Human Aldehyde Dehydrogenases: A Superfamily of Similar Yet Different Proteins Highly Related to Cancer.

The superfamily of human aldehyde dehydrogenases (hALDHs) consists of 19 isoenzymes which are critical for several physiological and biosynthetic processes and play a major role in the organism's detoxification via the NAD(P) dependent oxidation of numerous endogenous and exogenous aldehyde substrates to their corresponding carboxylic acids. Over the last decades, ALDHs have been the subject of several studies as it was revealed that their differential expression patterns in various cancer types are associated either with carcinogenesis or promotion of cell survival. Here, we attempt to provide a thorough review of hALDHs' diverse functions and 3D structures with particular emphasis on their role in cancer pathology and resistance to chemotherapy. We are especially interested in findings regarding the association of structural features and their changes with effects on enzymes' functionalities. Moreover, we provide an updated outline of the hALDHs inhibitors utilized in experimental or clinical settings for cancer therapy. Overall, this review aims to provide a better understanding of the impact of ALDHs in cancer pathology and therapy from a structural perspective.

Effectiveness of Hyperthermia as Monotherapy and Adjuvant Therapy Approaches Against an In Vitro Model of Colorectal Carcinoma.

BACKGROUND/AIM: Despite improvement in current therapies, the 5-year overall survival rate of colorectal carcinoma is still low especially in its metastatic form. On the other hand, hyperthermia has been utilized as a cancer treatment approach to improve overall therapeutic efficacy. In the present study, we have aimed to develop an optimized hyperthermic protocol against an in vitro model of human colon carcinoma, as a single and/or adjuvant treatment approach. MATERIALS AND METHODS: We have utilized an in vitro model of human colorectal carcinoma consisting of colorectal carcinoma (HT29, CaCo2) and normal colon epithelial (CCD841CoN) cell lines. Cells were exposed to 45°C, over 120 min, in the presence or absence of chemotherapeutic (5-Fluorouracil, Capecitabine) and targeted (Bevacizumab, Cetuximab) drugs. Cell viability levels were determined by the Alamar-blue assay while determination of cell death, reactive oxygen species (ROS) production, mitochondrial membrane depolarization (ΔΨµ) levels and cell cycle progression were performed by flow cytometry. RESULTS: CaCo2 and HT29 cells showed a differential response towards i) cell viability, ii) cell death, iii) ROS and ΔΨµ levels as well as iv) cell cycle distribution, in the presence of hyperthermia alone (monotherapy) or in combination with the above-mentioned drugs (adjuvant therapy). Finally, normal colon epithelial (CCD841CoN) cells remained minimally affected. CONCLUSION: We have developed an optimized experimental hyperthermic protocol, as a promising monotherapy and/or adjuvant therapy approach, with the capacity to potentiate chemotherapeutic as well as targeted drug-induced cytotoxicity against a model of colorectal carcinoma, to a variable degree.

Thermal activation of Ti(1-x)Au(x) thin films with enhanced hardness and biocompatibility.

The lifetime of orthopaedic implants can be extended by coating the softer Ti6Al4V alloy with harder biocompatible thin films. In this work, thin films of Ti(1-x)Au(x) are grown on Ti6Al4V and glass substrates by magnetron sputtering in the entire x = 0-1 range, before their key biomechanical properties are performance tuned by thermal activation. For the first time, we explore the effect of in-situ substrate heating versus ex-situ post-deposition heat-treatment, on development of mechanical and biocompatibility performance in Ti-Au films. A ∼250% increase in hardness is achieved for Ti-Au films compared to bulk Ti6Al4V and a ∼40% improvement from 8.8 GPa as-grown to 11.9 and 12.3 GPa with in-situ and ex-situ heat-treatment respectively, is corelated to changes in structural, morphological and chemical properties, providing insights into the origins of super-hardness in the Ti rich regions of these materials. X-ray diffraction reveals that as-grown films are in nanocrystalline states of Ti-Au intermetallic phases and thermal activation leads to emergence of mechanically hard Ti-Au intermetallics, with films prepared by in-situ substrate heating having enhanced crystalline quality. Surface morphology images show clear changes in grain size, shape and surface roughness following thermal activation, while elemental analysis reveals that in-situ substrate heating is better for development of oxide free Ti3Au ß-phases. All tested Ti-Au films are non-cytotoxic against L929 mouse fibroblast cells, while extremely low leached ion concentrations confirm their biocompatibility. With peak hardness performance tuned to >12 GPa and excellent biocompatibility, Ti-Au films have potential as a future coating technology for load bearing medical implants.

