Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.     

Reconstitution of EBV-directed T cell immunity by adoptive transfer of peptide-stimulated T cells in a patient after allogeneic stem cell transplantation for AITL

Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRβ) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.     

Osteopathy of the pectoral and pelvic limbs including pentadactyly in a young Kestrel (Falco t. tinnunculus)

Summary A five and a half weeks old female Kestrel exhibiting osteopathy of the pectoral and pelvic limbs, including symmetrical hyperdactyly, was investigated in order to clarify the pattern of the involved anatomical alterations and the possible causes of this developmental malformation. In the pectoral limb it consisted of a triplication of the alular digit, in the pelvic limb of a duplication of digit I. The live young Kestrel was observed for a period of two weeks to ascertain that it was unable to fly or procure prey on its own. After its death radiographs were taken and, apart from an eidonomic inspection including the wing claws, a detailed macroscopic dissection of the musculature of the pectoral and pelvic limbs was carried out using the in-water-method. Consecutive dissection steps were documented by a series of photographic slides. The relevant musculature, particularly that of the supernumerary digits, was recorded in proportional drawings. Subsequent to maceration of the limbs the isolated bones were reassembled according to the radiographs and also documented by means of photographs and drawings. This anatomical approach produced a reliable reconstruction of the skeletomuscular apparatus of the hyperdactylous limb parts. The eidonomic inspection revealed that at least young Kestrels may have two (alular and major digit) or even three wing claws per side. The proximal skeletal elements of both pectoral and pelvic limb were more sturdily built than in a typical Kestrel of comparable age. The proximal elements of the pelvic limb, the tarsometatarsus in particular, were shorter than in a typical Kestrel. In addition, the long axis of the tarsometatarsus was laterally bent in the transverse plane so that its proximal articular surfaces were medially inclined. Duplication of the cutaneous and osseous elements in the foot was accompanied by a duplication of some of the muscular and/or tendinous elements supplying digit I proper and the accessory digit I'. There were left-to-right asymmetries of the pedal musculature concerned. In contrast, the two accessory alular digits of each wing were almost completely devoid of musculature. Apart from atypical points of origin or insertion of the remaining distal musculture, left-to-right asymmetries and the two accessory alulae per wing, presumably, affected aerodynamic properties and resulted in flightlessness.A juvenile Kestrel of similar age and without hyperdactyly was dissected for comparison. In addition, the external appearance of the carpometacarpal region of two female Silkies, an obligatory pentadactylous breed of domestic fowl, was inspected and the skeletal parts of their pectoral and pelvic limbs compared with those of the hyperdactylous Kestrel. Our results and a literature review suggest that the symmetrical hyperdactyly in the Kestrel bears striking similarities to the hereditary hyperdactyly observed in certain breeds of domestic fowl. In addition, there is a striking resemblance between the hyperdactyly of the young Kestrel and certain forms of hyperdactyly induced by molecular genetical experiments of other authors on chicks. Comparison with these results taken from the literature suggest that the symmetrical hyperdactyly in the young Kestrel, including the alterations of the proximal skeletal elements, is caused by an unusually early expression of the Hoxd-11 gene group during embryological development. Most likely, this gene group is situated on the 2^nd chromosome in birds just as it is in mammals.

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Osteopathie der Vorder - und Hinterextremitäten, verbunden mit einer symmetrischen Hyperdactylie bei einem jungen Turmfalken (Falco tinnunculus)

