O-linked N-acetylglucosaminylation of Sp1 inhibits the human immunodeficiency virus type 1 promoter.
Human immunodeficiency virus type 1 (HIV-1) gene expression and replication are regulated by the promoter/enhancer located in the U3 region of the proviral 5' long terminal repeat (LTR). The binding of cellular transcription factors to specific regulatory sites in the 5' LTR is a key event in the replication cycle of HIV-1. Since transcriptional activity is regulated by the posttranslational modification of transcription factors with the monosaccharide O-linked N-acetyl-D-glucosamine (O-GlcNAc), we evaluated whether increased O-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the O-GlcNAcylation-enhancing agent glucosamine (GlcN) repressed viral transcription in a dose-dependent manner. Overexpression of O-GlcNAc transferase (OGT), the sole known enzyme catalyzing the addition of O-GlcNAc to proteins, specifically inhibited the activity of the HIV-1 LTR promoter in different T-cell lines and in primary CD4(+) T lymphocytes. Inhibition of HIV-1 LTR activity in infected T cells was most efficient (>95%) when OGT was recombinantly overexpressed prior to infection. O-GlcNAcylation of the transcription factor Sp1 and the presence of Sp1-binding sites in the LTR were found to be crucial for this inhibitory effect. From this study, we conclude that O-GlcNAcylation of Sp1 inhibits the activity of the HIV-1 LTR promoter. Modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation and links viral replication to the glucose metabolism of the host cell. Hence, the establishment of a metabolic treatment might supplement the repertoire of antiretroviral therapies against AIDS.


Mind-mindedness and maternal responsiveness in infant-mother interactions in mothers with severe mental illness.
Previous cross-diagnosis studies of interaction between mothers with severe mental illness and their babies have concluded that mothers with schizophrenia have deficits in interaction, but these studies have not included healthy controls. In-patients on a mother and baby unit, with diagnoses of schizophrenia (n=15), depressive mood disorders with or without psychosis (n=23), or mood disorders where mania was the predominant feature, with or without psychosis (n=12), were observed interacting with their infants on admission and discharge. Mothers' mind-mindedness and other measures of the quality of maternal and infant behaviour were coded. Findings from this sample were compared with those from healthy mothers and their infants (n=49). Compared with healthy controls, on admission depressed mothers were marginally less likely to comment appropriately on their infants' mental states. Both the depressed and mania groups were more likely to touch their babies and engage in attention-seeking behaviours. Interactional behaviours of mothers in the schizophrenia group were not markedly different from healthy controls. On discharge there were fewer differences between the clinical and healthy groups, although the depressed group continued to engage in more attention-seeking and touching behaviour and the mania group continued to touch their infants more. Only mothers in the schizophrenia group showed changes in interactional behaviours between admission and discharge, talking more to their infants. The findings challenge previous conclusions that mothers with schizophrenia have deficits in their interactions with their babies, and demonstrate that mothers with severe mental illness are able to respond appropriately to their infants' cues.


Vitamin D Status in Pregnant Women in Southern China and Risk of Preterm Birth: A Large-Scale Retrospective Cohort Study.
BACKGROUND The influence of maternal vitamin D on pregnancy outcomes, including preterm birth (PTB), is unclear due to different experimental designs and study populations (patient race and sample size) of previous studies. We aimed to investigate the relationship between 25-hydroxyvitamin D (25[OH] D) levels and PTB among pregnant women in southern China. MATERIAL AND METHODS A total of 11 641 pregnant women were retrospectively enrolled between January 2016 and April 2019. Vitamin D concentrations were evaluated by electrochemiluminescence immunoassay. Logistic regression analysis was used to analyze the association between vitamin D and PTB. RESULTS The average 25(OH) D concentration was 59.3±21.5 nmol/L; 34.8% of patients were vitamin D deficient, 43.0% were vitamin D insufficient (25[OH] D <50 nmol/L and 50-74.9 nmol/L, respectively). In total, 3.6% of newborns were born prematurely. Comparing the pre-term and full-term groups, 45.7% versus 42.9% and 29.8% versus 35% were vitamin D deficient and insufficient, respectively These differences were not significant (P>0.05). However, the mean vitamin D status was significantly different between the pre-term and full-term groups (61.3±21.3 and 59.1±21.5 nmol/L, respectively). No association was found between vitamin D deficiency/insufficiency and PTB in unadjusted or adjusted models, compared with vitamin D sufficiency (adjusted odds ratio, 1.016; 95% confidence interval, 0.794-1.301 and 0.842; 0.641-1.106, respectively). CONCLUSIONS Low maternal 25(OH) D levels are common in southern China. However, low vitamin D status in pregnant women appears to be unrelated to PTB. Measuring vitamin D level alone is therefore not sufficient to predict PTB.


Cannabis Microbiome and the Role of Endophytes in Modulating the Production of Secondary Metabolites: An Overview.
Plants, including cannabis (Cannabis sativa subsp. sativa), host distinct beneficial microbial communities on and inside their tissues and organs, including seeds. They contribute to plant growth, facilitating mineral nutrient uptake, inducing defence resistance against pathogens, and modulating the production of plant secondary metabolites. Understanding the microbial partnerships with cannabis has the potential to affect the agricultural practices by improving plant fitness and the yield of cannabinoids. Little is known about this beneficial cannabis-microbe partnership, and the complex relationship between the endogenous microbes associated with various tissues of the plant, and the role that cannabis may play in supporting or enhancing them. This review will consider cannabis microbiota studies and the effects of endophytes on the elicitation of secondary metabolite production in cannabis plants. The review aims to shed light on the importance of the cannabis microbiome and how cannabinoid compound concentrations can be stimulated through symbiotic and/or mutualistic relationships with endophytes.


Synergistic exacerbation of diastolic stiffness from short-term tachycardia-induced cardiodepression and angiotensin II.
Synergistic interaction between angiotensin II (Ang II) and evolving cardiodepression may play an important role in worsening chamber function, particularly in diastole. To test this hypothesis, Ang II was infused at 10 or 17 ng.kg(-1).min(-1) in 18 conscious dogs 4 days before and during induction of subacute cardiodepression by 48-hour tachypacing. The lower dose yielded negligible systemic pressure changes. Twelve additional animals served as paced-only controls. Pressure-dimension relations were recorded, and serial endocardial biopsies were obtained to assess histological and metalloproteinase (MMP) changes. Forty-eight-hour pacing alone depressed systolic function but had little effect on diastolic stiffness. Ang II alone only modestly raised diastolic stiffness at both doses and enhanced contractility at the higher dose. These changes recovered toward baseline after a 7-day infusion. However, Ang II (at either dose) combined with 48-hour pacing markedly increased ventricular stiffness (110+/-26% over baseline) and end-diastolic pressure (22+/-1.7 mm Hg). In contrast, pacing-induced inotropic and relaxation abnormalities were not exacerbated by Ang II. Zymography revealed MMP activation (72- and 92-kD gelatinases and 52-kDa caseinase) after a 4-day Ang II infusion (at both doses), which persisted during pacing. Tachypacing initiated 24 hours after cessation of a 7-day Ang II infusion also resulted in diastolic stiffening and corresponded with MMP reactivation. Ang II also induced myocyte necrosis, inflammation, and subsequent interstitial fibrosis, but these changes correlated less with chamber mechanics. Thus, Ang II amplifies and accelerates diastolic dysfunction when combined with evolving cardiodepression. This phenomenon may also underlie Ang II influences in late-stage cardiomyopathy, when chamber distensibility declines.


Normal human keratinocytes bind to the alpha3LG4/5 domain of unprocessed laminin-5 through the receptor syndecan-1.
Basal keratinocytes of the epidermis adhere to their underlying basement membrane through a specific interaction with laminin-5, which is composed by the association of alpha3, beta3, and gamma2 chains. Laminin-5 has the ability to induce either stable cell adhesion or migration depending on specific processing of different parts of the molecule. One event results in the cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) of the alpha3 chain. In this study, we recombinantly expressed the human alpha3LG4/5 fragment in mammalian cells, and we show that this fragment induces adhesion of normal human keratinocytes and fibrosarcoma-derived HT1080 cells in a heparan- and chondroitin sulfate-dependent manner. Immunoprecipitation experiments with Na2 35SO4-labeled keratinocyte and HT1080 cell lysates as well as immunoblotting experiments revealed that the major proteoglycan receptor for the alpha3LG4/5 fragment is syndecan-1. Syndecan-4 from keratinocytes also bound to alpha3LG4/5. Furthermore we could show for the first time that unprocessed laminin-5 specifically binds syndecan-1, while processed laminin-5 does not. These results demonstrate that the LG4/5 modules within unprocessed laminin-5 permit its cell binding activity through heparan and chondroitin sulfate chains of syndecan-1 and reinforce previous data suggesting specific properties for the precursor molecule.


Longitudinal development of secondary sexual characteristics in girls and boys between ages 91/2 and 151/2 years.
To identify ages when adolescents were in sexual maturity stages 2 through 5; to explain the relations between breast (girls), genital (boys), and pubic hair (girls and boys) development between ages 9(1/2) and 15(1/2) years; and to evaluate synchrony of pubertal development across characteristics. Annual pubertal assessments. Ten locations in the United States. A total of 859 adolescents (427 boys [49.7%] and 432 girls [50.3%]; 737 white [85.8%] and 122 black [14.2%]). Changes in the 5 stages of breast, genital, and pubic hair development. Girls were in breast maturity stages 2 and 3 earlier than comparable pubic hair stages. Although breast development in girls started earlier than pubic hair development, girls completed breast and pubic hair development at approximately the same age. Black girls were in all stages of breast and pubic hair development earlier than white girls. Boys were in stages 2, 3, 4, and 5 of genital development before the comparable pubic hair stage. In boys, genital development started earlier than pubic hair development, but pubic hair development was completed in less time. Black boys were in genital and pubic hair development about 7 months earlier than white boys. Black and white boys completed genital development in approximately 4(1/2) years, but black boys took approximately 6 months longer than white boys to complete pubic hair development. At stage 2, for 66.2% of girls, breast development preceded their pubic hair development; for 91.1% of boys, genital development preceded their pubic hair development. The results of this study are useful in understanding normative variation in the timing and change in the development of secondary sexual characteristics at puberty. They will help identify adolescents with atypical changes in sexual maturation and unusual progression of sexual maturation and growth disorders.


