Improving the Comparability and Predictability of a Pilot-Scale Model of Monoclonal Antibody Production Processes

The presentation discusses the methodology used to diagnose and address cell culture process performance offsets that existed in the pilot plant bioreactors. Data and discussion on the application of principles for process scale-up/ scale-down for cell culture are provided

Valuing a Culturally Diverse Work Force: An Examination of Intended Changes and Successes as Perceived by Upper and Middle Management, Supervisors, and Line Staff in a Health Care Organization

The growing diversity of the work force is having and will continue to have a significant impact on organizations. The diversity of cultures, lifestyles, and household compositions compels managers and organizational leaders to make a variety of organizational changes. Additionally, the globalization of business, the integration of the world economy, and the rapid changes in corporate life require a reexamination of how organizations are thought of, and how employees are managed (Copeland, 1988; Betters-Reed & Moore, 1988-89; Goldstein, 1988). Since in the past organizations have been based on white male European traditions, a revolutionary response is needed from organizational leaders and managers. Diversity in the work force is often defined as not only differences in cultural and ethnic backgrounds, but differences in gender, lifestyles, age, and career and developmental perspectives. However, for the purposes of this research, the definition of diversity is defined as the diversity of perspectives which arise from differences in cultural and ethnic backgrounds. Those managers and organizational leaders sensitive to the issues surfacing from a culturally diverse work force will have the competitive edge to succeed in a fast changing environment. Therefore those managers and leaders who are able to accurately identify and implement the strategies most successful in valuing a diverse work force will realize more successful organizational outcomes. The intent of this research was to identify, through the use of a survey instrument and review of the literature, appropriate methods for valuing a culturally diverse work force. One important issue which came from respondents had to do with their discomfort with recognizing cultural differences, preferring instead to refer to individual differences. Overall responses from upper management, middle management, supervisors, and line staff were fairly similar, although, as expected, management as group was better able to list the valuing cultural diversity strategies in the organization than line staff. Additionally, upper management, middle management, supervisors and line staff evaluated the success of the strategies somewhat differently. Finally, when it came to the organization\u27s values on cultural diversity and identification with those values, management as a group expressed greater agreement with the organization\u27s values than line staff

Degradation of MinD Oscillator Complexes by Escherichia coli ClpXP

MinD is a cell division ATPase in Escherichia coli that os- cillates from pole to pole and regulates the spatial position of the cell division machinery. Together with MinC and MinE, the Min system restricts assembly of the FtsZ-ring to midcell, oscillating between the opposite ends of the cell and preventing FtsZ-ring misassembly at the poles. Here, we show that the ATP-dependent bacterial proteasome complex ClpXP degrades MinD in reconstituted degradation reactions in vitro and in vivo through direct recognition of the MinD N-terminal region. MinD degradation is enhanced during stationary phase, suggesting that ClpXP regulates levels of MinD in cells that are not actively dividing. ClpXP is a major regulator of growth phase–dependent proteins, and these results suggest that MinD levels are also controlled during stationary phase. In vitro, MinC and MinD are known to coassemble into linear polymers; therefore, we monitored copolymers assembled in vitro after incubation with ClpXP and observed that ClpXP promotes rapid MinCD copolymer destabilization and direct MinD degradation by ClpXP. The N terminus of MinD, including residue Arg 3, which is near the ATP-binding site in sequence, is critical for degradation by ClpXP. Together, these results demonstrate that ClpXP degradation modifies conformational assemblies of MinD in vitro and depresses Min function in vivo during periods of reduced proliferation

An Essential Staphylococcus Aureus Cell Division Protein Directly Regulates FtsZ Dynamics

Binary fission has been well studied in rod-shaped bacteria, but the mechanisms underlying cell division in spherical bacteria are poorly understood. Rod-shaped bacteria harbor regulatory proteins that place and remodel the division machinery during cytokinesis. In the spherical human pathogen Staphylococcus aureus, we found that the essential protein GpsB localizes to mid-cell during cell division and co-constricts with the division machinery. Depletion of GpsB arrested cell division and led to cell lysis, whereas overproduction of GpsB inhibited cell division and led to the formation of enlarged cells. We report that S. aureus GpsB, unlike other Firmicutes GpsB orthologs, directly interacts with the core divisome component FtsZ. GpsB bundles and organizes FtsZ filaments and also stimulates the GTPase activity of FtsZ. We propose that GpsB orchestrates the initial stabilization of the Z-ring at the onset of cell division and participates in the subsequent remodeling of the divisome during cytokinesis

Proteolysis-Dependent Remodeling of the Tubulin Homolog FtsZ at the Division Septum in \u3ci\u3eEscherichia coli\u3c/i\u3e

