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Site-directed mutagenesis of the sensory [FeFe] hydrogenase from Thermotoga maritima reveals new insight into how the protein environment tunes the active site properties for its sensory role.

International audience; Gases like H2, N2, CO2, and CO are increasingly recognized as critical feedstock in “green” energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N2, CO2, and CO and the production of H2 require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N2 fixation by nitrogenase and H2 production by hydrogenase as well as CO2 and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere

Hydrogenases catalyze the reversible oxidation of H2. Carbon monoxide (CO) is known to be a competitive inhibitor of O2-sensitive [NiFe]-hydrogenases. Although the activities of some O2-tolerant [NiFe]-hydrogenases are unaffected by CO, the partially O2-tolerant [NiFe]-hydrogenase from Citrobacter sp. S-77 (S77-HYB) is inhibited by CO. In this work, the CO-bound state of S77-HYB was characterized by activity assays, spectroscopic techniques and X-ray crystallography. Electron paramagnetic resonance spectroscopy showed a diamagnetic Ni2+ state, and Fourier-transform infrared spectroscopy revealed the stretching vibration of the exogenous CO ligand. The crystal structure determined at 1.77 Å resolution revealed that CO binds weakly to the nickel ion in the Ni–Fe active site of S77-HYB. These results suggest a positive correlation between O2 and CO tolerance in [NiFe]-hydrogenases.

AimsL-type amino acid transporter 1 (LAT1) is a major Na+-independent neutral amino acid transporter, forming a complex with CD98hc. The aim of this study is to investigate the significance of LAT1 and CD98hc in invasive breast cancer.MethodsLAT1 and CD98hc expression was immunohistochemically assessed in 280 invasive breast cancers and analysed for association with clinicopathological features.ResultsHigh levels of LAT1 and CD98hc were observed in triple-negative breast cancers (TNBCs) possessing negative immunoreactivity with oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, compared with non-TNBCs (NTNBCs), and were associated with lymph-node metastasis and higher nuclear grade. The high-LAT1-expression group showed a poor prognosis in NTNBC and TNBC, however, high-CD98hc-expression group showed a poor prognosis only in NTNBC. LAT1 and CD98hc expression could be the prognostic factors in univariate analyses, but not in multivariate analyses. Further, we found that invasive tumour components showed higher LAT1 and CD98hc expression than non-invasive tumour components.ConclusionsLAT1 and CD98hc may possess prognostic values in invasive breast cancer. LAT1 may be linked with cancer cell activities and disease progression in breast cancer.

Background We examined the vascularity of mammary Paget disease histologically to confirm the increased blood flow observed previously by clinical imaging. The relationships among blood vessel density (BVD), histopathological parameters of blood flow in the nipple, and the expression of angiogenic factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) were examined. Methods We calculated the average CD34-positive BVD and podoplanin (D2–40)-positive lymphatic vessel density (LVD) and the proportion of proliferating of endothelial cells in 14 Paget disease, 3 dermatitis biopsy, and 14 age-matched control cases. As a parameter related to blood flow in the nipple, the total CD34-positive blood vessel lumen area relative to the entire nipple area was measured in each Paget disease and control case using an automated image analysis system. Immunohistochemical expression of bFGF and VEGFA in Paget cells was also examined. Results The average BVD and LVD were significantly higher in the Paget disease cases than in the dermatitis (p = 0.003) and control (p p = 0.003). The average BVD was correlated with the average LVD (r = 0.734, p r = 0.692, p p = 0.003) and control (p r = 0.818, p r = 0.503, p = 0.006). Conclusion These results provide direct histopathological evidence of a marked increase in nipple blood flow in Paget disease detected by clinical imaging. bFGF is considered to play a pivotal role in angiogenesis in mammary Paget disease.

Hydrogenases control the proton concentration in cells, which is an essential function for hydrogen metabolism in several microorganisms. Some [NiFe]-hydrogenases are catalytically active under air...