Zusammenfassung Ein fünfeinhalb Wochen alter weiblicher Turmfalke mit einer Osteopathie der Vorder- und Hinterextremitäten, verbunden mit einer symmetrischen Hyperdactylie, wurde untersucht, um das Muster der beteiligten anatomischen Veränderungen und die möglichen Ursachen dieser Mißbildung zu erkennen. An der Vorderextremität bestand sie aus einer Verdreifachung des Alula-Fingers, an der Hinterextremität aus einer Verdoppelung der Zehe I. Die Beobachtung des lebenden jungen Turmfalken während eines Zeitraumes von zwei Wochen ergab, dass er flugunfähig war und keine Beute schlagen konnte.Nach seinem Tod und einer Inspektion der Eidonomie, einschließlich der Flügelkrallen, wurden Röntgenaufnahmen angefertigt. Danach folgte eine detaillierte makroskopische Präparation der Flügel- und Beinmuskulatur unter Verwendung der In-Wasser-Methode. Die einzelnen Präparationsschritte wurden anhand von Dia-Serien dokumentiert. Die relevante Muskulatur, insbesondere die der überzähligen Digiti, wurde in proportionsgetreuen Zeichnungen festgehalten. Nach Mazeration der Extremitäten wurden die Einzelknochen, entsprechend den Röntgenbildern, wieder zusammengesetzt und ebenfalls mit Fotografien und Zeichnungen dokumentiert. Dieser anatomische Ansatz lieferte eine zuverlässige Rekonstruktion des Skelett-Muskel-Apparates der hyperdactylen Extremitätenanteile.Die eidonomische Inspektion ergab, dass zumindest junge Turmfalken zwei (Digitus alularis und majoris) oder sogar drei Flügelkrallen haben können. Die proximalen Skelettelemente der Vorder- und Hinterextremität waren deutlich robuster gebaut als bei einem typischen Turmfalken vergleichbaren Alters. Die proximalen Elemente der Hinterextremität, insbesondere der Tarsometatarsus, waren kürzer als bei einem typischen Turmfalken. Darüberhinaus war die Längsachse des Tarsometatarsus in der Transversalebene laterad gekrümmt, so dass sich seine proximalen Gelenkflächen schräg mediad richteten. Entsprechend der kutanen und knöchernen Doppelbildungen des Fußes waren auch einige der Muskeln und Sehnen doppelt vorhanden, welche die eigentliche erste Zehe und die akzessorische erste Zehe versorgten. Es traten Rechts-/Links-Asymmetrien der betreffenden Muskulatur auf. Im Gegensatz dazu waren die beiden akzessorischen Alula-Finger jedes Flügels fast vollständig ohne Muskulatur. Abgesehen von atypischen Ursprungs- und Insertionspunkten der verbleibenden distalen Muskulatur, beeinträchtigten Rechts-/Links-Asymmetrien und die beiden akzessorischen Alulae pro Flügel vermutlich die aerodynamischen Eigenschaften und führten zur Flugunfähigkeit.Ein junger Turmfalke ähnlichen Alters ohne Hyperdactylie wurde zum Vergleich präpariert. Zusätzlich wurde die äußere Erscheinung der Carpometacarpal-Region zweier Seidenhühner, einer obligatorisch pentadactylen Hühnerrasse, inspiziert und die Skelettelemente ihrer Vorder- und Hinterextremitäten mit denen des hyperdactylen Turmfalken verglichen. Unsere Ergebnisse und ein Überblick der Literatur lassen auffallende Übereinstimmungen zwischen der symmetrischen Hyperdactylie des jungen Turmfalken und der erblichen Hyperdactylie bestimmter Hühnerrassen erkennen. Darüberhinaus besteht eine auffallende übereinstimmung zwischen der Hyperdactylie des jungen Turmfalken und bestimmten Formen der Hyperdactylie, welche von anderen Autoren durch molekulargenetische Experimente an Hühnerküken induziert wurden. Ein Vergleich mit diesen Ergebnissen aus der Literatur legt nahe, dass die symmetrische Hyperdactylie des jungen Turmfalken, einschließlich der Veränderungen der proximalen Skelettelemente, durch eine ungewöhnlich frühe Expression der Hoxd-11 Gengruppe im Laufe der Embryonalentwicklung verursacht wurde. Sehr wahrscheinlich ist diese Gengruppe bei Vögeln auf dem zweiten Chromosom lokalisiert — ebenso wie bei Säugetieren.

     

Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines

Virulence in Toxoplasma gondii is strongly influenced by the genotype of the parasite. Type I strains uniformly cause rapid death in mice regardless of the host genotype or the challenge dose. In contrast, the outcome of infections with type II strains is highly dependent on the challenge dose and the genotype of the host. To understand the basis of acute virulence in toxoplasmosis, we compared low and high doses of the RH strain (type I) and the ME49/PTG strain (type II) of T. gondii in outbred mice. Differences in virulence were reflected in only modestly different growth rates in vivo, and both strains disseminated widely to different tissues. The key difference in the virulent RH strain was the ability to reach high tissue burdens rapidly following a low dose challenge. Lethal infections caused by type I (RH) or type II (PTG) strain infections were accompanied by extremely elevated levels of Th1 cytokines in the serum, including IFN-gamma, TNF-alpha, IL-12, and IL-18. Extensive liver damage and lymphoid degeneration accompanied the elevated levels of cytokines produced during lethal infection. Increased time of survival following lethal infection with the RH strain was provided by neutralization of IL-18, but not TNF-alpha or IFN-gamma. Nonlethal infections with a low dose of type II PTG strain parasites were characterized by a modest induction of Th1 cytokines that led to control of infection and minimal damage to host tissues. Our findings establish that overstimulation of immune responses that are normally necessary for protection is an important feature of acute toxoplasmosis.     

Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.     

Down-regulation by nuclear factor kappaB of human 25-hydroxyvitamin D3 1alpha-hydroxylase promoter

1,25-(OH)(2) vitamin D(3) is important for calcium homeostasis and cell differentiation. The key enzyme for the activation of liver-derived 25(OH) vitamin D(3) is 25-hydroxyvitamin D(3) 1alpha-hydroxylase. It is expressed mainly in the kidney but also in peripheral tissues. A 1413-bp fragment of the 1alpha-hydroxylase promoter was cloned into luciferase vectors pGL2basic and pGL3basic. Sequence analyses revealed four base exchanges and three base deletions compared with the published sequence which were identically found in five control persons. In silico promoter analyses revealed 17 putative nuclear factor (NF)kappaB sites, 10 of which were found to bind NFkappaB in EMSA experiments. Cotransfection of NFkappaB p50 and p65 subunits resulted in dramatic reduction of the promoter activity of the full-length construct as well as a series of 5'-deletion constructs. Deletion of the farmost 3'-situated NFkappaB-responsive element almost abolished NFkappaB responsiveness. Treatment of human embryonic kidney 293 cells with sulfasalazine, a NFkappaB inhibitor, resulted in enhanced 1alpha-hydroxylase mRNA production. Down-regulation of 1alpha-hydroxylase promoter through NFkappaB signaling may contribute to the pathogenesis of inflammation-associated osteopenia/osteoporosis.     

