Using deeply trapped intermediates to map the cytochrome c folding landscape

Replacement of iron with cobalt(III) selectively introduces a deep trap in the folding-energy landscape of the heme protein cytochrome c. Remarkably, neither the protein structure nor the folding thermodynamics is perturbed by this metal-ion substitution, as shown by data from spectroscopic and x-ray diffraction experiments. Through kinetics measurements, we have found parallel folding pathways involving several different misligated Co(III) species, and, as these folding intermediates persist for several hours under certain conditions, we have been able to elucidate fully their spectroscopic properties. The results, along with an analysis of the fluorescence energy-transfer kinetics during refolding, show that rapidly equilibrating populations of compact and extended polypeptide conformations are present until all molecules have reached the native structure. These measurements provide direct evidence that collapsed denatured structures are not substantially more stable than extended conformations of cytochrome c.     

Metal templated design of protein interfaces

Metal coordination is a key structural and functional component of a large fraction of proteins. Given this dual role we considered the possibility that metal coordination may have played a templating role in the early evolution of protein folds and complexes. We describe here a rational design approach, Metal Templated Interface Redesign (MeTIR), that mimics the time course of a hypothetical evolutionary pathway for the formation of stable protein assemblies through an initial metal coordination event. Using a folded monomeric protein, cytochrome cb₅₆₂, as a building block we show that its non-self-associating surface can be made self-associating through a minimal number of mutations that enable Zn coordination. The protein interfaces in the resulting Zn-directed, D₂-symmetrical tetramer are subsequently redesigned, yielding unique protein architectures that self-assemble in the presence or absence of metals. Aside from its evolutionary implications, MeTIR provides a route to engineer de novo protein interfaces and metal coordination environments that can be tuned through the extensive noncovalent bonding interactions in these interfaces.     

Increased P Wave Dispersion and Maximum P Wave Duration after Hemodialysis

Background: Atrial fibrillation is a frequent arrhythmia in patients undergoing hemodialysis. The consequences of hemodialysis on P wave durations and P wave dispersion have not been fully understood. The objective of this study was to study the effect of dialysis on P wave maximum (P_max), P wave minimum (P_min), and P wave dispersion (P_d). Methods: We studied P_max, P_min, and P_d in 32 patients (17 men and 15 women, mean age 54 ± 18 years) with chronic renal failure undergoing hemodialysis. The difference between maximum and minimum P wave duration was calculated and defined as P wave dispersion (P_d= P_max? P_min). Results: There was a significant increase in P_max at the end of dialysis compared to the beginning (98 ± 13 ms vs. 125 ± 12 ms, P < 0.001). P_min did not show any significant change (71 ± 11 ms vs. 73 ± 10 ms, P = 0.42). P_d was significantly increased at the end of dialysis (27 ± 9 ms vs. 52 ± 11 ms, P < 0.001). There was a negative correlation between serum potassium, magnesium, phosphate, blood urea nitrogen, and creatinin at the end of dialysis and P_max and P_d, respectively (P < 0.05). A weak positive correlation was found between serum calcium, bicarbonate at the end of dialysis and P_max and P_d (P < 0.05). Conclusion: Hemodialysis ends with significant increase in P wave maximum duration and P wave dispersion, which might be responsible for the increased occurrence of atrial fibrillation in these groups of patients.     

Bilateral thoracoscopic sympathectomy for primary hyperhydrosis: a review of 335 cases

Objective

The goal of this retrospective study was to evaluate the outcomes of bilateral video-assisted thoracoscopic sympathectomy for primary hyperhydrosis.

Methods

Between January 2007 and December 2011, a total of 335 patients (192 male, 143 female, mean age 28.3 years) who underwent bilateral thoracoscopic sympathectomy for primary hyperhydrosis were reviewed retrospectively.

Results

Hyperhydrosis occurred in the palmar and axillary region in 175 (52.23%) patients, in only the palmar region in 52 (15.52%), in the craniofacial region in 44 (13.13%), in only the axillary region in 42 (12.53%), and in the palmar and pedal regions in 22 (6.56%) patients. Bilateral thoracoscopic sympathectomy was performed in all patients. The mean follow-up period was 24 (6–48) months. The initial cure rate was 95% and the initial satisfaction rate was 93%. There was no mortality in this study. The complication rate was 15.82% in 53 patients.

Conclusion

Video-assisted thoracoscopic sympathectomy for the treatment of primary hyperhydrosis was effective, with low rates of morbidity and mortality. Despite the appearance of postoperative complications, such as compensatory sweating, patient satisfaction with the procedure was high and their quality of life improved.     

Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation

Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab' fragment) directed against the glycoprotein (GP) IIb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 30% to 40% in the presence of EDTA at 22 to 25 degrees C. Platelets stimulated by thrombin or PMA bound more fibrinogen than did those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with various platelet agonists. When the pool of GP IIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with the monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, stimulation with thrombin or PMA still induced substantial binding of antibody and fibrinogen, and aggregation ensued. Therefore, platelets exposed to "strong" agonists exhibit an increased number of surface-oriented epitopes associated with GP IIb/IIIa. The GP IIb/IIIa molecules bearing these newly exposed epitopes are functional in that they can bind fibrinogen and mediate platelet aggregation.     