Transcriptional activation of plant defense genes by fungal elicitor, wounding, and infection.
Activation of plant defense genes was investigated by analysis of transcripts completed in vitro by isolated nuclei. Elicitor treatment of suspension-cultured bean (Phaseolus vulgaris L.) cells caused marked transient stimulation of transcription of genes encoding apoproteins of cell wall hydroxyproline-rich glycoproteins (HRGP) and the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), concomitant with the onset of rapid accumulation of the respective mRNAs and hence expression of the phytoalexin (PAL, CHS), lignin (PAL), and HRGP defense responses. While there was a lag of 2 h prior to stimulation of HRGP gene transcription, induction of the transcription of PAL and CHS genes occurred within 5 min of elicitor treatment. Induction of transcription of PAL, CHS, and HRGP genes was also observed in wounded hypocotyls and in infected hypocotyls during race-cultivar-specific interactions with the fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. Transcriptional activation occurred not only in directly infected tissue but also in distant, hitherto uninfected tissue, indicating intercellular transmission of an endogenous signal for defense gene activation. It is concluded that transcriptional activation of defense genes characteristically underlies induction of the corresponding defense responses and expression of disease resistance.


[Aromatase inhibitors: pharmacological aspects].
Selective new aromatase inhibitors are a new class of agents that are of considerable interest in the treatment of hormone-dependent breast cancer in postmenopausal women. Aromatase is an enzymic complex that catalyses the conversion of the adrenal androgens androstenedione and testosterone to estrone. In postmenopausal women, the process of peripheral aromatisation accounts for the majority of circulating estrogens. The selective inhibition of estrogen production by aromatase inhibitors is an efficient strategy for breast cancer treatment. These compounds are classified as irreversible inhibitors of aromatase (type I), and comprise steroidal compounds. Reversible inhibitors of aromatase, which comprises non-steroidal compounds are type II aromatase inhibitors. Second and third generation aromatase inhibitors are considerably more potent and more specific in their ability to inhibit aromatase, as compared with first generation compounds (aminoglutethimide).


Impaired hypothalamic cocaine- and amphetamine-regulated transcript expression in lateral hypothalamic area and paraventricular nuclei of dehydration-induced anorexic rats.
Negative energy balance promotes physiological adaptations that ensure the survival of animals. The hypothalamic-pituitary-thyroid axis regulates basal energy expenditure and its down-regulating adaptation to negative energy balance is well described: in fasting, the serum content of thyrotrophin (TSH) and thyroid hormones (TH) decreases, enhancing the survival odds of individuals. By contrast, dehydration-induced anorexic (DIA) rats present an impaired hypothalamic-pituitary-thyroid (HPT) axis adaptation despite their negative energy balance: increased circulating TSH levels. The implication of cocaine- and amphetamine-regulated transcript (CART), an anorexic peptide, in HPT axis function impairment and food-avoidance behaviour displayed by DIA animals is unknown. Because CART is co-expressed with the peptide that regulates the HPT axis in hypophysiotrophic paraventricular nucleus (PVN) neurones (TSH-releasing hormone), we analysed CART expression and possible implications with respect to high TSH levels of DIA animals. We examined whether changes in CART expression from the lateral hypothalamic area (LHA) and arcuate nucleus (ARC) could participate in food-avoidance of DIA rats. DIA and forced-food restricted (FFR) animals reduced their body weight and food intake. FFR rats had a down-regulation of their HPT axis (reduced serum TH and TSH content), whereas DIA animals had reduced TH but increased TSH levels. CART mRNA expression in the ARC decreased similarly between experimental groups and diminished in anterior, medial PVN and in LHA of FFR animals, whereas DIA animals showed unchanged levels. This impaired CART mRNA expression in the anterior PVN and LHA could be related to the aberrant feeding behaviour of DIA rats but not to their deregulated HPT axis function.


Appendicitis. The diagnostic challenge continues.
The typical clinical manifestations of appendicitis are well described, but atypical presentations occur frequently. Uncertainty regarding the diagnosis may cause long delays before appropriate treatment is rendered. Several patients in whom the diagnosis was obscure are presented. Barium enema, ultrasonography, computerized tomography, and laparoscopy each may be helpful in diagnosis, but these studies should be used selectively and negative results can be misleading. It is wise to consider other causes of abdominal pain in patients suspected of having appendicitis, so that unnecessary operation can be avoided. It is more important to suspect appendicitis in patients who appear to have nonsurgical conditions of the abdomen, so that the incidence of perforation can be minimized.


Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I-[(14)Tyr]-orphanin FQ binding.
The recently discovered neuropeptide orphanin FQ (OFQ), and its opioid receptor-like (ORL1) receptor, exhibit structural features suggestive of the micro, kappa, and delta opioid systems. The anatomic distribution of OFQ immunoreactivity and mRNA expression has been reported recently. In the present analysis, we compare the distribution of orphanin receptor mRNA expression with that of orphanin FQ binding at the ORL1 receptor in the adult rat central nervous system (CNS). By using in vitro receptor autoradiography with (125)I-[(14)Tyr]-OFQ as the radioligand, orphanin receptor binding was analyzed throughout the rat CNS. Orphanin binding sites were densest in several cortical regions, the anterior olfactory nucleus, lateral septum, ventral forebrain, several hypothalamic nuclei, hippocampal formation, basolateral and medial amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, vestibular nuclear complex, and the spinal cord. By using in situ hybridization, cells expressing ORL1 mRNA were most numerous throughout multiple cortical regions, the anterior olfactory nucleus, lateral septum, endopiriform nucleus, ventral forebrain, multiple hypothalamic nuclei, nucleus of the lateral olfactory tract, medial amygdala, hippocampal formation, substantia nigra, ventral tegmental area, central gray, raphe complex, locus coeruleus, multiple brainstem motor nuclei, inferior olive, deep cerebellar nuclei, vestibular nuclear complex, nucleus of the solitary tract, reticular formation, dorsal root ganglia, and spinal cord. The diffuse distribution of ORL1 mRNA and binding supports an extensive role for orphanin FQ in a multitude of CNS functions, including motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression, and autonomic control of physiologic processes.


A flexible approach to training veterinarians in public health: an overview and early assessment of the DVM/MPH dual-degree program at the University of Minnesota.
As a result of the growing need for public-health veterinarians, novel educational programs are essential to train future public-health professionals. The University of Minnesota School of Public Health, in collaboration with the College of Veterinary Medicine, initiated a dual DVM/MPH program in 2002. This program provides flexibility by combining distance learning and on-campus courses offered through a summer public-health institute. MPH requirements are completed through core courses, elective courses in a focus area, and an MPH project and field experience. Currently, more than 100 students representing 13 veterinary schools are enrolled in the program. The majority of initial program graduates have pursued public-practice careers upon completion of the program. Strengths of the Minnesota program design include accessibility and an environment to support multidisciplinary training. Continued assessment of program graduates will allow for evaluation and adjustment of the program in the coming years.


High Centromere Protein-A (CENP-A) Expression Correlates with Progression and Prognosis in Gastric Cancer.
Recent studies have established the ability of centromere protein-A (CENP-A) to perform as an oncogene, regulating tumor progression. The aim of this research was to explore the relationship between CENP-A expression and clinical significance in gastric cancer (GC) patients. Experiments with a microarray were conducted using the Affymetrix U133 plus 2.0 GeneChip Array. Upregulated differentially expressed genes (DEGs) were identified via the GEO2R and intersected using a Venn diagram. Bioinformatic databases Omcomine, GEPIA, and Ualcan were applied to investigate the expression level of CENP-A in GC. The real-time quantitative RT-PCR (qRT-PCR) was used to validate the level of CENP-A mRNA in GC. Immunohistochemistry (IHC) was employed to verify the protein levels of CENP-A, while the relationship between CENP-A expression and patients' clinical parameters in GC was explored through the use of IHC. Kaplan-Meier analysis was conducted to evaluate the prognostic significance of CENP-A. Additionally, the Kaplan-Meier plotter database (KM plotter) was used to verify the prognostic function of CENP-A in GC patients. The results indicated that CENP-A was significantly overexpressed, both in protein and mRNA levels of GC tissues, compared to adjacent noncancerous tissues (P<0.05). Furthermore, we observed that CENP-A expression was positively associated with TNM stage, tumor classification, lymph node metastasis, distant metastasis, and Lauren type (P<0.05). Kaplan-Meier analysis showed that patients with an overexpression of CENP-A had significantly poorer overall survival (OS) times (P<0.05). Multivariate analysis suggested CENP-A may serve as an independent predicting factor for the poor outcome of GC patients. Our results show that CENP-A upregulation is significantly correlated with advanced tumor progression and poor prognosis. CENP-A may function as a novel potential biomarker for predicting the clinical outcomes of GC patients.


[The morphology of the regional lymph nodes in skin melanoma].
Morphometric analysis was used to study the architectonics of hyperplastic regional lymph nodes in malignant melanoma patients as offset by normal condition. Specific volume relationships between all basic structures of the organ were established practically in all study groups. The specific features established should be continued at morphologic examination of lymph nodes for skin melanoma diagnosis.


Higher levels of cardiovascular fitness are associated with better executive function and prefrontal oxygenation in younger and older women.
Many studies have suggested that physical exercise training improves cognition and more selectively executive functions. There is a growing interest to clarify the neurophysiological mechanisms that underlie this effect. The aim of the current study was to evaluate the neurophysiological changes in cerebral oxygenation associated with physical fitness level and executive functions. In this study, 22 younger and 36 older women underwent a maximal graded continuous test (i.e., [Formula: see text]O2max ) in order to classify them into a fitness group (higher vs. lower fit). All participants completed neuropsychological paper and pencil testing and a computerized Stroop task (which contained executive and non-executive conditions) in which the change in prefrontal cortex oxygenation was evaluated with near infrared spectroscopy (NIRS). Our findings revealed a Fitness × Condition interaction (p < 0.05) such that higher fit women scored better on measures of executive functions than lower fit women. In comparison to lower fit women, higher fit women had faster reaction times in the Executive condition of the computerized Stroop task. No significant effect was observed in the non-executive condition of the test and no interactions were found with age. In measures of cerebral oxygenation (ΔHbT and ΔHbO2), we found a main effect of fitness on cerebral oxygenation during the Stroop task such that only high fit women demonstrated a significant increase in the right inferior frontal gyrus. Higher fit individuals who demonstrate better cardiorespiratory functions (as measured by [Formula: see text]O2max ) show faster reaction times and greater cerebral oxygenation in the right inferior frontal gyrus than women with lower fitness levels. The lack of interaction with age, suggests that good cardiorespiratory functions can have a positive impact on cognition, regardless of age.