The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) fromThermotoga maritima(Tm) couples the endergonic reduction of protons (H+) by dihydronicotinamide adenine dinucleotide (NADH) (∆G0 ≈ 18 kJ mol−1) to the exergonic reduction of H+by reduced ferredoxin (Fdred) (∆G0 ≈ − 16 kJ mol−1). The specific mechanism by which HydABC functions is not understood. In the current study, we describe the biochemical and spectroscopic characterization ofTmHydABC recombinantly produced inEscherichia coliand artificially maturated with a synthetic diiron cofactor. We found thatTmHydABC catalyzed the hydrogen (H2)-dependent reduction of nicotinamide adenine dinucleotide (NAD+) in the presence of oxidized ferredoxin (Fdox) at a rate of ≈17 μmol NADH min−1 mg−1. Our data suggest that only one flavin is present in the enzyme and is not likely to be the site of electron bifurcation. FTIR and EPR spectroscopy, as well as FTIR spectroelectrochemistry, demonstrated that the active site for H2conversion, the H-cluster, inTmHydABC behaves essentially the same as in prototypical [FeFe] hydrogenases, and is most likely also not the site of electron bifurcation. The implications of these results are discussed with respect to the current hypotheses on the electron bifurcation mechanism of [FeFe] hydrogenases. Overall, the results provide insight into the electron-bifurcating mechanism and present a well-defined system for further investigations of this fascinating class of [FeFe] hydrogenases.Graphic abstract

Electron bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate-potential electron donor are split so that one is sent along a high-potential pathway to a high-potential acceptor and the other is sent along a low-potential pathway to a low-potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognized, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent ‘halves’ each made of two strongly interacting HydABC heterotrimers connected via a [4Fe–4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron–sulfur cluster domain: a ‘closed bridge’ and an ‘open bridge’ conformation, where a Zn2+ site may act as a ‘hinge’ allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD+ reduction/NADH oxidation site.

Sulfur isotope fractionation resulting from microbial sulfate reduction (MSR) provides some of the earliest evidence of life, and secular variations in fractionation values reflect changes in biogeochemical cycles. Here we determine the sulfur isotope effect of the enzyme adenosine phosphosulfate reductase (Apr), which is present in all known organisms conducting MSR and catalyzes the first reductive step in the pathway and reinterpret the sedimentary sulfur isotope record over geological time. Small fractionations may be attributed to low sulfate concentrations and/or high respiration rates, whereas fractionations greater than that of Apr require a low chemical potential at that metabolic step. Since Archean sediments lack fractionation exceeding the Apr value of 20‰, they are indicative of sulfate reducers having had access to ample electron donors to drive their metabolisms. Large fractionations in post-Archean sediments are congruent with a decline of favorable electron donors as aerobic and other high potential metabolic competitors evolved., Changes in S-isotope ratios over time provide clues to understanding the co-evolution of Earth and its biosphere. Here the authors determine the isotope effect of the first reductive enzyme in the sulfate respiration pathway and reinterpret sedimentary S-isotope records based on this biochemical constraint.

CASE REPORT, Fanconi syndrome (FS) is a rare disease characterized by a dysfunction of the proximal renal tubules. Drug-induced FS may be neglected or misdiagnosed, and most reported cases have occurred due to alkylating agents and platinum compounds. We encountered drug-induced FS in a patient with breast cancer using zoledronate. A 58-year-old woman underwent mastectomy for breast cancer at age 54. Two years later, bone metastases were detected and treated using a combination therapy of anastrozole and zoledronate. After 17 months, everolimus, exemestane, and zoledronate were prescribed. Then, she experienced hypocalcemia and proximal renal tubular dysfunction. FS induced by zoledronate was diagnosed, and zoledronate was immediately discontinued. This resulted in immediate and sustained improvement in renal function. In the past, three cases of zoledronate-induced FS were reported. All patients were prescribed zoledronate in combination with molecularly targeted drugs. Close monitoring of proximal tubular function is recommended for such patients.

