Alterations in the Salivary Proteome and N-Glycome of Sjögren's Syndrome Patients.
We used isobaric mass tagging (iTRAQ) and lectin affinity capture mass spectrometry (MS)-based workflows for global analyses of parotid saliva (PS) and whole saliva (WS) samples obtained from patients diagnosed with primary Sjögren's Syndrome (pSS) who were enrolled in the Sjögren's International Collaborative Clinical Alliance (SICCA) as compared with two control groups. The iTRAQ analyses revealed up- and down-regulation of numerous proteins that could be involved in the disease process (e.g., histones) or attempts to mitigate the ensuing damage (e.g., bactericidal/permeability increasing fold containing family (BPIF) members). An immunoblot approach applied to independent sample sets confirmed the pSS associated up-regulation of β2-microglobulin (in PS) and down-regulation of carbonic anhydrase VI (in WS) and BPIFB2 (in PS). Beyond the proteome, we profiled the N-glycosites of pSS and control samples. They were enriched for glycopeptides using lectins Aleuria aurantia and wheat germ agglutinin, which recognize fucose and sialic acid/N-acetyl glucosamine, respectively. MS analyses showed that pSS is associated with increased N-glycosylation of numerous salivary glycoproteins in PS and WS. The observed alterations of the salivary proteome and N-glycome could be used as pSS biomarkers enabling easier and earlier detection of this syndrome while lending potential new insights into the disease process.


Risk factors for the clinical course of cholecystitis in patients who undergo cholecystectomy.
The aims of this study were to evaluate risk factors for acute cholecystitis that have been previously acknowledged and to evaluate several co-morbidities, such as hypertension, diabetes mellitus, cardiovascular disease, cerebrovascular accident and end-stage renal disease for which the prevalence rate has increased in the elderly. We retrospectively reviewed 611 patients who underwent laparoscopic or open cholecystectomy for cholecystitis between January 2005 and January 2010. The relationships between the clinical outcomes and the clinico-demographic factors were analyzed by univariate and multivariate analyses. The diagnoses of the 611 patients who underwent laparoscopic cholecystectomy were acute cholecystitis (n=258; 42.2%) and chronic cholecystitis (n=353; 57.8%). Male gender (p<0.000), age >50 (p<0.000), fever (p<0.000), leukocytosis (p<0.000), AST elevation (p=0.009), alkaline phosphatase elevation (p<0.000) and an elevation of total bilirubin (p<0.000) were identified as risk factors for acute cholecystitis. The presence of diabetes mellitus (p=0.002) and hypertension (p=0.019) may be risk factors for acute cholecystitis. For patients with risk factors for acute cholecystitis, early management, that is, early checkup and diagnosis following early cholecystectomy, is recommended before the disease progresses to an acute form of cholecystitis.


Recurrence and 5-FU sensitivity of stage II/Dukes' B colorectal cancer with occult neoplastic cells in lymph node sinuses.
In 103 patients without occult neoplastic cells (ONCs) in the lymph node sinuses, the 5-year relapse-free survival (RFS) rate and overall survival (OS) rate were 90.2% and 91.8%, respectively. In 21 patients with ONCs, the 5-year RFS and OS rates were 34.9% and 62.3%, respectively. There were marked differences of survival between the two groups (p=0.0000 and p=0.0003). In the primary tumors of the 21 ONC-positive patients, high and low TS levels were found in 46.2% (6/13) and 53.8% (7/13) of the recurrence group (n=13), respectively. High and low DPD levels were found in 23.1% (3/13) and 76.9% (10/13), respectively. In the non-recurrence group (n=8), high and low TS or DPD levels were found in 75.0% (6/8) and 25.0% (2/8) versus 12.5% (1/8) and 87.5% (7/8), respectively. The percentage of patients with high TS and low DPD levels was 23.1% (3/13) in the recurrence group and 62.5% (5/8) in the non-recurrence group (p=0.07). These results suggest that the presence of ONCs had a clear association with the 5-year RFS and OS rates. The recurrence group of ONC-positive patients with stage II/Dukes' B colorectal cancer was unlikely to be highly responsive to 5-FU-based treatment, thus requiring multi-combination chemotherapy using CPT-11 and/or L-OHP.


Assessment of blind source separation techniques for video-based cardiac pulse extraction.
Blind source separation (BSS) aims at separating useful signal content from distortions. In the contactless acquisition of vital signs by means of the camera-based photoplethysmogram (cbPPG), BSS has evolved the most widely used approach to extract the cardiac pulse. Despite its frequent application, there is no consensus about the optimal usage of BSS and its general benefit. This contribution investigates the performance of BSS to enhance the cardiac pulse from cbPPGs in dependency to varying input data characteristics. The BSS input conditions are controlled by an automated spatial preselection routine of regions of interest. Input data of different characteristics (wavelength, dominant frequency, and signal quality) from 18 postoperative cardiovascular patients are processed with standard BSS techniques, namely principal component analysis (PCA) and independent component analysis (ICA). The effect of BSS is assessed by the spectral signal-to-noise ratio (SNR) of the cardiac pulse. The preselection of cbPPGs, appears beneficial providing higher SNR compared to standard cbPPGs. Both, PCA and ICA yielded better outcomes by using monochrome inputs (green wavelength) instead of inputs of different wavelengths. PCA outperforms ICA for more homogeneous input signals. Moreover, for high input SNR, the application of ICA using standard contrast is likely to decrease the SNR.


Serial enrichment of spermatogonial stem and progenitor cells (SSCs) in culture for derivation of long-term adult mouse SSC lines.
Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells(1-3). Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines(4). However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state. Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors(5,6). Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies(7). Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types(8). Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC(2,3). The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)(3). Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.


Latent development of occlusive coronary atherosclerosis as a cause of decompensation of non-ischemic dilated cardiomyopathy.
Four patients with chronically well-compensated, non-ischemic dilated cardiomyopathy (NIDC) presented with occlusive atherosclerotic coronary artery disease as the cause of subacute decompensation (FC III-IV heart failure) 8-13 years following the diagnosis of NIDC. In addition to the atherogenic condition of heart failure, 3 of the patients acquired major atherosclerotic risk factors (dyslipidemia, diabetes mellitus) during the interval between the diagnoses of NIDC and problematic atherosclerotic coronary disease. For each patient, dyspnea on exertion was the primary symptom during the subacute decompensation. Only 1 patient noted precordial chest pain in the form of atypical angina during some of the dyspneic events. The diagnosis of occlusive coronary artery disease was made by coronary angiography, followed by angioplasty-stent deployment in 3 patients and coronary artery bypass surgery in 1; all improved to their baseline FC I-II status following these coronary interventions. As survival of patients with NIDC increases, occlusive coronary artery disease may enter an otherwise stable clinical course to provoke unanticipated decompensation (principally dyspnea), and can do so without causing angina pectoris as a heralding symptom.


Extending the Transport Theorem to Rough Domains of Integration.
Transport theorems, such as that named after Reynolds, are an important tool in the field of continuum physics. Recently, Seguin and Fried used Harrison's theory of differential chains to establish a transport theorem valid for evolving domains that may become irregular. Evolving irregular domains occur in many different physical settings, such as phase transitions or fracture. Here, emphasizing concepts over technicalities, we present Harrison's theory of differential chains and the results of Seguin and Fried in a way meant to be accessible to researchers in continuum physics. We also show how the transport theorem applies to three concrete examples and approximate the resulting terms numerically. Furthermore, we discuss how the transport theorem might be used to weaken certain basic assumptions underlying the description of continua and the challenges associated with doing so.


Capsular types of Vibrio vulnificus: an analysis of strains from clinical and environmental sources.
Vibrio vulnificus produces a capsular polysaccharide (CPS) that is essential for virulence. CPS from V. vulnificus clinical strain MO6-24 has been purified and the structure determined. In preliminary screening with antisera raised to MO6-24 CPS, 4 (19%) of 21 clinical isolates (including MO6-24), but none of 67 environmental V. vulnificus isolates, agglutinated with anti-MO6-24 antisera (P = .003). CPS was isolated from a subset of 12 clinical and 7 environmental isolates and analyzed by high-performance anion-exchange chromatography and one-dimensional nuclear magnetic resonance. MO6-24 and 1 other serologically positive strain had identical CPS structures; the other 2 serologically positive strains had substitutions in two of four sugar residues. Thirteen other capsular types were identified among the remaining 15 strains from which CPS was extracted.


A Jun-binding protein related to a putative tumor suppressor.
A lambda gt11 cDNA library of chicken embryo fibroblasts was screened with biotinylated Jun protein to identify Jun-binding clones. Eight such clones were isolated; one contains a gene referred to as jif-1 that is homologous to the putative tumor suppressor gene QM. jif-1 codes for a protein of 25 kDa that binds to the leucine zipper of viral and cellular Jun. The Jif-1 protein also binds to itself. Jif-1 does not contain a leucine zipper, and it does not bind to the 12-O-tetradecanoylphorbol 13-acetate response element DNA sequence. Complex formation of Jif-1 with Jun inhibits DNA binding and reduces transactivation by Jun. Addition of Fos protein to Jun-Jif-1 complexes restores DNA-binding activity. These observations suggest that Jif-1 is a negative regulator of Jun.


Cerebrovascular changes in chronic hypertension. Protective effects of enalapril in rats.
Our recent study demonstrated that in chronic hypertension, hypertrophy of intracerebral arterioles was associated with an increase in the vascular extracellular matrix proteins: fibronectin, laminin, and collagen IV. An additional cerebral finding in chronic hypertension was hypertensive encephalopathy, in which breakdown of the blood-brain barrier (BBB) to serum proteins occurred in multifocal areas of the cortex and basal ganglia. This study was undertaken to determine which of these alterations were attenuated by antihypertensive therapy. Two weeks after the surgery to produce chronic renal hypertension, half the hypertensive rats were treated orally with enalapril (30 mg/kg), an angiotensin-converting enzyme inhibitor, for 5 weeks. Rats were perfusion-fixed, and their brains were removed and processed for morphological studies. The effect of treatment on vascular hypertrophy was assessed by quantitative morphometry and on the vascular extracellular matrix proteins and BBB permeability alterations by immunohistochemistry. There was increased immunoreactivity for laminin, fibronectin, and collagen IV in pial and intracerebral arterioles of untreated hypertensive rats. Immunoreactivity was greatest in arterioles in areas with breakdown of the BBB to serum proteins. Enalapril treatment for 5 weeks resulted in a significant reduction of the mean systolic blood pressure, which was accompanied by attenuation of vascular hypertrophy and attenuation of changes in the vascular extracellular matrix proteins. In addition, there was reduction in the magnitude of BBB breakdown after treatment. Enalapril treatment had a protective effect and attenuated vascular hypertrophy and the increase in vascular extracellular matrix proteins observed in chronic hypertension. In addition, there was reduction in the magnitude of BBB breakdown.


