Automized inline monitoring in perfused mammalian cell culture by MIR spectroscopy without calibration model building.

Process Analytical Technologies (PATs) are taking a key role in the run for automatization in the biopharmaceutical industry. Spectroscopic methods such as Raman spectroscopy or mid-infrared (MIR) spectroscopy are getting more recognition in the recent years for inline monitoring of bioprocesses due to their ability to measure various molecules simultaneously. However, their dependency on laborious model calibration making them a challenge to implement. In this study, a novel one-point calibration that requires a single reference point prior to the inline monitoring of glucose and lactate in bioprocesses with MIR spectroscopy is assessed with 22 mammalian cell perfusion (PER) processes in two different scales and four different products. Concentrations are predicted over all PERs runs with a root mean square error (RMSE) of 0.29 g/L for glucose and 0.24 g/L for lactate, respectively. For comparison conventional partial least square regression (PLSR) models were used and trained with spectroscopic data from six bioreactor runs in two different scales and three products. The general accuracy of those models (RMSE of 0.41 g/L for glucose and 0.16 g/L for lactate) are in the range of the accuracy of the one-point calibration. This shows the potential of the one-point calibration as an approach making spectroscopy more accessible for bioprocess development.

Underdiagnosed CKD in Geriatric Trauma Patients and Potent Prevention of Renal Impairment from Polypharmacy Risks through Individual Pharmacotherapy Management (IPM-III).

The aging global patient population with multimorbidity and concomitant polypharmacy is at increased risk for acute and chronic kidney disease, particularly with severe additional disease states or invasive surgical procedures. Because from the expertise of more than 58,600 self-reviewed medications, adverse drug reactions, drug interactions, inadequate dosing, and contraindications all proved to cause or exacerbate the worsening of renal function, we analyzed the association of an electronic patient record- and Summaries of Product Characteristics (SmPCs)-based comprehensive individual pharmacotherapy management (IPM) in the setting of 14 daily interdisciplinary patient visits with the outcome: further renal impairment with reduction of eGFR ≥ 20 mL/min (redGFR) in hospitalized trauma patients ≥ 70 years of age. The retrospective clinical study of 404 trauma patients comparing the historical control group (CG) before IPM with the IPM intervention group (IG) revealed a group-match in terms of potential confounders such as age, sex, BMI, arterial hypertension, diabetes mellitus, and injury patterns. Preexisting chronic kidney disease (CKD) > stage 2 diagnosed as eGFR < 60 mL/min/1.73 m2 on hospital admission was 42% in the CG versus 50% in the IG, although in each group only less than 50% of this was coded as an ICD diagnosis in the patients' discharge letters (19% in CG and 21% in IG). IPM revealed an absolute risk reduction in redGFR of 5.5% (11 of 199 CG patients) to 0% in the IPM visit IG, a relative risk reduction of 100%, NNT 18, indicating high efficacy of IPM and benefit in improving outcomes. There even remained an additive superimposed significant association that included patients in the IPM group before/beyond the 14 daily IPM interventions, with a relative redGFR risk reduction of 0.55 (55%) to 2.5% (5 of 204 patients), OR 0.48 [95% CI 0.438-0.538] (p < 0.001). Bacteriuria, loop diuretics, allopurinol, eGFR ≥ 60 mL/min/1.73 m2, eGFR < 60 mL/min/1.73 m2, and CKD 3b were significantly associated with redGFR; of the latter, 10.5% developed redGFR. Further multivariable regression analysis adjusting for these and established risk factors revealed an additive, superimposed IPM effect on redGFR with an OR 0.238 [95% CI 0.06-0.91], relative risk reduction of 76.2%, regression coefficient -1.437 including patients not yet visited in the IPM period. As consequences of the IPM procedure, the IG differed from the CG by a significant reduction of NSAIDs (p < 0.001), HCT (p = 0.028) and Würzburger pain drip (p < 0.001), and significantly increased prescription rate of antibiotics (p = 0.004). In conclusion, (1) more than 50% of CKD in geriatric patients was not pre-recognized and underdiagnosed, and (2) the electronic patient records-based IPM interdisciplinary networking strategy was associated with effective prevention of further periinterventional renal impairment and requires obligatory implementation in all elderly patients to urgently improve patient and drug safety.

Individual Pharmacotherapy Management (IPM)-IV: Optimized Usage of Approved Antimicrobials Addressing Under-Recognized Adverse Drug Reactions and Drug-Drug Interactions in Polypharmacy.