ORIGINAL ARTICLE, Introduction: Tertiary hyperparathyroidism (THPT) refers to the development of hypercalcemia and hyperparathyroidism following a prolonged period of secondary hyperparathyroidism. THPT is most commonly observed in patients after successful kidney transplantation (KTx). The treatment guidelines for post-KTx THPT are still unclear; thus, this study aimed to examine patients who underwent parathyroidectomy (PTx) for THPT and to assess the effects of PTx on long-term graft function. Methods: We performed a retrospective study of patients who underwent PTx for THPT between 2009 and 2017 at our institution. Levels of serum calcium (Ca) and intact-parathyroid hormone (i-PTH) were measured before and after PTx (one day, one month, three months, six months, one year, and three years) to evaluate the effects of PTx. Estimated glomerular filtration rate (eGFR) was measured to investigate the effect on graft function after PTx. Results: Seven males and six females were included in this study. The post-PTx levels of serum Ca and i-PTH significantly decreased at one day compared with the pre-PTx levels (p < .001). The mean eGFR level decreased at three and six months after PTx. However, at one year post-PTx, the eGFR level had improved to the pre-PTx level. There was no recurrence or loss of graft function during a 58-month follow-up period. Conclusions: After PTx, the levels of serum Ca and i-PTH significantly improved. The level of eGFR tended to decrease within the first year. We recommend that clinicians follow-up closely with PTx patients during the first year post-PTx due to unstable renal function.

Because the production of tumor‐associated antibodies (TAA) is a humoral immune response in cancer patients, serum autoantibodies may be detected even in patients with early‐stage tumors. Seventeen recombinant proteins with tags in Escherichia coli (p53, RalA, p90, NY‐ESO‐1, HSP70, c‐myc, galectin‐1, Sui1, KN‐HN‐1, HSP40, PrxVI, p62, cyclin B1, HCC‐22‐5, annexin II, HCA25a, and HER2) were applied as capturing antigens in sandwich ELISA to measure serum IgG levels. Sera from 73 healthy donors and 386 patients with breast cancer, including 182 stage 0/I patients, were evaluated using cutoff values for each TAA equal to the mean +3 SD of the serum levels of healthy controls. The positive TAA rates were relatively high for p53 (10%) and RalA (10%). The positive rates of all TAA of stage 0/I were similar to those of all patients. Even in the stage 0/I patients, 24% showed that two or more TAA were positive, and the positive rate of a five‐TAA combination assay was 37%. The positivity rate was significantly higher for the non–luminal type than for the luminal type (P = .003). Logistic analysis showed that seropositivity (positive for one or more TAA) in breast cancer patients was independent from any TNM factor or disease stage and was significantly associated with histological grade in the multivariate analysis (P = .007). TAA in breast cancer patients may be useful for early detection. However, seropositivity of breast cancer reflects the tumor characteristics but not the disease stage., We performed 17 tumor‐associated antibody (TAA) panel assays in 386 breast cancer patients’ serum. The tumor factor associated with the tumorigenesis regulated a humoral immune reaction that resulted in a diversity of TAA seropositivity.

Triple-negative breast cancer (TNBC) lacks an effective treatment target and is usually treated with chemotherapy. Treatment of older patients with TNBC, however, should be decided carefully because of the side effects of chemotherapy in this population. Some forms of TNBC are associated with a favorable prognosis and do not require chemotherapy. To optimize the treatment of older patients with TNBC, it is important to know the clinicopathological characteristics and a prognostic marker. In this study, classic clinicopathological factors, immunohistochemical characteristics (androgen receptor [AR], cytokeratin 5/6 [CK5/6], epidermal growth factor receptor), tumor-infiltrating lymphocytes (TILs), and the clinical outcome based on the status of each biomarker were compared among a consecutive series of female patients with TNBC aged ≥75 years (n = 75) and among those aged 55–64 years matched for the pathological stage (n = 47) who underwent surgery without neoadjuvant therapy. TNBC with special histology (particularly apocrine carcinoma, pleomorphic invasive lobular carcinoma, and metaplastic carcinoma) was more frequent in the older group than in the younger group (35/75, 57% versus 11/47, 23%, P = 0.010). The AR positivity rate was higher in older patients than in younger patients, whereas TILs and CK5/6 exhibited the opposite results. In multivariate analyses, AR positivity was an independent predictor of a favorable outcome in older patients (lower recurrence rate), whereas the high level of TILs was favorable in younger patients (lower recurrence and mortality rates). AR positivity or apocrine morphology was frequent and predicts a favorable clinical outcome in older patients with TNBC, suggesting the importance of AR examination in this population.

