Komagataella phaffii Erp41 is a protein disulfide isomerase with unprecedented disulfide bond catalyzing activity when coupled to glutathione.

In the methylotrophic yeast Komagataella phaffii, we identified an endoplasmic reticulum-resident protein disulfide isomerase (PDI) family member, Erp41, with a peculiar combination of active site motifs. Like fungal ERp38, it has two thioredoxin-like domains which contain active site motifs (a and a'), followed by an alpha-helical ERp29c C-terminal domain (c domain). However, while the a domain has a typical PDI-like active site motif (CGHC), the a' domain instead has CGYC, a glutaredoxin-like motif which confers to the protein an exceptional affinity for GSH/GSSG. This combination of active site motifs has so far been unreported in PDI-family members. Homology searches revealed ERp41 is present in the genome of some plants, fungal parasites, and a few nonconventional yeasts, among which are Komagataella spp. and Yarrowia lipolytica. These yeasts are both used for the production of secreted recombinant proteins. Here, we analyzed the activity of K. phaffii Erp41. We report that it is nonessential in K. phaffii, and that it can catalyze disulfide bond formation in partnership with the sulfhydryl oxidase Ero1 in vitro with higher turnover rates than the canonical PDI from K. phaffii, Pdi1, but slower activation times. We show how Erp41 has unusually fast glutathione-coupled oxidation activity and relate it to its unusual combination of active sites in its thioredoxin-like domains. We further describe how this determines its unusually efficient catalysis of dithiol oxidation in peptide and protein substrates.

Biochemical analysis of Komagataella phaffii oxidative folding proposes novel regulatory mechanisms of disulfide bond formation in yeast.

Oxidative protein folding in the endoplasmic reticulum (ER) is driven mainly by protein disulfide isomerase PDI and oxidoreductin Ero1. Their activity is tightly regulated and interconnected with the unfolded protein response (UPR). The mechanisms of disulfide bond formation have mainly been studied in human or in the yeast Saccharomyces cerevisiae. Here we analyze the kinetics of disulfide bond formation in the non-conventional yeast Komagataella phaffii, a common host for the production of recombinant secretory proteins. Surprisingly, we found significant differences with both the human and S. cerevisiae systems. Specifically, we report an inactive disulfide linked complex formed by K. phaffii Ero1 and Pdi1, similarly to the human orthologs, but not described in yeast before. Furthermore, we show how the interaction between K. phaffii Pdi1 and Ero1 is unaffected by the introduction of unfolded substrate into the system. This is drastically opposed to the previously observed behavior of the human pathway, suggesting a different regulation of the UPR and/or possibly different interaction mechanics between K. phaffii Pdi1 and Ero1.

A quantitative interpretation of oxidative protein folding activity in Escherichia coli.

BACKGROUND: Escherichia coli is of central interest to biotechnological research and a widely used organism for producing proteins at both lab and industrial scales. However, many proteins remain difficult to produce efficiently in E. coli. This is particularly true for proteins that require post translational modifications such as disulfide bonds. RESULTS: In this study we develop a novel approach for quantitatively investigating the ability of E. coli to produce disulfide bonds in its own proteome. We summarise the existing knowledge of the E. coli disulfide proteome and use this information to investigate the demand on this organism's quantitative oxidative folding apparatus under different growth conditions. Furthermore, we built an ordinary differential equation-based model describing the cells oxidative folding capabilities. We use the model to infer the kinetic parameters required by the cell to achieve the observed oxidative folding requirements. We find that the cellular requirement for disulfide bonded proteins changes significantly between growth conditions. Fast growing cells require most of their oxidative folding capabilities to keep up their proteome while cells growing in chemostats appear limited by their disulfide bond isomerisation capacities. CONCLUSION: This study establishes a novel approach for investigating the oxidative folding capacities of an organism. We show the capabilities and limitations of E. coli for producing disulfide bonds under different growth conditions and predict under what conditions excess capability is available for recombinant protein production.

Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness.

