ClC-3 deficiency prevents atherosclerotic lesion development in ApoE-/- mice.
Recent evidence suggested that ClC-3, encoding Cl(-) channel or Cl(-)/H(+) antiporter, plays a critical role in regulation of a variety of physiological functions. However, remarkably little is known about whether ClC-3 is involved in atherosclerosis. This study aims to establish the involvement and direct role of ClC-3 in atherogenesis and underlying mechanisms by using ClC-3 and ApoE double null mice. After a 16-week western-type high-fat diet, the ClC-3(+/+)ApoE(-/-) mice developed widespread atherosclerotic lesions in aorta. However, the lesion size was significantly reduced in aorta of ClC-3(-/-)ApoE(-/-) mice. Compared with the ClC-3(+/+) controls, there was significantly decreased ox-LDL binding and uptake in isolated peritoneal macrophages from ClC-3(-/-) mice. Moreover, the expression of scavenger receptor SR-A, but not CD36, was significantly decreased in both ClC-3(-/-) peritoneal macrophages and aortic lesions from ClC-3(-/-)ApoE(-/-) mice. These findings were further confirmed in ox-LDL-treated RAW264.7 macrophages, which showed that silence of ClC-3 inhibited SR-A expression, ox-LDL accumulation and foam cell formation, whereas overexpression of ClC-3 produced the opposite effects. In addition, ClC-3 siRNA significantly inhibited, whereas ClC-3 overexpression increased, the phosphorylation of JNK/p38 MAPK in ox-LDL-treated RAW264.7 foam cells. Pretreatment with JNK or p38 inhibitor abolished ClC-3-induced increase in SR-A expression and ox-LDL uptake. Finally, the increased JNK/p38 phosphorylation and SR-A expression induced by ClC-3 could be mimicked by reduction of [Cl(-)]i by low Cl(-) solution. Our findings demonstrated that ClC-3 deficiency inhibits atherosclerotic lesion development, possibly via suppression of JNK/p38 MAPK dependent SR-A expression and foam cell formation.


Carbon 11 labeling of the psychoactive drug o-methyl-bufotenine and its distribution in the animal organism.
A methylated derivative of serotonin, O-methyl-bufotenine has been labeled with 11C on the two methyl groups of the amine function. In order to avoid the cyclization which occurs during the Eschweiler-Clarke synthesis, we adopted a milder methylation procedure, based on Borch's method using [11C]formaldehyde and sodium cyanoborohydride. Several tens of millicuries of injectable product could be obtained in 50 min in a perfectly pure state and having a specific radioactivity of 50 to 100 mCi/mumol. The distribution study of O-methyl-bufotenine in the mouse and rabbit showed an accumulation of significant quantities of the compound in the brain, kidneys, lungs and liver. The study of the rapid kinetics of this hallucinogenic molecule is compatible with labeling by 11C, having a period of 20 min. The use of O-methyl-[11C]bufotenine to detect serotonin receptors in vivo in mental diseases, is considered.


Neuropsychological evaluation of high-risk children from birth to seven years of age.
High Risk Children (HRC) are those with an increased risk of abnormal development due to any factor affecting neurological growth. Those factors have been the focus of most studies in this area. However, little is known about their long-term consequences over the course of child development. The goal was to study the cognitive, emotional and academic outcomes of 7-year-old children diagnosed as HRC at birth. We compared 14 HRC and 20 healthy children using the WISC-IV, BASC and Brunet-Lezine tests. HRC showed cognitive, emotional and academic deficits compared with healthy children. However, Brunet-Lezine scores obtained over the course of development (6, 12, 18 and 24 months) were not predictive of the children's' current psychological status. Long-term follow-up with HRC should be maintained until 7 years of age, at which point an appropriate treatment should be implemented.


Synthesis and evaluation of gallocyanine dyes as potential agents for the treatment of Alzheimer's disease and related neurodegenerative tauopathies.
In search of safe and effective anti-Alzheimer disease agents a series of gallocyanine dyes have been synthesized and evaluated for their ability to inhibit LRPs/DKK1 interactions. Modulation of the interactions between LRPS and DKK1, regulate Wnt signaling pathway and affect Tau phosphorylation. The current efforts resulted in the identification of potent DKK1 inhibitors which are able to inhibit prostaglandin J2-induced tau phosphorylation at serine 396.


Chemotherapy for gastric cancer by finely tailoring anti-Her2 anchored dual targeting immunomicelles.
Micelles with high in vivo serum stability and intratumor accumulation post intravenous (i.v.) injection are highly desired for promoting chemotherapy. Herein, we finely synthesized and tailored well-defined anti-Her2 antibody Fab fragment conjugated immunomicelles (FCIMs), which showed interesting dual targeting function. The thermosensitive poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)(118) (PID(118)) shell with volume phase transition temperature (VPTT: 39 °C) and the anchored anti-Her2 Fab moiety contributed to the passive and active targeting, respectively. The doxorubicin (DOX) loading capacity of such FCIMs was successfully increased about 2 times by physically enhanced hydrophobicity of inner reservoir without structural deformation. The cellular uptake and intracellular accumulation of DOX by temperature regulated passive and antibody navigated active targeting was 4 times of Doxil. The cytotoxicity assay against Her2 overexpression gastric cancer cells (N87s) showed that the IC50 of the FCIMs was ≈ 9 times lower than that of Doxil under cooperatively targeting by Fab at T > VPTT. FCIMs showed high serum stability by increasing the corona PID(118) chain density (S(corona)/N(agg)). In vivo tissue distribution was evaluated in Balb/c nude mice bearing gastric cancer. As observed by the IVIS(®) imaging system, the intratumor accumulation of such finely tailored FCIMs system was obviously promoted 24 h post i.v. administration. Due to the high stability and super-targeting, the in vivo xenografted gastric tumor growth was significantly inhibited with relative tumor volume <2 which was much smaller than ≈ 5 of the control. Consequently, such finely tailored FCIMs with anti-Her2 active and temperature regulated passive dual tumor-targeting function show high potent in chemotherapy.


Redefining contraindications to laparoscopic colorectal resection for high-risk patients.
Patients with major comorbidities often are denied laparoscopic colorectal resections because they are thought to be at too "high risk." Paradoxically, these patients generally have the most to gain from a minimally invasive surgical approach. This study aimed to examine the feasibility and safety of laparoscopic colorectal resection to determine whether it is contraindicated for "high-risk" patients. From August 1996 to February 2004, 368 consecutive patients (95 men) undergoing a laparoscopic colorectal procedure by a single surgeon were prospectively studied with regard to pre-, peri-, and postoperative events. High-risk patients (n = 190) were defined as elderly (age, >80 years; n = 28), morbidly obese (body mass index [BMI], >30 kg/m(2); n = 55), American Society of Anesthesiology (ASA) 3 or 4 (n = 130), and recipients of preoperative radiotherapy (n = 54). Multiple risk factors were found for 67 patients, 7 of whom had three risk factors. The median age of the patients was 66 years (range, 19-92 years). The diagnoses included rectal cancer (n = 48), diverticulitis (n = 43), colon cancer (n = 34), benign polyp (n = 26), and other (n = 39). The following procedures were performed: colon resection (n = 114; left, 63; right, 41; total abdominal colectomy, 10), rectal resection (low anterior resection or pouch) (n = 49), coloanal anastomosis (n = 23), and other (n = 4). Data regarding intent to treat, operative events, morbidity, mortality, and outcomes were analyzed and form the basis of this report. No mortalities occurred. The major morbidity rate was 2%. There were no anastomotic leaks. The cases were laparoscopically performed (94%) or laparoscopically assisted, or were converted to open procedure (3%). The median estimated blood loss was 200 ml, and only 5% required perioperative transfusion. The perioperative course involved the following median periods: 2 days until flatus, 3 days until bowel movement, 1 day until clear liquid diet, 3 days until a regular diet, and 5 days until hospital discharge. In experienced hands, laparoscopic colorectal resection can be performed safely for "high-risk" surgical patients. The better than expected outcomes in this patient population reinforce the benefits of minimally invasive surgery for this patient group and argues against using parameters of increased age, morbid obesity, high ASA class, or preoperative radiation alone as contraindications to even complex laparoscopic colorectal procedures.


The microscopic structure of bone in normal children and patients with osteogenesis imperfecta: a survey using backscattered electron imaging.
The microstructure of iliac crest biopsies from normal children or from those afflicted with osteogenesis imperfecta (OI) has not previously been studied to determine the tissue histology in the context of the degree of mineralization. The material in this study comprised 112 iliac crest biopsies from children aged 1.9-22.9 years. Fifty-eight were reference biopsies taken from children with no bone disease and the remainder were biopsies from children diagnosed as having OI (23 were Type I, 8 Type III, 18 Type IV, and 5 Type V). The specimens, which had been embedded in polymethylmethacrylate (PMMA), were micromilled and carbon coated to permit backscattered electron imaging. Reference biopsies from very young children often contained densely mineralized cartilage, and evidence of rapid cortical drift. Circumferential lamellae became a prominent feature after the toddler stage, and active remodeling and slower cortical drift continued through childhood. The biopsies from older teenagers and young adults were indistinguishable. Occasional mineralized osteocyte lacunae were detected in even the youngest children. Bone from children with OI Type I often appeared normal in microstructure and amount, but in some there was a dearth of bone and an abundance of osteocytes. Compared with age-matched controls, cortical and trabecular bone from children with OI Types III and IV were markedly sparse and very cellular, and primary osteonal systems continued to be formed later than expected. A distinguishing feature of the bone from OI Type V patients was the failure of patches of bone to mineralize, especially adjoining a reversal line. Packets of bone tissue exhibiting either considerably higher than normal or deficient mineralization would contribute to the characteristic trait of mechanical weakness.


Acute aortic dissection in the Marfan syndrome during the COVID-19 epidemic.
A 17-year-old boy with a recent diagnosis of COVID-19 infection was admitted with acute chest pain due to type A aortic dissection and was subsequently diagnosed with the Marfan syndrome. Literature shows an increased rate of aortic dissection during flu season. The hypothesis is that a cytokine storm triggers the dissection.


