An AFM Approach Applied in a Study of α-Crystallin Membrane Association: New Insights into Lens Hardening and Presbyopia Development

The lens of the eye loses elasticity with age, while α-crystallin association with the lens membrane increases with age. It is unclear whether there is any correlation between α-crystallin association with the lens membrane and loss in lens elasticity. This research investigated α-crystallin membrane association using atomic force microscopy (AFM) for the first time to study topographical images and mechanical properties (breakthrough force and membrane area compressibility modulus (KA), as measures of elasticity) of the membrane. α-Crystallin extracted from the bovine lens cortex was incubated with a supported lipid membrane (SLM) prepared on a flat mica surface. The AFM images showed the time-dependent interaction of α-crystallin with the SLM. Force spectroscopy revealed the presence of breakthrough events in the force curves obtained in the membrane regions where no α-crystallin was associated, which suggests that the membrane’s elasticity was maintained. The force curves in the α-crystallin submerged region and the close vicinity of the α-crystallin associated region in the membrane showed no breakthrough event within the defined peak force threshold, indicating loss of membrane elasticity. Our results showed that the association of α-crystallin with the membrane deteriorates membrane elasticity, providing new insights into understanding the molecular basis of lens hardening and presbyopia

Interaction of β\u3csub\u3eL\u3c/sub\u3e- and γ-Crystallin with Phospholipid Membrane Using Atomic Force Microscopy

Highly concentrated lens proteins, mostly β- and γ-crystallin, are responsible for maintaining the structure and refractivity of the eye lens. However, with aging and cataract formation, β- and γ-crystallin are associated with the lens membrane or other lens proteins forming high-molecular-weight proteins, which further associate with the lens membrane, leading to light scattering and cataract development. The mechanism by which β- and γ-crystallin are associated with the lens membrane is unknown. This work aims to study the interaction of β- and γ-crystallin with the phospholipid membrane with and without cholesterol (Chol) with the overall goal of understanding the role of phospholipid and Chol in β- and γ-crystallin association with the membrane. Small unilamellar vesicles made of Chol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Chol/POPC) membranes with varying Chol content were prepared using the rapid solvent exchange method followed by probe tip sonication and then dispensed on freshly cleaved mica disk to prepare a supported lipid membrane. The βL- and γ-crystallin from the cortex of the bovine lens was used to investigate the time-dependent association of βL- and γ-crystallin with the membrane by obtaining the topographical images using atomic force microscopy. Our study showed that βL-crystallin formed semi-transmembrane defects, whereas γ-crystallin formed transmembrane defects on the phospholipid membrane. The size of semi-transmembrane defects increases significantly with incubation time when βL-crystallin interacts with the membrane. In contrast, no significant increase in transmembrane defect size was observed in the case of γ-crystallin. Our result shows that Chol inhibits the formation of membrane defects when βL- and γ-crystallin interact with the Chol/POPC membrane, where the degree of inhibition depends upon the amount of Chol content in the membrane. At a Chol/POPC mixing ratio of 0.3, membrane defects were observed when both βL- and γ-crystallin interacted with the membrane. However, at a Chol/POPC mixing ratio of 1, no association of γ-crystallin with the membrane was observed, which resulted in a defect-free membrane, and the severity of the membrane defect was decreased when βL-crystallin interacted with the membrane. The semi-transmembrane or transmembrane defects formed by the interaction of βL- and γ-crystallin on phospholipid membrane might be responsible for light scattering and cataract formation. However, Chol suppressed the formation of such defects in the membrane, likely maintaining lens membrane homeostasis and protecting against cataract formation

Interaction of Alpha-Crystallin with Four Major Phospholipids of Eye Lens Membranes

