Albumin Expands Albumin Reabsorption Capacity in Proximal Tubule Epithelial Cells through a Positive Feedback Loop between AKT and Megalin

Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1+ endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a phenomenon that we refer to as “albumin-induced albumin endocytosis”. Mice treated with L-lysine present decreased albumin endocytosis leading to proteinuria and albuminuria associated with inhibition of AKT activity. Renal cortex explants obtained from control mice treated with MK-2206 decreased albumin uptake and promoted megalin internalization. Our data highlight the mechanism behind the capacity of PTECs to adapt albumin reabsorption to physiologic fluctuations in its filtration, avoiding urinary excretion.

Differential Uptake of Murine and Human Exosomes by Normal and Inflamed Peripheral Tissues and Brain

Background: Exosomes function as an intercellular communication system conveying messages from donor to target cells in nearby or distant tissues. Many aspects of exosome trafficking remain unresolved, however. Here, we investigated uptake of ten radiolabeled murine or human exosomes of various cellular origins by the liver, kidney, spleen, and lung of male CD-1 mice. Methods: We radioactively labeled 10 exosomes from mouse or human cancerous or non-cancerous lines, injected them intravenously into male CD-1 mice, and studied their tissue uptake. We examined the ability of wheatgerm agglutinin (WGA), mannose-6 phosphate (M6P), and inflammation induced by lipopolysaccharide (LPS) to modulate uptake. We measured uptake rate using multiple-time regression analysis and used heat mapping and path analysis to correlate tissue and exosomal influences on uptake. Results: Except for the uptake of SCCVII exosomes by kidney, all exosomes were taken up by all tissues, although the uptake levels varied broadly among exosomes and tissues. The liver/serum uptake ratio for exosomes from primary human T-cells was the highest at 4,500 mL/g. Species of origin (mouse vs human) or source (cancerous vs noncancerous cells) did not influence tissue uptake. The uptake of some exosomes was altered by WGA and LPS but not by M6P, except for uptake inhibition of J774A.1 exosomes by liver, suggesting use of the M6P receptor. WGA or LPS treatments enhanced uptake of exosomes by brain and lung but inhibited uptake by liver and spleen. Response to LPS was not, however, predictive of response to WGA. No evidence for a universal binding site controlling exosome uptake was obtained. Applying path analysis and heat map analysis to the data, including our published results for brain, we found that exosome uptake patterns for lung and brain responded similarly to WGA or to LPS, whereas those for liver and spleen clustered together. In path analysis, the 10 exosomes clustered into distinct groups, suggesting that their bindings sites are similarly clustered. Conclusions: Uptake of exosomes by peripheral tissues is differentially regulated by both exosomes and target tissues and is dependent on the number and types of mutually interactive binding sites.

Design of D-amino acids SARS-CoV-2 Main protease inhibitors using the cationic peptide from rattlesnake venom as a scaffold

The C30 Endopeptidase (3C-like protease; 3CLpro) is essential for the life cycle of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) since it plays a pivotal role in viral replication and transcription and is hence a promising drug target. Molecules isolated from animals, insects, plants or microorganisms can serve as a scaffold for the design of novel biopharmaceutical products. Crotamine, a small cationic peptide from the venom of the rattlesnake Crotalus durissus terrificus has been the focus of many studies since it exhibits activities such as analgesic, in vitro antibacterial and hemolytic activities. The crotamine derivative L-peptides (L-CDP) that inhibit the 3CL protease in the low μM range were examined since they are susceptible to proteolytic degradation; we explored the utility of their D-enantiomers form. Comparative uptake inhibition analysis showed D-CDP as a promising prototype for a D-peptide-based drug. We also found that the D-peptides can impair SARS-CoV-2 replication in vivo, probably targeting the viral protease 3CLpro.

Different Withering Times Affect Sensory Qualities, Chemical Components, and Nutritional Characteristics of Black Tea

The present study emphasizes the effect of withering time set at 4 ± 0.5 h (WT4), 6 ± 0.5 h (WT6), 8 ± 0.5 h (WT8), 10 ± 0.5 h (WT10), and 12 ± 0.5 h (WT12) on the sensory qualities, chemical components, and nutritional characteristics of black tea. The sensory evaluation revealed high total quality scores at WT8 and WT10. Polysaccharides, amino acids, and soluble sugars significantly increased with an increase in withering time, and an apparent peak value was obtained at WT10. However, polyphenols, flavonoids, glycosides, organic acids, catechins, alkanoids, and theaflavins decreased with an increase in withering time. With an increase in withering time, the content of aromatic substances showed a trend of increasing first and then decreasing. The peaks of alcohols, aldehydes, and acids appeared at 10 ± 0.5 h, 10 ± 0.5 h, and 8 ± 0.5 h, respectively. The content of esters, ketones, and hydrocarbons showed a downward trend with an increase in withering time. Aroma analysis revealed that withering time could not exceed 10 ± 0.5 h. Black tea withered up to WT10 showed enhanced inhibition of α-glucosidase and α-amylase activity with good sensorial attributes. Glucose uptake inhibition capacity increased up 6 ± 0.5 h and then decreased, while antioxidant capacity decreased with an increase in withering time. The overall results show that the 8 ± 0.5 h to 10 ± 0.5 h withering time could improve black tea quality and nutritional characteristics.