Angiotensin II Type I Receptor (AT1R): The Gate towards COVID-19-Associated Diseases.
The binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein to its cellular receptor, the angiotensin-converting enzyme 2 (ACE2), causes its downregulation, which subsequently leads to the dysregulation of the renin-angiotensin system (RAS) in favor of the ACE-angiotensin II (Ang II)-angiotensin II type I receptor (AT1R) axis. AT1R has a major role in RAS by being involved in several physiological events including blood pressure control and electrolyte balance. Following SARS-CoV-2 infection, pathogenic episodes generated by the vasoconstriction, proinflammatory, profibrotic, and prooxidative consequences of the Ang II-AT1R axis activation are accompanied by a hyperinflammatory state (cytokine storm) and an acute respiratory distress syndrome (ARDS). AT1R, a member of the G protein-coupled receptor (GPCR) family, modulates Ang II deleterious effects through the activation of multiple downstream signaling pathways, among which are MAP kinases (ERK 1/2, JNK, p38MAPK), receptor tyrosine kinases (PDGF, EGFR, insulin receptor), and nonreceptor tyrosine kinases (Src, JAK/STAT, focal adhesion kinase (FAK)), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. COVID-19 is well known for generating respiratory symptoms, but because ACE2 is expressed in various body tissues, several extrapulmonary pathologies are also manifested, including neurologic disorders, vasculature and myocardial complications, kidney injury, gastrointestinal symptoms, hepatic injury, hyperglycemia, and dermatologic complications. Therefore, the development of drugs based on RAS blockers, such as angiotensin II receptor blockers (ARBs), that inhibit the damaging axis of the RAS cascade may become one of the most promising approaches for the treatment of COVID-19 in the near future. We herein review the general features of AT1R, with a special focus on the receptor-mediated activation of the different downstream signaling pathways leading to specific cellular responses. In addition, we provide the latest insights into the roles of AT1R in COVID-19 outcomes in different systems of the human body, as well as the role of ARBs as tentative pharmacological agents to treat COVID-19.


Stable dicentric duplication-deficiency chromosome 14 resulting from crossing-over within a maternal paracentric inversion.
We report on the extremely rare occurrence of a stable dicentric duplication-deletion chromosome 14 in a viable offspring with multiple malformations and developmental delay. This abnormality was derived from a maternal paracentric inversion in the long arm of chromosome 14. Both classical and molecular cytogenetic techniques were used to perform the chromosomal investigation of this structural abnormality. The immunofluorescent labeling of centromeric proteins shows only one functional centromere on the rearranged chromosome 14. The present observation confirms that paracentric inversions may lead to stable recombinant chromosomes.


[Clinical effects of packed red blood cells transfusion according to storage life and time of transfusion].
It was studied 66 males aged 39.5±5.3 years with hemorrhagic shock II degree. Gas composition of arterial and venous blood was studied twice (before and after transfusion). It was revealed that succinct transfusion of packed red blood cells (to 2 doses) with storage life to 3 days after bleeding stop and hypovolemia filling is the most effective correction of hemorrhagic shock II degree. Replacement therapy in operating room in condition of stopped bleeding and unrepaired hypovolemia is burdening factor because it does not conducive to transfer of oxygen at the tissue level and inhibits stimulation of bone marrow in response to hypoxia.


Cell culture dimensionality influences mesenchymal stem cell fate through cadherin-2 and cadherin-11.
The acquisition of a specific cell fate is one of the core aims of tissue engineering and regenerative medicine. Significant evidence shows that aggregate cultures have a positive influence on cell fate decisions, presumably through cell-cell interactions, but little is known about the specific mechanisms. To investigate the difference between cells cultured as a monolayer and as aggregates, we started by looking at cadherin expression, an important protein involved in cell adhesion, during the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in aggregate and monolayer cultures. We observed that proliferating hMSCs in monolayer culture expressed lower levels of cadherin-2 and increased cadherin-11 expression at cell-cell contact sites over time, which was not evident in the aggregate cultures. By knocking down cadherin-2 and cadherin-11, we found that both cadherins were required for adipogenic differentiation in a monolayer as well as aggregate culture. However, during osteogenic differentiation, low levels of cadherin-2 were found to be favorable for cells cultured as a monolayer and as aggregates, whereas cadherin- 11 was dispensable for cells cultured as aggregates. Together, these results provide compelling evidence for the important role that cadherins play in regulating the differentiation of hMSCs and how this is affected by the dimensionality of cell culture.


[Killer toxin and enzyme production by Candida albicans isolated from buccal mucosa in patients with cancer].
Opportunistic infections of the oral cavity are primarily caused by Candida and frequently occur in patients with cancer who are undergoing chemotherapy and antibiotic treatment. Of the specimens received from the oral mucosa of 44 patients with cancer, 25 (56.8%) yielded Candida on culture in Sabouraud agar. Twenty four of these isolates were identified as C. albicans (96%) and 1 as C. krusei (4%). The phenotypic characteristics of these isolates showed that all of them were strongly proteolytic, had a high ability to produce phospholipase, and presented the byotypes characterized as 811 (95.8%) and 511 (4.2%) in terms of susceptibility to killer toxins.


Phylogenetics, ancestral state reconstruction, and a new infrafamilial classification of the pantropical Ochnaceae (Medusagynaceae, Ochnaceae s.str., Quiinaceae) based on five DNA regions.
Ochnaceae s.str. (Malpighiales) are a pantropical family of about 500 species and 27 genera of almost exclusively woody plants. Infrafamilial classification and relationships have been controversial partially due to the lack of a robust phylogenetic framework. Including all genera except Indosinia and Perissocarpa and DNA sequence data for five DNA regions (ITS, matK, ndhF, rbcL, trnL-F), we provide for the first time a nearly complete molecular phylogenetic analysis of Ochnaceae s.l. resolving most of the phylogenetic backbone of the family. Based on this, we present a new classification of Ochnaceae s.l., with Medusagynoideae and Quiinoideae included as subfamilies and the former subfamilies Ochnoideae and Sauvagesioideae recognized at the rank of tribe. Our data support a monophyletic Ochneae, but Sauvagesieae in the traditional circumscription is paraphyletic because Testulea emerges as sister to the rest of Ochnoideae, and the next clade shows Luxemburgia+Philacra as sister group to the remaining Ochnoideae. To avoid paraphyly, we classify Luxemburgieae and Testuleeae as new tribes. The African genus Lophira, which has switched between subfamilies (here tribes) in past classifications, emerges as sister to all other Ochneae. Thus, endosperm-free seeds and ovules with partly to completely united integuments (resulting in an apparently single integument) are characters that unite all members of that tribe. The relationships within its largest clade, Ochnineae (former Ochneae), are poorly resolved, but former Ochninae (Brackenridgea, Ochna) are polyphyletic. Within Sauvagesieae, the genus Sauvagesia in its broad circumscription is polyphyletic as Sauvagesia serrata is sister to a clade of Adenarake, Sauvagesia spp., and three other genera. Within Quiinoideae, in contrast to former phylogenetic hypotheses, Lacunaria and Touroulia form a clade that is sister to Quiina. Bayesian ancestral state reconstructions showed that zygomorphic flowers with adaptations to buzz-pollination (poricidal anthers), a syncarpous gynoecium (a near-apocarpous gynoecium evolved independently in Quiinoideae and Ochninae), numerous ovules, septicidal capsules, and winged seeds with endosperm are the ancestral condition in Ochnoideae. Although in some lineages poricidal anthers were lost secondarily, the evolution of poricidal superstructures secured the maintenance of buzz-pollination in some of these genera, indicating a strong selective pressure on keeping that specialized pollination system.


Biomonitoring as an Underused Exposure Assessment Tool in Occupational Safety and Health Context-Challenges and Way Forward.
Recent advances in analytical chemistry have allowed a greater possibility of using quantitative approaches for measuring human exposure to chemicals. One of these approaches is biomonitoring (BM), which provides unequivocal evidence that both exposure and uptake of a chemical have taken place. BM has been a longstanding practice in occupational health for several reasons. BM integrates exposure from all routes. It can help identify unintentional and unexpected exposures and assess the effectiveness of existing risk-management measures. BM also provides relevant information to support policy development by delivering better evidence of workers' exposure to chemical substances, even within the framework of the present regulations. Thus, BM can allow for both the evaluation of the impact of regulation and identification of further needs for new or improved regulation. However, despite all these well-recognized advantages, BM is currently an underused exposure assessment tool. This paper provides an overview of the key aspects to be considered when using BM in the context of occupational health interventions. Additionally, this paper describes the potential of BM as an exposure assessment tool, distinguishing the role of BM in exposure assessment and health surveillance and clarifies ethical and communication aspects to guarantee that general data protection regulations are followed. In addition, actions and research needs are identified (particularly with reference to the European situation), which aim to encourage the increased use of BM as an exposure assessment tool.


