Detection of Functional Significance of Coronary Stenoses Using Dynamic 13N-Ammonia Stress-PET/CT with Absolute Values of Myocardial Blood Flow and Coronary Flow Reserve

Objectives. The aim of the study was to compare the values of myocardial blood flow (MBF) at stress, MBF at rest and coronary flow reserve (CFR) obtained by 13Nammonia stress-PET/CT in patients with various degrees of coronary stenosis and in healthy patients. And thus to estimate the possible contribution of the stress-PET/CT quantitative data to the detection of functionally significant coronary stenoses in patients with coronary artery disease (CAD). Materials and methods. 63 patients (mean age 64±9 years) with known CAD underwent dynamic 13N-ammonia stress-PET/CT followed by calculation of MBF both at stress and at rest in absolute units and CFR. We compared quantitative values in two groups of patients with coronary artery stenosis: 1) ≥75% (n = 36) and 2) <75% (n = 27) confirmed by invasive coronary angiography and in group of healthy patients (n = 11). Results. MBF at stress was significantly lower in group with ≥75% diameter stenoses (median 1,44 [1,21; 1,85] mL/min per g) compared with group with <75% diameter stenoses (2,42 [1,75; 2,89] mL/min/g) and the normal group (2,54 [2,31; 2,86] mL/min/g), (p <0,001). There was no reliable difference in MBF at rest between the three groups (p = NS). CFR was significantly lower in the group of patients with severe ≥75% stenoses (1,85 [1,54; 2,31]) in comparison with patients group with stenoses of intermediate <75% severity (2,73 [2,19; 3,21]), and also in comparison with the normal group (3,12 [2,75; 3,23]), (p <0,001). Conclusion. The values of MBF at stress and CFR are significantly lower in patients with severe coronary arteries stenoses comparing with the group of patients with mild and moderate stenoses. The value of MBF at rest used independently has no diagnostic utility for detection of functional significance of coronary artery stenoses. Keywords: myocardial blood flow, coronary flow reserve, PET/CT, 13N-ammonia, coronary stenosis

11C-Choline Pet/Ct in the Detection of Prostate Cancer Relapse in Patients After Radical Treatment With Psa Level < 10 Ng/Ml

Purpose: To evaluate the usefulness of 11C-Choline PET/CT in the detection of recurrent prostate cancer (PCa) in patients with biochemical relapse after radical treatment. Materials and methods: This retrospective study included 217 PCa patients who underwent 11C-Choline PET/CT in the Department of Nuclear Medicine of Bakoulev Scientific Centre. All patients had biochemical relapse 3±2 years after radical treatment for locally advanced PCa (T1–3 N0–1 M0): radical prostatectomy (n = 159) and radiation therapy (n = 58). The mean PSA value in the group was 2.1±2.5 (0.2–9.7) ng/ml, median – 1.9 ng/ml. Imaging was performed on PET/CT scanner (Biograph-64, Siemens) 10 min after injection of 11C-Choline (400–550 Mbq). Results: Overall, according to 11C-Choline PET/CT results PCa relapse was detected in 56% (121/217) of cases: in 50% (80/159) after radical prostatectomy and in 71% (41/58) after radiation therapy. The mean PSA value in PET-positive cases was 3.1±2.2 (0.2–9.7) ng/ml, while in PETnegative cases – 1.8±1.7 (0.2–4.6) ng/ml. The majority – 68% (65/96) patients with PET-negative scan had low PSA levels (&lt; 2 ng/ml). PET/CT results were positive in 43% (50/115) patients with PSA of &lt; 2 ng/ml, in 63% (45/72) with PSA of 2 to 5 ng/ml, and in 87% (26/30) with PSA of &gt; 5 ng/ml. Local relapse was detected in 51% (62/121) patients, distant metastases – in 28% (34/121) of cases, both local and distant metastases – in 21% (25/121) of cases. Lymph node metastases were detected in 38% (86/217) of all patients included in the analysis, of which 28% (24/86) had lesions in lymph node of normal size (median 7 mm). Of all PET-positive patients bone metastases were detected in 33% (40/121), of which 60% (24/40) had isolated skeletal involvement. Importantly, that 27% (11/40) of PETpositive patients with bone metastases had no structural abnormalities on CT images (CT-negative cases), corresponding to isolated involvement of bone marrow. And half of these CT-negative patients (5/11) had single lesions. The mean PSA value in patients with bone metastases was 5.0±3.7 (0.4–9.1) ng/ml, median – 3.8 ng/ml. According to 11C-Choline PET/CT results oligometastatic PCa recurrence was revealed in 38% (82/217) of all patients, of which 62% (51/82) had local relapse only. Distant oligometastatic lesions were detected in 38% (31/82), of which 13% (4/31) were presented by normal-size lymph nodes and 19% (6/31) – by early bone marrow metastases. 48% (58/121) of PET-positive results were confirmed by data of repeated PET/CT examinations. Conclusion: 11C-Choline PET/CT has been shown to be a single noninvasive accurate technique for detection of recurrent PCa in patients with rising PSA after radical treatment, which allows to differentiate patients with local and distant metastases in one study, as well as identify oligometastatic process, and therefore was useful in determining the further personalized therapeutic approach. Keywords: prostate cancer, PET/CT, 11C-Choline, biochemical recurrence, PSA

