Programming of Vascular Dysfunction in the Intrauterine Milieu of Diabetic Pregnancies.

With the rising global tide of obesity, gestational diabetes mellitus (GDM) burgeoned into one of the most common antenatal disorders worldwide. Macrosomic babies born to diabetic mothers are more likely to develop risk factors for cardiovascular disease (CVD) before they reach adulthood. Rodent studies in offspring born to hyperglycemic pregnancies show vascular dysfunction characterized by impaired nitric oxide (NO)-mediated vasodilation and increased production of contractile prostanoids by cyclooxygenase 2 (COX-2). Vascular dysfunction is a key pathogenic event in the progression of diabetes-related vascular disease, primarily attributable to glucotoxicity. Therefore, glucose-induced vascular injury may stem directly from the hyperglycemic intrauterine environment of GDM pregnancy, as evinced by studies showing endothelial activation and inflammation at birth or in childhood in offspring born to GDM mothers. This review discusses potential mechanisms by which intrauterine hyperglycemia programs dysfunction in the developing vasculature.

High-titer manufacturing of SARS-CoV-2 Spike-pseudotyped VSV in stirred-tank bioreactors.

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms have been rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus (S-VSV)-vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 ± 0.42 ×106 cells/mL in 1-L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 ± 0.58 ×108 plaque-forming units (PFUs)/mL at 3 days postinfection. When compared to growth in plate-based cultures, this was a 29-fold increase in virus production, meaning a 1-L bioreactor produces the same amount of virus as 1,284 plates of 15 cm. In addition, the omicron BA.1 S-VSV reached a peak titer of 5.58 ± 0.35 × 106 PFU/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-PFU ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Immunomodulatory activity of polyphenols derived from Cassia auriculata flowers in aged rats.

The immunomodulatory activity of Cassia auriculata (CA)-derived polyphenols was tested on aged rats. Rats (24-26 months old) were given CA polyphenols supplementation at doses of 25, 50, and 100 mg/kg for 28 days. Flow cytometry analysis of CA polyphenols-treated aged rats showed increased T and B cells percentage along with enhanced proliferation of splenocytes in both resting and LPS-stimulated cells. Increased percentage of pan T cells is further supported by an elevation of CD4+, CD8+, and CD4+CD25+ regulatory cells. In terms of innate immune cell activity, CA polyphenol supplementation reduced the oxidative burst activity of neutrophils in response to PMA and Escherichia coli activation. Our results collectively show that polyphenols derived from CA boost T cell immunity by increasing the number of T cells and its sensitivity towards stimulants and decreasing ROS production by neutrophils that could potentially harm multiple biological systems in aged individuals.

Insight on hole-hole interaction and magnetic order from dichroic auger-photoelectron coincidence spectra.

The absence of sharp structures in the Auger line shapes of partially filled bands has severely limited the use of electron spectroscopy in magnetic crystals and other correlated materials. By a novel interplay of experimental and theoretical techniques we achieve a combined understanding of the photoelectron, Auger, and Auger-photoelectron coincidence spectra (APECS) of the antiferromagnetic CoO. A recently discovered dichroic effect in angle resolved (DEAR) APECS reveals a complex pattern in the Auger line shape, which is here explained in detail, labeling the final states by their total spin. Since the dichroic effect exists in the antiferromagnetic state but vanishes at the Néel temperature, the DEAR-APECS technique detects the phase transition from its local effects, thus providing a unique tool to observe and understand magnetic correlations where the usual methods are not applicable.

Double renal chimney graft using only femoral approach.

Endovascular aneurysm repair (EVAR) is a minimally invasive treatment that can be offered to most patients with an aortic aneurysm. Patients who are rejected from standard EVAR often have more extensive aortic pathology and more medical comorbidities. The advent of fenestrated and branched stent grafts gives us an opportunity to treat the most demanding aortic aneurysms endovascularly. Fenestrated stent-grafts, however, are costly and time-consuming to manufacture, which limits their applicability, especially in the emergency setting. The chimney graft is a stent placed parallel to the aortic stent-graft to preserve flow to a vital aortic branch that was overstented to obtain an adequate seal. The technique can be used as a planned operation but also as a rescue procedure to salvage a side branch unintentionally covered during EVAR. As visceral branches of the aorta are usually directed caudally these vessels are, therefore, preferably catheterized from a brachial approach. We describe a case of a successful positioning of the chimney graft using only the femoral approach. The only femoral approach to position a renal chimney graft isn't recommended for the routine procedure but it is proved to be useful in selected case and when other treatment options are excluded.