A nonclinical model for predicting scaling and root planing case difficulty.
As students progress through their clinical education, the integration of patient types challenging the development of scaling and root planing skills is necessary. A clinical screening to determine case type is ideal; however, some programs do not have the time or resources for a clinical screening, and some patients do not want to take the time for the additional screening appointment. The intent of the screening process is to determine case type and/or treatment needs. In some cases, this information is used to match student educational requirements with patient needs. Even though an institution or program may have a clinical screening process, patient assignment can still be haphazard, resulting in an inequitable distribution of patient case types to students. It seems the problem is due, in part, to the absence of a mechanism for early identification and distribution of case types. The purpose of this study was to develop a model for initially predicting patient scaling and root planing case difficulty from nonclinical, patient variables obtained during a telephone interview. Dental hygiene student evaluation forms (n = 1,356) at the University of North Carolina at Chapel Hill School of Dentistry were gathered and categorized by patient case difficulty. A sample of charts was selected from each scaling classification. The scaling classifications were then subdivided into two samples for developing and testing the model. Variables selected from the patient charts as possible predictors were smoking status, race, age, gender, date of last prophylaxis, periodontal classification, and oral hygiene habits (brushing and flossing).(ABSTRACT TRUNCATED AT 250 WORDS)


Production of different phenotypes from the same genotype in the same environment by developmental variation.
The phenotype of an organism is determined by the genes, the environment and stochastic developmental events. Although recognized as a basic biological principle influencing life history, susceptibility to diseases, and probably evolution, developmental variation (DV) has been only poorly investigated due to the lack of a suitable model organism. This obstacle could be overcome by using the recently detected, robust and highly fecund parthenogenetic marbled crayfish as an experimental animal. Batch-mates of this clonal crayfish, which were shown to be isogenic by analysis of nuclear microsatellite loci, exhibited surprisingly broad ranges of variation in coloration, growth, life-span, reproduction, behaviour and number of sense organs, even when reared under identical conditions. Maximal variation was observed for the marmorated coloration, the pattern of which was unique in each of the several hundred individuals examined. Variation among identically raised batch-mates was also found with respect to fluctuating asymmetry, a traditional indicator of the epigenetic part of the phenotype, and global DNA methylation, an overall molecular marker of an animal's epigenetic state. Developmental variation was produced in all life stages, probably by reaction-diffusion-like patterning mechanisms in early development and non-linear, self-reinforcing circuitries involving behaviour and metabolism in later stages. Our data indicate that, despite being raised in the same environment, individual genotypes can map to numerous phenotypes via DV, thus generating variability among clone-mates and individuality in a parthenogenetic species. Our results further show that DV, an apparently ubiquitous phenomenon in animals and plants, can introduce components of randomness into life histories, modifying individual fitness and population dynamics. Possible perspectives of DV for evolutionary biology are discussed.


A model for smooth viewing and navigation of large 2D information spaces.
Large 2D information spaces, such as maps, images, or abstract visualizations, require views at various level of detail: close ups to inspect details, overviews to maintain (literally) an overview. Users often change their view during a session. Smooth animations enable the user to maintain an overview during interactive viewing and to understand the context of separate views. We present a generic model to handle smooth image viewing. The core of the model is a metric on the effect of simultaneous zooming and panning, based on an estimate of the perceived velocity. Using this metric, solutions for various problems are derived, such as the optimal animation between two views, automatic zooming, and the parametrization of arbitrary camera paths. Optimal is defined here as smooth and efficient. Solutions are based on the shortest paths of a virtual camera, given the metric. The model has two free parameters: animation speed and zoom/pan trade off. A user experiment to find good values for these is described. Finally, it is shown how the model can be extended to deal also with rotation and nonuniform scaling.


Novel tools for production and purification of recombinant adenoassociated virus vectors.
Standard protocols for the generation of adenoassociated virus type 2 (AAV-2)-based vectors for human gene therapy applications require cotransfection of cells with a recombinant AAV (rAAV) vector plasmid and a packaging plasmid that provides the AAV rep and cap genes. The transfected cells must also be overinfected with a helper virus, e.g., adenovirus (Ad), which delivers multiple helper functions necessary for rAAV production. Therefore, rAAV stocks produced using these protocols are contaminated with helper adenovirus. The generation of a novel packaging/helper plasmid, pDG, containing all AAV and Ad functions required for amplification and packaging of AAV vector plasmids, is described here. Cotransfection of cells with pDG and an AAV vector plasmid was sufficient for production of infectious rAAV, resulting in helper virus-free rAAV stocks. The rAAV titers obtained using pDG as packaging plasmid were up to 10-fold higher than those achieved using conventional protocols for rAAV production. Replacement of the AAV-2 p5 promoter by an MMTV-LTR promoter in pDG led to reduced expression of Rep78/68; however, expression of the VP proteins was significantly increased compared with VP levels from standard packaging plasmids. Immunofluorescence analyses showed that the strong accumulation of VP proteins in pDG-transfected cells resulted in enhanced AAV capsid assembly, which is limiting for efficient rAAV production. Furthermore, using a monoclonal antibody highly specific for AAV-2 capsids (A20), an rAAV affinity purification procedure protocol was established. The application of the tools described here led to a significant improvement in recombinant AAV vector production and purification.


Beckwith-Wiedemann and IMAGe syndromes: two very different diseases caused by mutations on the same gene.
Genomic imprinting is an epigenetically regulated mechanism leading to parental-origin allele-specific expression. Beckwith-Wiedemann syndrome (BWS) is an imprinting disease related to 11p15.5 genetic and epigenetic alterations, among them loss-of-function CDKN1C mutations. Intriguing is that CDKN1C gain-of-function variations were recently found in patients with IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, congenital adrenal hypoplasia, and genital anomalies). BWS and IMAGe share an imprinted mode of inheritance; familial analysis demonstrated the presence of the phenotype exclusively when the mutant CDKN1C allele is inherited from the mother. Interestingly, both IMAGe and BWS are characterized by growth disturbances, although with opposite clinical phenotypes; IMAGe patients display growth restriction whereas BWS patients display overgrowth. CDKN1C codifies for CDKN1C/KIP2, a nuclear protein and potent tight-binding inhibitor of several cyclin/Cdk complexes, playing a role in maintenance of the nonproliferative state of cells. The mirror phenotype of BWS and IMAGe can be, at least in part, explained by the effect of mutations on protein functions. All the IMAGe-associated mutations are clustered in the proliferating cell nuclear antigen-binding domain of CDKN1C and cause a dramatic increase in the stability of the protein, which probably results in a functional gain of growth inhibition properties. In contrast, BWS mutations are not clustered within a single domain, are loss-of-function, and promote cell proliferation. CDKN1C is an example of allelic heterogeneity associated with opposite syndromes.


New Mechanism for Release of Endosomal Contents: Osmotic Lysis via Nigericin-Mediated K+/H+ Exchange.
Although peptides, antibodies/antibody fragments, siRNAs, antisense DNAs, enzymes, and aptamers are all under development as possible therapeutic agents, the breadth of their applications has been severely compromised by their inability to reach intracellular targets. Thus, while macromolecules can often enter cells by receptor-mediated endocytosis, their missions frequently fail due to an inability to escape their entrapping endosomes. In this paper, we describe a general method for promoting release of any biologic material from any entrapping endosome. The strategy relies on the fact that all nascent endosomes contain extracellular (Na+-enriched) medium, but are surrounded by intracellular (K+-enriched) fluid in the cytoplasm. Osmotic swelling and rupture of endosomes will therefore be facilitated if the flow of K+ down its concentration gradient from the cytosol into the endosome can be facilitated without allowing downhill flow of Na+ from the endosome into the cytosol. While any K+ selective ionophore can promote the K+ specific influx, the ideal K+ ionophore will also exchange influxed K+ for an osmotically inactive proton (H+) in order to prevent buildup of an electrical potential that would rapidly halt K+ influx. The only ionophore that catalyzes this exchange of K+ for H+ efficiently is nigericin. We demonstrate here that ligand-targeted delivery of nigericin into endosomes that contain an otherwise impermeable fluorescent dye can augment release of the dye into the cell cytosol via swelling/bursting of the entrapping endosomes. We further show that nigericin-facilitated escape of a folate-targeted luciferase siRNA conjugate from its entrapping endosomes promotes rapid suppression of the intended luciferase reporter gene. Taken together, we propose that ionophore-catalyzed entry of K+ into endosomal compartments can promote the release of otherwise impermeable contents from their encapsulating endosomes.


Differential effects of leptin on the invasive potential of androgen-dependent and -independent prostate carcinoma cells.
Obesity has been linked with an increased risk of prostate cancer. The formation of toxic free oxygen radicals has been implicated in obesity mediated disease processes. Leptin is one of the major cytokines produced by adipocytes and controls body weight homeostasis through food intake and energy expenditure. The rationale of the study was to determine the impact of leptin on the metastatic potential of androgen-sensitive (LNCaP) cells as well as androgen-insensitive (PC-3 and DU-145) cells. At a concentration of 200 nm, LNCaP cells showed a significant increase (20% above control; P < .0001) in cellular proliferation without any effect on androgen-insensitive cells. Furthermore, exposure to leptin caused a significant (P < .01 to P < .0001) dose-dependent decrease in migration and invasion of PC3 and Du-145 prostate carcinoma cell lines. At the molecular level, exposure of androgen-independent prostate cancer cells to leptin stimulates the phosphorylation of MAPK at early time point as well as the transcription factor STAT3, suggesting the activation of the intracellular signaling cascade upon leptin binding to its cognate receptor. Taken together, these results suggest that leptin mediates the invasive potential of prostate carcinoma cells, and that this effect is dependent on their androgen sensitivity.


Diagnosing HIV infection in primary care settings: missed opportunities.
In the United States, 20% of HIV-infected persons are unaware of their diagnosis. Improved application of HIV screening recommendations in healthcare settings may facilitate diagnosis. Clinical patient data and previous healthcare visits were reviewed from medical records of newly diagnosed HIV-infected persons in Durham County, North Carolina, who initiated HIV care at Duke University Medical Center in 2008-2011. Comparisons were made to similar data from 2002-2004 using the Pearson's chi-square test and logistic regression. 101 consecutive newly diagnosed patients were identified: 67 males; 73 black, 20 white, and 8 Hispanic/Latino. Mean age was 39 years (range, 17-69), and 73 had health insurance. Median baseline CD4 count was 313 cells/μL (range, 4-1302), and HIV-1 viral load was 45,700 copies/mL (range, 165-10,000,000). One-third had a baseline CD4 count <50 cells/μL, and 15% presented with opportunistic infections. Compared to patients newly diagnosed in 2002-2004, significantly greater proportions were black and less immunocompromised in 2008-2011. Most had been seen at least once by a healthcare provider in the year prior to HIV diagnosis: 72 had ≥1 prior visits, and 47 had ≥2 visits. Among those with prior visits, 37/72 (51%) were seen in an emergency department on the first or second visit. Men were three times more likely than women to be diagnosed at their first healthcare encounter (p=0.03, OR=3.2). Despite CDC recommendations for widespread HIV screening in healthcare settings, HIV diagnosis remains delayed, even among those with frequent healthcare encounters. Educating providers and removing barriers to HIV screening may improve this problem.


[Minimally invasive operations for varicose disease of lower extremities].
The authors discuss specific using minimally invasive surgical technique of ablation of the saphenous vein trunks by a modified Oesch PIN stripper method proposed by the authors. An experience with surgical treatment of 245 patients with varicose veins using the same modified technique is analyzed. Postoperative complications and medium-term results are evaluated.