Patient: Male, 70-year-old Final Diagnosis: Esophageal cancer Symptoms: Muscle pain • weaknes of lower limbs Medication: — Clinical Procedure: Biopsy of the skin Specialty: Rheumatology Objective: Unknown ethiology Background: Dermatomyositis (DM) is occasionally associated with malignancy, which is so-called cancer-associated myositis. The cancer screening in patients with dermatomyositis is an important clinical issue. That is because malignant disease underlying dermatomyositis is potentially life-threatening. Transcriptional intermediary factor 1γ (TIF1γ) antibodies (anti-TIF1γ Abs) are one of the myositis-specific autoantibodies, which are investigated as potential predictors of malignancy in patients with dermatomyositis. However, the etiology of anti-TIF1γ Abs generations in various cancer patients is not known. Case Report: A 70-year-old male patient was admitted for muscle pain and weakness in both legs. Erythematous on the face, eruption, and a V sign were also observed. Laboratory tests showed the elevation of creatine kinase, myoglobin, and aldolase. He was diagnosed as dermatomyositis. Cancer screening was performed, and esophageal cancer was detected in the lower esophagus. Despite the symptoms of dermatomyositis were improved with steroid, methotrexate, and radical esophagectomy, he died with esophageal cancer 3 years after the onset of dermatomyositis. TIF1γ is frequently overexpressed in cancer tissues. Therefore, some cancer patients without dermatomyositis could be positive for anti-TIF1γ Abs. We retrospectively analyzed anti-TIF1γ Abs in cancer patients (n=131). However, the screening of anti-TIF1γ Abs in cancer patients without dermatomyositis (n=130) showed there were no seropositive patients. Only this cancer-associated myositis patient was positive for anti-TIF1γ Abs. Conclusions: Our result suggested the generation of anti-TIF1γ Abs is specific for cancer associated myositis, not for tumorigenesis.

Case Report, Serum p53 antibody (s-p53-Abs) is induced by p53 overexpression in cancer cells. Therefore, residual cancer cells may be the cause of seropositivity even after radical surgery. Only few reports have evaluated perioperative changes and long-term monitoring of s-p53-Abs titers in patients with breast cancer. This case report presents long-term monitoring of s-p53-Abs titers in a patient with locally advanced breast cancer who developed recurrence in the central nervous system after radical surgery. The s-p53-Abs titer was monitored for 3 years during neoadjuvant chemotherapy and radical surgery in a 38-year-old woman with clinical stage IIB (T2N1M0) breast cancer. The levels of both the carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3) were within the normal range, but s-p53-Abs test result (54.8 U/ml) was positive. Neither computed tomography nor bone scintigraphy showed any distant metastasis before treatment. The patient received neoadjuvant chemotherapy (epirubicin, 100 mg/m2; cyclophosphamide, 500 mg/m2; and fluorouracil, 500 mg/m2; followed by docetaxel, 75 mg/m2). One and a half year after surgery, s-p53-Abs results remained positive (5.66 U/ml), and brain metastasis was found. Despite resection for brain metastasis, s-p53-Abs titer remained high (5.88 U/ml), and carcinomatous meningitis was found. During the entire clinical course, although s-p53-Abs titers reflected the presence of residual cancer cells, both CEA and CA15-3 results were negative. The perioperative s-p53-Abs titer might be a useful marker for monitoring residual cancer cells in a patient with breast cancer. Recurrence in the central nervous system might be detected using s-p53-Abs monitoring.