Correlation-induced memory effects in transport properties of low-dimensional systems.

We demonstrate the remnant presence of initial correlations in the steady-state electrical current jS flowing between low-dimensional interacting leads. The leads are described as Luttinger liquids and electrons can tunnel via a quantum point contact. We derive an analytic result for the time-dependent current and show that ground-state correlations have a large impact on the relaxation and long-time behavior. In particular, the I-V characteristic is not reproduced by quenching the interaction in time. We further present a universal formula of jS for an arbitrary sequence of interaction quenches and it is established that jS is history dependent for nonsmooth switching process.

[Splenic artery pseudoaneurysm: a rare case of gastrointestinal haemorrhage in a patient with chronic pancreatitis]. / Pseudoaneurisma dell'arteria splenica: insolito caso di emorragia gastrointestinale in paziente con pancreatite cronica.

A bleeding pseudoaneurysm in patients with chronic pancreatitis is a rare and potentially lethal complication. This diagnosis may be very difficult and the optimal treatment remains controversial. We report the case of 80 years old female with calcific pancreatitis and severe intestinal bleeding due to a pseudoaneurysm of the splenic artery treated with interventional radiographic embolization.

G20210A prothrombin mutation and critical limb ischaemia in patients with peripheral arterial disease.

OBJECTIVES: To assess the possible association between inherited thrombophilic alterations and the severity of peripheral arterial disease (PAD). DESIGN: A case-control study. METHODS: We evaluated the presence of G20210A prothrombin (FII) and R506Q FV Leiden mutations, antithrombin, protein C and S deficiencies in 176 patients with PAD at Fontaine's stage II and in 106 patients with critical limb ischaemia (Fontaine's stage III/IV) consecutively referred to our unit. As control group, we studied 209 apparently healthy subjects. RESULTS: The prevalence of G20210A prothrombin mutation was similar in PAD patients and controls (odds ratio (OR): 1.361; 95% confidence interval (CI): 0.552-3.355; p=0.503 after adjustment for age, sex, smoking and presence of diabetes), but was significantly higher in patients with Fontaine's stage III/IV vs. those with stage II and controls (10.4% vs. 3.4% vs. 4.3%; p=0.02, respectively). According to a logistic multivariate model that included all patients with PAD, the presence of the FII G20210A mutation (OR: 4.621; 95% CI: 1.548-13.789; p=0.006) was associated with critical limb ischaemia after adjustment for age, sex, smoking, presence of diabetes and the use of platelet aggregation inhibitors. The prevalence of the other thrombophilic alterations was not different in patients with Fontaine's stage III/IV, in patients with stage II and in controls. CONCLUSION: These hypothesis-generating data suggest that the FII 20210A allele may be considered as a genetic marker predisposing critical ischaemia in patients with PAD, justifying larger longitudinal studies.

Comparison of five specific assays for determination of dabigatran plasma concentrations in patients enrolled in the START-Laboratory Register.

INTRODUCTION: Several specific assays are commercially available to determine dabigatran anticoagulant activity. Aims of this multicenter and multiplatform study were to compare five methods for dabigatran measurement and investigate their performances in the low concentration range. METHODS: Dabigatran levels were analyzed in 295 plasma samples from patients enrolled in the START-Laboratory Register by the following methods using dedicated calibrators and controls: STA-ECA II (Diagnostica Stago), standard and low range Hemoclot Thrombin Inhibitors (Hyphen BioMed), Direct Thrombin Inhibitor Assay (Instrumentation Laboratory), Direct Thrombin Inhibitor Assay (Siemens), Technoclot DTI (Technoclone). RESULTS: Methods showed variable agreement with the Hemoclot Thrombin Inhibitors assay used as reference test, with modest under- or overestimations (Bland-Altman bias from -17.3 to 4.0 ng/mL). Limits of detection and quantification varied depending on the assay (4-52 and 7-82 ng/mL, respectively). Between-run precision and accuracy were good for all methods for both quality control levels. Assay's repeatability assessed at very low dabigatran concentrations (from 10 to 60 ng/mL) was also acceptable, variability generally increased at lower drug levels. CONCLUSION: The five dabigatran-specific assays evaluated in this study provided reliable assessment of dabigatran plasma levels, although showing different performances.