Non-vascular claudication: a clinical conundrum.
This is a prospective study of patients referred to our department with symptoms of claudication unlikely to be of vascular origin. After clinical assessment these patients were referred to an orthopaedic physician. Of 1070 patients referred, 33 patients were diagnosed with a non-vascular problem. Of these, 21 were cases of spinal stenosis with or without nerve root irritation, seven had a combination of spinal pathology and peripheral vascular disease, and five were diagnosed with intervertebral disc prolapse (n=3), diabetic neuropathy (n=1) and chronic fatigue syndrome (n=1). The prognosis for patients with non-vascular claudication in respect of the development of premature vascular events is likely to be different from vascular claudicants and they should be counselled appropriately. Furthermore, the potentially less favourable outcome following reconstruction must be clearly explained to patients with a non-vascular contribution to their symptoms before any therapeutic vascular intervention. Failure to do this is likely to have significant medicolegal implications.


[Epidermoid carcinoma on Zenker's diverticulum].
Carcinoma is a rare but recognized complication of pharyngoesophageal diverticulum. We report the case of a 49-year-old man operated for a Zenker's diverticulum with an epidermoid carcinoma arising in the diverticulum. Since carcinoma is an uncommon complication of this pathology, we review the literature and discuss the therapeutic options.


The Mukaiyama type aldol reaction for the synthesis of trans-2,6-disubstituted tetrahydropyrans: synthesis of diospongin A and B.
An efficient synthesis protocol for the preparation of trans-2,6-disubstituted tetrahydropyrans by the reaction of 1-phenyl-1-triemthylsiloxyethylene with six membered cyclic hemiacetals in the presence of iodine is developed. This reaction proceeds smoothly under mild conditions employing a catalytic amount of molecular iodine. The feature of this novel conversion includes milder reaction conditions, broader substrate scope, functional group tolerance and good diastereoselectivity. The efficiency and practicality of the current method were successfully displayed in the total synthesis of diospongin A and B in good yields.


Dating the fungus-growing termites' mutualism shows a mixture between ancient codiversification and recent symbiont dispersal across divergent hosts.
The mutualistic symbiosis between fungus-growing termites and Termitomyces fungi originated in Africa and shows a moderate degree of interaction specificity. Here we estimate the age of the mutualism and test the hypothesis that the major splits have occurred simultaneously in the host and in the symbiont. We present a scenario where fungus-growing termites originated in the African rainforest just before the expansion of the savanna, about 31 Ma (19-49 Ma). Whereas rough age correspondence is observed for the four main clades of host and symbiont, the analysis reveals several recent events of host switching followed by dispersal of the symbiont throughout large areas and throughout different host genera. The most spectacular of these is a group of closely related fungi (the maximum age of which is estimated to be 2.4 Ma), shared between the divergent genera Microtermes, Ancistrotermes, Acanthotermes and Synacanthotermes (which diverged at least 16.7 Ma), and found throughout the African continent and on Madagascar. The lack of geographical differentiation of fungal symbionts shows that continuous exchange has occurred between regions and across host species.


Diabetes Mellitus is Associated With Low Secretion Rates of Immunoglobulin A in Saliva.
The association between diabetes mellitus (DM) and low secretory immunoglobulin A (s-IgA) secretion rates is one mechanism suspected of influencing susceptibility to infections among DM patients. However, several studies have shown contradictory results. We examined these two factors to seek evidence of an association among older people. We analyzed a prospective cohort of 2306 subjects (1209 men and 1097 women) around 64 years old from the New Integrated Suburban Seniority Investigation (NISSIN) Project in Nisshin, Japan. DM statuses were ascertained from levels of fasting plasma glucose and HbA1c, and s-IgA secretion rates were obtained from 5-min saliva samples. We used an analysis of covariance adjusted for possible confounders to compare s-IgA secretion rates according to DM status. s-IgA secretion rates in DM participants were lower than in those classified as normal (18.6 µg/min vs 15.0 µg/min, P = 0.03), even after elimination of the effects of possible confounders. DM was associated with lower s-IgA secretion rates. This suggests that lower s-IgA levels may be a mechanism of susceptibility to infection in individuals with DM.


[Relationship of vascular reactivity to blood lipid spectrum and lipid peroxidation in unstable angina].
To examine the relationship between the vascular response of the microcirculatory bed to vasoactive agents and lipid peroxidation (LPO) and antioxidative defense in patients with unstable angina pectoris. Thirty male patients (mean age 58.0 +/- 2.4 years) were examined. The patients were divided into 2 groups: 1) those with high alpha-cholesterolemia alone and 2) those with high alpha-cholesterolemia concurrent with hyperglyceridemia and elevated total cholesterol levels. Photoplethysmographic determination was used to evaluate vascular responses to norepinephrine (vasopressor function) and histamine (vasodilating function), the blood levels of cortisol, LPO products, antioxidative protection components, histamine, and serotonin. Vascular dilating function was found to be most pronounced in Group 1. Moreover, Group 1 patients had a significant depression of the antioxidative defense system and the highest blood levels of cortisol, and serotonin. The levels of LPO products did not differ significantly in both groups though they were significantly higher than those in healthy individuals. Impairment in the antioxidant system and in coordination of vasodilating function to histamine stimulation are typical of patients with unstable angina.


Exploiting the emergent nature of mixed methods designs: insights from a mixed methods impact evaluation in Malawi.
The application of mixed methods in Health Policy and Systems Research (HPSR) has expanded remarkably. Nevertheless, a recent review has highlighted how many mixed methods studies do not conceptualize the quantitative and the qualitative component as part of a single research effort, failing to make use of integrated approaches to data collection and analysis. More specifically, current mixed methods studies rarely rely on emergent designs as a specific feature of this methodological approach. In our work, we postulate that explicitly acknowledging the emergent nature of mixed methods research by building on a continuous exchange between quantitative and qualitative strains of data collection and analysis leads to a richer and more informative application in the field of HPSR. We illustrate our point by reflecting on our own experience conducting the mixed methods impact evaluation of a complex health system intervention in Malawi, the Results Based Financing for Maternal and Newborn Health Initiative. We describe how in the light of a contradiction between the initial set of quantitative and qualitative findings, we modified our design multiple times to include additional sources of quantitative and qualitative data and analytical approaches. To find an answer to the initial riddle, we made use of household survey data, routine health facility data, and multiple rounds of interviews with both healthcare workers and service users. We highlight what contextual factors made it possible for us to maintain the high level of methodological flexibility that ultimately allowed us to solve the riddle. This process of constant reiteration between quantitative and qualitative data allowed us to provide policymakers with a more credible and comprehensive picture of what dynamics the intervention had triggered and with what effects, in a way that we would have never been able to do had we kept faithful to our original mixed methods design.


Sentinel lymph node biopsy: is it indicated in patients with high-risk ductal carcinoma-in-situ and ductal carcinoma-in-situ with microinvasion?
Axillary lymph node status is the strongest prognostic indicator of survival for women with breast cancer. The purpose of this study was to determine the incidence of sentinel node metastases in patients with high-risk ductal carcinoma-in-situ (DCIS) and DCIS with microinvasion (DCISM). From November 1997 to November 1999, all patients who underwent sentinel node biopsy for high-risk DCIS (n = 76) or DCISM (n = 31) were enrolled prospectively in our database. Patients with DCIS were considered high risk and were selected for sentinel lymph node biopsy if there was concern that an invasive component would be identified in the specimen obtained during the definitive surgery. Patients underwent intraoperative mapping that used both blue dye and radionuclide. Excised sentinel nodes were serially sectioned and were examined by hematoxylin and eosin and by immunohistochemistry. Of 76 patients with high-risk DCIS, 9 (12%) had positive sentinel nodes; 7 of 9 patients were positive for micrometastases only. Of 31 patients with DCISM, 3 (10%) had positive sentinel nodes. 2 of 3 were positive for micrometastases only. Six of nine patients with DCIS and three of three with DCISM and positive sentinel nodes had completion axillary dissection; one patient with DCIS had an additional positive node detected by conventional histological analysis. This study documents a high incidence of lymph node micrometastases as detected by sentinel node biopsy in patients with high-risk DCIS and DCISM. Although the biological significance of breast cancer micrometastases remains unclear at this time, these findings suggest that sentinel node biopsy should be considered in patients with high-risk DCIS and DCISM.


[A new type of Clostridium thermocellum endoglucanase produced by the recombinant strain of E. coli. Some properties and identification in donor cells].
The properties of endoglucanase produced by the recombinant strain of E. coli carrying plasmid pCU 104 with a 2.9 kb insert of chromosomal DNA of C. thermocellum encoding the multiple forms of the 35.5 kD polypeptide (pI 4.3-4.7) were studied. The enzyme has a broad pH optimum of activity (6.0-7.5). The half-inactivation time for different forms of the enzyme at 65 degrees C is similar and is equal to 25-30 minutes. The enzyme is related to endoglucanases weakly adsorbed on cellulose (Kp = 0.065 1/g). Hydrolysis of microcrystalline cellulose is completed within 7 days (7-9%) and is accompanied by the formation of cellobiose and cellotriose. The enzyme splits dyed lichenan (mixed 1,3-1,4-beta-glucane) at a higher rate than the dyed CM-cellulose. A guinea pig antiserum to enzyme isoforms with a pI of 4.46-4.54 was obtained. Using direct solid phase immunoenzymatic analysis, it was demonstrated that all the enzyme isoforms under study (pI 4.3-4.7) are immunologically related (serum titers for different enzyme isoforms vary from 1:20,000 to 1:50,000). In the original culture fluid of C. thermocellum, the antigen related to the enzyme isolated from the recombinant strain was unobserved. However, SDS-PAAG electrophoresis of SDS- and mercaptoethanol-treated culture fluids revealed among 11 protein bands at least 4 antigens interacting with antibodies (Mr = 107, 76, 67 and 37 kD), although their antibody titers were far lower and did not exceed 1:300-1:500. The cumulative data suggest that the endoglucanase under study is not identical to the earlier described enzymes encoded by the cel A- and ceI B-genes of C. thermocellum.


