Flv3A facilitates O2 photoreduction and affects H2 photoproduction independently of Flv1A in diazotrophic Anabaena filaments.

The model heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a typical example of a multicellular organism capable of simultaneously performing oxygenic photosynthesis in vegetative cells and O2 -sensitive N2 -fixation inside heterocysts. The flavodiiron proteins have been shown to participate in photoprotection of photosynthesis by driving excess electrons to O2 (a Mehler-like reaction). Here, we performed a phenotypic and biophysical characterization of Anabaena mutants impaired in vegetative-specific Flv1A and Flv3A in order to address their physiological relevance in the bioenergetic processes occurring in diazotrophic Anabaena under variable CO2 conditions. We demonstrate that both Flv1A and Flv3A are required for proper induction of the Mehler-like reaction upon a sudden increase in light intensity, which is likely important for the activation of carbon-concentrating mechanisms and CO2 fixation. Under ambient CO2 diazotrophic conditions, Flv3A is responsible for moderate O2 photoreduction, independently of Flv1A, but only in the presence of Flv2 and Flv4. Strikingly, the lack of Flv3A resulted in strong downregulation of the heterocyst-specific uptake hydrogenase, which led to enhanced H2 photoproduction under both oxic and micro-oxic conditions. These results reveal a novel regulatory network between the Mehler-like reaction and the diazotrophic metabolism, which is of great interest for future biotechnological applications.

Pneumonitis after radiotherapy for lung cancer (PARALUC): an interventional study to create a symptom-based scoring system for identification of patients developing radiation pneumonitis.

BACKGROUND: Pneumonitis is a possible side effect of radiotherapy for lung cancer. Since it can occur up to several months following treatment, symptoms may not be associated with previous radiotherapy, and pneumonitis can become severe before diagnosed. This study aimed to develop a symptom-based scoring system to contribute to earlier detection of radiation pneumonitis requiring medical intervention (grade ≥ 2). METHODS: Patients irradiated for lung cancer complete a paper-based questionnaire (symptom-based score) during and up to 24 weeks following radiotherapy. Patients rate symptoms potentially associated with pneumonitis, and scoring points are assigned to severity of these symptoms. Sum scores are used to identify radiation pneumonitis. If radiation pneumonitis is suspected, patients undergo standard diagnostic procedures. If grade ≥ 2 pneumonitis is confirmed, medical intervention is indicated. The discriminative power of the score will be assessed by calculating the area under the receiver operating characteristic curve (AUC). If statistical significance of the AUC is reached, the optimal sum score to predict radiation pneumonitis will be established, which is defined as a cut-off value with sensitivity ≥90% and specificity ≥80%. Assuming a ratio between patients without and with pneumonitis of 3.63, a sample size of 93 patients is required in the full analysis set to yield statistical significance at the level of 5% with a power of 90% if the AUC under the alternative hypothesis is at least 0.9. Considering potential drop-outs, 98 patients should be recruited. If > 20% of patients are not satisfied with the score, modification is required. If the dissatisfaction rate is > 40%, the score is considered not useful. In 10 patients, functionality of a mobile application will be tested in addition to the paper-based questionnaire. DISCUSSION: If an optimal cut-off score resulting in sufficiently high sensitivity and specificity can be identified and the development of a symptom-based scoring system is successful, this tool will contribute to better identification of patients experiencing pneumonitis after radiotherapy for lung cancer. TRIAL REGISTRATION: Clinicaltrials.gov ( NCT04335409 ); registered on 2nd of April, 2020.

Alcohol improves cerebellar learning deficit in myoclonus-dystonia: A clinical and electrophysiological investigation.

