FEASIBILITY OF INSPIRATORY MUSCLE TRAINING FOR PATIENTS WITH PERSISTENT DYSPNOEA AFTER COVID-19 INFECTION: A PILOT STUDY.

Objective: This study investigates the feasibility of delivering inspiratory muscle training as part of the physical therapy treatment for patients with post-COVID dyspnoea. Design: Mixed-methods pilot study. Subjects/patients: Patients with complaints of dyspnoea after COVID-19 infection and their physical therapists. Methods: The Amsterdam University of Applied Sciences and the Amsterdam University Medical Centers conducted this study. Participants performed daily inspiratory muscle training at home for 6 weeks, consisting of 30 repetitions against a pre-set resistance. The primary outcome was feasibility assessed as acceptability, safety, adherence and patient- and professional experience obtained through diaries and semi-structured interviews. The secondary outcome was maximal inspiratory pressure. Results: Sixteen patients participated. Nine patients and 2 physical therapists partook in semi-structured interviews. Two patients dropped out before initiating the training. Adherence was 73.7%, and no adverse events occurred. Protocol deviations occurred in 29.7% of the sessions. Maximal inspiratory pressure changed from 84.7% of predicted at baseline to 111.3% at follow-up. Qualitative analysis identified barriers to training: 'Getting acquainted with the training material' and 'Finding the right schedule'. Facilitators were: 'Support from physical therapists' and 'Experiencing improvements'. Conclusion: Delivering inspiratory muscle training to patients with post-COVID dyspnoea seems feasible. Patients valued the simplicity of the intervention and reported perceived improvements. However, the intervention should be carefully supervised, and training parameters adjusted to individual needs and capacity.

Course of recovery of respiratory muscle strength and its associations with exercise capacity and handgrip strength: A prospective cohort study among survivors of critical illness.

BACKGROUND: Mechanical ventilation affects the respiratory muscles, but little is known about long-term recovery of respiratory muscle weakness (RMW) and potential associations with physical functioning in survivors of critical illness. The aim of this study was to investigate the course of recovery of RMW and its association with functional outcomes in patients who received mechanical ventilation. METHODS: We conducted a prospective cohort study with 6-month follow-up among survivors of critical illness who received ≥ 48 hours of invasive mechanical ventilation. Primary outcomes, measured at 3 timepoints, were maximal inspiratory and expiratory pressures (MIP/MEP). Secondary outcomes were functional exercise capacity (FEC) and handgrip strength (HGS). Longitudinal changes in outcomes and potential associations between MIP/MEP, predictor variables, and secondary outcomes were investigated through linear mixed model analysis. RESULTS: A total of 59 participants (male: 64%, median age [IQR]: 62 [53-66]) were included in this study with a median (IQR) ICU and hospital length of stay of 11 (8-21) and 35 (21-52) days respectively. While all measures were well below predicted values at hospital discharge (MIP: 68.4%, MEP 76.0%, HGS 73.3% of predicted and FEC 54.8 steps/2m), significant 6-month recovery was seen for all outcomes. Multivariate analyses showed longitudinal associations between older age and decreased MIP and FEC, and longer hospital length of stay and decreased MIP and HGS outcomes. In crude models, significant, longitudinal associations were found between MIP/MEP and FEC and HGS outcomes. While these associations remained in most adjusted models, an interaction effect was observed for sex. CONCLUSION: RMW was observed directly after hospital discharge while 6-month recovery to predicted values was noted for all outcomes. Longitudinal associations were found between MIP and MEP and more commonly used measures for physical functioning, highlighting the need for continued assessment of respiratory muscle strength in deconditioned patients who are discharged from ICU. The potential of targeted training extending beyond ICU and hospital discharge should be further explored.

Telerehabilitation Delivery in Canada and the Netherlands: Results of a Survey Study.

