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Background Effective stem cell therapy is dependent on the stem cell quality that is determined by their differentiation potential, impairment of which leads to poor engraftment and survival into the target cells. However, limitations in our understanding and the lack of reliable markers that can predict their maturation efficacies have hindered the development of stem cells as an effective therapeutic strategy. Our previous study identified CD10, a pro-adipogenic, depot-specific prospective cell surface marker of human adipose-derived stem cells (ASCs). Here, we aim to determine if CD10 can be used as a prospective marker to predict mature adipocyte quality and play a direct role in adipocyte maturation. Methods We first generated 14 primary human subject-derived ASCs and stable immortalized CD10 knockdown and overexpression lines for 4 subjects by the lentiviral transduction system. To evaluate the role of CD10 in adipogenesis, the adipogenic potential of the human subject samples were scored against their respective CD10 transcript levels. Assessment of UCP1 expression levels was performed to correlate CD10 levels to the browning potential of mature ASCs. Quantitative polymerase chain reaction (qPCR) and Western blot analysis were performed to determine CD10-dependent regulation of various targets. Seahorse analysis of oxidative metabolism and lipolysis assay were studied. Lastly, as a proof-of-concept study, we used CD10 as a prospective marker for screening nuclear receptor ligands library. Results We identified intrinsic CD10 levels as a positive determinant of adipocyte maturation as well as browning potential of ASCs. Interestingly, CD10 regulates ASC’s adipogenic maturation non-canonically by modulating endogenous lipolysis without affecting the classical peroxisome proliferator-activated receptor gamma (PPARγ)-dependent adipogenic pathways. Furthermore, our CD10-mediated screening analysis identified dexamethasone and retinoic acid as stimulator and inhibitor of adipogenesis, respectively, indicating CD10 as a useful biomarker for pro-adipogenic drug screening. Conclusion Overall, we establish CD10 as a functionally relevant ASC biomarker, which may be a prerequisite to identify high-quality cell populations for improving metabolic diseases.

Redox signaling and homeostasis are essential for cell survival and the immune response. Peroxiredoxin (Prx) modulates the level of H2O2 as a redox signal through H2O2 decomposition. The redox activity of thioredoxin (Trx) is required as a reducing equivalent to regenerate Prx. Edwardsiella piscicida is an opportunistic Gram-negative enteric pathogen that secretes a novel Trx-like effector protein, ETAE_2186 (Trxlp). Trxlp has unique structural properties compared with other Trx proteins. In enzymatic and binding assays, we confirmed Trxlp to be redox-inactive due to the low reactivity and flexibility of the resolving cysteine residue, C35, at the active site motif “31WCXXC35”. We identified key residues near the active site that are critical for reactivity and flexibility of C35 by site-directed mutagenesis analysis. NMR titration experiment demonstrated prolong inhibitory interaction of Trxlp with Prx1 resulting in the repression of Prx1–mediated H2O2 decomposition leading to increased ROS accumulation in infected host cells. Increased ROS in turn prevented nuclear translocation of NF-κB and inhibition of NF-κB target genes, leading to bacterial survival and enhanced replication inside host cells. Targeting Trxlp-mediated virulence promises to attenuate E. piscicida infection.

Adipose-derived stem cells (ASCs) have been increasingly used as a versatile source of mesenchymal stem cells (MSCs) for diverse clinical investigations. However, their applications often become complicated due to heterogeneity arising from various factors. Cellular heterogeneity can occur due to: (i) nomenclature and criteria for definition; (ii) adipose tissue depots (e.g., subcutaneous fat, visceral fat) from which ASCs are isolated; (iii) donor and inter-subject variation (age, body mass index, gender, and disease state); (iv) species difference; and (v) study design (in vivo versus in vitro) and tools used (e.g., antibody isolation and culture conditions). There are also actual differences in resident cell types that exhibit ASC/MSC characteristics. Multilineage-differentiating stress-enduring (Muse) cells and dedifferentiated fat (DFAT) cells have been reported as an alternative or derivative source of ASCs for application in regenerative medicine. In this review, we discuss these factors that contribute to the heterogeneity of human ASCs in detail, and what should be taken into consideration for overcoming challenges associated with such heterogeneity in the clinical use of ASCs. Attempts to understand, define, and standardize cellular heterogeneity are important in supporting therapeutic strategies and regulatory considerations for the use of ASCs.