It is well-studied that the significant factor in cataract formation is the association of α-crystallin, a major eye lens protein, with the fiber cell plasma membrane of the eye lens. The fiber cell plasma membrane of the eye lens consists of four major phospholipids (PLs), i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). Despite several attempts to study the interaction of α-crystallin with PLs of the eye lens membrane, the role of individual PL for the binding with α-crystallin is still unclear. We recently developed the electron paramagnetic resonance (EPR) spin-labeling method to study the binding of α-crystallin to the PC membrane (Mainali et al., 2020a). Here, we use the recently developed EPR method to explicitly measure the binding affinity (Ka) of α-crystallin to the individual (PE*, PS, and SM) and two-component mixtures (SM/PE, SM/PS, and SM/PC in 70:30 and 50:50 mol%) of PL membranes as well as the physical properties (mobility parameter and maximum splitting) of these membranes upon binding with α-crystallin. One of the key findings of this study was that the Ka of α-crystallin binding to individual PL membranes followed the trends: Ka(PC) \u3e Ka(SM) \u3e Ka(PS) \u3e Ka(PE*), indicating PE* inhibits binding the most whereas PC inhibits binding the least. Also, the Ka of α-crystallin binding to two-component mixtures of PL membranes followed the trends: Ka(SM/PE) \u3e Ka(SM/PS) \u3e Ka(SM/PC), indicating SM/PC inhibits binding the most whereas SM/PE inhibits binding the least. Except for the PE* membrane, for which there was no binding of α-crystallin, the mobility parameter for all other membranes decreased with an increase in α-crystallin concentration. It represents that the membranes become more immobilized near the headgroup regions of the PLs when more and more α-crystallin binds to them. The maximum splitting increased only for the SM and the SM/PE (70:30 mol%) membranes, with an increase in the binding of α-crystallin. It represents that the PL headgroup regions of these membranes become more ordered after binding of α-crystallin to these membranes. Our results showed that α-crystallin binds to PL membranes in a saturable manner. Also, our data suggest that the binding of α-crystallin to PL membranes likely occurs through hydrophobic interaction between α-crystallin and the hydrophobic fatty acid core of the membranes, and such interaction is modulated by the PL headgroup’s size and charge, hydrogen bonding between headgroups, and PL curvature. Thus, this study provides an in-depth understanding of α-crystallin interaction with the PL membranes made of individual and two-component mixtures of the four major PLs of the eye lens membranes

Association of Alpha-Crystallin with Human Cortical and Nuclear Lens Lipid Membrane Increases with the Grade of Cortical and Nuclear Cataract

Eye lens α-crystallin has been shown to become increasingly membrane-bound with age and cataract formation; however, to our knowledge, no studies have investigated the membrane interactions of α-crystallin throughout the development of cataracts in separated cortical membrane (CM) and nuclear membrane (NM) from single human lenses. In this study, four pairs of human lenses from age-matched male and female donors and one pair of male lenses ranging in age from 64 to 73 years old (yo) were obtained to investigate the interactions of α-crystallin with the NM and CM throughout the progression of cortical cataract (CC) and nuclear cataract (NC) using the electron paramagnetic resonance spin-labeling method. Donor health history information (diabetes, smoker, hypertension, radiation treatment), sex, and race were included in the data analysis. The right eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 1, NC: 2), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Similarly, left eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 2, NC: 3), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Analysis of α-crystallin binding to male and female eye lens CM and NM revealed that the percentage of membrane surface occupied (MSO) by α-crystallin increases with increasing grade of CC and NC. The binding of α-crystallin resulted in decreased mobility, increased order, and increased hydrophobicity on the membrane surface in male and female eye lens CM and NM. CM mobility decreased with an increase in cataracts for both males and females, whereas the male lens NM mobility showed no significant change, while female lens NM showed increased mobility with an increase in cataract grade. Our data shows that a 68 yo female donor (long-term smoker, pre-diabetic, and hypertension; grade 3 CC) showed the largest MSO by α-crystallin in CM from both the left and right lens and had the most pronounced mobility changes relative to all other analyzed samples. The variation in cholesterol (Chol) content, size and amount of cholesterol bilayer domains (CBDs), and lipid composition in the CM and NM with age and cataract might result in a variation of membrane surface mobility, membrane surface hydrophobicity, and the interactions of α-crystallin at the surface of each CM and NM. These findings provide insight into the effect of decreased Chol content and the reduced size and amount of CBDs in the cataractous CM and NM with an increased binding of α-crystallin with increased CC and NC grade, which suggests that Chol and CBDs might be a key component in maintaining lens transparency

Binding of β\u3csub\u3eL\u3c/sub\u3e-Crystallin with Models of Animal and Human Eye Lens-Lipid Membrane

