Cucumber grafting on indigenous cucurbit landraces confers salt tolerance and improves fruit yield by enhancing morpho-physio-biochemical and ionic attributes.
Pakistan is the 8th most climate-affected country in the globe along with a semi-arid to arid climate, thereby the crops require higher irrigation from underground water. Moreover, ~ 70% of pumped groundwater in irrigated agriculture is brackish and a major cause of secondary salinization. Cucumber (Cucumis sativus L.) is an important vegetable crop with an annual growth rate of about 3.3% in Pakistan. However, it is a relatively salt-sensitive crop. Therefore, a dire need for an alternate environment-friendly technology like grafting for managing salinity stress in cucumber by utilizing the indigenous cucurbit landraces. In this regard, a non-perforated pot-based study was carried out in a lath house to explore indigenous cucurbit landraces; bottle gourd (Lagenaria siceraria) (cv. Faisalabad Round), pumpkin (Cucurbit pepo. L) (cv. Local Desi Special), sponge gourd (Luffa aegyptiaca) (cv. Local) and ridge gourd (Luffa acutangula) (cv. Desi Special) as rootstocks for inducing salinity tolerance in cucumber (cv. Yahla F1). Four different salts (NaCl) treatments; T0 Control (2.4 dSm-1), T1 (4 dSm-1), T2 (6 dSm-1) and T3 (8 dSm-1) were applied. The grafted cucumber plants were transplanted into the already-induced salinity pots (12-inch). Different morpho-physio-biochemical, antioxidants, ionic, and yield attributes were recorded. The results illustrate that increasing salinity negatively affected the growing cucumber plants. However, grafted cucumber plants showed higher salt tolerance relative to non-grafted ones. Indigenous bottle gourd landrace (cv. Faisalabad Round) exhibited higher salt tolerance compared to non-grafted cucumber plants due to higher up-regulation of morpho-physio-biochemical, ionic, and yield attributes that was also confirmed by principal component analysis (PCA). Shoot and root biomass, chlorophylls contents (a and b), activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) enzymes, antioxidants scavenging activity (ASA), ionic (↑ K and Ca, ↓ Na), and yield-related attributes were found maximum in cucumber plants grafted onto indigenous bottle gourd landrace. Hence, the indigenous bottle gourd landrace 'cv. Faisalabad round' may be utilized as a rootstock for cucumber under a mild pot-based saline environment. However, indigenous bottle gourd landrace 'cv. Faisalabad round' may further be evaluated as rootstocks in moderate saline field conditions for possible developing hybrid rootstock and, subsequently, sustainable cucumber production.


Using Fuzzy Mathematical Model in the Differential Diagnosis of Pancreatic Lesions Using Ultrasonography and Echographic Texture Analysis.
Malignant tumors of the pancreas are the fourth leading cause of cancer-related deaths. This is mainly because they are often diagnosed at a late stage. One of the challenges in diagnosing focal lesions in the pancreas is the difficulty in distinguishing them from other conditions due to the unique location and anatomy of the organ, as well as the similarity in their ultrasound characteristics. One of the most sensitive imaging modalities of the pancreas is endoscopic ultrasonography. However, clinicians recognize that EUS is a difficult and highly operator-dependent method, while its results are highly dependent on the experience of the investigator. Hybrid technologies based on artificial intelligence methods can improve the accuracy and objectify the results of endosonographic diagnostics. Endoscopic ultrasonography was performed on 272 patients with focal lesions of the pancreatobiliary zone, who had been treated in the surgical section of the Kursk Regional Clinical Hospital in 2014-2023. The study utilized an Olympus EVIS EXERA II video information endoscopic system, along with an EU-ME1 ultrasound unit equipped with GF UM160 and GF UC140P-AL5 echo endoscopes. Out of the focal formations in the pancreatobiliary zone, pancreatic cancer was detected in 109 patients, accounting for 40.1% of the cases. Additionally, 40 patients (14.7%) were diagnosed with local forms of chronic pancreatitis. The reference sonograms displayed distinguishable focal pancreatic pathologies, leading to the development of hybrid fuzzy mathematical decision-making rules at the South-West State University in Kursk, Russian Federation. This research resulted in the creation of a fuzzy hybrid model for the differential diagnosis of chronic focal pancreatitis and pancreatic cancer. Endoscopic ultrasonography, combined with hybrid fuzzy logic methodology, has made it possible to create a model for differentiating between chronic focal pancreatitis and pancreatic ductal adenocarcinoma. Statistical testing on control samples has shown that the diagnostic model, based on reference endosonograms of the echographic texture of pancreatic focal pathology, has a confidence level of 0.6 for the desired diagnosis. By incorporating additional information about the contours of focal formations obtained through endosonography, the reliability of the diagnosis can be increased to 0.9. This level of reliability is considered acceptable in clinical practice and allows for the use of the developed model, even with data that is not well-structured.


Surgical correction of posterior leaflet tethering in ischemic mitral regurgitation.
Ischemic mitral regurgitation is a common complication in ischemic heart disease. It is generally accepted that intervention for the mitral valve is required if there is moderate to severe regurgitation. Generally, this would involve the placement of an annuloplasty ring or band to cinch down the dilated posterior annulus. More recently, attention has been drawn to the common problem of posterior leaflet tethering in these ischemic patients and the need to specifically address this pathology. A novel method has recently been described whereby the posteromedial papillary muscle is hitched up toward the annulus by a suture and then supported with a partially flexible annuloplasty ring. The purpose of this report is to show that this technique is easily performed and reproducible in other centres, and that it produces good short to medium term results. The report details the results achieved in six consecutive patients with at least moderate mitral regurgitation and posterior leaflet tethering.


Analysis of the origin of charge heterogeneity of Rauscher murine leukaemia virus p30.
Preparations of the 30 X 10(3) mol. wt. protein (p30) of Rauscher murine leukaemia virus (R-MuLV) which had been purified to homogeneity as judged by gel electrophoresis in the presence of SDS and by amino-terminal amino acid analysis, showed considerable isoelectric heterogeneity. It was found that R-MuLV p30 polypeptide chains are easily converted in vitro into chains with more acidic isoelectric points. R-MuLV p30 polypeptides with different isoelectric points displayed the same set of 125I-labelled tryptic peptides. It is concluded that the charge heterogeneity of R-MuLV p30, as revealed in isoelectric focusing experiments, is not caused by genetic heterogeneity of the virus genome but by post-translational modification.


Uncoupling anaphase-promoting complex/cyclosome activity from spindle assembly checkpoint control by deregulating polo-like kinase 1.
Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic phenotypes. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from spindle assembly checkpoint control.


Family Medicine Residencies: How Rural Training Exposure in GME Is Associated With Subsequent Rural Practice.
Rural US populations face a chronic shortage of physicians and an increasing gap in life expectancy compared to urban US populations, creating a need to understand how to increase residency graduates' desire to practice in such areas. This study quantifies associations between the amount of rural training during family medicine (FM) residencies and subsequent rural work. American Medical Association (AMA) Masterfile, AMA graduate medical education (GME) supplement, American Board of Family Medicine certification, Accreditation Council for Graduate Medical Education (ACGME), and Centers for Medicare and Medicaid Services hospital costs data were merged and analyzed. Multiple logistic regression measured associations between rural training and rural or urban practice in 2018 by all 12 162 clinically active physicians who completed a US FM residency accredited by the ACGME between 2008 and 2012. Analyses adjusted for key potential confounders (age, sex, program size, region, and medical school location and type) and clustering by resident program. Most (91%, 11 011 of 12 162) residents had no rural training. A minority (14%, 1721 of 12 162) practiced in a rural location in 2018. Residents with no rural training comprised 80% (1373 of 1721) of those in rural practice in 2018. Spending more than half of residency training months in rural areas was associated with substantially increased odds of rural practice (OR 5.3-6.3). Only 4% (424 of 12 162) of residents spent more than half their training in rural locations, and only 5% (26 of 436) of FM training programs had residents training mostly in rural settings or community-based clinics. There is a linear gradient between increasing levels of rural exposure in FM GME and subsequent rural work.


Plasma treatment as an efficient tool for controlled drug release from polymeric materials: A review.
One of the most actively developing fields in modern medicine is controlled drug delivery, an ability to keep optimal concentration of a drug at the desired body location. In particular, the most attention for potential use as drug delivery vehicles is drawn towards biodegradable polymeric materials. This is due to the versatility of tools for their fabrication, as well as due to the need to extract them after implantation being eliminated. In order to enhance polymer characteristics in terms of biocompatibility their surface can be functionalized. Plasma treatment is a method for the modification of material surface properties, which spans a wide range of applications in tissue engineering and regenerative medicine. The main advantage of this method is its ability to modify a polymeric surface without altering the bulk properties of materials, thus preserving original mechanical characteristics. Moreover, plasma modification is well-known for its speed, excluded need for solvents, and scalability. Recently, this approach has been gaining popularity for drug delivery applications. The applications of plasma treatment during the fabrication of drug delivery vehicles include surface activation, enhanced wettability, the fabrication of hydrophobic barrier layer, induced cross-linking and improved drug loading. This review covers the variety of approaches, applied to different polymeric biomaterials, including non-woven meshes, films, microparticles, microneedles and tablets, in order to achieve a controlled drug release. The applications of drug delivery devices with an implemented plasma treatment modification are also described.