During bacterial cell division a dynamic protein structure called the Z-ring assembles at the septum. The major protein in the Z-ring in Escherichia coli is FtsZ, a tubulin homolog that polymerizes with GTP. FtsZ is degraded by the two-component ATP-dependent protease ClpXP. Two regions of FtsZ, located outside of the polymerization domain in the unstructured linker and at the C-terminus, are important for specific recognition and degradation by ClpXP. We engineered a synthetic substrate containing green fluorescent protein (Gfp) fused to an extended FtsZ C-terminal tail (residues 317–383), including the unstructured linker and the C-terminal conserved region, but not the polymerization domain, and showed that it is sufficient to target a non-native substrate for degradation in vitro. To determine if FtsZ degradation regulates Z-ring assembly during division, we expressed a full length Gfp-FtsZ fusion protein in wild type and clp deficient strains and monitored fluorescent Z-rings. In cells deleted for clpX or clpP, or cells expressing protease-defective mutant protein ClpP(S97A), Z-rings appear normal; however, after photobleaching a region of the Z-ring, fluorescence recovers ~70% more slowly in cells without functional ClpXP than in wild type cells. Gfp-FtsZ(R379E), which is defective for degradation by ClpXP, also assembles into Z-rings that recover fluorescence ~2-fold more slowly than Z-rings containing Gfp-FtsZ. In vitro, ClpXP cooperatively degrades and disassembles FtsZ polymers. These results demonstrate that ClpXP is a regulator of Z-ring dynamics and that the regulation is proteolysis-dependent. Our results further show that FtsZ-interacting proteins in E. coli fine-tune Z-ring dynamics

Uropathogenic <i>Escherichia coli</i> metabolite-dependent quiescence and persistence may explain antibiotic tolerance during urinary tract infection

ABSTRACT In the present study, it is shown that although Escherichia coli CFT073, a human uropathogenic (UPEC) strain, grows in liquid glucose M9 minimal medium, it fails to grow on glucose M9 minimal medium agar plates seeded with ≤106 CFU. The cells on glucose plates appear to be in a “quiescent” state that can be prevented by various combinations of lysine, methionine, and tyrosine. Moreover, the quiescent state is characteristic of ~80% of E. coli phylogenetic group B2 multilocus sequence type 73 strains, as well as 22.5% of randomly selected UPEC strains isolated from community-acquired urinary tract infections in Denmark. In addition, E. coli CFT073 quiescence is not limited to glucose but occurs on agar plates containing a number of other sugars and acetate as sole carbon sources. It is also shown that a number of E. coli CFT073 mini-Tn5 metabolic mutants (gnd, gdhA, pykF, sdhA, and zwf) are nonquiescent on glucose M9 minimal agar plates and that quiescence requires a complete oxidative tricarboxylic acid (TCA) cycle. In addition, evidence is presented that, although E. coli CFT073 quiescence and persistence in the presence of ampicillin are alike in that both require a complete oxidative TCA cycle and each can be prevented by amino acids, E. coli CFT073 quiescence occurs in the presence or absence of a functional rpoS gene, whereas maximal persistence requires a nonfunctional rpoS. Our results suggest that interventions targeting specific central metabolic pathways may mitigate UPEC infections by interfering with quiescence and persistence. IMPORTANCE Recurrent urinary tract infections (UTIs) affect 10 to 40% of women. In up to 77% of those cases, the recurrent infections are caused by the same uropathogenic E. coli (UPEC) strain that caused the initial infection. Upon infection of urothelial transitional cells in the bladder, UPEC appear to enter a nongrowing quiescent intracellular state that is thought to serve as a reservoir responsible for recurrent UTIs. Here, we report that many UPEC strains enter a quiescent state when ≤106 CFU are seeded on glucose M9 minimal medium agar plates and show that mutations in several genes involved in central carbon metabolism prevent quiescence, as well as persistence, possibly identifying metabolic pathways involved in UPEC quiescence and persistence in vivo

Uropathogenic Escherichia coli Metabolite-Dependent Quiescence and Persistence May Explain Antibiotic Tolerance during Urinary Tract Infection

In the present study, it is shown that although Escherichia coli CFT073, a human uropathogenic (UPEC) strain, grows in liquid glucose M9 minimal medium, it fails to grow on glucose M9 minimal medium agar plates seeded with ≤106 CFU. The cells on glucose plates appear to be in a “quiescent” state that can be prevented by various combinations of lysine, methionine, and tyrosine. Moreover, the quiescent state is characteristic of ~80% of E. coli phylogenetic group B2 multilocus sequence type 73 strains, as well as 22.5% of randomly selected UPEC strains isolated from community-acquired urinary tract infections in Denmark. In addition, E. coli CFT073 quiescence is not limited to glucose but occurs on agar plates containing a number of other sugars and acetate as sole carbon sources. It is also shown that a number of E. coliCFT073 mini-Tn5 metabolic mutants (gnd, gdhA, pykF, sdhA, and zwf) are nonquiescent on glucose M9 minimal agar plates and that quiescence requires a complete oxidative tricarboxylic acid (TCA) cycle. In addition, evidence is presented that, although E. coli CFT073 quiescence and persistence in the presence of ampicillin are alike in that both require a complete oxidative TCA cycle and each can be prevented by amino acids, E. coli CFT073 quiescence occurs in the presence or absence of a functional rpoS gene, whereas maximal persistence requires a nonfunctional rpoS. Our results suggest that interventions targeting specific central metabolic pathways may mitigate UPEC infections by interfering with quiescence and persistence

