Three-Dimensional Printed Target Plates for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Three-dimensional printing (3D printing) is a fast-growing technology with high impact in industry, medicine, and the life sciences. Fused deposition modeling (FDM), which uses plastic filaments extruded through a heated nozzle, is the most common 3D printing technology for creation of objects. In this work, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) target plates printed by FDM technology using conductive plastic material were evaluated for their detection capability of proteins and peptides. The 3D printed MALDI targets were validated by analysis of different types of bacteria and compared with commercially available MBT BioTargets. The results indicate that 3D printed MALDI targets are comparable to standard MBT BioTargets and stainless-steel targets and may be used for different MALDI-TOF MS applications. The 3D printing allows easy manufacturing of MALDI targets with different dimensions and spot geometry. Moreover, the 3D printed MALDI targets are disposable, cheap, and easy to produce. These features make them a suitable cost-effective alternative to conventional targets for any MALDI MS analysis.

Low Density Lipoprotein Receptor Variants in the Beta-Propeller Subdomain and Their Functional Impact.

Background: Pathogenic variants in the low density lipoprotein receptor gene are associated with familial hypercholesterolemia. Some of these variants can result in incorrect folding of the LDLR protein, which is then accumulated inside the cell and cannot fulfill its function to internalize LDL particles. We analyzed the functional impact of 10 LDLR variants localized in the beta-propeller of epidermal growth factor precursor homology domain. The experimental part of the work was complemented by a structural analysis on the basis of 3D LDLR protein structure. Methods: T-Rex Chinese hamster ovary cells transfected with the human LDLR gene were used for live cell imaging microscopy, flow cytometry, and qRT-PCR analysis. Results: Our results showed that the analyzed LDLR protein variants can be divided into three groups. (1) The variants buried inside the 3D protein structure expressing proteins accumulated in the endoplasmic reticulum (ER) with no or reduced plasma membrane localization and LDL particle internalization, and associated with an increased gene expression of ER-resident chaperones. (2) The variants localized on the surface of 3D protein structure with slightly reduced LDLR plasma membrane localization and LDL particle internalization, and associated with no increased mRNA level of ER-resident chaperones. (3) The variants localized on the surface of the 3D protein structure but expressing proteins with cell responses similar to the group 1. Conclusion: All analyzed LDLR variants have been evaluated as pathogenic but with different effects on protein localization and function, and expression of genes associated with ER stress.

Soluble receptor for advanced glycation end products in patients with decreased renal function.

BACKGROUND: Advanced glycation end products (AGEs) accumulate in patients with decreased renal function and exert various toxic effects through the receptor for AGEs (RAGE). Soluble RAGE (sRAGE) is a naturally occurring inhibitor of AGE-RAGE action. The aim of the study is to describe the relationship of sRAGE to renal function and dialysis modalities. METHODS: The studied group consisted of 81 patients: 25 patients with various degrees of decreased renal function, 20 long-term hemodialysis (HD) patients, 15 peritoneal dialysis (PD) patients, and 21 healthy age-matched subjects. sRAGE was assessed immunochemically (enzyme-linked immunosorbent assay), and routine biochemical parameters were measured by means of certified methods. RESULTS: sRAGE level correlates positively with serum creatinine concentration (r = 0.50; P < 0.05), and its relationship to creatinine clearance is hyperbolic. sRAGE levels are elevated significantly, mainly in patients with end-stage renal disease (3,119.0 +/- 968.4 pg/mL in HD patients and 3,652.7 +/- 1,677.7 pg/mL in PD patients versus 1,405.1 +/- 426.1 pg/mL in controls; both P < 0.001 versus controls). In PD patients, sRAGE is detectable in spent dialysate (median, 75.8 pg/mL), correlates with its serum levels (r = 0.67; P < 0.05), and is related to protein losses in dialysate. In HD patients, sRAGE levels increase by 50% (P < 0.001) from 0 to 15 minutes during both HD and hemodiafiltration, and then decrease until the end of the session. CONCLUSION: Serum sRAGE levels increase in patients with decreased renal function, mainly patients with end-stage renal disease. It remains to be elucidated whether the increase is caused just by decreased renal function or whether sRAGE is upregulated to protect against toxic effects of AGEs.

Possible restorations of the upper extremity motion in tetraplegic patients - 5-year clinical experience.