Высокие значения исходных объемных ПЭТ-биомаркеров как предикторы неблагоприятного прогноза классической лимфомы Ходжкина

Purpose: To analyse the prognostic value of the initial volumetric PET biomarkers – the total metabolic tumor volume (MTV) and the total lesion glycolysis (TLG) – in classic Hodgkin's lymphoma (cHL) and determine their optimal threshold values for prognosis.Material and methods. This retrospective study included 62 cHL patients with different stages who underwent staging with 18F-FGD PET/CT. The follow-up period was from 6 to 61 months after the baseline PET/CT, 41 patients remained in remission, 10 patients had refractory course, 11 relapsed. The examinations were processed with automatic (multi-foci segmentation – MFS) method to obtain MTV and TLG using two fixed absolute thresholds (SUVmax ≥ 2.5 and SUVmax ≥ 4.0) and one relative threshold (41% of SUVmax).Results. In subgroups with disease remission (n = 41) and refractory course or relapse (n = 21), statistically significant differences between MTV and TLG with the two thresholds were found – SUVmax ≥ 2.5 and 41% of SUVmax (p &lt; 0.05). When using threshold of SUVmax ≥ 4.0 statistically differences between the mean of MTV and TLG were no detected.Univariate analysis revealed correlation between progression-free survival and volumetric PET biomarkers (MTV and TLG) with three thresholds (SUVmax ≥ 2.5, SUVmax ≥ 4.0, and 41% of SUVmax).Conclusion. In cHL high values of initial volumetric PET biomarkers – MTV and TLG – calculated with three thresholds (SUVmax ≥ 2.5, SUVmax ≥ 4.0, and 41% of SUVmax) are associated with unfavourable prognosis – a high probability of refractory disease course or relapse.The optimal prognostic thresholds values of MTV and TLG in the analysed group were determined respectively: SUVmax ≥ 2.5 – 204 cm3 and 961, at 41% of SUVmax – 105 cm3 and 620.Цель исследования: анализ прогностического значения исходных объемных ПЭТ-биомаркеров – общего метаболического объема опухоли (MTV) и общего уровня гликолиза (TLG) – при классической лимфоме Ходжкина (кЛХ) и определение их оптимальных пороговых прогностических значений.Материал и методы. Ретроспективно проанализированы результаты ПЭТ/КТ-исследований 62 пациентов с впервые выявленной кЛХ. Период наблюдения составил от 6 до 61 мес после исходного ПЭТ/ КТ-исследования. В течение указанного периода у 41 (66%) пациента сохранялась ремиссия заболевания, у 10 (16%) пациентов диагностировано рефрактерное течение, у 11 (18%) – рецидив. Показатели MTV и TLG рассчитывались автоматическим методом с использованием для каждого трех значений отсечки фона: двух абсолютных – SUVmax ≥ 2,5 и SUVmax ≥ 4,0 и одного относительного – 41% от SUVmax.Результаты. Для расчета пороговых прогностических значений MTV и TLG группа была разделена на две подгруппы: 1-я – с ремиссией заболевания в течение периода наблюдения (n = 41); 2-я – с рефрактерным или рецидивирующим течением (n = 21). В указанных подгруппах были выявлены статистически значимые различия между показателями MTV и TLG при двух используемых значениях отсечки фона – SUVmax ≥ 2,5 и 41% от SUVmax (р &lt; 0,05). При использовании отсечки фона – SUVmax ≥ 4,0 – значимых различий между указанными показателями в подгруппах получено не было.При однофакторном анализе параметры MTV и TLG с использованием трех пороговых значений отсечки фона (SUVmax ≥ 2,5, SUVmax ≥ 4,0 и 41% от SUVmax) коррелировали с выживаемостью без прогрессирования.Заключение. При кЛХ высокие значения исходных объемных ПЭТ-биомаркеров MTV и TLG, рассчитанные с использованием различных порогов отсечки фона (SUVmax ≥ 2,5, SUVmax ≥ 4,0 и 41% от SUVmax), ассоциируются с неблагоприятным прогнозом – высокой вероятностью рефрактерного течения заболевания или возникновения рецидива.Оптимальные прогностические пороговые значения MTV и TLG в анализируемой группе составили: при SUVmax ≥ 2,5 – 204 см3 и 961, при 41% от SUVmax – 105 см3 и 620 соответственно