Polyfluoroaromatic stavudine (d4T) ProTides exhibit enhanced anti-HIV activity.
Human Immunodeficiency Virus (HIV) damages the immune system and leads to the life-threatening acquired immunodeficiency syndrome (AIDS). Despite the advances in the field of antiretroviral treatment, HIV remains a major public health challenge. Nucleosides represent a prominent chemotherapeutic class for treating viruses, however their cellular uptake, kinase-mediated activation and catabolism are limiting factors. Herein, we report the synthesis and in vitro evaluation of stavudine (d4T) ProTides containing polyfluorinated aryl groups against two strains; HIV-1 (IIIB) and HIV-2 (ROD). ProTide 5d containing a meta-substituted pentafluorosulfanyl (3-SF5) aryl group showed superior antiviral activity over the parent d4T and the nonfluorinated analogue 5a. ProTide 5d has low nanomolar antiviral activity; (IC50 = 30 nM, HIV-1) and (IC50 = 36 nM, HIV-2) which is over tenfold more potent than d4T. Interestingly, ProTide 5d showed a significantly high selectivity indices with SI = 1753 (HIV-1) and 1461 (HIV-2) which is more than twice that of the d4T. All ProTides were screened in wild type as well as thymidine kinase deficient (TK-) cells. Enzymatic activation of ProTide 5d using carboxypeptidase Y enzyme and monitored using both 31P and 19F NMR is presented.


The true nature of Bell's palsy: analysis of 1,000 consecutive patients.
In a series of 1502 patients seen in our Facial Paralysis Research Clinic 1048 were diagnosed as having Bell's palsy. Review of clinical, epidemiologic, and laboratory data, plus review of the literature, has led to the conclusion that Bell's palsy is an acute benign cranial polyneuritis probably caused by reactivation of the herpes-simplex virus, and the dysfunction of the motor cranial nerves (V, VII, X) may represent inflammation and demyelinization rather than ischemic compression. Spinal fluid analysis suggests that the disease is a phenomenon of the central nervous system with secondary peripheral neural manifestations. With our presently available information, treatment of a viral disease with an anti-inflammatory agent is rational. Prednisone treatment started within the first week of the disease can restore better function to the paralyzed face than is achieved without such therapy, and facial nerve decompression has been unnecessary.


In vitro mutagenicity of gas-vapour phase extracts from flavoured and unflavoured heated tobacco products.
The in vitro mutagenic and genotoxic potential of Heated Tobacco Products (HTPs) has already been studied with the particulate phase and reported previously. This study has been designed to complement the in vitro assessment of the HTP and to determine whether the inclusion of potential flavourings would alter the in vitro response by testing the other phase of the aerosol, the gas-vapour phase (GVP). Both flavoured and unflavoured Neostik GVP samples did not show any sign of mutagenic activity in the Ames test but induced a mutagenic response in the mouse lymphoma assay (MLA), however, these responses were significantly less than those of the reference cigarette, 3R4F. The results demonstrated that GVP emissions of this HTP did not induce either new qualitative or quantitative mutagenic hazards compared to 3R4F, as assessed by the Ames test (no new responsive strains) and MLA (a lower mutagenic response), respectively. A statistical comparative analysis of the responses showed that the addition of flavourings that may thermally decompose under the conditions of use did not add to the in vitro baseline responses of the unflavoured Neostik.


Optimization of gas utilization efficiency for short-pulsed electron cyclotron resonance ion source.
Numerical analysis of (6)He atoms utilizing efficiency in the ion source with powerful gyrotron heating is performed in present work using zero-dimensional balanced model of ECR discharge in a magnetic trap. Two ways of creation of ion source with high gas utilization efficiency (up to 60%-90%) are suggested.


Genomic structure and alternative splicing of the insulin receptor tyrosine kinase substrate of 53-kDa protein.
Insulin receptor tyrosine kinase substrate of 53-kDa protein (IRSp53) is now known to be a key factor in cytoskeleton reorganization. The human IRSp53 was identified as a binding partner with DRPLA protein, a product of the gene responsible for a neurodegenerative disorder, dentatorubral pallidoluysian atrophy, as well as a binding partner with brain-specific angiogenesis inhibitor 1. Previous studies identified at least four isoforms (L-, M-, S- and T-forms) in human, where 511 amino acid residues from the N-terminus were identical, followed by unique sequences of 9-41 amino acid residues. As each isoform had a distinct function, the unique sequences at the C-terminus had a vital role in its function. Here we report that these isoforms were indeed generated by alternative splicing, which was established by experimental and computational studies on human and rodent genomes. Previous biochemical reports suggested that rodents may lack one of the isoforms (L-form). This study solved this issue, as a nucleotide substitution occurred at a splice donor site followed by a large deletion in the rodent genome compared with human, which made the generation of the L-form impossible. This study also revealed overlapping of the IRSp53 and AATK genes coded for by complementary strands.


Unique features of the TCR repertoire of reactivated memory T cells in the experimental mouse tumor model.
T cell engineering with T cell receptors (TCR) specific to tumor antigens has become a breakthrough towards personalized cancer adoptive cell immunotherapy. However, the search for therapeutic TCRs is often challenging, and effective strategies are strongly required for the identification and enrichment of tumor-specific T cells that express TCRs with superior functional characteristics. Using an experimental mouse tumor model, we studied sequential changes in TCR repertoire features of T cells involved in the primary and secondary immune responses to allogeneic tumor antigens. In-depth bioinformatics analysis of TCR repertoires showed differences in reactivated memory T cells compared to primarily activated effectors. After cognate antigen re-encounter, memory cells were enriched with clonotypes that express α-chain TCR with high potential cross-reactivity and enhanced strength of interaction with both MHC and docked peptides. Our findings suggest that functionally true memory T cells could be a better source of therapeutic TCRs for adoptive cell therapy. No marked changes were observed in the physicochemical characteristics of TCRβ in reactivated memory clonotypes, indicative of the dominant role of TCRα in the secondary allogeneic immune response. The results of this study could further contribute to the development of TCR-modified T cell products based on the phenomenon of TCR chain centricity.


No transmission of porcine endogenous retrovirus in an acute liver failure model treated by a novel hybrid bioartificial liver containing porcine hepatocytes.
A novel hybrid bioartificial liver (HBAL) was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs). This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses (PERVs) into canines with acute liver failure (ALF) undergoing HBAL. Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity. PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative. No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.


Generation of two human iPSC lines, HMGUi003-A and MRIi028-A, carrying pathogenic biallelic variants in the PPCS gene.
Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the de novo coenzyme A (CoA) synthesis starting from pantothenate. Mutations in PPCS cause autosomal-recessive dilated cardiomyopathy, often fatal, without apparent neurodegeneration, whereas pathogenic variants in PANK2 and COASY, two other genes involved in the CoA synthesis, cause Neurodegeneration with Brain Iron Accumulation (NBIA). PPCS-deficiency is a relatively new disease with unclear pathogenesis and no targeted therapy. Here, we report the generation of induced pluripotent stem cells from fibroblasts of two PPCS-deficient patients. These cellular models could represent a platform for pathophysiological studies and testing of therapeutic compounds for PPCS-deficiency.


Adrenal metastases by renal cell carcinoma. Incidence at nephrectomy.
Recently, there have been questions raised as to the results and feasibility of performing renal-sparing procedures for localized renal cell carcinoma. We reviewed the records of 72 patients treated with standard radical nephrectomy to determine the incidence of adrenal involvement and to determine if adrenal lesions were detectable by radiographic methods preoperatively. Four patients had evidence of adrenal involvement for an overall incidence of 5.5 per cent. All had upper pole lesions. Adrenal involvement in each case was apparent at surgery or on preoperative imaging studies. For patients with middle and lower pole localized renal cell carcinoma who have normal findings on preoperative computerized tomography scans, modified radical nephrectomy sparing the adrenal gland may be a surgical alternative to radical nephrectomy.


Natriuretic peptides for risk stratification of patients with acute coronary syndromes.
Natriuretic peptides (BNP and NT-proBNP) have been shown to be useful tools for risk stratification of patients with acute myocardial ischemia encompassing the whole spectrum of acute coronary syndromes (ACS), particularly for prediction of mortality. Both BNP and NT-proBNP possess several characteristics of the ideal biomarker, showing independent and incremental prognostic value above traditional clinical, electrocardiographic, and biochemical (particularly troponin) risk indicators. Specifically, in ACS patients, BNP and NT-proBNP have powerful prognostic value both in patients without a history of previous heart failure or without clinical or instrumental signs of left ventricular dysfunction on admission or during hospital stay. They can also be easily and rapidly measured in an emergency context. We have performed a meta-analysis of available studies concerning the prognostic value of natriuretic peptides. Our results show that the prognostic value of natriuretic peptides is similar: (1) both at short- and long-term; (2) when natriuretic peptides are measured at first patient contact or during hospital stay; (3) for BNP or NT-proBNP; and (4) in patients with ST elevation myocardial infarction or no ST elevation ACS. These data suggest that natriuretic peptide measurement should be integrated into routine evaluation of patients with an ACS.


Anti-adhesive and pro-apoptotic effects of 2-hydroxyethyl methacrylate on human gingival fibroblasts co-cultured with Streptococcus mitis strains.
To evaluate and observe the cellular reactions that occur during the interaction/integration between 2-hydroxyethyl methacrylate/host tissue/microbial environment, in a co-culture of human gingival fibroblasts (HGF) and Streptococcus mitis strains. Streptococcus mitis were cultured with strains in the presence of 3 mmol L(-1) HEMA for 48 h and 72 h. Cytotoxicity was evaluated by the trypan blue dye exclusion test. Apoptosis was evaluated by TUNEL analysis. Adhesion was evaluated by immunofluorescence and western blot analyses. Quantitative analyses of the results were acquired by Qwin Plus 3.5 and QuantityOne I-D analysis software, respectively. The statistical significance of the results was evaluated using t-tests and linear regression tests. The trypan blue dye test revealed 47.3% and 46.5% of dead fibroblasts after 48 and 72 h HEMA treatment, respectively, while bacterial viability was not influenced by the presence of HEMA and fibroblasts. The expression of pro-collagen I, involved in fibroblast adhesion, in untreated samples ranged from 12.49% to 6.91% of the positive area after 48 and 72 h, respectively, dropping to below 2% of the positive area in the other experimental conditions. Unlike the trypan blue test, co-cultured samples treated with HEMA showed 20% and 25% versus 17% and 21% (after 48 and 72 h, respectively) of apoptotic cells. The evidence for HEMA toxicity and anti-adhesive effects against eukaryotic cells was reduced in the presence of bacteria, suggesting that dental resins should be well polymerized to avoid the spread of toxic monomers within the mouth.