OBJECTIVE: To characterize neurophysiological subcortical abnormalities in myoclonus-dystonia and their modulation by alcohol administration. METHODS: Cerebellar associative learning and basal ganglia-brainstem interaction were investigated in 17 myoclonus-dystonia patients with epsilon-sarcoglycan (SGCE) gene mutation and 21 age- and sex-matched healthy controls by means of classical eyeblink conditioning and blink reflex recovery cycle before and after alcohol intake resulting in a breath alcohol concentration of 0.08% (0.8g/l). The alcohol responsiveness of clinical symptoms was evaluated by 3 blinded raters with a standardized video protocol and clinical rating scales including the Unified Myoclonus Rating Scale and the Burke-Fahn-Marsden Dystonia Rating Scale. RESULTS: Patients showed a significantly reduced number of conditioned eyeblink responses before alcohol administration compared to controls. Whereas the conditioning response rate decreased under alcohol intake in controls, it increased in patients (analysis of variance: alcohol state × group, p = 0.004). Blink reflex recovery cycle before and after alcohol intake did not differ between groups. Myoclonus improved significantly after alcohol intake (p = 0.016). The severity of action myoclonus at baseline correlated negatively with the conditioning response in classical eyeblink conditioning in patients. INTERPRETATION: The combination of findings of reduced baseline acquisition of conditioned eyeblink responses and normal blink reflex recovery cycle in patients who improved significantly with alcohol intake suggests a crucial role of cerebellar networks in the generation of symptoms in these patients. Ann Neurol 2017;82:543-553.

The First Survival Score for Patients Treated with Whole-Brain Radiotherapy Plus Simultaneous Integrated Boost for Brain Metastases.

A modern approach for brain metastases includes whole-brain radiotherapy plus simultaneous boost (WBRT+SIB). We developed a survival score in 128 patients treated with WBRT+SIB. Three models, each including three prognostic groups, were created. Positive predictive values (PPVs) for death ≤6 and survival ≥6 months were calculated. On multivariate analyses, performance score (KPS) and the number of brain metastases were significantly associated with survival. On univariate analyses, age showed a strong trend, and extra-cerebral cranial metastases a trend. In Model 1 (KPS, number of lesions), compared groups had 6-month survival rates of 15%, 38% and 57%. In Model 2 (KPS, lesions, age), rates were 17%, 33% and 75%, and in Model 3 (KPS, lesions, age, extra-cerebral metastases), 14%, 34% and 78%. PPVs for death ≤6 and survival ≥6 months were 85% and 57% (Model 1), 83% and 75% (Model 2), and 86% and 78% (Model 3). Thus, all models were accurate in predicting death ≤ 6 months; poor-prognosis patients may not benefit from SIB. Models 2 and 3 were superior in predicting survival ≥ 6 months. Given that Model 3 requires more data (including extensive staging), Model 2 is considered favorable for many patients. If extra-cerebral metastases are already known or extensive staging has been performed, Model 3 can also be used.

Treatment Outcomes After Higher-dose Fractionated Stereotactic Radiotherapy (FSRT) Alone for 1-4 Brain Metastases.

BACKGROUND/AIM: Fractionated stereotactic radiotherapy (FSRT) is increasingly used for brain metastases. We investigated higher-dose FSRT with biologically effective doses (BED) of 49.6-66.7 Gy12 Patients and Methods: Eleven characteristics were evaluated for local control (LC), overall survival (OS), and freedom from radiation necrosis (RN) in 69 patients with 1-4 brain metastases. Fifty-seven patients (83%) had extracranial metastases, 23 (33%) Karnofsky performance scores (KPS) ≤70, and 21 (30%) brain metastases ≥21 mm. RESULTS: At 1 and 2 years, LC-rates were 81% and 63%, OS-rates 66% and 43%, and freedom from RN-rates 98% and 87%, respectively. Median time to local progression was 35 months, median survival 19 months. KPS ≥90 was associated with better OS (p=0.048). BED of 49.6-57 Gy12 (vs. 63-66.7 Gy12) was associated with higher rates of freedom from RN (p=0.046), not with LC (p=0.78) or OS (p=0.55). CONCLUSION: Higher-dose FSRT appears feasible and effective in patients with 1-4 brain metastases. BED 63-66.7 Gy12 may not improve LC and OS but may increase RN risk.