Antimicrobial therapy is often a life-saving medical intervention for inpatients and outpatients. Almost all medical disciplines are involved in this therapeutic procedure. Knowledge of adverse drug reactions (ADRs) and drug-drug interactions (DDIs) is important to avoid drug-related harm. Within the broad spectrum of antibiotic and antifungal therapy, most typical ADRs are known to physicians. The aim of this study was to evaluate relevant pharmacological aspects with which we are not so familiar and to provide further practical guidance. Individual pharmacotherapy management (IPM) as a synopsis of internal medicine and clinical pharmacology based on the entirety of the digital patient information with reference to drug information, guidelines, and literature research has been continuously performed for over 8 years in interdisciplinary intensive care and trauma and transplant patients. Findings from over 52,000 detailed medication analyses highlight critical ADRs and DDIs, especially in these vulnerable patients with polypharmacy. We present the most relevant ADRs and DDIs in antibiotic and antifungal pharmacology, which are less frequently considered in relation to neurologic, hemostaseologic, hematologic, endocrinologic, and cardiac complexities. Constant awareness and preventive strategies help avoid life-threatening manifestations of these inherent risks and ensure patient and drug safety in antimicrobial therapy.

Individual Pharmacotherapy Management (IPM) - I: a group-matched retrospective controlled clinical study on prevention of complicating delirium in the elderly trauma patients and identification of associated factors.

BACKGROUND: Delirium is one of the most frequent complications in hospitalized elderly patients with additional costs such as prolongation of hospital stays and institutionalization, with risk of reduced functional recovery, long-term cognitive impairment, and increased morbidity and mortality. We analyzed the effect of individual pharmacotherapy management (IPM) in the University Hospital Halle in geriatric trauma patients on complicating delirium and aimed to identify associated factors. METHODS: In a retrospective controlled clinical study of 404 hospitalized trauma patients ≥70 years we compared the IPM intervention group (IG) with a control group (CG) before IPM implementation. Delirium was recorded from the hospital discharge letter. The medication review and data records included baseline data, all medications, diagnoses, electrocardiogram (ECG), laboratory and vital parameters during hospitalization. The IPM internist and the senior trauma physician guaranteed personnel and structural continuity in the implementation of the interdisciplinary patient rounds. RESULTS: There was a highly matched congruence between CG and IG in terms of age, gender, residency, BMI, most diagnoses, and injury patterns to compare the two groups. The total number of medications per patient was 11.1 ± 4.9 (CG) versus 10.4 ± 3.6 (IG). Our targeted IPM focus on 6 frontline aspects with reduction of antipsychotics, anticholinergic burden, benzodiazepines, serotonergic opioids, elimination of pharmacokinetic and pharmacodynamic drug interactions and overdosage reduced complicating delirium from 5% to almost zero at 0.5%. The association of IPM with a significant 10-fold reduction, OR = 0.09 [95% CI 0.01-0.7], in univariable regression, maintained of clinical relevance in multivariable regression OR = 0.1 [95% CI 0.01-1.1]. Factors most strongly associated with complicating delirium in univariable regression were cognitive dysfunction, nursing home residency, muscle relaxants, antiparkinsonian agents, xanthines, transient disorientation documented in the fall risk scale, antibiotic-requiring infections, antifungals, antipsychotics, and intensive care stay, the two latter maintaining significance in multivariable regression. CONCLUSIONS: IPM is associated with a highly effective prevention of complicating delirium in the elderly trauma patients. For patient safety it should be integrated as an essential preventative contribution. The associated factors help identify patients at risk.

[Development and feasibility of an intervention to reduce physical restraints in hospitals: A mixed methods study]. / Entwicklung und Machbarkeit einer Intervention zur Vermeidung freiheitsentziehender Maßnahmen im Krankenhaus.

Development and feasibility of an intervention to reduce physical restraints in hospitals: A mixed methods study Abstract. Background: Physical restraints (PR) are regularly used in people with dementia or delirium in general hospital settings. There is no clear evidence for the effectiveness of PR, but their use is associated with an increased risk for harm. Therefore, a restraint-free care is recommended. Aim: Development and feasibility test of an intervention to reduce PR in general hospital settings. Methods: Systematic literature reviews and theory-guided modelling of an intervention involving relevant clinical stakeholders and mixed methods study in two wards of a university hospital (geriatric traumatology and neurology). Results: The complex intervention comprises the following components: qualification of multipliers, interprofessional education about PR reduction, regular audit and feedback meetings, and the support regarding the aim of the intervention from nursing and medical leaders. The results of the feasibility test indicate that the intervention is feasible and helpful, but the interprofessional approach was not implemented as planned. An important barrier hampering PR reduction was the high workload. The results on the prevalence of PR could not be interpreted due to a very low number of measures applied. Conclusion: The complex intervention with a multiplier approach for preventing PR use was judged as feasible, but there is a need for further development to strengthen interprofessional cooperation. The feasibility of the intervention should also be tested in other departments.