Leukotriene B4 stimulation of phagocytes results in the formation of inositol 1,4,5-trisphosphate. A second messenger for Ca2+ mobilization.
Inositol trisphosphate (InsP3) production and cytosolic free Ca2+ ([Ca2+]i) elevations induced by leukotriene B4 (LTB4)-receptor activation were studied in the human promyelocytic-leukaemia cell line HL60, induced to differentiate by retinoic acid. The myeloid-differentiated HL60 cells respond to LTB4 by raising their [Ca2+]i with a dose-response relationship similar to that shown by normal human neutrophils. The observations of the LTB4 transduction mechanism were compared with those of the transduction mechanism of the chemotactic peptide fMet-Leu-Phe in HL60 cells differentiated with dimethyl sulphoxide. Both LTB4 and fMet-Leu-Phe triggered a rapid (less than 5 s) elevation of [Ca2+]i, which occurred in parallel with the InsP3 production from myo-[3H]inositol-labelled cells. The threshold concentrations of the agonists, for InsP3 production, were found at 10(-9) M, a slightly higher concentration than that required to detect [Ca2+]i elevations. No significant changes were noted in the phosphoinositide levels upon stimulation with LTB4. Exposure to Bordetella pertussis toxin before LTB4 stimulation abolished both the increased formation of InsP3 and the rise of [Ca2+]i. LTB4 and fMet-Leu-Phe elicited elevations of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with no detectable lag time, followed by slower and more sustained inositol 1,3,4-trisphosphate elevations. Stimulation with various leukotriene analogues revealed a good correlation between both total InsP3 as well as Ins(1,4,5)P3 formation and elevations of [Ca2+]1. Thus LTB4 receptor activation results in an increased production of Ins(1,4,5)P3 via a transduction mechanism also involving a nucleotide regulatory protein, as previously described for the fMet-Leu-Phe transduction mechanism.


Mammalian Llgl2 is necessary for proper branching morphogenesis during placental development.
Cell polarity plays a critical role in the development of all metazoans; however, the mechanisms of cell polarity and the specific role of cell polarity pathways in mammalian organisms are still poorly understood. Lethal giant larvae (Lgl) is an apical-basal polarity gene identified in Drosophila, where it functions as a tumor suppressor controlling self-renewal and differentiation of progenitor cells. There are two orthologs of Lgl in mammalian genomes: Llgl1 and Llgl2. While mammalian Lgls are assumed to be tumor suppressor genes, little is known about their function in vivo. Here we report the functional analysis of murine Llgl2. We generated Llgl2(-/-) mice and found that Llgl2 functions as a polarity protein required for proper branching morphogenesis during placental development. Llgl2(-/-) pups are born as runts but quickly catch up in size and grow into normal-size adults. Surprisingly, no prominent phenotypes or spontaneous tumors were observed in adult Llgl2(-/-) mice. Analyses of placental trophoblasts reveal a critical role for Llgl2 in cell polarization and polarized cell invasion. We conclude that mammalian Llgl2 is required for proper polarized invasion of trophoblasts and efficient branching morphogenesis during placental development, but, unlike its Drosophila ortholog, it does not function as a canonical tumor suppressor gene.


The evolution of drug resistance in clinical isolates of Candida albicans.
Candida albicans is both a member of the healthy human microbiome and a major pathogen in immunocompromised individuals. Infections are typically treated with azole inhibitors of ergosterol biosynthesis often leading to drug resistance. Studies in clinical isolates have implicated multiple mechanisms in resistance, but have focused on large-scale aberrations or candidate genes, and do not comprehensively chart the genetic basis of adaptation. Here, we leveraged next-generation sequencing to analyze 43 isolates from 11 oral candidiasis patients. We detected newly selected mutations, including single-nucleotide polymorphisms (SNPs), copy-number variations and loss-of-heterozygosity (LOH) events. LOH events were commonly associated with acquired resistance, and SNPs in 240 genes may be related to host adaptation. Conversely, most aneuploidies were transient and did not correlate with drug resistance. Our analysis also shows that isolates also varied in adherence, filamentation, and virulence. Our work reveals new molecular mechanisms underlying the evolution of drug resistance and host adaptation.


[The achievements and problems in controlling human echinococcosis in Bulgaria].
Progress in and problems of combatting human echinococcosis in Bulgaria are reviewed. It is emphasized that due to complex measures of the disease control started in 1960 considerable progress has been made in decreasing the morbidity and mortality rate of the population and animals. Along with the obvious progress there are also drawbacks and problems in the work performed. However, it is noteworthy that in Bulgaria there are all the necessary conditions for reducing the echinococcosis morbidity rate to just sporadic cases.


Regulation of a swelling-activated chloride current in bovine endothelium by protein tyrosine phosphorylation and G proteins.
1. The role of protein tyrosine phosphorylation and of G proteins in the activation of a swelling-activated Cl- current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells was studied using the whole-cell patch clamp technique. ICl,swell was activated by reducing the extracellular osmolality by either 12.5% (mild hypotonicity) or 25% (strong hypotonicity). 2. The protein tyrosine kinase (PTK) inhibitors tyrphostin B46, tyrphostin A25 and genistein inhibited ICl,swell with IC50 values of, respectively, 9.2 +/- 0.2, 61.4 +/- 1.7 and 62.9 +/- 1.3 microM. Tyrphostin A1, a tyrphostin analogue with little effect on PTK activity, and daidzein, an inactive genistein analogue, were without effect on ICl,swell. 3. The protein tyrosine phosphatase (PTP) inhibitors Na3VO4 (200 microM) and dephostatin (20 microM) potentiated ICl,swell activated by mild hypotonicity by 47 +/- 9 and 69 +/- 15%, respectively. 4. Intracellular perfusion with GTP gamma S (100 microM) transiently activated a Cl- current with an identical biophysical and pharmacological profile to ICl,swell. This current was inhibited by the tested PTK inhibitors and potentiated by the PTP inhibitors. Hypertonicity-induced cell shrinkage completely inhibited the GTP gamma S-activated Cl- current. 5. Intracellular perfusion with GDP beta S (1 mM) caused a time-dependent inhibition of ICl,swell, which was more pronounced when the current was activated by mild hypotonicity. 6. Our results demonstrate that the activity of endothelial swelling-activated Cl- channels is dependent on tyrosine phosphorylation and suggest that G proteins regulate the sensitivity to cell swelling.


Indigenous mothers' experiences of using primary care in Hamilton, Ontario, for their infants.
Access to primary care can help mitigate the negative impacts of social inequity that disproportionately affect Indigenous people in Canada. Despite this, however, Indigenous people cite difficulties accessing care. This study seeks to understand how Indigenous mothers-typically responsible for the health of their infants-living in urban areas, experience selecting and using health services to meet the health needs of their infants. Results provide strategies to improve access to care, which may lead to improved health outcomes for Indigenous infants and their families. This qualitative interpretive description study is guided by the Two-Eyed Seeing framework. Interviews were conducted with 19 Indigenous mothers and 5 primary care providers. The experiences of Indigenous mothers using primary care for their infants resulted in eight themes. Themes were organized according to three domains of primary care: structural, organizational and personnel. Primary care providers can develop contextual-awareness to better recognize and respond to the health and well-being of Indigenous families. Applying culturally safe, trauma and violence-informed and family-centred approaches to care can promote equitable access and positive health care interactions which may lead to improved health outcomes for Indigenous infants and their families.


The role of sex hormones in abdominal aortic aneurysms: a topical review.
Sex discrepancies have been reported for patients with abdominal aortic aneurysm (AAA) for decades. Men have a higher prevalence of disease, earlier onset, less morphological features obstructing eligibility for repair and better survival, both short and long term. In more recent years, several attempts have been made to identify the biologic or pathogenic factors contributing to these sex differences, including socioeconomic factors though all have failed. The greatest challenge is to reveal the variable mechanism for development of disease for both women and men, and secondly to identify the factors contributing to the progression of disease, and eventual rupture. Evaluations of diagnosed patients have failed to detect any factors associated with development of disease which would give a distinct explanation for the profound sex differences. Considering the obvious earlier trigger for development in men compared to women, excluding smoking, hypertension, hyperlipidemia as certified sole triggers, the remaining factors to explore are sex hormones or biological mechanisms. This topical review explores the contemporary publications on sex hormones and their association with AAA in women and men. The findings confirm the lack of scientific evidence for the influence of female and male sex hormones on development or progression of aneurysm disease. Weak indications support that women probably benefit from a longer reproductive history as a contributing protection against AAA development, influenced by smoking and heredity. There is some evidence that could support that, as for other manifestations of cardiovascular diseases, low testosterone levels in men, can contribute to an increased risk for AAA development. The influence of higher circulating levels of female sex hormones on risk development in men remains to be evaluated. In conclusion, this area will expand during the next decade, by combining registry-based and translational databases in stratified analysis for women and men, giving us more evidence that will contribute to important risk factor estimations for future cohorts at risk of AAA development.


Isoform-specific effects of the beta2 subunit on voltage-gated sodium channel gating.
Voltage-gated sodium channels (Nav) are complex glycoproteins comprised of an alpha subunit and often one to several beta subunits. We have shown that sialic acid residues linked to Nav alpha and beta1 subunits alter channel gating. To determine whether beta2-linked sialic acids similarly impact Nav gating, we co-expressed beta2 with Nav1.5 or Nav1.2 in Pro5 (complete sialylation) and in Lec2 (essentially no sialylation) cells. Beta2 sialic acids caused a significant hyperpolarizing shift in Nav1.5 voltage-dependent gating, thus describing for the first time an effect of beta2 on Nav1.5 gating. In contrast, beta2 caused a sialic acid-independent depolarizing shift in Nav1.2 gating. A deglycosylated mutant, beta(2-DeltaN), had no effect on Nav1.5 gating, indicating further the impact of beta2 N-linked sialic acids on Nav1.5 gating. Conversely, beta(2-DeltaN) modulated Nav1.2 gating virtually identically to beta2, confirming that beta2 N-linked sugars have no impact on Nav1.2 gating. Thus, beta2 modulates Nav gating through multiple mechanisms possibly determined by the associated alpha subunit. Beta1 and beta2 were expressed together with Nav1.5 or Nav1.2 in Pro5 and Lec2 cells. Together beta1 and beta2 produced a significantly larger sialic acid-dependent hyperpolarizing shift in Nav1.5 gating. Under fully sialylating conditions, the Nav1.2.beta1.beta2 complex behaved like Nav1.2 alone. When sialylation was reduced, only the sialic acid-independent depolarizing effects of beta2 on Nav1.2 gating were apparent. Thus, the varied effects of beta1 and beta2 on Nav1.5 and Nav1.2 gating are apparently synergistic and highlight the complex manner, through subunit- and sugar-dependent mechanisms, by which Nav activity is modulated.