Whole-brain Radiotherapy With or Without a Simultaneous Integrated Boost for Treatment of Brain Metastases.

BACKGROUND/AIM: Many patients with brain metastases receive whole-brain radiotherapy (WBRT), despite the increasing use of stereotactic radiotherapy alone. A more recent approach includes WBRT combined with simultaneous integrated boost (WBRT+SIB). This study compared WBRT alone and WBRT+SIB for unresected brain metastases. PATIENTS AND METHODS: One-hundred-and-three patients receiving WBRT+SIB were compared to 275 patients receiving WBRT alone for intracerebral control (IC) and overall survival (OS). RESULTS: Both treatment groups (WBRT alone and WBRT+SIB) were balanced with respect to patient characteristics. On multivariate analyses, WBRT+SIB (p=0.041), Karnofsky performance score (KPS) >70 (p<0.001), and 1-3 brain metastases (p=0.016) were significantly associated with IC. KPS >70 (p<0.001), favorable tumor type (p=0.011), 1-3 brain metastases (p=0.011), and absence of extracranial metastases (p<0.001) were significantly associated with OS. CONCLUSION: WBRT+SIB is associated with improved IC but similar OS when directly compared to WBRT for brain metastases. Selected patients with a high risk of intracerebral recurrence may benefit from SIB.

Radiation Necrosis Following Stereotactic Radiosurgery or Fractionated Stereotactic Radiotherapy with High Biologically Effective Doses for Large Brain Metastases.

In Radiation Therapy Oncology Group 90-05, the maximum tolerated dose of single-fraction radiosurgery (SRS) for brain metastases of 21-30 mm was 18 Gy (biologically effective dose (BED) 45 Gy12). Since the patients in this study received prior brain irradiation, tolerable BED may be >45 Gy12 for de novo lesions. We investigated SRS and fractionated stereotactic radiotherapy (FSRT) with a higher BED for radiotherapy-naive lesions. Patients receiving SRS (19-20 Gy) and patients treated with FSRT (30-48 Gy in 3-12 fractions) with BED > 49 Gy12 for up to 4 brain metastases were compared for grade ≥ 2 radiation necrosis (RN). In the entire cohort (169 patients with 218 lesions), 1-year and 2-year RN rates were 8% after SRS vs. 2% and 13% after FSRT (p = 0.73) in per-patient analyses, and 7% after SRS vs. 7% and 10% after FSRT (p = 0.59) in per-lesion analyses. For lesions ≤ 20 mm (137 patients with 185 lesions), the RN rates were 4% (SRS) vs. 0% and 15%, respectively, (FSRT) (p = 0.60) in per-patient analyses, and 3% (SRS) vs. 0% and 11%, respectively, (FSRT) (p = 0.80) in per-lesion analyses. For lesions > 20 mm (32 patients with 33 lesions), the RN rates were 50% (SRS) vs. 9% (FSRT) (p = 0.012) in both per-patient and per-lesion analyses. In the SRS group, a lesion size > 20 mm was significantly associated with RN; in the FSRT group, lesion size had no impact on RN. Given the limitations of this study, FSRT with BED > 49 Gy12 was associated with low RN risk and may be safer than SRS for brain metastases > 20 mm.

Early Identification of Pneumonitis in Patients Irradiated for Lung Cancer-Final Results of the PARALUC Trial.

