A novel single-base deletional mutation of MIP impairs protein distribution and cell-to-cell adhesion in autosomal dominant cataracts in a Chinese family.

Congenital cataracts are the leading cause of irreversible visual disability in children, and genetic factors play an important role in their development. In this study, targeted exome sequencing revealed a novel single-base deletional mutation of MIP (c.301delG; p.Ala101Profs*16) segregated with congenital punctate cataract in a Chinese family. The hydrophobic properties, and secondary and tertiary structures for truncated MIP were predicted to affect the function of protein by bioinformatics analysis. When MIP-WT and MIP-Ala101fs expression constructs were singly transfected into HeLa cells, it was found that the mRNA level showed no significant difference, while the protein level of the mutant was remarkably reduced compared to that of the wild-type MIP. Immunofluorescence images showed that the MIP-WT was principally localized to the plasma membrane, whereas the MIP-Ala101fs protein was aberrantly trapped in the cytoplasm. Furthermore, the cell-to-cell adhesion capability and the cell-to-cell communication property were both significantly reduced for MIP-Ala101fs compared to the MIP-WT (all *p < 0.05). This is the first report of the c.301delG mutation in the MIP gene associated with autosomal dominant congenital cataracts. We propose that the cataract is caused by the decreased protein expression and reduced cell-to-cell adhesion by the mutant MIP. The impaired trafficking or instability of the mutant protein, as well as compromised intercellular communication is probably a concurrent result of the mutation. The results expand the genetic and phenotypic spectra of MIP and help to better understand the molecular basis of congenital cataracts.

Mechanisms of microbial co-aggregation in mixed anaerobic cultures.

Co-aggregation of anaerobic microorganisms into suspended microbial biofilms (aggregates) serves ecological and biotechnological functions. Tightly packed aggregates of metabolically interdependent bacteria and archaea play key roles in cycling of carbon and nitrogen. Additionally, in biotechnological applications, such as wastewater treatment, microbial aggregates provide a complete metabolic network to convert complex organic material. Currently, experimental data explaining the mechanisms behind microbial co-aggregation in anoxic environments is scarce and scattered across the literature. To what extent does this process resemble co-aggregation in aerobic environments? Does the limited availability of terminal electron acceptors drive mutualistic microbial relationships, contrary to the commensal relationships observed in oxygen-rich environments? And do co-aggregating bacteria and archaea, which depend on each other to harvest the bare minimum Gibbs energy from energy-poor substrates, use similar cellular mechanisms as those used by pathogenic bacteria that form biofilms? Here, we provide an overview of the current understanding of why and how mixed anaerobic microbial communities co-aggregate and discuss potential future scientific advancements that could improve the study of anaerobic suspended aggregates. KEY POINTS: • Metabolic dependency promotes aggregation of anaerobic bacteria and archaea • Flagella, pili, and adhesins play a role in the formation of anaerobic aggregates • Cyclic di-GMP/AMP signaling may trigger the polysaccharides production in anaerobes.

A novel nonlocal partial differential equation model of endothelial progenitor cell cluster formation during the early stages of vasculogenesis.

The formation of new vascular networks is essential for tissue development and regeneration, in addition to playing a key role in pathological settings such as ischemia and tumour development. Experimental findings in the past two decades have led to the identification of a new mechanism of neovascularisation, known as cluster-based vasculogenesis, during which endothelial progenitor cells (EPCs) mobilised from the bone marrow are capable of bridging distant vascular beds in a variety of hypoxic settings in vivo. This process is characterised by the formation of EPC clusters during its early stages and, while much progress has been made in identifying various mechanisms underlying cluster formation, we are still far from a comprehensive description of such spatio-temporal dynamics. In order to achieve this, we propose a novel mathematical model of the early stages of cluster-based vasculogenesis, comprising of a system of nonlocal partial differential equations including key mechanisms such as endogenous chemotaxis, matrix degradation, cell proliferation and cell-to-cell adhesion. We conduct a linear stability analysis on the system and solve the equations numerically. We then conduct a parametric analysis of the numerical solutions of the one-dimensional problem to investigate the role of underlying dynamics on the speed of cluster formation and the size of clusters, measured via appropriate metrics for the cluster width and compactness. We verify the key results of the parametric analysis with simulations of the two-dimensional problem. Our results, which qualitatively compare with data from in vitro experiments, elucidate the complementary role played by endogenous chemotaxis and matrix degradation in the formation of clusters, suggesting chemotaxis is responsible for the cluster topology while matrix degradation is responsible for the speed of cluster formation. Our results also indicate that the nonlocal cell-to-cell adhesion term in our model, even though it initially causes cells to aggregate, is not sufficient to ensure clusters are stable over long time periods. Consequently, new modelling strategies for cell-to-cell adhesion are required to stabilise in silico clusters. We end the paper with a thorough discussion of promising, fruitful future modelling and experimental research perspectives.