Alterations in rheological properties and erythrocyte membrane proteins in cats with diabetes mellitus.
Many studies have shown that diabetes mellitus is associated with increased whole and blood viscosity and decreased erythrocyte deformability. It has been suggested that these abnormalities in blood rheology may play a causative role in the pathogenesis of diabetic vascular complications. However, less is known about the content and quality of membrane proteins which may contribute to abnormalities in membrane dynamic and decreased erythrocyte deformability. In the present study we analysed various rheological parameters (blood and plasma viscosity, erythrocyte deformability, haemotological parameters), in cats with non-insulin dependent diabetes mellitus (NIDDM). We also investigated alterations in erythrocyte membrane protein content by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We found that erythrocyte rigidity and plasma and whole blood viscosities were significantly higher in cats with NIDDM compared to controls. SDS-PAGE revealed that the band 5 corresponding to actin was weaker while band 4.5 corresponding to integral membrane proteins (glycophorin A, B and C) had disappeared. Also, band 4.9, which is composed of dematin (a protein with actin-bundling capacity) was lost. We suggest that the observed abnormalities in membrane proteins may play a role in reduced erythrocyte deformability associated with diabetes mellitus.


[Penetrating keratoplasty in children].
Penetrating keratoplasty in children is a high risk transplantation. Indications increased with improvement of surgical techniques and therapies. Our purpose is to study anatomic and functional results of penetrating keratoplasty in children. We report the results of a retrospective study including 23 penetrating keratoplasties (22 eyes) performed in 20 patients younger than 16 years old, between 1991 and 2000 at the department of ophthalmology at Charles Nicolle University Hospital-Tunis. Penetrating keratoplasty was performed for congenital dystrophies in 6 cases (17.3%), for acquired traumatic opacities in 4 cases (26%) and for non traumatic acquired opacities in 13 cases (56.7%). Non traumatic acquired opacities were keratoconus in 8 cases (61.5%), keratitis in 3 cases (23%) and graft rejection in 2 cases (15.5%). The mean follow-up was 41.8 months. Grafts remained clear in 20 cases (83.3%). Graft rejection occurred in 4 cases. Graft failure occurred in 3 cases (16.6%). Mean postoperative visual acuity was 3/10 (ranging from 1/20 to 7/10). Penetrating keratoplasty in children allows a satisfying anatomic success but a moderate visual improvement. Amblyopia is the major obstacle to success in children corneal grafting.


Internalized Cryptococcus neoformans Activates the Canonical Caspase-1 and the Noncanonical Caspase-8 Inflammasomes.
Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcosis in immunocompromised patients as well as immunocompetent individuals. Host cell surface receptors that recognize C. neoformans have been widely studied. However, intracellular sensing of this pathogen is still poorly understood. Our previous studies have demonstrated that both biofilm and acapsular mutant of C. neoformans are able to activate the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome. In the current study, it was found that opsonization-mediated internalization of encapsulated C. neoformans also activated the canonical NLRP3-apoptosis-associated speck-like protein containing a CARD (ASC)-caspase-1 inflammasome. In addition, the internalized C. neoformans activated the noncanonical NLRP3-ASC-caspase-8 inflammasome as well, which resulted in robust IL-1β secretion and cell death from caspase-1-deficient primary dendritic cells. Interestingly, we found that caspase-1 was inhibitory for the activation of caspase-8 in dendritic cells upon C. neorformans challenge. Further mechanistic studies showed that both phagolysosome membrane permeabilization and potassium efflux were responsible for C. neoformans-induced activation of either the canonical NLRP3-ASC-caspase-1 inflammasome or the noncanonical NLRP3-ASC-caspase-8 inflammasome. Moreover, challenge with zymosan also led to the activation of the noncanonical NLRP3-ASC-caspase-8 inflammasome in cells absent for caspase-1. Collectively, these findings uncover a number of novel signaling pathways for the innate immune response of host cells to C. neoformans infection and suggest that manipulating NLRP3 signaling may help to control fungal challenge.


Which approach is more effective in the selection of plants with antimicrobial activity?
The development of the present study was based on selections using random, direct ethnopharmacological, and indirect ethnopharmacological approaches, aiming to evaluate which method is the best for bioprospecting new antimicrobial plant drugs. A crude extract of 53 species of herbaceous plants collected in the semiarid region of Northeast Brazil was tested against 11 microorganisms. Well-agar diffusion and minimum inhibitory concentration (MIC) techniques were used. Ten extracts from direct, six from random, and three from indirect ethnopharmacological selections exhibited activities that ranged from weak to very active against the organisms tested. The strain most susceptible to the evaluated extracts was Staphylococcus aureus. The MIC analysis revealed the best result for the direct ethnopharmacological approach, considering that some species yielded extracts classified as active or moderately active (MICs between 250 and 1000 µg/mL). Furthermore, one species from this approach inhibited the growth of the three Candida strains. Thus, it was concluded that the direct ethnopharmacological approach is the most effective when selecting species for bioprospecting new plant drugs with antimicrobial activities.


Supraspinatus Muscle Tendon Lesion and Its Relationship with Long Head of the Biceps Lesion.
Objective To identify the clinical, radiological, and arthroscopic correlation of long head of the biceps tendon injuries and their influence on pain when associated with rotator cuff injuries. Methods Between April and December 2013, 50 patients were evaluated, including 38 (76%) women and 12 (24%) men, with a mean age of 65.1 years old. The patients were operated by the Shoulder and Elbow Group, Discipline of Sports Medicine, Orthopedics and Traumatology Department, Universidade Federal de São Paulo. The subjects underwent repair of the rotator cuff lesion with clinical, radiological and/or arthroscopic evidence of involvement of the long head of the biceps tendon. Results An association between pain at palpation of the intertubercular groove of the humerus and high-grade partial lesions (partial rupture of the tendon affecting more than 50% of its structure) was observed at the arthroscopy ( p = 0.003). There was also an association between the high-grade lesion of the long head of the biceps and injury to the supraspinatus muscle tendon ( p < 0.05). For each centimeter of the supraspinatus muscle tendon injury, the patient presented a 1.7 higher probability of having a high-grade lesion at the long head of the biceps. Conclusion Pain at the anterior shoulder region during palpation of the intertubercular groove of the humerus may be related to high-grade lesions to the long head of the biceps. Rotator cuff injury and its size are risk factors for high-grade injuries to the long head of the biceps tendon.


Tumor inhibitory triazenes. 3. Dealkylation within an homologous series and its relation to antitumor activity.
The in vivo antitumor activity and in vitro metabolic dealkylation have been measured for an homologous series of 3-alkyl-1-(4-carbamoylphenyl)-3-methyltriazenes and have been compared with their partition coefficients. This investigation has shown that the extent of oxidative metabolism in vitro and the antitumor activity in vivo of these compounds are dependent upon hydrophobicity. These findings provide confirmation for the relationship between metabolism and antitumor activity for aryldialkyltriazenes.


Jaguar (Panthera onca Linnaeus, 1758) roadkill in Brazilian Atlantic Forest and implications for species conservation.
We report the roadkill of a jaguar in one of the longest highways in Brazil (BR-101), in the stretch where this road crosses one of the most important Atlantic Forest remnants in the country: the Linhares-Sooretama block. The jaguar population present in this area represents the very last in entire Espírito Santo state. There is an approved project to the lines duplication of the entire BR-101 Highway and the company responsible by the work has already started the first activities in the state. However, there is no environmental impact assessment already done neither planning for the implementation of measures to avoid or reduce the roadkill risk in the region. Thus, to minimize the impacts associated with the BR-101, we do not recommend its lines duplication along the 15 km stretch traversing the Linhares-Sooretama block. In addition, alternatively, we suggest the deviation of the current route of the BR-101 Highway or the construction of overpasses to fauna in the most critical points, interspersing these overpasses with electronic speed monitoring devices and warning and educational plates.


Nothing is perfect: the safety issues of integrase inhibitor regimens.
Since the administration of the first integrase strand transfer inhibitor (INSTI) in 2007, most international treatment guidelines consider INSTI-based regimens to be the preferred antiretroviral combinations for HIV-1-infected patients as a result of their safety and efficacy profile. INSTIs are generally well tolerated, and reported rates of discontinuation due to drug-related adverse events (AEs) have been very low to date. However, recent reports indicate that physicians should be aware of potential INSTI-related AEs to ensure good clinical practice. The authors performed a critical review of the safety issues affecting INSTIs based on published evidence from original studies and new data from researchers. Almost all antiretroviral drugs, including INSTIs, are associated with undesirable AEs. Dolutegravir in particular has been associated with more frequent AEs such as neuropsychiatric disorders, neural tube defect in newborns, and weight gain. Data with bictegravir in routine practice are still scarce. While this association and its clinical relevance are not clear, physicians should be alert to the appearance of the aforementioned AEs and others in the future. In the meantime, INSTIs continue to be the preferred option in guidelines on antiretroviral therapy.