BACKGROUND: Following the onset of the COVID-19 pandemic, telerehabilitation (TR) has been expanding to address the challenges and risks of in-person delivery. It is likely that a level of TR delivery will continue after the pandemic because of its advantages, such as reducing geographical barriers to service. Many pandemic-related TR initiatives were put in place quickly. Therefore, we have little understanding of current TR delivery, barriers and facilitators, and how therapists anticipate integrating TR into current practice. Knowing this information will allow the incorporation of competencies specifically related to the use and provision of TR into professional profiles and entry-to-practice education, thereby promoting high-quality TR care. OBJECTIVE: This study aimed to obtain a descriptive overview of current TR practice among rehabilitation therapists in Canada and the Netherlands and identify perceived barriers to and facilitators of practice. METHODS: A web-based cross-sectional survey was conducted with occupational, physical, and respiratory therapists and dietitians in Canada (in French and English) and the Netherlands (in Dutch and English) between November 2021 and March 2022. Recruitment was conducted through advertisements on social media platforms and email invitations facilitated by regulatory and professional bodies. The survey included demographic and practice setting information; whether respondents delivered TR, and if so, components of delivery; confidence and satisfaction ratings with delivery; and barriers to and facilitators of use. TR satisfaction and uptake were measured using the Telehealth Usability Questionnaire and modified Technology Acceptance Model. Data were first summarized descriptively, and then, comparisons were conducted between professions. RESULTS: Overall, 723 survey responses were received, mostly from Canada (n=666, 92.1%) and occupational therapists (n=434, 60%). Only 28.1% (203/723) reported receiving specific training in TR, with 1.2% (9/723) indicating that it was part of their professional education. Approximately 19.5% (139/712) reported not using TR at all, whereas most participants (366/712, 51.4%) had been using this approach for 1 to 2 years. Services delivered were primarily teleconsultation and teletreatment with individuals. Respondents offering TR were moderately satisfied with their service delivery and found it to be effective; 90.1% (498/553) indicated that they were likely to continue offering TR after the pandemic. Technology access, confidence, and setup were rated the highest as facilitators, whereas technology issues and the clinical need for physical contact were the most common barriers. CONCLUSIONS: Professional practice and experience with TR were similar in both countries, suggesting the potential for common strategic approaches. The high prevalence of current practice and strong indicators of TR uptake suggest that therapists are likely to continue TR delivery after the pandemic; however, most therapists (461/712, 64.7%) felt ill prepared for practice, and the need to target TR competencies during professional and postprofessional education is critical. Future studies should explore best practice for preparatory and continuing education.

Experiences of interaction between people with cancer and their healthcare professionals: A systematic review and meta-synthesis of qualitative studies.

PURPOSE: This study investigates patients' experiences of interaction with their healthcare professionals (HCPs) during cancer treatment and identifies elements that HCPs can utilize to improve cancer care provision. METHODS: PubMed, CINAHL, PsycINFO, SCOPUS, and Embase were systematically searched for relevant studies published from January 2010 until February 2022. Qualitative studies investigating adult patients' perspectives on their interaction with HCPs during cancer treatment were included. Studies conducted during the diagnosis or end-of-life treatment phase were excluded. Duplicate removal, screening, and quality appraisal were independently performed by four reviewers using Covidence.org. We performed a thematic meta-synthesis of qualitative data extracted from studies meeting the quality criteria in three stages: excerpts coding, codes categorization, and theme identification by merging similar categories. RESULTS: Eighty-eight studies were included for quality appraisal, of which 50 papers met the quality inclusion criteria. Three themes were identified as essential to positively perceived patient-HCP interaction: "Support, respect and agency", "Quantity, timing, and clarity of information", and "Confidence, honesty, and expertise". Overall, patients experienced positive interaction with HCPs when the approach was person-centered and when HCPs possessed strong interpersonal skills. However, patients expressed negative experiences when their preferences regarding communication and the type of personal support needed were ignored. CONCLUSIONS: This meta-synthesis emphasizes the importance for HCPs to recognize all patients' needs, including communication and personal support preferences, to provide high-quality care. Consequently, healthcare professionals should continuously train their verbal and non-verbal communication, empathy, active listening, and collaboration skills during their undergraduate and continuing education.

