Mechanisms and Functions of Spatial Protein Quality Control.

A healthy proteome is essential for cell survival. Protein misfolding is linked to a rapidly expanding list of human diseases, ranging from neurodegenerative diseases to aging and cancer. Many of these diseases are characterized by the accumulation of misfolded proteins in intra- and extracellular inclusions, such as amyloid plaques. The clear link between protein misfolding and disease highlights the need to better understand the elaborate machinery that manages proteome homeostasis, or proteostasis, in the cell. Proteostasis depends on a network of molecular chaperones and clearance pathways involved in the recognition, refolding, and/or clearance of aberrant proteins. Recent studies reveal that an integral part of the cellular management of misfolded proteins is their spatial sequestration into several defined compartments. Here, we review the properties, function, and formation of these compartments. Spatial sequestration plays a central role in protein quality control and cellular fitness and represents a critical link to the pathogenesis of protein aggregation-linked diseases.

C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation.

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

Reactive astrocytes promote proteostasis in Huntington's disease through the JAK2-STAT3 pathway.

Huntington's disease is a fatal neurodegenerative disease characterized by striatal neurodegeneration, aggregation of mutant Huntingtin and the presence of reactive astrocytes. Astrocytes are important partners for neurons and engage in a specific reactive response in Huntington's disease that involves morphological, molecular and functional changes. How reactive astrocytes contribute to Huntington's disease is still an open question, especially because their reactive state is poorly reproduced in experimental mouse models. Here, we show that the JAK2-STAT3 pathway, a central cascade controlling astrocyte reactive response, is activated in the putamen of Huntington's disease patients. Selective activation of this cascade in astrocytes through viral gene transfer reduces the number and size of mutant Huntingtin aggregates in neurons and improves neuronal defects in two complementary mouse models of Huntington's disease. It also reduces striatal atrophy and increases glutamate levels, two central clinical outcomes measured by non-invasive magnetic resonance imaging. Moreover, astrocyte-specific transcriptomic analysis shows that activation of the JAK2-STAT3 pathway in astrocytes coordinates a transcriptional program that increases their intrinsic proteolytic capacity, through the lysosomal and ubiquitin-proteasome degradation systems. This pathway also enhances their production and exosomal release of the co-chaperone DNAJB1, which contributes to mutant Huntingtin clearance in neurons. Together, our results show that the JAK2-STAT3 pathway controls a beneficial proteostasis response in reactive astrocytes in Huntington's disease, which involves bi-directional signalling with neurons to reduce mutant Huntingtin aggregation, eventually improving disease outcomes.

Protein Loss in Peritoneal Effluent: Different Meaning for 24-h versus PET Samples.

INTRODUCTION: Quantification of peritoneal protein loss (PPL) may be expressed according to a timely collection (24-h measurement or 4-h PET assessment) and as a concentration. The aim of this study was to compare the quantification methods of 24-h and 4-h collections. METHODS: This study included 81 prevalent peritoneal dialysis patients. Demographics and clinical and bioelectrical impedance features were registered. PPL was measured (4-h PET and 24-h results) and peritoneal protein clearance was calculated. A linear regression model was performed. RESULTS: Age and continuous ambulatory peritoneal dialysis (compared to cycler) were positively associated with greater PPL on 24-h collections. Neither cardiovascular disease, hypertension, diabetes nor the comorbidity Charlson Index was significantly associated with PPL. There was a consistent univariable relationship with D/P creatinine, whichever sampling method was used. Only 24-h measurements of PPL correlated with body composition variables. In multiple linear regression analysis, D/P creatinine association with PPL stands out. On the other hand, 24-h determinations (in grams or clearance) were associated with overhydration. PET protein quantification was associated with peritoneal creatinine clearance. DISCUSSION/CONCLUSION: Different methods sign different pathophysiological pathways. PET protein loss quantification should be regarded as a marker of peritoneal membrane intrinsic permeability. Measurements of a 24-h sample might be closer to patients' clinical status and prognosis, signalizing opportunities for therapy intervention.

Relationship Between Peritoneal Protein Clearance and Hemoglobin in Peritoneal Dialysis Patients: A Prospective Cohort Study.