A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: a follow up.

With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan.

Variability of cut-off values for the detection of lupus anticoagulants: results of an international multicenter multiplatform study.

Essentials Between-lab variations of cut-off values in lupus anticoagulant detection are unknown. Cut-off values were calculated in 11 labs each testing plasma from 120 donors with 3 platforms. Major variation was observed even within the same platform. Cut-off values determined in different labs are not interchangeable. SUMMARY: Background Cut-off values for interpretation of lupus anticoagulant (LA) detection are poorly investigated. Aims (i) To assess whether results from healthy donors were normally distributed and (ii) the between-laboratories differences in cut-off values for screening, mixing and LA confirmation when calculated as 99th or 95th centiles, and (iii) to assess their impact on the detection rate for LA. Methods Each of 11 laboratories using one of the three widely used commercial platforms for LA detection was asked to collect plasmas from 120 healthy donors and to perform screening, mixing and LA confirmation with two methods (activated partial thromboplastin time [APTT] and dilute Russell viper venom [dRVV]). A common set of LA-positive or LA-negative freeze-dried plasmas was used to assess the LA detection rate. Results were centralized (Milano) for statistical analysis. Results and conclusions (i) Clotting times or ratios for healthy subjects were not normally distributed in the majority of cases. The take-home message is that cut-off values should be determined preferably by the non-parametric method based on centiles. (ii) There were relatively large inter-laboratory cut-off variations even within the same platform and the variability was marginally attenuated when results were expressed as ratios (test-to-normal pooled plasma). The take-home message is that cut-off values should be determined locally. (iii) There were differences between cut-off values calculated as 99th or 95th centiles that translate into a different LA detection rate (the lower the centile the greater the detection rate). The take-home message is that cut-off values determined as the 95th centile allow a better LA detection rate.

Poor comparability of coagulation screening test with specific measurement in patients receiving direct oral anticoagulants: results from a multicenter/multiplatform study.

Essentials Prothrombin and partial thromboplastin time (PT/PTT) measure direct oral anticoagulants (DOACs). PT, PTT and specific tests for DOACs were performed on patients treated for atrial fibrillation. Normal PT/PTT don't exclude DOAC activity and their prolongation doesn't confirm DOAC action. The use of PT or PTT to evaluate DOAC activity could cause dangerous misinterpretations. SUMMARY: Background Prothrombin time (PT) and activated partial thromboplastin time (APTT) have been proposed to measure the effect of oral anti-activated factor X (FXa) or anti-activated FII drugs, respectively. Aims To evaluate the relationships and responsiveness of PT and APTT versus direct oral anticoagulant (DOAC) concentrations measured with specific coagulation tests performed with different platforms in four Italian anticoagulation clinics. Methods Six hundred and thirty-five patients with atrial fibrillation participated in the study: 240 were receiving dabigatran, 264 were receiving rivaroxaban, and 131 were receiving apixaban. Blood was taken at trough and peak within the first month (15-25 days) of treatment. PT, APTT, diluted thrombin time (dTT) calibrated for dabigatran and anti-FXa calibrated for rivaroxaban or apixaban were determined. Results For dabigatran, the correlation between APTT and dTT ranged from r = 0.80 to r = 0.62. For rivaroxaban, the correlation between the anti-FXa assay and PT ranged from r = 0.91 to r = 0.73. For apixaban, the correlation between the anti-FXa assay and PT was lower than for the two other drugs (r = 0.81 to r = 0.54). Despite the above significant correlations, the responsiveness of PT or APTT was relatively poor. A discrepancy between global testing and DOAC plasma concentrations was shown in a considerable proportion of patients, depending on the platform and drug, with values ranging from 6% to 62%. Conclusions Overall, poor responsiveness of the screening tests to DOAC concentrations was observed. PT and APTT normal values cannot exclude DOAC anticoagulant activity, and PT or APTT prolongation is not always associated with DOAC anticoagulant effect as determined with specific tests.

Poor anticoagulation quality in the first 3 months after unprovoked venous thromboembolism is a risk factor for long-term recurrence.