Radiotherapy of lung cancer may cause pneumonitis that generally occurs weeks or months following therapy and can be missed. This prospective trial aimed to pave the way for a mobile application (app) allowing early diagnosis of pneumonitis. The primary goal was the identification of the optimal cut-off of a score to detect pneumonitis of grade ≥2 after radiotherapy for lung cancer. Based on the severity of symptoms (cough, dyspnea, fever), scoring points were 0−9. Receiver operating characteristic (ROC)-curves were used to describe the sensitivity and specificity. The area under the ROC-curve (AUC) was calculated to judge the accuracy of the score, Youden-index was employed to define the optimal cut-off. Until trial termination, 57 of 98 patients were included. Eight of 42 patients evaluable for the primary endpoint (presence or absence of radiation pneumonitis) experienced pneumonitis. AUC was 0.987 (0.961−1.000). The highest sensitivity was achieved with 0−4 points (100%), followed by 5 points (87.5%), highest specificity with 5−6 points (100%). The highest Youden-index was found for 5 points (87.5%). The rate of patient satisfaction with the symptom-based scoring system was 93.5%. A cut-off of 5 points was identified as optimal to differentiate between pneumonitis and no pneumonitis. Moreover, pneumonitis was significantly associated with an increase of ≥3 points from baseline (p < 0.0001). The scoring system provided excellent accuracy and high patient satisfaction. Important foundations for the development of a mobile application were laid.

A Prognostic Tool to Estimate the Risk of Pneumonitis in Patients Irradiated for Lung Cancer.

BACKGROUND/AIM: Radiotherapy of lung cancer can lead to pneumonitis. This study aimed to identify risk factors and create a prognostic tool. PATIENTS AND METHODS: Sixteen factors were evaluated in 169 patients irradiated for lung cancer including age, sex, lung function, primary tumor/nodal stage, histology, tumor location, surgery, systemic treatment, radiation volume, total dose, mean dose to ipsilateral lung, history of another malignancy, pack years, chronic inflammatory disease, and cardiovascular disease. RESULTS: Forty-one patients experienced pneumonitis. Significant associations were found for total doses >56 Gy (p=0.023), mean lung doses >20 Gy (p=0.002) or >13 Gy (p<0.001), and chronic inflammatory disease (p=0.034). Considering mean lung dose and chronic inflammatory disease, scores were 2, 3, 4, or 5 points. Pneumonitis rates were 0% (0/35), 24% (14/58), 32% (21/66), and 60% (6/10) (p=0.001), respectively. CONCLUSION: Based on significant risk factors, a prognostic tool was developed that can help estimate the risk of pneumonitis and contribute to personalized follow up of patients.

Prognostic Factors of Survival After Radiotherapy for Lung Cancer-The Impact of Smoking Pack Years.

BACKGROUND/AIM: The prognostic role of smoking pack years after thoracic irradiation for lung cancer needs further clarification, since previous studies showed conflicting results. Therefore, this study investigated potential prognostic factors for survival including pack years in 170 lung cancer patients receiving local radiotherapy. PATIENTS AND METHODS: Twelve factors were retrospectively evaluated for survival including age, sex, tumor site, histology, primary tumor stage, nodal stage, distant metastasis, radiation dose, upfront surgery or systemic treatment, pulmonary function, and number of pack years. RESULTS: On univariate analyses, absence of distant metastasis (p=0.049), radiation dose >56 Gy (p=0.019), and ≤40 pack years (p=0.005) were significantly associated with better survival. In the multivariate analysis, number of pack years (hazard ratio 2.18, 95% confidence interval 1.25-3.82, p=0.006) maintained significance; distant metastasis (p=0.34) and radiation dose (p=0.16) were not significant. CONCLUSION: Number of pack years was an independent predictor of survival after thoracic irradiation for lung cancer.

Estimating the Probability of Not Completing the Intended Course of Thoracic Radiotherapy for Lung Cancer.

