Predictors of In-Hospital Mortality in Older Inpatients with Suspected Infection.

OBJECTIVES: To determine the rate and predictors of death in older individuals with suspected infection at any time during hospital stay in a geriatric acute ward and the prognostic ability of different tools [quick Sequential (Sepsis-related) Organ Failure Assessment (qSOFA), Modified and National Early Warning Scores (MEWS) and (NEWS)] in such population. DESIGN: Prospective observational single-center cohort study. SETTING AND PARTICIPANTS: Among patients admitted to an acute geriatric unit of an Italian University Hospital with at least 1 sepsis risk factor, all subjects with suspected infection at admission or during hospital stay (defined as antibiotic prescription and associated culture test) were considered. METHODS: A geriatric assessment including comorbidity and social, functional, and cognitive status was performed for each patient. Clinical parameters were evaluated at least twice daily throughout hospital stay; qSOFA, MEWS, and NEWS were derived, with positive cutoffs set at ≥2, ≥5, and ≥7, respectively. RESULTS: Among 305 older inpatients (median age 86.0 years, 49.2% female), 21% died during hospital stay. Sepsis was diagnosed in 31.8% of the overall sample and in 64.1% of deceased patients. Deceased patients showed a significantly higher prevalence of prior institutionalization, functional dependence, cognitive impairment, and multimorbidity. The prognostic accuracy of the qSOFA score at infection onset was only fair (area under the receiver operating characteristic curve 0.72; 95% CI, 0.65-0.79, P < .001) and comparable with that of MEWS and NEWS. After multivariable analysis, in-hospital death was positively associated with male sex [odds ratio (OR), 2.11; 95% CI, 1.01-4.44; P = .048] and abnormal white blood cells count (OR, 4.93; 95% CI, 2.36-10.29; P < .001), platelet count (OR, 2.61; 95% CI, 1.10-6.16; P = .029) and serum creatinine (OR, 2.70; 95% CI, 1.30-5.61; P = .008), along with any of the score considered, and negatively associated with autonomy in instrumental activities (OR, 0.78; 95% CI, 0.68-0.90; P < .001). CONCLUSIONS: Prognosis in older inpatients with infection or sepsis appears to be determined both by the geriatric characteristics and by the severity of the acute event, expressed by recommended tools and blood test results.

Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc-fusion protein produced in CHO cells.

Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins.

Comparison of Diagnostic Accuracies of qSOFA, NEWS, and MEWS to Identify Sepsis in Older Inpatients With Suspected Infection.

OBJECTIVES: To determine and compare the accuracies of the quick Sequential (Sepsis-related) Organ Failure Assessment (qSOFA) and Modified and National Early Warning Scores (NEWS and MEWS) to identify sepsis in older inpatients with suspected infection. DESIGN: Prospective diagnostic accuracy study. SETTING AND PARTICIPANTS: Patients admitted to an acute geriatric unit of an Italian University Hospital with at least one sepsis risk factor and suspected infection defined as antibiotic prescription and associated culture test during hospital stay. METHODS: Sepsis diagnosis was defined as the presence on discharge documents of International Classification of Diseases, Ninth revision, Clinical Modification codes for severe sepsis, septic shock, or for infection and acute organ disfunction. For each patient, clinical parameters were evaluated at least twice daily throughout hospital stay; qSOFA, NEWS, and MEWS were derived, and worst scores recorded. Positive cutoffs were set at ≥2, ≥7, and ≥5, respectively. Sensitivity, specificity, positive and negative predictive values (PPV and NPV, respectively), and positive and negative likelihood ratios, as well as areas under the receiver operating characteristic curve (AUROCs) were calculated. RESULTS: Among 230 geriatric patients with suspected infection at risk for sepsis (median age 86 years, 49% women), 30.9% had a sepsis diagnosis. A qSOFA ≥2 was recorded in 111 (48.3%) patients, a MEWS ≥5 in 65 (28.3%), and a NEWS ≥7 in 115 (50.0%). The qSOFA showed the highest sensitivity [81.7%, 95% confidence interval (CI) 71.7%-89.5%], but low specificity (66.7%, 95% CI 59.1%-73.7%), resulting in a high NPV (89.1%; 95% CI 82.7%-93.8%) and poor PPV (52.3%, 95% CI 43.0%-61.4%). The AUROC for qSOFA was 0.76 (95% CI 0.69-0.83), comparable with that of NEWS (0.74, 95% CI 0.67-0.81, P = .44), but significantly higher than that of MEWS (0.70, 95% CI 0.63-0.77, P = .04). CONCLUSIONS AND IMPLICATIONS: Repeated qSOFA determinations are useful to rule out sepsis in geriatric inpatients with suspected infection, but poorly support its diagnosis due to low specificity. More complex MEWS and NEWS do not perform better. Implementation of clinical scores to reliably identify sepsis in older patients is urgently needed.

A comparative analysis of recombinant Fab and full-length antibody production in Chinese hamster ovary cells.

Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen-binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full-length antibodies are mammalian cell-based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small- and large-scale Fab production. Using a panel of full-length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full-length antibody format resulted in the recovery of fewer mini-pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody-producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full-length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats.

Defining lncRNAs Correlated with CHO Cell Growth and IgG Productivity by RNA-Seq.

How the long non-coding RNA (lncRNA) genome in recombinant protein producing Chinese hamster ovary (CHO) cell lines relates to phenotype is not well described. We therefore defined the CHO cell lncRNA transcriptome from cells grown in controlled miniature bioreactors under fed-batch conditions using RNA-Seq to identify lncRNAs and how the expression of these changes throughout growth and between IgG producers. We identify lncRNAs including Adapt15, linked to ER stress, GAS5, linked to mTOR signaling/growth arrest, and PVT1, linked to Myc expression, which are differentially regulated during fed-batch culture and whose expression correlates to productivity and growth. Changes in (non)-coding RNA expression between the seed train and the equivalent day of fed-batch culture are also reported and compared with existing datasets. Collectively, we present a comprehensive lncRNA CHO cell profiling and identify targets for engineering growth and productivity characteristics of CHO cells.

