Doughertypreston2779

Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.During aging, skeletal muscles become atrophic and lose contractile force. Aging can also impact the neuromuscular junction (NMJ), a synapse that transmits signals from motoneurons to muscle fibers to control muscle contraction. However, in contrast to muscle aging that has been studied extensively, less is known about the molecular mechanisms of NMJ aging although its structure and function are impaired in aged animals. To this end, we performed RNA sequencing (RNA-seq) analysis to identify genes whose expression in synapse-rich region is altered. Gene ontology (GO) analysis highlighted genes relating to nuclear structure or function. In particular, lamin A/C, an intermediate filament protein critical for the interphase nuclear architecture, was reduced. Remarkably, mutation of lamin A/C in muscles or motoneurons had no effect on NMJ formation in either sex of mice, but the muscle mutation caused progressive denervation, acetylcholine receptor (AChR) cluster fragmentation, and neuromuscular dysfunction. Interestingly, rapsyn, a protein critical to AChR clustering, was reduced in mutant muscle cells; and expressing rapsyn in muscles attenuated NMJ deficits of HSA-Lmna-/- mice. These results reveal a role of lamin A/C in NMJ maintenance and suggest that nuclear dysfunction or deficiency may contribute to NMJ deficits in aged muscles.SIGNIFICANCE STATEMENT This study provides evidence that lamin A/C, a scaffolding component of the nuclear envelope, is critical to maintaining the NMJ in mice. Its muscle-specific mutation led to progressive NMJ degeneration in vivo We showed that the mutation reduced the level of rapsyn, a protein necessary for acetylcholine receptor (AChR) clustering; and expression of rapsyn in muscles attenuated NMJ deficits of HSA-Lmna-/- mice. These results reveal a role of lamin A/C in NMJ maintenance and suggest that nuclear dysfunction or deficiency may contribute to NMJ deficits in aged muscles.The hippocampus supports episodic memory via interaction with a distributed brain network. Previous experiments using network-targeted noninvasive brain stimulation have identified episodic memory enhancements and modulation of activity within the hippocampal network. However, mechanistic insights were limited because these effects were measured long after stimulation and therefore could have reflected various neuroplastic aftereffects with extended time courses. In this experiment with human subjects of both sexes, we tested for immediate stimulation impact on encoding-related activity of the hippocampus and immediately adjacent medial-temporal cortex by delivering theta-burst transcranial magnetic stimulation (TBS) concurrent with fMRI, as an immediate impact of stimulation would suggest an influence on neural activity. We reasoned that TBS would be particularly effective for influencing the hippocampus because rhythmic neural activity in the theta band is associated with hippocampal memory processing. Firsing an accessible region of the hippocampal network via transcranial magnetic stimulation during concurrent fMRI. We reasoned that theta-burst stimulation would be particularly effective for impacting hippocampal function, as this stimulation rhythm should resonate with the endogenous theta-nested-gamma activity prominent in hippocampus. Indeed, theta-burst stimulation targeting the hippocampal network immediately impacted hippocampal activity during encoding, improving memory formation as indicated by enhanced later recollection. Rhythm- and location-control stimulation conditions had no such effects. These findings suggest a direct influence of noninvasive stimulation on hippocampal neural activity and highlight that the theta-burst rhythm is relatively privileged in its ability to influence hippocampal memory function.Endotoxin testing by recombinant factor C (rFC) is increasing with the addition of new suppliers of reagents. By use of a recombinantly produced factor C , based on the sequence of a coagulation enzyme present in horseshoe crab amebocyte lysates, the rFC tests are designed as substitutes for the traditional Limulus amebocyte lysate (LAL)/Tachypleus amebocyte lysate tests based on horseshoe crab blood. Comparative testing of samples with both the LAL and recombinant reagents has shown a high degree of correlation, suggesting that use of rFC is comparable to the more traditional LAL tests and may be technologically superior. Recombinant factor C does not recognize the factor G pathway, the alternate coagulation pathway that the lysate reagents detect. This feature allows rFC to detect endotoxin more selectively. As a recombinantly produced material, it avoids the use of the horseshoe crabs required for lysate production, thereby protecting this species, which is at risk in some parts of the world. Recombinant factor C is expected to further benefit from a more sustainable supply chain based upon a robust biotechnological production process. We summarize here the results of many studies that evaluated the use of recombinant technology for the detection of environmental endotoxin. Additionally, we include a review of the current compendia and regulatory status of the recombinant