Gene therapy for cartilage defects

Focal defects of articular cartilage are an unsolved problem in clinical orthopaedics. These lesions do not heal spontaneously and no treatment leads to complete and durable cartilage regeneration. Although the concept of gene therapy for cartilage damage appears elegant and straightforward, current research indicates that an adaptation of gene transfer techniques to the problem of a circumscribed cartilage defect is required in order to successfully implement this approach. In particular, the localised delivery into the defect of therapeutic gene constructs is desirable. Current strategies aim at inducing chondrogenic pathways in the repair tissue that fills such defects. These include the stimulation of chondrocyte proliferation, maturation, and matrix synthesis via direct or cell transplantation-mediated approaches. Among the most studied candidates, polypeptide growth factors have shown promise to enhance the structural quality of the repair tissue. A better understanding of the basic scientific aspects of cartilage defect repair, together with the identification of additional molecular targets and the development of improved gene-delivery techniques, may allow a clinical translation of gene therapy for cartilage defects. The first experimental steps provide reason for cautious optimism.     

Liquefaction of lignocellulose at high-solids concentrations

To improve process economics of the lignocellulose to ethanol process a reactor system for enzymatic liquefaction and saccharification at high-solids concentrations was developed. The technology is based on free fall mixing employing a horizontally placed drum with a horizontal rotating shaft mounted with paddlers for mixing. Enzymatic liquefaction and saccharification of pretreated wheat straw was tested with up to 40% (w/w) initial DM. In less than 10 h, the structure of the material was changed from intact straw particles (length 1-5 cm) into a paste/liquid that could be pumped. Tests revealed no significant effect of mixing speed in the range 3.3-11.5 rpm on the glucose conversion after 24 h and ethanol yield after subsequent fermentation for 48 h. Low-power inputs for mixing are therefore possible. Liquefaction and saccharification for 96 h using an enzyme loading of 7 FPU/g.DM and 40% DM resulted in a glucose concentration of 86 g/kg. Experiments conducted at 2%-40% (w/w) initial DM revealed that cellulose and hemicellulose conversion decreased almost linearly with increasing DM. Performing the experiments as simultaneous saccharification and fermentation also revealed a decrease in ethanol yield at increasing initial DM. Saccharomyces cerevisiae was capable of fermenting hydrolysates up to 40% DM. The highest ethanol concentration, 48 g/kg, was obtained using 35% (w/w) DM. Liquefaction of biomass with this reactor system unlocks the possibility of 10% (w/w) ethanol in the fermentation broth in future lignocellulose to ethanol plants.     

Mobile SARS‑CoV‑2 screening facilities for rapid deployment and university‐based diagnostic laboratory

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pandemic has created a public crisis. Many medical and public institutions and businesses went into isolation in response to the pandemic. Because SARS‐CoV‐2 can spread irrespective of a patient''s course of disease, these institutions’ continued operation or reopening based on the assessment and control of virus spread can be supported by targeted population screening. For this purpose, virus testing in the form of polymerase chain reaction (PCR) analysis and antibody detection in blood can be central. Mobile SARS‐CoV‐2 screening facilities with a built‐in biosafety level (BSL)‐2 laboratory were set up to allow the testing offer to be brought close to the subject group''s workplace. University staff members, their expertise, and already available equipment were used to implement and operate the screening facilities and a certified diagnostic laboratory. This operation also included specimen collection, transport, PCR and antibody analysis, and informing subjects as well as public health departments. Screening facilities were established at different locations such as educational institutions, nursing homes, and companies providing critical supply chains for health care. Less than 4 weeks after the first imposed lockdown in Germany, a first mobile testing station was established featuring a build‐in laboratory with two similar stations commencing operation until June 2020. During the 15‐month project period, approximately 33,000 PCR tests and close to 7000 antibody detection tests were collected and analyzed. The presented approach describes the required procedures that enabled the screening facilities and laboratories to collect and process several hundred specimens each day under difficult conditions. This report can assist others in establishing similar setups for pandemic scenarios.     

Impact of intraoral soft-tissue reconstruction on the development of quality of life after ablative surgery in patients with oral cancer

The aim of this prospective study was to assess the impact of intraoral soft-tissue reconstruction on the development of quality of life after ablative surgery for oral cancer. A total of 107 patients were enrolled in the study during the period between 1997 and 1999. Quality of life was assessed by using the quality-of-life core questionnaire and the head and neck module of the European Organization for Research and Treatment of Cancer. The questionnaires were distributed to the patients preoperatively on the day of hospital admission and 3 months, 6 months, and 12 months postoperatively. A total of 53 patients filled in all questionnaires and were available for complete longitudinal analysis. The changes in the scores and the impact of defect size, location, and anatomy, the extent of mandibular resection, and the mode of soft-tissue reconstruction were tested longitudinally for statistical significance by using repeated-measures analysis of variance procedures. Of all parameters tested, the mode of soft-tissue reconstruction had the most profound impact on the development of quality of life after ablative surgery for oral cancer in that it was associated with statistically significant changes in the most domains or items associated with postoperative quality of life. In contrast to local flaps, revascularized soft-tissue repair with forearm flaps was associated with an intermittent deterioration of physical and functional scores but was followed by improvement until the end of the first year, and it even surpassed the preoperative baseline level in oral functional and social domains. In large-volume defects, which required repair by myocutaneous grafts, quality of life was not restored to the same extent, and physical, functional, and social domains remained significantly lower.     

Einfluß unterschiedlicher Störsignale auf Mensch-maschine-Regelkreise

Dynamic behaviour of man has been investigated in a simple closed loop with a human controller. Either stochastic or deterministic disturbances act upon the output of the controlled element. A choice of the nearly 200 tests will be discussed concerning the learning ability, the variability of the results and the stationarity of the human controller. It will be shown that the quasi-linear frequency response of the open loop is independent of the type of disturbances provided the controlled elements do not contain dead-time. Conversely, dead-time within the controlled element affects the adaptibility of the human controller. Hence, in this case, the quasi-linear frequency response depends also on the type of the disturbance signal.     

Isolation, characterization and microsequence analysis of a small basic methylated DNA-binding protein from the Archaebacterium, Sulfolobus solfataricus

DNA-binding proteins have been extracted from the thermoacidophilic archaebacterium Sulfolobus solfataricus strain P1, grown at 86 degrees C and pH 4.5. These proteins, which may have a histone-like function, were isolated and purified under standard, non-denaturing conditions, and can be grouped into three molecular mass classes of 7, 8 and 10 kDa. We have purified to homogenity the main 7 kDa protein and determined its DNA-binding affinity by filter binding assays and electron microscopy. The Stokes radius of gyration indicates that the protein occurs as a monomer. The complete amino-acid sequence of this protein contains 14 lysine residues out of 63 amino acids and the calculated Mr is 7149. Five of the lysine residues are partially monomethylated to varying extents and the methylated residues are located exclusively in the N-terminal (positions 4 and 6) and the C-terminal (positions 60, 62 and 63) regions only. The protein is strongly homologous to the 7 kDa proteins of Sulfolobus acidocaldarius with the highest homology to protein 7d. Accordingly, the name of this protein from S. solfataricus was assigned as DNA-binding protein Sso7d.     

An artificial hydrophobic sequence functions as either an anchor or a signal sequence at only one of two positions within the Escherichia coli outer membrane protein OmpA

The 325-residue outer membrane protein, OmpA, of Escherichia coli, like most other outer membrane proteins with known sequence, contains no long stretch of hydrophobic amino acids. A synthetic oligonucleotide, encoding the sequence Leu-Ala-Leu-Val, was inserted four times between the codons for amino acid residues 153 and 154 and two, three, or four times between the codons for residues 228 and 229, resulting in the OmpA153-4, OmpA-228-2, -3, and -4 proteins, respectively. In the first case, the lipophilic sequence anchored the protein in the plasma membrane. In the OmpA228 proteins, 16 but not 12 or 8 lipophilic residues most likely also acted as an anchor. By removal of the NH2-terminal signal peptide, the function of the insert in OmpA153-4 was converted to that of a signal-anchor sequence. Possibly due to differences in amino acid sequences surrounding the insert, no signal function was observed with the insert in OmpA228-4. Production of the OmpA153-4 protein, with or without the NH2-terminal signal sequence, resulted in a block of export of chromosomally encoded OmpA. Clearly, long hydrophobic regions are not permitted within proteins destined for the bacterial outer membrane, and these proteins, therefore, have had to evolve another mechanism of membrane assembly.