Decreased Serum Stromal Cell-Derived Factor-1 in Patients with Familial Hypercholesterolemia and Its Strong Correlation with Lipoprotein Subfractions.

Stromal cell-derived factor-1 (SDF-1) is a chemokine that exerts multifaceted roles in atherosclerosis. However, its association with hyperlipidemia is contradictory. To date, serum SDF-1 and its correlations with lipid fractions and subfractions in heterozygous familial hypercholesterolemia (HeFH) have not been investigated. Eighty-one untreated patients with HeFH and 32 healthy control subjects were enrolled in the study. Serum SDF-1, oxidized LDL (oxLDL) and myeloperoxidase (MPO) were determined by ELISA. Lipoprotein subfractions were detected by Lipoprint. We diagnosed FH using the Dutch Lipid Clinic Network criteria. Significantly lower serum SDF-1 was found in HeFH patients compared to healthy controls. Significant negative correlations were detected between serum total cholesterol, triglycerides, LDL-cholesterol (LDL-C), apolipoprotein B100 (ApoB100) and SDF-1. Furthermore, serum SDF-1 negatively correlated with VLDL and IDL, as well as large LDL and large and intermediate HDL subfractions, while there was a positive correlation between mean LDL-size, small HDL and SDF-1. SDF-1 negatively correlated with oxLDL and MPO. A backward stepwise multiple regression analysis showed that the best predictors of serum SDF-1 were VLDL and oxLDL. The strong correlation of SDF-1 with lipid fractions and subfractions highlights the potential common pathways of SDF-1 and lipoprotein metabolism, which supports the role of SDF-1 in atherogenesis.

Assessment of amino-terminal C-type natriuretic peptide serum level and its correlation with high-density lipoprotein structure and function in patients with end stage renal disease before and after kidney transplantation.

We aimed to investigate serum amino-terminal C-type natriuretic peptide (NT-proCNP) and its relationship with quantitative and qualitative HDL-parameters in patients with end-stage renal disease (ESRD) before, then 1 and 6 months after kidney transplantation (TX). Seventy patients (47 males, 23 females, mean age 51.7 ± 12.4 years) were enrolled in a prospective follow-up study. We examined serum creatinine, C-reactive protein, procalcitonin, fasting glucose and lipid parameters before, then 1 and 6 months after TX. High-density lipoprotein- (HDL)-associated paraoxonase-1 (PON1) paraoxonase and arylesterase activities were measured spectrophotometrically. Lipoprotein subfractions were determined by Lipoprint. NT-proCNP and oxidized low-density lipoprotein (oxLDL) levels were measured by ELISA. Mean NT-proCNP was 45.8 ± 21.9 pmol/L before renal transplantation and decreased markedly 1 month and 6 months after transplantation (5.3 ± 2.5 and 7.7 ± 4.9 pmol/L, respectively, P = 1 × 10-4). During the 6 months' follow-up, PON1 arylesterase, paraoxonase and salt-stimulated paraoxonase activities improved. NT-proCNP positively correlated with procalcitonin and creatinine and negatively with GFR, LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C). There was a negative correlation between serum NT-proCNP and PON1 arylesterase activity. According to the multiple regression analysis, the best predicting variables of NT-proCNP were serum procalcitonin, creatinine and PON1 arylesterase activity. NT-proCNP might be a novel link between HDL dysfunction and impaired vascular function in ESRD, but not after kidney transplantation. Further studies in larger populations are needed to clarify the exact role of NT-proCNP in the risk prediction for cardiovascular comorbidities and complications in ESRD.

Position-dependent sequence motif preferences of SpCas9 are largely determined by scaffold-complementary spacer motifs.

Streptococcus pyogenes Cas9 (SpCas9) nuclease exhibits considerable position-dependent sequence preferences. The reason behind these preferences is not well understood and is difficult to rationalise, since the protein establishes interactions with the target-spacer duplex in a sequence-independent manner. We revealed here that intramolecular interactions within the single guide RNA (sgRNA), between the spacer and the scaffold, cause most of these preferences. By using in cellulo and in vitro SpCas9 activity assays with systematically designed spacer and scaffold sequences and by analysing activity data from a large SpCas9 sequence library, we show that some long (>8 nucleotides) spacer motifs, that are complementary to the RAR unit of the scaffold, interfere with sgRNA loading, and that some motifs of more than 4 nucleotides, that are complementary to the SL1 unit, inhibit DNA binding and cleavage. Furthermore, we show that intramolecular interactions are present in the majority of the inactive sgRNA sequences of the library, suggesting that they are the most important intrinsic determinants of the activity of the SpCas9 ribonucleoprotein complex. We also found that in pegRNAs, sequences at the 3' extension of the sgRNA that are complementary to the SL2 unit are also inhibitory to prime editing, but not to the nuclease activity of SpCas9.