Deficient Mechanical Activation of Anabolic Transcripts and Post-Traumatic Cartilage Degeneration in Matrilin-1 Knockout Mice.
Matrilin-1 (Matn1), a cartilage-specific peri-cellular and extracellular matrix (ECM) protein, has been hypothesized to regulate ECM interactions and transmit mechanical signals in cartilage. Since Matn1 knock-out (Matn1-/-) mice exhibit a normal skeleton, its function in vivo is unclear. In this study, we found that the anabolic Acan and Col2a transcript levels were significantly higher in wildtype (Matn1+/+) mouse cartilage than that of MATN1-/- mice in vivo. However, such difference was not observed between Matn1+/+ and MATN1-/- chondrocytes cultured under stationary conditions in vitro. Cyclic loading significantly stimulated Acan and Col2a transcript levels in Matn1+/+ but not in MATN1-/- chondrocytes. This suggests that, while Matn1+/+ chondrocytes increase their anabolic gene expression in response to mechanical loading, the MATN1-/- chondrocytes fail to do so because of the deficiency in mechanotransduction. We also found that altered elastic modulus of cartilage matrix in Matn1-/- mice, suggesting the mechanotransduction has changed due to the deficiency of Matn1. To understand the impact of such deficiency on joint disease, mechanical loading was altered in vivo by destabilization of medial meniscus. While Matn1+/+ mice exhibited superficial fissures and clefts consistent with mechanical damage to the articular joint, Matn1-/- mice presented more severe cartilage lesions characterized by proteoglycan loss and disorganization of cells and ECM. This suggests that Matn1 deficiency affects pathogenesis of post-traumatic osteoarthritis by failing to up-regulate anabolic gene expression. This is the first demonstration of Matn1 function in vivo, which suggests its protective role in cartilage degeneration under altered mechanical environment.


A toolkit for assembly of targeting clones for C. elegans transgenesis.
Transgenic worms are a key resource for C. elegans researchers dissecting molecular pathways using this simple metazoan model system. Transgenes provide an avenue to visualize developmental events, cellular processes as well as real-time signal events in live animals using genetically encoded sensors. Generation of these tools has become increasingly efficient with the advent of numerous integration methods including transposon, CRISPR and recombinase-mediated integration. A growing limitation in transgene production is the assembly of the targeting constructs used to direct insertion of sequences into the genome. Here we present a toolkit that facilitates rapid assembly of complex reporters using a Golden Gate (GG) cloning-based approach. Co-assembly of one to eight DNA segments into an integration vector can be routinely obtained at high efficiency using a library of entry plasmids. The toolkit consists of 20 SapI GG entry vectors and 100 SapI GG insert plasmids containing a variety of promoters, FPs, tags, linkers, ORFs, 3' UTRs and numerous components for bipartite expression systems that can be mixed to create a huge repertoire of reporter constructs. The assembly process also works well with PCR products and 5' phosphorylated double stranded oligonucleotides, and such DNAs can be used to supply novel genes, promoters, and tags into the pipeline. In addition, the toolkit also provides a series of 12 empty BsaI -based GG assembly vectors that facilitate the construction of additional SapI GG plasmids containing novel inserts. A manual outlining the entire approach is provided as an appendix as well as a Microsoft® Excel based assembly tool which allows the user to choose individual inserts among the libraries of clones at each position in the assembly template and output an annotated sequence. The assembly process can easily be multiplexed and is typically over 90% efficient. The approach is sufficiently efficient to make microinjection rather than clone generation the limiting factor in transgene generation.


L-carnitine for cognitive enhancement in people without cognitive impairment.
Safe interventions to enhance cognitive function in cognitively healthy people would be very valuable for several reasons, including a better quality of life and professional success. While L-carnitine has been reported to enhance cognitive function in some conditions, its efficacy is disputed. The evidence of its efficacy for cognitively healthy people has not previously been systematically reviewed. To assess the efficacy and safety of L-carnitine for the enhancement of cognitive function in people without cognitive impairment. We searched ALOIS, the Cochrane Dementia and Cognitive Improvement Group's Specialized Register, on 4 November 2016. We used the search terms 'L-carnitine' or 'acetyl-L-carnitine' or 'propionyl-L-carnitine' or 'ALC' or 'PLC' or 'ALCAR' or 'ALPAR'. We ran additional separate searches in several other sources to ensure that we retrieved the most up-to-date results. We also reviewed the bibliographies of the randomised controlled trials identified and contacted the authors and known experts in the field and pharmaceutical companies to identify additional published or unpublished data. Eligible trials were randomised controlled trials (RCTs) or quasi-RCTs, parallel-group or cross-over, that compared L-carnitine or its derivatives, acetyl-L-carnitine or propionyl-L-carnitine, at any dose and for any length of treatment, with placebo or no treatment in cognitively healthy people of any age and either gender. We used standard methodological procedures expected by Cochrane. Two review authors independently selected trials and evaluated the methodological quality, then extracted and analysed data from the included trials. Only two RCTs were eligible. One was a cross-over trial with 18 participants. The other randomised 400 participants to one of four treatments, of which two (L-carnitine and placebo) were relevant to this review, but the exact numbers of participants in these two treatment groups was not reported. All participants were young adults. Methodological details were poorly reported, and we considered the risk of bias in both studies to be unclear. The trials assessed different cognitive outcomes. We could extract cognitive data on approximately 200 participants from one trial. We found no evidence that L-carnitine has any effect on reaction time, vigilance, immediate memory, or delayed recall after three days of treatment. This trial report stated that there was a small number of adverse effects, none of which were serious. The small cross-over trial also reported no effect of L-carnitine on cognition, but did not provide data; no information was provided on adverse effects. We considered the available evidence to be of very low quality for all reported outcomes. Due to the limited number of included trials, short-term treatment, and inadequate reporting, we were unable to draw any conclusions about the efficacy or safety of L-carnitine for cognitive enhancement in healthy adults. Well-designed, randomised, placebo-controlled trials of L-carnitine for cognition enhancement in cognitively healthy people, with large samples and relatively long-term follow-up, are still needed.


Nuclear deadenylation/polyadenylation factors regulate 3' processing in response to DNA damage.
We previously showed that mRNA 3' end cleavage reaction in cell extracts is strongly but transiently inhibited under DNA-damaging conditions. The cleavage stimulation factor-50 (CstF-50) has a role in this response, providing a link between transcription-coupled RNA processing and DNA repair. In this study, we show that CstF-50 interacts with nuclear poly(A)-specific ribonuclease (PARN) using in vitro and in extracts of UV-exposed cells. The CstF-50/PARN complex formation has a role in the inhibition of 3' cleavage and activation of deadenylation upon DNA damage. Extending these results, we found that the tumour suppressor BARD1, which is involved in the UV-induced inhibition of 3' cleavage, strongly activates deadenylation by PARN in the presence of CstF-50, and that CstF-50/BARD1 can revert the cap-binding protein-80 (CBP80)-mediated inhibition of PARN activity. We also provide evidence that PARN along with the CstF/BARD1 complex participates in the regulation of endogenous transcripts under DNA-damaging conditions. We speculate that the interplay between polyadenylation, deadenylation and tumour-suppressor factors might prevent the expression of prematurely terminated messengers, contributing to control of gene expression under different cellular conditions.


The site of aromatization in the mouse cryptorchid testis.
Using the mouse cryptorchid model, degenerations of germ cells were observed as well as a reduced size of seminiferous tubules, while the area of the interstitial tissue increased. Aromatase, the enzyme responsible for the conversion of androgens into oestrogens, was immunolocalized in Leydig cells and in germ cells from both scrotal and abdominal testes, and in Sertoli cells only in a control testis. In the cryptorchid testis, aromatase was strongly expressed in a few tubules, including those spermatids that were still present. Other cells inside the tubules were negative for aromatase. In both testes, oestrogen receptors alpha were expressed only in Leydig cells. Strong aromatase expression in germ cells indicates an additional source of oestrogens in the testis besides the interstitial tissue.


Taxonomic reappraisal of the sphagesaurid crocodyliform Sphagesaurus montealtensis from the Late Cretaceous Adamantina Formation of São Paulo State, Brazil.
Sphagesaurus montealtensis is a sphagesaurid whose original description was based on a comparison with Sphagesaurus huenei, the only species of the clade described to that date. Better preparation of the holotype and the discovery of a new specimen have allowed the review of some characteristics and the identification of several synapomorphies of S. montealtensis with the genus Caipirasuchus: presence of antorbital fenestra; external nares bordered only by the premaxillae; premaxilla with four teeth and one diastema (between the 3rd-4th teeth); one diastema between the 4th premaxillary tooth and the 1" maxillary tooth; dentary with ten teeth and two diastemata (between the 4th-5th and 5th-6th teeth); nasal with a groove parallel to the suture with the frontal bone; nasal long, with an acute anterior margin touching anterolaterally the premaxilla, jugal is a straight bar in the lateral view; frontal is longer than wide; a dorsoventrally expanded and vertically oriented quadrate with a groove separating the medial and lateral condyles; the frontal has a discrete sagittal crest; dentary with six posterior sphagesauriform teeth and four anterior conical teeth, the first three are the smallest of the series and the fourth is slightly laterally compressed. The referral of S. montealtensis to the genus Caipirasuchus, as Caipirasuchus montealtensis comb. nov. is proposed here, based on the new taxonomic observations and the results of a phylogenetic analysis.


Relationship Between Endplate Defects, Modic Change, Facet Joint Degeneration, and Disc Degeneration of Cervical Spine.
The ''disc degeneration precedes facet joint osteoarthritis'' hypothesis and multidimensional analysis were actively discussed in lumbar spine. However, in cervical spine degeneration, the multifactorial analyzes of disc degeneration (DD), Modic changes (Mcs), facet degeneration, and endplate degeneration (ED) is still limited. In this cross-sectional study, we aimed to analyze the prevalence and interrelationship of cervical DD parameters. We retrospectively recruited 62 patients aged between 60 and 70 years. The disc height, segmental angle, ossified posterior longitudinal ligament (OPLL), ED, facet joint degeneration (FD), uncovertebral joint degeneration (UD), DD, spinal stenosis (SS), Mc, and cord signal change (CS) were evaluated using a previously well-known grading system. The prevalence of cervical degenerative parameters were DD (grade 1, 1.2%; grade 2, 13.3%; grade 3, 54.8%; grade 4, 19.0%; grade 5, 11.7%), OPLL (26.2%), SS (grade 0, 7.7%; grade 1, 42.3%; grade 2, 26.2%; grade 3, 23.8%), UD (39.1%), ED (normal, 69.0%; focal defect, 9.7%; corner defect, 11.7%; erosion, 6.9%; sclerosis, 2.8%), and FD (normal, 48.8%; narrowing, 27.0%; hypertrophied, 24.2%). The interrelationship of degenerative parameters showed close relation between UD, SS, DD, OPLL, Mc. ED, and CS has partial relation with degenerative finding. FD only has relation with UD, and Mc. Our results may indicate that FD is a degeneration that occurs independently, rather than as a result of other degenerative factors.


