Label-Free Techniques for Probing Biomolecular Condensates.

Biomolecular condensates play important roles in a wide array of fundamental biological processes, such as cellular compartmentalization, cellular regulation, and other biochemical reactions. Since their discovery and first observations, an extensive and expansive library of tools has been developed to investigate various aspects and properties, encompassing structural and compositional information, material properties, and their evolution throughout the life cycle from formation to eventual dissolution. This Review presents an overview of the expanded set of tools and methods that researchers use to probe the properties of biomolecular condensates across diverse scales of length, concentration, stiffness, and time. In particular, we review recent years' exciting development of label-free techniques and methodologies. We broadly organize the set of tools into 3 categories: (1) imaging-based techniques, such as transmitted-light microscopy (TLM) and Brillouin microscopy (BM), (2) force spectroscopy techniques, such as atomic force microscopy (AFM) and the optical tweezer (OT), and (3) microfluidic platforms and emerging technologies. We point out the tools' key opportunities, challenges, and future perspectives and analyze their correlative potential as well as compatibility with other techniques. Additionally, we review emerging techniques, namely, differential dynamic microscopy (DDM) and interferometric scattering microscopy (iSCAT), that have huge potential for future applications in studying biomolecular condensates. Finally, we highlight how some of these techniques can be translated for diagnostics and therapy purposes. We hope this Review serves as a useful guide for new researchers in this field and aids in advancing the development of new biophysical tools to study biomolecular condensates.

WITHDRAWN: Trace metals translocation from soil to plants: Health risk assessment via consumption of vegetables in the urban sprawl of a developing country.

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Effects of microplastic and heavy metals on coral reefs: A new window for analytical research.

In the modern world, plastic trash has been recognized as a global issue, and studies on microplastics (MPs) in the marine and inland environments have previously been conducted. Marine ecosystems act as a bio-diverse ecosystem where coral reefs contribute to make a sound living of the coastal people by gathering natural resources. The current study indicates that MPs and heavy metals (HMs) accumulation to biofilm and organic matter through sedimentation, precipitation, adsorption, and desorption that may have potential effect on growth and development of coral reefs in the marine ecosystems. However, the knowledge of distribution, impact, mechanism, degradation, and association mechanisms between MPs and HMs in the natural environment may open a new window for conducting analytical research from an ecological viewpoint. The current study thus summarizes the types of marine samples with the analytical techniques, polymers of MPs, and their impact on corals and other marine biota. This study also identifies existing knowledge gaps and recommends fresh lines of inquiry in light of recent developments in MPs and HMs research on the marine ecosystems. Overall, the present study suggests a sustainable intervention for reducing MPs and HMs from the marine ecosystems by demonstrating their existence in water, sediment, fish, corals, and other biota, and their impending ecotoxicological impacts on the environment and human health. The impacts of MPs and HMs on coral reefs are critically assessed in this study in light of the most recent scientific knowledge, existing laws, and new suggestions to minimize their contamination in the marine ecosystems.

Contamination and ecological risk assessment of Cr, As, Cd and Pb in water and sediment of the southeastern Bay of Bengal coast in a developing country.

Safe levels of heavy metals in the surface water and sediment of the eastern Bay of Bengal coast have not been universally established. Current study characterized heavy metals such as arsenic (As), chromium (Cr), cadmium (Cd) and lead (Pb) in surface water and sediments of the most important fishing resource at the eastern Bay of Bengal coast, Bangladesh. Both water and sediment samples were analyzed using inductively coupled plasma mass spectrometer. Considering both of the seasons, the mean concentrations of Cr, As, Cd, and Pb in water samples were 33.25, 8.14, 0.48, and 21.14 µg/L, respectively and in sediment were 30.47, 4.48, 0.20, and 19.98 mg/kg, respectively. Heavy metals concentration in water samples surpassed the acceptable limits of usable water quality, indicating that water from this water resource is not safe for drinking, cooking, bathing, and any other uses. Enrichment factors also directed minor enrichment of heavy metals in sediment of the coast. Other indexes for ecological risk assessment such as pollution load index (PLI), contamination factor (CF), geoaccumulation index (Igeo), modified contamination degree (mCd), and potential ecological risk index (PERI) also indicated that sediment of the coastal watershed was low contamination. In-depth inventorying of heavy metals in both water and sediment of the study area are required to determine ecosystem health for holistic risk assessment and management.

Label-free identification of protein aggregates using deep learning.