Role of Altered Metabolism of Triglyceride-Rich Lipoprotein Particles in the Development of Vascular Dysfunction in Systemic Lupus Erythematosus.

BACKGROUND: Impaired lipid metabolism contributes to accelerated inflammatory responses in addition to promoting the formation of atherosclerosis in systemic lupus erythematosus (SLE). We aimed to evaluate the lipid profile, inflammatory markers, and vascular diagnostic tests in active SLE patients to clarify the association between dyslipidemia and early vascular damage. PATIENTS AND METHODS: 51 clinically active SLE patients and 41 age- and gender-matched control subjects were enrolled in the study. Lipoprotein subfractions were detected by Lipoprint. Brachial artery flow-mediated dilation and common carotid intima-media thickness were detected by ultrasonography. Arterial stiffness indicated by augmentation index (Aix) and pulse wave velocity was measured by arteriography. RESULTS: We found significantly higher Aix, higher VLDL ratio, plasma triglyceride, ApoB100, and small HDL, as well as lower HDL-C, large HDL, and ApoA1 in patients with SLE. There was a significant positive correlation of Aix with triglyceride, VLDL, IDL-C, IDL-B, and LDL1. A backward stepwise multiple regression analysis showed IDL-C subfraction to be the best predictor of Aix. CONCLUSIONS: Our results indicate that in young patients with SLE, triglyceride-rich lipoproteins influence vascular function detected by Aix. These parameters may be assessed and integrated into the management plan for screening cardiovascular risk in patients with SLE.

Sphingosine 1-Phosphate and Apolipoprotein M Levels and Their Correlations with Inflammatory Biomarkers in Patients with Untreated Familial Hypercholesterolemia.

High-density lipoprotein (HDL)-bound apolipoprotein M/sphingosine 1-phosphate (ApoM/S1P) complex in cardiovascular diseases serves as a bridge between HDL and endothelial cells, maintaining a healthy endothelial barrier. To date, S1P and ApoM in patients with untreated heterozygous familial hypercholesterolemia (HeFH) have not been extensively studied. Eighty-one untreated patients with HeFH and 32 healthy control subjects were included in this study. Serum S1P, ApoM, sCD40L, sICAM-1, sVCAM-1, oxLDL, and TNFα concentrations were determined by ELISA. PON1 activities were measured spectrophotometrically. Lipoprotein subfractions were detected by Lipoprint. We diagnosed FH using the Dutch Lipid Clinic Network criteria. Significantly higher serum S1P and ApoM levels were found in HeFH patients compared to controls. S1P negatively correlated with large HDL and positively with small HDL subfractions in HeFH patients and the whole study population. S1P showed significant positive correlations with sCD40L and MMP-9 levels and PON1 arylesterase activity, while we found significant negative correlation between sVCAM-1 and S1P in HeFH patients. A backward stepwise multiple regression analysis showed that the best predictors of serum S1P were large HDL subfraction and arylesterase activity. Higher S1P and ApoM levels and their correlations with HDL subfractions and inflammatory markers in HeFH patients implied their possible role in endothelial protection.

Hand hygiene improvement of individual healthcare workers: results of the multicentre PROHIBIT study.

BACKGROUND: Traditionally, hand hygiene (HH) interventions do not identify the observed healthcare workers (HWCs) and therefore, reflect HH compliance only at population level. Intensive care units (ICUs) in seven European hospitals participating in the "Prevention of Hospital Infections by Intervention and Training" (PROHIBIT) study provided individual HH compliance levels. We analysed these to understand the determinants and dynamics of individual change in relation to the overall intervention effect. METHODS: We included HCWs who contributed at least two observation sessions before and after intervention. Improving, non-changing, and worsening HCWs were defined with a threshold of 20% compliance change. We used multivariable linear regression and spearman's rank correlation to estimate determinants for the individual response to the intervention and correlation to overall change. Swarm graphs visualized ICU-specific patterns. RESULTS: In total 280 HCWs contributed 17,748 HH opportunities during 2677 observation sessions. Overall, pooled HH compliance increased from 43.1 to 58.7%. The proportion of improving HCWs ranged from 33 to 95% among ICUs. The median HH increase per improving HCW ranged from 16 to 34 percentage points. ICU wide improvement correlated significantly with both the proportion of improving HCWs (ρ = 0.82 [95% CI 0.18-0.97], and their median HH increase (ρ = 0.79 [0.08-0.97]). Multilevel regression demonstrated that individual improvement was significantly associated with nurse profession, lower activity index, higher nurse-to-patient ratio, and lower baseline compliance. CONCLUSIONS: Both the proportion of improving HCWs and their median individual improvement differed substantially among ICUs but correlated with the ICUs' overall HH improvement. With comparable overall means the range in individual HH varied considerably between some hospitals, implying different transmission risks. Greater insight into improvement dynamics might help to design more effective HH interventions in the future.