AIM: The aim of the work is to objectify the functional effectiveness of these operations and their influence on the quality of life of handicapped patients. METHOD: The authors evaluate the results of reconstructive surgery restoring hand grip in a group of 15 tetraplegic patients (3 women and 12 men) with complete spinal cord lesion of C5-C7 segments. The average age of patients in the group is 33 (22-50) years old. The reconstructions were performed using tendon transfer and tenodesis in the forearm and hand area. The effectiveness of the transfer was assessed objectively with regard to muscle strength by measuring the restored "thumb-index finger" grip and "into fist" grip. The range of motion achieved was also evaluated. Transfer effectiveness was evaluated on the basis of subjective patients' evaluation. An ADL (activities of daily living) questionnaire by Mohammed's (1992) took into account the effect of the surgery in a whole range of common daily activities. RESULTS: An extended range of daily activities was evident mainly in the fields of: communication, eating and drinking and operations associated with increase in general selfcare of the patient. There was no deterioration of condition in any of the activities. CONCLUSIONS: Up to 80 % of tetraplegic patients are suitable candidates for transfers and, to a certain extent, it is possible to improve the upper limb function. In a partial function restoration of the upper limbs there is immense potential for improvement in the quality of life of these patients.

Arteriovenous graft for hemodialysis, graft venous anastomosis closure - current state of knowledge. Minireview.

UNLABELLED: Backround. The use of artificial vascular grafts (arteriovenous graft, AVG) is indicated in patients in hemodialysis programs if the subcutaneous venous bed is exhausted or unsuitable for arteriovenous fistula (AVF) creation. The native fistula should be the hemodialysis access of first choice: AVF has better results in terms of function and potential complications. However, the use of AVG is necessary in some patients. In these patients, extensive clinical examination, color duplex sonography and angiography should be performed prior to indication. The technique of graft implantation requires respect for geometric relations for the graft anastomoses to minimize the formation of intimal hyperplasia mainly on the venous anastomosis. The main complications of AVG are stenosis on the venous anastomosis (VAG), causing closure of graft and graft infection. The cumulative function of AVG is 59-90% in the first year and 50-82% in the second year. Arteriovenous graft stenosis leading to thrombosis is a major cause of complications in patients undergoing hemodialysis. The purpose of this review is to summarise current knowledge of the diagnostics and treatment of graft thrombosis and discuss the issue in combination with relevant publications via Pubmed database. CONCLUSION: The most frequent cause of failure of AVG for hemodialysis is stenosis and closure by VAG. AVG closure can be addressed surgically, endovascularly (amenable to thrombectomy by radiological or surgical means) and by hybrid performance.

Current treatment methods for long occlusions of the femoropopliteal segment in patients with intermittent claudication: Minireview.

UNLABELLED: Backround. Intermittent claudication is a classic symptom of peripheral arterial disease. It is mainly treated conservatively but if this fails, a form of revascularization is indicated. The revascularization in chronic occlusion of femoropopliteal region is currently performed by two basic methods: the standard method of surgical bypass and the newer miniinvasive alternative represented by the endovascular method. The treatment of patients with solely claudication and long occlusion of femoropopliteal region remains controversial. The aim of this minireview was to determine whether surgical bypass is still the best method of choice in a time of endovascular techniques. METHODS: A MEDLINE search for original and review articles using key terms, intermittent claudication and long femoropopliteal oclusion. RESULTS AND CONCLUSION: No ideal treatment for long occlusions of the femoropopliteal segment has been established to date. It is clear that the role of endovascular techniques in the treatment of SFA occlusions is increasing. It remains that, lower risk patients with claudication should be examined to assess the quality of veins suitable for revascularization and bypass should be selected as the first method of choice.

A Continuous Glucose Monitoring System (CGMS) - a promising approach for improving metabolic control in persons with type 1 Diabetes mellitus treated by insulin pumps.

This pilot study deals with the possibilities of a Continuous Glucose Monitoring System (CGMS, Minimed- Medtronic) to optimize insulin substitution. Ten persons with type 1 diabetes mellitus treated by means of an insulin pump entered the study and eight of them completed the protocol. CGMS was introduced for a period of 5 days. The standard dinner (60 g of carbohydrates) and overnight fasting were designed to ensure standard night conditions in all persons in the study while maintaining their usual daily eating routine, physical exercise and assessment of prandial insulin boluses. The only adaptation of basal rates of insulin pump was performed on day 3. Comparison of the mean plasma glucose concentration (0:00-24:00 hrs) between day 2 (before adaptation) and day 4 (following adaptation) was made. An independent comparison of the mean plasma glucose concentration between the night from day 2 till day 3 (22:00-6:00 hrs) and the night from day 4 till day 5 (22:00-6:00 hrs) was performed. The mean plasma glucose investigated by means of CGMS improved in the 24-hour period in 5 out of 8 persons and in the night fasting period (22:00 to 6 hrs) in 6 out of 8 persons. The CGMS is a useful means for assessment of the effectiveness of basal rate and prandial insulin doses in persons with type 1 diabetes treated by means of an insulin pump. However, further studies are necessary to improve the algorithm for insulin substitution.

Influence of delayed graft function and acute rejection on outcomes after kidney transplantation from donors after cardiac death.

