Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

Prostate-derived Sterile 20-like Kinases (PSKs/TAOKs) Phosphorylate Tau and are Activated in Tangle-bearing Neurons in Alzheimer's Disease

In Alzheimer disease (AD), the microtubule-associated protein tau is highly phosphorylated and aggregates into characteristic neurofibrillary tangles. Prostate-derived sterile 20-like kinases (PSKs/TAOKs) 1 and 2, members of the sterile 20 family of kinases, have been shown to regulate microtubule stability and organization. Here we show that tau is a good substrate for PSK1 and PSK2 phosphorylation with mass spectrometric analysis of phosphorylated tau revealing more than 40 tau residues as targets of these kinases. Notably, phosphorylated residues include motifs located within the microtubule-binding repeat domain on tau (Ser-262, Ser-324, and Ser-356), sites that are known to regulate tau-microtubule interactions. PSK catalytic activity is enhanced in the entorhinal cortex and hippocampus, areas of the brain that are most susceptible to Alzheimer pathology, in comparison with the cerebellum, which is relatively spared. Activated PSK is associated with neurofibrillary tangles, dystrophic neurites surrounding neuritic plaques, neuropil threads, and granulovacuolar degeneration bodies in AD brain. By contrast, activated PSKs and phosphorylated tau are rarely detectible in immunostained control human brain. Our results demonstrate that tau is a substrate for PSK and suggest that this family of kinases could contribute to the development of AD pathology and dementia

The Interaction between Enterobacteriaceae and Calcium Oxalate Deposits.

The role of calcium oxalate crystals and deposits in UTI pathogenesis has not been established. The objectives of this study were to identify bacteria present in pediatric urolithiasis and, using in vitro and in vivo models, to determine the relevance of calcium oxalate deposits during experimental pyelonephritis.Pediatric kidney stones and urine were collected and both cultured and sequenced for bacteria. Bacterial adhesion to calcium oxalate was compared. Murine kidney calcium oxalate deposits were induced by intraperitoneal glyoxalate injection and kidneys were transurethrally inoculated with uropathogenic Escherichia coli to induce pyelonephritis.E. coli of the family Enterobacteriaceae was identified in patients by calcium oxalate stone culture. Additionally Enterobacteriaceae DNA was sequenced from multiple calcium oxalate kidney stones. E. coli selectively aggregated on and around calcium oxalate monohydrate crystals. Mice inoculated with glyoxalate and uropathogenic E. coli had higher bacterial burdens, increased kidney calcium oxalate deposits and an increased kidney innate immune response compared to mice with only calcium oxalate deposits or only pyelonephritis.In a murine model, the presence of calcium oxalate deposits increases pyelonephritis risk, likely due to preferential aggregation of bacteria on and around calcium oxalate crystals. When both calcium oxalate deposits and uropathogenic bacteria were present, calcium oxalate deposit number increased along with renal gene transcription of inner stone core matrix proteins increased. Therefore renal calcium oxalate deposits may be a modifiable risk factor for infections of the kidney and urinary tract. Furthermore, bacteria may be present in calcium oxalate deposits and potentially contribute to calcium oxalate renal disease

Bacteria increases the murine CaOx deposit burden.

<p>A. The mean right (A), left (B) and combined mean (C) kidney bacterial burdens were lower with UPEC inoculation alone compared to kidneys with CaOx deposits and UPEC inoculation at 56,157±1.68X10⁵ versus 2.14X10⁶±3.34X10⁶; 24,843±69,527 versus 4.50X10⁶±7.83X10⁶ and 40,500±1.12X10⁵ versus 5.28X10⁶±1.78X10⁶ respectively. To present on a log scale graph, but not during statistical analysis, 0 values were assigned a value of 0.01 (D). Following glyoxalate injection, CaOx deposits are seen around the corticomedullary junction (arrows) (E) Following UPEC inoculation and glyoxalate injection, an increased distribution and number of CaOx deposits (arrows) is noted, extending into the medulla (arrowheads). D-E, representative 4X magnification image stitches, background cropped for clarity, scale bar = 1000μm. (F) CaOx deposit number per mean 4X imaging stitch cross-section area was significantly higher in the CaOx deposits and UPEC inoculation group compared to kidney stones alone. There was a higher percentage of CaOx deposit/total kidney cross section area (H) with CaOx deposits and UPEC than CaOx deposits alone. The location on the scatterplot for the representative images (D) and (E) are indicated by an arrowhead and arrow respectively.</p