Protein misfolding and aggregation play central roles in the pathogenesis of various neurodegenerative diseases (NDDs), including Huntington's disease, which is caused by a genetic mutation in exon 1 of the Huntingtin protein (Httex1). The fluorescent labels commonly used to visualize and monitor the dynamics of protein expression have been shown to alter the biophysical properties of proteins and the final ultrastructure, composition, and toxic properties of the formed aggregates. To overcome this limitation, we present a method for label-free identification of NDD-associated aggregates (LINA). Our approach utilizes deep learning to detect unlabeled and unaltered Httex1 aggregates in living cells from transmitted-light images, without the need for fluorescent labeling. Our models are robust across imaging conditions and on aggregates formed by different constructs of Httex1. LINA enables the dynamic identification of label-free aggregates and measurement of their dry mass and area changes during their growth process, offering high speed, specificity, and simplicity to analyze protein aggregation dynamics and obtain high-fidelity information.

Characterization of flat-fielding systems for quantitative microscopy.

Optical flat-fielding systems, such as field-mapping or integration-based beam shapers, are used to transform nonuniform illumination into uniform illumination. Thus, flat-fielding paves the way for imaging that is independent of position within a field of view and enables more quantitative analysis. Here, we characterize and compare three systems for homogenizing both widefield and multifocal illumination. Our analysis includes two refractive field-mapping beam shapers: PiShaper and TopShape, as well as one integration-based: Köhler integrator. The comparison is based on figures of merit including ISO-standard values, such as the plateau uniformity and edge steepness, transmission efficiency, stability of the beams along propagation and multifocal intensity, pitch, and point width. By characterizing and comparing existing beam shapers, we facilitate the choice of the appropriate flat-fielding solution and increase their accessibility for different applications.

Homogeneous multifocal excitation for high-throughput super-resolution imaging.

Super-resolution microscopies have become an established tool in biological research. However, imaging throughput remains a main bottleneck in acquiring large datasets required for quantitative biology. Here we describe multifocal flat illumination for field-independent imaging (mfFIFI). By integrating mfFIFI into an instant structured illumination microscope (iSIM), we extend the field of view (FOV) to >100 × 100 µm2 while maintaining high-speed, multicolor, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching adjacent images for fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM with ultrastructure expansion microscopy to collect three-dimensional (3D) images of hundreds of centrioles in human cells, or thousands of purified Chlamydomonas reinhardtii centrioles, per hour at an effective resolution of ~35 nm. Classification and particle averaging of these large datasets enables 3D mapping of posttranslational modifications of centriolar microtubules, revealing differences in their coverage and positioning.

Formulation, Evaluation and release rate characteristics of medicated jelly of vitamin C.

Medicated jelly formulations are patient friendly dosage form for pediatric, geriatric and dysphagic patients. These formulations offer rapid dissolution and absorption of drugs through oral mucosa therefore show the early onset of action. The objective of the study was to develop and evaluate oral jelly formulations of vitamin C. Slurry method was adopted using glucose 103gm, sugar 67gm, gelatin 10gm and sorbitol 6.56gm. Preformulation studies were performed including the organoleptic profile, pH, and solubility of both drugs. The medicated jelly of Vitamin C was prepared and evaluated for physical characteristics, weight variation, syneresis, pH, taste and palatability, drug content, release rate characteristics and stability studies. All the jellies were found to have patient welcoming taste and were palatable. All formulations showed more than 50% drug release within 15 minutes, while 93% drug was released in 30 minutes. The results of release kinetics showed that the formulation followed the zero order release kinetics. Thus the drug was released at constant rate independent of the drug concentration involved in the process. All the medicated jellies were found to remain stable stored for 60 days at different temperatures. The present study revealed that medicated jellies of vitamin C could be employed orally in an effective form as an alternative solid oral dosage form for special population such as pediatrics, geriatrics and patients with dysphagia.

Effect of Urea and Borate Plasticizers on Rheological Response of Corn Starch.

