Novel echocardiographic pixel intensity quantification method for differentiating transthyretin cardiac amyloidosis from light chain cardiac amyloidosis and other phenocopies.

AIMS: Differentiating cardiac amyloidosis (CA) subtypes is important considering the significantly different therapies for transthyretin (ATTR)-CA and light chain (AL)-CA. Therefore, an echocardiographic method to distinguish ATTR-CA from AL-CA would provide significant value. We assessed a novel echocardiographic pixel intensity method to quantify myocardial calcification to differentiate ATTR-CA from phenocopies of CA and from AL-CA, specifically. METHODS AND RESULTS: 167 patients with ATTR-CA (n=53), AL-CA (n=32), hypertrophic cardiomyopathy (n=37), and advanced chronic kidney disease (n=45) were retrospectively evaluated. The septal reflectivity ratio (SRR) was measured as the average pixel intensity of the visible anterior septal wall divided by the average pixel intensity of the visible posterior lateral wall. SRR and other myocardial strain-based echocardiographic measures were evaluated with receiver operator characteristic analysis to evaluate accuracy in distinguishing ATTR-CA from AL-CA and other forms of left ventricular hypertrophy. Mean septal reflectivity ratio (SRR) was significantly higher in the ATTR-CA cohort compared to the other cohorts (p <0.001). SRR demonstrated the largest AUC (0.91, p<0.0001) for distinguishing ATTR from all other cohorts and specifically for distinguishing ATTR-CA from AL-CA (AUC=0.90, p<0.0001, specificity 96%, sensitivity 63%). There was excellent inter- and intra-operator reproducibility with an ICC of 0.91 (p <0.001) and 0.89 (p <0.001), respectively. CONCLUSION: The SRR is a reproducible and robust parameter for differentiating ATTR-CA from other phenocopies of CA and specifically ATTR-CA from AL-CA.

Targeting Ultrasmall Gold Nanoparticles with cRGD Peptide Increases the Uptake and Efficacy of Cytotoxic Payload.

Cyclic arginyl-glycyl-aspartic acid peptide (cRGD) peptides show a high affinity towards αVß3 integrin, a receptor overexpressed in many cancers. We aimed to combine the versatility of ultrasmall gold nanoparticles (usGNP) with the target selectivity of cRGD peptide for the directed delivery of a cytotoxic payload in a novel design. usGNPs were synthesized with a modified Brust-Schiffrin method and functionalized via amide coupling and ligand exchange and their uptake, intracellular trafficking, and toxicity were characterized. Our cRGD functionalized usGNPs demonstrated increased cellular uptake by αVß3 integrin expressing cells, are internalized via clathrin-dependent endocytosis, accumulated in the lysosomes, and when loaded with mertansine led to increased cytotoxicity. Targeting via cRGD functionalization provides a mechanism to improve the efficacy, tolerability, and retention of therapeutic GNPs.

Distal Versus Proximal Radial Artery Access for Cardiac Catheterization and Intervention: Design and Rationale of the DIPRA Trial.

BACKGROUND: Radial artery (RA) catheterization is the access of choice over femoral artery access for most interventional vascular procedures given its safety and faster patient recovery. There has been growing interest in distal radial artery (dRA) access as an alternative to the conventional proximal radial artery (pRA) access. Preserving the RA is important which serves as a potential conduit for future coronary artery bypass surgery, dialysis conduit or preserve the artery for future cardiovascular procedures. The dRA runs in close proximity to the radial nerve, which raises the concern of potential detrimental effects on hand function. STUDY DESIGN: The Distal versus Proximal Radial Artery Access for cardiac catheterization and intervention (DIPRA) trial is a prospective, randomized, parallel-controlled, open-label, single center study evaluating the outcomes of hand function and effectiveness of dRA compared to pRA access in patients undergoing cardiac catheterization. The eligible subjects will be randomized to dRA and pRA access in a (1:1) fashion. The primary end point is an evaluation of hand function at one and twelve months follow-up. Secondary end points include rates of access site hematoma, access site bleeding, other vascular access complications, arterial access success rate, and RA occlusion at one and twelve months follow up. CONCLUSION: Effects of dRA on hand function remains unknown and it's use questionable in the presence of a widely accepted pRA. DIPRA trial is designed to determine the safety and effectiveness of dRA for diagnostic and interventional cardiovascular procedures compared to the standard of care pRA.