ПЭТ / КТ с 11С-холином в диагностике рецидива рака предстательной железы у пациентов с биохимическим прогрессированием

Objective. To evaluate the diagnostic impact of 11C–Choline PET / CT in the detection of recurrent prostate cancer (PCa) in patients with biochemical relapse after radical prostatectomy and to assess the correlation between PSA levels and PET / CT detection rate of PCa relapse.Subjects and methods. 85 patients with biochemical relapse (mean PSA 3.51 ± 3.87 ng / ml) after radical prostatectomy (n = 64) and radiotherapy (n = 21) underwent 11C–Choline PET / CT. According to PSA level, patients were divided into three groups: 2 ng / ml, 2 to 9 ng / ml and &gt; 9 ng / ml.Results. Overall, 11C–Choline PET / CT detected PCa relapse in 33 of 85 patients (39 %). The mean PSA value in PET-positive patients was 5.78 ± 4.95 (0.22–17.80) ng / ml, while in PET-negative patients – 1.43 ± 1.08 (0.28–4.57) ng / ml. Positive PET / CT results were obtained in 9 of 40 patients (22 %) with PSA of &lt; 2 ng / ml, in 17 of 38 patients (45 %) with PSA of 2 to 9 ng / ml, and in 7 of 7 patients (100 %) with PSA of &gt; 9 ng / ml. Local relapse was detected in 42 % (14 / 33) patients. Both local and distant metastases were diagnosed in 39 % (13 / 33) cases. Distant relapsewas identified in 19 % (6 / 33) cases. PET / CT allowed to assess the efficacy of treatment in 26 % (12 / 47) PET-negative patients under hormone therapy at the scan time. However, PET / CT wasn’t able to localize the site of PCa recurrence in these hormone-ensitive patients what might have affected the overall detection rate.Conclusion. 1) 11C–Choline PET / CT was able to detect and correctly identify the site of PCa relapse in 39 % cases and therefore was useful in determining the further therapeutic approach. 2) Our data confirmed the strong correlation between PSA levels and 11C–Choline PET / CT detection rate of PCa relapse (r = 0.9; p &lt; 0.001). 3) 11C–Choline PET / CT has limited utility in localizing the site of PCa recurrence in some patients under hormone therapy.Цель исследования – изучить возможности позитронно-эмиссионной томографии (ПЭТ), совмещенной с компьютерной томографией (ПЭТ / КТ) с 11С-холином в ранней диагностике прогрессирования рака предстательной железы (РПЖ) у больных после радикального лечения. Определить наличие корреляции между уровнем простатспецифического антигена (ПСА) и частотой выявления рецидива РПЖ при ПЭТ / КТ с 11С-холином.