Morphology of cells undergoing epithelial mesenchymal transition in microinvasive and early invasive oral squamous cell carcinom a - a light microscopic study.

The present study focuses on identification of cancer attributes of epithelial mesenchymal transition (EMT) at the earliest possible stage (microinvasion) under a light microscope by using hematoxylin and eosin stains, making it feasible for researchers to investigate such cases with ease without the use of extensive setups. The present study is the first in the English literature to define EMT features in micro-invasive and early invasive oral squamous cell carcinoma (OSCC) under a light microscope. This is a retrospective study of histological sections of 43 cases of OSCC from the Department of Oral Pathology and Microbiology. The data collected were later statistically analyzed. A total of 11 micro-invasive and 32 early invasive OSCC cases were assessed for core features of EMT. The predominant feature defining EMT found was dense inflammatory infiltrate in both microinvasive (91%) and early invasive OSCC (88%) followed by cell individualization in 82% of microinvasive and 75% of early invasive OSCC, which was then followed by other features. Reporting EMT in histopathological reports on a daily basis can aid in early diagnosis of OSCC as well as understanding carcinogenesis in early stages. Thereby, inclusion of EMT targeting therapeutics in early stages of OSCC can significantly alter the prognosis of cancer.

A predominant form of C-terminally end-cleaved AQP0 functions as an open water channel and an adhesion protein in AQP0ΔC/ΔC mouse lens.

The purpose of this investigation was to find out whether C-terminally end-cleaved aquaporin 0 (AQP0), that is present predominantly in the lens mature fiber cells of the WT, functions as a water channel and a cell-to-cell adhesion (CTCA) protein in a knockin (KI) mouse model (AQP0ΔC/ΔC) that does not express intact AQP0. A genetically engineered KI mouse model, AQP0ΔC/ΔC, expressing only end-cleaved AQP0 was developed. This model expresses 1-246 amino acids of AQP0, instead of the full length 1-263 amino acids. Lens transparency of postnatal day 10 (P10) was analyzed qualitatively by dark field imaging. WT, AQP0+/- and AQP0+/ΔC lenses were transparent; AQP0-/- and AQP0ΔC/ΔC mouse lenses displayed loss of transparency. Lens fiber cell membrane vesicles (FCMVs) were prepared from wild type (WT), AQP0 heterozygous (AQP0+/-), AQP0 knockout (AQP0-/-), AQP0+/ΔC and AQP0ΔC/ΔC; water permeability (Pf) was measured using the osmotic shrinking method. CTCA assay was performed using adhesion-deficient L-cells and FCMVs prepared from the abovementioned genotypes. FCMVs of AQP0+/- and AQP0-/- showed a statistically significant reduction (P < 0.001) in Pf and CTCA compared to those of WT. AQP0+/ΔC and AQP0ΔC/ΔC FCMVs exhibited no statistically significant alteration (P > 0.05) in Pf compared to those of WT. However, CTCA of AQP0+/ΔC AQP0ΔC/ΔC FCMVs was significantly higher (P < 0.001) than that of WT FCMVs. Our experiments clearly show that C-terminally end-cleaved AQP0 can function both as a water channel and a CTCA molecule in the lens fiber cell membranes. Also, end-truncation plays an important role in increasing the CTCA between fiber cells.

Molecular mechanism of Aquaporin 0-induced fiber cell to fiber cell adhesion in the eye lens.

Cell-to-cell adhesion (CTCA), which is key for establishing lens transparency, is a critical function of Aquaporin 0 (AQP0). The aim of this investigation was to find out the possible mechanism by which AQP0 exerts CTCA between fiber cells, since there are two proposals currently, either an AQP0-AQP0 interaction or an AQP0-lipid interaction. We studied the mechanism of AQP0-induced CTCA in intact AQP0 and C-terminally cleaved AQP0 (CTC-AQP0). Assays showed CTCA between L-cells transfected with intact AQP0 or CTC-AQP0 and parental L-cells indicating AQP0-membrane interaction. Both forms of AQP0 significantly (P < 0.001) promoted adhesion to negatively charged l-α-phosphatidylserine lipid vesicles signifying AQP0-lipid interaction. AQP0-expressing L-cells also promoted adhesion of WT and AQP0-KO mouse lens fiber cell membrane vesicles (FCMVs) significantly (P < 0.001). However, when FCMVs of WT or AQP0-KO were plated over parental L-cells, only WT vesicles adhered significantly, corroborating AQP0-membrane interaction. After incubating with extracellular domain-specific AQP0 antibody, L-cells expressing intact AQP0 or CTC-AQP0 showed a significant reduction (P < 0.001) in the adhesion of AQP0-KO FCMVs indicating extracellular loop involvement in CTCA. WT FCMVs from outer cortex and inner cortex promoted adhesion to parental L-cells, without any statistically significant difference in adhesion efficiency (P > 0.05). Ultrastructure studies of WT, AQP0-KO and transgenic lenses showed AQP0 is critical for fiber CTCA and compaction. The data collected clearly demonstrate that the positively charged amino acids in the AQP0 extracellular loop domains interact with the negatively charged lipids in the plasma membrane to promote CTCA for compaction of fiber cells.

