Improved Separation in Horizontal Protein SDS-PAGE with Double-Deck Flat Electrodes and a Field Inversion Gel Electrophoresis Module.

The horizontal flatbed electrophoresis method is employed to separate protein samples, providing greater flexibility for various electrophoretic applications and easier sample loading compared to its vertical counterpart. In the currently available equipment setup, cathode and anode electrodes are positioned on top of a gel at each end. Since an electric field enters the gel from the top, its strength gradually weakens from the top to the bottom of the gel. When examining the interior of gels following electrophoretic separation, the uneven electric field causes the protein bands to lie down forward in the direction of migration, leading to an increase in bandwidth. This issue has remained unaddressed for several decades. To address this problem, new clamp-shaped and double-deck electrodes were developed to apply an electric field simultaneously from both the top and bottom of the gel. Both of these new electrodes facilitated the formation of perpendicular protein band shapes and enhanced resolution at a comparable level. Due to their ease of use, double-deck electrodes are recommended. By combining these new electrodes with the field inversion gel electrophoresis (FIGE) technique, the protein bands could be focused and aligned nearly vertically, resulting in the highest level of electrophoretic resolution. Our electrodes are compatible with polyacrylamide gels of varying sizes, buffer systems, and sample well formats. They can be easily manufactured and seamlessly integrated into existing laboratory instruments for practical use.

Dyadic Associations Between Grandparent-Grandchild Familial Values and Successful Aging: Mediating Role of Quality of Life.

The current study aimed to determine the reciprocal associations between the familial values and successful aging of grandparents and grandchildren and the extent to which quality of life accounts for these reciprocal associations. Data was collected from 270 grandparent-grandchild pairs living together. Actor-Partner Independence Model indicated that familial values of both grandparents and grandchildren showed significant associations with their own successful aging (actor effects), whereas familial values of grandchildren showed significant associations with the successful aging of grandparents (partner effects). Grandchildren's quality of life mediated the actor and partner effects of familial values on the successful aging of grandchildren; whereas grandparents' quality of life only mediated the actor effect of familial values on the successful aging of grandparents. These findings showed the interdependence of grandparents and grandchildren while emphasizing the importance of grandparents-grandchildren familial values and quality of life in enhancing successful aging.

Janus Faced HMGB1 and Post-Aneurysmal Subarachnoid Hemorrhage (aSAH) Inflammation.

Aneurysmal subarachnoid hemorrhage (aSAH), resulting majorly from the rupture of intracranial aneurysms, is a potentially devastating disease with high morbidity and mortality. The bleeding aneurysms can be successfully secured; however, the toxic and mechanical impact of the blood extravasation into the subarachnoid space damages the brain cells leading to the release of different damage-associated molecular pattern molecules (DAMPs). DAMPs upregulate the inflammation after binding their cognate receptors on the immune cells and underlies the early and delayed brain injury after aSAH. Moreover, these molecules are also associated with different post-aSAH complications, which lead to poor clinical outcomes. Among these DAMPs, HMGB1 represents a prototypical protein DAMP that has been well characterized for its proinflammatory role after aSAH and during different post-aSAH complications. However, recent investigations have uncovered yet another face of HMGB1, which is involved in the promotion of brain tissue remodeling, neurovascular repair, and anti-inflammatory effects after SAH. These different faces rely on different redox states of HMGB1 over the course of time after SAH. Elucidation of the dynamics of these redox states of HMGB1 has high biomarker as well as therapeutic potential. This review mainly highlights these recent findings along with the conventionally described normal role of HMGB1 as a nuclear protein and as a proinflammatory molecule during disease (aSAH).

Spreader-CDR system for efficient, reproducible, and frugal western blot.

Efficient antibody incubation is a vital step for successful western blot. During the incubation, a thin antibody-depleted layer is created around the blotting membrane, which limits antibody binding. Although the conventional batch shaking method is ineffective against it, this layer can be easily disrupted by cyclic draining and replenishing (CDR) of the antibody solution during membrane incubation. Previously, we introduced a closed and rotating cylindrical chamber as a tool to implement CDR for western blots (rCDR). A new open bucket-style chamber was devised for easier operation and the possibility of process automation. Instead of rotation as in rCDR, rocking it back and forth achieved the CDR antibody incubation (R-CDR). The chamber was then equipped with a spreader-rod to facilitate the uniform movement of the antibody solution across the membrane surface. Hence, it was named spreader CDR (S-CDR). Compared to the batch incubation method, both the S-CDR and R-CDR devices produced significantly enhanced signals and developed faster results. There were several additional benefits of using the spreader-rod, which included uniform antibody binding across the membrane, reduced usage of antibodies, and the ability to recover results even from mishandled, creased membranes. The S-CDR device ensures better blots and can be easily implemented in existing western blot protocols.

