Structural basis for antigen recognition by methylated lysine-specific antibodies.
Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharacterized, partly owing to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to the development of a novel method to create such antibodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethylated Lys260 of MAP3K2 as a representative substrate. We isolated several methylsite-specific antibodies that contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methylated/nonmethylated antigens and structural motif that is responsible for recognition of the methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of Western blotting analysis suggests a critical antigen recognition mode to generate cross-reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our biophysical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design.


Complementing high-throughput X-ray powder diffraction data with quantum-chemical calculations: Application to piroxicam form III.
High-throughput crystallisation and characterisation platforms provide an efficient means to carry out solid-form screening during the pre-formulation phase. To determine the crystal structures of identified new solid phases, however, usually requires independent crystallisation trials to produce single crystals or bulk samples of sufficient quantity to carry out high-quality X-ray diffraction measurements. This process could be made more efficient by a robust procedure for crystal structure determination directly from high-throughput X-ray powder diffraction (XRPD) data. Quantum-chemical calculations based on dispersion-corrected density functional theory (DFT-D) have now become feasible for typical small organic molecules used as active pharmaceutical ingredients. We demonstrate how these calculations can be applied to complement high-throughput XRPD data by determining the crystal structure of piroxicam form III. These combined experimental/quantum-chemical methods can provide access to reliable structural information in the course of an intensive experimentally based solid-form screening activity or in other circumstances wherein single crystals might never be viable, for example, for polymorphs obtained only during high-energy processing such as spray drying or milling.


Developing New Oligo Probes to Distinguish Specific Chromosomal Segments and the A, B, D Genomes of Wheat (Triticum aestivum L.) Using ND-FISH.
Non-denaturing FISH (ND-FISH) technology has been widely used to study the chromosomes of Triticeae species because of its convenience. The oligo probes for ND-FISH analysis of wheat (Triticum aestivum L.) chromosomes are still limited. In this study, the whole genome shotgun assembly sequences (IWGSC WGA v0.4) and the first version of the reference sequences (IWGSC RefSeq v1.0) of Chinese Spring (T. aestivum L.) were used to find new tandem repeats. One hundred and twenty oligo probes were designed according to the new tandem repeats and used for ND-FISH analysis of chromosomes of wheat Chinese Spring. Twenty nine of the 120 oligo probes produce clear or strong signals on wheat chromosomes. Two of the 29 oligo probes can be used to conveniently distinguish wheat A-, B-, and D-genome chromosomes. Sixteen of the 29 oligo probes only produce clear or strong signals on the subtelomeric regions of 1AS, 5AS, 7AL, 4BS, 5BS, and 3DS arms, on the telomeric regions of 1AL, 5AL, 2BS, 3BL, 6DS, and 7DL arms, on the intercalary regions of 4AL and 2DL arms, and on the pericentromeric regions of 3DL and 6DS arms. Eleven of the 29 oligo probes generate distinct signal bands on several chromosomes and they are different from those previously reported. In addition, the short and long arms of 6D chromosome have been confirmed. The new oligo probes developed in this study are useful and convenient for distinguishing wheat chromosomes or specific segments of wheat chromosomes.


Splign: algorithms for computing spliced alignments with identification of paralogs.
The computation of accurate alignments of cDNA sequences against a genome is at the foundation of modern genome annotation pipelines. Several factors such as presence of paralogs, small exons, non-consensus splice signals, sequencing errors and polymorphic sites pose recognized difficulties to existing spliced alignment algorithms. We describe a set of algorithms behind a tool called Splign for computing cDNA-to-Genome alignments. The algorithms include a high-performance preliminary alignment, a compartment identification based on a formally defined model of adjacent duplicated regions, and a refined sequence alignment. In a series of tests, Splign has produced more accurate results than other tools commonly used to compute spliced alignments, in a reasonable amount of time. Splign's ability to deal with various issues complicating the spliced alignment problem makes it a helpful tool in eukaryotic genome annotation processes and alternative splicing studies. Its performance is enough to align the largest currently available pools of cDNA data such as the human EST set on a moderate-sized computing cluster in a matter of hours. The duplications identification (compartmentization) algorithm can be used independently in other areas such as the study of pseudogenes.


Clinical utility of perfusion (Q)-single-photon emission computed tomography (SPECT)/CT for diagnosing pulmonary embolus (PE) in COVID-19 patients with a moderate to high pre-test probability of PE.
We reviewed the clinical utility of perfusion (Q)-single-photon emission computed tomography (SPECT)/CT for diagnosing pulmonary embolus (PE) in patients hospitalized with severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). Following the World Health Organization's declaration of a global pandemic, our department policy recommended Q-only SPECT/CT for all patients undergoing nuclear medicine evaluation for suspected PE to reduce the risk of aerosolization of respiratory droplets. We performed a retrospective review of sequential patients admitted with COVID-19 imaged with Q-SPECT/CT between March 17, 2020, and June 30, 2020, at Memorial Sloan Kettering Cancer Center. We recorded patient demographics, clinical symptoms, Wells score (to stratify patients according to pre-test probability for PE prior to Q-SPECT/CT), and noted ancillary imaging findings on CT. Of the 33 patients imaged with Q-SPECT/CT, 6 patients (3 men, 3 women) had a laboratory confirmed diagnosis of COVID-19 (mean age, 55, ± 11.4 years, range 33-68). All patients had a current diagnosis of malignancy and had a moderate or high pre-test probability for PE (mean Wells score 2.8, range 2-4). Q-SPECT/CT was positive in 4/6 (67%) of patients. Distribution of pulmonary emboli was bilateral and segmental in 75% of patients. Ancillary acute findings on SPECT/CT included bilateral parenchymal ground glass opacities (n = 5), pleural effusions (n = 2), and pneumomediastinum (n = 1). Q-SPECT/CT has clinical utility for diagnosing PE in patients with COVID-19 where there is a contraindication for iodinated contrast media and a moderate or high pre-test probability for PE.


Reactive astrocytes in Alzheimer's disease: A double-edged sword.
Alzheimer's disease (AD) is a chronic and fatal disease, in which neuronal damage at its late stage cannot be easily reversed. Because AD progression is caused by multiple factors including diverse cellular processes, studies on AD pathogenesis at the molecular and cellular level are challenging. Based on the lessons from unsuccessful neuron-focused research for an AD cure, non-cell autonomous mechanisms including brain inflammation and reactive astrocytes have recently been in the spotlight as potential therapeutic targets for AD. Studies have shown that reactive astrocytes are not only the result of inflammatory defense reactions, but also an active catabolic decomposer that acts by taking up amyloid beta toxins. Here, we give an overview of the characteristics of reactive astrocytes as pathological features of AD. Reactive astrocytes exert biphasic effects, that is, beneficial or detrimental depending on multiple factors. Many efforts have been put forth for defining and characterizing molecular signatures for the beneficial and detrimental reactive astrocytes. In the foreseeable future, manipulating and targeting each established molecular signature should have profound therapeutic implications for the treatment of AD.


A novel lipoxygenase inhibitor Nordy attenuates malignant human glioma cell responses to chemotactic and growth stimulating factors.
Nordy is a chiral compound synthesized based on the structure of a natural lipoxygenase (LO) inhibitor nordihydroguaiaretic acid (NDGA) from plants. The aim of the present study is to investigate the effect of Nordy on malignant human glioma cell responses to chemoattractants and growth promoting signals. We found that Nordy, in a non-cytotoxic concentration range, potently inhibited the chemotaxis and calcium flux of a human glioblastoma cell line U87 induced by a formylpeptide receptor (FPR) agonist, formyl-methionyl-leucyl-phenylalanine (fMLF) and epidermal growth factor (EGF). U87 cells treated by Nordy also showed a significantly impaired proliferation and expression of mRNA for vascular endothelial growth factor (VEGF) induced by fMLF. The chemotactic and proliferation responses of Nordy treated U87 cells to EGF were concomitantly diminished. Further experiments revealed that Nordy did not significantly affect FPR gene expression in U87 cells, but attenuated the activation of a plethora of signaling molecules including ERK1/2, p38, JNK, and Akt when the cells were stimulated by fMLF. EGF-induced EGF receptor phosphorylation was also inhibited in Nordy-treated U87 cells. Moreover, Nordy significantly reduced the tumorigenicity of U87 cells in nude mice. Our results suggest that Nordy is capable of inhibiting glioma cell responses to signals that promote cell motility, growth and production of VEGF. Thus, Nordy may constitute a molecular basis for the development of novel anti-cancer drugs.


Drug screening in Drosophila; why, when, and when not?
The best global seller among oncology drugs in 2018 is lenalidomide, an analog of thalidomide. It took 53 years and a circuitous route from the discovery of thalidomide to approval of an analog for use in treatment of cancer. We understand now a lot more about the genetic and molecular basis of diseases than we did in 1953 when thalidomide was discovered. We have also no shortage of chemical libraries with hundreds of thousands of compounds, both synthetic and natural. What we need are better ways to search among these rich resources for compounds with the potential to do what we want them to do. This review summarizes examples from the literature that make Drosophila melanogaster a good model to screen for drugs, and discusses knowledge gaps and technical challenges that make Drosophila models not as widely used as they could or should be. This article is categorized under: Technologies > Analysis of Cell, Tissue, and Animal Phenotypes.