Eimeria stiedae causes most of the white-spotted liver lesions in wild European rabbits in Cambridgeshire, United Kingdom.
In rabbits, a white-spotted liver can be indicative of one of several disease processes, frequently caused by parasites. To date, the prevalence of white-spotted liver in wild rabbits, Oryctolagus cuniculus, in the United Kingdom is undetermined. We evaluated the prevalence and main parasitic etiologies of this entity in a U.K. population of wild rabbits. Wild rabbits (n = 87) were shot in Cambridgeshire for population control, and cadavers were donated for research. Postmortem examination was undertaken, including gross and histologic hepatic examination. Macroscopic lesions consistent with white-spotted liver were found in 46 of 87 (53%) rabbits examined; most of these lesions were considered to be mild. For 28 of 46 (59%) rabbits with gross hepatic lesions, an etiologic agent was apparent histologically. Eimeria stiedae was detected in 21 of 87 (24%) rabbits, and Calodium hepaticum (syn. Capillaria hepatica) was detected in 7 of 87 (8%). In the subset of rabbits killed in the summer, there was a significant association between white-spotted liver and juvenile age class. There was also an association between white-spotted liver caused by E. stiedae and juvenile age class. When restricting analysis to rabbits with white-spotted liver caused by E. stiedae and submitted in the summer, both juvenile age class and female had significant effects. E. stiedae and C. hepaticum can be transmitted to pet lagomorphs via contaminated vegetation, and to humans in the case of the latter, which demonstrates the importance of monitoring the prevalence of these parasitic diseases in wild rabbits.


Enhanced differentiation of adult bone marrow-derived stem cells to liver lineage in aggregate culture.
Hepatocyte-like cells derived from stem cells hold great potential for clinical and pharmaceutical applications, including high-throughput drug toxicity screening. We report a three-dimensional aggregate culture system for the directed differentiation of adult rat bone marrow-derived stem cells, rat multipotent adult progenitor cells, to hepatocyte-like cells. Compared to adherent monolayer cultures, differentiation in the aggregate culture system resulted in significantly higher expression level of liver-specific transcripts, including an increased albumin mRNA level, and higher levels of albumin and urea secretion. This coincides with the presence of significantly more cells that express intracellular albumin at levels found in primary hepatocytes. The differentiated cell aggregates exhibited cytochrome P450-mediated ethoxyresorufin-O-dealkylation and pentoxyresorufin-O-dealkylation activity. Consistent with these increased mature functions, cells within the aggregates were shown to have many ultrastructural features of mature hepatocytes by transmission electron microscopy. With the scalability of the aggregate culture system and the enhanced differentiation capability, this system may facilitate translation of generating hepatocytes from stem cells to technology.


Mangiferin, a dietary xanthone protects against mercury-induced toxicity in HepG2 cells.
Mercury is one of the noxious heavy metal environmental toxicants and is a cause of concern for human exposure. Mangiferin (MGN), a glucosylxanthone found in Mangifera indica, reported to have a wide range of pharmacological properties. The objective of this study was to evaluate the cytoprotective potential of MGN, against mercury chloride (HgCl(2) ) induced toxicity in HepG2 cell line. The cytoprotective effect of MGN on HgCl(2) induced toxicity was assessed by colony formation assay, while antiapoptotic effect by fluorescence microscopy, flow cytometric DNA analysis, and DNA fragmentation pattern assays. Further, the cytoprotective effect of MGN against HgCl(2) toxicity was assessed by using biochemical parameters like reduced glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) by spectrophotometrically, mitochondrial membrane potential by flowcytometry and the changes in reactive oxygen species levels by DCFH-DA spectrofluoremetric analysis. A significant increase in the surviving fraction was observed with 50 μM of MGN administered two hours prior to various concentrations of HgCl(2) . Further, pretreatment of MGN significantly decreased the percentage of HgCl(2) induced apoptotic cells. Similarly, the levels of ROS generated by the HgCl(2) treatment were inhibited significantly (P < 0.01) by MGN. MGN also significantly (P < 0.01) inhibited the HgCl(2) induced decrease in GSH, GST, SOD, and CAT levels at all the post incubation intervals. Our study demonstrated the cytoprotective potential of MGN, which may be attributed to quenching of the ROS generated in the cells due to oxidative stress induced by HgCl(2) , restoration of mitochondrial membrane potential and normalization of cellular antioxidant levels.


Simple method for maintaining serum lidocaine levels in the therapeutic range.
Lidocaine hydrochloride is commonly infused intravenously to prevent ventricular arrhythmias. In some patients, elevations in serum lidocaine levels can cause serious toxic effects. In a group of 19 patients given a constant infusion of lidocaine, we confirmed the observation that serum lidocaine levels rose significantly between four and 24 hours after initiation of therapy. One of these patients manifested a toxic reaction to lidocaine. The lidocaine infusion rate was modified in a second group of 32 patients on the basis of the four-hour serum level. In these patients, after dosage adjustment, the mean lidocaine level did not rise, and all levels remained within the 2- to 4-mg/L therapeutic range at 24 hours. Without dosage adjustment, half of these levels would have fallen outside the desired range. A simple formula can be used to adjust prophylactic lidocaine infusion rates to attain levels that remain therapeutic, yet nontoxic.


Screening for cognitive impairment in the elderly using the mini-mental state examination.
The use by non-medical staff of a brief screening instrument (the mini-mental state examination) to detect cognitive impairment among the elderly in primary care is described. Patients aged 75 years and over registered with nine general practices in north and north west London were invited by their general practitioners to take part in the study and 1170 patients participated, a response rate of 90%. The prevalence of possible or probable cognitive impairment (score below 25 on the mini-mental state examination) was 12.8%. Six per cent of patients scored below 19, at which score a high probability of dementia exists, although less than a third of this group had a diagnosis of dementia in their medical records. There was no significant difference between men and women or by social class in the proportion of patients with low scores, but the proportion with dementia rose from 2.5% in those aged 75-79 years to 29.0% among those aged 90 years and over. Under the new general practitioner contract there is a requirement to provide annual services to the elderly, including a 'mental assessment'. In a practice with a list size of 2000 around 130 patients are likely to be aged 75 years and over. Of these around 17 would require further assessment for possible dementia on the basis of results obtained using the mini-mental state examination.


Frequency and predictors of ventilator-associated pneumonia recurrence: a meta-analysis.
Large clinical series focusing on the risk factors associated with recurrence after the onset of an initial episode of ventilator-associated pneumonia (VAP) produced inconsistent results. A meta-analysis would be helpful to shed light on the issue. Our objective was to estimate the frequency of VAP recurrence and to identify risk factors associated with it. PubMed, Scopus, Current Contents, and references of retrieved articles were searched without language restrictions. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by using both the Mantel-Haenszel fixed-effect and the DerSimonian-Laird random effects models. The overall frequency of VAP recurrence in 969 patients of the seven eligible reports was 26.8%. Among the 20 evaluated risk factors, only acute lung injury/acute respiratory distress syndrome (OR, 1.76; 95% CI, 1.12-2.75) and shock (OR, 1.55; 95% CI, 1.01-2.41) at the day of diagnosis of the first VAP episode were found to be associated with VAP recurrence. There was also evidence, albeit inconsistent, that severity of illness at intensive care unit admission was associated with VAP recurrence. Recurrence involves almost one in four cases of VAP and is associated with acute lung injury/acute respiratory distress syndrome and shock, but not with first-episode causative pathogens. Recognition of these predictors may permit the timely implementation of measures to prevent recurrence of VAP.


Abnormal colonic accumulation of fluorine-18-FDG in pseudomembranous colitis.
A 51-yr-old man with a history of pancreatic carcinoma was studied with [18F]fluorodeoxyglucose ([18F]FDG) and PET as part of staging for residual disease after chemotherapy. The PET study was performed during a clostridium difficile-associated diarrheal illness. Striking [18F]FDG uptake was demonstrated in the wall of the colon over its entire length. Clostridium difficile associated diarrhea and mechanisms of [18F]FDG uptake in normal and abnormal tissues are briefly reviewed and a mechanism for FDG uptake in this patient is postulated.


Dietary fatty acids and oxidative stress in the heart mitochondria.
Our study compared the effects of different oils on oxidative stress in rat heart mitochondria, as well as on plasma parameters used as risk factors for cardiovascular disease. The rats were fed for 16 weeks with coconut, olive, or fish oil diet (saturated, monounsaturated, or polyunsaturated fatty acids, respectively). The cardiac mitochondria from rats fed with coconut oil showed the lowest concentration of oxidized proteins and peroxidized lipids. The fish oil diet leads to the highest oxidative stress in cardiac mitochondria, an effect that could be partly prevented by the antioxidant probucol. Total and LDL cholesterols decreased in plasma of rats fed fish oil, compared to olive and coconut oils fed rats. A diet enriched in saturated fatty acids offers strong advantages for the protection against oxidative stress in heart mitochondria.


Molecular Effects and Tissue Penetration Depth of Physical Plasma in Human Mucosa Analyzed by Contact- and Marker-Independent Raman Microspectroscopy.
Noninvasive epithelial tissue treatment with cold atmospheric plasma (CAP) is a promising option for local treatment of chronic inflammatory and precancerous lesions as well as various mucosal cancer diseases. Atmospheric pressure plasma jets (APPJ) are well-characterized and medically approved plasma sources. There are numbers of medically approved plasma sources for the treatment of epithelial diseases; however, little is known about the biochemical effects of CAP at the plasma-tissue interface. Furthermore, the actual penetration depth of CAP into tissue is currently unclear. Noninvasive and marker-independent Raman microspectroscopy was employed to assess the molecular effects of CAP on single cells and primary human cervical tissue samples. CAP treatment showed immediate and persisting changes of specific molecular tissue components determined by multivariate analysis. Raman imaging identified CAP-dependent changes in the morphology of the tissue, as well as molecular tissue components. The expression of the different components was not significantly altered within 24 h of incubation. DNA and lipids showed the strongest changes upon CAP treatment, which were traced to the basal cell layer of cervical epithelium, corresponding to an average functional plasma penetration depth of roughly 270 μm. In this study, Raman microspectroscopy is shown to be a promising method for molecular single-cell and solid tissue characterization. Regarding CAP treatment of tissues, Raman microspectroscopy could be suitable for the screening of biological mechanisms as well as for future contact- and marker-independent monitoring of plasma tissue effects.