A highly potent maytansinoid analogue and its use as a cytotoxic therapeutic agent in gold nanoparticles for the treatment of hepatocellular carcinoma.

Gold nanoparticles are promising drug delivery agents with the potential to deliver chemotherapeutic agents to tumour sites. The highly cytotoxic maytansinoid tubulin inhibitor DM1 has been attached to gold nanoparticles and shows tumour growth inhibition in mouse models of hepatocellular carcinoma. Attempting to improve the stability of the gold-cytotoxin bond led to the design and synthesis of novel maytansinoids with improved potency in cell viability assays and improved in vivo tolerability compared to the DM1 analogues. These novel maytansines may also have applications in other methods of drug delivery, for example as the cytotoxic component of antibody drug conjugates.

DM1 Loaded Ultrasmall Gold Nanoparticles Display Significant Efficacy and Improved Tolerability in Murine Models of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide with poor prognosis and limited options for treatment. Life expectancy after diagnosis is short; the currently available treatments are not well tolerated and have limited clinical benefit. There is a clear unmet clinical need for the development of new treatments. In this study, ultrasmall, 2 nm gold core nanoparticles (MidaCore) conjugated with the potent maytansine analogue DM1 (MTC-100038) were assessed as a systemic nanomedicine for the treatment of hepatocellular carcinoma. The platform improved overall tolerability of DM1, permitting ∼3-fold higher levels of drug to be administered compared to free drug. Dose for dose, MTC-100038 also facilitated delivery of ∼2.0-fold higher ( p = 0.039) levels of DM1 to the tumor compared to free DM1. MTC-100038 produced significant efficacy (tumor growth index ∼102%; p = <0.0001), in several murine xenograft models of HCC, and was superior to both free DM1 and the current standard of care, sorafenib. Furthermore, MTC-100038 displayed potent (nM) in vitro activity in various HCC primary patient derived cell lines and across various other different cancer cell types. These data demonstrate the potential of MidaCore nanoparticles to enhance tumor delivery of cytotoxic drugs and indicate MTC-100038 is worthy of further investigation as a potential treatment for HCC and other cancer types.

Pre-Ventricular Assist Device Palliative Care Consultation: A Qualitative Analysis.

INTRODUCTION: In 2013, the Centers for Medicare and Medicaid Services issued a mandate requiring that all patients undergoing destination therapy ventricular assist device (DT VAD) implantation have access to a palliative care team before surgery. Subsequently, many VAD programs implemented a mandatory preimplantation palliative care consultation for patients considering DT VAD. However, little is known about the quality of these consults. METHODS: All patients undergoing DT VAD implantation at Northwestern Memorial Hospital from October 30, 2013 (the Centers for Medicare and Medicaid Services decision date), through March 1, 2018, were included. Palliative care consultation notes were qualitatively analyzed for elements of "palliative care assessment" and preparedness planning. RESULTS: Sixty-eight preimplantation palliative care consultations were analyzed. Fifty-six percent of the consults occurred in the intensive care unit, and the median time from consult to VAD implant was six days. General palliative care elements were infrequently discussed. Furthermore, the elements of preparedness planning-device failure, post-VAD health-related quality of life, device complications, and progressive comorbidities-were discussed in only 10%, 54%, 49%, and 12% of consultations, respectively. CONCLUSIONS: One-time preimplantation palliative care consultations at our institution do not lead to completion of preparedness planning or even general palliative care assessment. Further work is needed to determine the most effective way to integrate palliative care into preimplantation care.

Monitoring wastewater discharge from the oil and gas industry using passive sampling and Danio rerio bioassay as complimentary tools.

Produced water (PW) represents the largest volume waste stream in oil and gas production operations from most offshore platforms. PW is difficult to monitor as releases are rapidly diluted and concentrations can reach trace levels. The use of passive samplers can over come this. Here polyethylene (PE) was calibrated for a diverse range of PW pollutants. Zebrafish were exposed to dilutions of PW and passive sampler extracts in order to investigate the relationship between freely dissolved chemical concentrations and acute toxic effects. The raw PW had an LC50 of 13% (percentage of PW in the standardized zebrafish medium). Observed non-viable deformations to embryos (at 5 hpf) included heart and yolk edema, head, spine and tail deformations. The dose-response relationship of lethal effects showed that if 0.0041 g of PE is exposed to this PW, then extracted, 50% of exposed D. rerio will suffer lethal effects. The sum of tested freely dissolved concentrations that led to 50% lethal effects (mortality and non-viable deformations) was 2.32 × 10-4 mg/L for PW and 7.92 × 10-2 mg/L for PE. This implies that exposure to raw PW was more toxic than exposure to PE extracts. This toxicity was attributed both to the presence of contaminants as well as PW salinity. Passive samplers are able to detect very low freely dissolved pollutant concentrations which is important for assessing the spatial dilution of PW releases. Bioassays provide complimentary information as they account for all toxic compounds including those that are not taken up by passive samplers.