Local tissue temperature increase of a generic implant compared to the basic restrictions defined in safety guidelines.
The objective of this study was to investigate if persons with implantable medical devices are intrinsically protected by the current electromagnetic safety guidelines. For inter-laboratory comparisons, the U.S. Food and Drug Administration has defined a generic implant as consisting of an insulated wire with noninsulated tips, simulating active implants composed of a metallic case, and insulated wires with electric contacts at the tip. In this study, we determined the amplitude of the uniform electric fields induced in body tissues that cause a local increase in the tissue temperature by 1 °C in the presence of this generic implant for a wide range of frequencies and wire lengths. The field amplitudes were compared to the basic restrictions of the current exposure guidelines for both occupational and uncontrolled exposure. Results showed that a 1 °C temperature increase in the tissues around the tips of the generic implant can be reached for field strengths much smaller than 1% of those in the basic restrictions. The simulated results were validated by experimental evaluations. The impact of perfusion was investigated and was found to lead to a reduction in the local temperature peak by only 1.6-3 times. Additional simulations inside an inhomogeneous anatomical model were performed to ascertain whether similar heating as in the generic model was observed. The significant temperature elevations due to the presence of a generic implant indicate that demonstrating compliance with the basic restrictions might not be sufficient for persons with implants. Special considerations may be required, especially in the case of novel, emerging technologies that feature strong near-fields at frequencies below 10 MHz (e.g., wireless power-transfer systems).


Seroprevalence of hepatitis C and associated risk factors among an urban population in Haiti.
The seroprevalence of hepatitis C varies substantially between countries and geographic regions. A better understanding of the seroprevalence of this disease, and the risk factors associated with seropositive status, supply data for the development of screening programs and provide insight into the transmission of the disease. The purpose of this investigation was to determine the seroprevalence of hepatitis C and associated risk factors in an urban population in Haiti. A prospective survey for hepatitis C antibodies was conducted among an urban outpatient population in Cap-Haitien, Haiti, with a sample size of 500 subjects. An anonymous 12 question survey, with inquiries related to demographic characteristics and risk factors for HCV acquisition, was concomitantly administered with testing. These demographic and behavioral risk factors were correlated with HCV antibody status using univariate and multivariate tests. The prevalence of positive HCV antibody was 22/500 (4.4%). Subjects that were anti-HCV positive had an average of 7 +/- 8.6 lifetime sexual partners, compared to average of 2.5 +/- 3.5 lifetime sexual partners among HCV-negative subjects (p = 0.02). In a multiple logistic regression model, intravenous drug use (OR 3.7, 1.52-9.03 95% CI) and number of sexual partners (OR 1.1, 1.04-1.20 95% CI) were independently associated with a positive HCV antibody result. A substantial number of subjects with HCV antibodies were detected in this population in Haiti. Further investigation into the correlation between the number of sexual partners and testing positive for hepatitis C antibodies is indicated.


The role of LARP1 in translation and beyond.
The LARP1 proteins form an evolutionarily homogeneous subgroup of the eukaryotic superfamily of La-Motif (LAM) containing factors. Members of the LARP1 family are found in most protists, fungi, plants, and animals. We review here evidence suggesting that LARP1 are key versatile messenger RNA (mRNA)-binding proteins involved in regulating important biological processes such as gametogenesis, embryogenesis, sex determination, and cell division in animals, as well as acclimation to stress in yeasts and plants. LARP1 proteins perform all these essential tasks likely by binding to key mRNAs and regulating their stability and/or translation. In human, the impact of LARP1 over cell division and proliferation is potentially under the control of the TORC1 complex. We review data suggesting that LARP1 is a direct target of this master signaling hub. TOR-dependent LARP1 phosphorylation could specifically enhance the translation of TOP mRNAs providing a way to promote translation, growth, and proliferation. Consequently, LARP1 is found to be significantly upregulated in many malignant cell types. In plants, LARP1 was found to act as a cofactor of the heat-induced mRNA degradation process, an essential acclimation strategy leading to the degradation of more than 4500 mRNAs coding for growth and development housekeeping functions. In Saccharomyces cerevisiae, the LARP1 proteins (Slf1p and Sro9p) are important, among other things, for copper resistance and oxidative stress survival. LARP1 proteins are therefore emerging as critical ancient mRNA-binding factors that evolved common as well as specific targets and regulatory functions in all eukaryotic lineages.


Online quantitative monitoring of live cell engineered cartilage growth using diffuse fiber-optic Raman spectroscopy.
Tissue engineering (TE) has the potential to improve the outcome for patients with osteoarthritis (OA). The successful clinical translation of this technique as part of a therapy requires the ability to measure extracellular matrix (ECM) production of engineered tissues in vitro, in order to ensure quality control and improve the likelihood of tissue survival upon implantation. Conventional techniques for assessing the ECM content of engineered cartilage, such as biochemical assays and histological staining are inherently destructive. Raman spectroscopy, on the other hand, represents a non-invasive technique for in situ biochemical characterization. Here, we outline current roadblocks in translational Raman spectroscopy in TE and introduce a comprehensive workflow designed to non-destructively monitor and quantify ECM biomolecules in large (>3 mm), live cell TE constructs online. Diffuse near-infrared fiber-optic Raman spectra were measured from live cell cartilaginous TE constructs over a 56-day culturing period. We developed a multivariate curve resolution model that enabled quantitative biochemical analysis of the TE constructs. Raman spectroscopy was able to non-invasively quantify the ECM components and showed an excellent correlation with biochemical assays for measurement of collagen (R2 = 0.84) and glycosaminoglycans (GAGs) (R2 = 0.86). We further demonstrated the robustness of this technique for online prospective analysis of live cell TE constructs. The fiber-optic Raman spectroscopy strategy developed in this work offers the ability to non-destructively monitor construct growth online and can be adapted to a broad range of TE applications in regenerative medicine toward controlled clinical translation.


Attitudes, awareness and barriers regarding evidence-based orthopedic surgery between health professionals from a Brazilian Public Health System (SUS) hospital: Study of 400 patients.
The fast-track concept refers to all phases of perioperative care: Preoperative, intraoperative, and postoperative strategies. Although most research has focused on adherence to medication, adherence also encompasses numerous health-related behaviors. The aim of this prospective study was to determine the attitudes and awareness among health professionals involved in the treatment of elderly patients with fractures of the femur and the results of 400 patients. The postoperative protocol acceleration was presented to various hospital departments through four seminars. Questionnaire with four ex-residents in the Department of Anesthesiology was conducted. Every 6 months, the results of project implementation to all departments were presented. It was considered adherence to the project when the professionals agreed with all the steps and routines of the project. Patients underwent spinal anesthesia with postoperative analgesia by lumbar plexus block. All departments involved in the treatment of elderly patients' adhered completely to the project and reported the importance of preanesthetic visit, the explanations of design, and reduction of fasting period. Just one anesthetist completely adhered to the project. No former resident of anesthesia joined the program. All parameters studied in 400 patients compared with the data before the project showed a reduction from 21.38% to 100%. Improving adherence requires a continuous and dynamic process. We can be inferred that the implementation of fast-track project Brazilian Public Health System (Sistema Único Saúde, SUS) costs decreased with elderly patients with hip fractures. The anesthesiologist was the major obstacle to deployment to all patients.


[Medical demography: the portrait of pathologic anatomy and cytology].
Assessment of care supply in terms of medical labor force is difficult because of the disparity of available data. Using the medical specialty of pathology as an example, we present for the first time a demographic analysis based on the confrontation of main data sources. This study underlines some of the structural and dynamic problems which threaten the demographic stability of the specialty. The public service suffers from a lack of practitioners, especially in regional hospitals. Whereas one third of French pathologists work in the larger hospitals around Paris, in some other regions private services constitute the main component of the supply. Projections based on the aging phenomenon and a reduction in entry indicate a future problem in the renewal of generations of pathologists. The consequences of this programmed decrease in the number of French pathologists will be further exacerbated by the movement of the sex ratio in favor of women.


Amphetamine's dose-dependent effects on dorsolateral striatum sensorimotor neuron firing.
Amphetamine elicits motoric changes by increasing the activity of central neurotransmitters such as dopamine and serotonin, but how these neurochemical signals are transduced into motor commands is unclear. The dorsolateral striatum (DLS), a component of the cortico-subcortical reentrant motor loop, contains abundant neurotransmitter transporters that amphetamine could affect. It has been hypothesized that DLS medium spiny neurons contribute to amphetamine's motor effects. To study striatal activity contributing to amphetamine-induced movements, activity of DLS neurons related to vertical head movement was recorded while tracking head movements before and after acute amphetamine injection. Relative to saline, all amphetamine doses induced head movements above pre-injection levels, revealing an inverted U-shaped dose-response function. Lower doses (1 mg/kg and 2 mg/kg, intraperitoneal) induced a greater number of long (distance and duration) movements than the high dose (4 mg/kg), which induced stereotypy. Firing rates (FR) of individual head movement neurons were compared before and after injection during similar head movements, defined by direction, distance, duration, and apex. Changes in FR induced by amphetamine were co-determined by dose and pre-injection FR of the neuron. Specifically, all doses increased the FRs of slower firing neurons but decreased the FRs of faster firing neurons. The magnitudes of elevation or reduction were greater at lower doses, but less pronounced at the high dose, forming an inverted U function. Modulation of DLS firing may interfere with sensorimotor processing. Furthermore, pervasive elevation of slow firing neurons' FRs may feed-forward and increase excitability in thalamocortical premotor areas, contributing to the increased movement initiation rate.