BACKGROUND/AIM: In some patients with lung cancer scheduled for thoracic radiotherapy (RT), treatment is discontinued before reaching the planned dose. For optimal treatment personalization, a tool estimating whether a patient can complete radiotherapy would be helpful. PATIENTS AND METHODS: Eleven pre-RT characteristics were analyzed in 170 patients receiving local RT for lung cancer. Characteristics included age, sex, tumor site, histology, tumor and nodal stage, distant metastasis, surgery, systemic treatment, pulmonary function, and smoking history. RESULTS: Age >75 years (p=0.038), distant metastasis (p=0.009), and forced expiratory volume in 1 second <1.2 l (p=0.038) were significantly associated with discontinuation of RT. A prognostic instrument was developed in 126 patients with complete data regarding these characteristics. It included three groups (0, 1, and 2-3 points) with non-completion rates of 33.3%, 55.0% and 75.0% (p=0.004). CONCLUSION: This new instrument can help estimating the probability that lung cancer patients assigned to local RT cannot complete the planned course of RT.

Identification and characterization of cellouronate (ß-1,4-linked polyglucuronic acid) lyase from the scallop Mizuhopecten yessoensis.

The semisynthetic polysaccharide cellouronate is a ß-1,4-linked polyglucuronic acid prepared from regenerated cellulose by chemical oxidation. Here, we isolated a novel enzyme, MyAly, as a cellouronate lyase from a scallop Mizuhopecten yessoensis. Its optimum temperature, pH, and NaCl concentration for cellouronate degradation were determined to be 30 °C, 6.9, and 200-500 mM, respectively. MyAly endolytically degraded cellouronate into unsaturated di-, tri-, and tetrasaccharides with kcat of 31.1 s-1. MyAly also showed an alginate-degradation activity with a kcat value of 0.58 s-1. However, there was no significant difference in Km values between cellouronate and alginate. MyAly consisted of 280 amino acids and shared 36.5-44.1 % identity with known marine gastropod alginate lyases belonging to the polysaccharide lyase family 14. This is the first study to identify and characterize a cellouronate-degrading lyase from a marine organism, providing a better understanding of the biodegradability of the industrially important polysaccharide, cellouronate, in marine environments.

Identification of New Chromosomal Loci Involved in com Genes Expression and Natural Transformation in the Actinobacterial Model Organism Micrococcus luteus.

Historically, Micrococcus luteus was one of the first organisms used to study natural transformation, one of the main routes of horizontal gene transfer among prokaryotes. However, little is known about the molecular basis of competence development in M. luteus or any other representative of the phylum of high-GC Gram-positive bacteria (Actinobacteria), while this means of genetic exchange has been studied in great detail in Gram-negative and low-GC Gram-positive bacteria (Firmicutes). In order to identify new genetic elements involved in regulation of the comEA-comEC competence operon in M. luteus, we conducted random chemical mutagenesis of a reporter strain expressing lacZ under the control of the comEA-comEC promoter, followed by the screening of dysregulated mutants. Mutants with (i) upregulated com promoter under competence-repressing conditions and (ii) mutants with a repressed com promoter under competence-inducing conditions were isolated. After genotype and phenotype screening, the genomes of several mutant strains were sequenced. A selection of putative com-influencing mutations was reinserted into the genome of the M. luteus reporter strain as markerless single-nucleotide mutations to confirm their effect on com gene expression. This strategy revealed mutations affecting com gene expression at genetic loci different from previously known genes involved in natural transformation. Several of these mutations decreased transformation frequencies by several orders of magnitude, thus indicating significant roles in competence development or DNA acquisition in M. luteus. Among the identified loci, there was a new locus containing genes with similarity to genes of the tad clusters of M. luteus and other bacteria.

Prevalence and Characteristics of Symptomatic Pneumonitis After Radiotherapy of Patients With Locally Advanced Lung Cancer.