Long-term reduction of renal interstitial hydrostatic pressure after neonatal renin-angiotensin system inhibition in the rat.
Neonatal inhibition of the renin angiotensin system (RAS) causes a decreased urinary concentrating ability, papillary atrophy, and tubulointerstitial inflammation long term. As a consequence of these morphological changes, we surmised that renal blood flow and renal interstitial hydrostatic pressure (RIHP) may be altered during and shortly after cessation of neonatal angiotensin-converting enzyme (ACE) inhibition, and that tentative changes of these variables would persist long after treatment withdrawal. Rats were given daily intraperitoneal injections of the ACE inhibitor, enalapril (10 mg/kg) or saline from days 3 to 23 postpartum, and the relationship between renal perfusion pressure (PP) and RIHP was investigated in 6- and 13-week-old anaesthetized rats. Neonatal ACE inhibition did not affect baseline RIHP short term, whereas RIHP was reduced at 13 weeks of age versus controls (11.6+/-1.6 vs 18.5+/-1.0 mmHg, P<0.05). Changes in RIHP correlated positively to changes in renal PP, independent of treatment and age (slope averaged 0.11+/-0.03). Ongoing ACE inhibition until 6 weeks of age neither affected baseline RIHP nor altered the reactivity to changes in perfusion pressure. Mild renal histopathological abnormalities were present already 3 weeks after cessation of treatment and were aggravated significantly in the 13-week-old rats, showing a complete loss of the papillary parenchyma. The reduced baseline RIHP in adult rats seemed to constitute a functional correlate to the major papillary atrophy. However, RIHP responses to changes in renal perfusion pressure was maintained, possibly indicating a compensatory effect of the remaining vasa recta and/or peritubular capillary network. Taken together, lack of neonatal angiotensin II type-1 (AT1) receptor stimulation induces not only irreversible abnormalities of the renal architecture, but causes alteration of intrarenal haemodynamics, such as a reduced RIHP, which may have implications for the regulation of pressure-natriuresis.


Protein thermal aggregation involves distinct regions: sequential events in the heat-induced unfolding and aggregation of hemoglobin.
Protein thermal aggregation plays a crucial role in protein science and engineering. Despite its biological importance, little is known about the mechanism and pathway(s) involved in the formation of aggregates. In this report, the sequential events occurring during thermal unfolding and aggregation process of hemoglobin were studied by two-dimensional infrared correlation spectroscopy. Analysis of the infrared spectra recorded at different temperatures suggested that hemoglobin denatured by a two-stage thermal transition. At the initial structural perturbation stage (30-44 degrees C), the fast red shift of the band from alpha-helix indicated that the native helical structures became more and more solvent-exposed as temperature increased. At the thermal unfolding stage (44-54 degrees C), the unfolding of solvent-exposed helical structures dominated the transition and was supposed to be responsible to the start of aggregation. At the thermal aggregation stage (54-70 degrees C), the transition was dominated by the formation of aggregates and the further unfolding of the buried structures. A close inspection of the sequential events occurring at different stages suggested that protein thermal aggregation involves distinct regions.


Omalizumab and the risk of malignancy: results from a pooled analysis.
Since initial registration, the omalizumab clinical trial database has expanded considerably, with a doubling of patients exposed in the clinical trial environment. Previous pooled data (2003) from phase I to III studies of omalizumab showed a numeric imbalance in malignancies arising in omalizumab recipients (0.5%) compared with control subjects (0.2%). The previous analysis was based on limited available data, warranting further investigation. We sought to examine the incidence of malignancy using comprehensive pooled data from clinical trials of omalizumab-treated patients. This pooled analysis included data from 67 phase I to IV clinical trials. The prespecified primary analysis assessed the incidence of primary malignancy in 32 randomized, double-blind, placebo-controlled (RDBPC) trials. There were 11,459 unique patients in all clinical trials (7,789 received omalizumab). The primary analysis identified malignancies in 25 patients (RDBPC trials): 14 in 4,254 omalizumab-treated patients and 11 in 3,178 placebo-treated patients. Incidence rates per 1,000 patient-years of observation time for omalizumab- and placebo-treated patients were 4.14 (95% CI, 2.26-6.94) and 4.45 (95% CI, 2.22-7.94), respectively; the corresponding rate ratio was 0.93 (95% CI, 0.39-2.27). Primary malignancies were of varying histologic type and occurred in a number of different organ systems; no cluster of histologies was identified. In this pooled analysis no association was observed between omalizumab treatment and risk of malignancy in RDBPC trials; the rate ratio was below unity. The data suggest that a causal relationship between omalizumab therapy and malignancy is unlikely.


[Post lumbar puncture headache in general pediatrics: a prospective multicenter study].
To investigate the frequency of post lumbar puncture headaches (PLPH) after diagnostic lumbar puncture (LP) and to search for influence factors. Conducted over 6 months into 8 paediatric services, the study concerned 2 to 15 years old children. The LP technic (cannula gauge, bevel orientation, child position, reinsertion of the stylet) was standardised. For each child questionnaires were filled with the parents 3 and 8 days after LP for inquiring headaches, postural characteristics, length and clinical consequences. Among 84 charts, 71 were exploitable. PLPH were observed in 25% of children. The mean duration was 5.9 days, mean bed rest was 1.4 day and mean antalgic treatment 1.7 day. The frequency of PLPH was not significantly influenced by age and sex, neither by the qualification of the physician performing the LP. The frequency was significantly lower when LP execution was easy (19.6 vs. 46.7%; P < 0.05), and when the child had viral meningitis (8 vs. 34.8%; P < 0.05). We have found a more important frequency of PLPH than in previous paediatric studies. Clinical consequences justify other researches to precise influence factors and reduce the frequency of this complication.


Total laparoscopic hysterectomy for very enlarged uteri.
To evaluate short-term outcome of total laparoscopic hysterectomy (TLH) performed in women with very enlarged uteri. Case control study (Canadian Task Force classification II-2). Hospital gynecologic service. Thirty-four consecutive women with very enlarged uteri. Total laparoscopic hysterectomy for benign pathology. Women with uterine enlargement (group 1) were compared with 68 women with uteri weighing 300 g or less (group 2) who underwent TLH during the same period. Matching was based on patient age +/- 2 years, surgeon (experienced senior, fellow), whether or not Burch operation was performed, and whether or not adnexectomy was performed. The groups were compared for complication rates, operating time, hospital stay, change in perioperative hemoglobin concentration, and vaginal and laparoscopic uterine morcellation. They did not differ statistically significantly in terms of indications for surgery, parity, postmenopausal status, and preoperative hemoglobin levels. No difference was seen in complication rates between groups. Operating time was significantly shorter (p <0.001) in women with smaller uteri than in those with very enlarged uteri, 108 +/- 35 and 156 +/- 50 minutes, respectively. The groups did not differ significantly in day 1 hemoglobin level drop, analgesia requirement (oral, intravenous opioid), time to passing gas and stool, or hospital stay. No conversion to laparotomy was required in either group. A very enlarged uterus should not be considered a contraindication for TLH. However, it may be necessary to undertake certain surgical steps to ensure optimal exposure of the operative field and more effective and safer excision of the uterine vascular pedicle.


Increasing the salience of fluency cues reduces the recognition memory impairment in amnesia.
The present study examined whether the recognition memory deficit in amnesia would be attenuated under conditions that increased the salience of study-induced fluency. Studied and unstudied items were drawn either from separate pools of letters (no-overlap condition) or from the same pool of letters (overlap condition). Study-induced fluency was more salient in the no-overlap than in the overlap condition, because in the no-overlap condition, such fluency occurred at the letter level as well as at the word level. The recognition memory impairment in amnesia was smaller in the no-overlap than in the overlap condition. These findings are consistent with the idea that enhancing the salience of fluency cues promotes reliance on a fluency heuristic that ordinarily is not fully engaged in amnesia, and reduces the recognition memory impairment in amnesia.


The expression and functional role of nicotinic acetylcholine receptors in rat adipocytes.
To clarify whether nicotine has a direct effect on the function of adipocytes, we evaluated nicotinic acetylcholine receptor (nAChR) expression in adipocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry and the direct effects of nicotine on the production of adipocytokines by enzyme-linked immunosorbent assay and Western blot analysis. Receptor binding assays were performed using [3H]nicotine. RT-PCR studies revealed that alpha1-7, 9, 10, beta1-4, delta, and epsilon subunit mRNAs are expressed in adipocytes. Immunocytochemical experiments also suggested the presence of alpha7 and beta2 subunits. The receptor binding assay revealed a binding site for nicotine (Kd = 39.2 x 10(-9) M) on adipocytes. Adipocytes incubated with nicotine for 12 and 36 h released tumor necrosis factor-alpha (TNF-alpha), adiponectin, and free fatty acid (FFA) into the medium in a dose-dependent manner with increasing nicotine concentration from 6 x 10(-8) to 6 x 10(-4) M. However, TNF-alpha protein levels in adipocytes incubated for 12 and 36 h decreased in a dose-dependent manner with increasing nicotine concentration from 6 x 10(-8) to 6 x 10(-4) M. These results show that adipocytes have functional nAChRs and suggest that nicotine reduces TNF-alpha protein production in adipocytes through the activation of nAChRs. Nicotine may temporarily lower insulin sensitivity by stimulating the secretion of TNF-alpha and FFA, whereas long-term direct stimulation of nAChRs by nicotine in addition to autonomic nervous system stimulation may contribute to better insulin sensitivity in vivo through a modulated secretion of adipocytokines.


Transcription of HERV-E and HERV-E-related sequences in malignant and non-malignant human haematopoietic cells.
Infection of a human T-cell leukaemia cell line (HSB-2) with HHV-6 led to the induction of exosome-like-particles attached to newly formed HHV-6 enveloped particles and to amplification of a 1642 bp molecule consisting of a partial human endogenous retrovirus (HERV)-E polymerase gene and repetitive sequences. We initiated an analysis of transcriptional patterns of predicted genes from HERV-E sequences in normal and malignant haematopoietic cells. Transcription patterns of regions corresponding to gag, pol and env genes at different chromosomal loci varied among cell types tested. Several specific transcripts were only observed in malignant haematopoietic cells and transcriptional activity varied among different malignant cell types. A transcript of 7.1 kb spanning the complete gag, pol and env gene region, originating from chromosome 8p23, was identified in normal peripheral blood cells and cells of the chronic myeloid leukaemia cell line K562. Our study describes new active HERV-E sequences and new loci throughout the human genome.