Toxicity Profiling of Biosurfactants Produced by Novel Marine Bacterial Strains.

Surface active agents (SAAs), currently used in modern industry, are synthetic chemicals produced from non-renewable sources, with potential toxic impacts on humans and the environment. Thus, there is an increased interest for the identification and utilization of natural derived SAAs. As such, the marine environment is considered a promising source of biosurfactants with low toxicity, environmental compatibility, and biodegradation compared to their synthetic counterparts. MARISURF is a Horizon 2020 EU-funded project aiming to identify and functionally characterize SAAs, derived from a unique marine bacterial collection, towards commercial exploitation. Specifically, rhamnolipids produced by Marinobacter MCTG107b and Pseudomonas MCTG214(3b1) strains were previously identified and characterized while currently their toxicity profile was assessed by utilizing well-established methodologies. Our results showed a lack of cytotoxicity in in vitro models of human skin and liver as indicated by alamar blue and propidium iodide assays. Additionally, the use of the single gel electrophoresis assay, under oxidative stress conditions, revealed absence of any significant mutagenic/anti-mutagenic potential. Finally, both 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) cell-free assays, revealed no significant anti-oxidant capacity for neither of the tested compounds. Consequently, the absence of significant cytotoxicity and/or mutagenicity justifies their commercial exploitation and potential development into industrial end-user applications as natural and environmentally friendly biosurfactants.

A novel methylated analogue of L-Mimosine exerts its therapeutic potency through ROS production and ceramide-induced apoptosis in malignant melanoma.

Melanoma is an aggressive and highly metastatic type of skin cancer where the design of new therapies is of utmost importance for the clinical management of the disease. Thus, we have aimed to investigate the mode of action by which a novel methylated analogue of L-Mimosine (e.g., L-SK-4) exerts its therapeutic potency in an in vitro model of malignant melanoma. Cytotoxicity was assessed by the Alamar Blue assay, oxidative stress by commercially available kits, ROS generation, caspase 3/7 activation and mitochondrial membrane depolarisation by flow cytometry, expression of apoptosis-related proteins by western immunoblotting and profiling of lipid biosynthesis by a metabolomic approach. Overall, higher levels of ROS, sphingolipids and apoptosis were induced by L-SK-4 suggesting that the compound's therapeutic potency is mediated through elevated ROS levels which promote the upregulation of sphingolipid (ceramide) biosynthesis thus leading to the activation of both extrinsic and intrinsic apoptosis, in an experimental model of malignant melanoma.

Allyl isothiocyanate regulates lysine acetylation and methylation marks in an experimental model of malignant melanoma.

OBJECTIVE(S): Isothiocyanates (ITCs) are biologically active plant secondary metabolites capable of mediating various biological effects including modulation of the epigenome. Our aim was to characterize the effect of allyl isothiocyanate (AITC) on lysine acetylation and methylation marks as a potential epigenetic-induced anti-melanoma strategy. METHODS: Our malignant melanoma model consisted of (1) human (A375) and murine (B16-F10) malignant melanoma as well as of human; (2) brain (VMM1) and lymph node (Hs 294T) metastatic melanoma; (3) non-melanoma epidermoid carcinoma (A431) and (4) immortalized keratinocyte (HaCaT) cells subjected to AITC. Cell viability, histone deacetylases (HDACs) and acetyltransferases (HATs) activities were evaluated by the Alamar blue, Epigenase HDAC Activity/Inhibition and EpiQuik HAT Activity/Inhibition assay kits, respectively, while their expression levels together with those of lysine acetylation and methylation marks by western immunoblotting. Finally, apoptotic gene expression was assessed by an RT-PCR-based gene expression profiling methodology. RESULTS: AITC reduces cell viability, decreases HDACs and HATs activities and causes changes in protein expression levels of various HDACs, HATs, and histone methyl transferases (HMTs) all of which have a profound effect on specific lysine acetylation and methylation marks. Moreover, AITC regulates the expression of a number of genes participating in various apoptotic cascades thus indicating its involvement in apoptotic induction. CONCLUSIONS: AITC exerts a potent epigenetic effect suggesting its potential involvement as a promising epigenetic-induced bioactive for the treatment of malignant melanoma.

