An Stomatin, Prohibitin, Flotillin, and HflK/C-Domain Protein Required to Link the Phage-Shock Protein to the Membrane in Bacillus subtilis.

Membrane surveillance and repair is of utmost importance to maintain cellular integrity and allow cellular life. Several systems detect cell envelope stress caused by antimicrobial compounds and abiotic stresses such as solvents, pH-changes and temperature in bacteria. Proteins containing an Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH)-domain, including bacterial flotillins have been shown to be involved in membrane protection and membrane fluidity regulation. Here, we characterize a bacterial SPFH-domain protein, YdjI that is part of a stress induced complex in Bacillus subtilis. We show that YdjI is required to localize the ESCRT-III homolog PspA to the membrane with the help of two membrane integral proteins, YdjG/H. In contrast to classical flotillins, YdjI resides in fluid membrane regions and does not enrich in detergent resistant membrane fractions. However, similarly to FloA and FloT from B. subtilis, deletion of YdjI decreases membrane fluidity. Our data reveal a hardwired connection between phage shock response and SPFH proteins.

Radiographic Evaluation of Keel Bone Damage in Laying Hens-Morphologic and Temporal Observations in a Longitudinal Study.

The keel bone of commercially kept laying hens is known to be frequently affected by morphologic changes such as fractures and deformations with important implications for animal welfare. To detect morphologic changes, various methods such as palpation, computed tomography, and ultrasound are available, though radiography allows for the greatest level of detail in combination with the most ease of use. To explore the benefits of radiography in providing objective data on keel fractures from the age of 22-61 weeks within a single laying period, the keel bones of 75 Lohmann Brown and 75 Lohmann Selected Leghorns were radiographed every 3 to 5 weeks. Type, location, angulation, dislocation, callus formation, and healing process were assessed descriptively for each lesion. Ninety-nine percent of the animals showed at least one keel bone lesion during the study and 97% of the animals had at least one keel bone fracture. In 77% of the cases, the caudal third of the keel bone was affected. The fracture types were transverse and oblique (88%), comminuted, and butterfly. Further lesions were sclerosis, new bone formation and angulation. For each keel bone, an average of three fractures (3.09 ± 1.80) was detected at the end of the study. The described radiographic protocol for keel bone lesions was suitable for longitudinal, on-site examinations in conscious laying hens. Our results also indicate that keel bone fractures are more frequent than reported in earlier studies. The described radiographic examination protocol can be used to perform comparative studies of palpatory findings, or to assess the clinical significance of different fracture types which require a high level of detail.

Keel bone fractures affect egg laying performance but not egg quality in laying hens housed in a commercial aviary system.

The aim of this study was to investigate the effect of keel bone fracture (KBF) severity and healing activity on individual productivity of laying hens. Focal hens (75 Lohmann Selected Leghorn (LSL), 75 Lohmann Brown (LB)) were housed alongside non-focal hens in 10 identical pens containing a commercial aviary system (15 focal hens per pen). Eggs of focal hens were identified by orally administering a dye on 3 consecutive days, resulting in a hen-specific color pattern in the yolk. Eggs were collected at 7 time points (37 to 61 weeks of age; WOA) for 5 d to determine individual laying performance and to assess egg quality. Radiographs were performed to score KBF severity on a continuous scale. Healing activity was scored as inactive, healing, or fresh. Linear mixed effects models were used for statistical analyses. We found an association between KBF severity and reduced egg laying performance with increasing age (P = 0.005). At 37 WOA, egg laying performance was similar across KBF severities, whereas at 61 WOA, performance in hens with the highest observed KBF severity was 16.2% lower than in hens without fractures. Hens with fresh fractures had a lower performance than hens with healing and inactive fractures at 37 WOA but higher performance at 61 WOA (P = 0.02). Egg quality parameters were not affected by fractures but were associated with an age × hybrid interaction (egg mass: P = 0.039, shell breaking strength: P = 0.03, shell width: P = 0.001). In conclusion, hens could maintain high performance irrespective of fracture severity until shortly after peak of lay, but seemed to redirect available resources towards fracture healing if a fresh fracture was present. At the end of lay, the negative effect of KBF fracture severity on individual production amplified indicating that hens were no longer able to cope with the physiological challenge of a fracture.

A Reliable Method to Assess Keel Bone Fractures in Laying Hens From Radiographs Using a Tagged Visual Analogue Scale.

Up to 97% of laying hens housed in aviary systems are affected by keel bone fractures. Due to the scope of the problem, multiple efforts investigating causes and consequences of fractures have been conducted. The most frequently used techniques to detect fractures lack accuracy and provide only vague information (palpation) or cannot be conducted longitudinally (dissection). Radiographic imaging overcomes these weaknesses as it allows longitudinal observations and provides detailed information for individual fractures of which a single keel may have several at different locations and of different origins. However, no standardized system exists to assess fracture severity from radiographs if multiple fractures are present. The aim of this study was therefore to test the reliability of a scoring system assessing the aggregate severity of multiple fractures, taking into account the characteristics of all present fractures (e.g., locations, callus formation, width of fracture gaps). We developed a scoring system based on a tagged visual analogue scale, ranging from score 0 (no fracture) to score 5 (extremely severe) with intermediate tags for scores 1, 2, 3, and 4. A catalog of example scores was provided to describe the range of each score visually. An online tutorial with an introduction, training and scoring session was completed by 14 participants with varying experience involving laying hens and keel bone damage. For inter-observer reliability, we found an Intraclass correlation coefficient (ICC) of 0.985 with a 95% confidence interval of 0.974 < ICC < 0.993 (average-rating, absolute-agreement, two-way random-effects model). Intraclass correlation coefficient for intra-observer reliability was 0.923 with a 95% confidence interval of 0.879 < ICC < 0.951 (single-rating, absolute-agreement, two-way mixed-effects model). Intra-observer reliability ranged from 0.704 to 1.0 indicating excellent agreement and similar ratings across and within participants. Further, high ICCs suggest that the introduction and the training sessions provided were adequate tools to prepare observers for the assessment task despite various backgrounds of the participants. Nonetheless, the validity of this scoring system needs to be investigated further in order to link responses of interest and biological relevance with the specific severity values resulting from our scoring system.

Single-Molecule Encapsulation: A Straightforward Route to Highly Stable and Printable Enzymes.

A mild, fast, and sequence-independent method for controlled enzyme immobilization is presented. This novel approach involves the encapsulation of single-enzyme molecules and the covalent attachment of these nanobiocatalysts onto surfaces. Fast and mild immobilization conditions, combined with low nonspecific adsorption on hydrophobic substrates, enables well-defined surface patterns via microcontact printing. The biohybrid materials show enhanced activity in organic solvents.