Original Article, Background: Ras-like GTPases, RalA, and RalB, are members of the Ras superfamily of small GTPases. Their aberrant activation is a major cause of human tumorigenesis induced by oncogenic Ras. Serum anti-RalA antibodies (s-RalA-Abs) are induced in patients with esophageal carcinoma, but there have been no reports regarding their presence in patients with breast cancer. Methods: Serum samples from 100 patients with breast cancer and 73 healthy individuals were analyzed using an enzyme-linked immunosorbent assay system specifically developed for s-RalA-Abs. Positive rates of s-RalA-Abs and serum p53 antibodies (s-p53-Abs) were evaluated for two cut-off optical density values of 0.26 and 0.33 (i.e., the control mean+2 and 3 standard deviations), respectively. Results: Overall positive rates for s-RalA-Abs at the two cut-offs were 24% and 11%, respectively, with no statistically significant differences between stage I and stage II cancers. The positive rate increased significantly in a combination assay of s-p53-Abs with s-RalA-Abs, and s-RalA-Abs was shown to be inversely associated with s-p53-Abs. Conclusions: s-RalA-Abs in combination with s-p53-Abs may be a candidate serum antibody marker for breast cancer.

Eight years after surgical treatment for breast cancer, serum p53 antibody levels suddenly increased in a 67-year-old woman without recurrent signs of cancer. Early esophageal squamous cell carcinoma was detected by endoscopic screening. After endoscopic submucosal dissection was completed, serum p53 antibody levels decreased to be in the normal range. Serum p53 antibody monitoring can be helpful for early detection of not only recurrence of primary carcinoma but also development of metachronous carcinoma. This case is the first to monitor the changing titers of serum p53 antibody at the early phase of esophageal cancer progression into lamina propria mucosae until the disappearance of tumor cells.

Iron-sulfur clusters form one of the largest and most diverse classes of enzyme cofactors in nature. They may serve as structural factors, form electron transfer chains between active sites and external redox partners, or form components of enzyme active sites. Their specific role is a consequence of the cluster type and the surrounding protein environment. The relative effects of these factors are not completely understood, and it is not yet possible to predict the properties of iron-sulfur clusters based on amino acid sequences or rationally tune their properties to generate proteins with new desirable functions. Here, we generate mutations in a [2Fe-2S] cluster protein, the TmHydC subunit of the trimeric [FeFe]-hydrogenase from Thermotoga maritima, to study the factors that affect its redox potential. Saturation mutagenesis of Val131 was used to tune the redox potential over a 135 mV range and revealed that cluster redox potential and electronic properties correlate with amino acid hydrophobicity and the ability to form hydrogen bonds to the cluster. Proline scanning mutagenesis between pairs of ligating cysteines was used to remove backbone amide hydrogen bonds to the cluster and decrease the redox potential by up to 132 mV, without large structural changes in most cases. However, substitution of Gly83 with proline caused a change of HydC to a [4Fe-4S] cluster protein with a redox potential of -526 mV. Together, these results confirm the importance of hydrogen bonding in tuning cluster redox potentials and demonstrate the versatility of iron-sulfur cluster protein folds at binding different types of clusters.

Case Report, Serum p53 antibody (s-p53-Ab) is the most recently developed biomarker for breast cancer. Only a few studies have evaluated perioperative s-p53-Ab titers in patients with breast cancer. S-p53-Ab titers were monitored for over 4 years, during the perioperative period and after surgery, in a 75-year-old woman with clinical stage IIA (T2N0M0) breast cancer. The results of screening tests were negative for cancer antigen 15-3, carcinoembryonic antigen (CEA), and National Cancer Center-Stomach-439; only s-p53-Ab level (10.1 U/ml) was positive preoperatively. S-p53-Ab titer remained positive (9.1 U/ml) after radical surgery. Pathologic analysis of surgically resected specimens showed a stage IIB tumor (pT2N1M0). Two years postoperatively, CEA level had increased to 5.9 ng/ml. At this time, liver metastases were detected by computed tomography. Neither chemotherapy nor hormone therapy were effective against this recurrence, and the patient died 3 years after surgery. Perioperative s-p53-Ab titer was a useful marker for long-term monitoring of residual cancer cells in a patient with stage IIA breast cancer.