Intact and N- or C-terminal end truncated AQP0 function as open water channels and cell-to-cell adhesion proteins: end truncation could be a prelude for adjusting the refractive index of the lens to prevent spherical aberration.

BACKGROUND: Investigate the impact of natural N- or C-terminal post-translational truncations of lens mature fiber cell Aquaporin 0 (AQP0) on water permeability (Pw) and cell-to-cell adhesion (CTCA) functions. METHODS: The following deletions/truncations were created by site-directed mutagenesis (designations in parentheses): Amino acid residues (AA) 2-6 (AQP0-N-del-2-6), AA235-263 (AQP0-1-234), AA239-263 (AQP0-1-238), AA244-263 (AQP0-1-243), AA247-263 (AQP0-1-246), AA250-263 (AQP0-1-249) and AA260-263 (AQP0-1-259). Protein expression was studied using immunostaining, fluorescent tags and organelle-specific markers. Pw was tested by expressing the respective complementary ribonucleic acid (cRNA) in Xenopus oocytes and conducting osmotic swelling assay. CTCA was assessed by transfecting intact or mutant AQP0 into adhesion-deficient L-cells and performing cell aggregation and adhesion assays. RESULTS: AQP0-1-234 and AQP0-1-238 did not traffic to the plasma membrane. Trafficking of AQP0-N-del-2-6 and AQP0-1-243 was reduced causing decreased membrane Pw and CTCA. AQP0-1-246, AQP0-1-249 and AQP0-1-259 mutants trafficked properly and functioned normally. Pw and CTCA functions of the mutants were directly proportional to the respective amount of AQP0 expressed at the plasma membrane and remained comparable to those of intact AQP0 (AQP0-1-263). CONCLUSIONS: Post-translational truncation of N- or C-terminal end amino acids does not alter the basal water permeability of AQP0 or its adhesive functions. AQP0 may play a role in adjusting the refractive index to prevent spherical aberration in the constantly growing lens. GENERAL SIGNIFICANCE: Similar studies can be extended to other lens proteins which undergo post-translational truncations to find out how they assist the lens to maintain transparency and homeostasis for proper focusing of objects on to the retina.

Functional characterization of an AQP0 missense mutation, R33C, that causes dominant congenital lens cataract, reveals impaired cell-to-cell adhesion.

Aquaporin 0 (AQP0) performs dual functions in the lens fiber cells, as a water pore and as a cell-to-cell adhesion molecule. Mutations in AQP0 cause severe lens cataract in both humans and mice. An arginine to cysteine missense mutation at amino acid 33 (R33C) produced congenital autosomal dominant cataract in a Chinese family for five generations. We re-created this mutation in wild type human AQP0 (WT-AQP0) cDNA by site-directed mutagenesis, and cloned and expressed the mutant AQP0 (AQP0-R33C) in heterologous expression systems. Mutant AQP0-R33C showed proper trafficking and membrane localization like WT-AQP0. Functional studies conducted in Xenopus oocytes showed no significant difference (P > 0.05) in water permeability between AQP0-R33C and WT-AQP0. However, the cell-to-cell adhesion property of AQP0-R33C was significantly reduced (P < 0.001) compared to that of WT-AQP0, indicated by cell aggregation and cell-to-cell adhesion assays. Scrape-loading assay using Lucifer Yellow dye showed reduction in cell-to-cell adhesion affecting gap junction coupling (P < 0.001). The data provided suggest that this mutation might not have caused significant alterations in protein folding since there was no obstruction in protein trafficking or water permeation. Reduction in cell-to-cell adhesion and development of cataract suggest that the conserved positive charge of Extracellular Loop A may play an important role in bringing fiber cells closer. The proposed schematic models illustrate that cell-to-cell adhesion elicited by AQP0 is vital for lens transparency and homeostasis.

Bacterial coaggregation in aquatic systems.