Correction: Low-Dose Irradiation Affects Expression of Inflammatory Markers in the Heart of ApoE -/- Mice.
[This corrects the article DOI: 10.1371/journal.pone.0119661.].


[Clostridium difficile reactive arthritis in a 7-year-old child].
Clostridium difficile reactive arthritis is a rare disease; only 5 pediatric cases have been reported in the literature. Its diagnosis is challenging. It manifests as asymmetric aseptic poly- or oligoarthritis, contemporary to infectious colitis, usually after a period of antibiotic therapy. We report a new case in a 7-year-old boy who presented with unusual polyarthritis affecting 12 joints 1 month after antibiotic therapy with amoxicillin-clavulanate. Punctures of both hip joints proved sterile but significantly improved symptoms. Diarrheic stool cultures during hospitalization provided the diagnosis. Antibiotic therapy using metronidazole completely resolved pain and joint swelling within a week. After 1 year of follow-up, there has been no recurrence. We present a review of the literature on this disease and underline the advantages of joint aspiration in this condition with the dual aim of not missing septic arthritis and effectively relieving pain.


Cell intrinsic and extrinsic activators of the unfolded protein response in cancer: Mechanisms and targets for therapy.
A variety of cell intrinsic or extrinsic stresses evoke perturbations in the folding environment of the endoplasmic reticulum (ER), collectively known as ER stress. Adaptation to stress and re-establishment of ER homeostasis is achieved by activation of an integrated signal transduction pathway called the unfolded protein response (UPR). Both ER stress and UPR activation have been implicated in a variety of human cancers. Although at early stages or physiological conditions of ER stress, the UPR generally promotes survival, when the stress becomes more stringent or prolonged, its role can switch to a pro-cell death one. Here, we discuss historical and recent evidence supporting an involvement of the UPR in malignancy, describe the main mechanisms by which tumor cells overcome ER stress to promote their survival, tumor progression and metastasis and discuss the current state of efforts to develop therapeutic approaches of targeting the UPR.


Risk factors in osteoporosis.
Osteoporosis is caused by a reduction in bone mass, but the cause of osteoporotic fractures is multifactorial because fractures arise following various degrees of trauma. There are therefore several different categories of risk factors for osteoporotic fracture. They may be related to the determinants of bone mineral density, capture the risk of falls, be related to skeletal fragility independently of bone mineral density or a combination of these. The identification of strong risk factors permits the identification of patients in whom further assessment and treatment can be offered in case finding strategies.


Experimental aspects and their clinical application.
After laboratory evaluation of nontoxic doses of intravitreal antibiotics, 26 cases of bacterial and fungal endophthalmitis were treated by intravitreal antibiotic or vitrectomy. In 46% of all cases, visual acuity was better than 20/100, whereas 27% had light perception to 20/300 visual acuity, 4% had no light perception, and 23% of the cases were enucleated or eviscerated. Best results have been achieved when the treatment began within 36 hours after symptomatic onset of infection and when the organism involved was not to virulent. We advocate intravitreal antibiotics immediately after intracameral and vitreous tap for culture, to be followed by vitrectomy 24 hours later if the culture is positive. In bacterial endophthalamitis when the vitreous is severely involved and in cases of fungal endophthalmitis, we advocate vitrectomy plus intravitreal antibiotics as the primary procedure.


In vitro bioactivity and phytochemical screening of Suaeda maritima (Dumort): a mangrove associate from Bhitarkanika, India.
To investigate the in vitro antioxidant and antimicrobial activities along with phytochemical screening of organic and aqueous extracts of leaf and stem of Suaeda maritima (Dumort), a mangrove associate from Bhitarkanika of Odisha, India. Antioxidant activity of the crude extracts was evaluated in terms of total antioxidant capacity, total phenol content, ascorbic acid content, DPPH radical scavenging, metal chelating, nitric oxide scavenging, and reducing power etc. The antimicrobial activity of the plant was determined by agar well diffusion method along with MIC and MBC carried out by microdilution techniques against 10 gram positive and gram negative human pathogenic bacteria. The qualitative and quantative phytochemical screening were carried out by standard biochemical assays. Out of the seven antioxidant bioassays, both the leaf and stem extracts were found to posses strong antioxidant properties of 70 % to 92 % for phenol, total antioxidant capacity, DPPH free radical scavenging activity and fairly good ascorbic acid content, metal chelating (1.33 %-22.55 %), reducing power (0.01-0.12) and nitric oxide scavenging (0.84 %-66.99 %) activities. Out of the four extracts evaluated for antimicrobial activity, two leaf extracts such as acetone and ethanol showed promising activity against four pathogenic bacteria and one stem methanol extracts against one pathogenic bacteria when compared with amoxcycillin as standard. The MIC and MBC values of the antimicrobial extracts ranged between 2.5 to 5.0 mg/mL. Screening of phytochemicals showed presence of carbohydrates, protein, tannins, alkaloids and flavonoids in comparatively higher amount than other phytochemicals tested. The present study reveals the presence of potential antioxidants and antimicrobial properties in the plant extract which could be exploited for pharmaceutical application.


MicroRNA-9 regulates osteoblast differentiation and angiogenesis via the AMPK signaling pathway.
MiR-9 has been found to be involved in the repair of spinal cord injury and regulates the proliferation and differentiation of mesenchymal stem cells. However, the role of miR-9 in repair of bone defects has not been well studied. The current study was designed to investigate its role and potential underlying mechanism in regulating osteoblast differentiation and angiogenesis. After treating the murine pre-osteoblast cell line MC3T3-E1 with BMP2, miR-9 expression was obviously down-regulated. Following transfection with miR-9 mimics, its overexpression enhanced the differentiation of MC3T3-E1 cells into osteoblasts as evidence that miR-9 up-regulated the mRNA levels of osteoblast differentiation-related protein, as well as increased differentiation and mineralization of osteoblasts. Further functional analysis has shown that miR-9 overexpression effectively increased human umbilical vein endothelial cell proliferation. Moreover, miR-9 up-regulation promoted cell migration, VEGF, and VE-cadherin concentrations, as well as tube formation in vitro. The mechanistic assay demonstrated that overexpression of miR-9-induced activation of the AMPK signaling pathway. Taken together, our findings suggested that miR-9 overexpression promoted osteoblast differentiation and angiogenesis via the AMPK signaling pathway, representing a novel and potential therapeutic target for the treatment of bone injury-related diseases.


The structural basis for cap binding by influenza virus polymerase subunit PB2.
Influenza virus mRNAs are synthesized by the trimeric viral polymerase using short capped primers obtained by a 'cap-snatching' mechanism. The polymerase PB2 subunit binds the 5' cap of host pre-mRNAs, which are cleaved after 10-13 nucleotides by the PB1 subunit. Using a library-screening method, we identified an independently folded domain of PB2 that has specific cap binding activity. The X-ray structure of the domain with bound cap analog m(7)GTP at 2.3-A resolution reveals a previously unknown fold and a mode of ligand binding that is similar to, but distinct from, other cap binding proteins. Binding and functional studies with point mutants confirm that the identified site is essential for cap binding in vitro and cap-dependent transcription in vivo by the trimeric polymerase complex. These findings clarify the nature of the cap binding site in PB2 and will allow efficient structure-based design of new anti-influenza compounds inhibiting viral transcription.


Multiband weights-induced periodic sparse representation for bearing incipient fault diagnosis.
Faulty impulses from incipient damaged bearings are typically submerged in harmonics, random shocks, and noise, making incipient fault diagnosis challenging. The prerequisite to this problem is the robust estimation of faulty impulses; thus, this paper proposes a multiband weights-induced periodic sparse representation (MwPSR) method. Firstly, a multiband weighted generalized minimax-concave induced sparse representation (MwGSR) approach is presented to accelerate the sparse approximation process and eliminate the interference components. A new indicator, coined the frequency-weighted energy operator spectrum's kurtosis-to-entropy ratio, is defined to construct the MwGSR's weights to accentuate faulty impulses. Secondly, to enhance the periodicity of the estimated impulses, a fault period decision strategy with an improved periodic target vector is developed and embedded into MwGSR to form MwPSR eventually. Detailed simulations and experiments demonstrate that MwPSR can achieve periodic sparsity with high accuracy and robustness and is reliable for incipient bearing fault diagnosis.


Split renal function in patients with suspected renal artery stenosis: a comparison between gamma camera renography and two methods of measurement with computed tomography.
To validate a method for calculating split renal function from computed tomography (CT) compared with gamma camera renography, and to test a new method for the measurement based on a volume-rendering technique. Thirty-eight patients, aged 65.7 +/- 11.6 (range 37.8-82.1) years, who had undergone both CT angiography and gamma camera renography for a suspected renal artery stenosis were included in this study. Split renal function was calculated from the CT examinations by measuring area and mean attenuation in the image slices of the kidneys, and also by measuring volume and mean attenuation from a 3D reconstruction of the kidneys. Gamma camera renography with 99mTc-MAG3 with or without captopril enhancement was used as a reference. The 2D CT method had good correlation with renography (r=0.93). Mean difference was 4.7 +/- 3.6 (0-12) percentage points per kidney. There was also excellent correlation between the two CT methods (r=1.00). CT is equivalent to renography in determining split renal function, and the measurement from the CT examination can be made more quickly and equally accurately with a 3D technique.


Expression profiles of circular RNAs in spermatozoa from aging men.
Advanced paternal age (APA) is associated with decreased fertility, but the mechanism underlying APA remains unknown. CircRNAs have been reported to be ideal candidate biomarkers for diagnostic and therapeutic applications in many diseases and are also involved in spermatogenesis. Hence, we aimed to assess the circRNA expression profile of spermatozoa from aging men. We recruited 6 subjects, including 3 in the younger group (men age < 40) and 3 in the APA group (men age ≥ 40). RNA sequencing was exploited to identify the expression profiles of circRNAs between the two groups. The expression levels of circRNAs were validated using real-time quantitative polymerase chain reaction (RT-qPCR). Kyoto Encyclopedia of Genes and Genomes biological pathway analysis and Gene Ontology analysis were performed to evaluate the functions of differentially expressed circRNAs (DE-circRNAs) between the two groups. In total, 18,787 circRNAs were sequenced in the spermatozoa of two groups. Our analysis revealed that there were 1056 downregulated circRNAs and 1228 upregulated circRNAs between the two groups, and KEGG analysis showed they were mainly involved in pathways including the DNA repair signaling pathway, meiotic recombination signaling pathway, and PI3K/AKT signaling pathway. In conclusion, our study suggested that circRNAs play a vital role in spermatozoa from aging men and provided a fresh perspective on the specific regulatory mechanism of spermatozoa from aging men.