With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X-press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (qS ) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy-chain single-domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X-press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and qS are used to control productivity and leakiness. In the X-press strain, extracellular SpA and VHH titer reach up to 349 and 19.6 mg g-1 , respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X-press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.

Safe application of extensive radiotherapy to a cardiac resynchronization device.

Patients with cardiac implantable electronic devices undergoing radiotherapy (RT) are prone to the risk of device failure. Guidelines and manufacturer's instructions are lacking practical recommendations for cumulative radiation doses to pacemakers or implantable cardioverter defibrillators. The present case demonstrates the effect of RT of a Merkel cell carcinoma near the location of a cardiac resynchronization therapy pacemaker. Despite guideline recommendations, surgical relocation or de novo implantation of the device on the contralateral side was avoided to prevent the dissemination of tumour cells, inflammation, and wound healing complications. A total dose of 47.25 Gy applied in very close proximity to the cardiac resynchronization therapy pacemaker was carried out safely without jeopardizing the patient and any device malfunction during and after treatment within >1.5 years of follow-up period. The present case demonstrates that high-dose RT near to a cardiac resynchronization therapy device can be carried out safely. Special precautions during RT as well as close device follow-up interrogations are mandatory. Large-scale studies are needed for the true frequency of adverse events.

Pre-Ablation rhTSH-Stimulated F-18 FDG PET/CT Changes Patient Management in Increased-Risk Thyroid Cancer.

The aim of the study was to evaluate the clinical impact of pre-ablation rhTSH-stimulated fluorine-18 fluorodeoxyglucose (F-18 FDG) PET/CT in addition to post-therapeutic whole body radioiodine scanning in patients with intermediate to high risk differentiated thyroid carcinoma (DTC). This was a retrospective single center study including 73 patients with thyroid cancer (44 females, mean age 43.2±16.2 years, 62% papillary, 31% follicular, 7% poorly differentiated). All patients underwent ablative radioiodine treatment (mean activity: 3661±673 MBq I-131) using rhTSH after thyroidectomy and lymph node (LN) dissection (01/2013-10/2016) and TSH-stimulated F-18 FDG PET/CT (4 MBq/kg body weight, low dose CT). Post-treatment I-131 whole body scan (I-131 WBS) was obtained 9 days afterwards in planar technique and in case of equivocal or abnormal findings using SPECT/CT. Thirty-one patients (42%) showed F-18 FDG avid lesions, 14 patients showed more FDG than iodine positive lesions and 5 patients more iodine positive lesions in I-131 WBS, respectively. Fifty-three patients showed identical F-18 FDG PET/CT and I-131 WBS. The initial treatment plan was changed from follow-up to therapy (surgery, systemic therapy using tyrosine-kinase inhibition) in 11 patients (15%) on the basis of F-18 FDG PET/CT imaging. Six of these 11 patients had papillary thyroid cancer. Three patients with histologically proven LN metastases had stimulated thyroglobulin-levels<2.0 ng/ml. Our study demonstrated a clinical benefit of pre-ablation rhTSH-stimulated F-18 FDG PET/CT imaging in about 20% of patients with intermediate to high risk DTC, leading to change in patient management in 15%.

Pulmonary function decreases moderately after accelerated high-dose irradiation for stage III non-small cell lung cancer.