Inhibition of Scavenger Receptor Class B Type 1 (SR-B1) Expression and Activity as a Potential Novel Target to Disrupt Cholesterol Availability in Castration-Resistant Prostate Cancer

There have been several studies that have linked elevated scavenger receptor class b type 1 (SR-B1) expression and activity to the development and progression of castration-resistant prostate cancer (CRPC). SR-B1 facilitates the influx of cholesterol to the cell from lipoproteins in systemic circulation. This influx of cholesterol may be important for many cellular functions, including the synthesis of androgens. Castration-resistant prostate cancer tumors can synthesize androgens de novo to supplement the loss of exogenous sources often induced by androgen deprivation therapy. Silencing of SR-B1 may impact the ability of prostate cancer cells, particularly those of the castration-resistant state, to maintain the intracellular supply of androgens by removing a supply of cholesterol. SR-B1 expression is elevated in CRPC models and has been linked to poor survival of patients. The overarching belief has been that cholesterol modulation, through either synthesis or uptake inhibition, will impact essential signaling processes, impeding the proliferation of prostate cancer. The reduction in cellular cholesterol availability can impede prostate cancer proliferation through both decreased steroid synthesis and steroid-independent mechanisms, providing a potential therapeutic target for the treatment of prostate cancer. In this article, we discuss and highlight the work on SR-B1 as a potential novel drug target for CRPC management.

Bacterial Engulfment Mechanism Is Strongly Conserved in Evolution Between Earthworm and Human Immune Cells

Invertebrates, including earthworms, are applied to study the evolutionarily conserved cellular immune processes. Earthworm immunocytes (so-called coelomocytes) are functionally similar to vertebrate myeloid cells and form the first line of defense against invading pathogens. Hereby, we compared the engulfment mechanisms of THP-1 human monocytic cells, differentiated THP-1 (macrophage-like) cells, and Eisenia andrei coelomocytes towards Escherichia coli and Staphylococcus aureus bacteria applying various endocytosis inhibitors [amantadine, 5-(N-ethyl-N-isopropyl) amiloride, colchicine, cytochalasin B, cytochalasin D, methyl-ß-cyclodextrin, and nystatin]. Subsequently, we investigated the messenger RNA (mRNA) expressions of immune receptor-related molecules (TLR, MyD88, BPI) and the colocalization of lysosomes with engulfed bacteria following uptake inhibition in every cell type. Actin depolymerization by cytochalasin B and D has strongly inhibited the endocytosis of both bacterial strains in the studied cell types, suggesting the conserved role of actin-dependent phagocytosis. Decreased numbers of colocalized lysosomes/bacteria supported these findings. In THP-1 cells TLR expression was increased upon cytochalasin D pretreatment, while this inhibitor caused a dropped LBP/BPI expression in differentiated THP-1 cells and coelomocytes. The obtained data reveal further insights into the evolution of phagocytes in eukaryotes. Earthworm and human phagocytes possess analogous mechanisms for bacterial internalization.

Anticancer and Structure Activity Relationship of Non-Symmetrical Choline Kinase Inhibitors

Choline kinase inhibitors are an outstanding class of cytotoxic compounds useful for the treatment of different forms of cancer since aberrant choline metabolism is a feature of neoplastic cells. Here, we present the most in-depth structure-activity relationship studies of an interesting series of non-symmetric choline kinase inhibitors previously reported by our group: 3a–h and 4a–h. They are characterized by cationic heads of 3-aminophenol bound to 4-(dimethylamino)- or 4-(pyrrolidin-1-yl)pyridinium through several linkers. These derivatives were evaluated both for their inhibitory activity on the enzyme and their antiproliferative activity in a panel of six human tumor cell lines. The compounds with the N-atom connected to the linker (4a–h) show the best inhibitory results, in the manner of results supported by docking studies. On the contrary, the best antiproliferative compounds were those with the O-atom bounded to the linker (3a–h). On the other hand, as was predictable in both families, the inhibitory effect on the enzyme is better the shorter the length of the linker. However, in tumor cells, lipophilicity and choline uptake inhibition could play a decisive role. Interestingly, compounds 3c and 4f, selected for both their ability to inhibit the enzyme and good antiproliferative activity, are endowed with low toxicity in non-tumoral cells (e.g., human peripheral lymphocytes) concerning cancer cells. These compounds were also able to induce apoptosis in Jurkat leukemic cells without causing significant variations of the cell cycle. It is worth mentioning that these derivatives, besides their inhibitory effect on choline kinase, displayed a modest ability to inhibit choline uptake thus suggesting that this mechanism may also contribute to the observed cytotoxicity.