Frequent ongoing T-cell receptor rearrangements in childhood B- precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease

Crosslineage T-cell receptor delta (TCR delta) rearrangements are widely used as tumor markers for the follow up of minimal residual disease in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR). The major drawback of this approach is the risk of false-negative results due to clonal evolution. We investigated the stability of V delta 2D delta 3 rearrangements in a group of 56 childhood B-precursor ALL patients by PCR and Southern blot analysis. At the PCR level, V delta 2D delta 3-to-J alpha rearranged subclones (one pathway for secondary TCR delta recombination) were demonstrated in 85.2% of V delta 2D delta 3-positive patients tested, which showed that small subclones are present in the large majority of patients despite apparently monoclonal TCR delta Southern blot patterns. Sequence analysis of V delta 2D delta 3J alpha rearrangements showed a biased J alpha gene usage, with HAPO5 and J alpha F in 26 of 32 and 6 of 32 clones, respectively. Comparison of V delta 2D delta 3 rearrangement status between diagnosis and first relapse showed differences in seven of eight patients studied. In contrast, from first relapse onward, no clonal changes were observed in six patients studied. To investigate the occurrence of crosslineage TCR delta rearrangements in normal B and T cells, fluorescence-activated cell sorter-sorted peripheral blood CD19+/CD3- and CD19-/CD3+ cell populations from three healthy donors were analyzed. V delta 2D delta 3 rearrangements were detected at low frequencies in both B and T cells, which suggests that V delta 2-to-D delta 3 joining also occurs during normal B-cell differentiation. A model for crosslineage TCR delta rearrangements in B-precursor ALL is deduced that explains the observed clonal changes between diagnosis and relapse and is compatible with multistep leukemogenesis of B-precursor ALL.     

British HIV Association (BHIVA) national cohort outcomes audit of patients commencing antiretrovirals from naïve

Objectives

The aim of this work was to audit the extent to which routine HIV care in the UK conforms with British HIV Association (BHIVA) guidelines and specifically the proportion of patients starting highly active antiretroviral therapy (HAART) who achieve the outcome of virological suppression below 50 HIV‐1 RNA copies/mL within 6 months.

Methods

A prospective cohort review of adults with HIV infection who started antiretroviral therapy (ART) for the first time between April and September 2006 was carried out using structured questionnaire forms.

Results

A total of 1170 adults from 122 clinical sites participated in the review. Of these patients, 699 (59.7%) started ART at CD4 counts <200 cells/μL and 193 (16.5%) had not been tested for HIV drug resistance. Excluding patients with valid reasons for stopping short‐term ART, 795 (73.5%) of 1081 patients had an undetectable viral load (VL) at follow‐up. Detectable VL was strongly associated with pretreatment CD4 count below 50 cells/μL and pretreatment VL above 100 000 copies/mL, and was not associated with clinic location or case load. About a quarter of patients did not have a VL measurement during the first 6 weeks after starting ART.

Conclusions

The majority of patients who initiated ART at sites participating in this UK national audit were managed within the BHIVA guidelines and achieved virological suppression below 50 copies/mL around 6 months after commencing treatment. Poor VL outcomes were associated with very low CD4 cell count and/or high VL at baseline but not with clinic case load or location. There is an urgent need to diagnose patients at an earlier stage of their HIV disease.     

Aortitis in a patient with psoriatic arthritis

Aortitis, inflammation of the aortic tissue, is most commonly caused by vasculitic rheumatic conditions, and less frequently infectious organisms. Involvement of the aorta is well defined in HLA-B27-associated spondyloarthropathies such as long-standing ankylosing spondylitis and Reiter's syndrome. However, unlike other spondyloarthropathies, aortic involvement or true aortitis is not a feature of psoriatic arthritis and has been reported in only a few cases. Herein, we report the case of a 22?year-old woman with psoriatic arthritis who developed descending aortitis while using tumor necrosis factor inhibitors.     

Evaluation of serum interleukin-6 (IL-6), IL-13, and IL-17 levels and computed tomography finding in interstitial lung disease associated with connective tissue disease patients

Objective

Interstitial lung disease (ILD) is one of the most severe complications which is associated with connective tissue disease (CTD) and causes to morbidity and mortality. So, we aimed to determine serum levels of interleukin-6 (IL-6), IL-13, and IL-17, to investigate whether these cytokines are related to CTD-ILD, and to find their possible contribution to determining the prognosis of the disease.

Methods

A total of 150 participants, 80 patients diagnosed with CTD-ILD (mean age, 58.21?±?12.36) and 70 healthy controls (mean age, 57.07?±?9.60) were recruited from the rheumatology department between January 2016 and June 2019 in the study. High-resolution computed tomography (HRCT) findings were scored as similarly to previous studies. Serum IL-6, IL 13, and IL-17 levels were measured by ELISA test kits.

Results

The levels of IL-6, IL-13, and IL-17 in CTD patients were significantly higher than the healthy individuals (p?<?001), but the HRCT score’s relation were not determined. IL-6 was associated with disease duration and disease activity scores of DAS28, ESDAII, and dSSc. There was a significant relation between dSSc, HCRT fibrosis, and total score.CRP, hemoglobin, and platelets were associated with the HRCT inflammation pattern.

Conclusion

At the study, it has been observed that serum IL-13, IL-6 and IL-17 levels are increased in patients with CTD-ILD. Besides, IL-6 was associated with disease activity scores of DAS28, ESDAII, and dSSc. Also, HRCT fibrosis score is associated with dSSc. Further and comprehensive studies are needed to understand better the complex intersection of lung disease with systemic autoimmunity.