Biochar improves maize growth by alleviation of nutrient stress in a moderately acidic low-input Nepalese soil.

We studied the role of biochar in improving soil fertility for maize production. The effects of biochar on the alleviation of three potential physical-chemical soil limitations for maize growth were investigated, i.e. water stress, nutrient stress and acid stress. Experiments involved soils with two dosages of biochar (0.5% and 2% w:w), as well as ones without biochar, in combination with four different dosages of NPK fertilizer, water and lime. Biochar was produced from the invasive shrubby weed Eupatorium adenophorum using flame curtain kilns. This is the first study to alleviate one by one the water stress, nutrient stress and acid stress in order to investigate the mechanisms of biochar effects on soil fertility. Biochar addition increased soil moisture, potassium (K) and plant available phosphorous (P-AL), which all showed significant positive relationship (p<0.001) with above ground biomass of maize. However, biochar was much more effective at abundant soil watering (+311% biomass) than at water-starved conditions (+67% biomass), indicating that biochar did increase soil moisture, but that this was not the main reason for the positive biomass growth effects. Biochar addition did have a stronger effect under nutrient-stressed conditions (+363%) than under abundant nutrient application (+132%). Biochar amendment increased soil pH, but liming and pH had no effect on maize dry biomass, so acidity stress alleviation was not the mechanism of biochar effects on soil fertility. In conclusion, the alleviation of nutrient stress was the probably the main factor contributing to the increased maize biomass production upon biochar addition to this moderately acidic Inceptisol.

The potential of biochar in improving drainage, aeration and maize yields in heavy clay soils.

Heavy clay soils are globally widespread but their poor drainage and poor aeration limit their use for agriculture. This study was designed to test the effect of the amendment of biochar (BC) from woody shrubs on drainage/saturated hydraulic conductivity (Ksat), soil aeration/air capacity, available water capacity and biomass and grain yields of maize. In a field experiment, BC from Gliricidia sepium was applied in planting basins or rip lines at 2.5% and 5% w/w in addition to a control without BC. The maize biomass and grain yields were higher in BC treated plots compared to control (p<0.05) during the 2012 and 2013 seasons. There was no significant difference in the yields between 2.5% and 5% BC treatments (e.g. grain yield were 6.6 and 8.1 t ha-1 in 2012 and 9.3 and 10.3 t ha-1 in 2013 compared to control with 4.2 and 6.7 t ha-1 in 2012 and 2013, respectively). Soil from the same field site was also mixed with a similar woody shrub BC from Eupatorium adenophorum in the laboratory at rates of 2.5%, 5% and 10% BC w/w and a control without BC. The mixtures were then incubated and subjected to two wet-dry cycles for two weeks. Core samples were taken from the incubated soil and tested for bulk density, Ksat and pF measurements. Total porosity and moisture at field capacity and wilting point were 72.3%, 43.7% and 23.7%, respectively, and not affected by BC amendment (p>0.05). In contrast, bulk density decreased linearly by 0.011±0.002 g cm-3 per percent BC added (p<0.001). Ksat and air capacity of the soil were 288 cm day-1 and 30.9%, respectively falling within the generally accepted optimal range. Both Ksat and air capacity followed a significant quadratic relation (p<0.05) upon BC addition, decreasing at low BC doses, reaching a minimum at 3-5% BC and increasing at higher doses. Results allowed a partial attribution of the yield increases to changes in soil physical properties such as changes in bulk density and not clearly to Ksat and air capacity.

Biochar from "Kon Tiki" flame curtain and other kilns: Effects of nutrient enrichment and kiln type on crop yield and soil chemistry.

