High resolution spatial profiling of kidney injury and repair using RNA hybridization-based in situ sequencing.

Emerging spatially resolved transcriptomics technologies allow for the measurement of gene expression in situ at cellular resolution. We apply direct RNA hybridization-based in situ sequencing (dRNA HybISS, Cartana part of 10xGenomics) to compare male and female healthy mouse kidneys and the male kidney injury and repair timecourse. A pre-selected panel of 200 genes is used to identify cell state dynamics patterns during injury and repair. We develop a new computational pipeline, CellScopes, for the rapid analysis, multi-omic integration and visualization of spatially resolved transcriptomic datasets. The resulting dataset allows us to resolve 13 kidney cell types within distinct kidney niches, dynamic alterations in cell state over the course of injury and repair and cell-cell interactions between leukocytes and kidney parenchyma. At late timepoints after injury, C3+ leukocytes are enriched near pro-inflammatory, failed-repair proximal tubule cells. Integration of snRNA-seq dataset from the same injury and repair samples also allows us to impute the spatial localization of genes not directly measured by dRNA HybISS.

High resolution mapping of the tumor microenvironment using integrated single-cell, spatial and in situ analysis.

Single-cell and spatial technologies that profile gene expression across a whole tissue are revolutionizing the resolution of molecular states in clinical samples. Current commercially available technologies provide whole transcriptome single-cell, whole transcriptome spatial, or targeted in situ gene expression analysis. Here, we combine these technologies to explore tissue heterogeneity in large, FFPE human breast cancer sections. This integrative approach allowed us to explore molecular differences that exist between distinct tumor regions and to identify biomarkers involved in the progression towards invasive carcinoma. Further, we study cell neighborhoods and identify rare boundary cells that sit at the critical myoepithelial border confining the spread of malignant cells. Here, we demonstrate that each technology alone provides information about molecular signatures relevant to understanding cancer heterogeneity; however, it is the integration of these technologies that leads to deeper insights, ushering in discoveries that will progress oncology research and the development of diagnostics and therapeutics.

The K(+) channel GIRK2 is both necessary and sufficient for peripheral opioid-mediated analgesia.

The use of opioid agonists acting outside the central nervous system (CNS) is a promising therapeutic strategy for pain control that avoids deleterious central side effects such as apnea and addiction. In human clinical trials and rat models of inflammatory pain, peripherally restricted opioids have repeatedly shown powerful analgesic effects; in some mouse models however, their actions remain unclear. Here, we investigated opioid receptor coupling to K(+) channels as a mechanism to explain such discrepancies. We found that GIRK channels, major effectors for opioid signalling in the CNS, are absent from mouse peripheral sensory neurons but present in human and rat. In vivo transgenic expression of GIRK channels in mouse nociceptors established peripheral opioid signalling and local analgesia. We further identified a regulatory element in the rat GIRK2 gene that accounts for differential expression in rodents. Thus, GIRK channels are indispensable for peripheral opioid analgesia, and their absence in mice has profound consequences for GPCR signalling in peripheral sensory neurons.

Cell-specific effects of variants of the 68-base pair tandem repeat on prodynorphin gene promoter activity.

A polymorphic 68-bp tandem repeat has been identified within the promoter of the human prodynorphin (PDYN) gene. We found that this 68-bp repeat in the PDYN promoter occurs naturally up to five times. We studied the effect of the number of 68-bp repeats, and of a SNP (rs61761346) found within the repeat on PDYN gene promoter activity. Thirteen promoter forms, different naturally occurring combinations of repeats and the internal SNP, were cloned upstream of the luciferase reporter gene, transfected into human SK-N-SH, H69, or HEK293 cells. Cells were then stimulated with TPA or caffeine. We found cell-specific effects of the number of 68-bp repeats on the transcriptional activity of the PDYN promoter. In SK-N-SH and H69 cells, three or four repeats led to lower expression of luciferase than did one or two repeats. The opposite effect was found in HEK293 cells. The SNP also had an effect on PDYN gene expression in both SK-N-SH and H69 cells; promoter forms with the A allele had significantly higher expression than promoter forms with the G allele. These results further our understanding of the complex transcriptional regulation of the PDYN gene promoter.

Recombinant production and properties of binding of the full set of mouse secreted phospholipases A2 to the mouse M-type receptor.

To date, 12 secreted phospholipases A2 (sPLA2s) have been identified in the mouse species and divided into three structural collections (I/II/V/X, III, and XII). On the basis of their different molecular properties and tissue distributions, each sPLA2 is likely to exert distinct functions by acting as an enzyme or ligand for specific soluble proteins or receptors, among which the M-type receptor is the best-characterized target. Here, we present the properties of binding of the full set of mouse sPLA2s to the mouse M-type receptor. All enzymes have been produced in Escherichia coli or insect cells, and their properties of binding to the cloned and native M-type receptor have been determined. sPLA2s IB, IIA, IIE, IIF, and X are high-affinity ligands (K0.5 = 0.3-3 nM); sPLA2s IIC and V are low-affinity ligands (K0.5 = 30-75 nM), and sPLA2s IID, III, XIIA, and XIIB bind only very weakly or do not bind to the M-type receptor (K0.5 > 100 nM). Three exogenous parvoviral group XIII PLA2s and two fungal group XIV sPLA2s do not bind to the receptor. Together, these results indicate that the mouse M-type receptor is selective for only a subset of mouse sPLA2s from the group I/II/V/X structural collection. Binding of mouse sPLA2s to a recombinant soluble mouse M-type receptor leads in all cases to inhibition of enzymatic activity, and the extent of deglycosylation of the receptor decreases yet does not abolish sPLA2 binding. The physiological meaning of binding of sPLA2 to the M-type receptor is discussed on the basis of our current knowledge of sPLA2 functions.