Nitrophorins (NPs) catalyze the nitrite dismutation reaction that is unprecedented in ferriheme proteins. Despite progress in studying the reaction mechanism, fundamental issues regarding the correlation of the structural features with the nitrite dismutase activity of NPs remain elusive. On the other hand, it has been shown that the nitrite complexes of NPs are unique among those of the ferriheme proteins since some of their electron paramagnetic resonance (EPR) spectra show significant highly anisotropic low spin (HALS) signals with large gmax values over 3.2. The origin of HALS signals in ferriheme proteins or models is not well understood, especially in cases where axial ligands other than histidine are present. In this study several mutations were introduced in NP4. The related nitrite coordination and dismutation reaction were investigated. As a result, the EPR spectra of the NP-nitrite complexes were found to be tightly correlated with the extent of heme ruffling and protonation state of the proximal His ligand-dictated by an extended H-bonding network at the heme active site. Furthermore, it is established that the two factors are essential in determining the nitrite dismutase activity of NPs. These results may provide a valuable guide for identifying or designing novel heme proteins with similar activity.

Nitrophorins (NP) 1–7 are NO-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. The isoform NP7 displays peculiar properties, such as an abnormally high isoelectric point, the ability to bind negatively charged membranes, and a strong pH sensitivity of NO affinity. A unique trait of NP7 is the presence of Glu in position 27, which is occupied by Val in other NPs. Glu27 appears to be important for tuning the heme properties, but its influence on the pH-dependent NO release mechanism, which is assisted by a conformational change in the AB loop, remains unexplored. Here, in order to gain insight into the functional role of Glu27, we examine the effect of Glu27 → Val and Glu27 → Gln mutations on the ligand binding kinetics using CO as a model. The results reveal that annihilation of the negative charge of Glu27 upon mutation reduces the pH sensitivity of the ligand binding rate, a process that in turn depends on the ionization of Asp32. We propose that Glu27 exerts a through-space electrostatic action on Asp32, which shifts the pKa of the latter amino acid towards more acidic values thus reducing the pH sensitivity of the transition between open and closed states.

To clarify the profile of adverse events from endocrine therapies in older patients. We surveyed 15 subjective symptoms including hot flashes, sweating, knuckle stiffness, knee/shoulder joint pain, limb numbness, lethargy, forgetfulness, depressive state, irritated state, genital bleeding, leukorrhea increase, vaginal dryness, bone fracture, and weight gain by a questionnaire among 2044 patients over 55 years old (total number of answered sheets, 8875) and compared the results according to age (56–69 years old vs. ≥ 70 years old) and type of therapy (aromatase inhibitors (AIs) vs. selective estrogen receptor modulators (SERMs)). Among patients 56–69 years old, 6093 and 314 responses were from patients treated with AIs (1477 patients) and SERMs (123 patients), respectively, and 2292 and 176 responses were from those ≥ 70 years old treated with AIs (581 patients) and SERMs (51 patients), respectively. In patients ≥ 70 years old, sweating, knuckle stiffness, knee/shoulder joint pain, limb numbness, and lethargy were significantly more frequent/severe with AIs than with SERMs. In those aged 56–69, knuckle stiffness and vaginal dryness were significantly more frequent with AIs than with SERMs, but the opposite occurred for hot flashes, leukorrhea increase, genital bleeding, and weight gain. Among patients ≥ 70 years old, many symptoms were significantly more frequent/severe with AIs than with SERMs, compared with those aged 56–69, which suggests a difference in the profile of adverse events according to the type of endocrine therapy and the patient’s age. It is important to consider the benefits and risks of each treatment to optimize endocrine therapy for older patients.