NAD(P)H‐dependent enzymes for reductive amination: active site description and carbonyl‐containing compound spectrum

International audienceThe biocatalytic asymmetric synthesis of amines from carbonyl compounds and amine precursors presents an important advance in sustainable synthetic chemistry. Oxidoreductases (ORs) that catalyze the NAD(P)H-dependent reductive amination of carbonyl compounds directly to amines using amine donors present advantages complementary to those of amine transaminases (ATAs) with respect to selectivity, stability and substrate scope. Indeed some ORs accept alkyl and aryl amines as reaction partners enabling access to chiral secondary amine products that are not directly accessible using ATAs. Moreover, superior atom economy can usually be achieved as no sacrificial amines are required as with ATAs. In recent years a number of ORs that apparently catalyze both imine formation and imine reduction in the reductive amination of carbonyls has been identified using structure informed protein engineering, sequence analysis from natural biodiversity and increasingly a mixture of both. In this review we summarize the development of such enzymes from the engineering of amino acid dehydrogenases (AADHs) and opine dehydrogenases (OpDHs) to become amine dehydrogenases (AmDHs), which are active toward ketones devoid of any requisite carboxylate and/or amine functions, through to the discovery of native AmDHs and reductive aminases (RedAms), and the engineering of all of these scaffolds for improved or altered activity. Structural and mechanistic studies have revealed similarities, but also differences in the determinants of substrate binding and mechanism in the enzymes. The survey reveals that a complementary approach to enzyme discovery that utilizes both natural genetic resources and engineering can be combined to deliver biocatalysts that have significant potential for the industrial synthesis of chiral amines

Speculated mechanisms for bacterial contribution to CaOx stones.

<p>(A) Figure key. (B) Bacteria bind to CaOx crystals that may provide a nidus for pyelonephritis or remain persist in a subclinical state (1) and bacterial communities form a biofilm (2). The biofilm results in crystal aggregation (3). (C) The bacterial enzymes citrate lyase splits citrate resulting in increased CaOx supersaturation (1). CaOx crystals form providing a key element of lithogenesis. (D) Bacteria bind to the urothelium (1) that results in secretion of innate immune proteins from recruited inflammatory cells (2) and the urothelium (3) The innate immune proteins are incorporated as stone matrix proteins.</p

UPEC selectively aggregate around CaOx monohydrate crystals: following incubation with GFP labeled UPEC, bacteria (green) could be seen aggregating around CaOx monohydrate (A, arrows) but not CaOx dihydrate (A, arrowheads) or silicon dioxide (B, arrows) crystals.

<p>At 12 hours, significantly more bacteria per crystal surface area were seen with CaOx monohydrate crystals than with CaOx dihydrate crystals, silicon dioxide crystals or background. There were no other significant differences between groups. Magnification 40X right panels, 100X left panels Scale bars = 20 microns. Incubation time = 6 hours for left panels and 12 hours for right panels.</p

Patient Characteristics.

<p>^{^} different stone from same patient than stone used for microbiome sequencing.</p><p>^# random urine sample. Citrate/creatinine (Cit/Cr) normal is > 0.18 mg/mg. Oxalate to creatinine normal is < 0.1 mg/mg. Calcium/creatinine (Ca/Cr) normal is < 0.21 mg/mg.</p><p>Patient Characteristics.</p

Induction of CaOx deposits and experimental pyelonephritis in mice.

<p>Induction of CaOx deposits and experimental pyelonephritis in mice.</p