LPS-Induced Inflammation Abolishes the Effect of DYRK1A on IkB Stability in the Brain of Mice.
Down syndrome is characterized by premature aging and dementia with neurological features that mimic those found in Alzheimer's disease. This pathology in Down syndrome could be related to inflammation, which plays a role in other neurodegenerative diseases. We previously found a link between the NFkB pathway, long considered a prototypical proinflammatory signaling pathway, and the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). DYRK1A is associated with early onset of Alzheimer's disease in Down syndrome patients. Here, we sought to determine the role of DYRK1A on regulation of the NFkB pathway in the mouse brain. We found that over-expression of Dyrk1A (on a C57BL/6J background) stabilizes IκBα protein levels by inhibition of calpain activity and increases cytoplasmic p65 sequestration in the mouse brain. In contrast, Dyrk1A-deficient mice (on a CD1 background) have decreased IκBα protein levels with an increased calpain activity and decreased cytoplasmic p65 sequestration in the brain. Taken together, our results demonstrate a role of DYRK1A in regulation of the NFkB pathway. However, decreased IκBα and DYRK1A protein levels associated with an increased calpain activity were found in the brains of mice over-expressing Dyrk1A after lipopolysaccharide treatment. Although inflammation induced by lipopolysaccharide treatment has a positive effect on calpastatin and a negative effect on DYRK1A protein level, a positive effect on microglial activation is maintained in the brains of mice over-expressing Dyrk1A.


Migration of nanosized layered double hydroxide platelets from polylactide nanocomposite films.
Melt-extruded L-polylactide (PLA) nanocomposite films were prepared from commercially available PLA and laurate-modified Mg-Al layered double hydroxide (LDH-C12). Three films were tested for total migration as well as specific migration of LDH, tin, laurate and low molecular weight PLA oligomers (OLLA). This is the first reported investigation on the migration properties of PLA-LDH nanocomposite films. The tests were carried out as part of an overall assessment of the suitability of such films for use as food contact materials (FCM). Total migration was determined according to a European standard method. All three films showed migration of nanosized LDH, which was quantified using acid digestion followed by inductively coupled plasma mass spectrometric (ICP-MS) detection of (26)Mg. Migration of LDH from the films was also confirmed by examining migrates using transmission electron microscopy (TEM) and was attributed indirectly to the significant PLA molecular weight reduction observed in extruded PLA-LDH-C12 films. Migration of tin was detected in two of the film samples prepared by dispersion of LDH-C12 using a masterbatch technique and migration of the laurate organomodifier took place from all three film types. The results indicate that the material properties are in compliance with the migration limits for total migration and specific lauric acid migration as set down by the EU legislation for FCM, at least if a reduction factor for fresh meat is taken into consideration. The tin detected arises from the use of organotin catalysts in the manufacture of PLA.


Expression of recombinant vaccinia virus-derived alphavirus proteins in mosquito cells.
A recombinant vaccinia virus strain which contains and expresses a 26S cDNA insert encoding Sindbis virus structural proteins (VV:3S) was used to infect a continuous line of Aedes albopictus mosquito cells. There were not visible cytopathic effects due to the virus infection and the cells continued to grow normally. However, examination of the proteins present in the cytoplasm of the infected cells with Sindbis virus-specific antisera revealed that Sindbis virus proteins were being synthesized and processed. These results are discussed with respect to vaccinia virus as a non-lethal expression vector to deliver and express eukaryotic genetic information in insect cell systems and using this system (VV:3S) to dissect various facets of togavirus-insect cell interactions.


Using Hormone Data and Age to Pinpoint Cycle Day within the Menstrual Cycle.
Background and Objectives: Menstrual cycle tracking is essential for reproductive health and overall well-being. However, there is still an over-reliance on estimations that standard cycles are 28 days long, divided evenly between the follicular and luteal phases. Due to the variability of cycle length and cycle phase lengths, common methods of identifying where an individual is in their cycle are often inaccurate. This study used daily hormone monitoring obtained through a remote hormone-monitoring platform to evaluate hormone levels across a menstrual cycle to identify nuances in the follicular and luteal phases in individuals of different age groups. Materials and Methods: This study used a remote fertility testing system that quantitatively tracks luteinizing hormone (LH) and pregnanediol-3-glucuronide (PdG) through urine tests read by an AI-powered smartphone app. The study analyzed cycle data from 1233 users with a total of 4123 evaluated cycles. Daily levels for LH and PdG were monitored across multiple cycles. Results: This study determined that calculated cycle lengths tended to be shorter than user-reported cycle lengths. Significant differences were observed in cycle phase lengths between age groups, indicating that follicular phase length declines with age while luteal phase length increases. Finally, the study found that if an individual's age, first cycle day, and current hormone levels are known, population-level hormone data can be used to pinpoint which cycle phase and cycle day they are in with 95% confidence. Conclusions: At-home hormone monitoring technologies can allow patients and clinicians to track their cycles with greater precision than when relying on textbook estimations. The study's findings have implications for fertility planning, clinical management, and general health monitoring. Prior to this study, no standard existed for pinpointing where a person was in their cycle through only one measure of LH and PdG. These findings have the potential to fill significant gaps within reproductive healthcare and beyond.


Expectations and Assumptions: Examining the Influence of Staff Culture on a Novel School-Based Intervention to Enable Risky Play for Children with Disabilities.
Risky play is challenging, exciting play with the possibility of physical, social, or emotional harm. Through risky play, children learn, develop, and experience wellbeing. Children with disabilities have fewer opportunities than their typically developing peers to engage in this beneficial type of play. Our team designed a novel, school-based intervention to address this disparity; however, our intervention yielded unexpected quantitative results. In the present study, we qualitatively examined divergent results at two of the five schools that participated in the intervention. Specifically, we aimed to explore how staff culture (i.e., shared beliefs, values, and practices) influenced the intervention. To explore this relationship, we employed a retrospective, qualitative, multiple case study. We used thematic analysis of evaluative interviews with staff members to elucidate the cultures at each school. Then, we used cross-case analysis to understand the relationships between aspects of staff culture and the intervention's implementation and results. We found that staff cultures around play, risk, disability influenced the way, and the extent to which, staff were willing to let go and allowed children to engage in risky play. Adults' beliefs about the purpose of play and recess, as well as their expectations for children with disabilities, particularly influenced the intervention. Furthermore, when the assumptions of the intervention and the staff culture did not align, the intervention could not succeed. The results of this study highlight the importance of (1) evaluating each schools' unique staff culture before implementing play-focused interventions and (2) tailoring interventions to meet the needs of individual schools.


Influence of Cation Size on the Structural Features and Interactions in Tertiary Alkylammonium Trifluoroacetates: A Density Functional Theory Investigation.
We present the results of electronic structure calculations based on density functional theory (DFT) in order to investigate the reactions of the interaction of tertiary alkylamines with alkyl groups of different sizes (triethyl, tributyl, dimethylethyl, and diisopropylethyl) with trifluoroacetic acid. We have obtained data on the affinity of the studied amines with a proton. It has been shown that amine interaction with the acid leads to proton transfer from the acid to the amine and formation of ions held together in the ion pair by electrostatic interaction and a very strong hydrogen bond. We have also investigated the energy profiles of the proton transfer from the tertiary alkylammonium cation to the trifluoroacetate anion within the ion pair and found different correlations between the geometric characteristics of the H-bond and the parameters obtained by the natural bond orbitals and quantum theory of atoms in molecules analyses. It has been established that the interionic interactions in these systems weaken as the length and the degree of cation alkyl chain branching increase. A good qualitative agreement between the theoretical results and the experimental data on physicochemical properties has been obtained.


Review of taxonomy, geographic distribution, and paleoenvironments of Azhdarchidae (Pterosauria).
The taxonomy, geographic distribution, and paleoenvironmental context of azhdarchid pterosaurs are reviewed. All purported pteranodontid, tapejarid, and azhdarchid specimens from the Cenomanian Kem Kem beds of Morocco are referred to a single azhdarchid taxon, Alanqa saharica. The four proposed autapomorphies of Eurazhdarcho langendorfensis from the lower Maastrichtian Sebeş Formation of Romania are based on misinterpretations of material and this taxon is likely a subjective junior synonym of Hatzegopteryx thambema. Among 54 currently reported azhdarchid occurrences (51 skeletal remains and 3 tracks) 13% are from lacustrine deposits, 17% from fluvial plain deposits, 17% from coastal plain deposits, 18% from estuarine and lagoonal deposits, and 35% from costal marine deposits. Azhdarchids likely inhabited a variety of environments, but were abundant near large lakes and rivers and most common in nearshore marine paleoenvironments.


Nocturnal hemodialysis is associated with restoration of early-outgrowth endothelial progenitor-like cell function.
Angiogenesis is a key response to tissue ischemia that may be impaired by uremia. Although early-outgrowth endothelial progenitor-like cells promote angiogenesis in the setting of normal renal function, cells from uremic patients are dysfunctional. When compared with conventional hemodialysis, it was hypothesized that nocturnal hemodialysis would improve the in vivo angiogenic activity of these cells in a well described model of ischemic vascular disease. Early-outgrowth endothelial progenitor-like cells were cultured from healthy controls (n = 5) and age- and gender-matched conventional hemodialysis (12 h/wk, n = 10) and nocturnal hemodialysis (30 to 50 h/wk, n = 9) patients. Cells (5 × 10(5)) or saline were injected into the ischemic hindlimb of athymic nude rats 1 day after left common iliac artery ligation. Although conventional dialysis cell injection had no effect versus saline, nocturnal hemodialysis and healthy control cell injection significantly improved ischemic hindlimb perfusion and capillary density. Nocturnal hemodialysis cell injection was also associated with significant increases in endogenous angiopoietin 1 expression in the ischemic hindlimb compared with saline and conventional dialysis cell injection. In contrast to a conventional dialytic regimen, nocturnal hemodialysis is associated with a significantly improved ability of early-outgrowth endothelial progenitor-like cells to promote angiogenesis and thus restore perfusion in a model of ischemic vascular disease.


