Evidence for the utilization of extracellular [γ-32P]ATP for the phosphorylation of intracellular proteins in the squid giant axon

Proteins in the squid giant axon were labeled with ³²P by in vitro incubation of isolated axoplasm with radioactive [γ-³²P]adenosine triphosphate (ATP) and separated by polyacrylamide sodium dodecyl sulfate gel electrophoresis. The two major phosphorylated regions on the gel had molecular weights of 400 000 and 200 000. These two peaks appear to be neurofilament proteins of squid axoplasm. The same set of proteins was phosphorylated in the axoplasm regardless of whether the [γ-³²P]ATP was applied in situ intracellularly or extracelarly. These results suggest that ATP in the extracellular space is, by some ATP-translocation mechanism, utilized in the process of intracellular phosphorylation. Measurements of the apparent influx of ATP across the squid axon membrane yielded results consistent with the view that ATP in the extracellular fluid could be transported into the axoplasm.     

Use of liposomes to aid intestinal absorption of entrapped insulin in normal and diabetic dogs

The effectiveness of liposomes in aiding intestinal absorption of entrapped insulin was studied in normal and diabetic dogs. Intraduodenal administration of free insulin (490 and 1630 U) or free insulin (88 U) plus empty liposomes to normal conscious dogs produced no change in plasma immunoreactive insulin or glucose Administration of 40–80 U insulin entrapped in liposomes composed of either phosphatidylcholine, distearoylphosphatidylcholine, or dipalmitoylphosphatidylcholine with cholesterol and dicetylphosophate ( in the ratio 10:2:1 by weight) to normal dogs produced substantial rises in peripheral plasma immunoreactive insulin after 45–60 min. However, the magnitude of these rises was neither reproducible nor dose-dependent. No fall in plasma glucose was observed. Intraduodenal administration of 50–100 U insulin entrapped in liposomes to diabetic dogs also produced rises in plasma immunoreactive insulin levels after 45–60 min but again these rises were not dose-related. However, unlike the results in normal dogs, a small fall in plasma glucose followed the plasma immunoreactive insulin rise in diabetic dogs. This glucose fall was not dose-dependent nor was it related to the magnitude of the rise in plasma immunoreactive insulin. In conclusion, it seems that administration of insulin in liposomes may allow absorption of partially degraded insulin into the circulation but the rise in plasma immunoreactive insulin observed in normal and diabetic dogs and the fall in plasma glucose in diabetic dogs are not influenced by the dose of insulin entrapped nor the lipid composition of the liposomes.     

Waste Rock Biogeochemistry in a Permafrost Environment: Examination of a Cover Design for a Low-Sulfide,Granitic Waste Rock

At the Diavik Waste Rock Project's mine-research site, the microbial colonization and oxidation of waste rock sulfide minerals are attenuated by the extreme freeze-thaw cycle of a permafrost environment. The closure design for the waste rock stockpile consists of a low-sulfide waste rock and low-permeability till, covering a relatively higher sulfide waste rock. This design was examined at the mine site through construction of experimental waste rock piles and active zone lysimeters with and without the till cover. Leachate from these experiments indicates variable pH and SO₄ concentrations that correlate with sulfide content and the thermal moderating influence of the till cover. The till initially provided a moderated environment for the production of acid, growth of acidophilic Fe- and S-oxidizing bacteria, and enhanced weathering until wet up and freezing of the till and underlying waste rock as a permafrost. Greater sulfide oxidation was observed above the till cover because of greater exposure to the annual freeze-thaw cycle. An examination of the bacterial communities at the genus level indicates the prevalence of Pseudomonas, Rhodanobacter, Sideroxydans, and Thiobacillus in the waste rock. Pseudomonas spp. were dominant in the drier and more extreme temperature environment above the till cover, while Thiobacillus spp. were dominant in the more sulfide-rich, wetter/frozen environment below the till. A decreasing trend in Thiobacillus spp. from the exterior to the interior and an opposing trend in Acidithiobacillus spp. suggest greater acid generation deeper in the waste rock further from the extreme temperature variation of the tundra climate. The presence of the till cover moderated temperature variations, enhanced the initial rate of sulfide oxidation, and allowed for greater microbial diversity, but the freezing of the till cover and underlying waste rock drastically reduced sulfide oxidation and the generation of acid rock drainage. These results highlight the importance of temperature on microbially catalyzed acid production and our ability to use the extreme temperatures of the tundra climate to minimize potential environmental effects from mining through formation of waste rock permafrost.     

Chitosan in Surface Modification for Bone Tissue Engineering Applications

Traditional methods of bone defect repair include autografts, allografts, surgical reconstruction, and metal implants that have several disadvantages such as donor site morbidity, rejection, risk of disease transmission, and repetitive surgery. Biomaterial‐based bone reconstructions can, therefore, be an efficient alternative due to the inherent properties of the materials. Chitosan (CS), the deacetylated form of chitin, is a biopolymer having a wide array of applicability in regenerative tissue applications owing to its biocompatible, in vitro degradative and bioresorbable nature. Extensive studies are being carried out using CS to augment the properties of the already existing methods and to also improve the applicability of CS‐based biocomposites in bone tissue repair. In this review, the suitability of CS as a surface modifier has been discussed in detail for the already existing implants, surface modifications of CS‐based natural biocomposites for bone tissue regeneration, and the wide range of techniques that can introduce these modifications. CS, being a natural polymer, possesses advantageous properties including surface modifier that makes it a suitable candidate for bone regeneration, and further research to investigate its osteogenic potential in vivo along with the molecular and signaling mechanisms involved in bone regeneration can aid in expanding its applicability in clinical trials.     