Although starch based materials have an array of fascinating industrial applications, the native starches do not show good mechanical strength, thermal stability, and rheological properties for their use in the mainstream processing industry. For example, the use of starches for producing controlled release fertilizers is a new research endeavor with detailed knowledge still to come. The thermal processing of native starches with water as a plasticizer results in poor physical and pasting properties of the final product. Therefore in this study, corn starch was thermally processed with urea and borate in a water medium. The pure starch (PS), starch-urea (SU), starch-borate (SB), and starch-urea-borate (SUB) samples were prepared and characterized for their rheological traits. The PS sample exhibited a peak viscosity of 299 cP after 17 min of thermal processing. Further heating of the suspension caused a decrease in viscosity of 38 points due to thermal cracking of the starch granules. A similar trend was depicted in the viscosity measurements of SU, SB, and SUB adhesives. However, the viscosity of these samples remained slightly higher than that for PS. Also, the reduction in viscosity after the peak value was not as notable as for PS. The modified starch behaved like a gel and its storage modulus was significantly higher than the loss modulus. The lower magnitudes of storage and loss moduli revealed that the modified starch was in the form of a weak gel and not a solid. The PS is more fluid in nature with dominating loss modulus at lower angular frequencies.

Preparation of Chito-Oligomers by Hydrolysis of Chitosan in the Presence of Zeolite as Adsorbent.

An increasing interest has recently been shown to use chitin/chitosan oligomers (chito-oligomers) in medicine and food fields because they are not only water-soluble, nontoxic, and biocompatible materials, but they also exhibit numerous biological properties, including antibacterial, antifungal, and antitumor activities, as well as immuno-enhancing effects on animals. Conventional depolymerization methods of chitosan to chito-oligomers are either chemical by acid-hydrolysis under harsh conditions or by enzymatic degradation. In this work, hydrolysis of chitosan to chito-oligomers has been achieved by applying adsorption-separation technique using diluted HCl in the presence of different types of zeolite as adsorbents. The chito-oligomers were retrieved from adsorbents and characterized by differential scanning calorimetry (DSC), liquid chromatography/mass spectroscopy (LC/MS), and ninhydrin test.

Laparoscopic cholecystectomy in the pediatric population: a single-center experience.

OBJECTIVE: We aimed to review our experience with laparoscopic cholecystectomy in the pediatric population to better understand the associated complications and outcomes. METHODS: We performed a retrospective chart review of children below 18 years of age who underwent laparoscopic cholecystectomy at a single academic institution between the years 1990 and 2010. RESULTS: Of the 325 cases of cholecystectomy, 202 (62.2%) were performed laparoscopically. The primary indication for surgery was symptomatic cholelithiasis (45.5%, n=92). Preoperative endoscopic retrograde cholangiopancreatography (ERCP) was performed in 25 (12.4%) patients. Variations in anatomy and technical difficulties (eg, presence of adhesions) were observed in 45 (22.3%) patients. Intraoperative cholangiogram was performed in 20 (9.9%) patients and 16 (7.9%) underwent concomitant splenectomy. Only 8 (4%) of the cases were converted to an open approach because of lack of anatomic clarity. There were zero common bile duct injuries; however, spillage of bile was present in 12 (5.9%) patients. Postoperative complications including wound infection, retained stones, abdominal abscess, and biloma, were observed in 9 patients (4.5%). The median operative time was 117.5 minutes. The median postoperative hospital stay was 1 day. Nineteen (9.4%) patients had recurrence of abdominal pain without associated pathology. Three patients (1.5%) required postoperative ERCP. The average follow-up period was 54 months. CONCLUSIONS: Laparoscopic cholecystectomy in the pediatric population results in short postoperative hospital stay and has low complication rates. In particular, zero bile duct injuries were noted.

Nodular fasciitis in the pediatric population: a single center experience.

PURPOSE: To describe our experience with nodular fasciitis in the pediatric population, in an attempt to contribute additional information that may be used to develop a more comprehensive characterization of the disease. METHODS: A twenty-year retrospective chart review of patients 18 years or younger diagnosed with nodular fasciitis at a single, large, academic institution. RESULTS: Five patients were treated for nodular fasciitis during the study period. The majority (60%) were male and all presented with rapidly enlarging masses that were excised surgically. The median age at presentation was 14 years. Most (60%) lesions were located in the upper extremities and 80% had no related symptomatology or disability. All lesions were less than 3 cm in greatest dimension and 4 patients had no signs of recurrence (one patient was lost to follow-up). Pathologic, immunohistochemical, and long-term follow-up data were lacking in our cohort. CONCLUSION: The characteristics of nodular fasciitis in children at our institution are slightly different than those which have been reported thus far based on no patients with head and neck disease or antecedent trauma to the affected area. Due to variability in clinical presentation and histologic findings, additional studies are necessary to provide more practical, consistent and effective management strategies.

Date palm (Phoenix dactylifera L.) leaves as biomonitors of atmospheric metal pollution in arid and semi-arid environments.

