, and 600°C as a function of the titanium overlayer thickness. A linear trend in the barrier thickness of the overlayer was observed between 400°C and 600°C substrate temperatures. The atomic force microscopy micrographs of the unannealed samples depicted layer-by-layer growth of elemental titanium on copper. At the Ti/CuO interface in such samples, the micrographs exhibited island formation of TiO₂ corresponding to the Volmer-Weber growth model. This formation has been interpreted as the relaxation in the strain energy. The percentage coverage of the underlying substrate by the TiO₂ islands showed a linear trend for the thicknesses of the titanium overlayer investigated. The average size of these islands also showed a linear trend as a function of the thickness of the overlayer.     

On the spectroscopic examination of printed documents by using a field emission scanning electron microscope with energy-dispersive X-ray spectroscopy (FE-SEM-EDS) and chemometric methods: application in forensic science

Analytical and Bioanalytical Chemistry - The detection of computer-generated document forgeries has always been a challenging task for forensic document examiners (FDE). With the aim to support the...     

Structural stability and electrical conductivity of amorphous Ge-metal alloy films

Stable homogeneous amorphous alloy¹ films of Ge with different concentrations of Al, Cu and Fe have been prepared by the simultaneous vapor deposition technique. Ge-Metal films are amorphous up to a concentration of ～ 40 at.% Al, ～ 20 at.% Cu and ～ 20 at.% Fe. The cyclic annealing and crystallization temperature of these films show that whereas Al increases the stability of the amorphous phase, the addition of Cu and Fe decreases it. The electrical resistivity decreases gradually with increasing Al content. In contrast, a rapid decrease in the electrical resistivity is observed for the Ge-Cu and Ge-Fe systems. The thermoelectric power (TEP) of Ge-Cu and Ge-Fe system assumes small values ～ few μV/deg for concentrations greater than few atomic percent. Ge-Al system exhibits large positive thermoelectric power at all compositions. The temperature dependence of the electrical resistivity of these alloy films show that the addition of Cu and Fe to Ge results in a drastic decrease in the activation energy of conduction whereas the addition of Al increases the activation energy. Ge-Al films exhibit intrinsic like conduction in the temperature range 100–300 K. The Ge-Cu and Ge-Fe films exhibit hopping conduction from 100–300 K and the related density of states is up to 100 times larger than in pure a-Ge films.     

Synthetic Heparanase Inhibitors Can Prevent Herpes Simplex Viral Spread

Herpes simplex virus (HSV-1) employs heparan sulfate (HS) as receptor for cell attachment and entry. During late-stage infection, the virus induces the upregulation of human heparanase (Hpse) to remove cell surface HS allowing viral spread. We hypothesized that inhibition of Hpse will prevent viral release thereby representing a new therapeutic strategy for HSV-1. A range of HS-oligosaccharides was prepared to examine the importance of chain length and 2-O-sulfation of iduronic moieties for Hpse inhibition. It was found that hexa- and octasaccharides potently inhibited the enzyme and that 2-O-sulfation of iduronic acid is tolerated. Computational studies provided a rationale for the observed structure–activity relationship. Treatment of human corneal epithelial cells (HCEs) infected with HSV-1 with the hexa- and octasaccharide blocked viral induced shedding of HS which significantly reduced spread of virions. The compounds also inhibited migration and proliferation of immortalized HCEs thereby providing additional therapeutic properties.     

Lactonisation--a degradation pathway for active pharmaceutical compounds: an in silico study in amorphous trehalose

The lactonisation of a CCR1 inhibitor (CC chemokine receptor 1, involved in autoimmune diseases) featuring a hydroxyl group in a gamma-position (gamma-OH) with respect to an amide group has been investigated in silico. The two key steps of the lactonisation reaction are (i) rearrangement to an optimal conformation and (ii) the formation of the lactone (ring closure) and expulsion of NH3. Quantum chemical calculations in the gas phase were employed to identify conformers of the molecule with favorable starting geometries for a lactonisation reaction. In total, calculations of 1296 conformers revealed that it is energetically feasible for an inhibitor molecule to adopt a conformation where the carbon atom of the amide group (C(amide)) is suitably close to the oxygen atom of the gamma-OH (O(gamma)) to facilitate a successful lactonisation reaction. Additionally, molecular dynamics methods were used to show that rearrangement to a suitable conformer for lactonisation to occur happens to a lesser extent when the CCR1 inhibitor was embedded in an amorphous trehalose matrix (a model carbohydrate excipient). The mechanism of the actual lactonisation was investigated using the complete Linear Synchronous Transit/Quadratic Synchronous Transit (LST/QST) method. This was performed in both the gas phase and in water and was found to be a concerted reaction.     

Identification of a potential general acid/base in the reversible phosphoryl transfer reactions catalyzed by tyrosine recombinases: Flp H305

Flp provides a unique opportunity to apply the tools of chemical biology to phosphoryl transfer reactions. Flp and other tyrosine recombinases catalyze site-specific DNA rearrangements via a phosphotyrosine intermediate. Unlike most related enzymes, Flp's nucleophilic tyrosine derives from a different protomer than the remainder of its active site. Because the tyrosine can be supplied exogenously, nonnatural synthetic analogs can be used. Here we examine the catalytic role of Flp's conserved H305. DNA cleavage was studied using a peptide containing either tyrosine (pKa congruent with 10) or 3-fluoro-tyrosine (pKa congruent with 8.4). Religation was studied using DNA substrates with 3'-phospho-cresol (pKa congruent with 10) or 3'-para-nitro-phenol (pKa congruent with 7.1). In both cases, the tyrosine analog with the lower pKa specifically restored the activity of an H305 mutant. These results provide experimental evidence that this conserved histidine functions as a general acid/base catalyst in tyrosine recombinases.