Dysregulation of Cell Envelope Homeostasis in Staphylococcus aureus Exposed to Solvated Lignin.

Lignin is an aromatic plant cell wall polymer that facilitates water transport through the vasculature of plants and is generated in large quantities as an inexpensive by-product of pulp and paper manufacturing and biorefineries. Although lignin's ability to reduce bacterial growth has been reported previously, its hydrophobicity complicates the ability to examine its biological effects on living cells in aqueous growth media. We recently described the ability to solvate lignin in Good's buffers with neutral pH, a breakthrough that allowed examination of lignin's antimicrobial effects against the human pathogen Staphylococcus aureus. These analyses showed that lignin damages the S. aureus cell membrane, causes increased cell clustering, and inhibits growth synergistically with tunicamycin, a teichoic acid synthesis inhibitor. In the present study, we examined the physiological and transcriptomic responses of S. aureus to lignin. Intriguingly, lignin restored the susceptibility of genetically resistant S. aureus isolates to penicillin and oxacillin, decreased intracellular pH, impaired normal cell division, and rendered cells more resistant to detergent-induced lysis. Additionally, transcriptome sequencing (RNA-Seq) differential expression (DE) analysis of lignin-treated cultures revealed significant gene expression changes (P < 0.05 with 5% false discovery rate [FDR]) related to the cell envelope, cell wall physiology, fatty acid metabolism, and stress resistance. Moreover, a pattern of concurrent up- and downregulation of genes within biochemical pathways involved in transmembrane transport and cell wall physiology was observed, which likely reflects an attempt to tolerate or compensate for lignin-induced damage. Together, these results represent the first comprehensive analysis of lignin's antibacterial activity against S. aureus. IMPORTANCE S. aureus is a leading cause of skin and soft tissue infections. The ability of S. aureus to acquire genetic resistance to antibiotics further compounds its ability to cause life-threatening infections. While the historical response to antibiotic resistance has been to develop new antibiotics, bacterial pathogens are notorious for rapidly acquiring genetic resistance mechanisms. As such, the development of adjuvants represents a viable way of extending the life span of current antibiotics to which pathogens may already be resistant. Here, we describe the phenotypic and transcriptomic response of S. aureus to treatment with lignin. Our results demonstrate that lignin extracted from sugarcane and sorghum bagasse restores S. aureus susceptibility to ß-lactams, providing a premise for repurposing these antibiotics in treatment of resistant S. aureus strains, possibly in the form of topical lignin/ß-lactam formulations.

Referred Orofacial Pain as an Initial Symptom of Distant, Nonmetastatic Cancer: Report of a Case and Review of the Literature.

Referred orofacial pain as the first symptom of an otherwise silent distant, nonmetastatic cancer has been reported, but there is sparse literature on the subject. Referred pain may not be considered in the orofacial pain differential diagnosis because of its rarity; however, this may delay a cancer diagnosis. The authors present a case report and a review of the English literature. Peer-reviewed publications were identified through a systematic search of MEDLINE, Embase, and Cochrane CENTRAL. Historic cases were reviewed, and available data regarding demographics, pain characteristics, treatment, cancer diagnosis, and outcome were extracted. Thirty-seven cases were identified. All cancers were intrathoracic. The average age was 54.1 years with a slight female predominance (3:2). Common pain characteristics were (1) diffuse location affecting the ear (76%), jaw (46%), and temple (30%); (2) constant duration (65%); (3) aching quality (74%); (4) severe intensity (94%); and (5) associated systemic symptoms (68%) such as weight loss and digital clubbing. The average time from the onset of facial pain to seeking medical attention was 9 months, and the average time from seeking medication attention to cancer diagnosis was 8 months. Orofacial pain was often attributed to odontogenic (35%) or neuropathic (25%) causes, and treatments for these conditions were also common. The impact of referred orofacial pain on the cancer prognosis was not possible because of the nature of the reviewed studies (case reports with no comparison group). Cancer-associated referred orofacial pain as a first symptom is rare but should be considered in cases with intractable pain and associated systemic symptoms.

Quantification of Staphylococcus aureus Biofilm Formation by Crystal Violet and Confocal Microscopy.

Most Staphylococcus aureus strains can grow as a multicellular biofilm, a phenotype of utmost importance to clinical infections such as endocarditis, osteomyelitis, and implanted medical device infection. As biofilms are inherently more tolerant to the host immune system and antibiotics, understanding the S. aureus genes and regulatory circuits that contribute to biofilm development is an active and on-going field of research. This chapter details a high-throughput and standardized way to grow S. aureus biofilms using a classical microtiter plate assay. Biofilms can be quantified using crystal violet or by confocal microscopy imaging and COMSTAT analysis.

Manufacturing process optimization of ADMA Biologics' intravenous immunoglobulin products, BIVIGAM® and ASCENIV™.