[The carbohydrate-resistant mechanism of the action of caries-prophylactic agents on the oral fluid in children].
Examinations of 176 children administered caries-preventing drugs for 2 years have shown that oral irrigation with 0.2% sodium fluoride solution, oral intake of fluorine in a dose of 1 mg, or of potassium orotate, or of calcium glucerophosphate improved the oral fluid resistance to carbohydrate action. Glycolytic enzyme activity of the oral fluid in these children was not augmenting during carbohydrate load, whereas in children not administered such preventive courses oral intake of 10% glucose solution resulted in essential elevation of salivary aldolase and lactate dehydrogenase activities, i.e. intensification of glycolytic processes, this leading to the development of acid potential in the oral cavity and, consequently, to a higher risk of caries development.


Timely care after an abnormal mammogram among low-income women in a public breast cancer screening program.
Since 1990, the National Breast and Cervical Cancer Early Detection Program (BCCEDP) has funded breast cancer screening and diagnostic services for low-income, underinsured women. Case management was implemented in 2001 to address barriers to follow-up after an abnormal mammogram, and free treatment was introduced in 2004. However, the effect of these policies on timeliness of care has not been empirically evaluated. Among 2252 BCCEDP participants in Massachusetts during 1998 through 2007, we conducted a time-to-event analysis with prepolicy-postpolicy comparisons to examine associations of case management and free treatment with diagnostic and treatment delays (>60 days and >90 days, respectively) after an abnormal mammogram. The proportion of women experiencing a diagnostic delay decreased from 33% to 23% after the introduction of case management (P < .001), with a significant reduction in the adjusted risk of diagnostic delay (relative risk [RR], 0.65; 95% confidence interval [CI], 0.53-0.79) that did not differ by race and ethnicity. However, case management was not associated with changes in treatment delay (RR, 0.93; 95% CI, 0.80-1.10). Free treatment was not associated with changes in the adjusted risk of diagnostic delay (RR, 0.61; 95% CI, 0.33-1.14) or treatment delay (RR, 0.77; 95% CI, 0.43-1.38) beyond improvements associated with case management. Case management to assist women in overcoming logistic and psychosocial barriers to care may improve time to diagnosis among low-income women who receive free breast cancer screening and diagnostic services. Programs that provide services to coordinate care, in addition to free screening and diagnostic tests, may improve population health.


MIMIC-SBDH: A Dataset for Social and Behavioral Determinants of Health.
Social and Behavioral Determinants of Health (SBDHs) are environmental and behavioral factors that have a profound impact on health and related outcomes. Given their importance, physicians document SBDHs of their patients in Electronic Health Records (EHRs). However, SBDHs are mostly documented in unstructured EHR notes. Determining the status of the SBDHs requires manually reviewing the notes which can be a tedious process. Therefore, there is a need to automate identifying the patients' SBDH status in EHR notes. In this work, we created MIMIC-SBDH, the first publicly available dataset of EHR notes annotated for patients' SBDH status. Specifically, we annotated 7,025 discharge summary notes for the status of 7 SBDHs as well as marked SBDH-related keywords. Using this annotated data for training and evaluation, we evaluated the performance of three machine learning models (Random Forest, XGBoost, and Bio-ClinicalBERT) on the task of identifying SBDH status in EHR notes. The performance ranged from the lowest 0.69 F1 score for Drug Use to the highest 0.96 F1 score for Community-Present. In addition to standard evaluation metrics such as the F1 score, we evaluated four capabilities that a model must possess to perform well on the task using the CheckList tool (Ribeiro et al., 2020). The results revealed several shortcomings of the models. Our results highlighted the need to perform more capability-centric evaluations in addition to standard metric comparisons.


Incidence of Odontogenic Disease in Patients With Pott's Puffy Tumor.
Osteomyelitis with subperiosteal abscess of the frontal bone, or Pott's puffy tumor (PPT), is a rare but life-threatening condition. The relationship of concurrent dental disease to PPT has not been well described. This study sought to delineate the incidence of odontogenic disease in PPT, especially in cases where there is no history of facial trauma or prior frontal sinus surgery. A retrospective chart review of patients diagnosed with PPT between 2010 and 2019 was carried out. Demographics, pertinent medical history, surgical procedures performed, and microbial cultures and antibiotics used were extracted for analysis. Maxillofacial computed tomography scans were reviewed for presence of odontogenic disease. A total of 17 patients were identified. Four had documented history of frontal bone trauma; 3 had previous frontal sinus surgery. Seven (41%) had documented odontogenic disease on initial radiology reports; however, upon dentist review, 16 (94%) had various pathology visible on their computed tomography scans. Twelve of these 16 patients had no previous frontal sinus trauma or surgery. Eight patients (47%) had only ipsilateral maxillary or ethmoid inflammation respective to PPT on computed tomography. Seventy-six percent of patients underwent endoscopic sinus surgery. Of 14 patients with cultures collected at the time of surgery, 7 (50%) had polymicrobial growth and 6 (43%) had anaerobic growth. The incidence of odontogenic disease in this population of PPT was 94%. In the absence of a history of frontal bone trauma or frontal sinus surgery, underlying dental origin should be suspected in cases of PPT. Initial radiology reports may not document all identifiable dental pathology. Therefore, patients presenting with PPT should undergo comprehensive evaluation, including an oral cavity and dental examination, and potential referral to dental colleagues.


Simulation of steady-state NMR of coupled systems using Liouville space and computer algebra methods.
A series of repeated pulses and delays applied to a spin system generates a steady state. This is relatively easy to calculate for a single spin, but coupled systems present real challenges. We have used Maple, a computer algebra program to calculate one- and two-spin symbolically, and larger systems numerically. The one-spin calculations illustrate and validate the methods and show how the steady-state free precession method converges to continuous wave NMR. For two-spin systems, we have derived a general formula for the creation of double-quantum signals as a function of irradiation strength, coupling constant, and chemical shift difference. The calculations on three-spin and larger systems reproduce and extend previously published results. In this paper, we have shown that the approach works well for systems in literature. However, the formalism is general and can be extended to more complex spin systems and pulses sequences.


[The medicalizing of mothers and children in Colonial Vietnam, 1860-1939].
Almost nothing has been written about the history of maternal and child care in South East Asia. The issue however has a great amount of interest. For instance, French Indochina under colonial rule (1860-1954) experienced significant advancements in both areas. Writing their history helps to better understand the international dissemination of medical intervention towards pregnant women and their children and some of the movement's national characteristics. Maternal and child care rapidly became the only area within the health policy Indochina received before and after the establishment of an official health program (1905). Its rise also occurred during a period of great scientific change which necessarily had influence on its trends and applications. Certainly, the French Republic wanted to exploit its new territory. Consequently, the colonial government in Hanoi would concentrate on infant morbidity and mortality. Nevertheless, within a few years and especially between the two world wars, the administrators would even provide social welfare, particularly to babies and grown children. Maternal and child care were priorities for the Vietnamese territories. However the colonial health system took local pathological, sociocultural, and economic contexts and peninsular medical traditions into account. The system was based everywhere on maternity wards and indigenous midwives to achieve colonial goals about fighting mortality and educating populations in hygiene. The data indicates that France's moves towards offering health care to Indochinese people improved morbidity and rates and accelerated medical acculturation.


NH2-terminal specificity and axonal localization of adrenocorticotropin binding sites in rat median eminence.
Adrenocorticotropin binding sites in the rat median eminence have been localized in vivo. These binding sites occur in the basalar zone, which is rich in axonal endings. Using competitive binding and quantitative light-microscope radioautography, we found that the median-eminence binding site, in contradistinction to the adrenal receptor, binds specifically the residue 4-10 region of the adrenocorticotropin molecule. Using quantitative electron-microscope radioautography and median-eminence deafferentation, we localized the binding sites to axon terminals in this region. In time-delayed uptake studies using light-microscope radioautography, we failed to observe concentration of radiolabel in neurons of the medial basal hypothalamus after the direct injection of radioiodinated adrenocorticotropin(1-24) into the median eminence.


SSC1, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein.
SSC1 is an essential member of the yeast HSP70 multigene family (E. Craig, J. Kramer, and J. Kosic-Smithers, Proc. Natl. Acad. Sci. USA 84:4156-4160, 1987). Analysis of the SSC1 DNA sequence revealed that it could encode a 70,627-dalton protein that is more similar to DnaK, an Escherichia coli hsp70 protein, than other yeast hsp70s whose sequences have been determined. Ssc1p was found to have an amino-terminal extension of 28 amino acids, in comparison with either Ssa1p, another hsp70 yeast protein, or Dnak. This putative leader is rich in basic and hydroxyl amino acids, characteristic of many mitochondrial leader sequences. Ssc1p that was synthesized in vitro could be imported into mitochondria and was cleaved in the process. The imported protein comigrated with an abundant mitochondrial protein that reacted with hsp70-specific antibodies. We conclude that Ssc1p is a mitochondrial protein and that hsp70 proteins perform functions in many compartments of the cell.