The study addresses an improved understanding on the vulnerability of the people living in urban slums and objects to helping in prioritisation of efforts and optimal utilisation of limited resources for better health outcome. The study was conducted in the city of “Bhubaneswar” the state capital of Odisha in India. Focused Group Discussions were conducted in 75 slums of the city followed by a detailed analysis of the data collected. The predominant vulnerability of people living in the slums under the study is water logging and unhygienic locality. This has direct impacts on the general health particularly of children who are prone to allergies resulting in colds, asthma etc. The study also revealed that no slum falling in the category of the most vulnerable while 24 slums falling in least vulnerable and rest of 51 slums are moderately vulnerable out of 75 slums assessed. The study has important implications for the planners, program managers and policy makers of urban health especially for better for the people living in urban slums

Figure S1. Correlation of adipogenic scores and adipogenic genes expression in 3T3-L1 cells. Figure S2. Sizes per lipid droplet from Nile Red staining were estimated with the modified method using background subtraction and circular Hough transform during adipogenesis of 3T3-L1 cells. Figure S3. Adipogenesis time course of human iPSC-derived MSCs. Figure S4. FATS analysis of browning in subcutaneous fat-derived ASCs. Figure S5. High-throughput adipogenic screening assay of nuclear receptor ligands using human iPSC-derived MSCs was analyzed by FATS. Table S1. List of all the nuclear receptor ligands that were tested for high-throughput screening assay for adipogenesis in 3T3-L1 cells. Table S2. Results of high-throughput screening assay for adipogenesis of human iPS-derived MSCs in a single plate using nuclear receptor ligands library. Table S3. List of RT-qPCR primers-Oligos 5’ to 3’.(DOCX 5748 kb)

Figure S1. Gene expression studies of additional ROS-related genes that are not included in Figure 1b. Figure S2. VS-ASCs have increased ROS when compared to SC-ASCs in the lower passage. Figure S3. Effects on adipogenesis are specifically mediated by ROS. Figure S4. Ascorbic acid treatment generally decreases both glycolytic and oxidative respirations. Table S1. List of subjects used for this study. Table S2. List of RT-qPCR primers-oligos 5⠲ to 3⠲. (DOCX 3731 kb)

The PhoP-PhoQ two-component system is commonly used by bacteria to sense environmental factors. Here we show that the PhoP-PhoQ system of Edwardsiella tarda detects changes in environmental temperature and Mg(2+) concentration as well as regulates the type III and VI secretion systems through direct activation of esrB. Protein secretion is activated from 23 to 35 °C or at low Mg(2+) concentrations, but it is suppressed at or below 20 °C, at or above 37 °C, or at high Mg(2+) concentrations. The effects of temperature and Mg(2+) concentration are additive. The PhoQ sensor domain has a low T(m) of 37.9 °C, and it detects temperatures through a conformational change of its secondary structure. Mutation of specific Pro or Thr residues increased the stability of the PhoQ sensor drastically, altering its temperature-sensing ability. The PhoQ sensor detects Mg(2+) concentration through the direct binding of Mg(2+) to a cluster of acidic residues (DDDSAD) and through changes that likely affect its tertiary structure. Here, we describe for the first time the use of PhoP-PhoQ as a temperature sensor for bacterial virulence control.

In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of a sensor kinase (usually a membrane protein) and a response regulator (usually a DNA binding protein). The EnvZ/OmpR two-component system responds to osmotic stress and regulates expression of outer membrane proteins. In Salmonella, EnvZ/OmpR also controls expression of another two-component system SsrA/B, which is located on Salmonella Pathogenicity Island (SPI) 2. SPI-2 encodes a type III secretion system, which functions as a nanomachine to inject bacterial effector proteins into eukaryotic cells. During the intracellular phase of infection, Salmonella switches from assembling type III secretion system structural components to secreting effectors into the macrophage cytoplasm, enabling Salmonella to replicate in the phagocytic vacuole. Major questions remain regarding how bacteria survive the acidified vacuole and how acidification affects bacterial secretion. We previously reported that EnvZ sensed cytoplasmic signals rather than extracellular ones, as intracellular osmolytes altered the dynamics of a 17-amino-acid region flanking the phosphorylated histidine. We reasoned that the Salmonella cytoplasm might acidify in the macrophage vacuole to activate OmpR-dependent transcription of SPI-2 genes. To address these questions, we employed a DNA-based FRET biosensor (“I-switch”) to measure bacterial cytoplasmic pH and immunofluorescence to monitor effector secretion during infection. Surprisingly, we observed a rapid drop in bacterial cytoplasmic pH upon phagocytosis that was not predicted by current models. Cytoplasmic acidification was completely dependent on the OmpR response regulator, but did not require known OmpR-regulated genes such as ompC, ompF, or ssaC (SPI-2). Microarray analysis highlighted the cadC/BA operon, and additional experiments confirmed that it was repressed by OmpR. Acidification was blocked in the ompR null background in a Cad-dependent manner. Acid-dependent activation of OmpR stimulated type III secretion; blocking acidification resulted in a neutralized cytoplasm that was defective for SPI-2 secretion. Based upon these findings, we propose that Salmonella infection involves an acid-dependent secretion process in which the translocon SseB moves away from the bacterial cell surface as it associates with the vacuolar membrane, driving the secretion of SPI-2 effectors such as SseJ. New steps in the SPI-2 secretion process are proposed., Rather than using the cadaverine operon to combat the acid conditions of the macrophage vacuole, Salmonella allows its cytoplasm to become acidified, which in turn drives secretion of effectors into the host cell., Author Summary The human pathogenic bacteria Salmonella encounters extreme and diverse conditions during the course of host infection. Survival and adaptation inside the host requires highly regulated virulence factors. When Salmonella is engulfed by a macrophage, it forms a vacuole-type structure that is actively acidified by the macrophage in an attempt to kill or neutralize the bacteria. However, the acidic pH in this “Salmonella-containing vacuole,” or SCV, does not kill the bacteria and is instead a major inducing signal for the expression of a specific class of virulence proteins. These proteins, encoded in the S almonella Pathogenicity Island 2 (SPI-2), function as a nanomachine that mediates the delivery of Salmonella effector proteins that disrupt host immune defenses. In this paper, we investigate unanswered questions regarding the ability of Salmonella to survive the low pH and its consequences for bacterial growth in the SCV. Using a fluorescent biosensor, we monitored the intracellular pH of the Salmonella cytoplasm while it resides in the SCV during macrophage infection. Our results indicate that the bacteria cytoplasm acidifies in response to SVC acidity; this acidification requires the transcription factor OmpR, a known regulator of SPI-2. OmpR represses the cadC/BA operon, which is involved in the recovery from acid stress, thus enabling Salmonella to assume the acidic pH of the macrophage vacuole. Acidification is required for the secretion of virulence factors; blocking acidification resulted in a neutralized cytoplasm that was defective for SPI-2 secretion. Our work challenges existing views that bacteria regulate their pH to maintain neutrality, and provides a new model for Salmonella virulence factor secretion and infection.