Determination of Serum Progranulin in Patients with Untreated Familial Hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant trait characterized by elevated LDL-C concentrations and is associated with an increased risk of premature atherosclerosis. Progranulin (PGRN) is a multifunctional protein that is known to have various anti-atherogenic effects. To date, the use of serum PGRN in patients with FH has not been studied. METHODS: In total, 81 untreated patients with heterozygous FH (HeFH) and 32 healthy control subjects were included in this study. Serum PGRN, sICAM-1, sVCAM-1, oxLDL and TNFα concentrations were determined by ELISA. Lipoprotein subfractions were detected by Lipoprint. We diagnosed FH using the Dutch Lipid Clinic Network criteria. RESULTS: We could not find a significant difference between the PGRN concentrations of the HeFH patients and controls (37.66 ± 9.75 vs. 38.43 ± 7.74 ng/mL, ns.). We found significant positive correlations between triglyceride, TNFα, sVCAM-1, the ratio of small HDL subfraction and PGRN, while significant negative correlations were found between the ratio of large HDL subfraction and PGRN both in the whole study population and in FH patients. PGRN was predicted by sVCAM-1, logTNFα and the ratio of small HDL subfraction. CONCLUSIONS: The strong correlations between HDL subfractions, inflammatory markers and PGRN suggest that PGRN may exert its anti-atherogenic effect in HeFH through the alteration of HDL composition and the amelioration of inflammation rather than through decreasing oxidative stress.

BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors.

Adenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.

A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs.

Detailed target-selectivity information and experiment-based efficacy prediction tools are primarily available for Streptococcus pyogenes Cas9 (SpCas9). One obstacle to develop such tools is the rarity of accurate data. Here, we report a method termed 'Self-targeting sgRNA Library Screen' (SLS) for assaying the activity of Cas9 nucleases in bacteria using random target/sgRNA libraries of self-targeting sgRNAs. Exploiting more than a million different sequences, we demonstrate the use of the method with the SpCas9-HF1 variant to analyse its activity and reveal motifs that influence its target-selectivity. We have also developed an algorithm for predicting the activity of SpCas9-HF1 with an accuracy matching those of existing tools. SLS is a facile alternative to the much more expensive and laborious approaches used currently and has the capability of delivering sufficient amount of data for most of the orthologs and variants of SpCas9.

Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases.

The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence. Here we have aimed to loosen these PAM constraints and have generated new PAM mutant variants of the four Cas12a orthologs that are active in mammalian and plant cells, by combining the mutations of their corresponding RR and RVR variants with altered PAM specificities. LbCas12a-RVRR showing the highest activity was selected for an in-depth characterization of its PAM preferences in mammalian cells, using a plasmid-based assay. The consensus PAM sequence of LbCas12a-RVRR resembles a TNTN motif, but also includes TACV, TTCV CTCV and CCCV. The D156R mutation in improved LbCas12a (impLbCas12a) was found to further increase the activity of that variant in a PAM-dependent manner. Due to the overlapping but still different PAM preferences of impLbCas12a and the recently reported enAsCas12a variant, they complement each other to provide increased efficiency for genome editing and transcriptome modulating applications.

[Capgras symptom associated parricides. 2 case reports]. / Capgras-féle tünettel összefüggo szülogyilkosságok. Két eset bemutatása.

Capgras symptom is characterized by the delusional belief that a person significant to the patient has been replaced by a 'double' or 'impostor'. Capgras symptom was discussed to be associated with violent behavior. We report here the cases of two male patients with schizophrenia paranoid type, where parricide was connected to Capgras delusion. It is important to emphasize that in our observed cases, non-adherence played an essential role in the development of violent behavior, parricide. Orv Hetil. 2019; 160(42): 1673-1676.

EAACI Guidelines on Allergen Immunotherapy: House dust mite-driven allergic asthma.

Allergen immunotherapy (AIT) has been in use for the treatment of allergic disease for more than 100 years. Asthma treatment relies mainly on corticosteroids and other controllers recommended to achieve and maintain asthma control, prevent exacerbations, and improve quality of life. AIT is underused in asthma, both in children and in adults. Notably, patients with allergic asthma not adequately controlled on pharmacotherapy (including biologics) represent an unmet health need. The European Academy of Allergy and Clinical Immunology has developed a clinical practice guideline providing evidence-based recommendations for the use of house dust mites (HDM) AIT as add-on treatment for HDM-driven allergic asthma. This guideline was developed by a multi-disciplinary working group using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. HDM AIT was separately evaluated by route of administration and children and adults: subcutaneous (SCIT) and sublingual AIT (SLIT), drops, and tablets. Recommendations were formulated for each. The important prerequisites for successful treatment with HDM AIT are (a) selection of patients most likely to respond to AIT and (b) use of allergen extracts and desensitization protocols of proven efficacy. To date, only AIT with HDM SLIT-tablet has demonstrated a robust effect in adults for critical end points (exacerbations, asthma control, and safety). Thus, it is recommended as an add-on to regular asthma therapy for adults with controlled or partially controlled HDM-driven allergic asthma (conditional recommendation, moderate-quality evidence). HDM SCIT is recommended for adults and children, and SLIT drops are recommended for children with controlled HDM-driven allergic asthma as the add-on to regular asthma therapy to decrease symptoms and medication needs (conditional recommendation, low-quality evidence).

