In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Experimental models have often been at the origin of immunological paradigms such as the M1/M2 dichotomy following macrophage polarization. However, this clear dichotomy in animal models is not as obvious in humans, and the separating line between M1-like and M2-like macrophages is rather represented by a continuum, where boundaries are still unclear. Indeed, human infectious diseases, are characterized by either a back and forth or often a mixed profile between the pro-inflammatory microenvironment (dominated by interleukin (IL)-1β, IL-6, IL-12, IL-23 and Tumor Necrosis Factor (TNF)-α cytokines) and tissue injury driven by classically activated macrophages (M1-like) and wound healing driven by alternatively activated macrophages (M2-like) in an anti-inflammatory environment (dominated by IL-10, Transforming growth factor (TGF)-β, chemokine ligand (CCL)1, CCL2, CCL17, CCL18, and CCL22). This review brews the complexity of the situation during infectious diseases by stressing on this continuum between M1-like and M2-like extremes. We first discuss the basic biology of macrophage polarization, function, and role in the inflammatory process and its resolution. Secondly, we discuss the relevance of the macrophage polarization continuum during infectious and neglected diseases, and the possibility to interfere with such activation states as a promising therapeutic strategy in the treatment of such diseases.

https://www.mdpi.com/1422-0067/19/6/1801/pdf

Role of Human Macrophage Polarization in Inflammation during Infectious Diseases

Evading persistent drug resistance in cancer and bacteria is quintessential to restore health in humans, and impels intervention strategies. A distinct property of the cancer phenotype is enhanced glucose metabolism and oxidative stress. Reactive oxygen species (ROS) are metabolic byproducts of aerobic respiration and are responsible for maintaining redox homeostasis in cells. Redox balance and oxidative stress are orchestrated by antioxidant enzymes, reduced thiols and NADP(H) cofactors, which is critical for cancer cells survival and progression. Similarly, Escherichia coli (E. coli) and life-threatening infectious pathogens such as Staphylococcus aureus (SA) and Mycobacterium tuberculosis (Mtb) are appreciably sensitive to changes in the intracellular oxidative environment. Thus, small molecules that modulate antioxidant levels and/or enhance intracellular ROS could disturb the cellular oxidative environment and induce cell death, and hence could serve as novel therapeutics. Presented here are a collec...

Role of Reactive Oxygen Species (ROS) in Therapeutics and Drug Resistance in Cancer and Bacteria

The mammary gland

B139 Oct-4 transcription factors play an important role in maintaining the pluripotent state of embryonic stem cells and may prevent expression of genes activated during differentiation. Human Oct-4 isoform mRNAs encode proteins that have identical POU DNA binding domains and C-terminal domains, but differ in their amino-terminal domains. We report here the cloning and characterization of the human Oct-4B isoform. Human Oct-4B cDNA encodes a 265-amino acid protein with a predicted molecular mass of 30 kDa. ES cell-based complementation assays using ZHBTc4 ES cells showed that unlike human Oct-4A, Oct-4B cannot sustain ES cell self-renewal. In addition, Oct-4B does not bind to a probe carrying the Oct-4 consensus binding sequence and we demonstrate that two separate regions of its amino terminal domain are responsible for inhibiting DNA binding. We also demonstrate that Oct-4B is mainly localized to the cytoplasm. Overexpression of Oct-4B did not activate transcription from Oct-4-dependent promoters, whereas Oct-4A did as previously reported. Furthermore, transcriptional activation by human Oct-4A was not inhibited by co-expression of Oct-4B. Taken together, these data suggest that the DNA binding, transactivation, and abilities to confer self-renewal of the human Oct-4 isoforms differ.

The human oct-4 isoforms differ in their ability to confer self-renewal.

Salmonella species can infect a diverse range of birds, reptiles, and mammals, including humans. The type III protein secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) delivers effector proteins required for intestinal invasion and the production of enteritis. The T3SS is regarded as the most important virulence factor of Salmonella. SPI-1 encodes transcription factors that regulate the expression of some virulence factors of Salmonella, while other transcription factors encoded outside SPI-1 participate in the expression of SPI-1-encoded genes. SPI-1 genes are responsible for the invasion of host cells, regulation of the host immune response, e.g., the host inflammatory response, immune cell recruitment and apoptosis, and biofilm formation. The regulatory network of SPI-1 is very complex and crucial. Here, we review the function, effectors, and regulation of SPI-1 genes and their contribution to the pathogenicity of Salmonella.

/pdf/salmonella-pathogenicity-island-1-spi-1-and-its-complex-51np54xldx.pdf

Salmonella Pathogenicity Island 1 (SPI-1) and Its Complex Regulatory Network.

Biofilms are communities of microorganisms that are attached to a biological or abiotic surface and are surrounded by a self-produced extracellular matrix. Cells within a biofilm have intrinsic characteristics that are different from those of planktonic cells. Biofilm resistance to antimicrobial agents has drawn increasing attention. It is well-known that medical device- and tissue-associated biofilms may be the leading cause for the failure of antibiotic treatments and can cause many chronic infections. The eradication of biofilms is very challenging. Many researchers are working to address biofilm-related infections, and some novel strategies have been developed and identified as being effective and promising. Nevertheless, more preclinical studies and well-designed multicenter clinical trials are critically needed to evaluate the prospects of these strategies. Here, we review information about the mechanisms underlying the drug resistance of biofilms and discuss recent progress in alternative therapies and promising strategies against microbial biofilms. We also summarize the strengths and weaknesses of these strategies in detail.