Inorganic phosphate (P(i)) and iron are essential nutrients that are depleted by vertebrates as a protective mechanism against bacterial infection. This depletion, however, is sensed by some pathogens as a signal to turn on the expression of virulence genes. Here, we show that the PhoB-PhoR two-component system senses changes in P(i) concentration, whereas the ferric uptake regulator (Fur) senses changes in iron concentration in Edwardsiella tarda PPD130/91 to regulate the expression of type III and VI secretion systems (T3SS and T6SS) through an E. tarda secretion regulator, EsrC. In sensing low P(i) concentration, PhoB-PhoR autoregulates and activates the phosphate-specific transport operon, pstSCAB-phoU, by binding directly to the Pho box in the promoters of phoB and pstS. PhoB also binds with EsrC simultaneously on the promoter of an E. tarda virulence protein, evpA, to regulate directly the transcription of genes from T6SS. In addition, PhoB requires and interacts with PhoU to activate esrC and suppress fur indirectly through unidentified regulators. Fur, on the other hand, senses high iron concentration and binds directly to the Fur box in the promoter of evpP to inhibit EsrC binding to the same region. In addition, Fur suppresses transcription of phoB, pstSCAB-phoU, and esrC indirectly via unidentified regulators, suggesting negative cross-talk with the Pho regulon. Physical interactions exist between Fur and PhoU and between Fur and EsrC. Our findings suggest that T3SS and T6SS may carry out distinct roles in the pathogenicity of E. tarda by responding to different environmental factors.

The recently identified type VI secretion system (T6SS) is implicated in the virulence of many Gram-negative bacteria. Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also gastro- and extra-intestinal infections in humans. The E . tarda virulent protein (EVP) gene cluster encodes a conserved T6SS which contains 16 open reading frames. EvpC is one of the three major EVP secreted proteins and shares high sequence similarity with Hcp1, a key T6SS virulence factor from Pseudomonas aeruginosa. EvpC contributes to the virulence of E. tarda by playing an essential role in functional T6SS. Here, we report the crystal structure of EvpC from E. tarda PPD130/91 at a 2.8 Å resolution, along with functional studies of the protein. EvpC has a β-barrel domain with extended loops. The β-barrel consists of 11 anti-parallel β-strands with an α-helix located on one side. In solution, EvpC exists as a dimer at low concentration and as a hexamer at higher concentration. In the crystal, the symmetry related EvpC molecules form hexameric rings which stack together to form a tube similar to Hcp1. Structure based mutagenesis revealed that N-terminal negatively charged residues, Asp4, Glu15 and Glu26, and C-terminal positively charged residues, Lys161, Lys162 and Lys163, played crucial roles in the secretion of EvpC. Moreover, the localization study indicates the presence of wild type EvpC in cytoplasm, periplasm and secreted fractions, whereas the N-terminal and C-terminal mutants were found mostly in the periplasmic region and was completely absent in the secreted fraction. Results reported here provide insight into the structure, assembly and function of EvpC. Further, these findings can be extended to other EvpC homologs for understanding the mechanism of T6SS and targeting T6SS mediated virulence in Gram-negative pathogens.