History of Overweight/Obesity as Predictor of Care Received at 1-year Follow-Up in Adolescents With Anorexia Nervosa or Atypical Anorexia Nervosa.
Previous research has indicated that patients with anorexia nervosa (AN) or atypical AN with premorbid history of overweight/obesity have greater weight loss and longer illness duration than patients with no such history. However, little is known about the association of premorbid overweight/obesity and receiving inpatient medical care during treatment for an eating disorder. Using logistic regression, we sought to determine if history of overweight/obesity was associated with receiving inpatient medical care in a sample of 522 patients (mean age 15.5 years, 88% female) with AN/atypical AN. Binary results demonstrated greater percent weight loss (27.4% vs. 16.2%) and higher percent median body mass index (%mBMI, 99.8% vs. 85.2%) at presentation in those with a history of overweight/obesity (p < .001) but no difference in duration of illness (p = .09). In models adjusted for demographics and percent weight loss, history of overweight/obesity was associated with lower odds of receiving inpatient medical care (odds ratio .60 [95% confidence interval: .45-.80]) at 1-year follow-up. However, these associations were no longer significant after adjusting for %mBMI. Mediation results suggest that %mBMI fully mediates the relationship between history of overweight/obesity and inpatient medical care, in that those with a history of overweight/obesity are less likely to receive care due to presenting at a higher weight. Our findings suggest that, despite greater degree of weight loss and no difference in duration of illness, participants with a history of overweight/obesity are less likely to receive inpatient medical care.


[Health checks in the Netherlands difficult to control].
The Dutch Health Inspectorate has reviewed 22 health-check providing organisations, which according to the Dutch 'Wet op het Bevolkingsonderzoek' (Population Screening Act) might need a license. 20 organisations did require a license, mainly because they used cancer tests or radiation. The providers offered a considerable variety of tests, many of which were not clearly evidence-based. Patients were insufficiently educated about the content and risks of the tests. Gaps in the legislation make it difficult to control proper compliance with the law. The Dutch Health Inspectorate recommends the following improvements: better communication with professional organisations, better patient education, writing a guideline on health checks and updating legislation to reflect the latest practices concerning health checks.


Cross-species protection: Schistosoma mansoni Sm-p80 vaccine confers protection against Schistosoma haematobium in hamsters and baboons.
The ability of the Schistosoma mansoni antigen, Sm-p80, to provide cross-species protection against Schistosoma haematobium challenge was evaluated in hamster and baboon models. Pronounced reduction in worm burden (48%) and in tissue egg load (64%) was observed in hamsters vaccinated with recombinant Sm-p80 admixed with glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). Similarly, in baboons, the Sm-p80/GLA-SE vaccine produced a 25% reduction in S. haematobium adult worms and decreased the egg load in the urinary bladder by 64%. A 40% and 53% reduction in fecal and urine egg output, respectively, was observed in vaccinated baboons. A balanced pro-inflammatory (Th17 and Th1) and Th2 type of response was generated after vaccination and appears indicative of augmented prophylactic efficacy. These data on cross-species protection coupled with the prophylactic, therapeutic and antifecundity efficacy against the homologous parasite, S. mansoni, reinforces Sm-p80 as a promising vaccine candidate. It is currently being prepared for GMP-compliant manufacture and for further pre-clinical development leading to human clinical trials. These results solidify the expectation that the Sm-p80 vaccine will provide relief for both the intestinal and the urinary schistosomiasis and thus will be greatly beneficial in reducing the overall burden of schistosomiasis.


Clinical implications of N-(2-hydroxypropyl)methacrylamide copolymers.
Different anticancer drugs, farmorubicin, doxorubicin, paclitaxel and cis-platin have been conjugated through a Gly-Phe-Leu-Gly tetrapeptide side chain to a water-soluble synthetic polymeric carrier based on N-(2-hydroxypropyl)methacryalmide (HPMA) non-targeted or targeted with galactosamine and/or human IVIg and used in Phase I clinical trials. Conjugation of the drugs to the polymeric carrier that is non-toxic and non-immunogenic in man significantly decreased their non-specific organ toxicities and increased maximum tolerated dose up to 5 times. Macromolecular therapeutics based on HPMA have radically different pharmacokinetics. Drugs are not released from their polymeric carrier and remain in the peripheral blood and urine of patients mostly in their polymer-bound form. A clinical response against some refractory cancers was recorded in Phase I clinical trials. It was also demonstrated that doxorubicin-HPMA copolymer conjugates containing an immunoglobulin moiety have both cytostatic and immunomobilizing activity.


Metformin: A Novel but Controversial Drug in Cancer Prevention and Treatment.
Metformin, a biguanide derivative that is widely used for treating type 2 diabetes mellitus, has recently been shown to exert potential anticancer effects. Many retrospective data and laboratory studies suggest the idea that metformin has antineoplastic activity, but some other studies reach conflicting conclusions. Although the precise molecular mechanisms by which metformin affects various cancers have not been fully elucidated, activation of AMPK-dependent and AMPK-independent pathways along with energy metabolism aberration, cell cycle arrest and apoptosis or autophagy induction have emerged as crucial regulators in this process. In this Review, we describe the role of metformin in the prevention and treatment of a variety of cancers and summarize the molecular mechanisms that are currently well documented in the ability of metformin as an anticancer agent. In addition, the scientific and clinical hurdles regarding the potential role of metformin in cancer will be discussed.


Effect of primrose oil on serum lipids and blood pressure in hyperlipidemic subjects.
Primrose oil (about 72% of fatty acids 18:2 omega 6 and 9% of 18:3 omega 6) was supplemented in addition to the used diet of 20 hyperlipidemic patients for 3 months in an open study. The dose of primrose oil was 2.4 ml, 4.8 ml and 7.2 ml during the first, second and third month, respectively. Primrose oil increased the proportion of gamma-linolenic acid in serum cholesteryl esters (1.81 vs. 2.13%, p less than 0.05), phospholipids (0.65 vs. 0.82%, p less than 0.01), triglycerides (0.70 vs. 0.99%, p less than 0.01) and free fatty acids (0.80 vs. 0.91%, NS). It did not, however, change serum cholesterol, HDL cholesterol or triglyceride mean values. Blood pressure mean values remained unchanged during the follow-up as well.


Trends in diabetic retinopathy and related medical practices among type 2 diabetes patients: Results from the National Insurance Service Survey 2006-2013.
The present study aimed to analyze the temporal changes in the prevalence, screening rate, visual impairments and treatment patterns of diabetic retinopathy in the Korean population over 8 years. This was a retrospective population-based study of Korean national health insurance beneficiaries aged 30 years or older with type 2 diabetes, obtained from the Korean National Health Insurance Claims database from 2006 to 2013 (n = 1,655,495 in 2006 and 2,720,777 in 2013). The annual prevalence rates of diabetes, diabetic retinopathy, dilated fundus examinations, visual impairment, laser treatment and vitrectomy, as determined based on diagnostic and treatment codes, were analyzed. There was a steady increase in the prevalence of diabetic retinopathy, from 14.3% in 2006 to 15.9% in 2013. However, the incidence of new diabetic retinopathy cases decreased from 6.7/100 person-years in 2006 to 5.6 in 2013. Approximately 98% of patients underwent at least one dilated fundus examination during the follow-up period. The prevalence of diabetic retinopathy peaked in the 60-69 years age group. The prevalence of diabetic retinopathy was higher in female than in male diabetes patients. The proportion of patients who underwent an annual dilated fundus examination improved from 24.3% in 2006 to 30.0% in 2013. Among patients with diabetic retinopathy, constant decreases in the proportions of those who received laser treatment (11.4% in 2006 to 6.9% in 2013) and who underwent vitrectomy (2.4% in 2006 to 1.7% in 2013) were noted. Additionally, a decreasing trend in the prevalence of visual impairment was noted among the patients with diabetic retinopathy, from 2% (4,820/237,267) in 2006 to 0.08% (3,572/431,964) in 2013. Although there was a rapid increase in the prevalence of diabetes in the Korean population in the past decade, the prevalence of diabetic retinopathy remained stable during the study period. However, just three out of 10 patients with diabetes underwent regular annual dilated fundus examinations. Thus, an improvement in the continuity of diabetic retinopathy screening among patients with diabetes is necessary to reduce the risk of visual impairment as a result of diabetic retinopathy.


Comparative evaluation of three FDA-approved HIV Ag/Ab combination tests using a genetically diverse HIV panel and diagnostic specimens.
HIV Ag/Ab combination assays are recommended by CDC for routine screening and several HIV Ag/Ab combination tests are now FDA-approved. Maintaining high specificity and consistent sensitivity across diverse HIV strains is critical for these assays to accurately detect HIV infection and expedite delivery of patient results. To evaluate performance of three FDA-approved HIV tests: ARCHITECT HIV Combo (Abbott), ADVIA Centaur HIV Combo (Siemens) and BioPlex HIV Ag-Ab (Bio-Rad). Sensitivity and specificity were evaluated using an extensive panel of 28 HIV infected human specimens and 17 cultured virus isolates representing multiple genotypes, 6 seroconversion panels, 4 human samples with acute infection, WHO p24 standard and 4020 clinical specimens. The p24 limit of detection (LOD) for the WHO standard was 0.19IU/ml, 0.70IU/ml, and 1.77IU/ml in BioPlex, ARCHITECT, and Centaur respectively. The distribution of LODs across 15 HIV-1 isolates was substantially narrower in ARCHITECT (5-33pg/ml) than in BioPlex (11-198pg/ml) and Centaur (6-384pg/ml). All assays detected antibodies to the majority of HIV-1 and HIV-2 variants. However, reduced sensitivity was observed for Centaur in detection of antibodies to HIV-1 group M (CRF02_AG), O and N variants. BioPlex and ARCHITECT showed better seroconversion sensitivity than Centaur, detecting one bleed (3-7 days) earlier in 4 (BioPlex) and 3 (ARCHITECT) of 6 seroconversion panels. ARCHITECT demonstrated the highest specificity (99.90-100%) compared to BioPlex (99.80%) and Centaur (99.42%). The overall performance of ARCHITECT and BioPlex was superior to Centaur, especially for detection of acute HIV infection.


