Achieving Molecular Recognition of Structural Analogues in Surface-Enhanced Raman Spectroscopy: Inducing Charge and Geometry Complementarity to Mimic Molecular Docking.

Molecular recognition of complex isomeric biomolecules remains challenging in surface-enhanced Raman scattering (SERS) spectroscopy due to their small Raman cross-sections and/or poor surface affinities. To date, the use of molecular probes has achieved excellent molecular sensitivities but still suffers from poor spectral specificity. Here, we induce "charge and geometry complementarity" between probe and analyte as a key strategy to achieve high spectral specificity for effective SERS molecular recognition of structural analogues. We employ 4-mercaptopyridine (MPY) as the probe, and chondroitin sulfate (CS) disaccharides with isomeric sulfation patterns as our proof-of-concept study. Our experimental and in silico studies reveal that "charge and geometry complementarity" between MPY's binding pocket and the CS sulfation patterns drives the formation of site-specific, multidentate interactions at the respective CS isomerism sites, which "locks" each CS in its analogue-specific complex geometry, akin to molecular docking events. Leveraging the resultant spectral fingerprints, we achieve > 97 % classification accuracy for 4 CSs and 5 potential structural interferences, as well as attain multiplex CS quantification with < 3 % prediction error. These insights could enable practical SERS differentiation of biologically important isomers to meet the burgeoning demand for fast-responding applications across various fields such as biodiagnostics, food and environmental surveillance.

The natural antisense transcript HAS2-AS1 regulates breast cancer cells aggressiveness independently from hyaluronan metabolism.

Hyaluronan (HA) is a ubiquitous extracellular matrix component playing a crucial role in the regulation of cell behaviors, including cancer. Aggressive breast cancer cells tend to proliferate, migrate and metastatize. Notably, triple-negative breast cancer cells lacking the expression of estrogen receptor (ER) as well as progesterone receptor and HER2 are more aggressive than ER-positive ones. As currently no targeted therapy is available for triple-negative breast cancer, the identification of novel therapeutic targets has a high clinical priority. In ER-negative cells, tumoral behavior can be reduced by inhibiting HA synthesis or silencing the enzymes involved in its metabolism, such as HA synthase 2 (HAS2). HAS2-AS1 is a long non-coding RNA belonging to the natural antisense transcript family which is known to favor HAS2 gene expression and HA synthesis, thus bolstering malignant progression in brain, ovary, and lung tumors. As the role of HAS2-AS1 has not yet been investigated in breast cancer, in this work we report that ER-positive breast cancers had lower HAS2-AS1 expression compared to ER-negative tumors. Moreover, the survival of patients with ER-negative tumors was higher when the expression of HAS2-AS1 was elevated. Experiments with ER-negative cell lines as MDA-MB-231 and Hs 578T revealed that the overexpression of either the full-length HAS2-AS1 or its exon 2 long or short isoforms alone, strongly reduced cell viability, migration, and invasion, whereas HAS2-AS1 silencing increased cell aggressiveness. Unexpectedly, in these ER-negative cell lines, HAS2-AS1 is involved neither in the regulation of HAS2 nor in HA deposition. Finally, transcriptome analysis revealed that HAS2-AS1 modulation affected several pathways, including apoptosis, proliferation, motility, adhesion, epithelial to mesenchymal transition, and signaling, describing this long non-coding RNA as an important regulator of breast cancer cells aggressiveness.

In Vitro Evaluation of Candidate Gene Targets for Cancer Therapy.

Discovery and development of gene targets for cancer therapeutics are lengthy and highly costly processes. Identification and evaluation of candidate gene targets are of fundamental importance. RNA interference allows candidate genes to be specifically and effectively knocked down in cancer cells. This tool can be easily incorporated into a loss-of-function approach in the initial evaluation of candidate gene targets for cancer treatment prior to moving on to animal studies and clinical trials. This chapter describes a relatively simple and straightforward protocol that makes use of small interfering RNA to achieve knockdown of the candidate gene target and to evaluate the resultant effects on four aspects of cancer cell behavior: migration, invasion, proliferation, and adhesion.

Salmonella effector SopD2 interferes with Rab34 function.

Many intracellular pathogens have evolved highly specialized mechanisms to isolate themselves from the host cell's innate immune response while still obtaining the necessary nutrients to survive. Salmonella utilizes type 3 secretion systems (T3SSs) to deliver bacterial proteins called effectors, across the encompassing Salmonella Containing vacuole (SCV) membrane, to subvert the host's membrane trafficking pathways and alter other cellular processes. The Salmonella Pathogenicity Island (SPI)-2 effector SopD2 has recently been demonstrated to modulate multiple members of the Rab GTPase family such as Rab7, Rab8, Rab10, and Rab32 (D'Costa et al., , Cell Reports, 12:1508-18; Spano et al., , Cell Host & Microbe, 19:216-26). Here, we demonstrate the additional capacity of SopD2 to bind Rab34 and modulate its function. Our data indicate that depletion of Rab34 delays maturation of the SCV, and consequently, inhibits intracellular Salmonella enterica serotype typhimurium (S. typhimurium) growth. Interestingly, intracellular growth of the S. typhimurium lacking SopD2 was severely impaired in Rab34-depleted cells, suggesting a compounding virulence effect. Overall this study reveals an additional member of the Rab GTPase family, Rab34, that is modulated by SopD2 and provides insight into its role in Salmonella biology.

Sortilin is associated with the chlamydial inclusion and is modulated during infection.

Chlamydia species are obligate intracellular pathogens that have a major impact on human health. The pathogen replicates within an intracellular niche called an inclusion and is thought to rely heavily on host-derived proteins and lipids, including ceramide. Sortilin is a transmembrane receptor implicated in the trafficking of acid sphingomyelinase, which is responsible for catalysing the breakdown of sphingomyelin to ceramide. In this study, we examined the role of sortilin in Chlamydia trachomatis L2 development. Western immunoblotting and immunocytochemistry analysis revealed that endogenous sortilin is not only associated with the inclusion, but that protein levels increase in infected cells. RNAi-mediated depletion of sortilin, however, had no detectable impact on ceramide delivery to the inclusion or the production of infectious progeny. This study demonstrates that whilst Chlamydia redirects sortilin trafficking to the chlamydial inclusion, RNAi knockdown of sortilin expression is insufficient to determine if this pathway is requisite for the development of the pathogen.