Differential proteomic analyses of green microalga Ettlia sp. at various dehydration levels.

Water deprivation could be a lethal stress for aquatic and aero-terrestrial organisms. Ettlia sp. is a unicellular photosynthetic freshwater microalga. In the present study, proteomic alterations and physiological characteristics of Ettlia sp. were analyzed to comprehend the molecular changes in dehydrated conditions. Varying levels of dehydration were achieved by incubating drained Ettlia sp. in different relative humidity environments for 24  hours. Using a comparative proteomic analysis, 52 differentially expressed protein spots were identified that could be divided into eight functional groups. The PCA analysis of normalized protein expression values demonstrated a clear segregation of protein expression profiles among different dehydration levels. Identified proteins could be grouped into four clusters based on their expression profiles. Proteins relating to photosynthesis comprised the largest group with 25% of the identified proteins that were decreased in dehydrated samples and belonged to cluster I. The photosynthetic activities were measured with rehydrated Ettlia sp. These results revealed that photosynthesis remained inhibited over extended time in response to dehydration. The expressions of reactive oxygen species (ROS) scavenger proteins increased in strong dehydration and were assigned to cluster III. Carbon metabolism proteins were suppressed, which might limit energy consumption, whereas glycolysis was activated at mild dehydration. The accumulation of desiccation-associated late embryogenesis proteins might inhibit the aggregation of housekeeping proteins. DNA protective proteins were expressed higher in the dehydrated state, which might reduce DNA damage, and membrane-stabilizing proteins increased in abundance in desiccation. These findings provide an understanding of Ettlia's adaptation and survival capabilities in a dehydrated state.

Rapid and efficient western blot assay by rotational cyclic draining and replenishing procedure.

Western blot is a principal technique for the detection of specific proteins in various biology disciplines. The antibody incubation is an imperative step in western blot. Antibody incubation by mild shaking on a rocker is generally used to facilitate mixing of antibodies. However, mild shaking is an inefficient process to remove antibody depletion layer on blotting membrane and requires hours of incubation time to achieve antibody binding to target proteins. We propose an alternative method of cyclic draining and replenishing (CDR) incubation of antibody solution using a rotational incubation chamber. The study demonstrated that rotational CDR incubation could shorten antibody incubation time with enhanced sensitivity. Moreover, rotational CDR incubation could achieve a stronger antibody binding with lower antibody concentration when compared with batch incubation. In addition, rotational CDR incubation significantly improved the detection of low abundance proteins. This simple modification in western blot procedure makes it rapid, sensitive, and cost-efficient.

Frequency of Dyslipidemia in patients with Lupus Nephritis.

OBJECTIVE: To determine the frequency of dyslipidemia in patients with lupus nephritis and its association with the degree of proteinuria. METHODS: This cross-sectional analytic study included 65 patients who fulfilled the ACR (American College of Rheumatology) criteria for SLE and had renal involvement, presenting to the Division of Rheumatology, Fatima Memorial Hospital (FMH), and Lahore from 21st Sep 2016 to 20th Dec 2016. After 12 hours overnight fast their blood samples were assessed for total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL). Patient demographic variables (age, sex) and disease characteristics (disease duration, degree of proteinuria, steroid dose) were noted. Patients were categorized into two groups on the basis of degree of proteinuria: having proteinuria >1gm or ≤ 1gm. Data was analyzed using SPSS version 22. Individual lipid profiles were correlated with the degree of proteinuria. RESULTS: Most common lipid abnormality found in our study was hypertriglyceridemia (58.5%). Total Cholesterol and LDL-C was high in 55.4% and 30.8% subjects respectively. Low HDL was found in 21.5% subjects. Increased frequency of dyslipidemia was noticed in those subjects who had proteinuria >1gm (P value < 0.05). CONCLUSION: Dyslipidemia was observed in a high frequency in patients with lupus nephritis and was strongly associated with their degree of proteinuria.