Humoral immunodeficiency patients require immunoglobulin replacement to prevent infection. Traditional intravenous immunoglobulin manufacturing methods have had the potential for containing impurities caused by physical, chemical and thermal stressors that alter proteins. Two intravenous immunoglobulin products, BIVIGAM® and ASCENIV™, are manufactured by a modified Cohn-Oncley fractionation method followed by chromatographic purification. These products have undergone a systematic quality by design optimization to identify critical manufacturing processes to produce the highest quality product. This data driven, small-scale approach has led to manufacturing enhancements that have yielded consistent product improvements. The systematic approach to optimizing manufacturing has guided process changes, in-process, procedural and engineering controls that have reduced protein shearing and aggregation, and improved purity resulting in products with lot-to-lot consistency.

Lignin-based polymers and nanomaterials.

Approximately 50 million tons of lignin are currently produced annually as a by-product of the pulp- and paper industry, and this amount is likely to double in the future with the anticipated production of renewable fuels and chemicals from lignocellulosic biomass, as a sustainable alternative to petroleum. The latter process can be expedited by valorizing lignin, which entails making products from lignin that generate additional revenues for biorefineries so that the production of biofuels becomes more competitive with gasoline. Industrially produced lignin is considered a low-value material that is used as a boiler fuel to generate heat and electricity, and as an ingredient of adhesives, cement, and drilling fluids for underwater oil wells. The aromatic nature of lignin, its ability to participate in radical-mediated cross-linking reactions, the many functional groups available for derivatization or chemical reactions, and its amenability to existing procedures for making thermoplastics, make it attractive as an additive to polymers to enhance UV-tolerance and/or other physico-chemical properties. Lignin can also be used as the basis for various nanomaterials, either per se or in combination with other polymers. This review summarizes recent developments in the synthesis of lignin-containing polymers and nanomaterials, whereby inherent variation in lignin subunit composition and structure, as a function of plant species and lignin extraction method, offer unique opportunities for fine-tuning material properties (e.g. tensile strength, hardness, elasticity) to match specific applications.

Altering physiological networks using drugs: steps towards personalized physiology.

BACKGROUND: The rise of personalized medicine has reminded us that each patient must be treated as an individual. One factor in making treatment decisions is the physiological state of each patient, but definitions of relevant states and methods to visualize state-related physiologic changes are scarce. We constructed correlation networks from physiologic data to demonstrate changes associated with pressor use in the intensive care unit. METHODS: We collected 29 physiological variables at one-minute intervals from nineteen trauma patients in the intensive care unit of an academic hospital and grouped each minute of data as receiving or not receiving pressors. For each group we constructed Spearman correlation networks of pairs of physiologic variables. To visualize drug-associated changes we split the networks into three components: an unchanging network, a network of connections with changing correlation sign, and a network of connections only present in one group. RESULTS: Out of a possible 406 connections between the 29 physiological measures, 64, 39, and 48 were present in each of the three component networks. The static network confirms expected physiological relationships while the network of associations with changed correlation sign suggests putative changes due to the drugs. The network of associations present only with pressors suggests new relationships that could be worthy of study. CONCLUSIONS: We demonstrated that visualizing physiological relationships using correlation networks provides insight into underlying physiologic states while also showing that many of these relationships change when the state is defined by the presence of drugs. This method applied to targeted experiments could change the way critical care patients are monitored and treated.

Identification of complex metabolic states in critically injured patients using bioinformatic cluster analysis.

INTRODUCTION: Advances in technology have made extensive monitoring of patient physiology the standard of care in intensive care units (ICUs). While many systems exist to compile these data, there has been no systematic multivariate analysis and categorization across patient physiological data. The sheer volume and complexity of these data make pattern recognition or identification of patient state difficult. Hierarchical cluster analysis allows visualization of high dimensional data and enables pattern recognition and identification of physiologic patient states. We hypothesized that processing of multivariate data using hierarchical clustering techniques would allow identification of otherwise hidden patient physiologic patterns that would be predictive of outcome. METHODS: Multivariate physiologic and ventilator data were collected continuously using a multimodal bioinformatics system in the surgical ICU at San Francisco General Hospital. These data were incorporated with non-continuous data and stored on a server in the ICU. A hierarchical clustering algorithm grouped each minute of data into 1 of 10 clusters. Clusters were correlated with outcome measures including incidence of infection, multiple organ failure (MOF), and mortality. RESULTS: We identified 10 clusters, which we defined as distinct patient states. While patients transitioned between states, they spent significant amounts of time in each. Clusters were enriched for our outcome measures: 2 of the 10 states were enriched for infection, 6 of 10 were enriched for MOF, and 3 of 10 were enriched for death. Further analysis of correlations between pairs of variables within each cluster reveals significant differences in physiology between clusters. CONCLUSIONS: Here we show for the first time the feasibility of clustering physiological measurements to identify clinically relevant patient states after trauma. These results demonstrate that hierarchical clustering techniques can be useful for visualizing complex multivariate data and may provide new insights for the care of critically injured patients.