BACKGROUND: Chemoradiotherapy (CRT) is the standard treatment for patients with inoperable stage III non-small cell lung cancer (NSCLC) stage III. With a median OS beyond 30 months, adequate pulmonary function (PF) is essential to ensure acceptable quality of life after treatment. Forced expiratory volume in 1 second (FEV1) and diffusing capacity of the lung for carbon monoxide (DLCO) are the most widely used parameters to assess lung function. The aim of the current study was to evaluate dose-volume effects of accelerated high-dose radiation on PF. METHODS: A total of 72 patients were eligible for the current analysis. After induction chemotherapy, all patients received dose-differentiated accelerated radiotherapy with intensity-modulated radiotherapy (IMRT-DART). PF tests were performed six weeks, three and six months after the end of radiotherapy. RESULTS: The median total dose to the tumor was 73.8 Gy (1.8 Gy bid) with a size dependent range between 61.2 and 90 Gy. In the whole cohort, 321 pulmonary function tests were performed. At six months, the median FEV1 relative to baseline was 0.95 (range: 0.56-1.36), and the relative median DLCO decreased to 0.98 (range: 0.64-1.50). The correlation between V20total lung and FEV1 decline was statistically significant (P = 0.023). A total of 13 of 34 (38%) COPD patients had a 4%-21% FEV1 decrease. CONCLUSION: Patients with a V20total lung < 21% are at a low risk for PF decrease after high dose irradiation treatment. Although overall short term FEV1 and DLCO differ only moderately from baseline these changes may be clinically important, especially in patients with COPD. KEY POINTS: Significant findings: Pulmonary function after high dose irradiation decreases only moderately. FEV1 and DLCO decrease depend on V20total lung . Small differences in lung function may be clinically important for COPD patients. KPS predicts minimal clinically important differences (MCID). WHAT THIS STUDY ADDS: This study shows that high-dose irradiation delivered with intensity-modulated techniques does not impair short-term lung function even in patients with compromised respiratory capacity before treatment. This is a pre-requisite for adequate quality of life after thoraco-oncological therapy.

In-Line Ultrasound-Enhanced Raman Spectroscopy Allows for Highly Sensitive Analysis with Improved Selectivity in Suspensions.

Raman spectroscopy is a nondestructive characterization method offering chemical-specific information. However, the cross-section of inelastically (Raman) scattered light is very low compared to elastically (Rayleigh) scattered light, resulting in weak signal intensities in Raman spectroscopy. Despite providing crucial information in off-line measurements, it usually is not sensitive enough for efficient, in-line process control in conjunction with low particle concentrations. To overcome this limitation, two custom-made 1.4404 stainless-steel prototype add-ons were developed for in-line Raman probes that enable ultrasound particle manipulation and thus concentration of particles in suspensions in the focus of the Raman excitation laser. Depending on size and density differences between particles and the carrier medium, particles are typically caught in the nodal planes of a quasi-standing wave field formed in an acoustic resonator in front of the sensor. Two arrangements were realized with regard to the propagation direction of the ultrasonic wave relative to the propagation direction of the laser. The parallel arrangement improved the limit of detection (LOD) by a factor of ≈30. In addition to increased sensitivity, the perpendicular arrangement offers increased selectivity: modifying the frequency of the ultrasonic wave field allows the liquid or solid phase to be moved into the focus of the Raman laser. The combination of in-line Raman spectroscopy with ultrasound particle manipulation holds promise to push the limits of conventional Raman spectroscopy, hence broadening its field of application to areas where previously Raman spectroscopy has not had sufficient sensitivity for accurate, in-line detection.

Kinetics and Predicted Structure of a Novel Xylose Reductase from Chaetomium thermophilum.

While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable.

Survival in early lung cancer patients treated with high dose radiotherapy is independent of pathological confirmation.

BACKGROUND: Approximately 15% of lung cancer patients are diagnosed in early stages. Microscopic proof of disease cannot always be obtained because of comorbidity or reluctance to undergo invasive diagnostic procedures. In the current study, survival data of patients with and without pathology are compared. METHODS: One hundred and sixty three patients with NSCLC I-IIb (T3 N0) treated between 2002 and 2016 were eligible: 123 (75%) had pathological confirmation of disease, whereas 40 (25%) did not. In accordance with international guidelines, both groups received radiotherapy. Comorbidity was assessed with the Charlson Comorbidity Index (CCI). RESULTS: The median follow-up was 28.6 months (range: 0.3-162): 66 (40%) patients are still alive, while 97 (59%) patients died: 48 (29%) cancer-related deaths and 49 (30%) from causes other than cancer. Median overall survival (OS) in patients without pathological confirmation was 58.6 months (range: 0.5-162), which did not differ from those with microscopic proof of disease (39.4 months, range: 0.3-147.5; logrank P = 0.481). Median cancer-specific survival (CSS) also did not differ at 113.4 months (range: 0.5-162) in the non-confirmation group (logrank P = 0.763) versus 51.5 months (range: 3.7-129.5) in patients with pathology. In Cox regression, a CCI of ≥ 3 was associated with poor OS (hazard ratio 2.0; range 1.2-3.4; P = 0.010) and CSS (hazard ratio 2.0; 1.0-4.0; P = 0.043). CONCLUSION: OS and CSS in early lung cancer patients depend on comorbidity rather than on pathological confirmation of disease.