Материалы и методы. Проанализированы результаты 85 ПЭТ / КТ- исследований с 11С-холином у больных с биохимическим рецидивом РПЖ после различных видов лечения: радикальной простатэктомии (n = 64) и лучевой терапии (n = 21). В зависимости от уровня ПСА пациенты были разделены на 3 группы: &lt; 2,0 нг / мл, 2,0–9,0 нг / мл и &gt; 9,0 нг / мл. Среднее значение ПСА на момент исследования составляло 3,51 ± 3,87 (0,22–17,80) нг / мл.Результаты. По результатам ПЭТ / КТ рецидив РПЖ был диагностирован в 39 % (33 / 85) случаев. Средний уровень ПСА у ПЭТ-положительных больных составил 5,78 ± 4,95 (0,22–17,80) нг / мл и был значимо выше, чем у ПЭТ-отрицательных: 1,43 ± 1,08 (0,28–4,57) нг / мл. Выявлена прямая корреляция между частотой диагностики рецидива РПЖ по данным ПЭТ / КТ и уровнем ПСА: у больных с уровнем ПСА &lt; 2 нг / мл ПЭТ-положительные результаты получены в 22 % (9 / 40), при ПСА 2–9 нг / мл – в 45 % (17 / 38) и при ПСА &gt;9 нг / мл – в 100 % (7 / 7) случаев. Локальный и сочетанный рецидивы диагностированы с одинаковой частотой. Местный рецидив выявлен в 42 % (14 / 33) случаях, локальный рецидив в сочетании с отдаленными метастазами – в 39 % (13 / 33), только отдаленные метастазы – в 19 % (6 / 33) случаев. В группе ПЭТ-отрицательных больных 26 % (12 / 47) исследований проводили на фоне гормонотерапии (ГТ) в короткие сроки (в течение 7 дней) после введения лекарственных препаратов, которые в дальнейшем были расценены как ложноотрицательные. Получение ПЭТ-отрицательных результатов у данных больных, вероятнее всего, свидетельствовало о снижении метаболической активности процесса на фоне проводимой ГТ, что не позволяло определить локализацию рецидивного процесса.Выводы. 1) Результаты ПЭТ / КТ подтвердили рецидив заболевания, позволили определить его локализацию и распространенность у 39 % пациентов, что имело принципиальное значение для последующего лечения. 2) Выявлена прямая сильная корреляция между частотой выявления рецидива РПЖ при ПЭТ / КТ с 11С-холином и уровнем ПСА (r = 0,9; р &lt; 0,001). 3) Выполнение ПЭТ / КТ в короткие сроки после введения гормональных препаратов не позволяет определить локализацию рецидива РПЖ

Optimisation of Over-Expression in E. coli and Biophysical Characterisation of Human Membrane Protein Synaptogyrin 1

