Real-time online monitoring of insect cell proliferation and baculovirus infection using digital differential holographic microscopy and machine learning

Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.     

Growth forms and life-history strategies predict the occurrence of aquatic macrophytes in relation to environmental factors in a shallow peat lake complex

Hydrobiologia - Aquatic ecosystems provide vital services, and macrophytes play a critical role in their functioning. Conceptual models indicate that in shallow lakes, plants with different growth...     

Independent Validation of an Existing Model Enables Prediction of Hearing Loss after Childhood Bacterial Meningitis

Objective

This study aimed external validation of a formerly developed prediction model identifying children at risk for hearing loss after bacterial meningitis (BM). Independent risk factors included in the model are: duration of symptoms prior to admission, petechiae, cerebral spinal fluid (CSF) glucose level, Streptococcus pneumoniae and ataxia. Validation helps to evaluate whether the model has potential in clinical practice.

Study design

116 Dutch school-age BM survivors were included in the validation cohort and screened for sensorineural hearing loss (>25 dB). Risk factors were obtained from medical records. The model was applied to the validation cohort and its performance was compared with the development cohort. Validation was performed by application of the model on the validation cohort and by assessment of discrimination and goodness of fit. Calibration was evaluated by testing deviations in intercept and slope. Multiple imputation techniques were used to deal with missing values.

Results

Risk factors were distributed equally between both cohorts. Discriminative ability (Area Under the Curve, AUC) of the model was 0.84 in the development and 0.78 in the validation cohort. Hosmer-Lemeshow test for goodness of fit was not significant in the validation cohort, implying good fit concerning the similarity of expected and observed cases. There were no significant differences in calibration slope and intercept. Sensitivity and negative predicted value were high, while specificity and positive predicted value were low which is comparable with findings in the development cohort.

Conclusions

Performance of the model remained good in the validation cohort. This prediction model might be used as a screening tool and can help to identify those children that need special attention and a long follow-up period or more frequent auditory testing.     

Analytical methods for structural ensembles and dynamics of intrinsically disordered proteins

Intrinsically disordered proteins, proteins that do not have a well-defined three-dimensional structure, make up a significant proportion of our proteome and are particularly prevalent in signaling and regulation. Although their importance has been realized for two decades, there is a lack of high-resolution experimental data. Molecular dynamics simulations have been crucial in reaching our current understanding of the dynamical structural ensemble sampled by intrinsically disordered proteins. In this review, we discuss enhanced sampling simulation methods that are particularly suitable to characterize the structural ensemble, along with examples of applications and limitations. The dynamics within the ensemble can be rigorously analyzed using Markov state models. We discuss recent developments that make Markov state modeling a viable approach for studying intrinsically disordered proteins. Finally, we briefly discuss challenges and future directions when applying molecular dynamics simulations to study intrinsically disordered proteins.     

Surviving in Changing Seascapes: Sediment Dynamics as Bottleneck for Long-Term Seagrass Presence

Changes in the seascape often result in altered hydrodynamics that lead to coinciding changes in sediment dynamics. Little is known on how altered sediment dynamics affect long-term seagrass persistence. We studied the thresholds of sediment dynamics in relation to seagrass presence by comparing sediment characteristics and seagrass presence data of seven separate seagrass meadows. All meadows had a long-term (>20 years) presence. Within these meadows, we distinguish so-called “hotspots” (areas within a meadow where seagrass was found during all mapping campaigns) and “coldspots” (with infrequent seagrass presence). We monitored static sediment characteristics (median grain size, bulk density, silt content) and sediment dynamics (that is, bed level change and maximum sediment disturbance depth), bioturbation (that is, lugworm densities and induced fecal pit and mound relief), and seagrass cover. We statistically analyzed which sediment characteristic best explains seagrass cover. Densely vegetated hotspots were shown to have lower sediment dynamics than sparsely vegetated hotspots and coldspots, whereas static sediment characteristics were similar (grain size, bulk density). The vegetation cover was either low (2–15%) or high (>30%) and sediment dynamics showed a threshold for vegetation cover. From this correlative finding, we postulate a self-sustaining feedback of relatively dense seagrass via sediment stabilization and accordingly a runaway feedback once the seagrass cover becomes too sparse. The sensitivity for sediment dynamics shown in our study implies that future existence of seagrass meadows may be at risk as ongoing climate change might directly (increased environmental extremes) or indirectly (changing seascapes) negatively affect seagrass beds.     