Impaired bone marrow B-cell development in mice with a bronchiolitis obliterans model of cGVHD.
Chronic graft-versus-host disease (cGVHD) causes significant morbidity and mortality in patients after allogeneic bone marrow (BM) or stem cell transplantation (allo-SCT). Recent work has indicated that both T and B lymphocytes play an important role in the pathophysiology of cGVHD. Previously, our group showed a critical role for the germinal center response in the function of B cells using a bronchiolitis obliterans (BO) model of cGVHD. Here, we demonstrated for the first time that cGVHD is associated with severe defects in the generation of BM B lymphoid and uncommitted common lymphoid progenitor cells. We found an increase in the number of donor CD4+ T cells in the BM of mice with cGVHD that was negatively correlated with B-cell development and the frequency of osteoblasts and Prrx-1-expressing perivascular stromal cells, which are present in the B-cell niche. Use of anti-DR3 monoclonal antibodies to enhance the number of donor regulatory T cells (Tregs) in the donor T-cell inoculum ameliorated the pathology associated with BO in this model. This correlated with an increased number of endosteal osteoblastic cells and significantly improved the generation of B-cell precursors in the BM after allo-SCT. Our work indicates that donor Tregs play a critical role in preserving the generation of B-cell precursors in the BM after allo-SCT. Approaches to enhance the number and/or function of donor Tregs that do not enhance conventional T-cell activity may be important to decrease the incidence and severity of cGVHD in part through normal B-cell lymphopoiesis.


Validation of cell culture models for the intestine and the blood-brain barrier and comparison of drug permeation.
Cell culture models are useful tools to study the uptake of drugs across the barriers of the human body, like the intestine, the skin or the blood-brain barrier. Cell-based in vitro models not only help to reduce the number of animals used but are also much faster to perform, more cost effective and give more reproducible data than animal studies. Given the increasing number of new drugs and chemicals under development, there is an urgent need for the establishment of such in vitro models. However, the validity of such in vitro models is reflected by its ability to accurately predict the behaviour of a substance at the corresponding in vivo barrier. Here, we compare a well-established cell culture model for the intestine, based on Caco-2 colon carcinoma cells, with a primary cell culture model of the blood-brain barrier. We find that Caco-2 cells and cells of the blood-brain barrier have different barrier properties. Therefore, cells used for cell-based assays should be derived from the corresponding tissue to reflect the in vivo barrier characteristics.


Post-stroke Visual Gait Measure for Developing Countries: A Reliability and Validity Study.
Visual gait assessment is a cost-effective clinical method to assess post-stroke gait deviations. The Rivermead Visual Gait Assessment (RVGA) is a one such measure that assesses the kinematic aspect of the gait deviations in stroke. However, the available information on psycho-clinocometric properties of the measure is not adequate. To establish reliability and validity of RVGA using walking-videos of the post-stroke subjects. Design: Observational study. A rehabilitation institute Participants: A convenience sample of 40 chronic stroke patients. RVGA, Fugl-Meyer assessment (lower extremity), 10-m walk test, Time up and go test, and Berg balance scale (BBS). Walking was video-taped from the anterior aspect, posterior aspect, affected side, and less-affected side. After coding the tapes, a research staff member provided them to four different raters in a random order. Each rater scored the coded video on the RVGA data collection sheet twice: one at the baseline and another after 1 month to eliminate any recollection of the initial assessment. The findings exhibit that there was good-to-excellent agreement between the scores of the raters and also between the assessments (correlation coefficient = 0.94 to 0.95; P < 0.001). The measure also exhibits acceptable validity when correlated with scores of BBS (r = 0.4; P < 0.001). Video-based RVGA is a reliable and valid tool to assess gait-related impairment in post-stroke hemiparesis. This cost-effective measure may be incorporated in the clinical and research practice to discern and quantify complex phenomenon of the gait deviation. RVGA may be considered as a useful tool, especially in developing countries where expensive gait analyzer is usually not available.


Cell migration: GAPs between membrane traffic and the cytoskeleton.
During cell migration, coordination between membrane traffic, cell substrate adhesion and actin reorganization is required for protrusive activity to occur at the leading edge. Actin organization is regulated by Rho family GTPases and, with a contribution from the endocytic cycle, serves to extend the cell front. The details of the molecular mechanisms that direct membrane traffic at sites of adhesion and rearrange actin at the cell edge are still unknown. However, recent findings show that a number of multi-domain proteins characterized by an ArfGAP domain interact with both actin-regulating and integrin-binding proteins, as well as affecting Rac-mediated protrusive activity and cell migration. Some of these proteins have been shown to localize to endocytic compartments and to have a role in regulating endocytosis. Given the participation of Arf proteins in regulating membrane traffic, one appealing hypothesis is that the ArfGAPs act as molecular devices that coordinate membrane traffic and cytoskeletal reorganization during cell motility.


[Drug therapy of atrial fibrillation].
Atrial fibrillation can, clinically, be differentiated into several subgroups. Paroxysmal atrial fibrillation lasts less than 24 hours and is often caused by an autonomic dysregulation. Chronic atrial fibrillation occurs in a reversible and an irreversible form. Atrial fibrillation is frequently observed transiently after cardiac surgery. The therapeutic goal must be clearly defined before treatment is initiated. In patients with a reversible form of chronic atrial fibrillation, sinus rhythm can be restored with class Ia, Ic and III antiarrhythmic drugs. These patients with sinus rhythm benefit from higher cardiac output, fewer thromboembolic complications and fewer symptoms (no palpitations). In patients suffering from an irreversible form of chronic atrial fibrillation the therapeutic aim is control of ventricular rate. Digoxin, beta-blockers (class II) and calcium antagonists (class IV) are successfully used to slow atrioventricular conduction. Patients aged over 65 years should be anticoagulated, since numerous studies have shown a marked decrease in thromboembolic complications with warfarin.


Transcriptional and functional complexity of Shank3 provides a molecular framework to understand the phenotypic heterogeneity of SHANK3 causing autism and Shank3 mutant mice.
Considerable clinical heterogeneity has been well documented amongst individuals with autism spectrum disorders (ASD). However, little is known about the biological mechanisms underlying phenotypic diversity. Genetic studies have established a strong causal relationship between ASD and molecular defects in the SHANK3 gene. Individuals with various defects of SHANK3 display considerable clinical heterogeneity. Different lines of Shank3 mutant mice with deletions of different portions of coding exons have been reported recently. Variable synaptic and behavioral phenotypes have been reported in these mice, which makes the interpretations for these data complicated without the full knowledge of the complexity of the Shank3 transcript structure. We systematically examined alternative splicing and isoform-specific expression of Shank3 across different brain regions and developmental stages by regular RT-PCR, quantitative real time RT-PCR (q-PCR), and western blot. With these techniques, we also investigated the effects of neuronal activity and epigenetic modulation on alternative splicing and isoform-specific expression of Shank3. We explored the localization and influence on dendritic spine development of different Shank3 isoforms in cultured hippocampal neurons by cellular imaging. The Shank3 gene displayed an extensive array of mRNA and protein isoforms resulting from the combination of multiple intragenic promoters and extensive alternative splicing of coding exons in the mouse brain. The isoform-specific expression and alternative splicing of Shank3 were brain-region/cell-type specific, developmentally regulated, activity-dependent, and involved epigenetic regulation. Different subcellular distribution and differential effects on dendritic spine morphology were observed for different Shank3 isoforms. Our results indicate a complex transcriptional regulation of Shank3 in mouse brains. Our analysis of select Shank3 isoforms in cultured neurons suggests that different Shank3 isoforms have distinct functions. Therefore, the different types of SHANK3 mutations found in patients with ASD and different exonic deletions of Shank3 in mutant mice are predicted to disrupt selective isoforms and result in distinct dysfunctions at the synapse with possible differential effects on behavior. Our comprehensive data on Shank3 transcriptional regulation thus provides an essential molecular framework to understand the phenotypic diversity in SHANK3 causing ASD and Shank3 mutant mice.


Molecular clones representing Sendai virus genes P, NP and M.
DNA copies of segments of Sendai virus genes P, NP and M, obtained by reverse transcription of virus mRNA species extracted from infected cells, were cloned in plasmid pBR322. Genes were identified by hybrid-arrested translation of viral mRNAs in vitro. Hybrid selection of NP mRNA confirmed the identity of an NP gene clone. Partial sequencing of this insert showed that it represents the 5'-terminal region of the gene, containing transcription termination signals. Hybridization of DNA inserts to blots of electrophoretically separated, denatured mRNA species indicated that the P, NP and M messages had sizes of 2400, 2100 and 1500 nucleotides, respectively. Specific T1 ribonuclease-resistant oligonucleotides, previously identified in the NP and M genes, were selected by hybridization to the respective inserts. Although most of the inserts are smaller than 500 base pairs, representing no more than 16% of the P gene and 24% of the NP gene, one of the M gene inserts, comprising 700 base pairs, represents almost half of that gene. These cloned virus gene segments will assist further investigations of the molecular biology of this model paramyxovirus.


Arginine vasopressin in hypothalamic paraventricular nucleus is transferred to the nucleus raphe magnus to participate in pain modulation.
Hypothalamic paraventricular nucleus (PVN) is one of the main sources of arginine vasopressin (AVP) synthesis and secretion. AVP is the most important bioactive substance in PVN regulating pain process. Our previous study has pointed that pain stimulation induced AVP increase in the nucleus raphe magnus (NRM), which plays a role in pain modulation. The present study was designed to investigate the source of AVP in the rat NRM during pain process using the methods of nucleus push-pull perfusion and radioimmunoassay. The results showed that pain stimulation increased the AVP concentration in the NRM perfusion liquid, PVN cauterization inhibited the role that pain stimulation induced the increase of AVP concentration in the NRM perfusion liquid, and PVN microinjection of L-glutamate sodium, which excited the PVN neurons, could increase the AVP concentration in the NRM perfusion liquid. The data suggested that AVP in the PVN might be transferred to the NRM to participate in pain modulation.


