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Background: The differentiation between Gaucher disease type 3 (GD3) and type 1 is challenging because pathognomonic neurologic symptoms may be subtle and develop at late stages. The ophthalmologist plays a crucial role in identifying the typical impairment of horizontal saccadic eye movements, followed by vertical ones. Little is known about further ocular involvement. The aim of this monocentric cohort study is to comprehensively describe the ophthalmological features of Gaucher disease type 3. We suggest recommendations for a set of useful ophthalmologic investigations for diagnosis and follow up and for saccadometry parameters enabling a correlation to disease severity.
Methods: Sixteen patients with biochemically and genetically diagnosed GD3 completed ophthalmologic examination including optical coherence tomography (OCT), clinical oculomotor assessment and saccadometry by infrared based video-oculography. Saccadic peak velocity, gain and latency were compared to 100 healthy controls, using parametric tests. Correlations between saccadic assessment and clinical parameters were calculated.
Results: Peripapillary subretinal drusen-like deposits with retinal atrophy (2/16), preretinal opacities of the vitreous (4/16) and increased retinal vessel tortuosity (3/16) were found. Oculomotor pathology with clinically slowed saccades was more frequent horizontally (15/16) than vertically (12/16). Saccadometry revealed slowed peak velocity compared to 100 controls (most evident horizontally and downwards). Saccades were delayed and hypometric. Best correlating with SARA (scale for the assessment and rating of ataxia), disease duration, mSST (modified Severity Scoring Tool) and reduced IQ was peak velocity (both up- and downwards). Motility restriction occurred in 8/16 patients affecting horizontal eye movements, while vertical motility restriction was seen less frequently. Impaired abduction presented with esophoria or esotropia, the latter in combination with reduced stereopsis.
Conclusions: Vitreoretinal lesions may occur in 25% of Gaucher type 3 patients, while we additionally observed subretinal lesions with retinal atrophy in advanced disease stages. Vertical saccadic peak velocity seems the most promising "biomarker" for neuropathic manifestation for future longitudinal studies, as it correlates best with other neurologic symptoms. Apart from the well documented abduction deficit in Gaucher type 3 we were able to demonstrate motility impairment in all directions of gaze.
Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection.
Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was revealed that, under optimal conditions, H. volcanii contains more non-coding sRNAs than protein-encoding mRNAs. However, the dRNA-Seq approach did not contain any length information. Therefore, a mixed RNA-Seq approach was used to determine transcript length and to identify additional transcripts, which are not present under optimal conditions. In total, 50 million paired end reads of 150 nt length were obtained. 1861 protein-coding RNAs (cdRNAs) were detected, which encoded 3092 proteins. This nearly doubled the coverage of cdRNAs, compared to the previous dRNA-Seq study. About 2/3 of the cdRNAs were monocistronic, and 1/3 covered more than one gene. In addition, 1635 non-coding sRNAs were identified. The highest fraction of non-coding RNAs were cis antisense RNAs (asRNAs). Analysis of the length distribution revealed that sRNAs have a median length of about 150 nt. Based on the RNA-Seq and dRNA-Seq results, genes were chosen to exemplify characteristics of the H. volcanii transcriptome by Northern blot analyses, e.g. 1) the transcript patterns of gene clusters can be straightforward, but also very complex, 2) many transcripts differ in expression level under the four analyzed conditions, 3) some genes are transcribed into RNA isoforms of different length, which can be differentially regulated, 4) transcripts with very long 5’-UTRs and with very long 3’-UTRs exist, and 5) about 30% of all cdRNAs have overlapping 3’-ends, which indicates, together with the asRNAs, that H. volcanii makes ample use of sense-antisense interactions. Taken together, this RNA-Seq study, together with a previous dRNA-Seq study, enabled an unprecedented view on the H. volcanii transcriptome.
Background: Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii.
Results: Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5′-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs).
Conclusion: This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated.