Anticancer activity of a novel methylated analogue of L-mimosine against an in vitro model of human malignant melanoma.

The anticancer activity of a series of novel synthesized, hydroxypyridone-based metal chelators (analogues of L-mimosine) was evaluated in an in vitro model of melanoma consisting of malignant melanoma (A375), non-melanoma epidermoid carcinoma (A431) and immortalized non-malignant keratinocyte (HaCaT) cells. More specifically, we have demonstrated that the L-enantiomer of a methylated analogue of L-mimosine (compound 22) can exert a potent anticancer effect in A375 cells when compared to either A431 or HaCaT cells. Moreover, we have demonstrated that this analogue has the ability to i) promote increased generation of reactive oxygen species (ROS), ii) activate both intrinsic and extrinsic apoptosis and iii) induce perturbations in cell cycle growth arrest. Our data highlights the potential of compound 22 to act as a promising therapeutic agent against an in vitro model of human malignant melanoma.

Iridium(iii) complexes of 1,2,4-triazines as potential bioorthogonal reagents: metal coordination facilitates luminogenic reaction with strained cyclooctynes.

In this paper we describe unprecedented Ir(iii) complexes of 5-(2-pyridyl)-1,2,4-triazine and their reactivity towards the strained cyclooctyne BCN. The coordination of a 1,2,4-triazine ring to an iridium(iii) ion drastically increases the speed of the reaction, showing the second order rate constant of 8 M-1 s-1, the record value to date for a triazine-BCN reaction.

DNA-dependent protein kinase: Epigenetic alterations and the role in genomic stability of cancer.

DNA-dependent protein kinase (DNA-PK), a member of phosphatidylinositol-kinase family, is a key protein in mammalian DNA double-strand break (DSB) repair that helps to maintain genomic integrity. DNA-PK also plays a central role in immune cell development and protects telomerase during cellular aging. Epigenetic deregulation due to endogenous and exogenous factors may affect the normal function of DNA-PK, which in turn could impair DNA repair and contribute to genomic instability. Recent studies implicate a role for epigenetics in the regulation of DNA-PK expression in normal and cancer cells, which may impact cancer progression and metastasis as well as provide opportunities for treatment and use of DNA-PK as a novel cancer biomarker. In addition, several small molecules and biological agents have been recently identified that can inhibit DNA-PK function or expression, and thus hold promise for cancer treatments. This review discusses the impact of epigenetic alterations and the expression of DNA-PK in relation to the DNA repair mechanisms with a focus on its differential levels in normal and cancer cells.

Hyperthermia Suppresses Post - In Vitro Proliferation and Tumor Growth in Murine Malignant Melanoma and Colon Carcinoma.

BACKGROUND: Several studies have highlighted hyperthermia's ability to enhance the effectiveness of radiation and chemotherapy in various in vitro and in vivo cancer models. MATERIALS AND METHODS: In vivo murine models of malignant melanoma and colon carcinoma were utilized for demonstrating hyperthermia's therapeutic effectiveness by examining levels of caspase 3, COX-2 and phospho-H2A.X (Ser139) as endpoints of apoptosis, proliferation and DNA damage respectively. RESULTS: Hyperthermia induced in vitro cytotoxicity in malignant melanoma (B16-F10) and colon carcinoma (CT26) cell lines. In addition, it reduced post-in vitro proliferation and suppression of tumor growth by inducing the expression of caspase-3 and phospho-H2A.X (Ser139) while reducing the expression of COX-2 in both murine cancer models. CONCLUSION: Hyperthermia can exert therapeutic effectiveness against melanoma and colon carcinoma by inhibiting a number of critical cellular cascades including apoptosis, proliferation and DNA damage.

Isothiocyanate-induced Cell Cycle Arrest in a Novel In Vitro Exposure Protocol of Human Malignant Melanoma (A375) Cells.