BACKGROUND/AIM: Radiotherapy of locally advanced lung cancer often requires high doses potentially leading to pneumonitis. This study evaluated the rate of symptomatic pneumonitis and characteristics in these patients. PATIENTS AND METHODS: This study included 278 patients irradiated for locally advanced lung cancer between 2016 and 2019. In patients experiencing symptomatic pneumonitis, patient and treatment characteristics were analyzed. RESULTS: Pneumonitis was diagnosed in 21 patients (7.6%) after a median of 9 (1-23) weeks. Ipsilateral lungs received mean doses >13 Gy in 21 (100%) and >20 Gy in 15 patients (71.4%). Seventeen patients (81.0%) received chemotherapy and/or immunotherapy, 12 (57.1%) had significant cardiovascular disease (all 21 patients had risk factors), 11 (52.4%) were heavy smokers (≥40 pack years), 7 (33.3%) were aged ≥74 years, 5 (23.8%) had chronic inflammatory disease and 4 (19.0%) had previous tumors. CONCLUSION: Overall pneumonitis rate was 7.6%. Frequent characteristics included high mean lung doses, systemic treatment, cardiovascular disease (and risk factors), heavy smoking, older age, chronic inflammatory disease and history of a previous tumor.

Prevalence and Characteristics of Pneumonitis Following Irradiation of Breast Cancer.

BACKGROUND/AIM: Pneumonitis is a serious complication after radiotherapy of breast cancer. This study aimed to identify its prevalence and potential risk factors. PATIENTS AND METHODS: A total of 606 patients irradiated following breast-conserving surgery or mastectomy were retrospectively analyzed. In patients developing pneumonitis, radiation and clinical parameters were investigated to identify potential risk factors. RESULTS: Eleven patients (1.8%) developed a pneumonitis grade ≥2. Mean doses to the ipsilateral lung were >7 Gy in 5 patients (45%). Of the other patients, 5 had a chronic inflammatory disease. Six patients (55%) had another malignancy (4 previous contralateral breast cancers, 1 previous ovarian and thyroid cancer, 1 synchronous carcinoma-in-situ (pTis) at the contralateral breast). Five patients (45%) received chemotherapy including taxanes and 4 patients (36%) received trastuzumab. CONCLUSION: The prevalence of pneumonitis was 1.8%. Potential risk factors included mean radiation dose to ipsilateral lung >7 Gy, systemic treatment with taxanes or trastuzumab, chronic inflammatory disease and history of another malignancy.

Influence of L-dopa on subtle motor signs in heterozygous Parkin- and PINK1 mutation carriers.

INTRODUCTION: A latent nigrostriatal deficit and its possible clinical consequences in asymptomatic heterozygous Parkin and PINK1 mutation carriers (AMC) have been a matter of investigation in recent years. Notably, mild Parkinsonian signs in heterozygous mutation carriers can be so subtle that they may be missed if not specifically investigated. METHODS: We studied 15 heterozygous Parkin and PINK1 AMC and 18 age- and sex-matched mutation-negative controls using a standardized video, instructing the probands to perform relevant parts of the UPDRS III to investigate fine motor movements at baseline and after first-time L-Dopa administration. Additionally, available UPDRS III scores of mutation carriers from the past ten years were reviewed. RESULTS: AMC showed a reduced number of fine motor movements per second compared to controls at baseline (p = 0.04). L-Dopa improved motor performance numerically but non-significantly in AMC (p = 0.2301), but significantly in healthy controls (p = 6.1·10-5). Although none of the AMC reported symptoms, nine showed rigidity, bradykinesia, tremor, and postural instability when the UPDRS III was applied. Mean UPDRSIII scores significantly decreased after L-Dopa administration (p = 0.005), but did not increase over the past ten years. CONCLUSIONS: (i) Heterozygous AMC show subtle motor abnormalities when a detailed, specialized motor examination is applied and compared to mutation-negative matched control subjects. (ii) The mild motor deficit present in a subgroup of heterozygous Parkin and PINK1 AMC appears to be non-progressive and responsive to L-dopa administration. (iii) Evaluating motor changes, their progression, and treatment response in AMC can provide valuable insights into possible early disease stages and compensatory mechanisms.