[Imaging of parathyroid adenomas with F­18 choline PET-CT]. / Darstellung und Lokalisation von Nebenschilddrüsenadenomen mit F­18 Cholin PET/CT.

BACKGROUND: Ultrasound and sestamibi scintigraphy are the recommended standard procedures for initial diagnosis in primary hyperparathyroidism (pHPT). Recently, F­18 choline positron emission tomography computed tomography (choline PET/CT) has been shown promising results for the diagnostic work up of primary hyperparathyroidism (pHPT) suggesting superiority over conventional scintigraphy using Tc99m sestamibi based protocols using planar dual-phase imaging, SPECT or SPECT/CT. METHODS: This review presents the results of F­18 choline PET/CT on the basis of a literature search using the keywords "primary hyperparathyroidism and choline", "primary hyperparathyroidism and PET", "parathyroid adenoma and choline" und "parathyroid adenoma and PET". RESULTS: 6 studies were identified dealing with the diagnostic impact of choline PET/CT. The studies included 5 to 151 patients. Localization of single gland adenomas can be achieved with choline PET/CT in 80 up to 96% of cases. A high sensitivity and accuracy of choline PET/CT imaging is documented even in cases of repeated surgery for recurrent pHPT, in coexistant nodular goiter or in the detection of adenoma in atypical localization. CONCLUSIONS: Using choline PET/CT parathyroid adenoma and probably parathyroid hyperplasia can be exactly localized in most patients with pHPT. Thus, a minimal-invasive surgical procedure is feasible with decreased risk of complications but high success rate in terms of biochemical cure. The diagnostic accuracy in multiglandular disease remains to be established.

Sequential immunotherapy in a patient with primary refractory Hodgkin lymphoma and novel mutations.

Primary resistant Hodgkin lymphoma is an aggressive disease with few treatment options and short survival. Neoplastic cells of classical Hodgkin lymphoma are heavily dependent on microenvironmental stimuli, regularly express PD-L1, and a relevant proportion of relapsed patients is sensitive to blocking of the PD1/PD-L1 axis. However, response duration is limited and further treatment options are unknown but urgently needed. We report a case of a patient without relevant response to five subsequent chemotherapy regimens who immediately and dramatically responded to an anti-PD1 mab. During the following two years she responded to the anti-CTLA-4 mab ipilimumab, the Jak2 inhibitor ruxolitinib, and a combination of lenalidomide plus cyclophosphamide given in subsequent relapses. A thorough genomic analysis demonstrated seven genomic alterations with six of them not previously described in this disease (i.e. BRIP1 G212fs*62, KRAS L19F, KDM5A R1239W, MYC A59T, ARIDA1A E1683fs*15 and TP53 277Y). Three alterations were considered actionable and one of them drugable. The number of mutations increased over time and the BRIP1 mutation was found to be a germline mutation.

Diagnostic accuracy and functional parameters of myocardial perfusion scintigraphy using accelerated cardiac acquisition with IQ SPECT technique in comparison to conventional imaging.