Purpose Paget disease of the breast is a rare cancer that originates from the nipple–areolar complex. It is often overlooked and misdiagnosed as benign chronic eczema of the nipple. We aimed to retrospectively verify whether blood flow analysis using Doppler sonography was useful for detecting the presence of Paget disease. Methods In this retrospective study, 12 patients with pathologically proven unilateral nipple eczematous lesions (seven with Paget disease and five with simple dermatitis) were included. Nipple blood flow signal was observed using Doppler sonography, and the detected blood flow signals were quantified using digitally recorded images. Quantified blood flow ratio and pathologically examined capillary density were evaluated between affected and unaffected nipples. Findings of mammography, grayscale sonography, and contrast-enhanced magnetic resonance imaging (CE-MRI) were reviewed. Results In patients with Paget disease, Doppler effects in the affected nipple were more clearly visible than those in the unaffected nipple. These effects were sufficiently visible to identify Paget disease. No obvious effects were observed in the affected and unaffected nipples of simple dermatitis. The quantified blood flow ratio and pathologically examined capillary density were significantly higher for the Paget lesion than those for the non-Paget lesion. The sensitivity of CE-MRI and Doppler sonography was markedly correlated, revealing blood flow changes in the nipple lesions of Paget disease. Conclusion Doppler sonography visualized the proliferation of blood vessels in Paget lesions. The visualization of increased nipple blood flow using Doppler sonography is a simple and low-cost method that provides useful data for identifying Paget disease during routine medical care.

Triple negative breast cancer (TNBC) is an aggressive histological subtype with limited treatment options. While rationally-derived regimens are emerging from the results of recent gene profiling studies, TNBC is an angiogenesis-dependent malignancy, and antiangiogenic therapies have been examined energetically in this field. Antiangiogenic agents in combination with chemotherapeutic platinum compounds have shown some benefit in recent randomized control studies for the treatment of TNBC. These combinations could be more safe and efficacious in patients, when pharmacotherapy schedule is modified taking into account its tolerability.

Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H2 level of the cellular environment. These hydrogenases contain the hydrogenase domain with distinct modifications in the active site pocket, followed by a Per-Arnt-Sim (PAS) domain. As yet, neither the physiological function nor the biochemical or spectroscopic properties of these enzymes have been explored. Here, we present the characterization of an artificially maturated, putative sensory [FeFe] hydrogenase from Thermotoga maritima (HydS). This enzyme shows lower hydrogen conversion activity than prototypical [FeFe] hydrogenases and a reduced inhibition by CO. Using FTIR spectroelectrochemistry and EPR spectroscopy, three redox states of the active site were identified. The spectroscopic signatures of the most oxidized state closely resemble those of the Hox state from the prototypical [FeFe] hydrogenases, while the FTIR spectra of both singly and doubly reduced states show large differences. The FTIR bands of both the reduced states are strongly red-shifted relative to the Hox state, indicating reduction at the diiron site, but with retention of the bridging CO ligand. The unique functional and spectroscopic features of HydS are discussed with regard to the possible role of altered amino acid residues influencing the electronic properties of the H-cluster.

A sub-angstrom-resolution X-ray crystal structure of [NiFe] hydrogenase, with direct detection of the products of the heterolytic splitting of dihydrogen into a hydride bridging the Ni and Fe and a proton attached to the sulphur of a cysteine ligand. [NiFe] hydrogenases use nickel and iron to catalyse the reversible oxidation of molecular hydrogen. They are the focus of much research worldwide because of their potential in biotechnology and in serving as natural models for biomimetic catalysts in the energy sector for hydrogen production and conversion. In protein X-ray crystallography it is notoriously difficult to detect hydrogens, a particularly significant problem in hydrogenases where hydrogens are involved directly in the reaction. Hideaki Ogata et al. have succeeded in obtaining a sub-angstrom resolution X-ray crystal structure of [NiFe] hydrogenase leading to detection of most of the hydrogens even close to the metal ions. Using their technique authors were able to detect the products of the heterolytic splitting of dihydrogen: a hydride that bridges the Ni and Fe ions, and a proton that is attached to the sulfur of a cysteine ligand. The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst1. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein2,3,4,5,6. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-angstrom resolution7. Here we report the crystal structure of a standard [NiFe] hydrogenase (∼91.3 kDa molecular mass) at 0.89 A resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H−) bridging the Ni and Fe and a proton (H+) attached to the sulphur of a cysteine ligand. The Ni–H− and Fe–H− bond lengths are 1.58 A and 1.78A, respectively. Furthermore, we can assign the Fe–CO and Fe–CN− ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis.