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Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective, 3D-printed coverslip holder that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that Pseudomonas aeruginosa wild type and a phenazine deletion mutant (Δphz) form similar biofilms. Extending this analysis to anoxic conditions, we reveal that microcolony and biofilm formation can only be observed under shaking conditions and are decreased in the Δphz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is not available. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for attachment to surfaces under static conditions, we demonstrate that H. volcanii mutants that lack archaella are negatively affected in their early stages of biofilm formation under shaking conditions.ImportanceDue to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.

Cell surfaces are critical for diverse functions across all domains of life, from cell-cell communication and nutrient uptake to cell stability and surface attachment. While certain aspects of the mechanisms supporting the biosynthesis of the archaeal cell surface are unique, likely due to important differences in cell surface compositions between domains, others are shared with bacteria or eukaryotes or both. Based on recent studies completed on a phylogenetically diverse array of archaea, from a wide variety of habitats, here we discuss advances in the characterization of mechanisms underpinning archaeal cell surface biogenesis. These include those facilitating co- and post-translational protein targeting to the cell surface, transport into and across the archaeal lipid membrane, and protein anchoring strategies. We also discuss, in some detail, the assembly of specific cell surface structures, such as the archaeal S-layer and the type IV pili. We will highlight the importance of post-translational protein modifications, such as lipid attachment and glycosylation, in the biosynthesis as well as the regulation of the functions of these cell surface structures and present the differences and similarities in the biogenesis of type IV pili across prokaryotic domains., This review discusses molecular pathways required for the biogenesis of archaeal cell surface proteins.

To evaluate the impact of postoperative interstitial brachytherapy with and without external radiotherapy in the treatment of primary and recurrent squamous cell carcinoma of the oral cavity and oropharynx. Patients and Methods: Between 1985 and 1997, a total of 318 patients were treated by interstitial Ir-192 low-dose-rate brachytherapy as part of their primary (n = 236) or recurrent treatment (n = 82). There were 263 male (83%) and 55 (17%) female patients. The distribution of UICC (1997) stages was as follows: I (61 patients, 19%), II (71 patients, 22%), III (58 patients, 18%), IV (128 patients, 40%). The primary tumor site was located in the oral cavity in 201 patients (63%), in the oropharynx in 86 patients (27%), lower lip in 19 patients (6%) and other regions in twelve cases (4%). Treatment concepts did not vary over the time and were dictated by the initial tumor extension: a total of 175 patients (55%) received a combination of surgery, interstitial brachytherapy (23–25 Gy) and external radiotherapy (50–60 Gy), 60 patients (19%) surgery and interstitial brachytherapy (45–55 Gy) alone. Advanced disease not amenable to primary surgery was either treated by radiochemotherapy and interstitial brachytherapy in 39 patients (12%) or a combination of interstitial brachytherapy, external radiotherapy and interstitial hyperthermia in 44 patients (14%). Results: Overall survival rates following primary and recurrent treatment were 50 ± 4% and 29 ± 5%, respectively, at 5 years (p < 0.0001). A significant impact on overall survival rate was noted for UICC stage: patients in stage I/II had survival rates of 64 ± 5% and 57 ± 10%, respectively, while patients in stage III/IV had survival rates of 39 ± 5% and 15 ± 5%, respectively, at 5 years (p < 0.0001). In addition, grading (p = 0.01) and hemoglobin levels (p = 0.05) had a significant influence on overall survival. Local tumor control rates for all patients were 74 ± 3% and 57 ± 7% at 5 years following primary and recurrent treatment (p = 0.01), respectively. The 145 patients treated for primary disease by a uniform concept of surgery, interstitial brachytherapy and external radiotherapy achieved excellent local control rates with 92 ± 4% (stage I/II) and 65 ± 6% (stage III/IV) at 5 years. Late treatment-related toxicity with soft tissue necrosis and/or osteonecrosis requiring mandibular resection was 7.5%. Conclusion: Local tumor excision followed by postoperative interstitial brachytherapy with and without external radiotherapy is associated with excellent locoregional control, a low risk of chronic sequelae and may therefore considered as a new approach avoiding mutilating radical surgery.

To elucidate the pathomechanism underlying persistent mirror movements (MM), we modelled the origin of electric brain activity associated with these movements. Movement-related cortical potentials (MRCP) in a group of subjects affected by persistent mirror movements were compared with those of a control group. The data of the normal subjects were best explained with two bilaterally active electric sources in the sensorimotor cortices with a clear preponderance of the hemisphere contralateral to the movement. In contrast, the MM subjects presented a fairly symmetric source activity in both hemispheres during unilateral intended movements. In the control group, the source representing the activity of the motor cortex ipsilateral to the moving finger reduced activity before the beginning of the movement; this was interpreted as an inhibition of the ipsilateral motor cortex during unilateral movement. In the MM group, however, this inhibition was not seen. Furthermore, while normal subjects demonstrated no relevant activity of an additional source placed near midline motor structures (supplementary motor area; SMA), subjects with MM showed considerable activity of this dipole source. These findings suggest that subjects with persistent MM have abnormal bilateral activation of the primary motor areas, probably together with an additional activation of mesial motor structures. This assumption fits well with the observation of an incomplete decussation of the pyramidal tract. The bilateral activation is then explained as a compensatory strategy in order to achieve sufficient force in the innervated target muscles.