Productivity and quality of recombinant proteins produced by stable CHO cell clones can be predicted by transient expression in HEK cells.

Selection of lead candidates in drug discovery is a complex and time-consuming process. Here, we describe an approach that allows prediction of the productivity and quality of recombinant proteins by stable producer cell clones with the help of transient transfection studies. This is exemplified for three distinct bispecific T cell engager (BiTE(®))-a new class of single-chain antibody-based therapeutics showing very promising results in the treatment of cancer. BiTE(®) titers of transiently transfected HEK cells showed a striking correlation with titers of selected stable CHO cell clones. Likewise, the percentage of the monomeric BiTE(®) fraction in cell culture supernatants correlated well between transiently expressing HEK and stably expressing CHO cell clones. This validates the use of transient transfection studies for the selection of biopharmaceutical lead candidates with desired pharmaceutical properties.

Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis.

Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis.

Choice of Plk1 docking partners during mitosis and cytokinesis is controlled by the activation state of Cdk1.

Spatial and temporal coordination of polo-like kinase 1 (Plk1) activity is necessary for mitosis and cytokinesis, and this is achieved through binding to phosphorylated docking proteins with distinct subcellular localizations. Although cyclin-dependent kinase 1 (Cdk1) creates these phosphorylated docking sites in metaphase, a general principle that explains how Plk1 activity is controlled in anaphase after Cdk1 inactivation is lacking. Here, we show that the microtubule-associated protein regulating cytokinesis (PRC1) is an anaphase-specific binding partner for Plk1, and that this interaction is required for cytokinesis. In anaphase, Plk1 creates its own docking site on PRC1, whereas in metaphase Cdk1 phosphorylates PRC1 adjacent to this docking site and thereby prevents binding of Plk1. Mutation of these Cdk1-sites results in a form of PRC1 that prematurely recruits Plk1 to the spindle during prometaphase and blocks mitotic progression. The activation state of Cdk1, therefore, controls the switch of Plk1 localization from centrosomes and kinetochores during metaphase, to the central spindle during anaphase.

Cooperation between mitotic kinesins controls the late stages of cytokinesis.

Cell division is regulated by protein kinases of the Cdk, Polo, and Aurora families. Although it has long been established that temporal control is central to the coordinated action of these kinases, the importance of spatial regulation has only recently been appreciated and is still poorly understood. The kinesin-6 family motor protein MKlp1 is a key regulator of cytokinesis and an ideal substrate for studying spatially regulated protein-phosphorylation events. MKlp1 is negatively regulated by Cdk1 phosphorylation during metaphase and becomes activated in anaphase when cleavage-furrow assembly commences. Aurora B phosphorylates MKlp1 during anaphase and is required for its function in cytokinesis. Another kinesin-6 family motor, MKlp2, mediates the relocation of Aurora B from the centromeres to the central spindle at the onset of anaphase. We now demonstrate that this process is required for the phosphorylation of MKlp1 at S911, an Aurora B consensus site overlapping a bipartite nuclear localization sequence (NLS). MKlp1(S911A) targets to the central spindle but is prematurely imported into the nucleus and fails to support cytokinesis. Spatial restriction of Aurora B to the central spindle by MKlp2 therefore regulates MKlp1 during cytokinesis in human cells.

KIF14 and citron kinase act together to promote efficient cytokinesis.