BACKGROUND: Delayed graft function (DGF) and acute rejection (AR) exert an adverse impact on graft outcomes after kidney transplantation using organs from donation after brain-stem death (DBD) donors. Here, we examine the impact of DGF and AR on graft survival in kidney transplants using organs from donation after cardiac death (DCD) donors. METHODS: We conducted a single-center retrospective study of DCD and DBD donor kidney transplants. We compared 1- and 4-year graft and patient survival rates, as well as death-censored graft survival (DCGS) rates, between the two groups using univariate analysis, and the impact of DGF and AR on graft function was compared using multivariate analysis. RESULTS: Eighty DCD and 206 DBD donor transplants were analyzed. Median follow-up was 4.5 years. The incidence of DGF was higher among DCD recipients (73% vs. 27%, P<0.001), and AR was higher among DBD recipients (23% vs. 9%, P<0.001). One-year and 4-year graft survival rates were similar (DCD 94% and 79% vs. DBD 90% and 82%). Among recipients with DGF, the 4-year DCGS rate was better for DCD recipients compared with DBD recipients (100% vs. 92%, P=0.04). Neither DGF nor AR affected the 1-year graft survival rate in DCD recipients, whereas in DBD recipients, the 1-year graft survival rate was worse in the presence of DGF (88% vs. 96%, P=0.04) and the 4-year DCGS rate was worse in the presence of AR (88% vs. 96%, P=0.04). CONCLUSION: Despite the high incidence of DGF, medium-term outcomes of DCD kidney transplants are comparable to those from DBD transplants. Short-term graft survival from DCD transplants is not adversely influenced by DGF and AR, unlike in DBD transplants.

Categorization of the hemodynamic response to hemodialysis: the importance of baroreflex sensitivity.

Intradialytic hypotension (IDH) remains an important cause of morbidity and mortality in hemodialysis (HD) patients. The baroreflex arc is under autonomic control and regulates blood pressure. This study aimed to investigate the contribution of impaired baroreflex sensitivity (BRS) to the pathophysiology of IDH. Thirty-four chronic HD (12 IDH-prone, 22 IDH-resistant) patients underwent BRS measurement during HD with relative blood volume monitoring. During analysis, patients were separated into four age-matched groups according to resting BRS>or=4.5 ms/mmHg and hemodynamic stability. Resting BRS was extremely heterogenous (geometric mean BRS 5.78+/-1.41 [range 1.76-41.41] ms/mmHg). Relative blood volume reduction was well matched in all groups (mean reduction in relative blood volume for all patients -6.74%+/-0.86%, P>0.05). Thirty-seven episodes of IDH occurred in the IDH prone, reduced BRS group. Patients with impaired resting BRS and prone to IDH had markedly different responses to HD as compared to the preserved BRS group, but the total peripheral resistance response was significantly lower than in the IDH-resistant patients (15.9%+/-2.1% vs. 42.4%+/-3.0%, respectively, P<0.001). In those patients prone to IDH and with impaired resting BRS, percentage reduction in cardiac output at the end of HD highly correlated with reduction in relative blood volume (r=0.94, P=0.006). Hypotension during dialysis may be an important source of recurrent cardiac injury and early recognition of those patients prone to relative symptomatic and asymptomatic hypotension remains important. Impaired resting BRS and recognition of a suboptimal peripheral pressor response, appear to predict those patients most likely to undergo hemodynamic instability and may assist in the pursuit of this elusive goal.

The haemodynamic and metabolic effects of hypertonic-glucose and amino-acid-based peritoneal dialysis fluids.

BACKGROUND: Continuous ambulatory peritoneal dialysis (CAPD) may exert significant effects on systemic haemodynamics. We have previously demonstrated that hypertonic glucose solutions are associated with higher blood pressure (BP) due to a rise in stroke volume (SV) and cardiac output (CO). However, the mechanisms underlying these changes have not been established. METHODS: Ten non-diabetic CAPD patients entered a randomized crossover study (eight completed) to compare conventional glucose-based fluid, biocompatible pH-neutral glucose-based fluid and 1.1% amino acid solution (lactate-buffered but completely free of glucose degradation products). BP and haemodynamic variables were measured using continuous arterial pulse wave analysis, and serial plasma glucose and insulin concentrations were collected. Left ventricular (LV) diameters were measured at the start and end of each dwell period using M-mode echocardiography. RESULTS: BP was similar during 1.36% glucose and 1.1% amino acid dwells, but was significantly higher during 3.86% glucose dwells with both conventional and biocompatible fluids (P < 0.001). This was associated with a significantly higher SV and CO (P < 0.001), although the haemodynamic response differed between conventional and biocompatible 3.86% solutions. Plasma glucose and insulin levels did not differ from baseline during 1.36% and amino acid dwells, but increased significantly during 3.86% glucose dwells. Despite a significantly higher ultrafiltration volume with 3.86% glucose, LV diameters were similar throughout. CONCLUSIONS: In conclusion, we have confirmed our previous findings demonstrating higher BP and adverse haemodynamics during 3.86% glucose dwells. These changes are associated with hyperglycaemia and hyperinsulinaemia, but are not related to differences in cardiac filling.