Biochar application to soils has been investigated as a means of improving soil fertility and mitigating climate change through soil carbon sequestration. In the present work, the invasive shrub "Eupatorium adenophorum" was utilized as a sustainable feedstock for making biochar under different pyrolysis conditions in Nepal. Biochar was produced using several different types of kilns; four sub types of flame curtain kilns (deep-cone metal kiln, steel shielded soil pit, conical soil pit and steel small cone), brick-made traditional kiln, traditional earth-mound kiln and top lift up draft (TLUD). The resultant biochars showed consistent pH (9.1 ± 0.3), cation exchange capacities (133 ± 37 cmolc kg-1), organic carbon contents (73.9 ± 6.4%) and surface areas (35 to 215 m2/g) for all kiln types. A pot trial with maize was carried out to investigate the effect on maize biomass production of the biochars made with various kilns, applied at 1% and 4% dosages. Biochars were either pretreated with hot or cold mineral nutrient enrichment (mixing with a nutrient solution before or after cooling down, respectively), or added separately from the same nutrient dosages to the soil. Significantly higher CEC (P< 0.05), lower Al/Ca ratios (P< 0.05), and high OC% (P<0.001) were observed for both dosages of biochar as compared to non-amended control soils. Importantly, the study showed that biochar made by flame curtain kilns resulted in the same agronomic effect as biochar made by the other kilns (P > 0.05). At a dosage of 1% biochar, the hot nutrient-enriched biochar led to significant increases of 153% in above ground biomass production compared to cold nutrient-enriched biochar and 209% compared to biochar added separately from the nutrients. Liquid nutrient enhancement of biochar thus improved fertilizer effectiveness compared to separate application of biochar and fertilizer.

The role of passive sampling in monitoring the environmental impacts of produced water discharges from the Norwegian oil and gas industry.

Stringent and periodic iteration of regulations related to the monitoring of chemical releases from the offshore oil and gas industry requires the use of ever changing, rapidly developing and technologically advancing techniques. Passive samplers play an important role in water column monitoring of produced water (PW) discharge to seawater under Norwegian regulation, where they are used to; i) measure aqueous concentrations of pollutants, ii) quantify the exposure of caged organisms and investigate PW dispersal, and iii) validate dispersal models. This article summarises current Norwegian water column monitoring practice and identifies research and methodological gaps for the use of passive samplers in monitoring. The main gaps are; i) the range of passive samplers used should be extended, ii) differences observed in absolute concentrations accumulated by passive samplers and organisms should be understood, and iii) the link between PW discharge concentrations and observed acute and sub-lethal ecotoxicological end points in organisms should be investigated.

Junctional Adhesion Molecule-A Is Highly Expressed on Human Hematopoietic Repopulating Cells and Associates with the Key Hematopoietic Chemokine Receptor CXCR4.

Hematopoietic stem/progenitor cells (HSPCs) reside in specialized bone marrow microenvironmental niches, with vascular elements (endothelial/mesenchymal stromal cells) and CXCR4-CXCL12 interactions playing particularly important roles for HSPC entry, retention, and maintenance. The functional effects of CXCL12 are dependent on its local concentration and rely on complex HSPC-niche interactions. Two Junctional Adhesion Molecule family proteins, Junctional Adhesion Molecule-B (JAM)-B and JAM-C, are reported to mediate HSPC-stromal cell interactions, which in turn regulate CXCL12 production by mesenchymal stromal cells (MSCs). Here, we demonstrate that another JAM family member, JAM-A, is most highly expressed on human hematopoietic stem cells with in vivo repopulating activity (p < .01 for JAM-A(high) compared to JAM-A(Int or Low) cord blood CD34(+) cells). JAM-A blockade, silencing, and overexpression show that JAM-A contributes significantly (p < .05) to the adhesion of human HSPCs to IL-1ß activated human bone marrow sinusoidal endothelium. Further studies highlight a novel association of JAM-A with CXCR4, with these molecules moving to the leading edge of the cell upon presentation with CXCL12 (p < .05 compared to no CXCL12). Therefore, we hypothesize that JAM family members differentially regulate CXCR4 function and CXCL12 secretion in the bone marrow niche. Stem Cells 2016;34:1664-1678.

CXCR2 modulates bone marrow vascular repair and haematopoietic recovery post-transplant.