[Usefulness of oral calcium test in renal lithiasis].
We report the results of an oral tolerance test performed in 317 patients with kidney stones. In order to avoid PTH or AMPc measurements, and therefore to reduce costs and time to get the results, we measured the tubular maxima of phosphate per glomerular filtration rate (TmP04/GFR, the phosphate threshold). Urine collections from 7 to 9 h and from 9 to 13 h were obtained. The samples were analyzed for calcium, creatinine and phosphorus content. All patients ingested 1 g of calcium mixed in a meal at 9 o'clock. Venous blood samples were obtained for calcium, creatinine and phosphorus measurements, previous to the calcium ingestion. Urinary calcium to creatinine ratio, before and after the calcium-load, as well as TmP04/GFR were calculated. In 97 subjects (30.8%) there were no calcium metabolism abnormalities. Idiopathic hypercalciuria was present in 183 (57%) and primary hyperparathyroidism in 37 (11.7%). Idiopathic hypercalciuria was classified in four subgroups: absorptive hypercalciuria with normal serum phosphorus, absorptive hypercalciuria with low serum phosphorus (renal phosphate leak), renal hypercalciuria with normal phosphorus and renal hypercalciuria with low serum phosphorus.


Preoperative Anemia Is Associated With Failure of Open Debridement Polyethylene Exchange in Acute and Acute Hematogenous Prosthetic Joint Infection.
Acute and acute hematogenous prosthetic joint infections (PJIs) are often treated with open debridement and polyethylene exchange (ODPE) in an effort to save the prosthesis, decrease morbidity, and reduce costs. However, failure of ODPE may compromise a subsequent 2-stage treatment. The purpose of this study is to identify patient factors that impact the success of ODPE for acute and acute hematogenous PJIs. A retrospective review examined comorbidities, preoperative laboratory values, and patient history for patients with successful and failed ODPE treatment for acute perioperative or acute hematogenous periprosthetic hip or knee joint infections. Successful treatment was defined as retaining a well-fixed implant without the need for additional surgery for a minimum of 6-month follow-up with or without lifelong oral maintenance antibiotics. Fifty-three of 72 patients (73.6%) underwent successful ODPE. Of the 19 failures, 14 completed 2-stage revision with one subsequent known failure for recurrent infection. Patients with a Staphylococcus aureus infection were more likely to fail ODPE (48.3% vs 11.6%, P = .0012, odds ratio 7.1, 95% confidence interval 2.3-25.3). Patients with a preoperative hematocrit ≤32.1 were also more likely to fail ODPE (55% vs 16%, P = .0013, odds ratio 6.7, 95% confidence interval 2.2-22.4). When neither risk factor was present, 97.1% of PJIs were successfully treated with ODPE. S aureus infection and preoperative hematocrit ≤32.1 are independent risk factors for ODPE failure. ODPE is a safe alternative to 2-stage revision in patients without preoperative anemia and without S aureus infection. Two-thirds of patients with a failed ODPE were successfully treated with a 2-stage reimplantation.


Sex differences in vocal learning ability in songbirds are linked with differences in flexible rhythm pattern perception.
Humans readily recognize familiar rhythmic patterns, such as isochrony (equal timing between events) across a wide range of rates. This reflects a facility with perceiving the relative timing of events, not just absolute interval durations. Several lines of evidence suggest this ability is supported by precise temporal predictions arising from forebrain auditory-motor interactions. We have shown previously that male zebra finches, Taeniopygia guttata, which possess specialized auditory-motor networks and communicate with rhythmically patterned sequences, share our ability to flexibly recognize isochrony across rates. To test the hypothesis that flexible rhythm pattern perception is linked to vocal learning, we ask whether female zebra finches, which do not learn to sing, can also recognize global temporal patterns. We find that females can flexibly recognize isochrony across a wide range of rates but perform slightly worse than males on average. These findings are consistent with recent work showing that while females have reduced forebrain song regions, the overall network connectivity of vocal premotor regions is similar to males and may support predictions of upcoming events. Comparative studies of male and female songbirds thus offer an opportunity to study how individual differences in auditory-motor connectivity influence perception of relative timing, a hallmark of human music perception.


Viral meningitis-associated hospitalizations in the United States, 1988-1999.
We used the National Hospital Discharge Survey and the Nationwide Inpatient Sample of the Health Care Cost and Utilization Project to estimate disease burden associated with viral meningitis hospitalizations in the United States. During 1988-1999, viral meningitis accounted for an estimated 434,000 hospitalizations (annual average, 36,000; average annual hospitalization rate, 14/100,000), and 2.1 million hospital days (annual average, 175,000). The estimated mean charge for viral meningitis-associated hospitalization during 1993-1997 varied between USD 6,562 and 8,313, resulting in annual estimated hospitalization costs between USD 234 and 310 million and a total estimated cost of nearly USD 1.3 billion for the 5-year period. In summary, viral meningitis remains an important cause of morbidity and financial burden and merits efforts to improve diagnostic, treatment, and prevention options.


Early responses to acute ozone exposure in two Fagus sylvatica clones differing in xeromorphic adaptations: photosynthetic and stomatal processes, membrane and epicuticular characteristics.
Two Fagus sylvatica L. clones were used to investigate the early responses to acute O3 exposure (150 nL L(-1), i.e., 1.35x ambient hourly peak in rural Italy) and whether xeromorphic adaptations affect gas exchange, membrane, and epicuticular responses. One clone originated in a wet and temperate climate in Central Italy (Tuscany); the other clone originated in a warmer and drier climate in the southern-most part of the F. sylvatica distribution (Sicily). Because of higher base gas exchange rates, the most negative effects of O3 exposure (gas exchange impairment, uncoupling between net photosynthesis and stomatal conductance, increased membrane lipid peroxidation) were found in the southern clone. Xeromorphic adaptations (higher epicuticular waxes and stomatal density, lower leaf wettability and size) were found in this clone. Our results suggest that xeromorphism may increase O3 sensitivity in species not adapted to face water stress, like the mesophilic F. sylvatica, when experiments are carried out with full irrigation. We present evidence describing the relationship between gas exchange and number and status of stomata. Stomatal density and the structural damage to stomata resulting from O3 exposure did not affect gas exchange: In fact, non-stomatal limitations to photosynthesis prevailed over stomatal limitations.


Blockchain, consent and prosent for medical research.
Recent advances in medical and information technologies, the availability of new types of medical data, the requirement of increasing numbers of study participants, as well as difficulties in recruitment and retention, all present serious problems for traditional models of specific and informed consent to medical research. However, these advances also enable novel ways to securely share and analyse data. This paper introduces one of these advances-blockchain technologies-and argues that they can be used to share medical data in a secure and auditable fashion. In addition, some aspects of consent and data collection, as well as data access management and analysis, can be automated using blockchain-based smart contracts. This paper demonstrates how blockchain technologies can be used to further all three of the bioethical principles underlying consent requirements: the autonomy of patients, by giving them much greater control over their data; beneficence, by greatly facilitating medical research efficiency and by reducing biases and opportunities for errors; and justice, by enabling patients with rare or under-researched conditions to pseudonymously aggregate their data for analysis. Finally, we coin and describe the novel concept of prosent, by which we mean the blockchain-enabled ability of all stakeholders in the research process to pseudonymously and proactively consent to data release or exchange under specific conditions, such as trial completion.


Phytochemical analysis of Saponaria officinalis L. shoots and flowers essential oils.
Phytochemical analysis by GC and GC/MS of the essential oil samples obtained from fresh shoots and flowers of Saponaria officinalis L. allowed the identification of 96 components in total, comprising 94.7% and 86.0% of the total oils compositions, respectively. Regarding the shoots essential oil, the major of 87 identified volatile compounds were phytol (14.1%), tricosane-6,8-dione (13.4%), patchouli alcohol (7.9%) and tricosane (7.2%), whereas patchouli alcohol (20.0%), heneicosane (11.5%) and tricosane (8.4%) were dominant among the 66 volatiles in the flower oil. Nonterpenoid compounds had the highest contribution in S. officinalis shoots essential oil (53.7%), while in the flower oil, constituents were almost evenly distributed between the oxygenated sesquiterpenoid (41.2%) and nonterpenoid compounds (39.5%).


Marangoni Bursting: Evaporation-Induced Emulsification of Binary Mixtures on a Liquid Layer.
Adjusting the wetting properties of water through the addition of a miscible liquid is commonly used in a wide variety of industrial processes involving interfaces. We investigate experimentally the evolution of a drop of water and volatile alcohol deposited on a bath of oil: The drop spreads and spontaneously fragments into a myriad of minute droplets whose size strongly depends on the initial concentration of alcohol. Marangoni flows induced by the evaporation of alcohol play a key role in the overall phenomenon. The intricate coupling of hydrodynamics, wetting, and evaporation is well captured by analytical scaling laws. Our scenario is confirmed by experiments involving other combinations of liquids that also lead to this fascinating phenomenon.


The efficacy and safety of tacrolimus monotherapy in adult-onset nephrotic syndrome caused by idiopathic membranous nephropathy.
The purpose of the study is to evaluate the efficiency and safety of tacrolimus (TAC) monotherapy in the treatment of nephrotic idiopathic membranous nephropathy (IMN) compared with the protocol of cyclophosphamide (CTX) combined with corticosteroids. In total, 58 patients with nephrotic syndrome and biopsy-proven IMN were included in this study. 30 patients received TAC monotherapy with an initial dose of 0.05-0.1 mg/kg/day. 28 patients received transvenous CTX at a dose of 0.5-0.75 g/m2 once in every month initially for 6 months and once in every 2 or 3 months for the later period, and the regimen was combined with corticosteroids (prednisone 1 mg/kg/d). All patients were observed for the treatment effects, recurrence and side effects. Twelve months after the initial treatment, a total of 24 (80%) patients in the TAC group and 23 (82.1%) patients in the CTX group achieved remission (either partial or complete remission). The survival curve of the probability of remission and complete remission were similar between the two groups (p > .05). Proteinuria (based on 24 h urinary protein excretion) was significantly decreased, and serum albumin was significantly increased after immunosuppressive treatment in both the groups. Estimated glomerular filtration rate (eGFR) was comparable between before and after treatment. The main adverse effects in TAC treatment were glucose intolerance, diabetes and abnormal aminotransferase. TAC monotherapy is an alternative therapeutic regimen for patients with nephrotic IMN. Its short-term efficiency and patient tolerance are both acceptable.