Solid-substrate fermentation of wheat grains by mycelia of indigenous species of the genus Ganoderma (higher Basidiomycetes) to enhance the antioxidant activities.
Species of the genus Ganoderma are a cosmopolitan wood decaying white rot fungi, which has been used by the Asians for therapeutic purposes for centuries. In the present study, solid-substrate fermentation (SSF) of wheat grains (Triticum aestivum L.) was carried out with indigenous Ganoderma australe (KUM60813) and G. neo-japonicum (KUM61076) selected based on ethnomycological knowledge. G. lucidum (VITA GL) (a commercial strain) was also included in the study. Antioxidant activities of the crude ethanol and aqueous extracts of the fermented and unfermented wheat grains were investigated by ferric reducing antioxidant power (FRAP), Trolox equivalent antioxidant capacity (TEAC), diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging ability, and lipid peroxidation assay. Among the six mycelia extracts tested, the ethanol extract from wheat fermented with KUM61076 mycelia showed the most potent antioxidant activities, whereas the ethanol extract of wheat grains fermented with KUM60813 mycelia has a good potential in protecting frying oils against oxidation. Total phenolic content (TPC) in the ethanol extracts were higher than that in the aqueous extract. The wheat grains fermented with G. australe (KUM60813) and G. neo-japonicum KUM61076 have greater antioxidant potential compared to the commercially available G. lucidum (VITA GL). The antioxidant activities of the mycelia extracts had a positive correlation with their phenolic contents. Thus phenolic compounds may play a vital role in the antioxidant activities of the selected Ganoderma spp.


Longitudinal Profiling of Plasma Cytokines and Its Association With Postoperative Delirium in Elderly Patients Undergoing Major Lower Limb Surgery: A Prospective Observational Study.
Surgery is accompanied by a systemic inflammatory response that may presage delirium in susceptible individuals. Little is known about the trajectory of plasma proinflammatory cytokines and their potential associations with postoperative delirium (POD). The current study longitudinally assessed both pro and anti-inflammatory plasma cytokine response and development of POD in older surgical patients to investigate associations with individual and/or clusters of cytokines that may indicate pathogenic mechanisms. A prospective longitudinal study sought to enroll patients >60 years old who were scheduled for major lower limb surgery under general anesthesia. Blood was obtained preoperatively and postoperatively from day 1 through postoperative day 4 for measurement of plasma interleukin-1β (IL-1β), IL-2, IL-4, IL-6, soluble IL-6 receptor (sIL-6R), IL-10, and tumor necrosis factor-α (TNF-α). Participants were assessed for POD twice daily for 4 days using the confusion assessment method. Trajectory of postoperative changes in plasma cytokines was determined by a group-based trajectory modeling analysis that was informed by distinct cytokines identified by time-dependent Cox regression model. One hundred eighty-eight patients were assessed for eligibility of whom 129 underwent major surgery and 126 had complete datasets for final analysis. POD was diagnosed in 31 of 126 patients (24.6%). Time-dependent Cox regression model identified that higher IL-6 and sIL-6R levels were associated with higher risk of developing POD. A two-cluster model (stable lower and fluctuating higher levels) was considered to be the most statistically appropriate model for IL-6 and sIL-6R trajectory. More participants with fluctuating higher IL-6 were delirious (73.3% vs 18.0%, P = .001) as were those with fluctuating higher sIL-6R (81.3% vs 16.4%, P = .001). As higher IL-6 and sIL-6R levels were significantly associated with higher risk of POD and the combination is required for IL-6 trans-signaling, it is possible that activation of this pathway may be associated with POD. Furthermore, it will be important to determine whether high levels of the combination of IL-6 and sIL-6R can be an early biomarker for the subsequent development of POD.


Evaluation of Nucleoside Analogs as Antimicrobials Targeting Unique Enzymes in Borrelia burgdorferi.
The first line therapy for Lyme disease is treatment with doxycycline, amoxicillin, or cefuroxime. In endemic regions, the persistence of symptoms in many patients after completion of antibiotic treatment remains a major healthcare concern. The causative agent of Lyme disease is a spirochete, Borrelia burgdorferi, an extreme auxotroph that cannot exist under free-living conditions and depends upon the tick vector and mammalian hosts to fulfill its nutritional needs. Despite lacking all major biosynthetic pathways, B. burgdorferi uniquely possesses three homologous and functional methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs: Bgp, MtnN, and Pfs) involved in methionine and purine salvage, underscoring the critical role these enzymes play in the life cycle of the spirochete. At least one MTAN, Bgp, is exceptional in its presence on the surface of Lyme spirochetes and its dual functionality in nutrient salvage and glycosaminoglycan binding involved in host-cell adherence. Thus, MTANs offer highly promising targets for discovery of new antimicrobials. Here we report on our studies to evaluate five nucleoside analogs for MTAN inhibitory activity, and cytotoxic or cytostatic effects on a bioluminescently engineered strain of B. burgdorferi. All five compounds were either alternate substrates and/or inhibitors of MTAN activity, and reduced B. burgdorferi growth. Two inhibitors: 5'-deoxy-5'-iodoadenosine (IADO) and 5'-deoxy-5'-ethyl-immucillin A (dEt-ImmA) showed bactericidal activity. Thus, these inhibitors exhibit high promise and form the foundation for development of novel and effective antimicrobials to treat Lyme disease.


A Simplified Vacuum Dressing System.
Negative pressure wound therapy (NPWT) may not necessarily require sophisticated equipment. In settings with limited medical resources, simplified vacuum dressings have been considered an effective alternative to the gold-standard vacuum-assisted closure system. The aim of this study was to evaluate the performance of a simplified vacuum dressing system in the treatment of complex wounds. This was a prospective study in which difficult-to-manage wounds were treated using a simplified vacuum dressing system. Thirty-nine patients with a total of 51 wounds were treated. The main complications were pain (15.7%), bleeding (9.6%), and extremity edema (5.2%). After NPWT, the wounds were closed by skin grafting or with a skin flap in 71.8% of patients. The results of NPWT were considered satisfactory in 87.2% of cases. The use of a simplified vacuum dressing system provided satisfactory results in the treatment of complex wounds in the studied population. The system proved easy to use, had a low complication rate and excellent costeffectiveness, and it may be considered as an effective alternative to the gold-standard vacuum-assisted closure system for patients requiring prolonged hospitalization due to the presence of extensive wounds that cannot be treated at home.


[Identification of Azospirillum genus bacteria isolated from the spring wheat root zone].
Bacteria with high N2-fixing activity were isolated from the root zone of spring wheat grown on leach chernozem and soddy podzolic soil in Ukrainian marshy woodlands. They were characterized by phenotypic signs and investigated with the help of molecular-genetic methods. On the basis of diagnostic signs the investigated strains were referred to Azospirillum brasilense from Azospirillum genus. Their 3'- and 5'-thermal 16S RNA hypervariable sites with length from 373 to 395 nucleotides were amplified and sequenced. The comparative analysis of results confirmed the 100% identity of 16S RNA sequences from investigated bacteria with the same sequences of A. brasilense from Gene Bank database. Thus the results of sequence analysis agree with results obtained during the investigation of phenotypic signs.


Novel treatment option for MUC16-positive malignancies with the targeted TRAIL-based fusion protein Meso-TR3.
The targeted delivery of cancer therapeutics represents an ongoing challenge in the field of drug development. TRAIL is a promising cancer drug but its activity profile could benefit from a cancer-selective delivery mechanism, which would reduce potential side effects and increase treatment efficiencies. We recently developed the novel TRAIL-based drug platform TR3, a genetically fused trimer with the capacity for further molecular modifications such as the addition of tumor-directed targeting moieties. MUC16 (CA125) is a well characterized biomarker in several human malignancies including ovarian, pancreatic and breast cancer. Mesothelin is known to interact with MUC16 with high affinity. In order to deliver TR3 selectively to MUC16-expressing cancers, we investigated the possibility of targeted TR3 delivery employing the high affinity mesothelin/MUC16 ligand/receptor interaction. Using genetic engineering, we designed the novel cancer drug Meso-TR3, a fusion protein between native mesothelin and TR3. The recombinant proteins were produced with mammalian HEK293T cells. Meso-TR3 was characterized for binding selectivity and killing efficacy against MUC16-positive cancer cells and controls that lack MUC16 expression. Drug efficacy experiments were performed in vitro and in vivo employing an intraperitoneal xenograft mouse model of ovarian cancer. Similar to soluble mesothelin itself, the strong MUC16 binding property was retained in the Meso-TR3 fusion protein. The high affinity ligand/receptor interaction was associated with a selective accumulation of the cancer drug on MUC16-expressing cancer targets and directly correlated with increased killing activity in vitro and in a xenograft mouse model of ovarian cancer. The relevance of the mesothelin/MUC16 interaction for attaching Meso-TR3 to the cancer cells was verified by competitive blocking experiments using soluble mesothelin. Mechanistic studies using soluble DR5-Fc and caspase blocking assays confirmed engagement of the extrinsic death receptor pathway. Compared to non-targeted TR3, Meso-TR3 displayed a much reduced killing potency on cells that lack MUC16. Soluble Meso-TR3 targets the cancer biomarker MUC16 in vitro and in vivo. Following attachment to the tumor via surface bound MUC16, Meso-TR3 acquires full activation with superior killing profiles compared to non-targeted TR3, while its bioactivity is substantially reduced on cells that lack the tumor marker. This prodrug phenomenon represents a highly desirable property because it has the potential to enhance cancer killing with fewer side-effects than non-targeted TRAIL-based therapeutics. Thus, further exploration of this novel fusion protein is warranted as a possible therapeutic for patients with MUC16-positive malignancies.


Docosahexaenoic acid attenuates mycotoxin-induced immunoglobulin a nephropathy, interleukin-6 transcription, and mitogen-activated protein kinase phosphorylation in mice.
The purpose of this investigation was to evaluate the dose-dependent effects of docosahexaenoic acid (DHA) on deoxynivalenol (DON)-induced IgA nephropathy in mice and their relation to proinflammatory gene expression and mitogen-activated protein kinase (MAPK) activation. Consumption of a modified AIN-93G diet containing 1, 5, and 30 g/kg DHA resulted in dose-dependent increases of DHA in liver phospholipids with concomitant decreases in arachidonic acid compared with control diets. DHA dose dependently inhibited increases in serum IgA and IgA immune complexes (IC) as well as IgA deposition in the kidney in DON-fed mice; the 30 g/kg DHA diet had the earliest detectable effects and maximal efficacy. Both splenic interleukin-6 (IL-6) mRNA and heterogeneous nuclear RNA (hnRNA), an indicator of IL-6 transcription, were significantly reduced in DON-fed mice that consumed 5 and 30 g/kg DHA; a similar reduction was observed for cyclooxygenase (COX-2) mRNA. In a subsequent study, acute DON exposure (25 mg/kg body weight) induced splenic IL-6 mRNA and hnRNA as well as COX-2 mRNA in mice fed the control diet, whereas induction of both RNA species was significantly inhibited in mice fed 30 g/kg DHA. These latter inhibitory effects corresponded to a reduction in DON-induced phosphorylation of p38, extracellular-signal related kinase 1/2, and c-Jun N-terminal kinase 1/2 MAPKs in the spleen. Taken together, the results indicate that DHA dose-dependently inhibited DON-induced IgA dysregulation and nephropathy, and that impairment of MAPK activation and expression of COX-2 and IL-6 are potential critical upstream mechanisms.