Several discoveries show that with age and cataract formation, β-crystallin binds with the lens membrane or associates with other lens proteins, which bind with the fiber cell plasma membrane, accompanied by light scattering and cataract formation. However, how lipids (phospholipids and sphingolipids) and cholesterol (Chol) influence β-crystallin binding to the membrane is unclear. This research aims to elucidate the role of lipids and Chol in the binding of β-crystallin to the membrane and the membrane’s physical properties (mobility, order, and hydrophobicity) with β-crystallin binding. We used electron paramagnetic resonance (EPR) spin-labeling methods to investigate the binding of βL-crystallin with a model of porcine lens-lipid (MPLL), model of mouse lens-lipid (MMLL), and model of human lens-lipid (MHLL) membrane with and without Chol. Our results show that βL-crystallin binds with all of the investigated membranes in a saturation manner, and the maximum parentage of the membrane surface occupied (MMSO) by βL-crystallin and the binding affinity (Ka) of βL-crystallin to the membranes followed trends: MMSO (MPLL) \u3e MMSO (MMLL) \u3e MMSO (MHLL) and Ka (MHLL) \u3e Ka (MMLL) ≈ Ka (MPLL), respectively, in which the presence of Chol reduces the MMSO and Ka for all membranes. The mobility near the headgroup regions of the membranes decreases with an increase in the binding of βL-crystallin; however, the decrease is more pronounced in the MPLL and MMLL membranes than the MHLL membrane. In the MPLL and MMLL membranes, the membranes become slightly ordered near the headgroup with an increase in βL-crystallin binding compared to the MHLL membrane. The hydrophobicity near the headgroup region of the membrane increases with βL-crystallin binding; however, the increase is more pronounced in the MPLL and MMLL membranes than the MHLL membrane, indicating that βL-crystallin binding creates a hydrophobic barrier for the passage of polar molecules, which supports the barrier hypothesis in cataract formation. However, in the presence of Chol, there is no significant increase in hydrophobicity with βL-crystallin binding, suggesting that Chol prevents the formation of a hydrophobic barrier, possibly protecting against cataract formation

Cholesterol and Cholesterol Bilayer Domains Inhibit Binding of Alpha-Crystallin to the Membranes Made of the Major Phospholipids of Eye Lens Fiber Cell Plasma Membranes

The concentration of α-crystallin decreases in the eye lens cytoplasm, with a corresponding increase in membrane-bound α-crystallin during cataract formation. The eye lens’s fiber cell plasma membrane consists of extremely high cholesterol (Chol) content, forming cholesterol bilayer domains (CBDs) within the membrane. The role of high Chol content in the lens membrane is unclear. Here, we applied the continuous-wave electron paramagnetic resonance spin-labeling method to probe the role of Chol and CBDs on α-crystallin binding to membranes made of four major phospholipids (PLs) of the eye lens, i.e., phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Small unilamellar vesicles (SUVs) of PC, SM*, and PS with 0, 23, 33, 50, and 60 mol% Chol and PE* with 0, 9, and 33 mol% Chol were prepared using the rapid solvent exchange method followed by probe-tip sonication. The 1 mol% CSL spin-labels used during SUVs preparation distribute uniformly within the Chol/PL membrane, enabling the investigation of Chol and CBDs’ role on α-crystallin binding to the membrane. For PC, SM*, and PS membranes, the binding affinity (Ka) and the maximum percentage of membrane surface occupied (MMSO) by α-crystallin decreased with an increase in Chol concentration. The Ka and MMSO became zero at 50 mol% Chol for PC and 60 mol% Chol for SM* membranes, representing that complete inhibition of α-crystallin binding was possible before the formation of CBDs within the PC membrane but only after the formation of CBDs within the SM* membrane. The Ka and MMSO did not reach zero even at 60 mol% Chol in the PS membrane, representing CBDs at this Chol concentration were not sufficient for complete inhibition of α-crystallin binding to the PS membrane. Both the Ka and MMSO were zero at 0, 9, and 33 mol% Chol in the PE* membrane, representing no binding of α-crystallin to the PE* membrane with and without Chol. The mobility parameter profiles decreased with an increase in α-crystallin binding to the membranes; however, the decrease was more pronounced for the membrane with lower Chol concentration. These results imply that the membranes become more immobilized near the headgroup regions with an increase in α-crystallin binding; however, the Chol antagonizes the capacity of α-crystallin to decrease the mobility near the headgroup regions of the membranes. The maximum splitting profiles remained the same with an increase in α-crystallin concentration, but there was an increase in the maximum splitting with an increase in the Chol concentration in the membranes. It implies that membrane order near the headgroup regions does not change with an increase in α-crystallin concentration but increases with an increase in Chol concentration in the membrane. Based on our data, we hypothesize that the Chol and CBDs decrease hydrophobicity (increase polarity) near the membrane surface, inhibiting the hydrophobic binding of α-crystallin to the membranes. Thus, our data suggest that Chol and CBDs play a positive physiological role by preventing α-crystallin binding to lens membranes and possibly protecting against cataract formation and progression