Topographic regulation of neuronal intermediate filaments by phosphorylation, role of peptidyl-prolyl isomerase 1: significance in neurodegeneration

The neuronal cytoskeleton is tightly regulated by phosphorylation and dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Defects in the relative kinase and phosphatase activities and/or deregulation of compartment-specific phosphorylation result in neurodegenerative disorders. The largest family of cytoskeletal proteins in mammalian cells is the superfamily of intermediate filaments (IFs). The neurofilament (NF) proteins are the major IFs. Aggregated forms of hyperphosphorylated tau and phosphorylated NFs are found in pathological cell body accumulations in the central nervous system of patients suffering from Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The precise mechanisms for this compartment-specific phosphorylation of cytoskeletal proteins are not completely understood. In this review, we focus on the mechanisms of neurofilament phosphorylation in normal physiology and neurodegenerative diseases. We also address the recent breakthroughs in our understanding the role of different kinases and phosphatases involved in regulating the phosphorylation status of the NFs. In addition, special emphasis has been given to describe the role of phosphatases and Pin1 in phosphorylation of NFs.     

Vascular changes precede tomographic changes in diabetic eyes without retinopathy and improve artificial intelligence diagnostics

The purpose of this study was to evaluate early vascular and tomographic changes in the retina of diabetic patients using artificial intelligence (AI). The study included 74 age‐matched normal eyes, 171 diabetic eyes without retinopathy (DWR) eyes and 69 mild non‐proliferative diabetic retinopathy (NPDR) eyes. All patients underwent optical coherence tomography angiography (OCTA) imaging. Tomographic features (thickness and volume) were derived from the OCTA B‐scans. These features were used in AI models. Both OCT and OCTA features showed significant differences between the groups (P < .05). However, the OCTA features indicated early retinal changes in DWR eyes better than OCT (P < .05). In the AI model using both OCT and OCTA features simultaneously, the best area under the curve of 0.91 ± 0.02 was obtained (P < .05). Thus, the combined use of AI, OCT and OCTA significantly improved the early diagnosis of diabetic changes in the retina.     

Deciphering PPARγ activation in cardiometabolic syndrome: studies by in silico and in vivo experimental assessment

Cardiometabolic syndrome (CMetS) is a consolidation of metabolic disorders characterized by insulin resistance, dyslipidemia and hypertension. Curcumin, a natural bioactive compound, has been shown to possess notable anti-oxidant activity and it has also been included as a super natural herb in the super natural herbs database. Most of the beneficial effects of Curcumin are possibly due to activation of the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ). The present study investigates molecular interactions of curcumin with PPARγ protein through molecular docking and molecular dynamics (MD) simulation studies. Further, effect of curcumin on high fat diet induced CMetS was studied in rats along with western blot for PPARγ and nuclear factor-κB (NF-κB) expressions and histopathological studies. Computational studies presented several significant molecular interactions of curcumin including Ser289, His323, His449 and Tyr473 of PPARγ. The in vivo results further confirmed that curcumin was able to ameliorate the abnormal changes and also, increased PPARγ expressions. The results confirm our hypothesis that activation of PPARγ by curcumin possesses the therapeutic potential to ameliorate the altered levels of metabolic changes in rats in the treatment of CMetS. This is the first report of CMetS treatment by curcumin and study of its underlying mechanism through in silico as well as in vivo experiments.     

Effect of thermostable mutations on the neurotensin receptor 1 (NTSR1) activation state

Abstract

Neurotensin (NTS) is a 13-amino acid neuropeptide with neuroendocrine and vasoactive functions that is widely expressed in the central nervous system and gastrointestinal tract. NTS is sensed by a multiple cell surface proteins including two G protein-coupling receptors (GPCRs): NTS receptors 1 and 2 (NTSR₁ and NTSR₂). Crystal structures of NTSR₁ have successfully elucidated agonist binding within the orthosteric pocket of receptor but have not revealed the full activation state of the receptor. Recent studies have attempted to address this challenge by improving NTSR₁ crystal formation via thermostable mutants; unfortunately, these mutations exhibit functional defects in the G protein coupling of NTSR₁. Here, we have used molecular dynamics simulations to gain greater insights into how the amino acid substitutions used in these thermostable mutants (E166A, L310A and F358A) impact receptor activation. Our simulations indicate that wild-type NTSR₁ in complex with NTS_8-13 shows more active-like features including a 17.7?Å shift in TM6, reflecting a network of polar and aromatic interactions orchestrating agonist-induced receptor conformational changes. We also provide evidence indicating that F358 is a precursor to the rotamer change observed in W321, and our collective analysis also suggests that mutations E166A and F358A are less impactful to G protein coupling than L310A. Furthermore, we believe that our findings can be used to design future NTSR₁ mutants that do not interfere with agonist-induced conformational changes and downstream G protein coupling and thus produce structures that will allow visualization of the fully activated receptor conformation.