Hierarchical Representation Learning in Graph Neural Networks With Node Decimation Pooling.
In graph neural networks (GNNs), pooling operators compute local summaries of input graphs to capture their global properties, and they are fundamental for building deep GNNs that learn hierarchical representations. In this work, we propose the Node Decimation Pooling (NDP), a pooling operator for GNNs that generates coarser graphs while preserving the overall graph topology. During training, the GNN learns new node representations and fits them to a pyramid of coarsened graphs, which is computed offline in a preprocessing stage. NDP consists of three steps. First, a node decimation procedure selects the nodes belonging to one side of the partition identified by a spectral algorithm that approximates the MAXCUT solution. Afterward, the selected nodes are connected with Kron reduction to form the coarsened graph. Finally, since the resulting graph is very dense, we apply a sparsification procedure that prunes the adjacency matrix of the coarsened graph to reduce the computational cost in the GNN. Notably, we show that it is possible to remove many edges without significantly altering the graph structure. Experimental results show that NDP is more efficient compared to state-of-the-art graph pooling operators while reaching, at the same time, competitive performance on a significant variety of graph classification tasks.


Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells.
After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt-/-). Lysates from Icmt-/- cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such as N-acetyl-S-geranylgeranyl-L-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt-/- cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt-/- cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt-/- cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells.


The Relationship between Feeding and Non-Nutritive Sucking Behaviours and Speech Sound Development: A Systematic Review.
Children with and without speech sound disorders (SSDs) are exposed to different patterns of infant feeding (breast/bottle-feeding) and may or may not engage in non-nutritive sucking (NNS) (pacifier/digit-sucking). Sucking and speech use similar oral musculature and structures, therefore it is possible that early sucking patterns may impact early speech sound development. The objective of this review is to synthesise the current evidence on the influence of feeding and NNS on the speech sound development of healthy full-term children. Electronic databases (PubMed, NHS CRD, EMBASE, MEDLINE) were searched using terms specific to feeding, NNS and speech sound development. All methodologies were considered. Studies were assessed for inclusion and quality by 2 reviewers. Of 1,031 initial results, 751 records were screened, and 5 primary studies were assessed for eligibility, 4 of which were included in the review. Evidence from the available literature on the relationship between feeding, NNS and speech sound development was inconsistent and inconclusive. An association between NNS duration and SSDs was the most consistent finding, reported by 3 of the 4 studies. Quality appraisal was carried out using the Appraisal Tool for Cross-Sectional Studies (AXIS). The included studies were found to be of moderate quality. Key Messages: This review found there is currently limited evidence on the relationship between feeding, NNS and speech sound development. Exploring this unclear relationship is important because of the overlapping physical mechanisms for feeding, NNS and speech production, and therefore the possibility that feeding and/or sucking behaviours may have the potential to impact on speech sound development. Further high-quality research into specific types of SSD using coherent clinically relevant assessment measures is needed to clarify the nature of the association between feeding, NNS and speech sound development, in order to inform and support families and health care professionals.


Tips and Traps: Lessons From Codesigning a Clinician E-Monitoring Tool for Computerized Cognitive Behavioral Therapy.
Computerized cognitive behavioral therapy (cCBT) is an acceptable and promising treatment modality for adolescents with mild-to-moderate depression. Many cCBT programs are standalone packages with no way for clinicians to monitor progress or outcomes. We sought to develop an electronic monitoring (e-monitoring) tool in consultation with clinicians and adolescents to allow clinicians to monitor mood, risk, and treatment adherence of adolescents completing a cCBT program called SPARX (Smart, Positive, Active, Realistic, X-factor thoughts). The objectives of our study were as follows: (1) assess clinicians' and adolescents' views on using an e-monitoring tool and to use this information to help shape the development of the tool and (2) assess clinician experiences with a fully developed version of the tool that was implemented in their clinical service. A descriptive qualitative study using semistructured focus groups was conducted in New Zealand. In total, 7 focus groups included clinicians (n=50) who worked in primary care, and 3 separate groups included adolescents (n=29). Clinicians were general practitioners (GPs), school guidance counselors, clinical psychologists, youth workers, and nurses. Adolescents were recruited from health services and a high school. Focus groups were run to enable feedback at 3 phases that corresponded to the consultation, development, and postimplementation stages. Thematic analysis was applied to transcribed responses. Focus groups during the consultation and development phases revealed the need for a simple e-monitoring registration process with guides for end users. Common concerns were raised in relation to clinical burden, monitoring risk (and effects on the therapeutic relationship), alongside confidentiality or privacy and technical considerations. Adolescents did not want to use their social media login credentials for e-monitoring, as they valued their privacy. However, adolescents did want information on seeking help and personalized monitoring and communication arrangements. Postimplementation, clinicians who had used the tool in practice revealed no adverse impact on the therapeutic relationship, and adolescents were not concerned about being e-monitored. Clinicians did need additional time to monitor adolescents, and the e-monitoring tool was used in a different way than was originally anticipated. Also, it was suggested that the registration process could be further streamlined and integrated with existing clinical data management systems, and the use of clinician alerts could be expanded beyond the scope of simply flagging adolescents of concern. An e-monitoring tool was developed in consultation with clinicians and adolescents. However, the study revealed the complexity of implementing the tool in clinical practice. Of salience were privacy, parallel monitoring systems, integration with existing electronic medical record systems, customization of the e-monitor, and preagreed monitoring arrangements between clinicians and adolescents.


Effect of n-Caproate Concentration on Chain Elongation and Competing Processes.
Chain elongation is an open-culture fermentation process that facilitates conversion of organic residues with an additional electron donor, such as ethanol, into valuable n-caproate. Open-culture processes are catalyzed by an undefined consortium of microorganisms which typically also bring undesired (competing) processes. Inhibition of competing processes, such as syntrophic ethanol oxidation, will lead to a more selective n-caproate production process. In this study, we investigated the effect of n-caproate concentration on the specific activity of chain elongation and competing processes using batch inhibition assays. With "synthetic medium sludge" (originally operating at 3.4 g/L n-caproate), syntrophic ethanol oxidation was proportionally inhibited by n-caproate until 45% inhibition at 20 g/L n-caproate. Hydrogenotrophic methanogenesis was for 58% inhibited at 20 g/L n-caproate. Chain elongation of volatile fatty acids (volatile fatty acid upgrading; the desired process), was completely inhibited at 20 g/L n-caproate with all tested sludge types. "Adapted sludge" (operating at 23.2 g/L n-caproate) showed a 10 times higher volatile fatty acid upgrading activity at 15 g/L n-caproate compared to "nonadapted sludge" (operating at 7.1 g/L n-caproate). This shows that open cultures do adapt to perform chain elongation at high n-caproate concentrations which likely inhibits syntrophic ethanol oxidation through hydrogenotrophic methanogenesis. As such, we provide supporting evidence that the formation of n-caproate inhibits syntrophic ethanol oxidation which leads to a more selective medium chain fatty acid production process.


An alternative aerosol delivery system for amiloride.
The advent of novel treatments such as aerosolized amiloride are potentially useful additions to the therapeutic options available for the treatment of cystic fibrosis. Unfortunately, amiloride and other aerosolized drugs such as antibiotics are generally administered via jet nebulisers which are time consuming to use, and thus limit the acceptance and efficacy of these forms of treatment. In vitro experiments were performed in order to determine whether amiloride could be administered in dry powder form using a Turbohaler. Amiloride was micronised and loaded into 200 micrograms Turbohalers. The dose delivered per actuation and particle size distribution of the generated aerosol were assessed using a flow of 60 l/min through the Turbohaler. The dose of amiloride delivered was measured by collecting the aerosol on a filter and the quantity of drug was assayed by an ultraviolet spectrophotometric method. The particle size distribution was assessed using a Malvern MasterSizer laser particle sizer and compared with that generated by a commercially available 200 micrograms budesonide Turbohaler. The mean (SD) dose delivered per actuation was 246.3 (40.4) micrograms. The volume median diameter of the amiloride aerosol was 3.80 (0.68) micron compared with 3.07 (1.47) microns for budesonide. These results suggest that therapeutic doses of micronised amiloride could be delivered effectively and conveniently as a dry powder aerosol using a Turbohaler.


Anatomical and functional brain abnormalities in drug-naive first-episode schizophrenia.
The authors sought to explore whether anatomical and functional brain deficits are present in similar or different brain regions early in the course of schizophrenia, before antipsychotic treatment, and whether these deficits are more severe or otherwise different in patients with prominent negative symptoms. A total of 100 drug-naive first-episode schizophrenia patients and 100 matched healthy comparison subjects underwent structural and resting-state functional MRI scanning. Gray matter volume and amplitude of low-frequency fluctuations during resting-state functional studies were measured. Group comparisons of gray matter volume showed significant differences mainly in thalamo-cortical networks, while alterations in the amplitude of low-frequency fluctuations were observed in fronto-parietal and default mode networks. Thus, different brain regions had alterations in gray matter volume and resting state physiology. These changes did not correlate with the duration of untreated illness, nor with acute clinical symptom severity. Patients with prominent negative symptoms had greater regional alterations in brain anatomy, particularly in the left dorsolateral prefrontal cortex, while the pattern of functional alterations was unrelated to severity of negative symptoms. Anatomical and resting-state functional deficits were observed in different brain regions, indicating that anatomical and functional brain abnormalities are significantly dissociated in the early course of schizophrenia. The lack of association of these abnormalities with illness duration and episode severity suggests that these anatomical and functional changes may be early-evolving features of the illness that are relatively stable early in the course of illness. The different structural deficits of regional gray matter observed in patients with prominent negative symptoms may provide unique insight into the early regional neuropathology of this symptom dimension in schizophrenia.