BACKGROUND/AIM: Several studies have documented the effects of isothiocyanates (ITCs) on cancer prevention by inducing oxidative stress, activating apoptosis, affecting cell-cycle regulation, etc. Previously, we have shown that ITCs, administered at low concentrations by the means of double-bolus are capable of potentiating cytotoxicity in human malignant melanoma (A375) cells by inducing apoptosis. The aim of the present study was to further investigate the effect of the treatment of A375 cells with ITCs on cell-cycle progression and the levels of various cell cycle regulators. MATERIALS AND METHODS: Cell-cycle analysis was performed by means of flow cytometry whereas western immunoblotting was used to determine the expression levels of these protein regulators. RESULTS: Our data showed an increase in the number of cells in the G2/M phase accompanied by a decrease in the G0/G1 phase, while several cell-cycle regulators were shown to be differentially expressed upon exposure to ITCs. CONCLUSION: ITCs induced cell-cycle arrest in A375 cells.

Novel Docosahexaenoic Acid Ester of Phloridzin Inhibits Proliferation and Triggers Apoptosis in an In Vitro Model of Skin Cancer.

Skin cancer is among the most common cancer types accompanied by rapidly increasing incidence rates, thus making the development of more efficient therapeutic approaches a necessity. Recent studies have revealed the potential role of decosahexaenoic acid ester of phloridzin (PZDHA) in suppressing proliferation of liver, breast, and blood cancer cell lines. In the present study, we investigated the cytotoxic potential of PZDHA in an in vitro model of skin cancer consisting of melanoma (A375), epidermoid carcinoma (A431), and non-tumorigenic (HaCaT) cell lines. Decosahexaenoic acid ester of phloridzin led to increased cytotoxicity in all cell lines as revealed by cell viability assays. However, growth inhibition and induction of both apoptosis and necrosis was more evident in melanoma (A375) and epidermoid carcinoma (A431) cells, whereas non-tumorigenic keratinocytes (HaCaT) appeared to be more resistant as detected by flow cytometry. More specifically, PZDHA-induced cell cycle growth arrest at the G2/M phase in A375 and A431 cells in contrast to HaCaT cells, which were growth arrested at the G0/G1 phase. Elevated intracellular generation of reactive oxygen species ROS was detected in all cell lines. Overall, our findings support the potential of PZDHA as a novel therapeutic means against human skin cancer.

Human aldehyde dehydrogenase 3A1 (ALDH3A1) exhibits chaperone-like function.

Aldehyde dehydrogenase 3A1 (ALDH3A1) is a metabolic enzyme that catalyzes the oxidation of various aldehydes. Certain types of epithelial tissues in mammals, especially those continually exposed to environmental stress (e.g., corneal epithelium), express ALDH3A1 at high levels and its abundance in such tissues is perceived to help to maintain cellular homeostasis under conditions of oxidative stress. Metabolic as well as non-metabolic roles for ALDH3A1 have been associated with its mediated resistance to cellular oxidative stress. In this study, we provide evidence that ALDH3A1 exhibits molecular chaperone-like activity further supporting its multifunctional role. Specifically, we expressed and purified the human ALDH3A1 in E. coli and used the recombinant protein to investigate its in vitro ability to protect SmaI and citrate synthase (from precipitation and/or deactivation) under thermal stress conditions. Our results indicate that recombinant ALDH3A1 exhibits significant chaperone function in vitro. Furthermore, over-expression of the fused histidine-tagged ALDH3A1 confers host E. coli cells with enhanced resistance to thermal shock, while ALDH3A1 over-expression in the human corneal cell line HCE-2 was sufficient for protecting them from the cytotoxic effects of both hydrogen peroxide and tert-butyl hydroperoxide. These results further support the chaperone-like function of human ALDH3A1. Taken together, ALDH3A1, in addition to its primary metabolic role in fundamental cellular detoxification processes, appears to play an essential role in protecting cellular proteins against aggregation under stress conditions.

Gold nanoparticles, radiations and the immune system: Current insights into the physical mechanisms and the biological interactions of this new alliance towards cancer therapy.