Inhibition of basal and interleukin-1-induced VCAM-1 expression by phospholipid hydroperoxide glutathione peroxidase and 15-lipoxygenase in rabbit aortic smooth muscle cells

Cytokines or hydroperoxides upregulate cell adhesion molecules (CAM) in early stages of atherosclerosis. VCAM-1 expression was therefore investigated in rabbit aortic smooth muscle cells (SMC) stably transfected either with phospholipid hydroperoxide glutathione peroxidase (PHGPx; SMCPHGPx) as a hydroperoxide-reducing enzyme or with 15-lipoxygenase (15-LOX; SMCLOX) as a hydroperoxide-producing enzyme. Transfected cells showed up to 3-fold enhanced PHGPx and a marked LOX activity, respectively, that was absent in controls. Intracellular hydroperoxides were 6-fold higher in SMCLOX than in SMC or SMCPHGPx. Intracellular protein thiols were decreased by 50 and 90% in SMCPHGPx and SMCLOX, respectively. Glutathione mixed disulfides were tentatively increased from SMC via SMCPHGPx to SMCLOX, accordingly. Thiol reduction with tris(2-carboxyethyl)phosphine completely restored protein thiols in SMCPHGPx, whereas in SMCLOX only 60% of control values were recovered. Basal VCAM-1 mRNA levels were decreased by 50% in SMCPHGPx and 75% in SMCLOX. VCAM-1-inducibility was abrogated in SMCLOX but not in SMCPHGPx. Accordingly, NFkappaB-driven reporter gene activation by IL-1 was unaffected in SMCPHGPx but abolished in SMCLOX. The data confirm that PHGPx overexpression dampens CAM expression either by lowering stimulatory hydroperoxides or by using hydroperoxides for protein modification. But hydroperoxides, when constitutively overproduced as in SMCLOX, inhibit CAM expression and render cells refractory to IL-1 stimulation likely due to oxidation of protein thiols of the signaling system.     

Enantiomeric resolution of kielcorin derivatives by HPLC on polysaccharide stationary phases using multimodal elution

Analytical HPLC methods using carbamate chiral stationary phases of polysaccharide derivatives were developed for the enantiomeric resolution of five racemic mixtures of xanthonolignoids: rac-trans-kielcorin C, rac-cis-kielcorin C, rac-trans-kielcorin D, rac-trans-isokielcorin D, and rac-trans-kielcorin E. The separations were evaluated with the stationary phases cellulose tris-3,5-dimethylphenylcarbamate, amylose tris-3,5-dimethylphenylcarbamate, amylose tris-(S)-1-phenylethylcarbamate, and amylose tris-3,5-dimethoxyphenylcarbamate under normal, reversed-phase, and polar organic elution conditions. Chiral recognition of those chiral stationary phases, the influence of mobile phases on the enantiomers separation, and the effects of structural features of the solutes on the chiral discrimination observed are discussed. The best performance was achieved on an amylose tris-3,5-dimethylphenylcarbamate phase. Polar organic conditions gave shorter retention factors and better resolutions and were a valuable alternative to the alcohol-hexane or reversed-phase conditions.     

Bone marrow stromal cells improve cardiac performance in healed infarcted rat hearts

Postinfarct congestive heart failure is one of the leading causes of morbidity and mortality in developed and developing countries. The main purpose of this study was to investigate whether transplantation of bone marrow stromal cells (BMSC) directly into the myocardium could improve the performance of healed infarcted rat hearts. Cell culture medium with or without BMSC was injected into borders of cardiac scar tissue 4 wk after experimental infarction. Cardiac performance was evaluated 2 wk after cellular (n = 10) or medium (n = 10) injection by electro- and echocardiography. Histological study was performed 3 wk after treatment. Electrocardiography of BMSC-treated infarcted rats showed electrical and mechanical parameters more similar to those in control than in medium-treated animals: a normal frontal QRS axis in 6 of 10 BMSC-treated and all control rats and a rightward deviation of the QRS axis in all 10 medium-treated animals. BMSC treatment, assessed by echocardiography, improved fractional shortening (39.00 +/- 4.03%) compared with medium-treated hearts (18.20 +/- 0.74%) and prevented additional changes in cardiac geometry. Immunofluorescence microscopy revealed colocalization of 4',6-diamidino-2-phenylindole-labeled nuclei of transplanted cells with cytoskeletal markers for cardiomyocytes and smooth muscle cells, indicating regeneration of damaged myocardium and angiogenesis. These data provide strong evidence that BMSC implantation can improve cardiac performance in healed infarctions and open new promising therapeutic opportunities for patients with postinfarction heart failure.