The value of effective public tuberculosis treatment: an analysis of opportunity costs associated with multidrug resistant tuberculosis in Latvia.
A challenge to effective protection against tuberculosis is to sustain expensive and complex treatment public programs. Potential consequences of program failure include acquired drug resistance, poor patient outcomes, and potentially much higher system costs, however. In contrast, effective efforts have value illustrated by impacts they prevent. We compared the healthcare costs and treatment outcomes among multidrug-resistant tuberculosis (MDR-TB) and non MDR-TB patients in Latvia to identify benefits or costs associated with both. We measured and compared costs, healthcare utilization, and outcomes for patients who began treatment through Latvia's TB control program in 2002 using multivariate regression analysis and negative binomial regression. We analyzed data for 92 MDR-TB and 54 non MDR-TB patients. Most (67%) MDR-TB patients had history of prior tuberculosis treatment. MDR-TB was associated with lower cure rates (71% vs. 91%) and greater resource utilization. MDR-TB treatment cost almost $20,000 more than non MDR-TB. Up to 2/3 of MDR-TB treated in our sample was preventable at a potential savings of over $1.3 million in healthcare resources as well as substantial individual health.


Brugada ECG Pattern Unmasked by IV Flecainide in an Individual with Idiopathic Fascicular Ventricular Tachycardia.
A 45-year old man presents with stable monomorphic ventricular tachycardia. He had previously been diagnosed with idiopathic fascicular ventricular tachycardia. Intravenous flecainide results in termination of his tachycardia but unmasks a latent type 1 Brugada ECG pattern not seen on his resting ECG. We discuss his subsequent management and the need to consider an alternative diagnosis in individuals with a Brugada type ECG pattern who present with stable monomorphic ventricular tachycardia.


Macroamylase in the pleural fluid of a patient with lymphoma.
A patient with a macroamylase in pleural fluid is described. A diagnosis of recurrent pleural effusion due to chronic relapsing pancreatitis had been made on the basis of hyperamylasemia, high pleural fluid amylase, and abdominal pain. The amylase in the pleural fluid as well as the serum was ultimately characterized as a macroamylase on electrophoresis and chromatography. A diagnosis of lymphoma subsequently became obvious. The pathogenesis of the macroamylase in the pleural effusion and the association of macroamylasemia with malignant disease is discussed.


Rhabdomyosarcoma xenotransplants in zebrafish embryos.
Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcomas. High-risk and metastatic disease continues to be associated with very poor prognosis. RMS model systems that faithfully recapitulate the human disease and provide rapid, cost-efficient estimates of antitumor efficacy of candidate drugs are needed to facilitate drug development and personalized medicine approaches. Here, we present a new zebrafish-based xenotransplant model allowing for rapid and easily accessible drug screening using low numbers of viable tumor cells and relatively small amounts of water-soluble chemicals. Under optimized temperature conditions, embryonal RMS xenografts were established in zebrafish embryos at 3 h postfertilization (hpf). In proof-of-principle experiments, chemotherapy drugs with established clinical anti-RMS efficacy (vincristine, dactinomycin) and the mitogen-activated protein kinase kinase inhibitor trametinib were shown to significantly reduce the cross-sectional area of the tumors by 120 hpf. RMS xenograft models in zebrafish embryos henceforth could serve as a valuable addition to cell culture and mammalian models of RMS and represent a rapid and cost-effective solution for preclinical candidate drug testing.


Effect of Xuebijing injection on systemic lupus erythematosus in mice.
To observe the effects of Xuebijing injection on dendritic cells (DCs) and T lymphocytes, and the potential mechanisms of its therapeutic effect on systemic lupus erythematosus (SLE). A widely used mouse model, SLE-prone BLLF1 mice aged 8-10 weeks, was employed. Mice were randomly divided into 4 groups: a normal group, a model group and two treatment groups treated with Xuebijing Injection with a dose of 6.4 mL/kg via intraperitoneal administration for SLE-prone BLLF1 mice aged 8 weeks (treatment A group) and 10 weeks (treatment B group). Renal tissue sections were stained with Masson's trichrome and periodic acid-silver methenamine. Histopathological changes in the kidney were evaluated by a light microscopy. The capacity of the DCs isolated from the spleen to stimulate the T cell proliferation in response to concanavalin A (Con A) was determined. Compared with the model group, levels of anti-dsDNA antibodies in the two treatment groups decreased remarkablly (P<0.01, P<0.05), and levels of serum creatinine and blood urea nitrogen increased (P<0.01, P<0.05). Pathological changes were found in the kidney in the model group. Histopathological abnormalities were alleviated in the two treatment groups. Treatment with Xuebijing injection also significantly upregulated the expression of CD80, CD86 and major histocompatibility class II by DCs compared with the model group (P<0.05). When splenic T lymphocytes from BLLF1 mice were co-cultured with DCs at ratios of 1:100, 1:150 and 1:200 for 3 and 5 days, the proliferation of T lymphocytes was suppressed compared with the normal group (P<0.05), but this was restored by Xuebijing Injection under the same conditions. In the model group, levels of tumor necrosis factor (TNF)-α in supernatants were significantly elevated compared with the normal group (P<0.01), interleukin-2 levels decreased (P<0.05), while these changes were significantly alleviated in the Xuebijing treatment groups. Xuebijing Injection alleviated renal injury in SLE-prone BLLF-1 mice. The mechanism might be through influencing T cell polarization mediated by DCs, and Xuebijing Injection might be a potential drug that suppresses immune dysfunction in patients with SLE.


Clinically stable disease is associated with a lower risk of both income loss and disability pension for patients with multiple sclerosis.
To assess the risk of losing income from salaries and risk disability pension for multiple sclerosis patients with a clinically stable disease course 3 years after the start of disease-modifying therapy (DMT). Data from the Danish Multiple Sclerosis Registry were linked to other Danish nationwide population-based databases. We included patients who started treatment with a DMT between 2001 and 2014. Patients were categorised into a clinically stable group (No Evidence of Disease Activity (NEDA-2)) and a clinically active group (relapse activity or 6-month confirmed Expanded Disability Status Scale worsening). Outcomes were: (1) loss of regular income from salaries and (2) a transfer payment labelled as disability pension. We used a Cox proportional hazards model to estimate confounder-adjusted HRs, and absolute risks were plotted using cumulative incidence curves accounting for competing risks. We included 2406 patients for the income analyses and 3123 patients for the disability pension analysis. Median follow-up from index date was ~5 years in both analyses. The NEDA-2 group had a 26% reduced rate of losing income (HR 0.74; 95% CI 0.60 to 0.92). HRs were calculated for 5-year intervals in the disability pension analysis: year 0-5: a 57% reduced rate of disability pension for the NEDA-2 group (HR 0.43; 95% CI 0.33 to 0.55) and year 5-10: a 36% reduced rate (HR 0.64; 95% CI 0.40 to 1.01). Clinically stable disease course (NEDA-2) is associated with a reduced risk of losing income from salaries and a reduced risk of disability pension.


Potential for gulls to transport bacteria from human waste sites to beaches.
Contamination of recreational beaches due to fecal waste from gulls complicates beach monitoring and may pose a risk to public health. Gulls that feed at human waste sites may ingest human fecal microorganisms associated with that waste. If these gulls also visit beaches, they may serve as vectors, transporting fecal microorganisms to the beach where they may subsequently contaminate sand and water. In this study, samples collected from landfills, treated wastewater storage lagoons, and public beaches demonstrated a spatial and temporal overlap of markers for gull and human-associated microorganisms. In addition, markers for gull, fecal indicator bacteria, and the human-associated marker, HF183, were detected in gull feces and cloacae samples. Further, HF183 was detected in cloacae samples from gulls that were documented by radio-telemetry traveling between human waste sites and public beaches. This study highlights the potential for gulls that visit human waste sites to disperse human-associated microorganisms in the beach landscape.


A Selective Autophagy Pathway for Phase-Separated Endocytic Protein Deposits.
Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargo to the membrane-bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we report a selective autophagy pathway for protein condensates formed by endocytic proteins in yeast. In this pathway, the endocytic protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis and does not involve other known selective autophagy receptors but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates, at the plasma membrane and in autophagic bodies, shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy route. Our data suggest a model for autophagic degradation of macromolecular protein complexes by the action of intrinsic autophagy receptors.


Hematopoietic niches, erythropoiesis and anemia of chronic infection.
Anemia is a significant co-morbidity of chronic infections, as well as other inflammatory diseases. Anemia of chronic infection results from defective bone marrow erythropoiesis. Although the limitation of iron availability has been considered a key factor, the exact mechanisms underlying blockade in erythroid generation during infection are not fully understood. Erythropoiesis is a tightly regulated process that is very sensitive to environmental changes. During the last decade, the importance of the bone marrow hematopoietic niche has been progressively acknowledged. Several bone marrow cell types (such as macrophages, mesenchymal stem cells, and progenitor cells) and molecular mediators (such as CXCL12) have been identified as fundamental for both the maintenance of hematopoietic stem cell pluripotency and their most adequate differentiation into each hematopoietic cell lineage. Importantly, both niche-supporting cells and hematopoietic progenitors were found to be able to sense local and systemic cues to adapt the hematopoietic output to needs of the organism. Here, we review how hematopoietic progenitors and niche-supporting cells sense and respond to stress cues and suggest a potential role for the hematopoietic niche in the development of anemia of chronic infection.


Investigating the Ustilago maydis/Zea mays pathosystem: transcriptional responses and novel functional aspects of a fungal calcineurin regulatory B subunit.
The sustainable control of basidiomycete biotrophic plant pathogenesis requires an understanding of host responses to infection, as well as the identification and functional analysis of fungal genes involved in disease development. The creation and analysis of a suppressive subtractive hybridization (SSH) cDNA library from Ustilago maydis-infected Zea mays seedlings enabled the identification of fungal and plant genes expressed during disease development, and uncovered new insights into the interactions of this model system. Candidate U. maydis pathogenesis genes were identified by using the current SSH cDNA library analysis, and by knowledge generated from previous cDNA microarray and comparative genomic analyses. These identifications were supported by the independent determination of transcript level changes in different cell-types and during pathogenic development. The basidiomycete specific um01632, the highly in planta expressed um03046 (zig1), and the calcineurin regulatory B subunit (um10226, cnb1), were chosen for deletion experiments. um01632 and zig1 mutants showed no difference in morphology and did not have a statistically significant impact on pathogenesis. cnb1 mutants had a distinct cell division phenotype and reduced virulence in seedling assays. Infections with reciprocal wild-type×Δcnb1 haploid strain crosses revealed that the wild-type allele was unable to fully compensate for the lack of a second cnb1 allele. This haploinsufficiency was undetected in other fungal cnb1 mutational analyses. The reported data improves U. maydis genome annotation and expands on the current understanding of pathogenesis genes in this model basidiomycete.