Tetracyclines and photosensitive skin reactions: A narrative review.
Tetracyclines are a group of broad-spectrum antibiotics largely employed in infectious, dermatological and surgical fields. Some adverse events may occur during treatment, including photosensitivity reactions, which are divided in phototoxic or photoallergic. We performed a systematic search on Pubmed, Cochrane and Embase from database inception to August 9, 2020 aim to summarize all available papers on photosensitive reactions related to tetracyclines in all clinical settings where they are used on human being. On the basis of our inclusion criteria, we selected only randomized controlled trials, open comparative trials and prospective cohort studies performed on both volunteers and patients, moreover we included a pharmacovigilance register. Thirty-eight articles met inclusion criteria, describing photo-sensitive effects due to doxycycline, minocycline, tetracycline, lymecycline, sarecycline, demethylchlortetracycline, chlortetracycline and metacycline, across six diagnoses (acne, Lyme disease, Gulf Veteran Illness, adbominal aortic aneurysms, traveler's diarrhea and pterygium) and several volunteers who were deliberately exposed to natural or artificial light sources. Not all drugs belonging to tetracyclines class are available to date, moreover the studies included lacked a homogeneous design and most of them involved a scarce number of patients, including reactions induced in volunteers during photo-testing. Available data on incidence, severity and clinical relevance of tetracyclines-related photo-sensitive reactions are scarce, heterogeneous and weak. What we can extrapolate is that some tetracyclines are more often related to phototoxic skin reactions than others and some of those seem to have a very low risk of phototoxicity.


The mechanism of [3H]dexamethasone uptake into prolactin producing rat pituitary cells (GH3 cells) in culture.
Glucocorticosteroids stimulate growth hormone (GH) synthesis and inhibit prolactin (PRL) synthesis and cell growth in cultured GH3 cells, a clonal cell strain derived from a rat pituitary tumour. This model system was used to study the mechanism by which glucocorticosteroids enter target cells. The cellular uptake of [3H]dexamethasone was temperature dependent and was further inhibited by addition of an excess amount of cold dexamethasone. Half maximal uptake was obtained after about 5 min at 37 degrees C. The initial rates of [3H]dexamethasone uptake were a linear function of the extracellular hormone concentration. The uptake of [3H]dexamethasone in intact cells studied at different temperatures resulted in linear Arrhenius plots, with a calculated energy of activation of 91.0 kJ x mole-1 x degree-1. Scatchard analysis of specifically cell bound [3H]dexamethasone at equilibrium (0 degrees C) showed a straight line with a calculated dissociation constant (Kd) of 1.6 x 10(-9) M and a maximal uptake of 180 x 10(-15) mole/mg cell protein. Specific binding of [3H]dexamethasone to cytosol proteins could only be demonstrated at 0 degrees C. These results indicate that [3H]dexamethasone diffuses passively into the cell, and binds to specific receptors in an energy dependent way.


Variability in reproductive success viewed from a life-history perspective in baboons.
Nonhuman primates, like humans, mature slowly and have low fertility during a relatively long life. As data have accumulated on life-history patterns of nonhuman primates, comparative studies have yielded important insights into the evolution of this slow life-history style of primates. However, in order to understand selection pressures and evolutionary potential within species, it is important to complement comparative studies with detailed studies of life-history variability within species and to identify sources of this variability. Here we present a summary of how foraging environment, social status, and group size (a measure of population density) contribute to within-population variance in reproductive success for savannah baboons. We also discuss the extent to which savannah baboons, with their highly flexible and adaptable behavior, change their foraging environments by shifting home ranges and seeking rich food sources and how low-ranking females, which disproportionately bear the costs of social life, may mitigate those costs.


Recent advances in Clostridium difficile-associated disease.
The main purpose of this article is to review recent developments in the management of acute and recurrent Clostridium difficile-associated disease, with consideration of existing and new antibiotic and non-antibiotic agents for treatment. Details of the current developmental stage of new agents are provided and the role of surgery in the management of severe disease is discussed. Infection control measures considered comprise prudent use of antimicrobials, prevention of cross-infection and surveillance. Other topics that are covered include the recent emergence of an epidemic hypervirulent strain, pathogenesis, clinical presentation and approaches to rapid diagnosis and assessment of the colonic disease.


Validation of reference genes for normalization of real-time quantitative RT-PCR data in traumatic brain injury.
Careful validation of reference genes used for the normalization of real-time RT-PCR data is required to obtain accurate results regarding gene expression. We evaluated the stability of seven commonly used reference genes in the cerebral cortex and hippocampus of rats 3 days following traumatic brain injury (TBI). HPRT, SDHA, and GUSB were found to be the most stable reference genes in the cerebral cortex, whereas B2MG, TBP, and GAPDH were the most stable in the hippocampus. The use of three reference genes was determined to be the optimal number for accurate normalization of data. To illustrate this point, when our gene of interest, substance P (SP), was normalized against the three most stable reference genes in both brain areas, we found no significant difference between injured and uninjured rats at the 3-day time point. However, when our SP data were normalized to each reference gene individually, SP mRNA level was highly variable depending on the reference gene chosen. The results of the present study highlight the importance of validating reference genes to be used for real-time RT-PCR analysis. The use of the most stable reference genes presented here will allow more accurate normalization of gene expression data in TBI.


Bilateral Macular Edema: A New Ocular Feature of Dandy-Walker Syndrome.
To describe a case of bilateral cystoid macular edema in a patient with Dandy-Walker syndrome. An 18-year-old male was referred to our tertiary referral center for evaluation of his decreased visual acuity. Detailed ophthalmic examination and imaging revealed the presence of bilateral cystoid macular edema, which was successfully treated with intravitreal triamcinolone injections (2 mg in 0.05 ml). Recurrence of macular edema developed after a period of approximately four months. This is an unusual ophthalmic manifestation of Dandy-Walker syndrome. Cystoid macular edema should be included in the differential diagnosis of subjects with Dandy-Walker syndrome presenting with decreased vision. The pathogenetic mechanism for the development macular edema in this case is not clear. Intravitreal triamcinolone is an effective treatment, but edema was recurrent in our case. Other approaches (such as oral Acetazolamide or intravitreal Anti-VEGF) have to be considered as well.


Linking microbial oxidation of arsenic with detection and phylogenetic analysis of arsenite oxidase genes in diverse geothermal environments.
The identification and characterization of genes involved in the microbial oxidation of arsenite will contribute to our understanding of factors controlling As cycling in natural systems. Towards this goal, we recently characterized the widespread occurrence of aerobic arsenite oxidase genes (aroA-like) from pure-culture bacterial isolates, soils, sediments and geothermal mats, but were unable to detect these genes in all geothermal systems where we have observed microbial arsenite oxidation. Consequently, the objectives of the current study were to measure arsenite-oxidation rates in geochemically diverse thermal habitats in Yellowstone National Park (YNP) ranging in pH from 2.6 to 8, and to identify corresponding 16S rRNA and aroA genotypes associated with these arsenite-oxidizing environments. Geochemical analyses, including measurement of arsenite-oxidation rates within geothermal outflow channels, were combined with 16S rRNA gene and aroA functional gene analysis using newly designed primers to capture previously undescribed aroA-like arsenite oxidase gene diversity. The majority of bacterial 16S rRNA gene sequences found in acidic (pH 2.6-3.6) Fe-oxyhydroxide microbial mats were closely related to Hydrogenobaculum spp. (members of the bacterial order Aquificales), while the predominant sequences from near-neutral (pH 6.2-8) springs were affiliated with other Aquificales including Sulfurihydrogenibium spp., Thermocrinis spp. and Hydrogenobacter spp., as well as members of the Deinococci, Thermodesulfobacteria and beta-Proteobacteria. Modified primers designed around previously characterized and newly identified aroA-like genes successfully amplified new lineages of aroA-like genes associated with members of the Aquificales across all geothermal systems examined. The expression of Aquificales aroA-like genes was also confirmed in situ, and the resultant cDNA sequences were consistent with aroA genotypes identified in the same environments. The aroA sequences identified in the current study expand the phylogenetic distribution of known Mo-pterin arsenite oxidase genes, and suggest the importance of three prominent genera of the order Aquificales in arsenite oxidation across geochemically distinct geothermal habitats ranging in pH from 2.6 to 8.


Contributions of mangrove conservation and restoration to climate change mitigation in Indonesia.
Mangrove forests are important carbon sinks and this is especially true for Indonesia where about 24% of the world's mangroves exist. Unfortunately, vast expanses of these mangroves have been deforested, degraded or converted to other uses resulting in significant greenhouse gas emissions. The objective of this study was to quantify the climate change mitigation potential of mangrove conservation and restoration in Indonesia. We calculated the emission factors from the dominant land uses in mangroves, determined mangrove deforestation rates and quantified the total emissions and the potential emission reductions that could be achieved from mangrove conservation and restoration. Based upon our analysis of the carbon stocks and emissions from land use in mangroves we found: (1) Indonesia's mangrove ecosystem carbon stocks are amongst the highest of any tropical forest type; (2) mangrove deforestation results in greenhouse gas emissions that far exceed that of upland tropical deforestation; (3) in the last decade the rates of deforestation in Indonesian mangroves have remained high; and (4) conservation and restoration of mangroves promise to sequester significant quantities of carbon. While mangroves comprise only ≈2.6% of Indonesia's total forest area, their degradation and deforestation accounted for ≈10% of total greenhouse gas emissions arising from the forestry sector. The large source of greenhouse gas emissions from a relatively small proportion of the forest area underscores the value for inclusion of mangroves as a natural climate solution (NCS). Mangrove conservation is far more effective than mangrove restoration in carbon emissions reductions and an efficient pathway to achieve Indonesia's nationally determined contribution (NDC) targets. The potential emission reduction from halting deforestation of primary and secondary mangroves coupled with restoration activities could result in an emission reduction equivalent to 8% of Indonesia's 2030 NDC emission reduction targets from the forestry sector.