ABCB1 and cytochrome P450 genotypes and phenotypes: influence on methadone plasma levels and response to treatment.
The in vivo implication of various cytochrome P450 (CYP) isoforms and of P-glycoprotein on methadone kinetics is unclear. We aimed to thoroughly examine the genetic factors influencing methadone kinetics and response to treatment. Genotyping for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, ABCB1, and UGT2B7 polymorphisms was performed in 245 patients undergoing methadone maintenance treatment. To assess CYP3A activity, the patients were phenotyped with midazolam. The patients with lower CYP3A activity presented higher steady-state trough (R,S)-methadone plasma levels (4.3, 3.0, and 2.3 ng/mL x mg for low, medium, and high activity, respectively; P = .0002). As previously reported, CYP2B6*6/*6 carriers had significantly higher trough (S)-methadone plasma levels (P = .0001) and a trend toward higher (R)-methadone plasma levels (P = .07). CYP2D6 ultrarapid metabolizers presented lower trough (R,S)-methadone plasma levels compared with the extensive or intermediate metabolizers (2.4 and 3.3 ng/mL x mg, respectively; P = .04), whereas CYP2D6 poor metabolizer status showed no influence. ABCB1 3435TT carriers presented lower trough (R,S)-methadone plasma levels (2.7 and 3.4 ng/mL . mg for 3435TT and 3435CC carriers, respectively; P = .01). The CYP1A2, CYP2C9, CYP2C19, CYP3A5, and UGT2B7 genotypes did not influence methadone plasma levels. Only CYP2B6 displayed a stereoselectivity in its activity. In vivo, CYP3A4 and CYP2B6 are the major CYP isoforms involved in methadone metabolism, with CYP2D6 contributing to a minor extent. ABCB1 genetic polymorphisms also contribute slightly to the interindividual variability of methadone kinetics. The genetic polymorphisms of these 4 proteins had no influence on the response to treatment and only a small influence on the dose requirement of methadone.


Hemoglobin switching in sheep: a comparison of the erythropoietin-induced switch to HbC and the fetal to adult hemoglobin switch.
Stimulation of sheep erythropoietic progenitor cells by erythropoietin (epo) has been studied with regard to its effect on the pattern of hemoglobin production. An analysis of hemoglobin (Hb) synthesis in BFU-E- and CFU-E-derived colonies from fetuses either homozygous for HbA (AA) (homozygous also for the beta c gene responsible for HbC production) or HbB (BB) (lacking the beta c gene) indicated the following. Colonies derived from precursor cells from 51- and 89-day fetuses exhibited small but detectable increments of HbB synthesis with prolonged incubation in vitro. This response was not dependent on the epo concentration. Erythropoietic precursor cells from a 124-day BB fetus were already committed to HbB synthesis, since HbF production was replaced by HbB on successive days in vitro as erythroid colonies matured; this switch was not affected by varying the epo concentration. In contrast, progenitor cells from a 124-day AA fetus responded to higher doses of epo by forming colonies in which more HbC was made at the expense of both HbF and HbA. Erythropoietic stress did not result in induction of HbF in vivo or in erythroid colonies derived from CFU-E in young adult BB sheep, whereas our prior studies had shown induction of HbC synthesis under analogous conditions in colonies derived from young adult AA sheep. We conclude that the epo-induced HbF (or HbA) to HbC switch and the fetal to adult hemoglobin switch are regulated by different mechanisms.


Cognitive maps beyond the hippocampus.
We present a conceptual framework for the role of the hippocampus and its afferent and efferent structures in rodent navigation. Our proposal is compatible with the behavioral, neurophysiological, anatomical, and neuropharmacological literature, and suggests a number of practical experiments that could support or refute it. We begin with a review of place cells and how the place code for an environment might be aligned with sensory cues and updated by self-motion information. The existence of place fields in the dark suggests that location information is maintained by path integration, which requires an internal representation of direction of motion. This leads to a consideration of the organization of the rodent head direction system, and thence into a discussion of the computational structure and anatomical locus of the path integrator. If the place code is used in navigation, there must be a mechanism for selecting an action based on this information. We review evidence that the nucleus accumbens subserves this function. From there, we move to interactions between the hippocampal system and the environment, emphasizing mechanisms for learning novel environments and for aligning the various subsystems upon re-entry into familiar environments. We conclude with a discussion of the relationship between navigation and declarative memory.


Early chordate origin of the vertebrate integrin αI domains.
Half of the 18 human integrins α subunits have an inserted αI domain yet none have been observed in species that have diverged prior to the appearance of the urochordates (ascidians). The urochordate integrin αI domains are not human orthologues but paralogues, but orthologues of human αI domains extend throughout later-diverging vertebrates and are observed in the bony fish with duplicate isoforms. Here, we report evidence for orthologues of human integrins with αI domains in the agnathostomes (jawless vertebrates) and later diverging species. Sequence comparisons, phylogenetic analyses and molecular modeling show that one nearly full-length sequence from lamprey and two additional fragments include the entire integrin αI domain region, have the hallmarks of collagen-binding integrin αI domains, and we show that the corresponding recombinant proteins recognize the collagen GFOGER motifs in a metal dependent manner, unlike the α1I domain of the ascidian C. intestinalis. The presence of a functional collagen receptor integrin αI domain supports the origin of orthologues of the human integrins with αI domains prior to the earliest diverging extant vertebrates, a domain that has been conserved and diversified throughout the vertebrate lineage.


Advances in the molecular neurobiology of posttraumatic stress disorder from global contexts: A systematic review of longitudinal studies.
Trauma exposure is prevalent globally and is a defining event for the development of posttraumatic stress disorder (PTSD), characterised by intrusive thoughts, avoidance behaviours, hypervigilance and negative alterations in cognition and mood. Exposure to trauma elicits a range of physiological responses which can interact with environmental factors to confer relative risk or resilience for PTSD. This systematic review summarises the findings of longitudinal studies examining biological correlates predictive of PTSD symptomology. Databases (Pubmed, Scopus and Web of Science) were systematically searched using relevant keywords for studies published between 1 January 2021 and 31 December 2022. English language studies were included if they were original research manuscripts or meta-analyses of cohort investigations that assessed longitudinal relationships between one or more molecular-level measures and either PTSD status or symptoms. Eighteen of the 1,042 records identified were included. Studies primarily included military veterans/personnel, individuals admitted to hospitals after acute traumatic injury, and women exposed to interpersonal violence or rape. Genomic, inflammation and endocrine measures were the most commonly assessed molecular markers and highlighted processes related to inflammation, stress responding, and learning and memory. Quality assessments were done using the Systematic Appraisal of Quality in Observational Research, and the majority of studies were rated as being of high quality, with the remainder of moderate quality. Studies were predominantly conducted in upper-income countries. Those performed in low- and middle-income countries were not broadly representative in terms of demographic, trauma type and geographic profiles, with three out of the four studies conducted assessing only female participants, rape exposure and South Africa, respectively. They also did not generate multimodal data or use machine learning or multilevel modelling, potentially reflecting greater resource limitations in LMICs. Research examining molecular contributions to PTSD does not adequately reflect the global burden of the disorder.


Glass-phase coordination polymer displaying proton conductivity and guest-accessible porosity.
We describe the preparation of the crystalline and glassy state of a coordination polymer displaying proton conduction and guest-accessible porosity. EXAFS and solid-state NMR analyses indicated that pyrophosphate and phosphate ions are the main proton transporters in the glass and that homogeneously distributed 5-chloro-1H-benzimidazole in the glass provides the porosity.


Diabetic euglycemic ketoacidosis in newly diagnosed type 1 diabetes mellitus during Ramadan fasting.
Real euglycemic diabetic ketoacidosis [DKA; blood glucose <200 mg/dL (11.1 mmol/L)] is rare, and long-lasting starvation conditions due to intervening diseases in type 1 diabetes mellitus patients may also cause it. Euglycemic DKA is also reported in insulin-dependent diabetics with depression, alcoholics, glycogen storage diseases, and chronic liver disease apart from pregnant cases. This case report is presented to emphasize the importance of evaluation of acid-base state, urine glucose, and ketone values at the application in all newly diagnosed type 1 diabetic patients with normal glucose levels by defining euglycemic DKA that resulted from long-lasting starvation during Ramadan fasting in a newly diagnosed 14-year-old male patient.


The impact of housing-price-related indices on suicide rates in Taiwan.
While certain socioeconomic factors have been studied in relation to suicide, housing-price-related indexes have rarely been investigated. This article aims to examine the impact of housing-price-related indexes on suicide rates in the general population of Taiwan, a country with high housing costs and suicide rates. The study utilized three national housing-price-related indexes from 2012 to 2019: (1) housing price index, (2) housing price to income ratio, and (3) housing rental index. Cause of Death Data was employed to calculate suicide rate. A linear regression model with autoregressive errors was used to analyze the association between housing-price-related indexes and suicide rates among different sex and age groups. The findings revealed that higher housing rental index values were associated with increased suicide rates in young and middle-aged adults compared to the elderly population, regardless of sex. However, this association was not observed with the other two housing-price-related indexes (i.e. housing price index and housing price to income ratio). These results offer valuable insights for policymakers, mental health professionals, and housing advocates to improve housing affordability and reduce the burden of suicide in the general population, particularly among younger generations.


Strikingly different localization of galectin-3 and galectin-4 in human colon adenocarcinoma T84 cells. Galectin-4 is localized at sites of cell adhesion.
Two beta-galactoside-binding proteins were found to be prominently expressed in the human colon adenocarcinoma T84 cell line. Cloning and sequencing of one, a 36-kDa protein, identified it as the human homolog of galectin-4, a protein containing two carbohydrate binding domains and previously found only in the epithelial cells of the rat and porcine alimentary tract. The other, a 29-kDa protein, is galectin-3, containing a single carbohydrate binding domain, previously found in a number of different cell types including human intestinal epithelium. Despite the marked similarities in the carbohydrate binding domains of these two galectins, their cellular distribution patterns are strikingly different and vary with cellular conditions. In confluent T84 cells, galectin-4 is mostly cytosolic and concentrated at the basal membrane, whereas galectin-3 tends to be concentrated in large granular inclusions mostly at the apical membrane. In subconfluent T84 cells, each galectin is distributed to specific domains of lamellipodia, with galectin-4 concentrated in the leading edge and galectin-3 more proximally. Such different localization of galectins-4 and -3 within T84 cells implies different targeting mechanisms, ligands, and functions. The localization of galectin-4 suggests a role in cell adhesion which is also supported by the ability of immobilized recombinant galectin-4 to stimulate adhesion of T84 cells.