OBJECTIVE: The relationship between higher peritoneal protein clearance (PPCl) and hemoglobin (Hb) levels in peritoneal dialysis (PD) patients is unknown. We explored this relationship and interaction on all-cause mortality in this prospective cohort study with a large number of PD patients. METHODS: We enrolled prevalent PD patients in a single PD center. Demographic characteristics and clinical and biochemical data were collected. The total amount of protein loss in the dialysate and PPCl corrected for serum albumin were calculated. The primary study endpoint was all-cause mortality. We examined the relationship between PPCl, Hb, and all-cause mortality in the Cox regression model. RESULTS: We included a total of 487 PD patients (58.3% males, mean age 49.5 ± 14.9 years). The median PD duration at enrollment was 30.1 (15.8-48.3) months. Mean Hb level was 11.1 ± 1.9 g/dL, and 221 (45.3%) patients had Hb levels <11 g/dL. Patients with Hb < 11 g/dL had lower serum albumin, lower residual renal creatinine clearance, and higher PPCl. In a multilinear regression model, PPCl (ß = -0.12, P = .015) had an independent negative linear association with Hb levels. In the logistic regression model, higher PPCl was independently associated with lower Hb (<11 g/dL) (odds ratio = 1.02; 95% confidence interval [CI]: 1.01-1.03). In the overall cohort, after adjusting for confounders in the Cox regression model, decrease in Hb level was independently associated with increased risk (hazard ratio: 0.86, 95% CI: 0.77-0.95) of all-cause mortality. Interaction-effect test showed that PPCl influenced the relationship between Hb level and all-cause mortality (P = .011). After adjusting for confounders, lower Hb level was independently associated with a higher risk (hazard ratio: 0.85, 95% CI: 0.74-0.97) of all-cause mortality only in patients with PPCl ≥59.5 mL/day and not in patients with lower PPCl. CONCLUSIONS: Higher PPCl was an independent predictive factor of lower Hb levels in PD patients. Therefore, PPCl influenced the relationship between Hb level and all-cause mortality in PD patients.

Association of Lean Body Mass Index and Peritoneal Protein Clearance in Peritoneal Dialysis Patients.

BACKGROUND/AIMS: The relationship between peritoneal protein clearance (PPCl) and nutritional status in peritoneal dialysis (PD) population have not been clarified. This study aims to investigate the relationship between PPCl and nutritional status in PD population. METHODS: Prevalent PD patients were enrolled in the cross-sectional survey in a single center from April to November 2013. The total amount of protein loss in the dialysate was calculated. PPCl reflects the individual differences of peritoneal protein loss, and is calculated by the formula, that PPCl (ml/day)=24-h dialysate protein loss / (albumin/0.4783). Nutritional status measured by lean body mass index (LBMI) was assessed by multi-frequency bioelectrical impedance analysis (BIA). RESULTS: Totally 351 PD patients (55% male, 17.1% with diabetes, mean age 47.7±14.3 years) were included. The median PPC l was 58 ml/day. Patients were divided into four groups for comparison according to the PPC quartiles. Compared with lower PPCl quartiles, patients with higher PPCl had higher body mass index (BMI) (P< 0.001), body surface area (BSA) (P < 0 .001), LBMI (P<0.001), 4-hour D/P creatinine ratio (P< 0.001), and lower residual renal CCl (P<0.001). Compared with conventional body index (BMI and BSA) in ROC analysis, LBMI (area under curve: 0.71, 95% confidence interval [CI]: 0.66-0.77) had better performance in predicting higher PPCl. After adjustment in logistic regression models, each 1 kg/m2 increase of LBMI (odd ratio[OR] =1.37; 95% CI: 1.17-1.60), each 0.1 increase of 4-hour D/P creatinine ratio (OR =1.47; 95% CI: 1.11-1.93), and every 1 L/week/1.73m2 decrease of residual renal CCl (OR =0.98; 95% CI: 0.96-0.99) were independently associated with higher PPCl (> 58 ml/day). CONCLUSION: Higher LBMI was independently associated with higher , indicating that better nutritional status dominates peritoneal protein metabolism in PD patients.