Progress in functional and structural studies of integral membrane proteins (IMPs) is lacking behind their soluble counterparts due to the great challenge in producing stable and homogeneous IMPs. Low natural abundance, toxicity when over-expressed and potential lipid requirements of IMPs are only a few reasons for the limited progress. Here, we describe an optimised workflow for the recombinant over-expression of the human tetraspan vesicle protein (TVP) synaptogyrin in Escherichia coli and its biophysical characterisation. TVPs are ubiquitous and abundant components of vesicles. They are believed to be involved in various aspects of the synaptic vesicle cycle, including vesicle biogenesis, exocytosis and endocytotic recycling. Even though TVPs are found in most cell types, high-resolution structural information for this class of membrane proteins is still missing. The optimisation of the N-terminal sequence of the gene together with the usage of the recently developed Lemo21(DE3) strain which allows the balancing of the translation with the membrane insertion rate led to a 50-fold increased expression rate compared to the classical BL21(DE3) strain. The protein was soluble and stable in a variety of mild detergents and multiple biophysical methods confirmed the folded state of the protein. Crosslinking experiments suggest an oligomeric architecture of at least four subunits. The protein stability is significantly improved in the presence of cholesteryl hemisuccinate as judged by differential light scattering. The approach described here can easily be adapted to other eukaryotic IMPs

The Crystal Structure of the Human Co-Chaperone P58IPK

P58IPK is one of the endoplasmic reticulum- (ER-) localised DnaJ (ERdj) proteins which interact with the chaperone BiP, the mammalian ER ortholog of Hsp70, and are thought to contribute to the specificity and regulation of its diverse functions. P58IPK, expression of which is upregulated in response to ER stress, has been suggested to act as a co-chaperone, binding un- or misfolded proteins and delivering them to BiP. In order to give further insights into the functions of P58IPK, and the regulation of BiP by ERdj proteins, we have determined the crystal structure of human P58IPK to 3.0 Å resolution using a combination of molecular replacement and single wavelength anomalous diffraction. The structure shows the human P58IPK monomer to have a very elongated overall shape. In addition to the conserved J domain, P58IPK contains nine N-terminal tetratricopeptide repeat motifs, divided into three subdomains of three motifs each. The J domain is attached to the C-terminal end via a flexible linker, and the structure shows the conserved Hsp70-binding histidine-proline-aspartate (HPD) motif to be situated on the very edge of the elongated protein, 100 Å from the putative binding site for unfolded protein substrates. The residues that comprise the surface surrounding the HPD motif are highly conserved in P58IPK from other organisms but more varied between the human ERdj proteins, supporting the view that their regulation of different BiP functions is facilitated by differences in BiP-binding

Structure of nitrilotriacetate monooxygenase component B from Mycobacterium thermoresistibile

The 1.6 Å resolution crystal structure of nitrilotriacetate monooxygenase component B (NTA-MoB) from M. thermoresistibile is presented, revealing a highly conserved C-terminal tail that may modulate the activity of NTA-MoB in mycobacteria

Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region.Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 A resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs.The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics

SAD phasing using iodide ions in a high-throughput structural genomics environment

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) focuses on the structure elucidation of potential drug targets from class A, B, and C infectious disease organisms. Many SSGCID targets are selected because they have homologs in other organisms that are validated drug targets with known structures. Thus, many SSGCID targets are expected to be solved by molecular replacement (MR), and reflective of this, all proteins are expressed in native form. However, many community request targets do not have homologs with known structures and not all internally selected targets readily solve by MR, necessitating experimental phase determination. We have adopted the use of iodide ion soaks and single wavelength anomalous dispersion (SAD) experiments as our primary method for de novo phasing. This method uses existing native crystals and in house data collection, resulting in rapid, low cost structure determination. Iodide ions are non-toxic and soluble at molar concentrations, facilitating binding at numerous hydrophobic or positively charged sites. We have used this technique across a wide range of crystallization conditions with successful structure determination in 16 of 17 cases within the first year of use (94% success rate). Here we present a general overview of this method as well as several examples including SAD phasing of proteins with novel folds and the combined use of SAD and MR for targets with weak MR solutions. These cases highlight the straightforward and powerful method of iodide ion SAD phasing in a high-throughput structural genomics environment