Five inhibitory receptors display distinct vesicular distributions in T cells.
T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3 and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and a biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.


Partial reversibility of growth hormone (GH) deficiency in the GH-releasing hormone (GHRH) knockout mouse by postnatal treatment with a GHRH analog.
The proliferation of pituitary somatotroph cells and the synthesis and secretion of GH require the hypothalamic peptide GH-releasing hormone (GHRH). Accordingly, we have shown that mice with targeted disruption [knockout (KO)] of the GHRH gene (GHRHKO) have isolated GH deficiency (GHD) and anterior pituitary hypoplasia. The weight of GRHRKO mice is about 60% that of normal mice by 12 wk of age. The phenotype is strikingly similar to that observed in the mouse with mutated GHRH receptor (little). It is not known whether exposure to endogenous GHRH during intrauterine growth is necessary for postnatal GH secretion, and whether GHD due to congenital lack of GHRH activity would be reversible by treatment with GHRH during the postnatal period. To answer this question, we treated GHRHKO mice with a long-acting superactive GHRH analog (JI-38) at two ages: from wk 2-6 (2 microg, twice a day) and from wk 12-16 (4 microg, twice a day). Normal littermates served as controls. At both ages JI-38 caused growth acceleration, increase in size of the pituitary gland, increase in pituitary GH mRNA and GH protein levels and serum GH, and significant increase in liver IGF-I mRNA, although none of these parameters was fully normalized. Our findings demonstrate that GHD and pituitary hypoplasia in GHRHKO mice may be partially reversed by long-term treatment with a GHRH analog, and that somatotroph cells maintain responsiveness to GHRH even if this factor is absent during intrauterine development.


Women of Reproductive Age Living with HIV in Argentina: Unique Challenges for Reengagement in Care.
This study evaluated the reasons for not taking antiretroviral treatment (ART) among women of reproductive age who are disengaged from HIV care (have missed pharmacy pickups and physician visits), with the goal of identifying strategies for reengagement in HIV care. Participants were cisgender women (n = 162), 18 to 49 years of age, and who completed sociodemographic, medical history, reasons why they were not taking ART, mental health, motivation, and self-efficacy assessments. Latent class analysis was used for analysis. Women who reported avoidance-based coping (avoid thinking about HIV) had higher depression (U = 608.5, z = -2.7, P = .007), lower motivation (U = 601, z = -2.8, P = .006), and lower self-efficacy (U = 644.5, z = -2.4, P = .017) than those not using this maladaptive strategy. As women living with HIV experience a disproportionate burden of poor health outcomes, interventions focused on the management of depression may improve HIV outcomes and prevent HIV transmission.


[Folic acid reduces the risk of neural tube defects: awareness and folate intake among pregnant women in 2006].
Folic acid plays an important role in proliferating cells and tissues of the fetus. A randomized control trial demonstrated in 1991 that 4 mg of folic acid supplements successfully prevented 72% of recurrence of neural tube defects (NTDs) in women who had had afflicted pregnancy. In 2000, the Japanese Government recommended women of childbearing age to take 400 microgram of folate supplements per day from 4 weeks prior to and 12 weeks after conception. A questionnaire study was performed in pregnant women by post on their awareness of the role folic acid plays, their life style, and folate intake by dietary consumption. Thirty-five percent of 1,251 pregnant women were aware of the important role of folic acid in the critical stage of fetal development and 31% actually took the supplement. Information on folic acid was obtained through mass media in 47% of the women, through the internet in 17%, through healthcare providers in 13% and so forth. The food record analysis revealed that the dietary intake of folic acid averaged 341 microg/day that was 60 microg less than what was recommended by the Government and that 33 of 86 women took the supplement. Overall, a half of pregnant women are required to take 400 microg folate supplement per day. It is to be stressed that primary prevention of NTDs by periconceptional intake of folic acid is a major public health opportunity and that prevention is more important than cure in the management of NTDs.


[Methicillin-resistant Staphylococcus aureus in the Americas: systematic review and metanalysis of prevalence in food-producing animalsStaphylococcus aureus resistente a la meticilina en la Región de las Américas: revisión sistemática y metanálisis de la prevalencia en la actividad agropecuaria].
To determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in animals used to produce foods in the Americas. A systematic literature review was performed in the following databases: Scopus, Web of Science, MEDLINE, EMBASE, and Virtual Health Library. Articles published in the past 10 years, without language limits, were selected. The outcome of interest was the prevalence of MRSA in food-producing animals. Prevalence rates were meta-analyzed in grouped random effects models using the DerSimonian and Laird method. The geographic distribution of MRSA and the time trend of resistance were also analyzed. Of 19 studies included, 11 were performed in the United States and 11 analyzed pig samples. Five studies were performed in South America. The samples analyzed in the studies were collected in farming, processing, and retail sites. MRSA prevalence in the Americas was 7.6% (95%CI: 5.6-9.5%), and was higher in pigs [12.6% (95%CI: 7.0-18.2%)] followed by bovine cattle [2.4% (95%CI: 1.2-3.7%)] and poultry [1.8% (95CI%: 0.3-3.4%)]. MRSA prevalence was higher in pigs in North America and bovine cattle in Latin America. There was no significant variation in MRSH prevalence along the 10-year period analyzed. MRSA prevalence in food-producing animals in the Americas was higher in pigs, without significant changes across time.


Microbiological analysis of food contact surfaces in child care centers.
A study of six child care centers was conducted to assess the microbiological quality of three food contact surfaces (one food serving surface and two food preparation surfaces) and one non-food contact surface (diaper changing surface) to determine the effectiveness of cleaning and sanitization procedures within the facilities. Aerobic plate counts (APCs) and Escherichia coli/coliform counts of 50-cm(2) areas on all surfaces were determined using standard microbiological swabbing methods. Samples were taken three times a day (preopening, lunchtime, and following final cleanup) twice per month for 8 months in each child care center (n = 288 sampling times). Mean log APCs over the survey period were 1.32, 1.71, 1.34, 1.96, 1.50, and 1.81 log CFU/50 cm(2) for the six centers. Mean log coliform counts were 0.15, 0.40, 0.33, 1.41, 0.28, and 1.12 CFU/50 cm(2) for the same centers. Coliforms were detected in 283 of 1,149 (24.7%) samples, with counts ranging from 1 to 2,000 CFU/50 cm(2), while E. coli was detected in 18 of 1,149 (1.6%) samples, with counts ranging from 1 to 35 CFU/50 cm(2). The findings of this study demonstrated that the extent of bacterial contamination was dependent on the center, time of day, and the area sampled. While no direct correlation between contamination and illness can be made, given the high risk of food-borne illness associated with children, microbial contamination of food contact or non-food contact surfaces is an aspect of food safety that requires more attention. Emphasis on training and the development of modified standard sanitation operating procedures for child care centers are needed to reduce potential hazards.


Linkage of the gene for an X-linked mental retardation disorder to a hypervariable (AGAT)n repeat motif within the human hypoxanthine phosphoribosyltransferase (HPRT) locus (Xq26).
We recently reported a new X-linked mental retardation (XLMR) disorder in a four-generation family of Dutch descent. Features included Dandy-Walker malformation, basal ganglia disease, and seizures. Twenty-six family members, including two living affected males and two obligate carriers, were available for study. No evidence of linkage was observed between the disease locus and RFLPs from several X-chromosome regions, including Xp21-p22 (13 markers), proximal Xq (four markers), and Xq28 (three markers). However, a new hypervariable short tandem repeat (STR) within the HPRT gene at Xq26 showed positive linkage to the disease locus, with a maximum lod score of 2.19 at a recombination fraction of 0. A second hypervariable marker in Xq26, the dinucleotide repeat XL90A3 (DXS425), showed a lod score of .84 at a recombination fraction of .11. Both the HPRT and DXS425 markers were typed in 40 CEPH families, and subsequent multipoint linkage analysis showed the following order: Xcen-DXS425-(HPRT,XLMR)-F9-qter. HPRT and these flanking markers are therefore useful for carrier detection and prenatal diagnosis in this family. This study illustrates that hypervariable STRs will be powerful tools for linkage analysis and genetic diagnosis, particularly when relatively small families are involved.


The burden of occupational injury attributable to high temperatures in Australia, 2014-19: a retrospective observational study.
To assess the population health impact of high temperatures on workplace health and safety by estimating the burden of heat-attributable occupational injury in Australia. Retrospective observational study; estimation of burden of occupational injury in Australia attributable to high temperatures during 2014-19, based on Safe Work Australia (work-related traumatic injury fatalities and workers' compensation databases) and Australian Institute of Health and Welfare data (Australian Burden of Disease Study and National Hospital Morbidity databases), and a meta-analysis of climate zone-specific risk data. Burden of heat-attributable occupational injuries as disability-adjusted life years (DALYs), comprising the numbers of years of life lived with disability (YLDs) and years of life lost (YLLs), nationally, by Köppen-Geiger climate zone, and by state and territory. During 2014-19, an estimated 42 884 years of healthy life were lost to occupational injury, comprising 39 485 YLLs (92.1%) and 3399 YLDs (7.9%), at a rate of 0.80 DALYs per 1000 workers per year. A total of 967 occupational injury-related DALYs were attributable to heat (2.3% of occupational injury-related DALYs), comprising 890 YLLs (92%) and 77 YLDs (8%). By climate zone, the heat-attributable proportion was largest in the tropical Am (12 DALYs; 3.5%) and Aw zones (34 DALYs; 3.5%); by state and territory, the proportion was largest in New South Wales and Queensland (each 2.9%), which also included the largest numbers of heat-attributable occupational injury-related DALYs (NSW: 379 DALYs, 39% of national total; Queensland: 308 DALYs; 32%). An estimated 2.3% of the occupational injury burden in Australia is attributable to high ambient temperatures. To prevent this burden increasing with global warming, adaptive measures and industry-based policies are needed to safeguard workplace health and safety, particularly in heat-exposed industries, such as agriculture, transport, and construction.