Agrobiodiversity and Its Conservation in Nepal

Nepal is a part of the world\u27s biodiversity hotspot and ranks the 49th in the world for biodiversity. Agrobiodiversity and its conservation status were studied through literature review, field survey, key informant survey and focus group discussion. Results of field implementation of some good practices and action research were also documented. Among 24,300 total species in the country, 28% are agricultural genetic resources (AGRs), termed as agrobiodiversity. Agrobiodiversity has six components (crops, forages, livestock, aquatic, insects and microorganisms) and four sub-components (domesticated, semi-domesticated, wild relatives and wild edible) in Nepal. Agrobiodiversity on each component exists at agroecosystem, species, variety/breed/biotype/race/strain, genotype and allele levels, within an altitude range from 60 to 5,000 masl. There are 12 agroecosystems supporting 1026 species under crop component, 510 under forage, 35 under livestock, 250 under the aquatic animal, 17 under aquatic plant, 3,500 under insect and 800 under microorganism. An estimated loss of agrobiodiversity is 40%, however, farmers have reported up to 100% loss of AGRs in some areas for a particular species. Conservation of agrobiodiversity has been initiated since 1986. Four strategies namely ex-situ, on-farm, in-situ and breeding have been adopted for conservation and sustainable utilization of AGRs. Eighty good practices including process, methods and actions for managing agrobiodiversity have been in practice and these practices come under five conservation components (sensitization, method and approach, accelerator, value and enabling environment). Within the country, 18,765 accessions of AGRs have been conserved in different kinds of banks. A total of 24,683 accessions of Nepalese crops, forages and microbes have been conserved in different International and foreign genebanks. Some collections are conserved as safety duplication and safety backup in different CGIARs\u27 banks and World Seed Vault, Korea. Two global databases (GENESYS and EURISCO) have maintained 19,200 Nepalese accessions. Geographical Information System, Climate Analog Tool and biotechnological tools have been applied for better managing AGRs. Many stakeholders need to further concentrate on the conservation and utilization of AGRs. Global marketing of some native AGRs is necessary for sustaining agriculture and attracting young generations as well as conserving them through use

Vision and perception of community on the use of recycled water for household laundry: A case study in Australia

This study investigates the community perception of household laundry as a new end use of recycled water in three different locations of Australia through a face to face questionnaire survey (n=478). The study areas were selected based on three categories of (1) non-user, (2) perspective user and (3) current user of recycled water. The survey results indicate that significantly higher number (70%) of the respondents supported the use of recycled water for washing machines (χ2=527.40, df=3; p=0.000). Significant positive correlation between the overall support for the new end use and the willingness of the respondents to use recycled water for washing machine was observed among all users groups (r=0.43, p=0.000). However, they had major concerns regarding the effects of recycled water on the aesthetic appearance of cloth, cloth durability, machine durability, odour of the recycled water and cost along with the health issues. The perspective user group had comparatively more reservations and concerns about the effects of recycled water on washing machines than the non-users and the current users (χ2=52.73, df=6; p=0.000). Overall, community from all three study areas are willing to welcome this new end use as long as all their major concerns are addressed and safety is assured. © 2013 Elsevier B.V

Minimally Invasive Plate Osteosynthesis In The Treatment Of Isolated Ulnar Bone Fractures

BACKGROUND: Minimally invasive internal fixation is also called “biological internal fixation”. This concept is used widely in the treatment of various fractures but to date, there have been no report about such application in the repair of isolated ulnar bone fractures. METHODS: Eleven patients with fracture of isolated ulnar bone (four AO type 22A11 and seven AO type 22A12), mean age 43.16 (range, 24- 59y), were treated using closed reduction and locking compression plate with minimally invasive plate osteosynthesis (MIPO) with the aim of minimising soft tissue damage. RESULTS: Fractures healed at an average of 7.8 weeks with good to excellent clinical outcomes. There were no complications such as nonunion, implant failure or neurovascular injuries. CONCLUSIONS: MIPO seems to be advantageous for soft tissue and bone biology. Good union was seen and fracture complications were also prevented by early mobilisation