Infection in the intensive care unit alters physiological networks.

BACKGROUND: Physicians use clinical and physiological data to treat patients every day, and it is essential for treating a patient appropriately. However, medical sources of clinical physiological data are only now starting to find use in bioinformatics research. RESULTS: We collected 29 types of physiological and clinical data on a minute-by-minute basis from trauma patients in the intensive care unit along with whether they contracted an infection during their stay. Dividing the patients into two groups based on this criterion, we determined that the correlational network amongst pairs of physiological variables changes based on whether the patient contracted an infection. CONCLUSION: Examining the variable pairs with the largest change in correlation across groups reveals potential changes in the way our treatments affect the patient's physiology and in how our bodies react to physiological insults. These findings highlight the usefulness of physiological informatics and suggest new relationships to study while also validating previously reported relationships.

Field application of activated carbon amendment for in-situ stabilization of polychlorinated biphenyls in marine sediment.

We report results on the first field-scale application of activated carbon (AC) amendment to contaminated sediment for in-situ stabilization of polychlorinated biphenyls (PCBs). The test was performed on a tidal mud flat at South Basin, adjacent to the former Hunters Point Naval Shipyard, San Francisco Bay, CA. The major goals of the field study were to (1) assess scale up of the AC mixing technology using two available, large-scale devices, (2) validate the effectiveness of the AC amendment at the field scale, and (3) identify possible adverse effects of the remediation technology. Also, the test allowed comparison among monitoring tools, evaluation of longer-term effectiveness of AC amendment, and identification of field-related factors that confound the performance of in-situ biological assessments. Following background pretreatment measurements, we successfully incorporated AC into sediment to a nominal 30 cm depth during a single mixing event, as confirmed by total organic carbon and black carbon contents in the designated test plots. The measured AC dose averaged 2.0-3.2 wt% and varied depending on sampling locations and mixing equipment. AC amendment did not impact sediment resuspension or PCB release into the water column over the treatment plots, nor adversely impactthe existing macro benthic community composition, richness, or diversity. The PCB bioaccumulation in marine clams was reduced when exposed to sediment treated with 2% AC in comparison to the control plot Field-deployed semi permeable membrane devices and polyethylene devices showed about 50% reduction in PCB uptake in AC-treated sediment and similar reduction in estimated pore-water PCB concentration. This reduction was evident even after 13-month post-treatment with then 7 months of continuous exposure, indicating AC treatment efficacy was retained for an extended period. Aqueous equilibrium PCB concentrations and PCB desorption showed an AC-dose response. Field-exposed AC after 18 months retained a strong stabilization capability to reduce aqueous equilibrium PCB concentrations by about 90%, which also supports the long-term effectiveness of AC in the field. Additional mixing during or after AC deployment, increasing AC dose, reducing AC-particle size, and sequential deployment of AC dose will likely improve AC-sediment contact and overall effectiveness. The reductions in PCB availability observed with slow mass transfer under field conditions calls for predictive models to assess the long-term trends in pore-water PCB concentrations and the benefits of alternative in-situ AC application and mixing strategies.

Measurement of activated carbon and other black carbons in sediments.

Black carbon in sediment, present natively or added as a treatment amendment in the form of activated carbon, reduces contaminant bioavailability. Field evaluation of activated carbon effectiveness in reducing contaminant bioavailability requires accurate methods to measure the amendment in sediments. The most commonly used method to separate black carbon from natural organic matter in soils and sediments is low temperature (375 degrees C) thermal oxidation which resulted in significant losses of activated carbons. A method was developed to isolate activated carbon using a solution of concentrated sulfuric acid and potassium dichromate to oxidize the natural organic matter while preserving the activated carbon. The chemical oxidation method was applied to assess the delivery of activated carbon to sediments in a pilot-scale demonstration project carried out in Grasse River, NY. Using this method on sediment from the Grasse River, over 98% of the natural organic matter was removed while preserving at least 95% of the activated carbon. The method was also demonstrated on other carbonaceous geosorbents and native black carbon in several sediment samples.

A probabilistic algorithm for estimating the intention of computer users with movement disorders.

Standard computer interfaces are inefficient for many users who have movement disorders. We believe this stems from the standard view that interface devices accurately represent the intention of the user. For those who have movement disorders, such as dystonia, it may be more effective to view the computer interface as an instrument used to estimate the user's intent. We develop a probabilistic algorithm to design an estimation device for computer input. We also implement the algorithm as part of text-entry software and perform a preliminary case study on a patient with dystonia, using a joystick as a text input device.