Different patterns of inflammatory and angiogenic factors are associated with peritoneal small solute transport and peritoneal protein clearance in peritoneal dialysis patients.

BACKGROUND: Both peritoneal small solute transport and peritoneal protein clearance are closely linked to outcomes in peritoneal dialysis (PD) patients. However, the associated factors of these two components are not fully understood so far. This study aimed to investigate the association between a panel of systemic and peritoneal inflammatory and angiogenic factors and peritoneal solute transport properties. METHODS: Stable PD patients in PD center of Renji Hospital, School of Medicine, Shanghai Jiao Tong University were enrolled in present study. Serum and overnight effluent markers including angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), sTie-2, VEGF, IL-6 and IL-10 were determined. Mass transfer area coefficient of creatinine (MTACcr) and peritoneal protein clearance (Prcl) were calculated. Multivariable linear regression was used to examine the association between these markers and MTACcr as well as Prcl. RESULTS: A total of 320 patients were enrolled in present study, which consisted of 166 (51.9%) males with a mean age of 56.8 ± 14.2 years and a median PD duration of 32.5 (9.0-56.3) months. Multiple regression analyses showed that BSA, history glucose exposure, dialysate IL-6 AR and dialysate Ang-1 AR were independent associated factors of MTACcr, while BSA and serum Ang-1 were independent associated factors of Prcl. CONCLUSIONS: MTACcr representing peritoneal small-solute transport and Prcl representing peritoneal large molecular transport are associated with slightly different panels of inflammatory and angiogenic factors.

Evidence for lymphatic Aß clearance in Alzheimer's transgenic mice.

Evidence has shown that lymphatic drainage contributes to removal of debris from the brain but its role in the accumulation of amyloid ß peptides (Aß) has not been demonstrated. We examined the levels of various forms of Aß in the brain, plasma and lymph nodes in a transgenic model of Alzheimer's disease (AD) at different ages. Herein, we report on the novel finding that Aß is present in the cervical and axillary lymph nodes of AD transgenic mice and that Aß levels in lymph nodes increase over time, mirroring the increase of Aß levels observed in the brain. Aß levels in lymph nodes were significantly higher than in plasma. At age 15.5months, there was a significant increase of monomeric soluble Aß40 (p=0.003) and Aß42 (p=0.05) in the lymph nodes over the baseline values measured at 6months of age. In contrast, plasma levels of Aß40 showed no significant changes (p=0.68) and plasma levels Aß42 significantly dropped (p=0.02) at the same age. Aß concentration was low to undetectable in splenic lymphoid tissue and several other control tissues including heart, lung, liver, kidneys and intestine of the same animals, strongly suggesting that Aß peptides in lymph nodes are derived from the brain.

Improved clearance of host cell protein impurities at the polishing purification step using multimodal chromatography.

In biotherapeutic protein production, host cell proteins (HCPs) are one of the main process related impurities which must be cleared and controlled through downstream processing. In this paper, we studied a novel therapeutic protein molecule which had a high level of HCP co-purification throughout the downstream process. Here, we focused on the polishing purification step and developed an effective strategy for improving HCP clearance using multimodal chromatography (MMC) resin, Nuvia cPrime. A high throughput process development (HTPD) workflow was used to identify the resin and process conditions which could enable significant HCP clearance while maintaining acceptable product quality and process performance. HCP analysis of gradient elution fractions on multimodal chromatography found that HCPs eluted at the beginning of the gradient, at a lower salt concentration than the therapeutic protein. Based on these findings, a step elution process involving an intermediate low salt wash was developed to clear weak-binding HCPs, while retaining the therapeutic protein on the column. This strategy was highly effective and enabled 80 % reduction in total HCP content, including some problematic and difficult to remove candidates such as Peroxiredoxin-1, Serine protease HTRA1, Clusterin and Lipoprotein lipase.

Echinometra lucunter molecules reduce Aß42-induced neurotoxicity in SH-SY5Y neuron-like cells: effects on disaggregation and oxidative stress.