Murine models of bone marrow transplantation show that pre-conditioning regimens affect the integrity of the bone marrow endothelium and that the repair of this vascular niche is an essential pre-requisite for successful haematopoietic stem and progenitor cell engraftment. Little is known about the angiogenic pathways that play a role in the repair of the human bone marrow vascular niche. We therefore established an in vitro humanized model, composed of bone marrow stromal and endothelial cells and have identified several pro-angiogenic factors, VEGFA, ANGPT1, CXCL8 and CXCL16, produced by the stromal component of this niche. We demonstrate for the first time that addition of CXCL8 or inhibition of its receptor, CXCR2, modulates blood vessel formation in our bone marrow endothelial niche model. Compared to wild type, Cxcr2(-/-) mice displayed a reduction in bone marrow cellularity and delayed platelet and leucocyte recovery following myeloablation and bone marrow transplantation. The delay in bone marrow recovery correlated with impaired bone marrow vascular repair. Taken together, our data demonstrate that CXCR2 regulates bone marrow blood vessel repair/regeneration and haematopoietic recovery, and clinically may be a therapeutic target for improving bone marrow transplantation.

Prevalence of diabetic retinopathy within a national diabetic retinopathy screening service.

AIMS: Determine the prevalence and severity of diabetic retinopathy (DR) and risk factors in a large community based screening programme, in order to accurately estimate the future burden of this specific and debilitating complication of diabetes. METHODS: A cross-sectional analysis of 91,393 persons with diabetes, 5003 type 1 diabetes and 86,390 type 2 diabetes, at their first screening by the community based National Diabetic Retinopathy Screening Service for Wales from 2005 to 2009. Image capture used 2×45° digital images per eye following mydriasis, classified by qualified retinal graders with final grading based on the worst eye. RESULTS: The prevalence of any DR and sight-threatening DR in those with type 1 diabetes was 56.0% and 11.2%, respectively, and in type 2 diabetes was 30.3% and 2.9%, respectively. The presence of DR, non-sight-threatening and sight-threatening, was strongly associated with increasing duration of diabetes for either type 1 or type 2 diabetes and also associated with insulin therapy in those with type 2 diabetes. CONCLUSIONS: Prevalence of DR within the largest reported community-based, quality assured, DR screening programme, was higher in persons with type 1 diabetes; however, the major burden is represented by type 2 diabetes which is 94% of the screened population.

Equilibrium partition coefficients of diverse polar and nonpolar organic compounds to polyoxymethylene (POM) passive sampling devices.

Equilibrium passive samplers (EPS) based on polyoxymethylene (POM) are increasingly used for determining freely dissolved water and pore water concentrations of hydrophobic organic compounds in the environment. Unlike other polymeric materials commonly used as EPS, namely poly(dimethylsiloxane) (PDMS) and low-density polyethylene (PE), POM is a polar polymer, containing repeating H-bond accepting ether units. Thus, POM is expected to be a more sensitive EPS than PDMS and PE for polar, H-bond donating compounds, such as many hormones, pharmaceuticals, and biocides. To better characterize the sorption capacity of POM for diverse polar and apolar compounds, equilibrium POM-water partition coefficients, K(POM/w), were measured for 56 compounds, including several classes of polar compounds and organochlorine pesticides. Using this data set and literature data, various POM-partitioning models were calibrated and validated for their ability to predict K(POM/w). The best performing models tested were an Abraham descriptor based polyparameter linear free energy relationship (PP-LFER) (SD = 0.24 log units) and COSMOthermX (SD = 0.37 log units). The performance of SPARC (SD = 0.61 log units) and log-log correlations with K(ow) (SD = 0.49 log units) were lower. A comparison with PDMS and PE confirmed expectations that POM exhibits a higher sensitivity for H-bond donating polar compounds than PDMS and PE do for these compounds. These findings expand the domain of chemicals for which POM can be used as an EPS sampler, and demonstrate that POM is as suitable a passive sampler for many polar organic compounds as it is for hydrophobic organic compounds.

Negative regulation of HIF-1α by an FBW7-mediated degradation pathway during hypoxia.