Physiotherapy With Telerehabilitation in Patients With Complicated Postoperative Recovery After Esophageal Cancer Surgery: Feasibility Study.

BACKGROUND: Improvement of functional status with physiotherapy is an important goal for patients with postoperative complications and an increased length of hospital stay (LoS) after esophagectomy. Supervised physiotherapy with telerehabilitation instead of conventional face-to-face care could be an alternative to treat these patients in their home environment after hospital discharge (T0), but its feasibility has not yet been investigated in detail. OBJECTIVE: The aim of this study was to investigate the feasibility of a 12-week supervised postoperative physiotherapy intervention with telerehabilitation for patients with esophageal cancer who underwent esophagectomy and had postoperative complications or who had an increased LoS. The secondary objective was to investigate the preliminary effectiveness of telerehabilitation on functional recovery compared with usual care. METHODS: A prospective feasibility study with a matched historical comparison group was performed. Feasibility outcomes included willingness and adherence to participate, refusal rate, treatment duration, occurrence of adverse events, and patient satisfaction. Secondary outcome measures were measurements of musculoskeletal and cardiovascular functions and activities according to the domains of the International Classification of Functioning, Disability and Health. RESULTS: A total of 22 patients with esophageal cancer who underwent esophagectomy and had postoperative complications or an increased LoS were included. The mean age at surgery was 64.55 (SD 6.72) years, and 77% (17/22) of patients were male. Moreover, 15 patients completed the intervention. Patient adherence was 99.8% in the first 6 weeks and dropped to 75.6% in the following 6 weeks, with a mean difference of -24.3% (95% CI 1.3 to 47.2; P=.04). At 3 months post operation, no differences in functional status were found between the intervention group and the matched historical comparison group. CONCLUSIONS: This study showed that a postoperative physiotherapeutic intervention with telerehabilitation is feasible for patients with postoperative complications or an increased LoS after esophageal cancer surgery up to 6 weeks after T0.

Influence of body composition and muscle strength on outcomes after multimodal oesophageal cancer treatment.

BACKGROUND: Influence of sarcopenia in combination with other body composition parameters and muscle strength on outcomes after oesophageal surgery for oesophageal cancer remains unclear. The objectives were (i) to describe the incidence of sarcopenia in relation to adipose tissue quantity and distribution and muscle strength; (ii) to evaluate if neoadjuvant chemoradiation (nCRTx) influences body composition and muscle strength; and (iii) to evaluate the influence of body composition and muscle strength on post-operative morbidity and long-term survival. METHODS: This retrospective study included patients with oesophageal cancer who received nCRTx followed by surgery between January 2011 and 2016. Skeletal muscle, visceral, and subcutaneous adipose tissue cross-sectional areas were calculated based on computed tomography scans, and muscle strength was measured using hand grip tests, 30 seconds chair stand tests, and maximal inspiratory and expiratory pressure tests prior to nCRTx and after nCRTx. RESULTS: A total of 322 patients were included in this study. Sarcopenia was present in 55.6% of the patients prior to nCRTx and in 58.2% after nCRTx (P = 0.082). Patients with sarcopenia had a significantly lower muscle strength and higher fat percentage. The muscle strength and incidence of sarcopenia increased while the mean body mass index and fat percentage decreased during nCRTx. A body mass index above 25 kg/m2 was associated with anastomotic leakage (P = 0.032). Other body composition parameters were not associated with post-operative morbidity. A lower handgrip strength prior to nCRTx was associated with pulmonary and cardiac complications (P = 0.023 and P = 0.009, respectively). In multivariable analysis, a lower number of stands during the 30 seconds chair stand test prior to nCRTx (hazard ratio 0.93, 95% confidence interval 0.87-0.99, P = 0.017) and visceral adipose tissue of >128 cm2 after nCRTx (hazard ratio 1.81, 95% confidence interval 1.30-2.53, P = 0.001) were associated with worse overall survival. CONCLUSIONS: Sarcopenia occurs frequently in patients with oesophageal cancer and is associated with less muscle strength and a higher fat percentage. Body composition changes during nCRTx did not influence survival. Impaired muscle strength and a high amount of visceral adipose tissue are associated with worse survival. Therefore, patients with poor fitness might benefit from preoperative nutritional and muscle strengthening guidance, aiming to increase muscle strength and decrease visceral adipose tissue. However, this should be confirmed in a large prospective study.