Background: Echinometra lucunter is a sea urchin commonly found on America's rocky shores. Its coelomic fluid contains molecules used for defense and biological processes, which may have therapeutic potential for the treatment of amyloid-based neurodegenerative diseases, such as Alzheimer's, that currently have few drug options available. Methods: In this study, we incubated E. lucunter coelomic fluid (ELCF) and fractions obtained by solid phase extraction in SH-SY5Y neuron-like cells to evaluate their effect on cell viability caused by the oligomerized amyloid peptide 42 (Aß42o). Moreover, the Aß42o was quantified after the incubation with ELCF fractions in the presence or not of cells, to evaluate if samples could cause amyloid peptide disaggregation. Antioxidant activity was determined in ELCF fractions, and cells were evaluated to check the oxidative stress after incubation with samples. The most relevant fraction was analyzed by mass spectrometry for identification of molecules. Results: ELCF and certain fractions could prevent and treat the reduction of cell viability caused by Aß42o in SH-SY5Y neuron-like cells. We found that one fraction (El50) reduced the oligomerized Aß42 and the oxidative stress caused by the amyloid peptide through its antioxidant molecules, which in turn reduced cell death. Mass spectrometry analysis revealed that El50 comprises small molecules containing flavonoid antioxidants, such as phenylpyridazine and dihydroquercetin, and two peptides. Conclusion: Our results suggest that sea urchin molecules may interact with Aß42o and oxidative stress, preventing or treating neurotoxicity, which may be useful in treating dementia.

The Role of VCP Mutations in the Spectrum of Amyotrophic Lateral Sclerosis-Frontotemporal Dementia.

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two neurological diseases which, respectively, and primarily affect motor neurons and frontotemporal lobes. Although they can lead to different signs and symptoms, it is now evident that these two pathologies form a continuum and that hallmarks of both diseases can be present within the same person in the so-called ALS-FTD spectrum. Many studies have focused on the genetic overlap of these pathologies and it is now clear that different genes, such as C9orf72, TARDBP, SQSTM1, FUS, and p97/VCP can be mutated in both the diseases. VCP was one of the first genes associated with both FTD and ALS representing an early example of gene overlapping. VCP belongs to the type II AAA (ATPases Associated with diverse cellular activities) family and is involved in ubiquitinated proteins degradation, autophagy, lysosomal clearance and mitochondrial quality control. Since its numerous roles, mutations in this gene lead to different pathological features, first and foremost TDP-43 mislocalization. This review aims to outline recent findings on VCP roles and on how its mutations are linked to the neuropathology of ALS and FTD.

Peritoneal Protein Clearance Is Associated With Cardiovascular Events but Not Mortality in Peritoneal Dialysis Patients.

Introduction: Association of peritoneal protein clearance (Pcl) with outcomes in patients with peritoneal dialysis (PD) is uncertain. Thus, we aimed to investigate its impact on cardiovascular events and all-cause mortality in patients with PD and factors associated with Pcl. Methods: Prevalent patients with PD from January 2014 to April 2015 in the center of Renji Hospital were enrolled. At the time of enrollment, serum and dialysate samples were collected to detect biochemical parameters and Angiopoietin-2-Tie2 system cytokines. Mass transfer area coefficient of creatinine (MTACcr) and Pcl were calculated. Patients were dichotomized into two groups by the median Pcl level (68.5 ml/day) and were followed up prospectively until the end of the study (1 October 2018). Results: A total of 318 patients with PD [51.2% men, mean age 56.7 ± 14.3 y, median PD duration 31.5 (12.1-57.2) months] were enrolled. Among them, 25.7% were comorbid with diabetes and 28.6% had a history of cardiovascular disease (CVD). After being followed up for up to 43.9 (24.2-50.3) months, 63 had developed cardiovascular events, and 81 patients were died. Among them, the high Pcl group had occurred 39 cardiovascular events and 51 deaths, and the low Pcl group had 24 cardiovascular events and 30 deaths. Kaplan-Meier analysis showed that both the occurrence of cardiovascular events and all-cause mortality were increased in patients with high Pcl. However, after adjusting for important confounders and serum Angiopoietin-2 (Angpt-2) level, Pcl was still an independent risk factor for cardiovascular events [hazard ratio (HR) = 1.006 (1.000-1.012), p = 0.038] but not mortality. On multivariate regression analysis, serum albumin, MTACcr, and body mass index (BMI) were found to be independently associated with Pcl. Conclusion: High Pcl is an independent risk factor for cardiovascular events but not all-cause mortality. The prediction of cardiovascular events by Pcl was independent of serum Angpt-2.