Intraoperative ischemia of the distal end of colon anastomoses as detected with visible light spectroscopy causes reduction of anastomotic strength.
To explore new methods for intraoperative evaluation of tissue oxygenation, we evaluated the use of visible light spectroscopy as a predictor of anastomotic strength in an experimental model with ischemic murine colon anastomoses. Male rats (n = 34) were divided into 2 groups (ischemia and nonischemia). In the ischemia group the arteries of the distal colon were ligated until tissue oxygen saturation (StO2) dropped below 55%. A segment of the proximal part of the colon was resected until a well-perfused area was reached and an anastomosis was performed. In the nonischemia group, resection of a segment of descending colon and a colon anastomosis was performed. The animals were sacrificed on the 3rd or 7th postoperative d. The anastomosis was tested for bursting pressure and breaking strength. After ligation of the relevant mesenteric arteries, StO2 of the distal part of the colon decreased (54.6% SD 6.4% versus 71.2% SD 7.4%, P <or= 0.05). On the 3rd or 7th postoperative d StO2 had normalized. Adhesion score in the ischemia group was higher compared to the nonischemia group (1.6 versus 0.4, P <or= 0.05). There were no differences in bursting pressure between both groups. Breaking strength was lower in the ischemia group on the 3rd postoperative d (162.3 SD 47.3 versus 212.6 SD 41.2, P <or= 0.05). Ischemia can intraoperatively accurately be detected by visible light spectroscopy. Partially ischemic anastomoses showed more adhesions and diminished breaking strength in the early phase of healing, whereas bursting pressure was not affected. Low StO2 of a distal colon anastomosis appeared to be a risk factor for anastomotic dehiscence at d 3 and beyond.


Surface thermodynamics of phagocytic ingestion of non-opsonized bacteria by granulocytes in liquids of different surface tensions.
The free energy of engulfment of four bacterial species by human granulocytes is calculated from contact angle data as a function of the surface tension gamma LV of the suspending liquid. The resulting curves predict that at low liquid surface tensions gamma LV, the phagocytic ingestion increases with decreasing hydrophobicity of the bacteria while at high surface tensions gamma LV, it increases with increasing hydrophobicity. Furthermore, these curves reach a minimum at values of gamma LV equal to the surface tension gamma LV of the bacteria. The experimental results support these predictions. Thus, the determination of the surface tension of the suspending medium at which phagocytic ingestion becomes minimum represents a novel technique to establish the surface tension of ingested particles, such as bacteria. The results obtained in this fashion for the four bacterial species are in good agreement with those obtained from contact angles, as well as those derived from bacterial adhesion experiments.


Measuring Glucose Consumption and Gluconeogenesis in 3D Human Tissue Cultures with Nanoliter Input Volumes.
Organotypic and microphysiological culture of primary human tissues and cancers has emerged as a powerful set of technologies that allow to faithfully mimic cellular metabolism and functions ex vivo. The predominant 3D culture methods include spheroids and microfluidic chips. These cultures use low cell numbers and culture volumes, which, however, poses important limitations for the available amounts of sample for downstream analyses. Here, we describe a detailed method for the measurement of glucose consumption dynamics in organotypic culture using a bienzymatic colorimetric assay that accurately quantifies glucose levels using nanoliter input volumes. As an example we utilize spheroids consisting of primary human hepatocytes. The assay has been carefully optimized and benchmarked and is compatible with both longitudinal and high-throughput screening in both static and perfused conditions. The method is straightforward and only requires a microplate reader capable of running absorbance kinetic measurements.


Age-associated decreases in human DNA repair capacity: Implications for the skin.
Multiple pathways are involved in accurate synthesis and distribution of DNA during replication, repair and maintenance of genomic integrity. An increased error rate, abovethe spontaneous mutation baseline, has been implicated in carcinogenesis and aging. Moreover, cytogenetic abnormalities are increased in Down's, Edwards', Patau's, and Klinefelter's syndromes with increasing maternal age, and in Marfan's and Apert's syndromes with paternal age. In response to DNA damage, multiple overlapping systems of DNA repair have evolved, preferentially repairing the transcribed strand within transcriptionally-active regions of the genome. These include direct reversal of dimers and specific adducts and pathways for base excision, nucleotide excision, and mismatch repair. A consensus has emerged that some DNA repair capacities decline with organism age, contradictory reports notwithstanding. As is the case for inborn defects in humans, knockout mice lacking components of nucleotide excision repair or DNA-damage checkpoint arrest have increased frequencies of skin and internal cancers, whereas mice overexpressing DNA repair genes have fewer spontaneous cancers. Oxidative stress and resultant free radicals can damage genomic and mitochondrial DNA; damage increases with age but decreases with caloric restriction. We review recent studies of long-lived C. elegans mutants which appear to involve metabolic attenuation, the role of telomere shortening and telomerase in cellular senescence, and the genetic bases of progeroid syndromes in humans. Finally, we discuss roles of extrinsic and intrinsic factors in skin aging, and their association with DNA damage, emphasizing preventive and protective measures and prospects for intervention by modulating DNA repair pathways in the skin.


Influence of different thermal treatment methods on the processing qualities of sesame seeds and cold-pressed oil.
This study investigated the impact of multiple thermal treatments (explosion-puffing, microwave, and roasting) on the processing qualities of sesame seeds and cold-pressed oil. The scanning electron microscopy (SEM) showed fissures and cavities of sesame seed surface upon thermal treatments. The microwave treatment promoted the maximum conversion of sesamolin into sesamol in the sesame oil. Compared with other treatments, explosion-puffing treatment resulted in most significant increases in the multiple beneficial phytochemicals, as well as in vitro antioxidant properties determined by 2,2-dipheny1-1-picrylhydrazyl radical (DPPH) radical scavenging activity, Ferric reducing antioxidant power (FRAP) and oxidative stability index (OSI). Additionally, thermal treatment processing caused varying degrees of damage of crude protein, total amino acids (TAA) and protein structure (tertiary and second structure). In which, explosion-puffing achieved minimal reduction in the first two indicators. Collectively, explosion-puffing might be a preferable thermal treatment method for industrial sesame processing with improved quality specifications.


Immunogenicity, safety and tolerability of inactivated trivalent influenza vaccine in overweight and obese children.
Obesity may be a risk factor for increased hospitalization and deaths from infections due to respiratory pathogens. Additionally, obese patients appear to have impaired immunity after some vaccinations. To evaluate the immunogenicity, safety and tolerability of an inactivated trivalent influenza vaccine (TIV) in overweight and obese children, 28 overweight/obese pediatric patients and 23 healthy normal weight controls aged 3-14 years received a dose of TIV. Four weeks after vaccine administration, significantly higher seroprotection rates against the A/H1N1 strain were observed among overweight/obese children compared with normal weight controls (p<0.05). Four months after vaccination, similar or slightly higher seroconversion and seroprotection rates against the A/H1N1 and A/H3N2 strains were detected in overweight/obese than in normal weight children, whereas significantly higher rates of seroconversion and seroprotection against the B strain were found in overweight/obese patients than in normal weight controls (p<0.05 for seroconversion and seroprotection). Geometric mean titers (GMTs) and fold increase against B strains were significantly higher in overweight/obese patients than in normal weight controls 4 months after vaccine administration (p<0.01 for GMT values and p<0.05 for fold increase). The frequency of local and systemic reactions was similar between the groups, and there were no serious adverse events. The results of this study indicate that in overweight and obese children, antibody response to TIV administration is similar or slightly higher than that evidenced in normal weight subjects of similar age and this situation persists for at least 4 months after vaccine administration in the presence of a favorable safety profile.


The fibrillin microfibril/elastic fibre network: A critical extracellular supramolecular scaffold to balance skin homoeostasis.
Supramolecular networks composed of fibrillins (fibrillin-1 and fibrillin-2) and associated ligands form intricate cellular microenvironments which balance skin homoeostasis and direct remodelling. Fibrillins assemble into microfibrils which are not only indispensable for conferring elasticity to the skin, but also control the bioavailability of growth factors targeted to the extracellular matrix architecture. Fibrillin microfibrils (FMF) represent the core scaffolds for elastic fibre formation, and they also decorate the surface of elastic fibres and form independent networks. In normal dermis, elastic fibres are suspended in a three-dimensional basket-like lattice of FMF intersecting basement membranes at the dermal-epidermal junction and thus conferring pliability to the skin. The importance of FMF for skin homoeostasis is illustrated by the clinical features caused by mutations in the human fibrillin genes (FBN1, FBN2), summarized as "fibrillinopathies." In skin, fibrillin mutations result in phenotypes ranging from thick, stiff and fibrotic skin to thin, lax and hyperextensible skin. The most plausible explanation for this spectrum of phenotypic outcomes is that FMF regulate growth factor signalling essential for proper growth and homoeostasis of the skin. Here, we will give an overview about the current understanding of the underlying pathomechanisms leading to fibrillin-dependent fibrosis as well as forms of cutis laxa caused by mutational inactivation of FMF-associated ligands.


Anticoagulant Rodenticides, Islands and Animal Welfare Accountancy.
Anticoagulant rodenticides are used to manage rodents in domestic, municipal, agricultural, and conservation settings. In mammals and birds, anticoagulant poisoning causes extensive hemorrhagic disruption, with the primary cause of death being severe internal bleeding occurring over days. The combined severity and duration of these effects represent poor welfare outcomes for poisoned animals. Noting a lack of formal estimates of numbers of rodents and nontarget animals killed by anticoagulant poisoning, the ready availability and worldwide use of anticoagulants suggest that very large numbers of animals are affected globally. Scrutiny of this rodent control method from scientific, public, and regulatory perspectives is being driven largely by mounting evidence of environmental transfer of residual anticoagulants resulting in harmful exposure in wild or domestic animals, but there is also nascent concern for the welfare of targeted rodents. Rodent control incurs a cumulative ledger of animal welfare costs over time as target populations reduced by poisoning eventually recover to an extent requiring another reduction. This 'rolling toll' presents a critical contrast to the animal welfare accountancy ledger for eradication scenarios, where rodent populations can be completely removed by methods including anticoagulant use and then kept from coming back (e.g., on islands). Successful eradications remove any future need to control rodents and to incur the associated animal welfare costs.