technologies for use in the quality control of pharmaceutical manufacturing. Our analysis confirms that the recombinant technologies are comparable in protecting patient safety.Quality is defined by the American Society for Quality (ASQ) as ″the totality of features and characteristics of a product or service that bears on its ability to satisfy given needs″. Therefore, quality is applicable to processes that supply outcomes which values can be measured. The statistical control is an effective methodology which provides the outcome of quality of goods, bringing an added value that other methods like the quality by inspection do not bring about. The statistical methods applied to process control have been thoroughly developed and the mathematics that supports them have been broadly demonstrated. Artificial Intelligence is a field where mathematics, statistics and programming play a joint role and its results could also be applied to disciplines like quality control. Nevertheless, its utilization is subordinated to the qualification of implemented algorithms. This research presents a standard procedure to qualify Artificial Intelligence algorithms, allowing their usage in regulated environments to grant the quality of the delivered products or services (e.g., in drugs and medicines manufacturing). The regulated principles are defined by the concept of Quality by Design (QbD), which is a notion introduced in the pharmaceutical industry as a good practice for process management under multivariate analysis. This study is intended to provide a guidance to qualify AI algorithms taking the QbD guidelines as foundation for this purpose.For steam sterilization load validation of porous/ hard goods loads, regulatory guidelines state to identify and validate the worst-case challenge which is most difficult to sterilize. The maximum load and the minimum load are usually considered as worst-case challenge, and both are validated. This paper demonstrates with example how the control strategy determines the worst-case load in the steam sterilizer. It further establishes with examples how a suitably designed control strategy can nullify the impact of varying load characteristics (e.g., mass, configuration and air removal challenges) in porous/ hard goods loads, and make it load size independent.Given the surging interest in developing prefilled syringe and autoinjector combination products, investment in an early compatibility assessment is critical to prevent unwarranted drug/container closure interactions and avoid potential reformulation during late stages of drug development. In addition to the standard evaluation of drug stability, it is important to consider container closure functionality and overall device performance changes over time, due to drug-container closure component interaction. This study elucidates the mechanisms that cause changes in syringe glide force over time and its impact on the injection duration. It is an expansion of the previous work, which indicated that drug formulation variables such as formulation excipients and pH affect syringe functionality over time. The current study describes an investigative process for troubleshooting prolonged and variable autoinjector injection time caused by an increased syringe glide force variability over time. This increase in glide force variability stems from two root causes namely plunger dimensional variation and syringe silicone oil change over time. The results demonstrate (a) the underlying factors of silicone oil change in the presence of drug formulation matrices, (b) accelerated stability of syringe glide force as a good indicator of long-term, real-time stability, and (c) that buffer matrix filled syringes can be used to predict the syringe functionality and stability of drug product filled syringes. Based on the experimental findings of a variety of orthogonal characterization techniques including contact angle, interfacial tension and calculation of Hansen Solubility Parameters, it is proposed that silicone oil change is caused by formulation excipients and a complex set of phenomena summarized as ″wet, wash, and de-lube″ processes.A validated bioassay is used to measure the potency of commercial lots, and as such, must be accurate, precise, and fit for its intended purpose. Regulatory guidelines for the validation of a bioassay include a characterization of features, such as accuracy, precision, linearity, and range. The journey of a validated bioassay typically starts in a development lab, where it may be eventually be qualified to measure the potency of clinical lots. Prior to validation, as the bioassay is transferred to new laboratories, the bioassay is studied and assessed across a series of small experiments. In this work, instead of considering each study as a separate, independent report, it is proposed that, beginning with the qualification study, the accuracy and precision of the bioassay be continuously characterized, with each subsequent study result building from the ones that preceded. We call this continuous qualification. Such a proposition is naturally carried out using Bayesian statistical methods in which the historical study data is used to construct prior knowledge that is blended with the current study data.