A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (³H or ¹⁴C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 μl of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.     

Molecular Modeling of Calixarenes with Group I Metal Ions

Molecular mechanics calculations have been used to model the geometries of the complexes of Group I metal ions with calix[n]arenes (n = 4,5). A simple procedure in which the calixarene atoms are assigned partial charges on the basis of AM1 calculations and the metal ions are allowed to bind electrostatically to the calixarenes produces surprising good results when the resulting structures are compared to known crystallographic data on the complexes. Encapsulated solvent molecules and/or counterions can be included in the calculations and, indeed, are necessary to reproduce the X-ray data. Electronic Supplementary Material available.     

Arabinose 5-phosphate isomerase as a target for antibacterial design: Studies with substrate analogues and inhibitors

Structural requirements of d-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Pseudomonas aeruginosa were analysed in detail using advanced NMR techniques. We performed epitope mapping studies of the binding between the enzyme and the most potent KdsD inhibitors found to date, together with studies of a set of newly synthesised arabinose 5-phosphate (A5P) mimetics. We report here the first experimental evidence that KdsD may bind the furanose form of A5P, suggesting that catalysis of ring opening may be an important part of KdsD catalysis.     

Length–weight relationships of seven fish species in the Yangtze River Estuary and its adjacent waters,Eastern China

The length–weight relationships (LWRs) for seven fish species belonging to five families in the Yangtze River Estuary and its adjacent waters are presented. Cynoglossus gracilis, Cynoglossus macrolepidotus, Ctenotrypauchen chinensis, Collichthys niveatus, Ophichthus apicalis and Erisphex potti were collected monthly in 2009 using trammel nets (the size of smaller mesh net was 1.5 m × 15 m × 4 panels with mesh sizes of 2.5, 3.4, 4.3 and 5.8 cm; the size of larger-mesh net was 2.4 m × 30 m × 4 panels with mesh sizes of 5.0, 6.0, 7.0 and 8.0 cm; soaking overnight). Lophiogobius ocellicauda were collected December in 2020 using shrimp trawl nets (mesh size 2.0 cm). The precision of measurement for the fish specimens is 0.1 cm total length and 0.1 g total weight. The estimated ranges of the parameters a and b for the seven fish species were from 0.0001 to 0.0289 and 2.718 to 3.541, respectively. Two new maximum total length were recorded for Ctenotrypauchen chinensis and Ophichthus apicalis.     

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.     

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis¹. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity² and geometry³ of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments⁴. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).     

Roles of Long-range Electrostatic Domain Interactions and K+ in Phosphoenzyme Transition of Ca2+-ATPase

Sarcoplasmic reticulum Ca²⁺-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca²⁺ transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 m inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K⁺ is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.     

UHPLC-MS/MS-GNPS based phytochemical investigation of Equisetum arvense L. And evaluation of cytotoxicity against human melanoma and ovarian cancer cells

Equisetum arvense L. is widely used as a traditional medicine for the management of inflammation and cancer. In the present study, phyto-chemical analysis of E. arvense was carried out and its cytotoxic potential against human melanoma (MDA-MB-435) and ovarian cancer cells (OVCAR3) was evaluated. Phyto-chemical profile of E. arvense methanolic extract and its fractions was established employing UHPLC-MS/MS and Global Natural Product Social molecular networking. Cytotoxic activity was evaluated using absorbance assay (CellTiter-Blue® Cell Viability Assay). Overall, 22 compounds were identified in the crude extract and polarity-based fractions of E. arvense. Flavonoids, flavonoid-O-glycosides and phenolic acids were found to be the major classes of phyto-chemicals. In addition, the crude extract of E. arvense and its fractions were found active against the tested cell lines. The highest anti-cancer activity against OVCAR3 cells was exhibited by the n-hexane fraction. These results indicated that E. arvense is rich in flavonoids and might be used for the development of anti-cancer drugs against melanoma and ovarian cancers.