A New Zebrafish Model to Measure Neuronal α-Synuclein Clearance In Vivo.

The accumulation and aggregation of α-synuclein (α-SYN) is a common characteristic of synucleinopathies, such as Parkinson's Disease (PD), Dementia with Lewy Bodies (DLB) or Multiple System Atrophy (MSA). Multiplications of the wildtype gene of α-SYN (SNCA) and most point mutations make α-SYN more aggregate-prone, and are associated with mitochondrial defects, trafficking obstruction, and impaired proteostasis, which contribute to elevated neuronal death. Here, we present new zebrafish models expressing either human wildtype (wt), or A53T mutant, α-SYN that recapitulate the above-mentioned hallmarks of synucleinopathies. The appropriate clearance of toxic α-SYN has been previously shown to play a key role in maintaining cell homeostasis and survival. However, the paucity of models to investigate α-SYN degradation in vivo limits our understanding of this process. Based on our recently described imaging method for measuring tau protein clearance in neurons in living zebrafish, we fused human SNCA to the photoconvertible protein Dendra2 which enabled analyses of wt and A53T α-SYN clearance kinetics in vivo. Moreover, these zebrafish models can be used to investigate the kinetics of α-SYN aggregation and to study the mechanisms, and potential new targets, controlling the clearance of both soluble and aggregated α-SYN.

Impact of 40 Hz Transcranial Alternating Current Stimulation on Cerebral Tau Burden in Patients with Alzheimer's Disease: A Case Series.

BACKGROUND: Alzheimer's disease (AD) is characterized by diffuse amyloid-ß (Aß) and phosphorylated Tau (p-Tau) aggregates as well as neuroinflammation. Exogenously-induced 40 Hz gamma oscillations have been showing to reduce Aß and p-Tau deposition presumably via microglia activation in AD mouse models. OBJECTIVE: We aimed to translate preclinical data on gamma-induction in AD patients by means of transcranial alternating current stimulation (tACS). METHODS: Four participants with mild-to-moderate AD received 1 h of daily 40 Hz (gamma) tACS for 4 weeks (Monday to Friday) targeting the bitemporal lobes (20 h treatment duration). Participant underwent Aß, p-Tau, and microglia PET imaging with [11C]-PiB, [18F]-FTP, and [11C]-PBR28 respectively, before and after the intervention along with electrophysiological assessment. RESULTS: No adverse events were reported, and an increase in gamma spectral power on EEG was observed after the treatment. [18F]-FTP PET revealed a significant decrease over 2% of p-Tau burden in 3/4 patients following the tACS treatment, primarily involving the temporal lobe regions targeted by tACS and especially mesial regions (e.g., entorhinal cortex). The amount of intracerebral Aß as measured by [11C]-PiB was not significantly influenced by tACS, whereas 1/4 reported a significant decrease of microglia activation as measured by [11C]-PBR28. CONCLUSION: tACS seems to represent a safe and feasible option for gamma induction in AD patients, with preliminary evidence of a possible effect on protein clearance partially mimicking what is observed in animal models. Longer interventions and placebo control conditions are needed to fully evaluate the potential for tACS to slow disease progression.

Overhydration May Be the Missing Link between Peritoneal Protein Clearance and Mortality.