Secreted phospholipase A2 inhibitors are also potent blockers of binding to the M-type receptor.

Mammalian secreted phospholipases A(2) (sPLA(2)s) constitute a family of structurally related enzymes that are likely to play numerous biological roles because of their phospholipid hydrolyzing activity and binding to soluble and membrane-bound proteins, including the M-type receptor. Over the past decade, a number of competitive inhibitors have been developed against the inflammatory-type human group IIA (hGIIA) sPLA(2) with the aim of specifically blocking its catalytic activity and pathophysiological functions. The fact that many of these inhibitors, including the indole analogue Me-Indoxam, inhibit several other sPLA(2)s that bind to the M-type receptor prompted us to investigate the impact of Me-Indoxam and other inhibitors on the sPLA(2)-receptor interaction. By using a Ca(2+) loop mutant derived from a venom sPLA(2) which is insensitive to hGIIA inhibitors but still binds to the M-type receptor, we demonstrate that Me-Indoxam dramatically decreases the affinity of various sPLA(2)s for the receptor, yet an sPLA(2)-Me-Indoxam-receptor complex can form at very high sPLA(2) concentrations. Me-Indoxam inhibits the binding of iodinated mouse sPLA(2)s to the mouse M-type receptor expressed on live cells but also enhances binding of sPLA(2) to phospholipids. Because Me-Indoxam and other competitive inhibitors protrude out of the sPLA(2) catalytic groove, it is likely that the inhibitors interfere with the sPLA(2)-receptor interaction by steric hindrance and to different extents that depend on the type of sPLA(2) and inhibitor. Our finding suggests that the various anti-inflammatory therapeutic effects of sPLA(2) inhibitors may be due not only to inhibition of enzymatic activity but also to modulation of binding of sPLA(2) to the M-type receptor or other as yet unknown protein targets.

Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity.

Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, a homologous but nontoxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1-22) of OS2, but not the central one (residues 58-89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102-119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera, which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity.

Structure and function comparison of Micropechis ikaheka snake venom phospholipase A2 isoenzymes.

Comparison of the crystal structures of three Micropechis ikaheka phospholipase A2 isoenzymes (MiPLA2, MiPLA3 and MiPLA4, which exhibit different levels of pharmacological effects) shows that their C-terminus (residues 110-124) is the most variable. M-Type receptor binding affinity of the isoenzymes has also been investigated and MiPLA4 binds to the rabbit M-type receptor with high affinity. Examination of surface charges of the isoenzymes reveals a trend of increase in positive charges with potency. The isoenzymes are shown to oligomerize in a concentration-dependent manner in a semi-denaturing gel. The C-termini of the medium (MiPLA4) and highly potent (MiPLA2) isoenzyme molecules cluster together, forming a highly exposed area. A BLAST search using the sequence of the most potent MiPLA2 results in high similarity to Staphylococcus aureus clotting factor A and cadherin 11. This might explain the myotoxicity, anticoagulant and hemoglobinuria effects of MiPLA2s.

Novel mammalian group XII secreted phospholipase A2 lacking enzymatic activity.

An increasing number of mammalian secreted phospholipases A(2) (sPLA(2)s) has been identified over the past few years. Here, we report the identification and recombinant expression of a novel sPLA(2)-like protein in mouse and human species that has been called group XIIB (GXIIB). The mature protein has a molecular mass of 19.7 kDa and structural features similar to those of the previously identified GXII sPLA(2), now called GXIIA. Strikingly, the GXIIB sPLA(2) has a mutation in the active site, replacing the canonical histidine by a leucine, suggesting that this sPLA(2) is catalytically inactive. Recombinant expression of human (hGXIIB) and mouse (mGXIIB) sPLA(2)s in Escherichia coli indicates that GXIIB sPLA(2)s display no measurable lipolytic activity on various types of phospholipid substrates. Furthermore, these sPLA(2)-like proteins display relatively weak affinity to phospholipid vesicles. Binding experiments indicate that these proteins are also unable to bind to the well-known M-type sPLA(2) receptor. The RNA tissue distribution of GXIIB sPLA(2)s is distinct from that of other sPLA(2)s including the homologous GXIIA. Strong expression was observed in liver, small intestine, and kidney in both human and mouse species. Interestingly, the expression of the novel sPLA(2) is dramatically decreased in human tumors from the same tissues. The absence of enzymatic activity suggests that the GXIIB sPLA(2)-like proteins probably exert their biological roles by acting as ligands for as yet unidentified receptors.

Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2.

Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A(2) (sPLA(2)s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA(2)s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1-225 microm range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA(2)s, with Me-Indoxam being the most generally potent sPLA(2) inhibitor. A dramatic correlation exists between the ability of the sPLA(2)s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA(2)s are uniquely efficient in this regard.