Project PAVE (Personality And Vision Experimentation): role of personal and interpersonal resilience in the perception of emotional facial expression.
The aim of the proposed theoretical model is to illuminate personal and interpersonal resilience by drawing from the field of emotional face perception. We suggest that perception/recognition of emotional facial expressions serves as a central link between subjective, self-related processes and the social context. Emotional face perception constitutes a salient social cue underlying interpersonal communication and behavior. Because problems in communication and interpersonal behavior underlie most, if not all, forms of psychopathology, it follows that perception/recognition of emotional facial expressions impacts psychopathology. The ability to accurately interpret one's facial expression is crucial in subsequently deciding on an appropriate course of action. However, perception in general, and of emotional facial expressions in particular, is highly influenced by individuals' personality and the self-concept. Herein we briefly outline well-established theories of personal and interpersonal resilience and link them to the neuro-cognitive basis of face perception. We then describe the findings of our ongoing program of research linking two well-established resilience factors, general self-efficacy (GSE) and perceived social support (PSS), with face perception. We conclude by pointing out avenues for future research focusing on possible genetic markers and patterns of brain connectivity associated with the proposed model. Implications of our integrative model to psychotherapy are discussed.


Defining Cyberbullying.
Is cyberbullying essentially the same as bullying, or is it a qualitatively different activity? The lack of a consensual, nuanced definition has limited the field's ability to examine these issues. Evidence suggests that being a perpetrator of one is related to being a perpetrator of the other; furthermore, strong relationships can also be noted between being a victim of either type of attack. It also seems that both types of social cruelty have a psychological impact, although the effects of being cyberbullied may be worse than those of being bullied in a traditional sense (evidence here is by no means definitive). A complicating factor is that the 3 characteristics that define bullying (intent, repetition, and power imbalance) do not always translate well into digital behaviors. Qualities specific to digital environments often render cyberbullying and bullying different in circumstances, motivations, and outcomes. To make significant progress in addressing cyberbullying, certain key research questions need to be addressed. These are as follows: How can we define, distinguish between, and understand the nature of cyberbullying and other forms of digital conflict and cruelty, including online harassment and sexual harassment? Once we have a functional taxonomy of the different types of digital cruelty, what are the short- and long-term effects of exposure to or participation in these social behaviors? What are the idiosyncratic characteristics of digital communication that users can be taught? Finally, how can we apply this information to develop and evaluate effective prevention programs?


The effects of environmental temperature on the properties of myofibrillar adenosine triphosphatase from various species of fish.
1. Myofibrillar adenosine triphosphatase (ATPase) activities were measured for white myotomal muscle of 19 species of fish. 2. The activity was measured at different temperatures and after periods of preincubation at 37 degrees C. 3. The inactivation half-life at 37 degrees C depended on environmental temperature, increasing as the temperature increased. 4. Cold-water fish had higher myofibrillar adenosine triphosphatase activity at low temperatures than had warm-water fish. 5. The significance of these results is discussed.


Duration, Pitch, and Loudness in Kunqu Opera Stage Speech.
Kunqu is a special type of opera within the Chinese tradition with 600 years of history. In it, stage speech is used for the spoken dialogue. It is performed in Ming Dynasty's mandarin language and is a much more dominant part of the play than singing. Stage speech deviates considerably from normal conversational speech with respect to duration, loudness and pitch. This paper compares these properties in stage speech conversational speech. A famous, highly experienced female singer's performed stage speech and reading of the same lyrics in a conversational speech mode. Clear differences are found. As compared with conversational speech, stage speech had longer word and sentence duration and word duration was less variable. Average sound level was 16 dB higher. Also mean fundamental frequency was considerably higher and more varied. Within sentences, both loudness and fundamental frequency tended to vary according to a low-high-low pattern. Some of the findings fail to support current opinions regarding the characteristics of stage speech, and in this sense the study demonstrates the relevance of objective measurements in descriptions of vocal styles.


Anesthetic strategy for cesarean section in a patient with factor XI deficiency. Case report.
Factor XI deficiency is a rare hematologic disorder. Hemophilia C (factor XI deficiency) affects both genders and it is usually asymptomatic, manifesting only as postoperative hemorrhage. It is an autosomal recessive, homozygous or heterozygous, disorder, and its severity depends on the levels of factor XI. The objective of this report was to present the anesthetic strategy in a patient with hemophilia C. This is a 32 years old female, gravida 1/para 0, on the 39th week of pregnancy, scheduled for elective cesarean section. Physical and laboratorial exams did not show any abnormalities. According to the recommendations of the hematologist, on the day of the procedure, the patient was given promethazine, 25 mg, hydrocortisone, 500 mg, due to prior transfusion reaction, and plasma, 10 mL x kg(-1) for a total of 700 mL. Two hours later, the patient underwent subarachnoid block under routine monitoring. Ringer's lactate, 2000 mL, was administered for hydration. The anesthetic-surgical procedure proceeded without intercurrences. Postoperatively, the patient was doing well when, on the 3rd PO day, fresh frozen plasma (FFP), 10 mL x kg(-1), was administered to prevent late postoperative bleeding. The objective of this report was to present the anesthetic protocol for patients with hemophilia C and to alert for the need of investigation in patients with a history of postoperative bleeding, when a coagulation study should e be done before any invasive procedure and, in the case of prolonged aPTT, one should investigate the presence of factor XI deficiency.


Psychological flexibility as a moderator of the relationship between HIV-related stigma and resilience among HIV/AIDS patients.
HIV-related stigmatisation is common in many parts of the world and is experienced by all categories of people living with HIV and AIDS (PLWHA). Although the negative consequences of HIV-related stigmatisation on the resilience of PLWHA is well documented, little is known about the plausible role of certain personal characteristics in moderating the stigma-resilience relationship. In addition to investigating the direct association of HIV-related stigma (personalised stigma, disclosure concern, concern about public attitude and negative self-image) with resilience, the present study examined whether psychological flexibility (PF) moderates the HIV-related stigmaresilience relationship among PLWHA. Participants included 280 PLWHA (M = 39.48; SD = 9.03) selected from Sacred Heart Catholic Hospital (SHCH), Obudu, Cross River State, Nigeria. Participants completed relevant self-report measures. Results showed that patients reported moderately high levels of resilience (M = 59.13; SD = 13.98). Hierarchical multiple regression analysis showed that HIV-related stigma (personalised stigma, disclosure concern and concern about public attitudes) were not significantly associated with resilience (p = 0.230; p = 0.747; p = 0.528). HIV-related negative self-image and PF were independently and significantly associated with resilience (p = 0.024; p = 0.000). Results of moderation hypothesis revealed that PF did not moderate the relationship between HIV-related disclosure concern and resilience (p = 0.903), and between HIV-related concern about public attitudes and resilience (p = 0.905), but PF moderated the relationship of HIV-related personalised stigma and resilience (p = 0.023), and the relationship of HIV-related negative self-image and resilience (p = 0.004). Therefore, interventions to promote resilience abilities in PLWHA should consider facilitating patients' psychological flexibility skills as it is critical in decreasing the hazardous effect of HIV-related stigma on the patients.


Impact of Asymmetric Weight Update on Neural Network Training With Tiki-Taka Algorithm.
Recent progress in novel non-volatile memory-based synaptic device technologies and their feasibility for matrix-vector multiplication (MVM) has ignited active research on implementing analog neural network training accelerators with resistive crosspoint arrays. While significant performance boost as well as area- and power-efficiency is theoretically predicted, the realization of such analog accelerators is largely limited by non-ideal switching characteristics of crosspoint elements. One of the most performance-limiting non-idealities is the conductance update asymmetry which is known to distort the actual weight change values away from the calculation by error back-propagation and, therefore, significantly deteriorates the neural network training performance. To address this issue by an algorithmic remedy, Tiki-Taka algorithm was proposed and shown to be effective for neural network training with asymmetric devices. However, a systematic analysis to reveal the required asymmetry specification to guarantee the neural network performance has been unexplored. Here, we quantitatively analyze the impact of update asymmetry on the neural network training performance when trained with Tiki-Taka algorithm by exploring the space of asymmetry and hyper-parameters and measuring the classification accuracy. We discover that the update asymmetry level of the auxiliary array affects the way the optimizer takes the importance of previous gradients, whereas that of main array affects the frequency of accepting those gradients. We propose a novel calibration method to find the optimal operating point in terms of device and network parameters. By searching over the hyper-parameter space of Tiki-Taka algorithm using interpolation and Gaussian filtering, we find the optimal hyper-parameters efficiently and reveal the optimal range of asymmetry, namely the asymmetry specification. Finally, we show that the analysis and calibration method be applicable to spiking neural networks.


The value of aortic valve replacement in elderly patients: an economic analysis.
Economists have designed frameworks to measure the economic value of improvements in health and longevity. Heart valve replacement surgery has significantly prolonged life expectancy and quality of life. For the example of aortic valve replacement, what is its economic value according to this framework? From 1961 through 2003, a total of 4617 adult patients underwent aortic valve replacement by one team of cardiac surgeons. These patients were provided with a prospective lifetime follow-up service. As of 2005, observed follow-up was 31,671 patient-years, with a maximum of 41 years. A statistical model was used to generate the future life-years of patients currently alive. The value of life-years proposed by economists was applied to determine the economic value of the additional life given to these patients by aortic valve replacement. The total life-years after aortic valve replacement were 53,323, with a gross value of 14.6 billion dollars. The total expected life-years without surgery were 10,157, with an estimated value of 3.0 billion dollars. Thus the net life-years gained by AVR were 43,166, worth 11.6 billion dollars. Subtracting the 451 million dollars total lifetime cost of surgery, the net value of the life-years gained by AVR was 11.2 billion dollars. The mean net value decreases according to age at surgery but is still worth 600,000 dollars for octogenarians and 200,000 dollars for nonagenarians. According to the economic concept of the value of a statistical life, the return on the investment for aortic valve replacement is enormous for patients of all ages, even very elderly patients.