TCF4-mediated Fuchs endothelial corneal dystrophy: Insights into a common trinucleotide repeat-associated disease.
Fuchs endothelial corneal dystrophy (FECD) is a common cause for heritable visual loss in the elderly. Since the first description of an association between FECD and common polymorphisms situated within the transcription factor 4 (TCF4) gene, genetic and molecular studies have implicated an intronic CTG trinucleotide repeat (CTG18.1) expansion as a causal variant in the majority of FECD patients. To date, several non-mutually exclusive mechanisms have been proposed that drive and/or exacerbate the onset of disease. These mechanisms include (i) TCF4 dysregulation; (ii) toxic gain-of-function from TCF4 repeat-containing RNA; (iii) toxic gain-of-function from repeat-associated non-AUG dependent (RAN) translation; and (iv) somatic instability of CTG18.1. However, the relative contribution of these proposed mechanisms in disease pathogenesis is currently unknown. In this review, we summarise research implicating the repeat expansion in disease pathogenesis, define the phenotype-genotype correlations between FECD and CTG18.1 expansion, and provide an update on research tools that are available to study FECD as a trinucleotide repeat expansion disease. Furthermore, ongoing international research efforts to develop novel CTG18.1 expansion-mediated FECD therapeutics are highlighted and we provide a forward-thinking perspective on key unanswered questions that remain in the field.


Docking with PIPER and refinement with SDU in rounds 6-11 of CAPRI.
Our approach to protein-protein docking includes three main steps. First we run PIPER, a new rigid body docking program. PIPER is based on the Fast Fourier Transform (FFT) correlation approach that has been extended to use pairwise interactions potentials, thereby substantially increasing the number of near-native structures generated. The interaction potential is also new, based on the DARS (Decoys As the Reference State) principle. In the second step, the 1000 best energy conformations are clustered, and the 30 largest clusters are retained for refinement. Third, the conformations are refined by a new medium-range optimization method SDU (Semi-Definite programming based Underestimation). SDU has been developed to locate global minima within regions of the conformational space in which the energy function is funnel-like. The method constructs a convex quadratic underestimator function based on a set of local energy minima, and uses this function to guide future sampling. The combined method performed reliably without the direct use of biological information in most CAPRI problems that did not require homology modeling, providing acceptable predictions for targets 21, and medium quality predictions for targets 25 and 26.


Linear behavior of a preformed microbubble containing light absorbing nanoparticles: insight from a mathematical model.
Microbubbles are used as ultrasonic contrast agents in medical imaging because of their highly efficient scattering properties. Gold nanoparticles absorb specific wavelengths of optical radiation very effectively with the subsequent generation of thermo-acoustic waves in the surrounding medium. A theoretical and numerical analysis of the possibility of inducing radial oscillations in a pre-existing spherical microbubble, through the laser excitation of gold nanoparticles contained within, is presented. A description of such a system can be obtained in terms of a confined two-phase model, with the nanoparticles suspended in a confined region of gas, surrounded by a liquid. The Rayleigh-Plesset equation is assumed to be valid at the boundary between the gas and the liquid. The confined two-phase model is solved in linear approximation. The system is diagonalized and the general solution is obtained. This solution is in the form of exponentially decaying oscillatory functions for the temperature and pressure inside the bubble, and radial oscillations of the bubble boundary. It was found that, for the right size of bubbles, the oscillatory behavior takes place in the low megahertz range, which is ideal for medical applications. This study suggests the possibility of new applications of microbubbles in photoacoustic imaging.


Molecular basis of osmotic regulation.
Cells almost universally respond to the stress of long-term hyperosmolality by accumulating compatible organic osmolytes. This allows them to maintain normal cell volume without a deleterious increase in intracellular inorganic ion concentration. Cells in the renal inner medulla are exposed to variable concentrations of salt and urea that may reach molal levels. The organic osmolytes that they accumulate include sorbitol, betaine, inositol, taurine, and glycerophosphocholine (GPC). This review considers recent advances in understanding osmotic regulation of these substances. Sorbitol is synthesized from glucose catalyzed by aldose reductase. Hypertonicity elevates the abundance of this enzyme by increasing transcription of its gene. Betaine is taken up via a specialized transporter. Hypertonicity raises the number of transporters by increasing their transcription. Current studies demonstrate that the 5' regions flanking the aldose reductase and betaine transporter genes contain osmotic response elements that increase transcription in response to hypertonicity. Osmotic regulation of inositol and taurine uptake also involves increased expression of specific transporter genes. GPC is unique in that its level rises in response to high urea, as well as hypertonicity. GPC accumulation is mainly regulated by changes in its degradation to choline, catalyzed by GPC:choline phosphodiesterase. Numerous other genes, including those for heat shock proteins, are also induced by hypertonicity. Their regulation and their role in osmotic regulation are the subject of considerable ongoing research.


Effects of enamel matrix derivative and transforming growth factor-beta1 on human periodontal ligament fibroblasts.
The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.


An update on the anticancer effects of a combination of chemotherapy and hyperthermia.
Hyperthermia combined with chemotherapy is a complicated and poorly understood area, but one that portends great biological and clinical interest. The interrelationship of heat to vascular supply of tumor and normal organs that metabolize chemotherapy agents is complicated. The evocation of thermotolerance by chemical agents is a largely unexplored area. A class of chemotherapy agents that increase in cytotoxicity linearly with increasing temperature are the alkylating agents cisplatinum, mitomycin C, and mitoxantrone. A class of chemotherapy agents that increase in cytotoxicity only after a threshold temperature above 42 degrees are Adriamycin, bleomycin, and actinomycin D. Time sequencing of heat and Adriamycin affects cytotoxicity, implying a membrane effect of the heat and drug interaction. A new class of drugs thought not to be chemotherapy drugs because they are not cytotoxic at 37 degrees but are cytotoxic at greater than 41.5 degrees provides a new area of research. Chemotherapy agents that do not change in cytotoxicity with heat include the Vinca alkaloids, amsacrine, and 5-fluorouracil. In order to examine the complex interaction of heat and chemotherapy agents, whole animal models of tumor and normal tissue effects are extremely important.


Screening Hamstring Injury Risk Factors Multiple Times in a Season Does Not Improve the Identification of Future Injury Risk.
To determine if eccentric knee flexor strength and biceps femoris long head (BFlh) fascicle length were associated with prospective hamstring strain injury (HSI) in professional Australian Football players, and if more frequent assessments of these variables altered the association with injury risk. Across two competitive seasons, 311 Australian Football players (455 player seasons) had their eccentric knee flexor strength during the Nordic hamstring exercise and BFlh architecture assessed at the start and end of preseason and in the middle of the competitive season. Player age and injury history were also collected in preseason. Prospective HSIs were recorded by team medical staff. Seventy-four player seasons (16%) sustained an index HSI. Shorter BFlh fascicles (<10.42 cm) increased HSI risk when assessed at multiple time points only (relative risk [RR], 1.9; 95% confidence interval [CI], 1.2-3.0). Neither absolute (N) nor relative (N·kg-1) eccentric knee flexor strength was associated with HSI risk, regardless of measurement frequency (RR range, 1.0-1.1); however, between-limb imbalance (>9%), when measured at multiple time points, was (RR, 1.8; 95% CI, 1.1-3.1). Prior HSI had the strongest univariable association with prospective HSI (RR, 2.9; 95% CI, 1.9-4.3). Multivariable logistic regression models identified a combination of prior HSI, BFlh architectural variables and between-limb imbalance in eccentric knee flexor strength as optimal input variables; however, their predictive performance did not improve with increased measurement frequency (area under the curve, 0.681-0.726). More frequent measures of eccentric knee flexor strength and BFlh architecture across a season did not improve the ability to identify which players would sustain an HSI.


Psychosocial Barriers of Public Transport Use and Social Exclusion among Older Adults: Empirical Evidence from Lahore, Pakistan.
Transport planning and public health have been intertwined historically. The health impact of public transport services, such as social exclusion, is a widely discussed research topic. Social exclusion is a paramount concern for older adults' health in the wake of emerging global challenges. However, there remains a significant research gap on how psychosocial barriers faced by older adults in using public transport services influence the social exclusion behavior. The present research provides empirical evidence and shows the impact of certain psychosocial barriers of public transportation on older adults' social exclusion. A total of 243 Pakistani older adults (aged 60-89 years old) voluntarily participated in this cross-sectional study. The participants provided self-reports on their psychosocial barriers (including perceived norms, attitude, personal ability, habits, neighborhood social constraints, and intention) and the corresponding social exclusion. Partial Least Square Structural Equation Modeling (PLS-SEM) was utilized for the data analysis. The structural path model supported the significant associations between psychosocial barriers and social exclusion. Except for perceived descriptive norms, all other psychosocial barriers predicted older adults' social exclusion. The research portrays the significance of the psychosocial factors to examine social exclusion and offers practical implications for urban and transport planners. The concerned policymakers can use the research findings to develop age-sensitive, socially sustainable, and healthy cities.