Considering both cancer's serious impact on public health and the side effects of cancer treatments, strategies towards targeted cancer therapy have lately gained considerable interest. Employment of gold nanoparticles (GNPs), in combination with ionizing and non-ionizing radiations, has been shown to improve the effect of radiation treatment significantly. GNPs, as high-Z particles, possess the ability to absorb ionizing radiation and enhance the deposited dose within the targeted tumors. Furthermore, they can convert non-ionizing radiation into heat, due to plasmon resonance, leading to hyperthermic damage to cancer cells. These observations, also supported by experimental evidence both in vitro and in vivo systems, reveal the capacity of GNPs to act as radiosensitizers for different types of radiation. In addition, they can be chemically modified to selectively target tumors, which renders them suitable for future cancer treatment therapies. Herein, a current review of the latest data on the physical properties of GNPs and their effects on GNP circulation time, biodistribution and clearance, as well as their interactions with plasma proteins and the immune system, is presented. Emphasis is also given with an in depth discussion on the underlying physical and biological mechanisms of radiosensitization. Furthermore, simulation data are provided on the use of GNPs in photothermal therapy upon non-ionizing laser irradiation treatment. Finally, the results obtained from the application of GNPs at clinical trials and pre-clinical experiments in vivo are reported.

Development of a Novel Experimental In Vitro Model of Isothiocyanate-induced Apoptosis in Human Malignant Melanoma Cells.

BACKGROUND: Isothiocyanates are constituents of cruciferous vegetables which have been associated with reduced cancer risk partially through their ability to induce apoptosis in malignant cells including melanoma. MATERIALS AND METHODS: We have utilized human malignant melanoma (A375), epidermoid carcinoma (A431) and immortalized keratinocyte (HaCaT) cells exposed to various isothiocyanates, under different experimental conditions. RESULTS: An experimental in vitro model utilizing low isothiocyanate concentrations (0.1-5 µM for 48 h with all treatments being refreshed after 24h) was shown to be (i) most efficient in exerting an anti-cancer effect when compared to higher concentrations (5-100 µM for 24 or 48 h added as a single bolus) and (ii) specific to A375 cells while A431 and HaCaT cells remained unaffected. Such effect involved the activation of several caspases including (iii) initiator caspases 8, 9, 4 (indicating the involvement of intrinsic, extrinsic and endoplasmic reticulum-based pathways) and (iv) effector caspases 3, 7 and 6. CONCLUSION: Utilization of low isothiocyanate concentrations (under the conditions described herein) exerts an anti-cancer effect specific to human malignant melanoma cells thus providing a therapeutic basis for their utilization in management of the disease.

Aldehyde dehydrogenase 3A1 promotes multi-modality resistance and alters gene expression profile in human breast adenocarcinoma MCF-7 cells.

Aldehyde dehydrogenases participate in a variety of cellular homeostatic mechanisms like metabolism, proliferation, differentiation, apoptosis, whereas recently, they have been implicated in normal and cancer cell stemness. We explored roles for ALDH3A1 in conferring resistance to chemotherapeutics/radiation/oxidative stress and whether ectopic overexpression of ALDH3A1 could lead to alterations of gene expression profile associated with cancer stem cell-like phenotype. MCF-7 cells were stably transfected either with an empty vector (mock) or human aldehyde dehydrogenase 3A1 cDNA. The expression of aldehyde dehydrogenase 3A1 in MCF-7 cells was associated with altered cell proliferation rate and enhanced cell resistance against various chemotherapeutic drugs (4-hydroxyperoxycyclophosphamide, doxorubicin, etoposide, and 5-fluorouracil). Aldehyde dehydrogenase 3A1 expression also led to increased tolerance of MCF-7 cells to gamma radiation and hydrogen peroxide-induced stress. Furthermore, aldehyde dehydrogenase 3A1-expressing MCF-7 cells exhibited gene up-regulation of cyclins A, B1, B2, and down-regulation of cyclin D1 as well as transcription factors p21, CXR4, Notch1, SOX2, SOX4, OCT4, and JAG1. When compared to mock cells, no changes were observed in mRNA levels of ABCA2 and ABCB1 protein pumps with only a minor decrease of the ABCG2 pump in the aldehyde dehydrogenase 3A1-expressing cells. Also, the adhesion molecules EpCAM and CD49F were also found to be up-regulated in aldehyde dehydrogenase 3A1expressing cells. Taken together, ALDH3A1 confers a multi-modality resistance phenotype in MCF-7 cells associated with slower growth rate, increased clonogenic capacity, and altered gene expression profile, underlining its significance in cell homeostasis.

Phytochemicals in regulating fatty acid ß-oxidation: Potential underlying mechanisms and their involvement in obesity and weight loss.