The pre- and postoperative course of functional status in patients undergoing esophageal cancer surgery.

INTRODUCTION: To optimally target physiotherapy treatment, knowledge of the pre- and postoperative course of functional status in patients undergoing esophagectomy is required. The aim of this prospective longitudinal study was to investigate the course of functional status in patients with esophageal cancer before and after esophagectomy. MATERIALS AND METHODS: Functional status outcome measures of patients with esophageal cancer who underwent surgery between March 2012 and June 2016 were prospectively measured at 3 months and at 1 day before surgery and at 1 week and at 3 months after surgery. Analysis of repeated measurements with the mixed model approach was used to study changes over time. RESULTS: Hundred fifty-five patients were measured at 3 months and at 1 day before surgery, of which 109 (70.3%) at 1 week and 60 (38.7%) at 3 months after surgery. Mean (SD) age at surgery was 63.5 years (9.3), and 122 patients (78.7%) were male. The incidence of postoperative complications was 83 (53.5%). Three months postoperatively, functional status measures returned to baseline levels, except from handgrip strength (beta [95% CI] -6.2 [-11.3 to -1.1]; P = 0.02) and fatigue (4.7 [0.7to 8.7]; P = 0.02). No differences were observed in the course of functional status between patients with and without postoperative complications. CONCLUSION: Functional status of patients undergoing esophagectomy returned to baseline values three months after surgery, despite the high incidence of postoperative complications. This requires rethinking the concept of prehabilitation, where clearly not all patients benefit from high functional status to prevent postoperative complications.

Muscle Strength Is Associated With Muscle Mass in Patients With Esophageal Cancer Awaiting Surgery.

BACKGROUND AND PURPOSE: Decreased muscle mass and muscle strength are independent predictors of poor postoperative recovery in patients with esophageal cancer. If there is an association between muscle mass and muscle strength, physiotherapists are able to measure muscle strength as an early predictor for poor postoperative recovery due to decreased muscle mass. Therefore, in this cross-sectional study, we aimed to investigate the association between muscle mass and muscle strength in predominantly older patients with esophageal cancer awaiting esophagectomy prior to neoadjuvant chemoradiation. METHODS: In patients with resectable esophageal cancer eligible for surgery between March 2012 and October 2015, we used computed tomographic scans to assess muscle mass and compared them with muscle strength measures (handgrip strength, inspiratory and expiratory muscle strength, 30 seconds chair stands test). We calculated Pearson correlation coefficients and determined associations by multivariate linear regression analysis. RESULTS AND DISCUSSION: A tertiary referral center referred 125 individuals to physiotherapy who were eligible for the study; we finally included 93 individuals for statistical analysis. Multiple backward regression analysis showed that gender (95% confidence interval [CI], 2.05-33.82), weight (95% CI, 0.39-1.02), age (95% CI, -0.91 to -0.04), left handgrip strength (95% CI, 0.14-1.44), and inspiratory muscle strength (95% CI, 0.08-0.38) were all independently associated with muscle surface area at L3. All these variables together explained 66% of the variability (R) in muscle surface area at L3 (P < .01). CONCLUSIONS: This study shows an independent association between aspects of muscle strength and muscle mass in patients with esophageal cancer awaiting surgery, and physiotherapists could use the results to predict muscle mass on the basis of muscle strength in preoperative patients with esophageal cancer.

Characterization of CNPY5 and its family members.