Rhizobium inoculation enhances copper tolerance by affecting copper uptake and regulating the ascorbate-glutathione cycle and phytochelatin biosynthesis-related gene expression in Medicago sativa seedlings.
Despite numerous reports that legume-rhizobium symbiosis alleviates Cu stress in plants, the possible roles of legume-rhizobium symbiosis and the regulatory mechanisms in counteracting Cu toxicity remain unclear. Here, Sinorhizobium meliloti CCNWSX0020 was used for analyzing the effects of rhizobium inoculation on plant growth in Medicago sativa seedlings under Cu stress. Our results showed that rhizobium inoculation alleviated Cu-induced growth inhibition, and increased nitrogen concentration in M. sativa seedlings. Moreover, the total amount of Cu uptake in inoculated plants was significantly increased compared with non-inoculated plants, and the increase in the roots was much higher than that in the shoots, thus decreasing the transfer coefficient and promoting Cu phytostabilization. Cu stress induced lipid peroxidation and reactive oxygen species production, but rhizobium inoculation reduced these components' accumulation through altering antioxidant enzyme activities and regulating ascorbate-glutathione cycles. Furthermore, legume-rhizobium symbiosis regulated the gene expression involved in antioxidant responses, phytochelatin (PC) biosynthesis, and metallothionein biosynthesis in M. sativa seedlings under Cu stress. Our results demonstrate that rhizobium inoculation enhanced Cu tolerance by affecting Cu uptake, regulating antioxidant enzyme activities and the ascorbate-glutathione cycle, and influencing PC biosynthesis-related gene expression in M. sativa. The results provide an efficient strategy for phytoremediation of Cu-contaminated soils.


All-optical single-sideband frequency upconversion utilizing the XPM effect in an SOA-MZI.
An all-optical single sideband (OSSB) frequency upconverter based on the cross-phase modulation (XPM) effect is proposed and experimentally demonstrated to overcome the power fading problem caused by the chromatic dispersion of fiber in radio-over-fiber systems. The OSSB frequency upconverter consists of an arrayed waveguide grating (AWG) and a semiconductor optical amplifier Mach-Zehnder interferometer (SOA-MZI) and does not require an extra delay line used for phase noise compensation. The generated OSSB radio frequency (RF) signal transmitted over single-mode fibers up to 20 km shows a flat electrical RF power response as a function of the fiber length. The upconverted electrical RF signal at 48 GHz shows negligible degradation of the phase noise even without an extra delay line. The measured phase noise of the upconverted RF signal (48 GHz) is -74.72 dBc/Hz at an offset frequency of 10 kHz. The spurious free dynamic range (SFDR) measured by a two-tone test to estimate the linearity of the OSSB frequency upconverter is 72.5 dB·Hz2/3.


In vivo effect of piperine on serum and tissue glycoprotein levels in benzo(a)pyrene induced lung carcinogenesis in Swiss albino mice.
In recent years, considerable emphasis has been focused on identifying new cancer chemopreventive agents, which could be useful for the human population. Piperine is a pure, pungent alkaloid constituent of black and long peppers (Piper nigrum and Piper longum), that acts as an antioxidant and anticancer agent by its numerous macromolecules associated with them. In the present study, piperine was found to suppress benzo(a)pyrene (B(a)p) induced lung cancer in Swiss albino mice. In lung cancer bearing mice, altered levels of total protein and protein bound carbohydrate components (hexose, hexosamine and sialic acid) were observed in serum, lung and liver tissues. Dietary supplementation of piperine (50 mg/kg body weight) to B(a)p administered animals decreased the total protein and protein bound carbohydrate levels of lung cancer bearing animals in during initiation and post-initiation phases. Our data suggest that piperine may extend its chemopreventive effect through modulating the protein bound carbohydrate levels, as they are one of the indicators of tumorigenesis.


Decision making in professional associations.
Processes for decision making by large groups such as professional associations have not adapted rapidly to the growing complexity of the situations those associations face. This paper describes a ten-step procedure which has enabled professional bodies to engage a large sample of their constituency in extensive planning exercises. These planning workshops addressed a wide range of complex issues, many of which were sensitive and threatening. The steps comprised identifying the problems, collating the agenda, analysis of the issues raised, deriving a structured sequence of tasks, pre-circulating papers, forming working groups, providing adequate time and facilities in a suitable setting for effective workshop activity, ensuring communication among groups within the workshop, managing the plenary reporting and consensus seeking, plus recording and circulating the report back to members. Groups worked cooperatively and constructively without evidence of intragroup competition, which reflects both the homogeneity of purpose within those professions and the effect of strong task structuring.


Mucin Secretion in Cystic Fibrosis: A Systematic Review.
Mucus protects the epithelium against invaders and toxic materials. Sticky and thick mucus is characteristic of CF. The aim of this systematic review is to characterize the specific mucins secreted in the lung and intestinal tract of CF patients. A systematic literature search was conducted up to December 31, 2019. The following terms were used: "cystic fibrosis" AND "mucin." Case-control studies comparing mucin expression in CF patients to healthy controls were included. We found 741 eligible studies, 694 studies were rejected because they were performed in animals and not in full text, and 32 studies were excluded being editorials, duplications, review articles, meta-analysis, or not in English. Fifteen studies were eligible for our study, including 150 CF patients compared to 82 healthy controls, all fulfilled the inclusion criteria. The main mucin types expressed in the sinus submucosal glands, sputum, tracheobronchial surface epithelium, and lung submucosal glands were MUC5AC and MUC5B. Increase in the number of sinusoidal submucosal glands and expression of MUC5B was found in CF patients, but no such difference from healthy controls was found for the number of goblet cells in the surface epithelium nor in the expression of -MUC5AC. The opposite was found in the tracheobronchial surface epithelium and in the lungs. Increased expression of MUC5AC in the surface epithelium and of MUC5B in the subepithelial glands may be the result of higher secretion rate of mucin into the lumen of the respiratory tract, causing mucus plaque, infection, and inflammation.


A 3D pancreatic tumor model to study T cell infiltration.
The desmoplastic nature of the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) prevents the infiltration of T cells and the penetration of chemotherapeutic drugs, posing a challenge to the validation of targeted therapies, including T cell immunotherapies. We present an in vitro 3D PDAC-TME model to observe and quantify T cell infiltration across the vasculature. In a three-channel microfluidic device, PDAC cells are cultured in a collagen matrix in the central channel surrounded, on one side, by endothelial cells (ECs) to mimic a blood vessel and, on the opposite side, by pancreatic stellate cells (PSCs) to simulate exocrine pancreas. The migration of T cells toward the tumor is quantified based on their activation state and TME composition. The presence of EC-lining drastically reduces T cell infiltration, confirming the essential role of the vasculature in controlling T cell trafficking. We show that activated T cells migrate ∼50% more than the not-activated ones toward the cancer cells. Correspondingly, in the absence of cancer cells, both activated and not-activated T cells present similar migration toward the PSCs. The proposed approach could help researchers in testing and optimizing immunotherapies for pancreatic cancer.


Do estrogens improve bone mass in osteoporotic women over ten years of menopause.
A retrospective analysis of 24 patients with established osteoporosis and with ten or more years of menopause treated with conjugated estrogen, progesterone and calcium followed for one year has been performed. Treated women received 0.625 mg/day of conjugated estrogen from day 1 to 25, 5 mg/day of medroxiprogesterone from day 13 to 25, of each cycle, plus calcium (500-1000 mg/day), during one year (12 cycles). As control group was used 18 age-matched that received only calcium (500 a 1000 mg/day). All patients had at least two dual-photon spine and proximal femur (neck, Ward's triangle and trocanter) densities measurements performed 12 months apart. Estrogen treatment was associated with increased bone mineral density at spine and trocanter. Control group did not present any statistically change after one year in any site studied. We concluded that women with ten or more years of menopause and established osteoporosis treated with replacement hormonal therapy and calcium results in improvement of bone mineral density. These data support that women with ten or more years of menopause respond to estrogen replacement therapy with absolute increments in bone density similar to those seen in younger women, in the early menopause.


Mother-infant interactions and infant development are altered by maternal iron deficiency anemia.
The aim of this study was to determine whether iron deficiency anemia (IDA) in young South African mothers alters mother-infant interactions and the infant's development. The study was a prospective, randomized, controlled intervention trial with 3 groups of mothers: nonanemic controls and anemic mothers administered either placebo (25 mg ascorbic acid and 10 microg folate) or daily iron treatment (125 mg FeSO(4) plus ascorbate and folate). Mothers of full-term, normal birth weight infants (n = 81) were followed from 10 wk to 9 mo postpartum. Maternal iron status, socioeconomic level, mother-infant interaction [Parent/Caregiver Involvement Scale (PCIS scale)], and infant development (Griffiths scale) were assessed. At baseline, anemic mothers tended (P < 0.10) to be less responsive to, and more controlling of, their infants. Infants of anemic mothers were developmentally delayed at 10 wk in hand-eye movement and overall quotient. Despite normalization of maternal iron status with supplementation in some mothers, the developmental delays were not diminished at 9 mo. At 9 mo, anemic mothers were significantly more "negative" towards their babies, engaged less in goal setting, and were less "responsive" than control mothers. In contrast, the behavior of anemic mothers given iron treatment toward their children was similar to that of the control mothers on all 11 scales of the PCIS. In conclusion, IDA altered mother-child interactions at both 10 wk and 9 mo postpartum. Additionally, infants whose mothers were anemic in the early postpartum scored worse on developmental tests at 10 wk and 9 mo of age.


A Rapid-Test for Screening Biochar Effects on Seed Germination.
We developed a rapid-test to screen for effects of biochar on seed germina- tion and soils. Crop seeds were placed in containers and covered with 15 g of soil with 1% biochar by weight. Two agricultural soils from South Carolina USA were used. Eighteen biochars were produced from six primary feedstocks [pine chips (PC), poultry litter (PL), swine solids (SS), switchgrass (SG); and two blends of PC and PL, 50% PC/50% PL (55), and 80% PC/20% PL (82)]. Each feedstock was pyrolyzed at 350, 500 and 700°C. There were few biochar effects on seed germination. Shoot dry weight was increased for carrot, cucumber, lettuce, oat, and tomato; primarily with biochars containing PL. Soil pH, electrical conductivity and extractable phosphorus primarily increased with PL, SS, 55, and 82 treatments for both soil types and across species. This method can be an early indicator of biochar effects on seed germination and soil health.


