Cathepsin K Is Present in Invasive Oral Tongue Squamous Cell Carcinoma In Vivo and In Vitro

Objectives

Cathepsin K, a lysosomal cysteine protease, is expressed in the tumor microenvironment (TME) of skin carcinoma, but nothing is known about cathepsin K in oral tongue squamous cell carcinoma (OTSCC). Our aim was to describe the expression of cathepsin K in invasive OTSCC in vitro and in a series of clinical cancer specimens.

Materials and Methods

OTSCC invasion in vitro was studied using invasive HSC-3 tongue carcinoma cells in 3D organotypic models. In total, 121 mobile tongue OTSCCs and 10 lymph node metastases were analyzed for cathepsin K expression. The association between cathepsin K expression and clinicopathological factors was evaluated.

Results

Cysteine protease inhibitor E64 and cathepsin K silencing significantly (p<0.0001) reduced HSC-3 cell invasion in the 3D models. Cathepsin K was expressed in a majority of carcinoma and metastatic cells, but the expression pattern in carcinoma cells did not correlate with clinical parameters. Instead, the weak expression of cathepsin K in the invasive TME front correlated with increased overall recurrence (p<0.05), and in early-stage tumors this pattern predicted both cancer recurrence and cancer-specific mortality (p<0.05 and p<0.005, respectively).

Conclusions

Cathepsin K is expressed in OTSCC tissue in both carcinoma and TME cells. Although the diminished activity and expression in aggressive tongue HSC-3 cells reduced 3D invasion in vitro, the amount of cathepsin K in carcinoma cells was not associated with the outcome of cancer patients. Instead, cathepsin K in the invasive TME front seems to have a protective role in the complex progression of tongue cancer.     

Large-Scale Genomic Analysis Suggests a Neutral Punctuated Dynamics of Transposable Elements in Bacterial Genomes

Insertion sequences (IS) are the simplest and most abundant form of transposable DNA found in bacterial genomes. When present in multiple copies, it is thought that they can promote genomic plasticity and genetic exchange, thus being a major force of evolutionary change. The main processes that determine IS content in genomes are, though, a matter of debate. In this work, we take advantage of the large amount of genomic data currently available and study the abundance distributions of 33 IS families in 1811 bacterial chromosomes. This allows us to test simple models of IS dynamics and estimate their key parameters by means of a maximum likelihood approach. We evaluate the roles played by duplication, lateral gene transfer, deletion and purifying selection. We find that the observed IS abundances are compatible with a neutral scenario where IS proliferation is controlled by deletions instead of purifying selection. Even if there may be some cases driven by selection, neutral behavior dominates over large evolutionary scales. According to this view, IS and hosts tend to coexist in a dynamic equilibrium state for most of the time. Our approach also allows for a detection of recent IS expansions, and supports the hypothesis that rapid expansions constitute transient events—punctuations—during which the state of coexistence of IS and host becomes perturbated.     

Mapping of the Neisseria meningitidis NadA cell-binding site: relevance of predicted {alpha}-helices in the NH2-terminal and dimeric coiled-coil regions

NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH(2)-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH(2)-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH(2) globular head domain and the NH(2) dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.     

Hepatic deficiency of low density lipoprotein receptor-related protein-1 reduces high density lipoprotein secretion and plasma levels in mice

The low density lipoprotein receptor-related protein-1 (LRP1) is known to serve as a chylomicron remnant receptor in the liver responsible for the binding and plasma clearance of apolipoprotein E-containing lipoproteins. Previous in vitro studies have provided evidence to suggest that LRP1 expression may also influence high density lipoprotein (HDL) metabolism. The current study showed that liver-specific LRP1 knock-out (hLrp1(-/-)) mice displayed lower fasting plasma HDL cholesterol levels when compared with hLrp1(+/+) mice. Lecithin:cholesterol acyl transferase and hepatic lipase activities in plasma of hLrp1(-/-) mice were comparable with those observed in hLrp1(+/+) mice, indicating that hepatic LRP1 inactivation does not influence plasma HDL remodeling. Plasma clearance of HDL particles and HDL-associated cholesteryl esters was also similar between hLrp1(+/+) and hLrp1(-/-) mice. In contrast, HDL secretion from primary hepatocytes isolated from hLrp1(-/-) mice was significantly reduced when compared with that observed with hLrp1(+/+) hepatocytes. Biotinylation of cell surface proteins revealed decreased surface localization of the ATP-binding cassette, subfamily A, member 1 (ABCA1) protein, but total cellular ABCA1 level was not changed in hLrp1(-/-) hepatocytes. Finally, hLrp1(-/-) hepatocytes displayed reduced binding capacity for extracellular cathepsin D, resulting in lower intracellular cathepsin D content and impairment of prosaposin activation, a process that is required for membrane translocation of ABCA1 to facilitate cholesterol efflux and HDL secretion. Taken together, these results documented that hepatic LRP1 participates in cellular activation of lysosomal enzymes and through this mechanism, indirectly modulates the production and plasma levels of HDL.     

Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of Muscleblind-like-1 isoforms

Muscleblind-like-1 (MBNL1) is a splicing regulatory factor controlling the fetal-to-adult alternative splicing transitions during vertebrate muscle development. Its capture by nuclear CUG expansions is one major cause for type 1 myotonic dystrophy (DM1). Alternative splicing produces MBNL1 isoforms that differ by the presence or absence of the exonic regions 3, 5, and 7. To understand better their respective roles and the consequences of the deregulation of their expression in DM1, here we studied the respective roles of MBNL1 alternative and constitutive exons. By combining genetics, molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1; and finally (iv) the exon 7 region enhances MBNL1-MBNL1 dimerization properties. Consequently, the abnormally high inclusion of the exon 5 and 7 regions in DM1 is expected to enhance the potential of MBNL1 of being sequestered with nuclear CUG expansions, which provides new insight into DM1 pathophysiology.     

Why Have Small Multidrug Resistance Proteins Not Evolved into Fused,Internally Duplicated Structures?

The increasing number of solved membrane protein structures has led to the recognition of a common feature in a large fraction of the small-molecule transporters: inverted repeat structures, formed by two fused homologous membrane domains with opposite orientation in the membrane. An evolutionary pathway in which the ancestral state is a single gene encoding a dual-topology membrane protein capable of forming antiparallel homodimers has been posited. A gene duplication event enables the evolution of two oppositely orientated proteins that form antiparallel heterodimers. Finally, fusion of the two genes generates an internally duplicated transporter with two oppositely orientated membrane domains. Strikingly, however, in the small multidrug resistance (SMR) family of transporters, no fused, internally duplicated proteins have been found to date. Here, we have analyzed fused versions of the dual-topology transporter EmrE, a member of the SMR family, by blue-native PAGE and in vivo activity measurements. We find that fused constructs give rise to both intramolecular inverted repeat structures and competing intermolecular dimers of varying activity. The formation of several intramolecularly and intermolecularly paired species indicates that a gene fusion event may lower the overall amount of active protein, possibly explaining the apparent absence of fused SMR proteins in nature.     

Dynamic Eye Tracking Based Metrics for Infant Gaze Patterns in the Face-Distractor Competition Paradigm

Objective

To develop new standardized eye tracking based measures and metrics for infants’ gaze dynamics in the face-distractor competition paradigm.

Method

Eye tracking data were collected from two samples of healthy 7-month-old (total n = 45), as well as one sample of 5-month-old infants (n = 22) in a paradigm with a picture of a face or a non-face pattern as a central stimulus, and a geometric shape as a lateral stimulus. The data were analyzed by using conventional measures of infants’ initial disengagement from the central to the lateral stimulus (i.e., saccadic reaction time and probability) and, additionally, novel measures reflecting infants gaze dynamics after the initial disengagement (i.e., cumulative allocation of attention to the central vs. peripheral stimulus).

Results

The results showed that the initial saccade away from the centrally presented stimulus is followed by a rapid re-engagement of attention with the central stimulus, leading to cumulative preference for the central stimulus over the lateral stimulus over time. This pattern tended to be stronger for salient facial expressions as compared to non-face patterns, was replicable across two independent samples of 7-month-old infants, and differentiated between 7 and 5 month-old infants.

Conclusion

The results suggest that eye tracking based assessments of infants’ cumulative preference for faces over time can be readily parameterized and standardized, and may provide valuable techniques for future studies examining normative developmental changes in preference for social signals.

Significance

Standardized measures of early developing face preferences may have potential to become surrogate biomarkers of neurocognitive and social development.