Light and oxygen are factors that are very much entangled in the reactive oxygen species (ROS) stress response network in plants, algae and cyanobacteria. The first obligatory step in understanding the ROS network is to separate these responses. In this study, a LC-MS/MS based quantitative proteomic approach was used to dissect the responses of Chlamydomonas reinhardtii to ROS, light and oxygen employing an interlinked experimental setup. Application of novel bioinformatics tools allow high quality retention time alignment to be performed on all LC-MS/MS runs increasing confidence in protein quantification, overall sequence coverage and coverage of all treatments measured. Finally advanced hierarchical clustering yielded 30 communities of co-regulated proteins permitting separation of ROS related effects from pure light effects (induction and repression). A community termed redox(II) was identified that shows additive effects of light and oxygen with light as the first obligatory step. Another community termed 4-down was identified that shows repression as an effect of light but only in the absence of oxygen indicating ROS regulation, for example, possibly via product feedback inhibition because no ROS damage is occurring. In summary the data demonstrate the importance of separating light, O₂ and ROS responses to define marker genes for ROS responses. As revealed in this study, an excellent candidate is DHAR with strong ROS dependent induction profiles.

Bacteriophage P22 recognizes O-antigen polysaccharides of Salmonella enterica subsp. enterica (S.) with its tailspike protein (TSP). In the serovars S. Typhimurium, S. Enteritidis, and S. Paratyphi A, the tetrasaccharide repeat units of the respective O-antigens consist of an identical main chain trisaccharide but different 3,6-dideoxyhexose substituents. Here, the epimers abequose, tyvelose and paratose determine the specific serotype. P22 TSP recognizes O-antigen octasaccharides in an extended binding site with a single 3,6-dideoxyhexose binding pocket. We have isolated S. Paratyphi A octasaccharides which were not available previously and determined the crystal structure of their complex with P22 TSP. We discuss our data together with crystal structures of complexes with S. Typhimurium and S. Enteritidis octasaccharides determined earlier. Isothermal titration calorimetry showed that S. Paratyphi A octasaccharide binds P22 TSP less tightly, with a difference in binding free energy of similar to 7 kJ mol(-1) at 20 degrees C compared with S. Typhimurium and S. Enteritidis octasaccharides. Individual protein-carbohydrate contacts were probed by amino acid replacements showing that the dideoxyhexose pocket contributes to binding of all three serotypes. However, S. Paratyphi A octasaccharides bind in a conformation with an energetically unfavorable phi/epsilon glycosidic bond angle combination. In contrast, octasaccharides from the other serotypes bind as solution-like conformers. Two water molecules are conserved in all P22 TSP complexes with octasaccharides of different serotypes. They line the dideoxyhexose binding pocket and force the S. Paratyphi A octasaccharides to bind as nonsolution conformers. This emphasizes the role of solvent as part of carbohydrate binding sites.

Electric stimulation of the median nerve at the wrist evokes a series of electric potentials that can be recorded from the scalp or directly from the cortex. These somatosensory evoked potentials (SEP) include a parietal negativity with a maximum 20 ms after the stimulus, which originates in the somatosensory cortex, probably area 3b (Allison et al. [1991a], Brain 114:2465–2503 and Desmedt et al. [1987], Electroenceph Clin Neurophysiol 68:1–19). Thirty milliseconds after the stimulus, a negative potential (N30) occurs at frontal recording sites. Recently it was observed that the amplitude of this potential is altered in patients with dystonia, Parkinson's disease, and Huntington's chorea. It has been argued that the N30 potential stems from cortical areas other than the somatosensory cortex, for example, the supplementary motor area. We used multichannel recordings to investigate the scalp distribution of the N20 and the N30 potentials in healthy subjects. We found that the N20 as well as the N30 potentials were accompanied by a corresponding positivity at frontal and parietal recording sites, respectively. The N20/P20 and the N30/P30 potential fields had a mirrorlike appearance, and both showed a polarity reversal near the central sulcus. This and the results of correlation analyses led us to the conclusion that the N30 generator is located near the central sulcus. © 1995 Wiley-Liss, Inc.

The cerebral events related to limb movements can be studied noninvasively with the method of evoked potentials. In this study, a brain potential is analysed that follows the onset of a simple finger movement. Because this potential occurred after active as well as after passive movements, its previously alleged reafferent somatosensory nature is confirmed in this study. Detailed topographic analysis revealed that this potential has the same polarity and merges with the preceeding Bereitschaftspotential (BP; in the active movement) at central electrodes, whereas at parietal electrodes polarity is opposite to the BP. In individual subjects, the maximum of the BP and the peak of the reafferent potential are separated by a small gap, previously described as pre-motion positivity. A comparison with the N20 potential of the electrically evoked somatosensory potential showed similar potential topography, albeit opposite polarity. The dipole analysis supported the view that the reafferent and the electrically evoked potentials are likely to arise from the same cortical area, namely the primary somatosensory cortex.

Several reports have described that a positive vertex peak of an evoked potential varied in amplitude and latency specifically when images of faces were the eliciting stimulus. The scalp topography and the possible underlying dipole sources of this peak are the subject of this report. We presented black-and-white photographs of human faces, flowers and leaves to 16 healthy subjects and recorded the evoked brain potentials from 31 scalp electrodes. We found the previously described higher amplitude of the positive vertex peak when faces were the crucial stimulus, but the latency of this peak was the same (180 ms) for all three categories of stimulus. At the posterior temporal electrodes, the face waveforms showed a negative peak at 175 ms, which was only rudimentary in the waveforms elicited by the other stimuli. Since in most previous reports a mastoid reference was used, it is most likely that the previously described latency shift of the positive vertex peak associated with face stimuli was due to the interaction with this posterior temporal peak. The dipole analysis of the possible generators of the recorded potentials suggested the sequential activation of occipital, lateral temporal and mesio-temporal brain structures during the perception of a human face.