INTRODUCTION: Peritoneal protein loss (PPL) has been associated with mortality. Inflammation was assumed a putative cause with malnutrition as a consequence. Hydrostatic convection is a major drive for microvascular protein transport, but most studies in peritoneal dialysis (PD) patients overlooked this mechanism. An association between peritoneal protein clearance (PPCl) and venous congestion has been reported recently. The aim of this study was to explore the importance of fluid overload in PPCl in PD. METHODS: Sixty-seven prevalent PD patients were assessed with peritoneal equilibration test and multifrequency bioelectrical impedance assessment (BIA). PPL and PPCl were calculated from simultaneously obtained 24-h peritoneal effluent. RESULTS: PPL averaged 5.2 g/24 h. It was higher in patients on continuous treatment than in those without a long dwell. Significant associations between PPCl and BIA parameters of overhydration were found in both univariable and multivariable analyses. Lean mass index, partly dependent on hydration status, was associated with PPCl in univariable but not in multivariable analysis. A multiple linear model identified extracellular water excess and higher D/P creatinine as predictors of higher PPCl, independent of PD duration, type of PD, age, gender, albumin, cardiovascular disease, C-reactive protein, or lean mass index. CONCLUSIONS: The uni- and multivariable strong associations between fluid overload and PPCl support the importance of hydrostatic pressure-induced convection for PPCl. Also, peritoneal small solute transport was associated with PPCl. Both are amenable by adjusted dialysis prescription, especially focused on fluid status and avoidance of overhydration. The assumption of an association with inflammation and malnutrition was not confirmed.

Dysregulated Tear Film Proteins in Macular Edema Due to the Neovascular Age-Related Macular Degeneration Are Involved in the Regulation of Protein Clearance, Inflammation, and Neovascularization.

Macular edema and its further complications due to the leakage from the choroidal neovascularization in course of the age-related macular degeneration (AMD) is a leading cause of blindness among elderly individuals in developed countries. Changes in tear film proteomic composition have been reported to occur in various ophthalmic and systemic diseases. There is an evidence that the acute form of neovascular AMD may be reflected in the tear film composition. Tear film was collected with Schirmer strips from patients with neovascular AMD and sex- and age-matched control patients. Two-dimensional electrophoresis was performed followed by MALDI-TOF mass spectrometry for identification of differentially expressed proteins. Quantitative analysis of the differential electrophoretic spots was performed with Delta2D software. Altogether, 11 significantly differentially expressed proteins were identified; of those, 8 were downregulated, and 3 were upregulated in the tear film of neovascular AMD patients. The differentially expressed proteins identified in tear film were involved in signaling pathways associated with impaired protein clearance, persistent inflammation, and neovascularization. Tear film protein analysis is a novel way to screen AMD-related biomarkers.

Determining the Clearance of Degraded Protein via a Monoclonal Antibody Purification Process in Support of Cleaning Carryover Limits in Multiproduct Facilities.

Cleaning validation acceptance criteria in multiproduct facilities are established using maximum allowable carryover calculations. Carryover calculations incorporate the shared equipment surface area between two products to ensure that an acceptable limit for residue from the previously manufactured product to the subsequent product is determined. The shared surface area can be limited to areas where carryover presents the highest risk to product quality or patient safety. In these cases, specifically for biologic drug substance manufacturing, the shared surface area is limited to equipment after the purification process based on the assumption that the purification process would remove potential product fragment residues from the previous product. Until now, this assumption has been based on empirical knowledge without experimental data quantifying the clearance or removal of potential residues. We present a three-part study that determined the effects of cleaning conditions on selected monoclonal antibodies (mAbs) and the generation of degraded fragments and evaluated the clearance of both the degraded mAb1 in a laboratory setting and the degraded fragments in the presence of a subsequent product, assessing the risk of co-purification. Several analytical techniques were used, including gel electrophoresis, capillary zone electrophoresis/laser-induced florescence detection, and liquid chromatography-mass spectrometry. Protein fragment generation was demonstrated for five different mAbs from different immunoglobulin G subclasses. The clearance of the degraded fragments in the absence and presence of the subsequent product was demonstrated by calculating fold clearance and log reduction value (LRV) for each chromatography step. The data showed that the fragments generated during cleaning could be removed by the purification process. The fold clearances were determined to be values of 5400 (3.7 LRV) in the absence of subsequent product and 4428 (3.6 LRV) in the presence of subsequent product. The results supported the removal of product residues from shared surface areas by the purification process in multiproduct biologic drug substance manufacturing facilities.

BAG3 and BAG6 differentially affect the dynamics of stress granules by targeting distinct subsets of defective polypeptides released from ribosomes.