Diese Arbeit behandelt das Thema der Darstellung und der Simulation von Pflanzen mit Lindenmayer-Systemen. Zur Darstellung der aus Lindenmayer- Systemen entwickelten Strukturen wird das Programm Linde 3D entwickelt, welches dem Benutzer das Erstellen und die Simulation von Objekten unter Verwendung von deterministischen, geschachtelten, stochastischen, kontextsensitiven, umgebungssensitiven und offenen Lindenmayer-Systemen ermöglicht.
Neben der Entwicklung des Programms Linde 3D liegt ein weiterer Schwerpunkt dieser Arbeit auf der Simulation der biologischen Prozesse Vernalisation und Stratifikation. Für die Simulation dieser Prozesse werden Lindenmayer- Systeme entwickelt, welche die Grundfunktionalität der Prozesse simulieren und in Abhängigkeit dieser dreidimensionale Modelle der Pflanze erzeugen.
Das Programm Linde 3D ist so konzipiert, dass es allgemein eingesetzt werden kann. Neben dem Verständnis für die abstrakten Modelle der Lindenmayer-Systeme werden keine speziellen Kenntnisse des Anwenders vorausgesetzt. Die Eingabe der L-Systeme erfolgt entweder über die Auswahl vordefinierter Lindenmayer-Systeme und Umweltdaten oder durch Komposition von Lindenmayer-Systemen und Umweltdaten durch den Anwender. Die graphische Interpretation der Lindenmayer-Systeme erfolgt unter Verwendung des Schildkröten-Modells. Die Ausgabe des Programms Linde 3D besteht zum einen aus einer direkten Darstellung der generierten Szene im Programm und zum anderen aus der Ausgabe der Szene in Form einer oder mehrerer Dateien für den POVRay Raytracer. Die erzeugten Dateien können durch externe Programme aufbereitet und zu einer Animation zusammengefügt werden.
Die vorliegende Arbeit beginnt mit einer kurzen Einführung in das Thema der Fraktale und Lindenmayer-Systeme, sowie den nötigen Grundlagen für das Verständnis der biologischen Hintergründe. Im Anschluss werden dem Leser die notwendigen theoretischen Grundkenntnisse zu Lindenmayer-Systemen und ein Einblick in aktuelle Anwendungen und Entwicklungen vermittelt. Nach einer Beschreibung der Anforderungen, des Aufbaus und der Implementierung des Programms Linde 3D werden die erworbenen Grundkenntnisse im Bereich der Lindenmayer-Systeme und das Programm Linde 3D eingesetzt, um Schritt für Schritt Lindenmayer-Systeme für die Simulation der biologischen Prozesse Vernalisation und Stratifikation zu entwickeln. Nach der Konstruktion der L-Systeme werden die erworbenen theoretischen Grundlagen für den Bereich des Testens von Parser und Schildkröten-Modell auf Funktionalität benötigt. Im Ausblick werden Ideen für Anwendungen und Erweiterungen des Programms Linde 3D beschrieben.
We report on posttransplant relapsed pediatric patients with B-precursor acute lymphoblastic leukemia with no further standard of care therapy who were treated with the T-cell engaging CD19/CD3-bispecific single-chain antibody construct blinatumomab on a compassionate use basis. Blast load was assessed prior to, during and after blinatumomab cycle using flow cytometry to detect minimal residual disease, quantitative polymerase chain reaction for rearrangements of the immunoglobulin or T-cell receptor genes, and bcr/abl mutation detection in one patient with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dosage of 5 or 15 μg/m2/day. Nine patients received a total of 18 cycles. Four patients achieved complete remission after the first cycle of treatment; 2 patients showed a complete remission from the second cycle after previous reduction of blast load by chemotherapy. Three patients did not respond, of whom one patient proceeded to a second cycle without additional chemotherapy and again did not respond. Four patients were successfully retransplanted in molecular remission from haploidentical donors. After a median follow up of 398 days, the probability of hematologic event-free survival is 30%. Major toxicities were grade 3 seizures in one patient and grade 3 cytokine release syndrome in 2 patients. Blinatumomab can induce molecular remission in pediatric patients with posttransplant relapsed B-precursor acute lymphoblastic leukemia and facilitate subsequent allogeneic hematopoietic stem cell transplantation from haploidentical donor with subsequent long-term leukemia-free survival.