Hypoxia inducible factor-1α (HIF-1α) stimulates expression of genes associated with angiogenesis and is associated with poor outcomes in ovarian and other cancers. In normoxia, HIF-1α is ubiquitinated and degraded through the E3 ubiquitin ligase, von Hippel-Lindau; however, little is known about the regulation of HIF-1α in hypoxic conditions. FBW7 is an E3 ubiquitin ligase that recognizes proteins phosphorylated by glycogen synthase kinase 3ß (GSK3ß) and targets them for destruction. This study used an ovarian cancer cell model to test the hypothesis that HIF-1α phosphorylation by GSK3ß in hypoxia leads to interaction with FBW7 and ubiquitin-dependent degradation. Expression of constitutively active GSK3ß reduced HIF-1α protein and transcriptional activity and increased ubiquitination of HIF-1α in hypoxia, whereas pharmacologic inhibition of GSK3 or expression of siGSK3ß promoted HIF-1α stabilization and activity. A mechanism through FBW7 was supported by the observed decrease in HIF-1α stabilization when FBW7 was overexpressed and both the elevation of HIF-1α levels and decrease in ubiquitinated HIF-1α when FBW7 was suppressed. Furthermore, HIF-1α associated with FBW7γ by co-immunoprecipitation, and the interaction was weakened by inhibition of GSK3 or mutation of GSK3ß phosphorylation sites. The relevance of this pathway to angiogenic signaling was supported by the finding that endothelial cell tube maturation was increased by conditioned media from hypoxic SK-OV-3 cell lines expressing suppressed GSK3ß or FBW7. These data introduce a new mechanism for regulation of HIF-1α during hypoxia that utilizes phosphorylation to target HIF-1α for ubiquitin-dependent degradation through FBW7 and may identify new targets in the regulation of angiogenesis.

Interactions between Kar2p and its nucleotide exchange factors Sil1p and Lhs1p are mechanistically distinct.

Kar2p, an essential Hsp70 chaperone in the endoplasmic reticulum of Saccharomyces cerevisiae, facilitates the transport and folding of nascent polypeptides within the endoplasmic reticulum lumen. The chaperone activity of Kar2p is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, namely Sil1p and Lhs1p. Here, we demonstrate that the binding requirements for Lhs1p are complex, requiring both the nucleotide binding domain plus the linker domain of Kar2p. In contrast, the IIB domain of Kar2p is sufficient for binding of Sil1p, and point mutations within IIB specifically blocked Sil1p-dependent activation while remaining competent for activation by Lhs1p. Taken together, these results demonstrate that the interactions between Kar2p and its two nucleotide exchange factors can be functionally resolved and are thus mechanistically distinct.

Partitioning of organochlorine pesticides from water to polyethylene passive samplers.

The mass transfer rates and equilibrium partitioning behaviour of 14 diverse organochlorine pesticides (OCP) between water and polyethylene (PE) passive samplers, cut from custom made PE sheets and commercial polyethylene plastic bags, were quantified. Overall mass transfer coefficients, k(O), estimated PE membrane diffusion coefficients, D(PE), and PE-water partitioning coefficients, K(PE-water,) are reported. In addition, the partitioning of three polycyclic aromatic hydrocarbons (PAHs) from water to PE is quantified and compared with literature values. K(PE-water) values agreed mostly within a factor of two for both passive samplers and also with literature values for the reference PAHs. As PE is expected to exhibit similar sorption behaviour to long-chain alkanes, PE-water partitioning coefficients were compared to hexadecane-water partitioning coefficients estimated with the SPARC online calculator, COSMOtherm and a polyparameter linear free energy relationship based on the Abraham approach. The best correlation for all compounds tested was with COSMOtherm estimated hexadecane-water partitioning coefficients.

Nucleotide binding by Lhs1p is essential for its nucleotide exchange activity and for function in vivo.

Protein translocation and folding in the endoplasmic reticulum of Saccharomyces cerevisiae involves two distinct Hsp70 chaperones, Lhs1p and Kar2p. Both proteins have the characteristic domain structure of the Hsp70 family consisting of a conserved N-terminal nucleotide binding domain and a C-terminal substrate binding domain. Kar2p is a canonical Hsp70 whose substrate binding activity is regulated by cochaperones that promote either ATP hydrolysis or nucleotide exchange. Lhs1p is a member of the Grp170/Lhs1p subfamily of Hsp70s and was previously shown to function as a nucleotide exchange factor (NEF) for Kar2p. Here we show that in addition to this NEF activity, Lhs1p can function as a holdase that prevents protein aggregation in vitro. Analysis of the nucleotide requirement of these functions demonstrates that nucleotide binding to Lhs1p stimulates the interaction with Kar2p and is essential for NEF activity. In contrast, Lhs1p holdase activity is nucleotide-independent and unaffected by mutations that interfere with ATP binding and NEF activity. In vivo, these mutants show severe protein translocation defects and are unable to support growth despite the presence of a second Kar2p-specific NEF, Sil1p. Thus, Lhs1p-dependent nucleotide exchange activity is vital for ER protein biogenesis in vivo.