The leaves of date palms were evaluated as a possible biomonitor of heavy metal contamination in Ma'an city, Jordan. Concentrations of (Fe), (Pb), (Zn), (Cu), (Ni), and (Cr) were determined in washed and unwashed leaves and soil samples collected from different sites with different degrees of metal contamination (urban, suburban, industrial, highway and rural sites); separate leaves were taken from outside the city to be used as a control sample. Samples collected from industrial sites were found to have high concentrations of all metals except those of Cu, Ni and Pb, which were found at high levels in the highway site samples which is associated with the road traffic. The difference between unwashed and washed samples showed that metal pollutants exist as contaminants, particularly Pb, Zn and Ni, which varied in concentration, depending on the source of the metal.

Preparation of highly selective solid-phase extractants for Cibacron reactive dyes using molecularly imprinted polymers.

Selective polymeric extractants were prepared for preconcentration of Cibacron reactive red dye, a dye that is often applied with Cibacron reactive blue and Cibacron reactive yellow for dyeing of fabrics. The best extractant was fabricated (in chloroform) using methacrylic acid (as monomer), ethylene glycol dimethacrylate (as crosslinker), AIBN (as initiator for polymerization), and red dye as template molecule, with a molar stoichiometric ratio of 8.0:40.0:2.5:0.63, respectively. The structure of the molecularly imprinted polymer (MIP) was robust, and resisted dissolution up to 260 degrees C. Compared with the un-imprinted polymer, the imprinted product has a large specific surface area which improved its adsorption capacity. The effect of imprinting was obvious from the adsorption capacity measured at pH 4 for red dye (the imprinted molecule), which was increased from 24.0 to 79.3 mg g(-1) after imprinting. Equilibrium adsorption studies revealed that the dye-imprinted-polymer enables efficient extraction of red dye even in the presence of blue and yellow dyes which have similar chemical natures to the red dye. The selectivity coefficients S (red dye/dye), were 13.9 and 17.1 relative to the yellow and blue dyes, respectively. The MIP was found to be effective for red dye preconcentration, with a preconcentration factor of 100, from tap water and treated textile wastewater. The factors affecting extraction of red dye by the MIP were studied and optimized. Under the optimized extraction conditions, red dye was selectively quantified in the presence of other competing dyes at a concentration of 20 microg L(-1) from different water systems with satisfactory recoveries (91-95%) and RSD values (approximately 5.0%).

The results of arthroscopic versus mini-open repair for rotator cuff tears at mid-term follow-up.

BACKGROUND: To prospectively evaluate patients who underwent a "mini-open" repair versus a completely arthroscopic technique for small to large size rotator cuff tears. METHODS: Fifty-two patients underwent "mini-open" or all arthroscopic repair of a full thickness tear of the rotator cuff. Patients who complained of shoulder pain and/or weakness and who had failed a minimum of 6 weeks of physical therapy and had at least one sub-acromial injection were surgical candidates. Pre and post-operative clinical evaluations included the following: 1) demographics; 2) Simple Shoulder Test (SST); 3) University of California, Los Angeles (UCLA) rating scale; 4) visual analog pain assessment (VAS); and 5) pre-op SF12 assessment. Descriptive analysis was performed for patient demographics and for all variables. Pre and post outcome scores, range of motion and pain scale were compared using paired t-tests. Analysis of variance (ANOVA) was used to evaluate any effect between dependent and independent variables. Significance was set at p is less than or equal to 0.05. RESULTS: There were 31 females and 21 males. The average follow-up was 50.6 months (27 - 84 months). The average age was similar between the two groups [arthroscopic x = 55 years/mini-open x = 58 years, p = 0.7]. Twenty-seven patients underwent arthroscopic repair and 25 underwent repair with a mini-open incision. The average rotator cuff tear size was 3.1 cm (range: 1-5 centimeters). There was no significant difference in tear size between the two groups (arthroscopic group = 2.9 cm/mini-open group = 3.2 cm, p = 0.3). Overall, there was a significant improvement from pre-operative status in shoulder pain, shoulder function as measured on the Simple Shoulder test and UCLA Shoulder Form. Visual analog pain improved, on average, 4.4 points and the most recent Short Shoulder Form and UCLA scores were 8 and 26 respectively. Both active and passive glenohumeral joint range of motion improved significantly from pre-operatively. CONCLUSION: Based upon the number available, we found no statistical difference in outcome between the two groups, indicating that either procedure is efficacious in the treatment of small and medium size rotator cuff tears. LEVEL OF EVIDENCE: Type III.