Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Version 4.2020, NCCN Clinical Practice Guidelines in Oncology.

Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are characterized by a progressive accumulation of leukemic cells in the peripheral blood, bone marrow, and lymphoid tissues. Treatment of CLL/SLL has evolved significantly in recent years because of the improved understanding of the disease biology and the development of novel targeted therapies. In patients with indications for initiating treatment, the selection of treatment should be based on the disease stage, patient's age and overall fitness (performance status and comorbid conditions), and cytogenetic abnormalities. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with CLL/SLL.

NCCN Guidelines Insights: Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Version 2.2019.

Chronic lymphocytic leukemia (CLL) is generally characterized by an indolent disease course. Histologic transformation (also known as Richter's transformation) to more aggressive lymphomas, such as diffuse large B-cell lymphoma or Hodgkin lymphoma, occurs in approximately 2% to 10% of patients and is associated with a poor prognosis. These NCCN Guidelines Insights discuss the recommendations for the diagnosis and management of patients with histologic transformation.

Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose-regulated GTH1 promoter of Pichia pastoris.

Protein production in Pichia pastoris is often based on the methanol-inducible P AOX1 promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose-regulated P GTH1 promoter which we reported recently. To further increase its potential, we investigated its regulation in more details by the screening of promoter variants harboring deletions and mutations. Thereby we could identify the main regulatory region and important putative transcription factor binding sites of P GTH1 . Concluding from that, yeast metabolic regulators, monomeric Gal4-class motifs, carbon source-responsive elements, and yeast GC-box proteins likely contribute to the regulation of the promoter. We engineered a P GTH1 variant with greatly enhanced induction properties compared with that of the wild-type promoter. Based on that, a model-based bioprocess design for high volumetric productivity in a limited time was developed for the P GTH1 variant, to employ a glucose fed-batch strategy that clearly outperformed a classical methanol fed-batch of a P AOX1 strain in terms of titer and process performance.

Hairy Cell Leukemia, Version 2.2018, NCCN Clinical Practice Guidelines in Oncology.

Hairy cell leukemia (HCL) is a rare type of indolent B-cell leukemia, characterized by symptoms of fatigue and weakness, organomegaly, pancytopenia, and recurrent opportunistic infections. Classic HCL should be considered a distinct clinical entity separate from HCLvariant (HCLv), which is associated with a more aggressive disease course and may not respond to standard HCL therapies. Somatic hypermutation in the IGHV gene is present in most patients with HCL. The BRAF V600E mutation has been reported in most patients with classic HCL but not in those with other B-cell leukemias or lymphomas. Therefore, it is necessary to distinguish HCLv from classic HCL. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of classic HCL.

Highly efficient production of rare sugars D-psicose and L-tagatose by two engineered D-tagatose epimerases.

Rare sugars are monosaccharides that do not occur in nature in large amounts. However, many of them demonstrate high potential as low-calorie sweetener, chiral building blocks or active pharmaceutical ingredients. Their production by enzymatic means from broadly abundant epimers is an attractive alternative to synthesis by traditional organic chemical means, but often suffers from low space-time yields and high enzyme costs due to rapid enzyme degradation. Here we describe the detailed characterization of two variants of d-tagatose epimerase under operational conditions that were engineered for high stability and high catalytic activity towards the epimerization of d-fructose to d-psicose and l-sorbose to l-tagatose, respectively. A variant optimized for the production of d-psicose showed a very high total turnover number (TTN) of up to 10(8) catalytic events over a catalyst's lifetime, determined under operational conditions at high temperatures in an enzyme-membrane reactor (EMR). Maximum space-time yields as high as 10.6 kg L(-1) d(-1) were obtained with a small laboratory-scale EMR, indicating excellent performance. A variant optimized for the production of l-tagatose performed less stable in the same setting, but still showed a very good TTN of 5.8 × 10(5) and space-time yields of up to 478 g L(-1) d(-1) . Together, these results confirm that large-scale enzymatic access to rare sugars is feasible.

A separation-integrated cascade reaction to overcome thermodynamic limitations in rare-sugar synthesis.

Enzyme cascades combining epimerization and isomerization steps offer an attractive route for the generic production of rare sugars starting from accessible bulk sugars but suffer from the unfavorable position of the thermodynamic equilibrium, thus reducing the yield and requiring complex work-up procedures to separate pure product from the reaction mixture. Presented herein is the integration of a multienzyme cascade reaction with continuous chromatography, realized as simulated moving bed chromatography, to overcome the intrinsic yield limitation. Efficient production of D-psicose from sucrose in a three-step cascade reaction using invertase, D-xylose isomerase, and D-tagatose epimerase, via the intermediates D-glucose and D-fructose, is described. This set-up allowed the production of pure psicose (99.9%) with very high yields (89%) and high enzyme efficiency (300 g of D-psicose per g of enzyme).

Clinical outcomes of amniotic membrane transplantation in the management of acute ocular chemical injury.