Hospital and outpatient clinic utilization among older people in the 3-5 years following the initiation of continuing care: a longitudinal cohort study

Background: Few studies have investigated the subsequent rate of hospital and outpatient clinic utilization in those who receive continuing care and have documented frequent usage over one year. Such knowledge may be helpful in identifying those who would benefit from preventive interventions. The aim of this study was to investigate and compare the subsequent rate of hospital and outpatient clinic utilization among older people with 0, 1, 2, 3 or more hospital stays in the first year following the initiation of continuing care. A further aim was to compare these groups regarding demographic data, health complaints, functional and cognitive ability, informal care and mortality. Methods: A total of 1079 people, aged 65 years or older, who received a decision regarding the initiation of continuing care during the years 2001, 2002 or 2003 were investigated. Four groups were created based on whether they had 0, 1, 2 or >= 3 hospital stays in the first year following the initiation of continuing care and were investigated regarding the rate of hospital and outpatient clinic utilization in the subsequent 3-5 years. Results: Fifty seven percent of the sample had no hospital stay during the first year following the initiation of continuing care, 20% had 1 stay, 10% had 2 stays and 13% had three or more hospital stays (range: 3-13). Those with >= 3 hospital stays in the first year continued to have the significantly highest rate of hospital and outpatient care utilization in the subsequent years. This group accounted for 57% of hospital stays in the first year, 27% in the second year and 18% in the third year. In this group the risk of having >= 3 hospital stays in the second year was 27% and 12% in the third year. Conclusions: There is a clear need for interventions targeted on prevention of frequent hospital and outpatient clinic utilization among those who are high users of hospital care in the first year after the initiation of continuing care. Perhaps an increased availability of medically skilled staff in the day to day care of these people in the municipalities could prevent frequent hospital and outpatient clinic utilization, especially hospital readmissions

AB Toxins: A Paradigm Switch from Deadly to Desirable

To ensure their survival, a number of bacterial and plant species have evolved a common strategy to capture energy from other biological systems. Being imperfect pathogens, organisms synthesizing multi-subunit AB toxins are responsible for the mortality of millions of people and animals annually. Vaccination against these organisms and their toxins has proved rather ineffective in providing long-term protection from disease. In response to the debilitating effects of AB toxins on epithelial cells of the digestive mucosa, mechanisms underlying toxin immunomodulation of immune responses have become the focus of increasing experimentation. The results of these studies reveal that AB toxins may have a beneficial application as adjuvants for the enhancement of immune protection against infection and autoimmunity. Here, we examine similarities and differences in the structure and function of bacterial and plant AB toxins that underlie their toxicity and their exceptional properties as immunomodulators for stimulating immune responses against infectious disease and for immune suppression of organ-specific autoimmunity

Association between mental health conditions and rehospitalization, mortality, and functional outcomes in patients with stroke following inpatient rehabilitation

<p>Abstract</p> <p>Background</p> <p>Limited evidence exists regarding the association of pre-existing mental health conditions in patients with stroke and stroke outcomes such as rehospitalization, mortality, and function. We examined the association between mental health conditions and rehospitalization, mortality, and functional outcomes in patients with stroke following inpatient rehabilitation.</p> <p>Methods</p> <p>Our observational study used the 2001 VA Integrated Stroke Outcomes database of 2162 patients with stroke who underwent rehabilitation at a Veterans Affairs Medical Center.</p> <p>Separate models were fit to our outcome measures that included 6-month rehospitalization or death, 6-month mortality post-discharge, and functional outcomes post inpatient rehabilitation as a function of number and type of mental health conditions. The models controlled for patient socio-demographics, length of stay, functional status, and rehabilitation setting.</p> <p>Results</p> <p>Patients had an average age of 68 years. Patients with stroke and two or more mental health conditions were more likely to be readmitted or die compared to patients with no conditions (OR: 1.44, p = 0.04). Depression and anxiety were associated with a greater likelihood of rehospitalization or death (OR: 1.33, p = 0.04; OR:1.47, p = 0.03). Patients with anxiety were more likely to die at six months (OR: 2.49, p = 0.001).</p> <p>Conclusions</p> <p>Patients with stroke with pre-existing mental health conditions may need additional psychotherapy interventions, which may potentially improve stroke outcomes post-hospitalization.</p