The Canopy (CNPY) family consists of four members predicted to be soluble proteins localized to the endoplasmic reticulum (ER). They are involved in a wide array of processes, including angiogenesis, cell adhesion, and host defense. CNPYs are thought to do so via regulation of secretory transport of a diverse group of proteins, such as immunoglobulin M, growth factor receptors, toll-like receptors, and the low-density lipoprotein receptor. Thus far, a comparative analysis of the mammalian CNPY family is missing. Bioinformatic analysis shows that mammalian CNPYs, except the CNPY1 homolog, have N-terminal signal sequences and C-terminal ER-retention signals and that mammals have an additional member CNPY5, also known as plasma cell-induced ER protein 1/marginal zone B cell-specific protein 1. Canopy proteins are particularly homologous in four hydrophobic alpha-helical regions and contain three conserved disulfide bonds. This sequence signature is characteristic for the saposin-like superfamily and strongly argues that CNPYs share this common saposin fold. We showed that CNPY2, 3, 4, and 5 (termed CNPYs) localize to the ER. In radioactive pulse-chase experiments, we found that CNPYs rapidly form disulfide bonds and fold within minutes into their native forms. Disulfide bonds in native CNPYs remain sensitive to low concentrations of dithiothreitol (DTT) suggesting that the cysteine residues forming them are relatively accessible to solutes. Possible roles of CNPYs in the folding of secretory proteins in the ER are discussed.

Folding-function relationship of the most common cystic fibrosis-causing CFTR conductance mutants.

Cystic fibrosis is caused by mutations in the CFTR gene, which are subdivided into six classes. Mutants of classes III and IV reach the cell surface but have limited function. Most class-III and class-IV mutants respond well to the recently approved potentiator VX-770, which opens the channel. We here revisited function and folding of some class-IV mutants and discovered that R347P is the only one that leads to major defects in folding. By this criterion and by its functional response to corrector drug VX-809, R347P qualifies also as a class-II mutation. Other class-IV mutants folded like wild-type CFTR and responded similarly to VX-809, demonstrating how function and folding are connected. Studies on both types of defects complement each other in understanding how compounds improve mutant CFTR function. This provides an attractive unbiased approach for characterizing mode of action of novel therapeutic compounds and helps address which drugs are efficacious for each cystic fibrosis disease variant.

Correction: Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers.

[This corrects the article DOI: 10.1371/journal.pone.0206692.].

Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers.

As an integral membrane protein, purification and characterization of phospho-N- acetylmuramyl- pentapeptide translocase MraY have proven difficult. Low yield and concerns of retaining stability and activity after detergent solubilization have hampered the structure-function analysis. The recently developed detergent-free styrene-maleic acid (SMA) co-polymer system offers an alternative approach that may overcome these disadvantages. In this study, we used the detergent free system to purify MraY from Bacillus subtilis. This allowed efficient extraction of MraY that was heterologously produced in Escherichia coli membranes into SMA-wrapped nanodiscs. The purified MraY embedded in these nanodiscs (SMA-MraY) was comparable to the micellar MraY extracted with a conventional detergent (DDM) with regard to the yield and the purity of the recombinant protein but required significantly less time. The predominantly alpha-helical secondary structure of the protein in SMA-wrapped nanodiscs was also more stable against heat denaturation compared to the micellar protein. Thus, this detergent-free system is amenable to extract MraY efficiently and effectively while maintaining the biophysical properties of the protein. However, the apparent activity of the SMA-MraY was reduced compared to that of the detergent-solubilized protein. The present data indicates that this is caused by a lower accessibility of the enzyme in SMA-wrapped nanodiscs towards its polyisoprenoid substrate.

Preparation and Practical Applications of 2',7'-Dichlorodihydrofluorescein in Redox Assays.