BACKGROUND: IQ SPECT consists of a new pinhole-like collimator, cardio-centric acquisition, and advanced 3D iterative SPECT reconstruction. The aim of this paper was to compare diagnostic accuracy and functional parameters obtained with IQ SPECT versus conventional SPECT in patients undergoing myocardial perfusion scintigraphy with adenosine stress and at rest. METHODS: Eight patients with known or suspected coronary artery disease underwent [99mTc] tetrofosmin gated SPECT. Acquisition was performed on a Symbia T6 equipped with IQ SPECT and on a conventional gamma camera system. Gated SPECT data were used to calculate functional parameters. Scores analysis was performed on a 17-segment model. Coronary angiography and clinical follow-up were considered as diagnostic reference standard. RESULTS: Mean acquisition time was 4 minutes with IQ SPECT and 21 minutes with conventional SPECT. Agreement degree on the diagnostic accuracy between both systems was 0.97 for stress studies, 0.91 for rest studies and 0.96 for both studies. Perfusion abnormalities scores obtained by using IQ SPECT and conventional SPECT were not significant different: SSS, 9.7±8.8 and 10.1±6.4; SRS, 7.1±6.1 and 7.5±7.3; SDS, 4.0±6.1 and 3.9±4.3, respectively. However, a significant difference was found in functional parameters derived from IQ SPECT and conventional SPECT both after stress and at rest. Mean LVEF was 8% lower using IQ SPECT. Differences in LVEF were found in patients with normal LVEF and patients with reduced LVEF. CONCLUSIONS: Functional parameters using accelerated cardiac acquisition with IQ SPECT are significantly different to those obtained with conventional SPECT, while agreement for clinical interpretation of myocardial perfusion scintigraphy with both techniques is high.

Diagnostic performance of [18F] FDG PET-CT compared to bone scintigraphy for the detection of bone metastases in lung cancer patients.

BACKGROUND: Accurate staging of lung cancer is essential for effective patient management and selection of appropriate therapeutic strategy. The aim of this paper was to compare the value of bone scintigraphy and FDG PET-CT for detecting bone metastases in lung cancer patients and the impact of these modalities in disease staging. METHODS: One hundred sixty-four lung cancer patients who had undergone both FDG PET-CT and bone scintigraphy within 14 days were included into this study. The analysis of FDG PET-CT and bone scintigraphy was carried out patient- and lesion-based. RESULTS: One hundred twenty-one patients were negative and 43 patients positive for bone metastases. FDG PET-CT found bone metastases in 42/43 patients and bone scintigraphy in 38/43 patients. Sensitivity, specificity and accuracy of FDG PET-CT and bone scintigraphy for detecting bone metastases were 97.7%, 100% and 99.4%, and 87.8%, 97.5% and 94.2%, respectively. FDG PET-CT identified 430 bone metastases and bone scintigraphy 246 bone metastases. Skull was the only region where bone scintigraphy identified more lesions than FDG PET-CT. Based on both scintigraphic modalities disagreement concerning disease stage was found in 3 patients. CONCLUSION: FDG PET-CT yielded a higher sensitivity, specificity and accuracy than bone scintigraphy for identifying bone metastases in lung cancer patients. FDG PET-CT thus can be recommended for initial staging of lung cancer patients without applying bone scintigraphy for the detection of bone metastases.

The value of (18)F-fluoride PET/CT in the assessment of screw loosening in patients after intervertebral fusion stabilization.

PURPOSE: We evaluated (18)F-fluoride PET/CT for the diagnosis of screw loosening after intervertebral fusion stabilization and compared the results with those from functional radiography. METHODS: A group of 59 patients with pain in the region of previous intervertebral fusion stabilization and suspicion of implant instability due to screw loosening were investigated with (18)F-fluoride PET/CT and functional radiography, 30.1 ± 3.4 and 29.3 ± 3.2 months, respectively, after surgery. The criterion for loosening was increased focal uptake surrounding the screw entry point and shaft. SUVmax and SUVmean were measured in a region of interest (ROI) drawn around each screw (334 screws analysed). The final diagnosis was established by surgical exploration in 27 patients and clinical follow-up after intervertebral fusion stabilization in 32 patients. RESULTS: Of the 59 patients, 20 were proven positive for implant failure due to screw loosening and 39 were confirmed negative. The sensitivity, specificity and accuracy of (18)F-fluoride PET/CT were 75%, 97.4% and 89.8% in the patient-based analysis, and 45.6%, 100% and 80% in the screw-based analysis, respectively. The positive and negative predictive values were 93.8% and 100 % in the patient-based analysis, and 88.4 and 76% in the screw-based analysis, respectively. CT signs in PET/CT allowed screw breakage to be detected in three patients. SUVmax, SUVmean and SUVmax/SUVmean ratios in screw ROIs and respective values in reference regions were all found to be significantly different between screws positive for loosening (58 screws) and screws negative for loosening (276 screws). The ratio between SUVmax in screw ROIs and the values in reference regions was the most significant parameter for distinguishing screws positive and screws negative for loosening. CONCLUSION: (18)F-Fluoride PET/CT imaging is useful for the diagnosis of screw loosening in patients with persistent symptoms after intervertebral fusion stabilization.