Changing Paradigms in Seagrass Restoration

Sharing experiences and results among scientists and managers working on seagrass restoration was the main objective of the first European Seagrass Restoration Workshop that gathered researchers from around Europe. The meeting was the first forum in Europe that allowed for scientists, NGOs, and managers to interact and share their experiences relating to seagrass restoration and management. The results show that none of the seagrass restoration programs developed in Europe by the participants during the last 10 years was successful. Furthermore, an informal review of data published in seagrass restoration success, showed that the results reported were biased because they were mostly based on a very short monitoring period (i.e. < 1 year). Numerous decision trees, guidelines, and restoration models have been developed to aid seagrass restoration management, but the results of this workshop point toward a new paradigm in seagrass restoration were efforts should shift to give priority to natural restoration potential, with an emphasis on the fact that restoration should never be considered the first alternative when planning for the mitigation of coastal development projects or to justify mitigation as a compensation measure for economic activities.     

Antroduodenal motility in chronic pancreatitis: are abnormalities related to exocrine insufficiency?

In patients with chronic pancreatitis (CP) the relation among exocrine pancreatic secretion, gastrointestinal hormone release, and motility is disturbed. We studied digestive and interdigestive antroduodenal motility and postprandial gut hormone release in 26 patients with CP. Fifteen of these patients had pancreatic insufficiency (PI) established by urinary para-aminobenzoic acid test and fecal fat excretion. Antroduodenal motility was recorded after ingestion of a mixed liquid meal. The effect of pancreatic enzyme supplementation was studied in 8 of the 15 CP patients with PI. The duration of the postprandial antroduodenal motor pattern was significantly (P < 0.01) prolonged in CP patients (324 +/- 20 min) compared with controls (215 +/- 19 min). Antral motility indexes in the first hour after meal ingestion were significantly reduced in CP patients. The interdigestive migrating motor complex cycle length was significantly (P < 0.01) shorter in CP patients (90 +/- 8 min) compared with controls (129 +/- 8 min). These abnormalities were more pronounced in CP patients with exocrine PI. After supplementation of pancreatic enzymes, these alterations in motility reverted toward normal. Digestive and interdigestive antroduodenal motility are abnormal in patients with CP but significantly different from controls only in those with exocrine PI. These abnormalities in antroduodenal motility in CP are related to maldigestion.     

Two similar enhanced root-colonizing Pseudomonas strains differ largely in their colonization strategies of avocado roots and Rosellinia necatrix hyphae

Pseudomonas alcaligenes AVO73 and Pseudomonas pseudoalcaligenes AVO110 were selected previously as efficient avocado root tip colonizers, displaying in vitro antagonism towards Rosellinia necatrix, causal agent of avocado white root rot. Despite the higher number of antagonistic properties shown in vitro by AVO73, only AVO110 demonstrated significant protection against avocado white root rot. As both strains are enhanced root colonizers, and as colonization is crucial for the most likely biocontrol mechanisms used by these strains, namely production of non-antibiotic antifungal compounds and competition for nutrients and niches, we decided to compare the interactions of the bacterial strains with avocado roots as well as with R. necatrix hyphae. The results indicate that strain AVO110 is superior in biocontrol trait swimming motility and establishes on the root tip of avocado plants faster than AVO73. Visualization studies, using Gfp-labelled derivatives of these strains, showed that AVO110, in contrast to AVO73, colonizes intercellular crevices between neighbouring plant root epidermal cells, a microhabitat of enhanced exudation. Moreover, AVO110, but not AVO73, also colonizes root wounds, described to be preferential penetration sites for R. necatrix infection. This result strongly suggests that AVO110 meets, and can attack, the pathogen on the root. Finally, when co-inoculated with the pathogen, AVO110 utilizes hyphal exudates more efficiently for proliferation than AVO73 does, and colonizes the hyphae more abundantly than AVO73. We conclude that the differences between the strains in colonization levels and strategies are likely to contribute to, and even can explain, the difference in disease-controlling abilities between the strains. This is the first report that shows that two similar bacterial strains, selected by their ability to colonize avocado root, use strongly different root colonization strategies and suggests that in addition to the total bacterial root colonization level, the sites occupied on the root are important for biocontrol.     

The Most Prevalent Freeman-Sheldon Syndrome Mutations in the Embryonic Myosin Motor Share Functional Defects

The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/β cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5–9-fold), leading to a longer lived detached state and a slowed V_max of the ATPase (2–35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function.