Medical Algorithms: Diagnosis and treatment of Hymenoptera venom allergy.

Diagnosis of Hymenoptera venom allergy (HVA) is straightforward in the majority of patients, but can be challenging in double positive and test negative patients. Test results sometimes can be confusing as patients with high skin test reactivity and high specific IgE (sIgE) levels are not at risk for severe systemic sting reactions (SSR), and conversely, patients with weakly positive or even negative tests can experience severe SSR. Venom immunotherapy (VIT) is safe, highly effective, and recommended in patients with moderate to severe SSR and in patients with SSR confined to generalized skin symptoms if quality of life is impaired.

Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants.

Cpf1s, the RNA-guided nucleases of the class II clustered regularly interspaced short palindromic repeats system require a short motive called protospacer adjacent motif (PAM) to be present next to the targeted sequence for their activity. The TTTV PAM sequence of As- and LbCpf1 nucleases is relatively rare in the genome of higher eukaryotic organisms. Here, we show that two other Cpf1 nucleases, Fn- and MbCpf1, which have been reported to utilize a shorter, more frequently occurring PAM sequence (TTN) when tested in vitro, carry out efficient genome modification in mammalian cells. We found that all four Cpf1 nucleases showed similar activities and TTTV PAM preferences. Our approach also revealed that besides their activities their PAM preferences are also target dependent. To increase the number of the available targets for Fn- and MbCpf1 we generated their RVR and RR mutants with altered PAM specificity and compared them to the wild-type and analogous As- and LbCpf1 variants. The mutants gained new PAM specificities but retained their activity on targets with TTTV PAMs, redefining RR-Cpf1's PAM-specificities as TTYV/TCCV, respectively. These variants may become versatile substitutes for wild-type Cpf1s by providing an expanded range of targets for genome engineering applications.

[A short overview on the changes of the content of delusions - the impact of technological innovations, historical events and culture].

The content aspect of delusions has been going through fast and great changes over the past hundred years. In this paper we would like to lead the reader through these changes with the brief overview of the related literature, thus giving a better understanding of the underlying causes. In recent years several studies have been published on the impact of technological and cultural changes in delusions, as opposed to the stability of delusional themes over longer periods of time, proved by long-term comparisons and comparative transcultural studies on delusions, allowing for only a certain degree of variability.

Erratum to: Allergen immunotherapy for allergic rhinoconjunctivitis: protocol for a systematic review.

[This corrects the article DOI: 10.1186/s13601-016-0099-6.].

Erratum to: Allergen immunotherapy for insect venom allergy: protocol for a systematic review.

[This corrects the article DOI: 10.1186/s13601-016-0095-x.].

EAACI guidelines on allergen immunotherapy: Prevention of allergy.

Allergic diseases are common and frequently coexist. Allergen immunotherapy (AIT) is a disease-modifying treatment for IgE-mediated allergic disease with effects beyond cessation of AIT that may include important preventive effects. The European Academy of Allergy and Clinical Immunology (EAACI) has developed a clinical practice guideline to provide evidence-based recommendations for AIT for the prevention of (i) development of allergic comorbidities in those with established allergic diseases, (ii) development of first allergic condition, and (iii) allergic sensitization. This guideline has been developed using the Appraisal of Guidelines for Research & Evaluation (AGREE II) framework, which involved a multidisciplinary expert working group, a systematic review of the underpinning evidence, and external peer-review of draft recommendations. Our key recommendation is that a 3-year course of subcutaneous or sublingual AIT can be recommended for children and adolescents with moderate-to-severe allergic rhinitis (AR) triggered by grass/birch pollen allergy to prevent asthma for up to 2 years post-AIT in addition to its sustained effect on AR symptoms and medication. Some trial data even suggest a preventive effect on asthma symptoms and medication more than 2 years post-AIT. We need more evidence concerning AIT for prevention in individuals with AR triggered by house dust mites or other allergens and for the prevention of allergic sensitization, the first allergic disease, or for the prevention of allergic comorbidities in those with other allergic conditions. Evidence for the preventive potential of AIT as disease-modifying treatment exists but there is an urgent need for more high-quality clinical trials.