BACKGROUND: Amniotic membrane transplantation (AMT) has been used in the management of acute ocular chemical burns to promote epithelialisation, reduce inflammation and restore ocular surface integrity. The aim of this study is to analyse the morphological and functional outcomes of patients receiving AMT after ocular chemical burn. METHODS: We performed a retrospective analysis of all patients treated for acute ocular chemical burn between 1998 and 2008 in two participating centres (University of Duisburg-Essen, Germany and Royal Victoria Infirmary, Department of Ophthalmology, Newcastle University, UK). Ocular chemical burns were classified by Roper-Hall and Dua classifications. RESULTS: 72 eyes of 54 consecutive patients aged 37.3 years (±SD 11.6 years) were included in this cohort study. 7 chemical burns were acid burns, 61 were alkaline and 4 were of unknown origin. In 37 eyes (51.4%), AMT was applied within the first 6 days after injury. Mean follow-up time was 36.4 months (median 18.5; 1.3-117.3  months). Overall, 29 eyes (40.3%) achieved a best-corrected visual acuity of LogMAR 0.2 (0.63 decimal) or better at final visit. Complete 360° limbal stem cell deficiency (LSCD) occurred in 33 eyes (45.8%), while partial LSCD occurred in 21 eyes (29.2%). CONCLUSION: AMT is an effective adjunctive treatment in the management of acute ocular chemical burns to support epithelial healing and restore ocular surface integrity with potential to improve vision. However, long-term debilitated vision remained in those with severe burns complicated by LSCD.

Use of flow-cytometric analysis to optimize cell banking strategies for production of biopharmaceuticals from mammalian cells.

Production of biopharmaceuticals from mammalian cells requires generation of master, working and post-production cell banks of high quality under GMP conditions. An optimal cryopreservation strategy is needed for each new production cell line, particularly with regard to establishing production processes that are completely devoid of serum or even any animal components and to ensuring robust thaw performance for reliable production. Here, we describe a novel strategy employing flow-cytometric (FC) analysis of Annexin V-stained cells for high-throughput characterization of cell banks. Our data show that this method enables predictive evaluation of a cryopreservation strategy as early as 6h after thawing of cells. Furthermore, a broad study is presented characterizing various factors that may influence the quality of serum-free production cell banks from NSO and CHO cell lines. These results demonstrate how FC-based analysis can be used for development of future state-of-the-art cryopreservation strategies.

Analysis of RegA, a pathway-specific regulator of the friulimicin biosynthesis in Actinoplanes friuliensis.

The rare actinomycete Actinoplanes friuliensis is the producer of the lipopeptide antibiotic friulimicin, which is active against a broad range of Gram-positive bacteria such as methicillin-resistant Enterococcus spec. and Staphylococcus aureus (MRE, MRSA) strains. Friulimicin consists of a decapeptide core and an acyl residue linked to an exocyclic amino acid. The complete biosynthetic gene cluster consisting of 24 open reading frames was characterized by sequence analysis and the transcription units were subsequently determined by RT-PCR experiments. In addition to several genes for biosynthesis, self-resistance and transport four different regulatory genes (regA, regB, regC and regD) were identified within the cluster. To analyse the role of the pathway-specific regulatory protein RegA in the friulimicin biosynthesis, the corresponding gene was inactivated resulting in friulimicin non-producing mutants. Furthermore, several protein-binding sites within the friulimicin gene cluster were identified by gel retardation assays. By real-time RT-PCR experiments, it was shown that the majority of the friulimicin biosynthetic genes is positively regulated by RegA.

Comparative analysis of transcriptional activities of heterologous promoters in the rare actinomycete Actinoplanes friuliensis.

Manipulation of secondary metabolite production in the rare actinomycete Actinoplanes friuliensis, the producer of the lipopeptide antibiotic friulimicin, is hampered by the lack of sophisticated genetic tools. Since no expression vectors have been developed from endogenous Actinoplanes plasmids and expression signals, engineering of antibiotic biosynthesis relies on the use of vector systems derived from Streptomyces. While PhiC31 derived vectors were shown to integrate efficiently into the chromosome of Actinoplanes, information on promoter activity is missing. The manuscript describes the investigation of several different promoter systems which are widely used in Streptomyces in A. friuliensis by promoter probe experiments using eGFP as a reporter. These experiments indicated that promoter strength in A. friuliensis did not correlate to activity in Streptomyces lividans. The ermE* promoter regarded as one of the strongest promoter in Streptomyces has only low activity in A. friuliensis. In contrast, the promoter of the apramycin resistance gene aac(3)IV, originating from the Gram-negative Escherichia coli had the highest activity. By real-time RT-PCR experiments the transcription activity of ermE* promoter in comparison to a native promoter of the friulimicin biosynthetic gene cluster was analysed. This confirmed the results of the promoter probe experiments that indicated quite weak promoter activity of P-ermE* in Actinoplanes.

Abnormal motor cortex excitability in congenital stroke.

The aim of the present study was to investigate corticospinal and intracortical excitability in patients with congenital stroke. In adults, stroke sequelae reduce corticospinal excitability, as indicated by an elevated threshold for motor evoked potentials (MEP), and increase intracortical excitability, as indicated by reduced intracortical inhibition. Ten patients with pre- or perinatally acquired, unilateral cortico-subcortical infarctions in the middle cerebral artery territory were studied with single pulse transcranial magnetic stimulation (TMS) to measure motor threshold (MT) and with paired pulse TMS to study short interval intracortical inhibition (SICI) and intracortical facilitation (ICF). Eight healthy, age-matched subjects served as controls. MT over the affected hemisphere of patients compared with the dominant hemisphere of controls was significantly elevated, reflecting reduced corticospinal excitability, and SICI was significantly reduced, reflecting increased intracortical excitability. No such differences were found for ICF. Findings in patients with congenital stroke were comparable with adulthood stroke. Thus, similar assumptions can be made: reduced corticospinal excitability is probably a consequence of neuronal damage. Reduced intracortical inhibition might represent deficient inhibitory cortical properties or might reflect a compensational mechanism, dispositioning for use-dependent plasticity.