The sensitivity to replacement and displacement of the eyes region in early adolescence, young and later adulthood.
Recent evidence suggests a rather gradual developmental trajectory for processing vertical relational face information, lasting well into late adolescence (de Heering and Schlitz, 2008). Results from another recent study (Tanaka et al., 2014) indicate that children and young adolescents use a smaller spatial integration field for faces than do adults, which particularly affects assessment of long-range vertical relations. Here we studied sensitivity to replacement of eyes and eyebrows (F), variation of inter-eye distance (H), and eye height (V) in young adolescents (11-12 years), young (21-25 years), and middle-age adults (51-62 years). In order to provide a baseline for potential age effects the sensitivity to all three types of face manipulations was calibrated to equal levels for the young adults group. Both the young adolescents and the middle-age adults showed substantially lower sensitivity compared to young adults, but only the young adolescents had selective impairment for V relational changes. Their inversion effects were at similar levels for all types of face manipulations, while in both adult groups the inversion effects for V were considerably stronger than for H or F changes. These results suggest that young adolescents use a limited spatial integration field for faces, and have not reached a mature state in processing vertical configural cues. The H-V asymmetry of inversion effects found for both adult groups indicates that adults integrate across the whole face when they view upright stimuli. However, the notably lower sensitivity of middle-age adults for all types of face manipulations, which was accompanied by a strong general "same" bias, suggests early age-related decline in attending cues for facial difference.


ON THE NATURE OF THE DYE PENETRATING THE VACUOLE OF VALONIA FROM SOLUTIONS OF METHYLENE BLUE.
When uninjured cells of Valonia are placed in methylene blue dissolved in sea water it is found, after 1 to 3 hours, that at pH 5.5 practically no dye penetrates, while at pH 9.5 more enters the vacuole. As the cells become injured more dye enters at pH 5.5, as well as at pH 9.5. No dye in reduced form is found in the sap of uninjured cells exposed from 1 to 3 hours to methylene blue in sea water at both pH values. When uninjured cells are placed in azure B solution, the rate of penetration of dye into the vacuole is found to increase with the rise in the pH value of the external dye solution. The partition coefficient of the dye between chloroform and sea water is higher at pH 9.5 than at pH 5.5 with both methylene blue and azure B. The color of the dye in chloroform absorbed from methylene blue or from azure B in sea water at pH 5.5 is blue, while it is reddish purple when absorbed from methylene blue and azure B at pH 9.5. Dry salt of methylene blue and azure B dissolved in chloroform appears blue. It is shown that chiefly azure B in form of free base is absorbed by chloroform from methylene blue or azure B dissolved in sea water at pH 9.5, but possibly a mixture of methylene blue and azure B in form of salt is absorbed from methylene blue at pH 5.5, and azure B in form of salt is absorbed from azure B in sea water at pH 5.5. Spectrophotometric analysis of the dye shows the following facts. 1. The dye which is absorbed by the cell wall from methylene blue solution is found to be chiefly methylene blue. 2. The dye which has penetrated from methylene blue solution into the vacuole of uninjured cells is found to be azure B or trimethyl thionine, a small amount of which may be present in a solution of methylene blue especially at a high pH value. 3. The dye which has penetrated from methylene blue solution into the vacuole of injured cells is either methylene blue or a mixture of methylene blue and azure B. 4. The dye which is absorbed by chloroform from methylene blue dissolved in sea water is also found to be azure B, when the pH value of the sea water is at 9.5, but it consists of azure B and to a less extent of methylene blue when the pH value is at 5.5. 5. Methylene blue employed for these experiments, when dissolved in sea water, in sap of Valonia, or in artificial sap, gives absorption maxima characteristic of methylene blue. Azure B found in the sap collected from the vacuole cannot be due to the transformation of methylene blue into this dye after methylene blue has penetrated into the vacuole from the external solution because no such transformation detectable by this method is found to take place within 3 hours after dissolving methylene blue in the sap of Valonia. These experiments indicate that the penetration of dye into the vacuole from methylene blue solution represents a diffusion of azure B in the form of free base. This result agrees with the theory that a basic dye penetrates the vacuole of living cells chiefly in the form of free base and only very slightly in the form of salt. But as soon as the cells are injured the methylene blue (in form of salt) enters the vacuole. It is suggested that these experiments do not show that methylene blue does not enter the protoplasm, but they point out the danger of basing any theoretical conclusion as to permeability on oxidation-reduction potential of living cells from experiments made or the penetration of dye from methylene blue solution into the vacuole, without determining the nature of the dye inside and outside the cell.


Morphological characterization of immortalized hypothalamic neurons synthesizing luteinizing hormone-releasing hormone.
An immortalized LHRH cell line has recently been developed by genetically targeting these neurons for tumorigenesis. One of the subclones, the GT1-7 cells, was characterized at both the light and electron microscopic levels to study the cellular and subcellular organization of these cells, particularly as they relate to biosynthesis, processing, and secretion. The cells were fixed onto slides 18-36 h after plating. LHRH and GnRH-associated peptide (GAP) immunoreactivities (IR) were detected by immunocytochemistry using colloidal gold labeling. These cultured cells exhibited the classical neuronal appearance of LHRH neurons, and they established numerous interconnections. Neighboring neurons were coupled by tight junctions, while more distant cells were interconnected with neural axon-like processes and collaterals. This cellular organization is suggestive of a neural network where neuronal activity is coordinated. At the ultrastructural level, the nondividing cells possessed indented nuclei, well developed Golgi complexes, and abundant numbers of ribosomes and secretory granules. Clathrin-coated vesicles were found in fusion with the plasma membrane. The ribosomes and secretory vesicles were particularly prominent, suggestive of high rates of protein biosynthesis and secretion. All of the cells immunostained for both LHRH and GAP; however, GAP IR was always more pronounced than that for LHRH. This finding was corroborated by biochemical data reported in a companion paper. The GAP IR was associated with ribosomes and secretory vesicles. By comparison, LHRH IR was restricted mainly to the secretory vesicles. Using colloidal gold particles of different sizes to denote LHRH or GAP IR, it was determined that both GAP and LHRH IR were colocalized within the same secretory vesicle. Taken together, these data suggest that pro-LHRH is biosynthesized on the ribosomes, packaged as an intact protein into the secretory vesicles, processed to LHRH and GAP-(1-56) within these vesicles, and transported to the periphery of the cell in preparation for secretion. These morphological data emphasize the utility of using these immortalized LHRH neuronal cells to dissect the cellular and subcellular architecture involved in biosynthesis, processing, and secretion. In addition, our results provide the first detailed evidence for the intracellular pathway involved in pro-LHRH biosynthesis, processing, and secretion in these cultured neuronal cells.


Factors influencing surgical delay after hip fracture in hospitals of Emilia Romagna Region, Italy: a multilevel analysis.
Hip fracture is becoming a major public health concern, with associated mortality and morbidity particularly in the elderly. This study aims to investigate factors (i.e. patient factors and hospital variables) associated with increased risk for delaying surgery after hip fractures, and to assess whether and to what extent timing was associated with mortality risk. All patients aged 65 and over, resident in Emilia Romagna Region (Italy) and admitted to hospital for hip fracture (2009 - 2010) were selected. Data on surgical delay were adjusted using multilevel logistic regression model. A Cox proportional hazard model was fitted to a propensity score matched sample to assess mortality between delayed and early treated patients. Of the 10, 995 patients included (mean age: 83.3 years), 44.9% underwent surgery within 2 days. Significant risk factors for delayed surgery were: gender (OR: 1.16), comorbidity (OR: 1.29), anticoagulant (OR: 7.64) ,antiplatelet medication (OR: 2.43) , type of procedure (OR: 1.37) and day of admission (OR: Thu-Fri: 6.05; Sat-Sun: 1.17). Type of hospital and annual volume of hip fracture surgeries were not sufficient to explain hospital variability. A significant difference in mortality rate between early and delayed surgery emerged six months post surgery.


A Stable Silylene with a σ2, π- Butadiene Ligand.
The synthesis of a new type of silylene 1 is reported. It adopts a bicyclo[2.1.1]hexene structure in which a hafnocene group is incorporated. The silylene is stabilized by homoconjugation with the remote C═C double bond. This is indicated by its highly shielded 29Si NMR chemical shift (δ29Si = -155) and is firmly established by its experimental molecular structure from XRD analysis. The results of a detailed bonding analysis based on DFT calculations suggest for model compounds of silylene 1 and for its heavier germanium, tin, and lead homologues uniformly electronic structures of carbene analogues that are stabilized by homoconjugation. This stabilization mode is equivalent to a σ2, π-coordination of the butadiene ligand to the element atom as it is typical for zirconocene or hafnocene butadiene complexes.