Isolation of Quartz Grains for Optically Stimulated Luminescence (OSL) Dating of Quaternary Sediments for Paleoenvironmental Research.
Optically stimulated luminescence (OSL) dating quantifies the time since mineral grains were deposited and shielded from additional light or heat exposure, which effectively resets the luminescence clock. The systematics of OSL dating is based on the dosimetric properties of common minerals, like quartz and feldspar. The acquired luminescence with exposure to natural ionizing radiation after burial provides a depositional age for many Quaternary sedimentary systems, spanning the past 0.5 Ma. This contribution details the procedures for separating pure quartz grains of a known range of particle sizes to facilitate luminescence analysis with small or single grain aliquots. Specifically, protocols are given for the needed data and interpretations for effective OSL dating of terrestrial sediment cores or sample tubes from exposures. These cores, 5-20 m long in 1.2 m sections, are split lengthwise and crown-cut leaving 80% of core volume undisturbed, which facilitates sampling of light-protected sediment for OSL dating deep within the core. Sediment samples are then subjected to a series of physical separations to obtain a certain grain-size interval (e.g., 150-250 µm). Magnetic minerals are removed in wet and dry states using magnets. A series of chemical digestions starts with soaking in H2O2 to remove organic matter, followed by HCl exposure to remove carbonate minerals, followed by density separation. Subsequently, grains are soaked in HF for 80 min and after in HCl to render solely quartz grains. The mineralogic purity (>99%) of the quartz extract is quantified with grain petrographic assessment and Raman spectroscopy. Repeating this quartz isolation procedure may be necessary with sediment that contains <15% quartz grains. Excitation of the purified quartz grains by LED-derived blue and IR light allows calculations of the fast and IR depletion ratios, which are metrics to assess the dominance of luminescence emissions from quartz.


HIV vaccine strategies.
Traditional methods of vaccine development have not produced effective vaccines for several prevalent infectious diseases, including AIDS, malaria and tuberculosis. These difficult diseases call attention to the importance of new approaches that profit from modern technologies. Successful efforts in the past have typically taken advantage of naturally occurring, protective immune responses, but this avenue is not readily available in certain cases, such as in HIV infection, where the immune system rarely confers protective immunity. However, there are alternative strategies and areas of research that may facilitate the development of highly effective vaccines. These include the identification of immunogens that elicit broadly neutralizing antibodies, determination of the molecular and cellular basis for immune responses to the components of the infectious agent, the identification of relevant forms of viral proteins for antigen presentation, stimulation of relevant T-cell types, and enhancement of antigen-presenting, dendritic cell function. Answering these basic research questions will aid in rational vaccine design. It is also extremely important to optimize techniques for the testing and production of new vaccines including the quantitation of immune responses in animals and in humans, identification of surrogate markers of immune protection, streamlined vaccine production, and rapid evaluation of candidate vaccines for testing in clinical trials. We have put these ideas into practice in two recent studies in which we generated enhanced cytotoxic T lymphocyte (CTL) responses, while retaining robust humoral responses, to wild-type viral proteins by immunizing mice with genetically modified forms of HIV-1 Env, Gag and Pol delivered in the form of plasmid DNA expression vectors.


Malonyl and palmityl transferase-less mutants of the yeast fatty-acid-synthetase complex.
146 independently isolated mutants of the fatty acid synthetase gene locus fas 1 were subdivided into six different complementation groups. Three of these groups, Va, Vb and Vd, have not been described before. The mutant fatty acid synthetases isolated from representatives of complementation group Vb were specifically deficient in two component enzymes at the same time, the malonyl and palmityl transferases. Among more than 180 fas 1 and fas 2 mutants systematically screened for malonyl and palmityl transferase activities no mutant was found affected in only one of these two fatty acid synthetase component enzymes. From this it is concluded that both transfer reactions are catalyzed by the same enzyme. In any malonyl transferase-less fatty acid synthetase, neither of the two known malonyl binding sites, i.e. enzyme-bound pantetheine and the non-thiol binding site, accepts malonate. This indicates that malonate is transferred to both groups by the same enzyme. So far, no acetyl transferase-less fas mutants have been characterized. On the other hand, the mutants of two fas 1 complementation groups, Va and Vd, though negative in overall fatty acid synthetase activity had no deficiency in any of the known component enzymes which can be tested in vitro. A possible interrelationship between both findings is discussed.


Dietary intake in young adults born small or appropriate for gestational age: data from the Haguenau cohort.
Compare the dietary intake of young adults born small for gestational age (SGA) versus those born appropriate for gestational age (AGA). Cross-sectional analysis. Data at the 8-year follow-up Haguenau cohort (France). Data from 229 AGA-born adults and 172 SGA-born adults with available dietary information are presented. Dietary intake was based on a food questionnaire including 19 items. The χ(2) test was run to compare intake between SGA-born and AGA-born individuals. An a priori score was calculated based on the adherence to recommendations from the French Nutrition and Health Program and included 8 components with the lowest value indicating a lower adherence to recommendations. The score was then divided into quartiles. Relative risks and 95% CIs, controlling for age and sex in multivariate analysis, were calculated in order to determine the risk of belonging to the first versus the second to the fourth quartiles in SGA-born and AGA-born individuals. Intakes of SGA-born adults indicated that they consumed more meat, sugar and less fish than AGA-born individuals (all p<0.05). Multivariate analyses with adjustment for age and sex showed that the relative risk of belonging to the first quartile versus the other three quartiles did not disclose any significant difference in SGA-born versus AGA-born participants: RR=0.92 (95% CI 0.65 to 1.30). Aside from the differences found by univariate analyses, no significant differences were obtained in multivariate analyses. Findings suggest that parameters of fetal programming are more associated with the development of metabolic syndrome in adulthood rather than dietary patterns.


Epidemiology of drug-drug interactions as a cause of hospital admissions.
The purpose of this review was to focus on hospital admissions caused by a specific type of adverse drug reaction (ADR) that can be assumed to be avoidable in almost all cases: the drug-drug interaction (D-DI). To determine the epidemiology of D-DIs in hospital admissions, a review of the adverse drug reaction literature was undertaken to answer several questions: (a) what is the incidence of hospital admissions attributable to D-DIs?; (b) what percentage of drug-related hospital admissions are attributable to D-DIs?; (c) are there any patterns to the above findings, i.e. are some D-DIs or specific drugs more likely to have been associated with hospital admissions?; and (d) are there certain patient risk factors (e.g. age) that are associated with D-DIs that led to a hospital admission? Nine ADR studies were found that either included a D-DI category as a cause for hospital admissions, or provided sufficient information so that a causal relationship could be inferred. The incidence of hospital admissions due to D-DIs ranged from 0 to 2.8%. The data found in the studies we reviewed, however, were insufficient to allow meaningful quantification of specific drugs as usual causes for D-DI-related admissions, and because of the very small numbers of patients for which a D-DI was believed to be the cause it is not possible to provide a meaningful summary of risk factors specific for D-DI admissions. We cannot conclude that D-DIs are a significant problem. There is a need to view the quantification of D-DIs in relation to the number of medications prescribed by physicians, dispensed by pharmacists and taken by patients.


Exploring willingness to participate in future Human Infection Studies in Lusaka, Zambia: A nested qualitative exploratory study.
Human Infection Studies (HIC) involve intentional infection of volunteers with a challenge agent or pathogen with the aim of understanding and developing vaccines as well as understanding the disease pathophysiology in a well-controlled environment. Though Africa carries the highest burden of vaccine-preventable diseases, the region is only now being primed to conduct HIC relevant to its population. Given the imminent introduction of HIC in Zambia, we sought to understand potential participants' willingness to volunteer for such studies. We used a qualitative exploratory approach to understand the potential participants' perceptions on willingness to participate in HIC using the example of typhoid. Healthy adults, recruited using random selection and purposive sampling from higher learning institutions in Lusaka, participated in 15 in-depth interviews (IDIs) and 5 Focus Group Discussions (FGDs) respectively. Participants considered typhoid a serious disease with potential for life-long consequences and death. After sharing audio-visual materials introducing the concepts of HIC, some participants expressed open willingness to participate or alternatively the need to consult parents and professors, and expressed fear of death and illness. Though willing to be quarantined for up to six months, participants expressed concerns regarding separation from family and duties, having insufficient information to decide, inadequate access to care, severe disease, life-long injury or side-effects, death, and vaccine failure. These concerns along with possibility of underlying conditions that compromise individual immunity, competing priorities, parental refusal, and distrust of study or vaccine efficacy could lead to refusal to participate. Reasons for willingness to participate included monetary compensation, altruism and being part of a team that comes up with a vaccine. Though afraid of deliberate typhoid infection, potential participants are willing to consider participation if given adequate information, time to consult trusted persons, compensation and assurance of adequate care.


Successful catheter ablation of incessant atrial tachycardia in pregnancy using three-dimensional electroanatomical mapping with minimal radiation.
Arrhythmias during pregnancy are not an infrequent problem and present a difficult therapeutic challenge to physicians. Anti-arrhythmic medication is used with some trepidation given concerns for the unborn foetus. Catheter ablation is typically avoided due to concerns regarding foetal exposure to radiation and delayed until the post-partum period. With the availability of three-dimensional mapping systems, catheter ablation may be performed with minimal radiation. We report a pregnant woman who underwent successful ablation of focal atrial tachycardia using three-dimensional electroanatomical mapping with minimal radiation exposure.