Oxidative stress, a state in which intra- or extracellular oxidant production outweighs the antioxidative capacity, lies at the basis of many diseases. DCFH2-DA (2',7'-dichlorodihydrofluorescein diacetate) is the most widely used fluorogenic probe for the detection of general oxidative stress. However, the use of DCFH2-DA, as many other fluorogenic redox probes, is mainly confined to the detection of intracellular oxidative stress in vitro. To expand the applicability of the probe, an alkaline hydrolysis and solvent extraction procedure was developed to generate high-purity DCFH2 (2',7'-dichlorodihydrofluorescein) from DCFH2-DA using basic laboratory equipment. Next, the utility of DCFH2 was exemplified in a variety of cell-free and in vitro redox assay systems, including oxidant production by transition metals, photodynamic therapy, activated macrophages, and platelets, as well as the antioxidative capacity of different antioxidants. In cells, the concomitant use of DCFH2-DA and DCFH2 enabled the measurement and compartmentalized analysis of intra- and extracellularly produced oxidants, respectively, using a single read-out parameter. Furthermore, hepatocyte-targeted liposomes were developed to deliver the carboxylated derivative, 5(6)-carboxy-DCFH2, to hepatocytes in vivo. Liposome-delivered 5(6)-carboxy-DCFH2 enabled real-time visualization and measurement of hepatocellular oxidant production during liver ischemia-reperfusion. The liposomal 5(6)-carboxy-DCFH2 can be targeted to other tissues where oxidative stress is important, including cancer.

Photodynamic Therapy with Liposomal Zinc Phthalocyanine and Tirapazamine Increases Tumor Cell Death via DNA Damage.

The efficacy of photodynamic therapy (PDT) in some solid tumors is limited by the poor biodistributive properties of conventional photosensitizers and a natural predisposition of tumor cells to survive hypoxia and oxidative stress. This study investigated the therapeutic potential of a third-generation photosensitizer, liposomal zinc phthalocyanine (ZnPC), in combination with the hypoxic cytotoxin tirapazamine (TPZ). TPZ induces DNA double strand breaks (DSBs) under hypoxic conditions and subsequent apoptosis via p53 signaling. Experiments were performed in tumor cells with functional p53 (Sk-Cha1) and dysfunctional p53 (A431). The combination therapy of TPZ and PDT induced DNA DSBs and cell cycle stalling and enhanced the cytotoxicity of PDT by exacerbating apopotic and non-apoptotic tumor cell death. These phenomena occurred regardless of oxygen tension and the mechanism of cell death differed per cell line. Liposomes containing both ZnPC and TPZ exhibited no dark toxicity but were more lethal to both cell types after PDT compared to ZnPC-liposomes lacking TPZ­an effect that was more pronounced under hypoxic conditions. In conclusion, TPZ is a suitable pharmaceutical compound to increase PDT efficacy by exploiting the post-PDT tumor hypoxia. The inclusion of TPZ and ZnPC into a single liposomal delivery system was feasible. The PDT strategy described in this study may be valuable for the treatment of PDT-recalcitrant tumors.

New Insight into the Catalytic Mechanism of Bacterial MraY from Enzyme Kinetics and Docking Studies.

Phospho-MurNAc-pentapeptide translocase (MraY) catalyzes the synthesis of Lipid I, a bacterial peptidoglycan precursor. As such, MraY is essential for bacterial survival and therefore is an ideal target for developing novel antibiotics. However, the understanding of its catalytic mechanism, despite the recently determined crystal structure, remains limited. In the present study, the kinetic properties of Bacillus subtilis MraY (BsMraY) were investigated by fluorescence enhancement using dansylated UDP-MurNAc-pentapeptide and heptaprenyl phosphate (C35-P, short-chain homolog of undecaprenyl phosphate, the endogenous substrate of MraY) as second substrate. Varying the concentrations of both of these substrates and fitting the kinetics data to two-substrate models showed that the concomitant binding of both UDP-MurNAc-pentapeptide-DNS and C35-P to the enzyme is required before the release of the two products, Lipid I and UMP. We built a model of BsMraY and performed docking studies with the substrate C35-P to further deepen our understanding of how MraY accommodates this lipid substrate. Based on these modeling studies, a novel catalytic role was put forward for a fully conserved histidine residue in MraY (His-289 in BsMraY), which has been experimentally confirmed to be essential for MraY activity. Using the current model of BsMraY, we propose that a small conformational change is necessary to relocate the His-289 residue, such that the translocase reaction can proceed via a nucleophilic attack of the phosphate moiety of C35-P on bound UDP-MurNAc-pentapeptide.