/pdf/promising-therapeutic-strategies-against-microbial-biofilm-3ul58d9273.pdf

Promising Therapeutic Strategies Against Microbial Biofilm Challenges.

Biochanin A (BCA) is an isoflavone mainly found in red clover with poor solubility and oral absorption that is known to have various effects, including anti-inflammatory, estrogen-like, and glucose and lipid metabolism modulatory activity, as well as cancer preventive, neuroprotective, and drug interaction effects. BCA is already commercially available and is among the main ingredients in many types of supplements used to alleviate postmenopausal symptoms in women. The activity of BCA has not been adequately evaluated in humans. However, the results of many in vitro and in vivo studies investigating the potential health benefits of BCA are available, and the complex mechanisms by which BCA modulates transcription, apoptosis, metabolism, and immune responses have been revealed. Many efforts have been exerted to improve the poor bioavailability of BCA, and very promising results have been reported. This review focuses on the major effects of BCA and its possible molecular targets, potential uses, and limitations in health maintenance and treatment.

/pdf/perspectives-regarding-the-role-of-biochanin-a-in-humans-1dsssp38ns.pdf

Perspectives Regarding the Role of Biochanin A in Humans

Spinal cord injury (SCI) is a serious condition that affects bodily function; however, there is no effective therapy in clinical practice. MCC950, a selective NOD-like receptor protein-3 (NLRP3) inflammasome inhibitor, has been reported to alleviate canonical and non-canonical NLRP3 inflammasome activation of the inflammatory response in vitro and in vivo. However, the effect of MCC950 treatment on neurological post-SCI recovery remains unclear. In this study, we assessed the pharmacological effect of MCC950 on an experimental SCI model in vivo and neuronal injury in vitro. We found that MCC950 improved the grip strength, hind limb movements, spinal cord edema, and pathological injury in the SCI mice. We demonstrated that it exerted this effect by blocking NLRP3 inflammasome assembly, including NLRP3-ASC and NLRP3-Caspase-1 complexes, as well as the release of pro-inflammatory cytokines TNF-α, IL-1β, and IL-18. Moreover, we found that MCC950 reduced spinal neuron injury and NLRP3 inflammasome activation, which had been induced by oxygen-glucose deprivation (OGD) or lipopolysaccharides (LPS) in vitro. In conclusion, our findings indicate that MCC950 alleviates inflammatory response and improves functional recovery in the acute mice model of SCI by blocking NLRP3 inflammasome assembly and alleviating downstream neuroinflammation. Therefore, these findings could prove useful in the development of effective therapeutic strategies for the treatment and prognosis of SCI.

/pdf/mcc950-a-selective-inhibitor-of-nlrp3-inflammasome-reduces-1bx03ka9cg.pdf

MCC950, a Selective Inhibitor of NLRP3 Inflammasome, Reduces the Inflammatory Response and Improves Neurological Outcomes in Mice Model of Spinal Cord Injury.

The present study investigated the characteristic features of cancer stem cells (CSCs) using an aggressive human osteosarcoma cell line OS-65. Hoechst 33342 dye exclusion was used to distinguish the cancer stem-like side population (SP) cells from OS-65 cells. Furthermore, the SP cells were characterized via chemoresistance and cell death assays, reverse transcription-quantitative polymerase chain reaction and immunofluorescence. The present study identified ~3.3% of cancer stem-like SP cells from OS-65 cells whose prevalence is reduced significantly (0.9%) following treatment with verapamil. It was demonstrated that osteosarcoma SP cells are highly efficient at generating additional sarcospheres as transcriptional regulation of stemness genes, including SOX2, OCT-4 and NANOG, is highly upregulated. Notably, these SP cells demonstrated high resistance against chemotherapeutic drugs and apoptosis via elevated transcriptional regulation of several ATPase binding cassette (ABC) transporter and anti-apoptotic proteins, including ABCG2, ABCB1/MDR1 ABCB5, B cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein, respectively. The results of the present study suggested that CSCs may be a novel therapeutic target for the prevention of tumor relapse.

/pdf/increased-multi-drug-resistance-and-reduced-apoptosis-in-3sstwdotzx.pdf

Yang Wang

Papers

Salmonella Pathogenicity Island 1 (SPI-1) and Its Complex Regulatory Network.

Promising Therapeutic Strategies Against Microbial Biofilm Challenges.

Perspectives Regarding the Role of Biochanin A in Humans

MCC950, a Selective Inhibitor of NLRP3 Inflammasome, Reduces the Inflammatory Response and Improves Neurological Outcomes in Mice Model of Spinal Cord Injury.

Increased multi-drug resistance and reduced apoptosis in osteosarcoma side population cells are crucial factors for tumor recurrence.