The Long Non-Coding RNA Transcriptome Landscape in CHO Cells Under Batch and Fed-Batch Conditions.

The role of non-coding RNAs in determining growth, productivity, and recombinant product quality attributes in Chinese hamster ovary (CHO) cells has received much attention in recent years, exemplified by studies into microRNAs in particular. However, other classes of non-coding RNAs have received less attention. One such class are the non-coding RNAs known collectively as long non-coding RNAs (lncRNAs). The authors have undertaken the first landscape analysis of the lncRNA transcriptome in CHO using a mouse based microarray that also provided for the surveillance of the coding transcriptome. The authors report on those lncRNAs present in a model host CHO cell line under batch and fed-batch conditions on two different days and relate the expression of different lncRNAs to each other. The authors demonstrate that the mouse microarray is suitable for the detection and analysis of thousands of CHO lncRNAs and validated a number of these by qRT-PCR. The authors then further analyzed the data to identify those lncRNAs whose expression changed the most between growth and stationary phases of culture or between batch and fed-batch culture to identify potential lncRNA targets for further functional studies with regard to their role in controlling growth of CHO cells. The authors discuss the implications for the publication of this rich dataset and how this may be used by the community.

RTN3 Is a Novel Cold-Induced Protein and Mediates Neuroprotective Effects of RBM3.

Cooling and hypothermia are profoundly neuroprotective, mediated, at least in part, by the cold shock protein, RBM3. However, the neuroprotective effector proteins induced by RBM3 and the mechanisms by which mRNAs encoding cold shock proteins escape cooling-induced translational repression are unknown. Here, we show that cooling induces reprogramming of the translatome, including the upregulation of a new cold shock protein, RTN3, a reticulon protein implicated in synapse formation. We report that this has two mechanistic components. Thus, RTN3 both evades cooling-induced translational elongation repression and is also bound by RBM3, which drives the increased expression of RTN3. In mice, knockdown of RTN3 expression eliminated cooling-induced neuroprotection. However, lentivirally mediated RTN3 overexpression prevented synaptic loss and cognitive deficits in a mouse model of neurodegeneration, downstream and independently of RBM3. We conclude that RTN3 expression is a mediator of RBM3-induced neuroprotection, controlled by novel mechanisms of escape from translational inhibition on cooling.

Characterization of durum wheat high molecular weight glutenin subunits Bx20 and By20 sequences by a molecular and proteomic approach.

Wheat high molecular weight glutenin subunit variation is important because of its great influence on glutenin polymer structure, that is related to dough technological properties. Among the different subunits, the pair Bx20 and By20 is known to have a negative effect on quality, but the reasons are not clear: Bx20 has two cysteines, which theoretically make this subunit a chain extender of the glutenin polymer, just like the other Bx subunits, showing four cysteines, two of which should be involved in intra-molecular disulfide bonds. By20 has never been characterized so far at molecular level. Here we report the nucleotide sequences of Bx20 and By20 genes isolated from the durum wheat cultivar 'Lira 45' and the validation of the corresponding deduced amino acid sequences by using MALDI-TOF and LC-MS/MS. Four nucleotide differences were identified in the Bx20 gene with respect to the deduced sequence present in NCBI, causing two amino acid substitutions. For the By20 subunit, nucleotide and amino acid sequences revealed a great similarity to By15, both at gene and protein levels, showing five nucleotide changes generating two amino acid differences. No evidence of post-translational modifications has been found. Hypotheses are formulated in regard to relationships with technological quality. Copyright © 2016 John Wiley & Sons, Ltd.

Atrophy and lower regional perfusion of temporo-parietal brain areas are correlated with impairment in memory performances and increase of EEG upper alpha power in prodromal Alzheimer's disease.

BACKGROUND: Temporo-parietal cortex thinning is associated with mild cognitive impairment (MCI) due to Alzheimer's disease (AD). The increase of the EEG upper/low alpha power ratio has been associated with MCI due to AD subjects and to the atrophy of temporo-parietal brain areas. Moreover, subjects with a higher alpha3/alpha2 frequency power ratio showed lower brain perfusion than in the low alpha3/alpha2 group. The two groups have significantly different hippocampal volumes and correlation with the theta frequency activity. METHODS: 74 adult subjects with MCI underwent clinical and neuropsychological evaluation, electroencephalogram (EEG) recording, and high resolution 3D magnetic resonance imaging (MRI). 27 of them underwent EEG recording and perfusion single-photon emission computed tomography (SPECT) evaluation. The alpha3/alpha2 power ratio as well as cortical thickness was computed for each subject. The difference in cortical thickness between the groups was estimated. Pearson's r was used to assess the correlation topography between cortical thinning as well as between brain perfusion and memory impairment. RESULTS: In the higher upper/low alpha group, memory impairment was more pronounced both in the MRI group and the SPECT MCI group. Moreover, it was correlated with greater cortical atrophy and lower perfusional rate in temporo-parietal cortex. CONCLUSION: High EEG upper/low alpha power ratio was associated with cortical thinning lower perfusion in temporo-parietal. Moreover, atrophy and lower perfusional rate were both significantly correlated with memory impairment in MCI subjects. The increase of EEG upper/low alpha frequency power ratio could be useful for identifying individuals at risk for progression to AD dementia and may be of value in the clinical context.