Isolation of lipids from biological samples.

Isolation of the lipid fraction from biological samples has been a crucial part of countless studies over the last century. This considerable research interest has led to the development of a number of methods for isolating a range of molecular species that fall under the umbrella term "lipid". Such methods vary in popularity, complexity, specificity and even toxicity. In this review, we explore examples of published methods (1952-2014) for isolating lipids from biological samples and attempt to assess the limits of techniques both from a chemical and biological perspective. We also suggest how a suitable method might be chosen for a novel application.

Potato Patatin Generates Short-Chain Fatty Acids from Milk Fat that Contribute to Flavour Development in Cheese Ripening.

The potato lipase, patatin, has long been thought of as essentially inactive towards triacylglycerols. Recently, technology has been developed to isolate potato proteins in native form as food ingredients at industrial scale. Characterisation of native patatin obtained in this way revealed that this enzyme activity towards triacylglycerols has been underestimated. This enables the application of patatin in cheese ripening, which is described in this study. When patatin is added to milk during cheese making, the lipase preferentially releases short-chain fatty acids that contribute to cheese flavour in a dose-dependent manner. Fortuitously, the lipase activity is found mainly in the curd. The release of the short-chain fatty acids matches the activity profile of patatin towards homotriacylglycerols of defined chain length. Residual patatin in the whey fraction can be inactivated effectively by heat treatment that follows Arrhenius kinetics. The results are discussed in terms of cheese making, patatin substrate preference and implications for the use of patatin more generally in food emulsions.

Furan fatty acids efficiently rescue brain cells from cell death induced by oxidative stress.

Treatment of rat brain C6 astroglioma cells with furan fatty acid F6 prior to exposure to hydrogen peroxide shows a strong protective effect of F6 against cell death resulting from oxidative stress. This protective effect is obtained only for F6 administered as a free fatty acid and with an intact furan ring. It is proposed that brain cells are rescued by F6 scavenging radicals elicited by lipid peroxidation within the cell membrane. Oxidative processes outside the cell membrane, such as protein carbonylation, are not affected by F6. Furan fatty acids such as those present in fish oils and marine organisms are likely beneficial for consumption in reducing the risk of diseases that have been implicated to arise from oxidative stress, such as Alzheimer's disease.

Hyperacidification of trans-Golgi network and endo/lysosomes in melanocytes by glucosylceramide-dependent V-ATPase activity.

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H(+) -translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent K(i) for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.

Novel Hsp90 partners discovered using complementary proteomic approaches.

Hsp90 is an essential eukaryotic molecular chaperone that stabilizes a large set of client proteins, many of which are involved in various cellular signaling pathways. The current list of Hsp90 interactors comprises about 200 proteins and this number is growing steadily. In this paper, we report on the application of three complementary proteomic approaches directed towards identification of novel proteins that interact with Hsp90. These methods are coimmunoprecipitation, pull down with biotinylated geldanamycin, and immobilization of Hsp90beta on sepharose. In all, this study led to the identification of 42 proteins, including 18 proteins that had not been previously characterized as Hsp90 interactors. These novel Hsp90 partners not only represent abundant protein species, but several proteins were identified at low levels, among which signaling kinase Cdk3 and putative transcription factor tripartite motif-containing protein 29. Identification of tetratricopeptide-repeat-containing mitochondrial import receptor protein Tom34 suggests the involvement of Hsp90 in the early steps of translocation of mitochondrial preproteins. Taken together, our data expand the knowledge of the Hsp90 interactome and provide a further step in our understanding of the Hsp90 chaperone system.