BACKGROUND AND AIM: Several factors are associated with an increased risk of recurrent venous thromboembolism (VTE). The aim of the study was to investigate whether the quality of oral anticoagulation therapy (OAT) is a long-term risk factor for recurrence of VTE after OAT interruption. METHODS AND RESULTS: A total of 297 patients (170 males) with a recent acute unprovoked VTE episode were prospectively monitored during OAT in our anticoagulation clinic and followed up for 21 months after OAT interruption. Recurrent events were recorded in 42 subjects for 493 years of follow-up [14.1% of patients; 8.5% patient-years (pt-y)] after OAT withdrawal. The rate of recurrence was not correlated to OAT duration. Subjects experiencing recurrence after OAT interruption had spent significantly more time at markedly subtherapeutic international normalized ratio (INR) levels (<1.5) and less time within the therapeutic range (2.0-3.0 INR) during OAT. Relative risk (RR) of recurrence was significantly higher [2.77 (95% confidence interval (CI) 1.49-5.18; P = 0.001) and 2.70 (95% CI 1.39-5.25; P = 0.003) at univariate and multivariate analysis, respectively] in those who spent more time (upper quintile) at INR values <1.5, being especially evident in the first 90 days of OAT. RR was significantly higher at univariate [2.05 (95% CI 1.07-3.96; P = 0.031)] but not at multivariate [1.98 (95% CI 0.98-4.0; P = 0.056)] analysis when the entire OAT period was considered. Subjects in the upper quintile of time spent at INR values <1.5 had significantly higher D-dimer values when OAT was stopped and after 3 months. CONCLUSIONS: The amount of time that subjects with an acute unprovoked VTE event spend at near-normal INR values (<1.5) during the first 3 months of treatment is associated with higher D-dimer values measured during OAT and after its interruption and is a significant risk factor for late VTE recurrence.

Studies on lipid peroxidation and protein oxidation in the aging brain.

Lipid peroxidation (LP) and protein oxidation (PO) were investigated in hippocampus and frontal cortex homogenates from young (5 months), mature (13 months) and old (24 months) Wistar rats and young (5 months) and old (24 months) Brown Norway rats. LP and PO were determined in basal conditions and after incubation without iron (spontaneous condition) or with iron (stimulated condition). LP was measured as HPLC-assayed malondialdehyde (MDA) and PO as protein carbonyl (CO) content. Brain homogenates formed considerable amounts of MDA and CO spontaneously and, to an even greater extent, in the presence of Fe2+. Old rats showed greater iron-stimulated LP in the cortex than young rats, but the difference was not significant. Basal (but not spontaneous or stimulated) PO was significantly increased (19%) in the hippocampus of old compared to young rats. This study does not confirm the age-related increase in LP reported in the literature and only partially confirms the findings concerning PO.

Amiodarone induced phospholipidosis. Biochemical, morphological and functional changes in the lungs of rats chronically treated with amiodarone.

Amiodarone, an antiarrhythmic drug, causes pulmonary fibrosis in some patients during chronic treatment but the mechanism is unknown. We studied the effects of amiodarone on pulmonary biochemistry, morphology and function at doses of 25 and 50 mg/kg/12 hr given to rats by gavage for four weeks. Plasma and pulmonary phospholipids were significantly augmented, 13% and 88% respectively, in the group given amiodarone 50 mg/kg/12 hr compared to pair-fed controls. Typical phospholipidosis-like light and electron microscopic alterations were seen in the lung, their severity related to the extent of biochemical changes induced by amiodarone. Pulmonary function tests revealed mild but not significant changes in O2 and CO2 alveolar exchange efficiency and lung compliance (P-V curve) of treated animals in comparison to pair fed controls. Plasma average concentrations of amiodarone and its main metabolite, desethylamiodarone, after four weeks were 2.46 +/- 0.18 and 0.73 +/- 0.13 micrograms/ml, respectively, in the 50 mg/kg/12 hr group. In the same group amiodarone and desethylamiodarone concentrations in lung were 163 +/- 26 and 569 +/- 153 times higher than those in plasma. A highly significant correlation was found between amiodarone concentrations in plasma and lung and phospholipid content in the lung. A subgroup of animals received amiodarone 50 mg/kg/12 hr for 8 weeks. The pulmonary phospholipidosis-like lesions were similar to those observed after one month of treatment, no fibrosis was evident on light microscopic examination.