The establishment of a sessile community is believed to occur in a sequence of steps where genetically distinct bacteria can become attached to partner cells via specific molecules, in a process known as coaggregation. The presence of bacteria with the ability to autoaggregate and coaggregate has been described for diverse aquatic systems, particularly freshwater, drinking water, wastewater, and marine water. In these aquatic systems, coaggregation already demonstrated a role in the development of complex multispecies sessile communities, including biofilms. While specific molecular aspects on coaggregation in aquatic systems remain to be understood, clear evidence exist on the impact of this mechanism in multispecies biofilm resilience and homeostasis. The identification of bridging bacteria among coaggregating consortia has potential to improve the performance of wastewater treatment plants and/or to contribute for the development of strategies to control undesirable biofilms. This study provides a comprehensive analysis on the occurrence and role of bacterial coaggregation in diverse aquatic systems. The potential of this mechanism in water-related biotechnology is further described, with particular emphasis on the role of bridging bacteria.

Significance of E-cadherin and CD44 expression in patients with unresectable metastatic colorectal cancer.

The loss of adhesion molecules is reported to be associated with tumor invasion and metastasis in numerous types of cancer. Epithelial (E)-cadherin is an important molecule for cell-to-cell adhesion, while cluster of differentiation (CD)44 is an important molecule for cell-to-extracellular matrix adhesion. The focus of the present study was to evaluate the significance of the expression of E-cadherin and CD44 in patients with the unresectable metastatic colorectal cancer (CRC) who are undergoing palliative chemotherapy. Formalin-fixed, paraffin-embedded samples were obtained from 49 patients who underwent primary tumor resection and who were receiving palliative chemotherapy for unresectable metastatic CRC. The expression of E-cadherin and CD44 was evaluated using immunohistochemistry. The expression of E-cadherin was not significantly associated with progression-free survival (PFS; P=0.2825) or overall survival (OS; P=0.6617). The expression of CD44 was not associated with PFS (P=0.4365), but it did exhibit a certain level of association with OS (P=0.0699). However, the combined low expression of E-cadherin and CD44 demonstrated a significant association with decreased PFS (P=0.0101) and OS (P=0.0009). The combined loss of E-cadherin and CD44 expression also led to a reduction in the objective response rate and disease control rate (P=0.0076 and P=0.0294, respectively). A univariate analysis indicated that the combined low expression of E-cadherin and CD44 (P=0.0474) and sex (P=0.0330) were significantly associated with decreased PFS, and multivariate analysis confirmed combined low expression of E-cadherin and CD44 as an independent risk factor for decreased PFS [hazard ratio (HR), 8.276; 95% confidence interval (CI), 1.383-43.311; P=0.0227]. Univariate and multivariate analyses also indicated that the combined low expression of E-cadherin and CD44 expression was a significant prognostic factor for poor OS (HR, 15.118; 95% CI, 2.645-77.490; P=0.0039). Therefore the current study suggests that the combined low expression of E-cadherin and CD44 is an effective independent predictor of decreased chemotherapeutic outcome and survival in patients with unresectable metastatic CRC.

Immunohistochemical study of CD44s expression in oral squamous cell carcinoma-its correlation with prognostic parameters.

BACKGROUND AND OBJECTIVES: The study aims at the observation of the immunohistochemical expression of CD44s in Oral Squamous Cell Carcinoma (OSCC) and to correlate its expression with prognostic parameters. MATERIALS AND METHODS: A total of 30 cases of OSCC, - 10 cases of each well differentiated (WD SCC), moderately differentiated (MD SCC) and poorly differentiated squamous cell carcinomas (PD SCC) were included in the study. The sections were subjected to immunohistochemical study using CD44s antigen marker. The degree of intensity and distribution of CD44s immunostaining was assessed and correlated with prognostic markers such as tumor stage (tumor size), tumor grade (Broder's histological grading), tumor site, tumor thickness (histological depth of invasion) and nodal status. RESULTS: CD44s expression by tumor cells in OSCCs is statistically correlated with tumor grade i.e. Higher mean of CD44s immunoexpression was observed in WD SCC group (10.80 ± 3.97), followed by MD SCC group (5.90 ± 3.38) and PD SCC group showed least CD44s immunoexpression (3.70 ± 4.64). There was no statistical significance observed with respect to the other prognostic markers. CONCLUSION: Based on these observations it can be suggested that the decrease in expression of CD44s in OSCC cells may be due to the reduced cell-to-cell and cell-to-matrix adhesion, resulting in easy detachment from the rigid constitution. Low expression of CD44s in OSCC tissues may be an indicator of tumor invasion and high metastatic potential.