Stress granules (SGs) are dynamic ribonucleoprotein granules induced by environmental stresses. They play an important role in the stress response by integrating mRNA stability, translation, and signaling pathways. Recent work has connected SG dysfunction to neurodegenerative diseases. In these diseases, SG dynamics are impaired because of mutations in SG proteins or protein quality control factors. Impaired SG dynamics and delayed SG dissolution have also been observed for SGs that accumulate misfolding-prone defective ribosomal products (DRiPs). DRiP accumulation inside SGs is controlled by a surveillance system referred to as granulostasis and encompasses the molecular chaperones VCP and the HSPB8-BAG3-HSP70 complex. BAG3 is a member of the BAG family of proteins, which includes five additional members. One of these proteins, BAG6, is functionally related to BAG3 and able to assist degradation of DRiPs. However, whether BAG6 is involved in granulostasis is unknown. We report that BAG6 is not recruited into SGs induced by different types of stress, nor does it affect SG dynamics. BAG6 also does not replace BAG3's function in SG granulostasis. We show that BAG3 and BAG6 target different subsets of DRiPs, and BAG3 binding to DRiPs is mediated by HSPB8 and HSP70. Our data support the idea that SGs are sensitive to BAG3-HSP70-bound DRiPs but not to BAG6-bound DRiPs. Additionally, only BAG3 is strongly upregulated in the stress recovery phase, when SGs dissolve. These data exclude a role for BAG6 in granulostasis and point to a more specialized function in the clearance of a specific subset of DRiPs.

Prescribing high-quality peritoneal dialysis: Moving beyond urea clearance.

Urea removal in peritoneal dialysis (PD) has been a primary measure of dialysis adequacy, but its utility remains limited due to its poor correlation with the clearance of other important uraemic retention solutes and the low certainty of evidence relating peritoneal urea clearance and survival of individuals doing PD. Indeed, clearances of other uraemic solutes, electrolyte imbalances, hypoalbuminaemia and nutritional status, may provide a more holistic measure of dialysis adequacy when evaluating individuals on PD in addition to focusing on person-centred outcomes. Here, we review the history of the urea and creatinine-centric approach to dialysis adequacy and explore the potential importance of other uraemic retention solutes, electrolyte disturbances, phosphorus control, peritoneal protein losses and hypoalbuminaemia, as well as nutritional management to promote a broader multidimensional concept of clearance for PD.

Relationships Between Peritoneal Protein Clearance and Parameters of Fluid Status Agree with Clinical Observations in Other Diseases that Venous Congestion Increases Microvascular Protein Escape.

BACKGROUND: Peritoneal effluent from peritoneal dialysis (PD) patients contains proteins, mainly transported from the circulation through large pores in the venular part of the peritoneal microvessels. Hydrostatic convection is the major driver for peritoneal protein transport, although in PD there is additional diffusion. Consequently, venous pressure may have a role in peritoneal protein transport. The aim of the study was to investigate the importance of venous congestion on the magnitude of peritoneal protein clearance in incident PD patients using non-invasive measurements. METHODS: A total of 316 adult PD patients, on PD for 8 - 12 weeks and collecting 24-hour urine and dialysate for total protein determination, underwent standard peritoneal equilibration testing (PET) along with measurement of N terminal pro-brain natriuretic peptide (NT-proBNP) and C-reactive protein (CRP), multifrequency bioimpedance analysis, and a transthoracic echocardiogram. RESULTS: Statistically significant univariate relationships for peritoneal protein clearance with a Spearman correlation coefficient > 0.25 were present for 4-hour dialysate/plasma (D/P) creatinine, NT-proBNP, extracellular/total body water, extracellular water excess, left ventricular mass, and right atrial area. Negative correlations were found with serum total protein and residual renal function. On multivariate analysis, logNTproBNP (ß 0.11, p = 0.007) and right atrial area (ß 0.01, p = 0.03) were significant independent predictors of peritoneal protein clearance. CONCLUSION: Indicators of venous congestion showed the most important relationships with peritoneal protein clearance. These indicators have not been assessed in previous studies on the presence or absence of relationships between peritoneal protein clearance and mortality.