(R,S)-3,4-dichlorobenzoylalanine (FCE 28833A) causes a large and persistent increase in brain kynurenic acid levels in rats.

Kynurenic acid is an endogenous excitatory amino-acid receptor antagonist with neuroprotective and anticonvulsant properties. We demonstrate here that systemic administration of the new and potent kynurenine 3-hydroxylase inhibitor (R,S)-3,4-dichlorobenzoylalanine (FCE 28833A) causes a dose-dependent elevation in endogenous kynurenine and kynurenic acid levels in rat brain tissue. In hippocampal microdialysates, peak increases of 10- and 80-fold above basal kynurenic acid concentrations, respectively, were obtained after a single oral or intraperitoneal administration of 400 mg/kg FCE 28833A. After intraperitoneal treatment with FCE 28833A, extracellular brain kynurenic acid levels remained significantly elevated for at least 22 h, rendering this compound a far more effective enhancer of kynurenic acid levels than the previously described kynurenine 3-hydroxylase blocker m-nitrobenzoylalanine. FCE 28833A and similar molecules may have therapeutic value in diseases which are linked to a hyperfunction of excitatory amino-acid receptors.

Brain concentrations of kynurenic acid after a systemic neuroprotective dose in the gerbil model of global ischemia.

1. Kynurenic acid (KYNA) is a kynurenine metabolite and a broad spectrum excitatory amino acid antagonist that has been shown to be neuroprotective in models of cerebral ischemia, when administered exogenously. However, the actual concentration required in the CNS to evoke significant neuroprotection has never been assessed. 2. The purpose of this study was to address this question in the gerbil model of forebrain ischemia. KYNA (400-1600 mg/kg) or vehicle were administered i.p. 15 min before 5 min bilateral carotid occlusion. 3. Seven days after reperfusion, ischemia-induced hippocampal nerve cell loss (95% in vehicle-treated) was significantly lower in KYNA-treated gerbils (65% and 52% at 1000 and 1200 mg/Kg, respectively, P < 0.01). Treatment with 1000 mg/kg produced brain KYNA concentrations that were dramatically elevated (135.9 and 42.3 microM in CSF and whole brain, vs 0.032 and 0.16 microM in controls, at 15 min after ischemia), as measured in a separate group of transcardially-perfused gerbils. Cerebral KYNA concentrations tended to return to basal values 2 hours after reperfusion. 4. These results indicate that KYNA has a marked neuroprotective effect in a model of forebrain ischemia. This activity is associated with KYNA concentrations in the brain and CSF that are compatible with the in vitro affinity of the compound for ionotropic glutamate receptors.

The effects of aging on MAO activity and amino acid levels in rat brain.

The possibility that age-related changes in amino acid levels in various rat brain areas might correlate with modifications of monoamine oxidase (MAO) activity, already found with aging, has been examined. Taurine, aspartic acid and glutamic acid levels were found to be unchanged or decreased with age, whereas GABA and glutamine concentrations increased or remained unchanged. Serine and glycine (except in pons-medulla) levels were found to be unaffected by age. The increase in total MAO activity with aging in some brain areas might contribute to the changes in amino acid levels. Likewise, the possible influence of age-induced changes in activity of various enzymes involved in H2O2 and NH3 detoxication and in amino acid biosynthesis on rat brain amino acid levels are considered. Oral administration of clorgyline or 1-deprenyl to young rats did not significantly modify the concentrations of most brain amino acids.

Determination of retinoids in galenicals by column liquid chromatography with fluorescence and diode-array detection.

Simple and rapid reversed-phase gradient column liquid chromatography (LC) with fluorescence detection at different wavelengths was developed for the simultaneous analysis of all-trans, 13-cis, 9-cis retinoic acids, vitamin A palmitate and beta-carotene in galenicals. The assay results agreed with those obtained by an LC method with diode-array UV detection. A post-column on-line photochemical reactor (irradiation at 254 and 366 nm) was inserted between the LC column and the fluorescence detector to enhance the performance of the method. Two fluorescence spectra (photoreactor on and off) were obtained for each analyte which proved useful for the unambiguous identification of the various analytes.