Specific protein homeostatic functions of small heat-shock proteins increase lifespan.
During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat-shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress-denatured substrates and/or to prevent aggregation of disease-associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70-dependent refolding of stress-denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70-independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.


Genome-wide identification of the cation/proton antiporter (CPA) gene family and functional characterization of the key member BnaA05.NHX2 in allotetraploid rapeseed.
Rapeseed (Brassica napus L.) is susceptible to nutrient stresses during growth and development; however, the CPA (cation proton antiporter) family genes have not been identified in B. napus and their biological functions remain unclear. This study was aimed to identify the molecular characteristics of rapeseed CPAs and their transcriptional responses to multiple nutrient stresses. Through bioinformatics analysis, 117 BnaCPAs, consisting of three subfamilies: Na+/H+ antiporter (NHX), K+ efflux antiporter (KEA), and cation/H+ antiporter (CHX), were identified in the rapeseed genome. Transcriptomic profiling showed that BnaCPAs, particularly BnaNHXs, were transcriptionally responsive to diverse nutrient stresses, including Cd toxicity, K starvation, salt stress, NH4+ toxicity, and low Pi. We found that the salt tolerance of the transgenic rapeseed lines overexpressing BnaA05.NHX2 was significantly higher than that of wild type. Subcellular localization showed that BnaA05.NHX2 was localized on the tonoplast, and TEM combined with X-ray energy spectrum analysis revealed that the vacuolar Na+ concentrations of the BnaA05.NHX2-overexpressing rapeseed plants were significantly higher than those of wild type. The findings of this study will provide insights into the complexity of the BnaCPA family and a valuable resource to explore the in-depth functions of CPAs in B. napus.


Deglycosylation of SLAMF7 in breast cancers enhances phagocytosis.
N-linked glycosylation of proteins is one of the post-translational modifications (PTMs) that shield tumor antigens from immune attack. Signaling lymphocytic activation molecule family 7 (SLAMF7) suppresses cancer cell phagocytosis and is an ideal target under clinical development. PTM of SLAMF7, however, remains less understood. In this study, we investigated the role of N-glycans on SLAMF7 in breast cancer progression. We identified seven N-linked glycosylation motifs on SLAMF7, which are majorly occupied by complex structures. Evolutionally conserved N98 residue is enriched with high mannose and sialylated glycans. Hyperglycosylated SLAMF7 was associated with STT3A expression in breast cancer cells. Inhibition of STT3A by a small molecule inhibitor, N-linked glycosylation inhibitor-1 (NGI-1), reduced glycosylation of SLAMF7, resulting in enhancing antibody affinity and phagocytosis. To provide an on-target effect, we developed an antibody-drug conjugate (ADC) by coupling the anti-SLAMF7 antibody with NGI-1. Deglycosylation of SLAMF7 increases antibody recognition and promotes macrophage engulfment of breast cancer cells. Our work suggests deglycosylation by ADC is a potential strategy to enhance the response of immunotherapeutic agents.


Preferential expression of the complement regulatory protein decay accelerating factor at the fetomaternal interface during human pregnancy.
The expression of decay accelerating factor (DAF) was investigated in human fetal and extra-fetal tissues using a panel of mAb directed against different epitopes on the DAF molecule. By immunostaining, extensive reactivity was observed on the placental trophoblast epithelium and this was confined exclusively to sites of direct contact with maternal blood and tissues at the fetomaternal interface. Within the fetus, by contrast, very little staining was observed especially on hemopoietic cell populations in the developing liver. The antibodies identified a component of 70,000 m.w., comigrating on SDS-PAGE with red cell DAF, on isolated trophoblast membranes and an apparently quantitative increase in the expression of DAF was observed during placental development. Northern analysis using a DAF cDNA clone revealed 1.5-, 1.6-, and 2.2-kb mRNA transcripts typical of DAF in mRNA prepared from whole term placentae and from purified trophoblast cells. DAF appears to be preferentially expressed at the fetomaternal interface during development and may function specifically to inhibit amplification convertases formed at this site either directly or indirectly as a result of maternal complement activation. This molecule may play an important role in protecting the semiallogeneic human conceptus from maternal C-mediated attack.


Surface enhanced Raman scattering and plasmon enhanced fluorescence in zinc-tellurite glass.
We report significant enhancements in Er(3+) luminescence as well as in Raman intensity in silver nanoparticles embedded zinc-tellurite glass. Surface enhanced Raman scattering effect is highlighted for the first time in tellurite glass containing silver NPs resulting in an enhanced Raman signal (~10 times). SAED manifest the growth of Ag(0) nanoparticles along the (111) and (200) crystallographic planes having average diameter in the range 14-36 nm. Surface plasmon resonance bands are observed in the range 484-551 nm. Furthermore, four prominent photoluminescence bands undergo significant enhancements up to 3 times. The enhancement is majorly attributed to the local field effect of silver NPs.


A pilot randomised controlled trial of abrupt versus gradual smoking cessation in combination with vaporised nicotine products for people receiving alcohol and other drug treatment.
Vaporised nicotine products (VNPs) may be a potential quit smoking strategy. Most research has permitted participants to use VNPs ad libitum. This is the first study to examine combining the use of a VNP with a gradual or abrupt cessation guideline. This study aims to test the potential feasibility of a quit smoking strategy (abrupt verses gradual cessation) in combination with vaporised nicotine products among people in AOD treatment. We conducted a pilot randomised controlled trial between April 2018 and July 2019. Participants were recruited from AOD programs located within one area health service in Australia. Participants were provided with two VNPs, a 12-week supply of nicotine e-liquid and randomised to either the abrupt (assigned a quit date the day they were provided their VNP) or gradual quit smoking strategy (reduce baseline number of cigarettes per day by 25% over a 4 week period), no further behavioral support was provided. Feasibility was assessed through successful recruitment rates, retention, and adherence to study conditions. Participant perceived helpfulness and satisfaction assessed acceptability. Among 80 interested individuals, 66 were eligible and consented (100% recruitment rate). From the 66 participants that consented and completed the baseline survey, 60 received the intervention assigned at a 1:1 ratio with 30 in the gradual cessation and 30 in the abrupt cessation group. Retention was 86.4% (n = 52) at 12-weeks post-intervention commencement. Ninety-six percent (n = 25) of participants in the gradual and 95.8% (n = 23) of participants in the abrupt group were using the VNPs at 12-weeks (p = 0.66). There was no difference in adherence to the assigned quit plan between gradual cessation 44% (n = 11) and abrupt cessation 71% (n = 17) groups (p = 0.117). Median perceived helpfulness of VNPs was high for both gradual (10/10) and abrupt (9/10) groups (p = 0.813). Similarly, median perceived satisfaction of VNPs was high for both gradual (9 /10) and abrupt (8/10) groups (p = 0.414). AOD participants found an intervention that involved VNPs to be satisfying and helpful. Future large scale trials are needed to elucidate whether a gradual or abrupt cessation guideline is more beneficial in main a quit attempt with a VNP.


Malposition of peripherally inserted central catheter: experience from 3012 cancer patients.
The objectives of the study are to observe and analyze the causes of 237 patients' malposition peripherally inserted central catheter (PICC) from 3012 patients in clinical practice and to further explore the methods of its prevention and remedies, which can help to improve the skills of placing tubes, to efficiently avoid and reduce the occurrence of complications, and to alleviate the pains and cost of patients. From August 2000 to March 2012, 3012 cases receiving PICC were reviewed. The locations of the tube tips were recorded by chest X ray and summarized for analysis. Malposition was observed in 237 cases (7.87%). The most frequently occurring site was in the jugular vein followed by the axillary vein. By taking different remedies, all the malpositioned PICCs were relocated in the superior vena cava or subclavian vein. In order to secure the safety usage of PICC, the strict rules of handling, the skill and experience of operator, and cooperation patients are necessary.


Breast cancer: advances in surgical management.
The surgical management of breast cancer has changed dramatically from a deforming ablative procedure to an approach that for the majority of breast cancer patients can preserve the breast and axillary anatomy. The current approach to the diagnosis of breast cancer and the evolution of the more limited approach to surgical resection are discussed. The technique of sentinel lymph node biopsy, originally developed for melanoma patients, has now been adopted for use in the treatment of breast cancer. The methodology and advantages of this approach to the axillary lymph nodes in both tumor recognition and reduced risk to the patient are detailed.


Laser-induced fluorescence (LIF) recognition of the structural composition of porcine heart valves.
Reconstruction and replacement of heart valves with grafts fro pig tissue is a common procedure. However, bioprosthetic valves wear out in a shorter time span than mechanical valves. Bioprosthetic valve structure may contribute to degenerative changes that lead to valve failure. There is, at present, no method to examine the structure of a tissue valve prior to implant. Laser-induced fluorescence (LIF) of natural fluorophores is an elegant method developed for the detection of tumors, dermal lesions and atherosclerosis. We have studied LIF as a potential diagnostic technique for analysis of valvular tissue. Using excimer laser excitation, we examined natural fluorescence recorded from porcine aortic, mitral and pulmonary valves. All three valve outflow surface tissue layers are less fluorescent at 390-450 nm than the inflow layers. Immunohistochemical analysis of collagen I and elastin content in inflow and outflow surface layers of all three valves correlated well with LIF intensities and dI/d lambda values at selected wavelengths. In conclusion, the differences observed in emitted LIF from valve surface layers are found to correlate well with diversity in the structural protein content. The LIF spectroscopic measurements may provide an appropriate tool for examination of tissue valve structure prior to use for implantation.