Excessive accumulation of fat as the result of more energy intake and less energy expenditure is known as obesity. Lipids are essential components in the human body and are vital for maintaining homeostasis and physiological as well as cellular metabolism. Fatty acid synthesis and catabolism (by fatty acid oxidation) are normal part of basic fuel metabolism in animals. Fatty acids are degraded in the mitochondria by a biochemical process called ß-oxidation in which two-carbon fragments are produced in each cycle. The increase in fatty acid ß-oxidation is negatively correlated with body mass index. Although healthy life style, avoiding Western diet, dieting and strenuous exercise are the commonly used methods to lose weight, they are not considered a permanent solution in addition to risk attenuation of basal metabolic rate (BMR). Pharmacotherapy offers benefits of weight loss by altering the satiety and lowering absorption of fat from the food; however, its side effects may outweigh the benefits of weight loss. Alternatively, dietary phytochemicals and natural health products offer great potential as an efficient weight loss strategy by modulating lipid metabolism and/or increasing BMR and thermogenesis. Specifically, polyphenols such as citrus flavonoids, green tea epigallocatechin gallate, resveratrol, capsaicin and curcumin, have been reported to increase lipolysis and induce fatty acid ß-oxidation through modulation of hormone sensitive lipase, acetyl-coA carboxylase, carnitine acyl transferase and peroxisome proliferator-activated receptor gamma coactivator-1. In this review article, we discuss selected phytochemicals in relation to their integrated functionalities and specific mechanisms for weight loss.

Effects of hyperthermia as a mitigation strategy in DNA damage-based cancer therapies.

Utilization of thermal therapy (hyperthermia) is defined as the application of exogenous heat induction and represents a concept that is far from new as it goes back to ancient times when heat was used for treating various diseases, including malignancies. Such therapeutic strategy has gained even more popularity (over the last few decades) since various studies have shed light into understanding hyperthermia's underlying molecular mechanism(s) of action. In general, hyperthermia is applied as complementary (adjuvant) means in therapeutic protocols combining chemotherapy and/or irradiation both of which can induce irreversible cellular DNA damage. Furthermore, according to a number of in vitro, in vivo and clinical studies, hyperthermia has been shown to enhance the beneficial effects of DNA targeting therapeutic strategies by interfering with DNA repair response cascades. Therefore, the continuously growing evidence supporting hyperthermia's beneficial role in cancer treatment can also encourage its application as a DNA repair mitigation strategy. In this review article, we aim to provide detailed information on how hyperthermia acts on DNA damage and repair pathways and thus potentially contributing to various adjuvant therapeutic protocols relevant to more efficient cancer treatment strategies.

Efficient E. coli expression strategies for production of soluble human crystallin ALDH3A1.

Aldehyde dehydrogenase 3A1 (ALDH3A1) is a recently characterized corneal crystallin with its exact functions still being unclear. Expressing recombinant human ALDH3A1 has been difficult in Escherichia coli (E. coli) because of low solubility, yield and insufficient purity issues. In this report, we compared different E. coli expression strategies (namely the maltose binding protein; MBP- and the 6-his-tagged expression systems) under conditions of auto-induction and co-expression with E. coli's molecular chaperones where appropriate. Thus, we aimed to screen the efficiency of these expression strategies in order to improve solubility of recombinant ALDH3A1 when expressed in E. coli. We showed that the MBP- tagged expression in combination with lower-temperature culture conditions resulted in active soluble recombinant ALDH3A1. Expression of the fused 6-his tagged-ALDH3A1 protein resulted in poor solubility and neither lowering temperature culture conditions nor the auto-induction strategy improved its solubility. Furthermore, higher yield of soluble, active native form of 6-his tagged-ALDH3A1 was facilitated through co-expression of the two groups of E. coli's molecular chaperones, GroES/GroEL and DnaK/DnaJ/GrpE. Convenient one step immobilized affinity chromatography methods were utilized to purify the fused ALDH3A1 hybrids. Both fusion proteins retained their biological activity and could be used directly without removing the fusion tags. Taken together, our results provide a rational option for producing sufficient amounts of soluble and active recombinant ALDH3A1 using the E. coli expression system for conducting functional studies towards elucidating the biological role(s) of this interesting corneal crystallin.