Is it possible to diagnose Posterior Semicircular Canal BPPV from the sitting position? The role of the Head Pitch Test and the upright tests along the RALP and LARP planes.
The diagnosis of benign paroxysmal positional vertigo (BPPV) involving the posterior semicircular canal (PSC) is traditionally entrusted to positioning tests where patients are rapidly brought in the supine position. This prospective study aims to define the role of a diagnostic protocol for PSC-BPPV including only upright tests. 109 patients with PSC-BPPV were enrolled. The Head Pitch Test (HPT) was carried out first. If uneventful, the patient's head was turned 45° to each side and bent back-and-forth along the plane aligning either with the right anterior-left posterior (RALP) or left anterior-right posterior (LARP) canals, thus performing the upright RALP / upright LARP (uRALP/uLARP) test. Nystagmus observed was used to predict the diagnosis, which was therefore confirmed by Dix-Hallpike tests. PSC-BPPV could be correctly diagnosed in 75.2% of cases with the sole HPT and in 87.2% of cases by adding the uRALP/uLARP test (Upright Protocol). The time elapsed from symptoms onset was closely related to the protocol sensitivity, as it reached 100% (64/64) in acute patients while decreased to 68.9% (31/45) in cases evaluated after 7 days (p < 0.001). Upright maneuvers could correctly diagnose PSC-BPPV in most cases. uRALP/uLARP test demonstrated to improve the sensitivity of the HPT, mainly in recent-onset BPPV.


Developing antiepileptic drugs in children: balancing protection and access.
This chapter addresses the issue of the balance between protection of children submitted to research and children's access to new treatments, focusing on the field of antiepileptic drugs. Due to the lack of interest pharmaceutical companies have for such an unrewarding market, ethical and legal problems, difficulties recruiting patients, and in Europe insufficient public resources, many infants and children with epilepsy are still exposed to off-label drugs. The incentives and regulations specifically directed toward research in children in the United States and EU provided the first step to change this condition. The challenge is to perform trials determined by the needs of children and to obtain benefits in the same order of magnitude as for adults but with reduced risks. In order to optimize the development of new AEDs in pediatrics, the new European guidelines (2010) recommend the identification of adult clinical trial results that can be extrapolated to the pediatric population (i.e., those in partial epilepsies) and the use of innovative strategies that help limit the number of pediatric patients enrolled in trials (i.e., those with epileptic encephalopathies). A key step will be to develop international networks of pediatric epilepsy centers with the shared purpose of optimizing development and execution of clinical trials.


Identification of an E-box motif as a transcriptional repressor element in the proximal promoter region of the GCLC gene in rat lung epithelial L2 cells.
Glutathione (GSH) is a critical antioxidant for protecting the airway epithelium from oxidant injury and its levels are mainly controlled by glutamate-cysteine ligase (GCL), which is the rate-limiting enzyme in GSH synthesis. A full understanding of the gene regulation mechanism of this important enzyme may disclose the role it plays in respiratory diseases. GCL is made up of two differentially regulated subunits, a catalytic or heavy subunit (GCLC) and a modifier or light subunit (GCLM). Many studies in this field led to the findings of important positive regulatory regions of the GCLC promoter. For a detailed analysis of this gene regulation in the respiratory system, we cloned a 1.76-kb 5'-flanking region of the rat GCLC gene, inserted into a luciferase reporter vector. Exonuclease III was used to cut the 5'-flanking region of the rat GCLC gene unidirectionally into deletion mutants of different lengths. Sequential deletion analysis revealed that regions from -403 to -111 and from -705 to -613 are involved in positive regulation and the region from -745 to -705 is involved in negative regulation of the GCL gene in rat lung epithelial L2 cells. Specific proteins binding to these regions were confirmed by electrophoretic mobility-shift assays (EMSAs) and antibody supershift assays. An E-box motif was found in the negative regulatory region -745 to -705. Site-directed mutagenesis proved that the functional element in this negative regulatory region was a putative E-box element. EMSA and supershift assays showed that USF1 and USF2 can specifically bind to the E-box element. Overexpression of USFs in L2 cells led to a decreased activity of the GCLC promoter. Western blotting demonstrated that the expression of GCLC protein was decreased in the retroviral USFs-expressing cells than in nontransfected (no DNA added) cells, suggesting that USF binding to the E-box at -729/-724 serves to trans-repress GCLC gene expression. These findings indicate that the E-box is an important transcriptional suppressor element in the GCLC promoter in rat lung epithelial L2 cells. Inhibition of interaction between the E-box and the USF may provide an effective means of ameliorating oxidant injury of the lung.


Concurrent capecitabine with external beam radiotherapy versus radiotherapy alone in painful bone metastasis of breast cancer origin.
In breast cancer, painful bone metastases are common. Local radiotherapy is the standard treatment of painful bone metastases. Pain control and overall response rateswere low in radiotherapy alone.The objectives of this study were to compare the safety and efficacy of external beam radiotherapy with concurrent capecitabine vs. external beam radiotherapy alone in pain control of painful bone metastases in breast cancer patients. Eighty-four patients with painful bone metastases from breast cancer participated in this prospective study. We randomized the patients into two groups: group A treated with radiotherapy 30 Gy in 10 fractions and group B treated with capecitabine 825 mg/m2 every 12 hrs. concurrently with the same radiotherapy dose. There was no statistically significant difference between the two groups regarding early treatment toxicity. Most of the toxicity was gastrointestinal (diarrhea and nausea) and mild (grade I or II). The median pain score decreased from week one, and there was a marked response at week4. The difference in median pain score between both groups was statistically significant with p-value = 0.045. The median analgesic score in both groups was statistically significant with a p-value = 0.032 at week 12. A complete response to pain at week 4 was 19% and 42.9% in groups A and B, respectively. Concurrent chemoradiation in painful bone metastases from breast cancer origin was tolerable and safe; it had a higher overall response rate and pain palliation than radiotherapy alone.


Insights into thermal hydrolyzed sludge liquor - Identification of plant-growth-promoting compounds.
This study proposes a new path to utilize thermal hydrolyzed sludge (TH sludge) as fertilizer given high value chemical compounds that can promote plant growth were identified in the liquid fraction of TH sludge (TH liquor). Together with micro- and macro-nutrients released/synthesized during thermal hydrolysis, the feasibility of using TH liquor as organic fertilizer was evaluated. Besides high contents of N, P and K, total free amino acids (FAAs) and plant-growth-promoting FAAs (including glutamic acid, leucine and cystine) also presented in high concentration (4.98-6.48 and 1.12-2.73 g/100 g) in the TH liquor. For the first time, phytohormone compound, indole-3-acetic acid, was observed and the content was the highest in TH liquor with 165 °C treatment (165 °C TH liquor). Meantime, 165 °C TH liquor did not have negative impact on the growth of soil microbes, and this product, instead, demonstrated stimulating effect on the plant growth. These results suggest that 165 °C TH liquor has a great potential to be an organic fertilizer. The remaining solids of TH sludge could be converted to valuable biochar. The holistic approach of using TH liquor as organic fertilizer and producing biochar could realize nearly zero-waste discharge in sludge management.


Evaluation of risk factors, long-term outcomes, and immediate and delayed autotransplantation to minimize postsurgical hypoparathyroidism in multiple endocrine neoplasia type 1 (MEN1): A retrospective cohort study.
Postoperative hypoparathyroidism from inadequate parathyroid hormone is of concern after multigland resections in multiple endocrine neoplasia type 1-related primary hyperparathyroidism. We evaluated risk factors, long-term outcomes, and roles of autotransplantation and cryopreservation in postoperative hypoparathyroidism in multiple endocrine neoplasia type 1. Retrospective cohort study of patients with multiple endocrine neoplasia type 1 and parathyroidectomy who were evaluated at MD Anderson from 1990 to 2020. We included 206 patients. Median follow-up after the last operation (index 65%, reoperation 35%) was 8 years. Index parathyroidectomy was subtotal in 47%, less than subtotal in 42%, and total in 12%; hypoparathyroidism was more frequent after total parathyroidectomy. Forty-seven patients (23%) had hypoparathyroidism ≥6 months; odds were significantly higher when cumulative ≥4 glands were resected (odds ratio 6 [2.96-12.24]) or when immediate postoperative parathyroid hormone was <15 pg/mL (odds ratio 13.10 [3.61-47.47]). After median 26 months postoperatively, 30% recovered parathyroid function spontaneously; this was less likely when ≥4 glands were resected (odds ratio 0.19 [0.05-0.72]). None of the 4 patients who were aparathyroid (parathyroid hormone undetectable or ≤3 pg/mL) at 6 months postoperatively recovered parathyroid function. Immediate autotransplantation success rate was 72%. Cryopreservation was performed in 96 operations with delayed autotransplantation in 10 patients (10% utilization), of whom 5 recovered parathyroid function (time to recovery 12-93 months). Odds of prolonged hypoparathyroidism are higher when cumulative ≥4 glands are resected or postoperative parathyroid hormone is <15 pg/mL. Spontaneous recovery occurred but was less likely when ≥4 glands were resected or patients were aparathyroid at 6 months postoperatively. Cryopreservation should be sparingly used, but there is value in select high-risk patients such as reoperative parathyroidectomy/cervical surgery.


Targeting papain-like protease for broad-spectrum coronavirus inhibition.
The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics. Drugging the multi-functional papain-like protease (PLpro) domain of the viral nsp3 holds promise. However, none of the known coronavirus PLpro inhibitors has been shown to be in vivo active. Herein, we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity, including against the Sarbecoviruses (SARS-CoV-1 and SARS-CoV-2), Merbecovirus (MERS-CoV), as well as the Alphacoronavirus (hCoV-229E and hCoV-OC43). Importantly, F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice. F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage, as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity. Despite the significant difference of substrate recognition, mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue, whereas an allosteric inhibitor of MERS-PLpro interacting with its 271E position. Our proof-of-concept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anti-coronavirus agents. The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.