Multiple mitotic kinesins and microtubule-associated proteins (MAPs) act in concert to direct cytokinesis (Glotzer, M. 2005. Science. 307:1735-1739). In anaphase cells, many of these proteins associate with an antiparallel array of microtubules termed the central spindle. The MAP and microtubule-bundling protein PRC1 (protein-regulating cytokinesis 1) is one of the key molecules required for the integrity of this structure (Jiang, W., G. Jimenez, N.J. Wells, T.J. Hope, G.M. Wahl, T. Hunter, and R. Fukunaga. 1998. Mol. Cell. 2:877-885; Mollinari, C., J.P. Kleman, W. Jiang, G. Schoehn, T. Hunter, and R.L. Margolis. 2002. J. Cell Biol. 157:1175-1186). In this study, we identify an interaction between endogenous PRC1 and the previously uncharacterized kinesin KIF14 as well as other mitotic kinesins (MKlp1/CHO1, MKlp2, and KIF4) with known functions in cytokinesis (Hill, E., M. Clarke, and F.A. Barr. 2000. EMBO J. 19:5711-5719; Matuliene, J., and R. Kuriyama. 2002. Mol. Biol. Cell. 13:1832-1845; Kurasawa, Y., W.C. Earnshaw, Y. Mochizuki, N. Dohmae, and K. Todokoro. 2004. EMBO J. 23:3237-3248). We find that KIF14 targets to the central spindle via its interaction with PRC1 and has an essential function in cytokinesis. In KIF14-depleted cells, citron kinase but not other components of the central spindle and cleavage furrow fail to localize. Furthermore, the localization of KIF14 and citron kinase to the central spindle and midbody is codependent, and they form a complex depending on the activation state of citron kinase. Contrary to a previous study (Di Cunto, F., S. Imarisio, E. Hirsch, V. Broccoli, A. Bulfone, A. Migheli, C. Atzori, E. Turco, R. Triolo, G.P. Dotto, et al. 2000. Neuron. 28:115-127), we find a general requirement for citron kinase in human cell division. Together, these findings identify a novel pathway required for efficient cytokinesis.

Assay and functional properties of Rabkinesin-6/Rab6-KIFL/MKlp2 in cytokinesis.

Here we describe methods for the characterization of the kinesin-9 family motor protein Rabkinesin-6/Rab6-KIFL/MKlp2 in cytokinesis. Here we outline biochemical assays for studying the interaction of Rabkinesin-6/Rab6-KIFL/MKlp2 with microtubules, and its regulation and interaction with the mitotic polo-like kinase 1 using recombinant proteins expressed in and purified from insect cells. Protocols for the in vivo functional analysis of Rabkinesin-6/Rab6-KIFL/MKlp2 using depletion with small interfering RNA duplexes are described.

Relocation of Aurora B from centromeres to the central spindle at the metaphase to anaphase transition requires MKlp2.

Mitotic kinases of the Polo and Aurora families are key regulators of chromosome segregation and cytokinesis. Here, we have investigated the role of MKlp1 and MKlp2, two vertebrate mitotic kinesins essential for cytokinesis, in the spatial regulation of the Aurora B kinase. Previously, we have demonstrated that MKlp2 recruits Polo-like kinase 1 (Plk1) to the central spindle in anaphase. We now find that in MKlp2 but not MKlp1-depleted cells the Aurora B-INCENP complex remains at the centromeres and fails to relocate to the central spindle. MKlp2 exerts dual control over Aurora B localization, because it is a binding partner for Aurora B, and furthermore for the phosphatase Cdc14A. Cdc14A can dephosphorylate INCENP and may contribute to its relocation to the central spindle in anaphase. We propose that MKlp2 is involved in the localization of Plk1, Aurora B, and Cdc14A to the central spindle during anaphase, and that the integration of signaling by these proteins is necessary for proper cytokinesis.

Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis.

We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

Identification of the human PHLDA1/TDAG51 gene: down-regulation in metastatic melanoma contributes to apoptosis resistance and growth deregulation.

To identify molecules involved in the progression of human melanoma to metastatic disease, autologous primary and metastatic melanoma cells were compared by differential mRNA display. One cDNA, expressed in primary but not in autologous metastatic cells in three different patients, was cloned and characterized, and shown to be the human homologue of the inducible, immediate early TDAG51/PHLDA1 (pleckstrin-homology-like domain family A, member1) gene. Monoclonal antibodies produced against the PHLDA1 protein revealed homogeneous strong expression by benign melanocytic nevi, and progressively reduced expression in primary and metastatic melanomas in vivo. Analysis of stable cDNA transfectants in two different cell lines revealed that constitutive PHLDA1 expression is associated with reduced cell growth, cloning efficiency, and colony formation but not with alterations in cell cycle parameters. However, PHLDA1 expression was associated with increased basal apoptosis as assessed by live cell annexin V binding, terminal deoxynucleotidyltransferase-dependent nucleotide incorporation, and with increased cleavage of poly(ADP-ribose) polymerase and caspase-9. Constitutive PHLDA1 expression greatly enhances the sensitivity of human melanoma cells to the chemotherapeutic agents doxorubicin and camptothecin. These results suggest that PHLDA1 is constitutively expressed by melanocytic nevi where it may contribute to their benign phenotype. The progressive loss of PHLDA1 expression in melanomas may play a role in deregulated cell growth and apoptosis resistance in these tumors.