Changes in the viscosity of the plasma membrane of flounder (Platichthys flesus L.) erythrocyte induced by varying the content of membrane cholesterol or by benzyl alcohol. Correlation of the activity of the intrinsic Mg2+-ATPase and the viscosity.
The order parameter S and the correlation time tau from the ESR-spectra of 5-DS and 5-DD, respectively, are used as measures of the viscosity of the plasma membrane. It is suggested that the membrane undergoes a thermotropic phase transition which correlates with a change of the activation energy of the (Na+ + K+)-ATPase but not of the Mg2+-ATPase. The viscosity varies almost proportionally with the amount of cholesterol in the membrane when 0 less than mg C/mg PL less than 0.5. It is suggested that cholesterol is uniformly distributed in the membrane when mg C/mg PL less than 0.5 and that cholesterol-rich domains appear when mg C/mg PL greater than 0.5. Benzyl alcohol decreases the viscosity of the membrane. The effect depends on the presence of cholesterol in the membrane. The onset of the effect of benzyl alcohol on the native membrane is delayed--in terms of alcohol concentration--relative to the cholesterol-depleted membrane. Benzyl alcohol affects only the outer part of the membrane bilayer. Changes of the viscosity and of the activity of the Mg2+-ATPase are proportional. The effect of cholesterol-depletion on the activity of the Mg2+-ATPase cannot be ascribed only to the simultaneous lowering of the viscosity.


On the taxonomic structure and variability of Macropsidius involutus Dlabola, 1963 (Homoptera: Cicadellidae: Macropsinae)-morphology and calling signal pattern.
Variability of genitalia shape and male calling signal pattern in Macropsidius involutus from 10 localities in South-eastern Europe (the Crimea, the Lower Volga Region), Kazakhstan, and Kyrgyzstan was investigated. Variability of pygofer processes, penis shape, and signal pattern clearly has a geographic component, but none of these traits clearly supports subdividing this species into different subspecies unequivocally. M. involutus was found at altitudes from 0 up to ca. 2000 m a. s. l. in steppes and deserts in the plains and in steppe zones of mountains. Apparently, different environmental conditions in different parts of its range led to divergence of local populations, but the absence of zoogeographical barriers between them makes possible gene exchange and thus prevents emergence of clearly delineated subspecific taxa.


[Investigation methods of cerebral perfusion].
Brain energy metabolism is essentially oxydative, through the glycolysis and the Krebs cycle. Brain tissue cannot survive more than a few minutes if the substrate (glucose, for which there is no endogenous reserve) or oxygen supply is abolished. Otherwise, there is a precise matching between perfusion (cerebral blood flow-CBF) and oxydative (oxygen consumption-CMRO2) and glycolytic regional metabolism. When intraarterial pressure is decreased (due to a systemic arterial pressure impairment or an arterial obstructive lesion), CBF is maintained constant through an increase of the pial vessels caliber and therefore an enhancement of the cerebral blood volume (CBV); this is cerebral circulatory autoregulation. In physiologic conditions, any increase of the local metabolic demand (during a motor, sensory, or cognitive activation) is supplied through a local enhancement of perfusion, and reversely. Therefore, the measurement of brain perfusion is useful not only to assess the consequences of cerebrovascular or some other diseases, but also to observe the functions involved in the normal working brain. This measurement is most frequently focused on CBF (flow) parameter, but in some clinical circumstances the access to CBV will be also of major importance for a correct understanding of the physiological or pathophysiological situation. We shall describe the different methods available both in clinical and experimental practice, and shall indicate for each one its characteristics, advantages and pitfalls.


Optimal design and fabrication of three-dimensional calibration specimens for scanning probe microscopy.
Micro-/nano-scale roughness specimens are highly demanded to synthetically calibrate the scanning probe microscopy (SPM) instrument. In this study, three-dimensional (3D) specimens with controllable main surface evaluation parameters were designed. In order to improve the design accuracy, the genetic algorithm was introduced into the conventional digital filter method. A primary 3D calibration specimen with the dimension of 10 μm × 10 μm was fabricated by electron beam lithography. Atomic force microscopy characterizations demonstrated that the statistical and spectral parameters of the fabricated specimen match well with the designed values. Such a kind of 3D specimens has the potential to calibrate the SPM for applications in quantitative surface evaluations.


Adjuvant effect of the human metapneumovirus (HMPV) matrix protein in HMPV subunit vaccines.
The human metapneumovirus (HMPV) fusion (F) protein is the most immunodominant protein, yet subunit vaccines containing only this protein do not confer complete protection. The HMPV matrix (M) protein induces the maturation of antigen-presenting cells in vitro. The inclusion of the M protein into an F protein subunit vaccine might therefore provide an adjuvant effect. We administered the F protein twice intramuscularly, adjuvanted with alum, the M protein or both, to BALB/c mice at 3 week intervals. Three weeks after the boost, mice were infected with HMPV and monitored for 14 days. At day 5 post-challenge, pulmonary viral titres, histopathology and cytokine levels were analysed. Mice immunized with F+alum and F+M+alum generated significantly more neutralizing antibodies than mice immunized with F only [titres of 47 ± 7 (P<0.01) and 147 ± 13 (P<0.001) versus 17 ± 2]. Unlike F only [1.6 ± 0.5 × 10(3) TCID50 (g lung)(-1)], pulmonary viral titres in mice immunized with F+M and F+M+alum were undetectable. Mice immunized with F+M presented the most important reduction in pulmonary inflammation and the lowest T-helper Th2/Th1 cytokine ratio. In conclusion, addition of the HMPV-M protein to an F protein-based vaccine modulated both humoral and cellular immune responses to subsequent infection, thereby increasing the protection conferred by the vaccine.


An improved system for the evaluation of antiviral compounds against herpes simplex virus type 2.
Infection with herpes simplex virus type 2 (HSV-2) can result in lesions in reproductive organs, along with long-term latency. In this work, a non-lethal strain of HSV-2 which was isolated clinically was used to infect female mice intravaginally. Body weight, vulval lesions, histological examination of vaginal tissue, and viral load were monitored and used as indices for evaluating antiviral drugs against HSV-2 infection. The results indicated that mice infected with HSV-2 exhibited significant reduction in body weight, serious vulval lesions, massive lymphocyte invasion of vaginal tissue, and approximately 10⁴ copies/μl of HSV-2 were found in vaginal and uterine tissues. Aciclovir (ACV) treatment inhibited loss in body weight, genital pathology and virus replication (reduced to 10⁰·³ copies/μl) effectively. The study provides a simple, reproducible and feasible animal model for anti-HSV-2 drugs evaluation and HSV-2 vaccine research.


Safety and immunogenicity of a combined diphtheria-tetanus-acellular pertussis-hepatitis B vaccine administered according to two different primary vaccination schedules. Multicenter Working Group.
The reactogenicity and immunogenicity of a tetravalent diphtheria-tetanus-acellular pertussis-hepatitis B (DTPa-HB) vaccine (SmithKline Beecham) were studied in 565 infants immunized according to one of two different schedules, at 2, 4 and 6 months of age (group A n = 208) or at 3, 5 and 11 months of age (group B n = 357). The incidences of local and general reactions within the first 8 days after vaccination were similar in the two groups of infants, the vast majority being mild in intensity and occurring within 2-3 days of vaccine administration. Severe local symptoms were rare: pain after 0.6% of all doses, redness after 0.5% and 1.3%, and swelling after 0.3% and 1.5%, in group A and B, respectively. Only one infant in group A and one in group B had a temperature > 39.0 degrees C. Both schedules proved satisfactory in obtaining high levels of antibodies against all antigens. The rates of serologic response against the different antigens reached 100% in both groups. Antibody titres against all vaccine components were elevated following both schedules, but after the third dose of vaccine geometric mean antibody titres (GMTs) against D toxoid, filamentous haemagglutinin (FHA), pertactin (PRN) and hepatitis B (HB) were significantly higher in the 3, 5, 11 group than after the 2, 4, 6 schedule. Antibody titres measured at 7 months of age in the group immunized at 2, 4 and 6 months were higher than those reached at 6 months of age in infants immunized at 3, 5 and 11 months, but FHA and PRN were within the range of DTPa vaccine with proven efficacy. We conclude that DTPa-HB vaccine was safe, well tolerated and highly immunogenic. Both vaccination schedules (2, 4, 6 and 3, 5, 11) can be considered suitable for mass immunization programmes.


The relation between hiding skill and judgment of eye direction in preschool children.
This study examines J. H. Flavell, S. G. Shipstead, and K. Croft's (1978) finding that 2 1/2-year-old children can hide an object behind a screen but cannot achieve the same result by placing the screen in front of the object. Experiment 1 replicated this finding alongside a task in which children judged what a person in a picture was looking at. Performance on the move-object task approached ceiling; performances on the move-screen and looking-where tasks were highly correlated even after age and control task performance were partialed out (r = .54, p < .01). Experiment 2 examined whether the finding resulted because the object was more interesting to manipulate than the screen. The move-object task remained easier than the move-screen task with an interesting screen and a dull object. The move-screen task again correlated specifically with the looking-where task. Results are explained in terms of engagement, a precursor to a mature understanding of attention.


Stoichiometric food chain model on discrete time scale.
Stoichiometry-based models can yield many new insights into producer - grazer systems. Many interesting results can be obtained from models continuous in time. There raises the question of how robust the model predictions are to time discretization. A discrete stoichiometric food-chain model is analyzed and compared with a corresponding continuous model. Theoretical and numerical results show that the discrete and continuous models have many properties in common but differences also exist. Stoichiometric impacts of producer nutritional quality also persist in the discrete system. Both types of models can exhibit qualitatively different behaviors with the same parameter sets. Discretization enlarges the parameter ranges for the existence of chaotic dynamics. Our results suggest that the stoichiometric mechanisms are robust to time discretization and the nutritional quality of the producer can have dramatic and counterintuitive impacts on population dynamics, which agrees with the existing analysis of pelagic systems.


Guide to development of compound files for PBPK modeling in the Simcyp population-based simulator.
The Simcyp Simulator is a software platform for population physiologically-based pharmacokinetic (PBPK) modeling and simulation. It links in vitro data to in vivo absorption, distribution, metabolism, excretion and pharmacokinetic/pharmacodynamic outcomes to explore clinical scenarios and support drug development decisions, including regulatory submissions and drug labels. This tutorial describes the different input parameters required, as well as the considerations needed when developing a PBPK model within the Simulator, for a small molecule intended for oral administration. A case study showing the development and application of a PBPK model for ondansetron is herein used to aid the understanding of different PBPK model development concepts.