[Diagnosis of viral respiratory infections].
More than 80% of the cases of respiratory infections in children are of viral origin. Viral culture has been the reference method for the diagnosis of viral respiratory infections for years, but there is now a tendency to replace viral culture by molecular biology techniques, notably real-time PCR-based assay, because of its excellent sensitivity and good feasibility. Currently in most laboratories, however, diagnosis of viral respiratory infections is still done using techniques based on detection of viral antigens, especially immunofluorescence assays. Rapid diagnostic tests for use outside of laboratories are now available on the open market, and even if their sensitivity remains lower than that of other techniques, it is likely that they will become widely used, especially in doctors' offices, in the near future. New methods for the diagnosis of viral infections based on DNA microarray technologies are currently under investigation and appear to very promising.


The diversity of benthic diatoms affects ecosystem productivity in heterogeneous coastal environments.
The current decrease in biodiversity affects all ecosystems, and the impacts of diversity on ecosystem functioning need to be resolved. So far, marine studies about diversity-ecosystem productivity-relationships have concentrated on small-scale, controlled experiments, with often limited relevance to natural ecosystems. Here, we provide a real-world study on the effects of microorganismal diversity (measured as the diversity of benthic diatom communities) on ecosystem productivity (using chlorophyll a concentration as a surrogate) in a heterogeneous marine coastal archipelago. We collected 78 sediment cores at 17 sites in the northern Baltic Sea and found exceptionally high diatom diversity (328 observed species). We used structural equation models and quantile regression to explore relationships between diatom diversity and productivity. Previous studies have found contradictory results in the relationship between microorganismal diversity and ecosystem productivity, but we showed a linear and positive basal relationship between diatom diversity and productivity, which indicates that diatom diversity most likely forms the lowest boundary for productivity. Thus, although productivity can be high even when diatom diversity is low, high diatom diversity supports high productivity. The trait composition was more effective than taxonomical composition in showing such a relationship, which could be due to niche complementarity. Our results also indicated that environmental heterogeneity leads to substantial patchiness in the diversity of benthic diatom communities, mainly induced by the variation in sediment organic matter content. Therefore, future changes in precipitation and river runoff and associated changes in the quality and quantity of organic matter in the sea, will also affect diatom communities and, hence, ecosystem productivity. Our study suggests that benthic microorganisms are vital for ecosystem productivity, and together with the substantial heterogeneity of coastal ecosystems, they should be considered when evaluating the potential productivity of coastal areas.


Rational design of FRET-based ratiometric chemosensors for in vitro and in cell fluorescence analyses of nucleoside polyphosphates.
Ratiometric fluorescence sensing is a useful technique for the precise and quantitative analysis of biological events occurring under complex conditions, such as those inside cells. We report herein the design of new ratiometric chemosensors for nucleoside polyphosphates such as ATP that are based on binding-induced modulation of fluorescence resonance energy transfer (FRET) coupled with a turn-on fluorescence-sensing mechanism. We designed these new FRET-based ratiometric chemosensors by utilizing spectral overlap changes to modulate the FRET efficiency. Introduction of coumarin fluorophores as the FRET donors into a binuclear zinc complex as the FRET acceptor provided the ratiometric chemosensors. These chemosensors exhibited a clear dual-mission signal change upon binding with strong affinity (K(app) ≈ 10(6)-10(7) M(-1)) to nucleoside polyphosphates in aqueous solution, whereas no detectable emission change was observed with monophosphates and phosphodiester species or various other anions. These chemosensors were used for real-time fluorescence monitoring of enzyme reactions such as saccharide synthesis by glycosyltransferase and phosphorylation by protein kinase, both of which involve nucleoside polyphosphates as substrates. The utility of ratiometric sensing by chemosensors was further demonstrated in a fluorescence-imaging study of the nucleoside polyphosphates inside living cells, wherein we ratiometrically visualized the stimulus-responsive concentration change of ATP, an indicator of the cellular energy level.


Biotin synthase from Escherichia coli: isolation of an enzyme-generated intermediate and stoichiometry of S-adenosylmethionine use.
A cell-free extract from Escherichia coli containing an E. coli biotin synthase that was expressed to approx. 1% of soluble cell protein by cloning the E. coli bioB gene was used to investigate the biotin synthase reaction. The pH optimum was between 8 and 8.5, and the reaction velocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine. The catalytic-centre activity of the enzyme in vitro was estimated to be 0.95 h-1, and each molecule of enzyme turned over less than one molecule of dethiobiotin, i.e. the enzyme was not acting catalytically. HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate: (1) it was labelled with 14C, and therefore derived from the [14C]dethiobiotin substrate; (2) it was present only in reaction mixtures containing biotin synthase; (3) it was not derived from [14C]biotin; (4) 35S from [35S]cystine was incorporated into the intermediate during the reaction; (5) its synthesis was dependent on the presence of S-adenosylmethionine, and was decreased when free cysteine was omitted from the reaction; (6) it could be isolated from the reaction mixture by chromatography and then re-introduced into an assay as the substrate, whereupon it was converted to biotin; (7) this conversion to biotin was S-adenosylmethionine-dependent. During the reaction S-adenosylmethionine was cleaved to methionine and presumably 5'-deoxyadenosine. Observation of the intermediate allowed us to perform experiments to determine the stoichiometry of S-adenosylmethionine use. We propose that two molecules of S-adenosylmethionine are used to synthesize one molecule of biotin, i.e. one from dethiobiotin to the intermediate, and a second from the intermediate to biotin.


Hypothalamic astrocytes respond to transforming growth factor-alpha with the secretion of neuroactive substances that stimulate the release of luteinizing hormone-releasing hormone.
Previous studies demonstrated the involvement of transforming growth factor-alpha (TGF alpha), a member of the epidermal growth factor (EGF) family, in the developmental regulation of hypothalamic LHRH release. Although both TGF alpha and EGF stimulate LHRH release, they do not appear to act directly on LHRH neurons, as no EGF/TGF alpha receptors are detected on these cells in vivo. Instead, the stimulatory effect of TGF alpha on LHRH release seems to require a glial intermediacy. The present study identifies one of the glial molecules involved in this process. In vitro exposure of purified hypothalamic astrocytes to TGF alpha or EGF in a defined medium led to activation of the cyclooxygenase-mediated pathway of arachidonic acid metabolism, as indicated by an increase in PGE2 release, but failed to affect lipooxygenase-mediated metabolism, as assessed by the lack of increase in leukotriene C4 production; addition of TGF alpha- (T-CM) or EGF-conditioned medium to cultures of LHRH-producing GT1-1 cells stimulated LHRH release. In contrast, direct exposure of GT1-1 cells to the growth factors was ineffective. Incubation of the cells in medium conditioned by untreated astrocytes (CM) was also ineffective. Blockade of either EGF receptor signal transduction or cyclooxygenase activity in the astrocytic cultures prevented both TGF alpha-induced PGE2 formation in astrocytes and the stimulatory effect of T-CM on LHRH release. Immunoneutralization of PGE2 actions or selective removal of the PG from T-CM also prevented T-CM-induced LHRH release. Addition of exogenous PGE2 restored the effect. Thus, PGE2 is one of the glial molecules involved in mediating the stimulatory effect of TGF alpha on LHRH release. The effectiveness of PGE2 in eliciting LHRH release was, however, greatly reduced when PG was delivered to GT1-1 cells in astrocyte-defined medium instead of CM. Thus, astrocytes appear to produce a yet to be identified substance(s) that facilitates the stimulatory effect of PGE2 on LHRH output. We postulate that the ability of TGF alpha to enhance LHRH release depends on the potentiating interaction of PGE2 with these additional glial-derived molecules.


Skin Conductance as a Viable Alternative for Closing the Deep Brain Stimulation Loop in Neuropsychiatric Disorders.
Markers from local field potentials, neurochemicals, skin conductance, and hormone concentrations have been proposed as a means of closing the loop in Deep Brain Stimulation (DBS) therapy for treating neuropsychiatric and movement disorders. Developing a closed-loop DBS controller based on peripheral signals would require: (i) the recovery of a biomarker from the source neural stimuli underlying the peripheral signal variations; (ii) the estimation of an unobserved brain or central nervous system related state variable from the biomarker. The state variable is application-specific. It is emotion-related in the case of depression or post-traumatic stress disorder, and movement-related for Parkinson's or essential tremor. We present a method for closing the DBS loop in neuropsychiatric disorders based on the estimation of sympathetic arousal from skin conductance measurements. We deconvolve skin conductance via an optimization formulation utilizing sparse recovery and obtain neural impulses from sympathetic nerve fibers stimulating the sweat glands. We perform this deconvolution via a two-step coordinate descent procedure that recovers the sparse neural stimuli and estimates physiological system parameters simultaneously. We next relate an unobserved sympathetic arousal state to the probability that these neural impulses occur and use Bayesian filtering within an Expectation-Maximization framework for estimation. We evaluate our method on a publicly available data-set examining the effect of different types of stress on peripheral signal changes including body temperature, skin conductance and heart rate. A high degree of arousal is estimated during cognitive tasks, as are much lower levels during relaxation. The results demonstrate the ability to decode psychological arousal from neural activity underlying skin conductance signal variations. The complete pipeline from recovering neural stimuli to decoding an emotion-related brain state using skin conductance presents a promising methodology for the ultimate realization of a closed-loop DBS controller. Closed-loop DBS treatment would additionally help reduce unnecessary power consumption and improve therapeutic gains.