Assessment of response to neoadjuvant radiochemotherapy with F-18 FLT and F-18 FDG PET/CT in patients with rectal cancer.

OBJECTIVE: The comparison of 2-deoxy-2-[18F]fluoro-D-glucose (F-18 FDG) and 3'-deoxy-3'-[18F]fluorothymidine (F-18 FLT) imaging in patients with rectal cancer before and after neoadjuvant radiochemotherapy (RCT) in relation to histopathology and immunohistochemistry obtained from surgery. METHODS: 20 consecutive patients (15 m, 5 f), mean age of 65 ± 10 years were included into this prospective study with a mean follow-up of 4.1 ± 0.8 years. RESULTS: Among histopathological responders (n = 8 out of 20), posttreatment F-18 FLT and F-18 FDG scans were negative in 75 % (n = 6) and 38 % (n = 3), respectively. The mean response index (RI) was 61.0 % ± 14.0 % for F-18 FLT and 58.7 % ± 14.6 % for F-18 FDG imaging. Peritumoral lymphocytic infiltration (CD3 positive cells) was significantly related to posttreatment SUVmax in F-18 FDG but not F-18 FLT studies. CONCLUSION: A significant decrease of SUVmax in F-18 FDG and F-18 FLT studies could be seen after RCT. Negative posttreatment F-18 FLT studies identified more histopathological responders.

Sentinel lymph node biopsy is precise after primary systemic therapy in stage II-III breast cancer patients.

BACKGROUND: Sentinel lymph node biopsy (SLNB) without axillary lymph node dissection (ALND) in SLN negative patients is a standard of care for most breast cancer patients. SLNB for axillary staging after primary systemic therapy (PST) is still under discussion because of possibly reduced accuracy, while data are lacking. The purpose of this study was to evaluate the accuracy of SLNB after PST. MATERIALS AND METHODS: A total of 185 breast cancer patients were treated with PST; 160 patients received preoperative chemotherapy, and 25 patients received preoperative endocrine therapy. Thus, 143 of 160 patients with preoperative chemotherapy and 22 of 25 patients with preoperative endocrine therapy were eligible for evaluation. The combination of blue dye and radioactive tracer was used for identification of SLNs. All patients received SLNB and axillary lymph node dissection (ALND). Pathologic assessment of SLNs was performed and compared to non-SLN status. RESULTS: Pathologic complete response rates and breast conserving therapy rates were 15.4 and 78.3% in the preoperative chemotherapy group and 0 and 77.3% in the preoperative endocrine therapy group, respectively. Identification rate, sensitivity, overall accuracy, and false-negative rate were 81.1% (116 of 143), 91.7% (55 of 60), 95.7% (111 of 116), and 8.3% (5 of 60) in the preoperative chemotherapy group and 77.3% (17 of 22), 90.0% (9 of 10), 94.1% (16 of 17), and 10.0% (1 of 10) in the preoperative endocrine therapy group, respectively. DISCUSSION: SLNB after primary systemic therapy is accurate, and the results are comparable to those of primary SLNB. SLNB after PST could spare ALND in up to 40% of patients with primary positive axillary lymph nodes and should be considered as a standard for axillary staging in those patients.