Quantitative chromosome map of Coix lacryma-jobi L. by imaging methods.
Prometaphase chromosomes of Coix lacryma-jobi L. were quantitatively analyzed based on their distribution patterns of DAPI signals. The DAPI signals showed prominent uneven distribution along the chromosomes. Based on the DAPI signal patterns, a quantitative chromosome map was constructed for the first time in C. lacryma-jobi. The quantitative chromosome map will offer the foundation for genome analysis of C. lacryma-jobi.


Gold nanoparticles functionalised by Gd-complex of DTPA-bis(amide) conjugate of glutathione as an MRI contrast agent.
The work is directed toward the synthesis of gold nanoparticles (Au NPs) coated with paramagnetic Gd-complex of DTPA-bis(amide) conjugate of glutathione (GdL) for use as a highly efficient MRI contrast agent. Well-dispersed spherical Au NPs coated with gadolinium complexes, abbreviated as Au@GdL, have been obtained; the mean size of Au@GdL is 5-7 nm, and the numbers of GdL are 1.36x10(4) per Au NP. Au@GdL exhibits high longitudinal (r1) and transverse (r2) relaxivities of 1.87x10(5) and 3.02x10(5) mM(-1) s(-1), respectively.


RNA-seq analysis of Pichia anomala reveals important mechanisms required for survival at low pH.
The product yield and titers of biological processes involving the conversion of biomass to desirable chemicals can be limited by environmental stresses encountered by the microbial hosts used for the bioconversion. One of these main stresses is growth inhibition due to exposure to low pH conditions. In order to circumvent this problem, understanding the biological mechanisms involved in acid stress response and tolerance is essential. Characterisation of wild yeasts that have a natural ability to resist such harsh conditions will pave the way to understand the biological basis underlying acid stress resistance. Pichia anomala possesses a unique ability to adapt to and tolerate a number of environmental stresses particularly low pH stress giving it the advantage to outcompete other microorganisms under such conditions. However, the genetic basis of this resistance has not been previously studied. To this end, we isolated an acid resistant strain of P. anomala, performed a gross phenotypic characterisation at low pH and also performed a whole genome and total RNA sequencing. By integrating the RNA-seq data with the genome sequencing data, we found that several genes associated with different biological processes including proton efflux, the electron transfer chain and oxidative phosphorylation were highly expressed in P. anomala cells grown in low pH media. We therefore present data supporting the notion that a high expression of proton pumps in the plasma membrane coupled with an increase in mitochondrial ATP production enables the high level of acid stress tolerance of P. anomala. Our findings provide insight into the molecular and genetic basis of low pH tolerance in P. anomala which was previously unknown. Ultimately, this is a step towards developing non-conventional yeasts such as P. anomala for the production of industrially relevant chemicals under low pH conditions.


De novo mutation in the GNAL gene causing seemingly sporadic dystonia in a Serbian patient.
Mutations in GNAL (DYT25) have recently been established as the first confirmed cause of focal or segmental adult-onset dystonia. Mutation carriers show craniocervical involvement; however, the GNAL mutational and phenotypic spectrum remain to be further characterized, and guidelines for diagnostic testing need to be established. The authors used Sanger sequencing to test for changes in the GNAL coding or splice-site regions in 236 Serbian patients suffering from isolated dystonia with craniocervical involvement. One novel likely pathogenic substitution (c.1061T>C; p.Val354Ala) in GNAL was detected in a sporadic cervical dystonia patient (mutation frequency: 0.4%). This mutation was not present in the DNA of either parent, despite confirmed parentage. This is the first report of a de novo GNAL mutation causing genetically proven, seemingly sporadic DYT25 dystonia. Our finding highlights the importance of genetic testing for GNAL mutations in establishing the molecular diagnosis even for patients with a negative family history.


Screening study of anti-emetics to improve GDF15-induced malaise and anorexia: Implications for emesis control.
Considerable preclinical and clinical attention has focused on the food intake and body weight suppressive effects of growth differentiation factor 15 (GDF15) and its elevated blood levels as a consequence of disease states and disease treatment therapeutics. We have previously reported that exogenous administration of GDF15 induces anorexia through nausea and emesis in multiple species. Importantly, GDF15 signaling as a meditator of chemotherapy-induced anorexia and emesis has recently been demonstrated in both murine and nonhuman primate models. The mechanism, however, by which GDF15 induces malaise and the utility of existing therapeutic targets to counteract its effects remain largely unknown. Using a dose of GDF15 that mimics stimulated levels following chemotherapy administration and reliably induces malaise, we sought to screen anti-emetics that represent distinct pharmacotherapeutic classes hypothesized to reduce GDF15-induced effects in rats. Strikingly, our results showed that none of the tested compounds were effective at preventing GDF15-induced malaise. These results illustrate the complexity of GDF15 signaling mechanism and may have important implications for medical conditions characterized by elevated GDF15 levels and incomplete symptom control, such as chemotherapy-induced nausea and vomiting.


cDNA sequence and spatio-temporal expression of prolactin in the orange-spotted grouper, Epinephelus coioides.
The cDNA sequence encoding the prolactin (PRL) gene of the orange-spotted grouper was obtained by randomly sequencing its pituitary cDNA library. The full-length cDNA of orange-spotted grouper PRL consists of 1342 bp. The open reading frame encodes a protein of 212 amino acids, which consists of a putative signal peptide of 24 residues and a mature protein of 188 amino acids. Northern blot analysis identified a single transcript of 1.4 kb in the pituitary gland, indicating perhaps the presence of only one form of PRL in the orange-spotted grouper. The expression of PRL gene in different tissues and during embryonic development of orange-spotted grouper was determined using one-step RT-PCR followed by Southern blot analysis. In addition to the pituitary gland, high levels of PRL gene expression were detected in the olfactory bulb, forebrain, midbrain, hypothalamus, hindbrain, medulla, gill, spleen, and adipose tissue. Very low levels of expression were detected in the kidney, liver, and blood cells. The PRL gene was also expressed in the gonads, with the higher level of expression observed in the stage 3 as compared to stage 2 ovary. During embryonic development, mRNA for PRL was detected at all time points examined except at 2h 15min post-fertilization, and its expression peaked at 9h post-fertilization, at the time of neurulation. Results from the current study suggest that PRL may have important paracrine or autocrine roles in diverse tissue types, including the brain and the gonads, and during embryonic development of the orange-spotted grouper.


Economic analysis of recombinant activated factor VII versus plasma-derived activated prothrombin complex concentrate in mild to moderate bleeds: haemophilia registry data from the Czech Republic.
Several studies suggest that recombinant activated factor VII (rFVIIa) is more cost-effective than plasma-derived activated prothrombin complex concentrate (pd-aPCC) in haemophilia with inhibitors. However, most do not consider differences between treated patients. This study compared the pharmacoeconomics of rFVIIa versus pd-aPCC treatment of mild to moderate bleeds in inhibitor patients, taking co-variables into account. The HemoRec and HemIS registries capture exhaustive bleeding data in inhibitor patients in the Czech Republic. For each bleed, patient and bleed characteristics, treatment outcomes and bypassing agent use were retrospectively analysed, and direct costs of care per bleed calculated. Generalised Linear Model regression methods with cluster effect were employed to account for the possibility of several bleedings from the same patient. There were 108 and 53 mild to moderate bleeds in the rFVIIa and pd-aPCC groups, respectively. Although re-bleeding rates were similar in both groups, deeper analyses revealed significant differences in time to bleed resolution: 93.8% of bleeds treated with rFVIIa were resolved within ≤ 12 h, versus 60.4% with pd-aPCC (P < 0.001). Mean total cost/bleed was lower with rFVIIa (336,852 [median, 290,696] CZK; €12,760 [11,011]) than pd-aPCC (522,768 [341,310] CZK; €19,802 [12,928]) (P = 0.002). Results were maintained after controlling for potential co-variables (bleed nature, time to treatment, target joints). The lower total treatment costs per bleed with rFVIIa than pd-aPCC suggest that first-line rFVIIa is more cost-effective than pd-aPCC in mild to moderate bleeds. Time to bleed resolution was also significantly shorter with rFVIIa. These results were maintained when controlled for potential confounders.


Protein kinase A and C are "Gatekeepers" of capacitative Ca2+ entry in the prothoracic gland cells of the silkworm, Bombyx mori.
Application of protein kinases A and C inhibitors to the prothoracic glands cells of the silkworm, Bombyx mori, resulted in slow and gradual increases in intracellular Ca(2+) ([Ca(2+)](i)). Pharmacological manipulation of the Ca(2+) signalling cascades in the prothoracic gland cells of B. mori suggests that these increases of [Ca(2+)](i) are mediated neither by voltage-gated Ca(2+) channels nor by intracellular Ca(2+) stores. Rather they result from slow Ca(2+) leak from plasma membrane Ca(2+) channels that are sensitive to agents that inhibit capacitative Ca(2+) entry and are abolished in the absence of extracellular Ca(2+). Okadaic acid, an inhibitor of PP1 and PP2A phosphatases, blocked the increase in [Ca(2+)](i) produced by the inhibitors of protein kinase A and C. The combined results indicate that the capacitative Ca(2+) entry channels in prothoracic gland cells of B. mori are probably modulated by protein kinases A and C.


Membrane and synaptic effects of corticotropin-releasing factor on periaqueductal gray neurons of the rat.
Corticotropin-releasing factor (CRF) has been identified as a major component of the hypothalamic-pituitary-adrenal (HPA) axis. By stimulating the release of adrenocorticotropin hormone (ACTH), CRF acts as a key mediator of the stress response. However, CRF receptors and neuronal elements are present in many extrahypothalamic regions of the brain. A region that contains both CRF-ergic neurons and CRF receptors is the midbrain periaqueductal gray (PAG). The physiological effects of CRF in the PAG are unknown. In this study, an in vitro preparation, extracellular and intracellular patch-clamp recordings, were used to examine the effects of CRF, applied through an injecting electrode, on PAG neurons. Recordings were made from 147 neurons in the PAG. CRF injecting electrode concentrations of 0.05 and 1 microM were tested. At the higher concentration, CRF had a predominant excitatory effect on the neurons, and at the lower concentration, CRF produced no significant effect on the neurons. The excitatory effect was dose dependent and was often associated with a depolarization in membrane potential in intracellular recordings. Application of the CRF antagonist, alpha-helical CRF, blocked this excitatory effect. It is concluded that CRF has a predominant excitatory effect on PAG neurons. It is also concluded that CRF is not acting presynaptically. This excitatory effect of CRF on PAG neurons may lead to activation of a descending analgesic pathway.