Contrasting the seasonal and elevational prevalence of generalist avian haemosporidia in co-occurring host species.
Understanding the ecology and evolution of parasites is contingent on identifying the selection pressures they face across their infection landscape. Such a task is made challenging by the fact that these pressures will likely vary across time and space, as a result of seasonal and geographical differences in host susceptibility or transmission opportunities. Avian haemosporidian blood parasites are capable of infecting multiple co-occurring hosts within their ranges, yet whether their distribution across time and space varies similarly in their different host species remains unclear. Here, we applied a new PCR method to detect avian haemosporidia (genera Haemoproteus, Leucocytozoon, and Plasmodium) and to determine parasite prevalence in two closely related and co-occurring host species, blue tits (Cyanistes caeruleus, N = 529) and great tits (Parus major, N = 443). Our samples were collected between autumn and spring, along an elevational gradient in the French Pyrenees and over a three-year period. Most parasites were found to infect both host species, and while these generalist parasites displayed similar elevational patterns of prevalence in the two host species, this was not always the case for seasonal prevalence patterns. For example, Leucocytozoon group A parasites showed inverse seasonal prevalence when comparing between the two host species, being highest in winter and spring in blue tits but higher in autumn in great tits. While Plasmodium relictum prevalence was overall lower in spring relative to winter or autumn in both species, spring prevalence was also lower in blue tits than in great tits. Together, these results reveal how generalist parasites can exhibit host-specific epidemiology, which is likely to complicate predictions of host-parasite co-evolution.


CYLD Regulates Noscapine Activity in Acute Lymphoblastic Leukemia via a Microtubule-Dependent Mechanism.
Noscapine is an orally administrable drug used worldwide for cough suppression and has recently been demonstrated to disrupt microtubule dynamics and possess anticancer activity. However, the molecular mechanisms regulating noscapine activity remain poorly defined. Here we demonstrate that cylindromatosis (CYLD), a microtubule-associated tumor suppressor protein, modulates the activity of noscapine both in cell lines and in primary cells of acute lymphoblastic leukemia (ALL). Flow cytometry and immunofluorescence microscopy reveal that CYLD increases the ability of noscapine to induce mitotic arrest and apoptosis. Examination of cellular microtubules as well as in vitro assembled microtubules shows that CYLD enhances the effect of noscapine on microtubule polymerization. Microtubule cosedimentation and fluorescence titration assays further reveal that CYLD interacts with microtubule outer surface and promotes noscapine binding to microtubules. These findings thus demonstrate CYLD as a critical regulator of noscapine activity and have important implications for ALL treatment.


Components of methacholine-initiated conducted vasodilation are unaffected by arteriolar pressure.
Conducted vasodilation occurs remotely from a site of microapplication of a drug. Intravascular pressure is required for a conducted response in vivo, yet in vitro studies in unpressurized arterioles show pressure is not essential. To determine how pressure affects conducted vasodilation, intra-arteriolar pressure was controlled within an in situ isolated segment (average length 950 +/- 96 microns, average baseline diameter 28 +/- 2.1 microns) of arterioles in the hamster cheek pouch. Methacholine (10(-4) M, 5 s) was microapplied either onto the isolated segment or remotely, with local and conducted vasodilation measured at both locations. Increasing pressure in the lumen of the segment (0-80 cmH2O) increased the segment local dilation to methacholine, and the segment-conducted dilation plateaued (at 4.1 +/- 0.8 micron) when segment pressure reached 20 cmH2O. Any local (16 +/- 1.5 microns) and conducted (4.4 +/- 1.3 microns) dilations viewed outside the segment were unaffected by segment pressure and persisted in its absence. Thus segment pressure affected only electromechanical transduction of the conducted response. Thus vasomotor signals move throughout the vasculature regardless of tone, but tone is essential to transduce the response.


How alcohol intake affects visual temporal processing.
Alcohol affects vision. However, the influence of alcohol on visual processing is largely unknown. Here, we investigated the effects of alcohol on visual spatiotemporal processing. We employed a visual paradigm, the shine through backward masking paradigm, in which a vernier is either presented alone or followed by a variety of mask. We investigated performance for women at blood alcohol levels of 0mg/kg, 400mg/kg and 600 mg/kg and for men at 0mg/kg, 400mg/kg and 800 mg/kg. When the vernier was presented alone, vernier offset discrimination was not affected by alcohol. When the vernier was followed by a mask, stimulus onset asynchronies (SOAs) between target and mask were significantly longer after alcohol intake. However, as a second experiment showed, spatial and temporal processing per se were not impaired by alcohol. In addition, spatial processing was not affected by moderate alcohol consumption. Hence, moderate consumption of alcohol does not affect visual processing per se. We propose that the longer SOAs after alcohol intake are related to changes in mechanisms of target stabilization rather than changes in spatial and temporal sensitivity as has been previously suggested.


Survival after bilateral risk-reducing mastectomy in healthy BRCA1 and BRCA2 mutation carriers.
In healthy BRCA1/2 mutation carriers, bilateral risk-reducing mastectomy (BRRM) strongly reduces the risk of developing breast cancer (BC); however, no clear survival benefit of BRRM over BC surveillance has been reported yet. In this Dutch multicenter cohort study, we used multivariable Cox models with BRRM as a time-dependent covariable to estimate the associations between BRRM and the overall and BC-specific mortality rates, separately for BRCA1 and BRCA2 mutation carriers. During a mean follow-up of 10.3 years, 722 out of 1712 BRCA1 (42%) and 406 out of 1145 BRCA2 (35%) mutation carriers underwent BRRM. For BRCA1 mutation carriers, we observed 52 deaths (20 from BC) in the surveillance group, and 10 deaths (one from BC) after BRRM. The hazard ratios were 0.40 (95% CI 0.20-0.90) for overall mortality and 0.06 (95% CI 0.01-0.46) for BC-specific mortality. BC-specific survival at age 65 was 93% for surveillance and 99.7% for BRRM. For BRCA2 mutation carriers, we observed 29 deaths (7 from BC) in the surveillance group, and 4 deaths (no BC) after BRRM. The hazard ratio for overall mortality was 0.45 (95% CI 0.15-1.36). BC-specific survival at age 65 was 98% for surveillance and 100% for BRRM. BRRM was associated with lower mortality than surveillance for BRCA1 mutation carriers, but for BRCA2 mutation carriers, BRRM may lead to similar BC-specific survival as surveillance. Our findings support a more individualized counseling based on BRCA mutation type.


Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.


Mechanisms of platelet activation by thrombin: a short history.
Platelet activation by thrombin is relevant to arterial thrombosis, therefore it is an attractive target for the development of new antithrombotic drugs. In the 1970s the platelet membrane complex glycoprotein (GP) Ib-V-IX was shown to have a high affinity binding site for thrombin on GPIbα and a substrate cleaved by thrombin, GPV. For several years it was considered to be involved in platelet activation by thrombin. The discovery of the protease activated receptors (PARs) in 1991 was a major breakthrough in the field. The first member of this family of receptors to be discovered was PAR1, a seven transmembrane G-protein coupled receptor which, upon cleavage by thrombin, unmasks a new amino-terminus able to bind intramolecularly to PAR1 itself thus inducing signaling. On human platelets PAR1 and, later PAR4, were demonstrated to mediate most of the platelet responses to thrombin. However, after the discovery of PARs, different groups demonstrated that GPIbα is required to stimulate a full platelet activation by thrombin. A model where thrombin binds to the GPIb receptor prior to proteolysis of the PAR receptors was supported by several lines of evidence. A role for GPV as inhibitor of GPIbα signaling has been shown by using GPV knock-out mice. Crystallographic data suggested that thrombin bound to GPIbα might be able to interact with other GPIbα molecules on the same or other platelets, shedding light on a new role for thrombin binding to GPIbα. Finally, anti-PAR1 molecules were developed which are now in phase II and III clinical studies as antithrombotic drugs.


'CFTR-opathies': disease phenotypes associated with cystic fibrosis transmembrane regulator gene mutations.
Cystic fibrosis is a genetic disease that is associated with abnormal sweat electrolytes, sino-pulmonary disease, exocrine pancreatic insufficiency, and male infertility. Insights into genotype/phenotype relations have recently been gained in this disorder. The strongest relationship exists between 'severe' mutations in the gene that encodes the cystic fibrosis transmembrane regulator (CFTR) and pancreatic insufficiency. The relationship between 'mild' mutations, associated with residual CFTR function, and expression of disease is less precise. Atypical 'mild' mutations in the CFTR gene have been linked to late-onset pulmonary disease, congenital bilateral absence of the vas deferens, and idiopathic pancreatitis. Less commonly, sinusitis, allergic bronchopulmonary aspergillosis, and possibly even asthma may also be associated with mutations in the CFTR gene, but those syndromes predominantly reflect non-CFTR gene modifiers and environmental influences.


A Review of Clinical Outcomes for Gait and Other Variables in the Surgical Treatment of Idiopathic Normal Pressure Hydrocephalus.
Idiopathic normal pressure hydrocephalus (INPH) is a treatable cause of gait disturbance, cognitive impairment, and urinary incontinence. This clinical triad of symptoms occurs in association with ventriculomegaly and normal cerebrospinal fluid (CSF) pressure. Although the treatment outcomes after CSF shunting for INPH have improved significantly since its first description in 1965, shortcomings in our understanding still remain. Not all INPH patients exhibit clinical improvement after shunting, and it is challenging to identify patients who are more likely to benefit from shunting. The Cochrane Library, Medline, Embase, and PubMed databases were searched for English-language publications between 1965 and October 2015. Reference lists of publications were also manually searched for additional publications. The findings of this review indicate that, despite efforts to improve patient selection, the degree of clinical improvement after shunting continues to demonstrate significant variability both within and between studies. These discrepancies in treatment outcomes are the result of controversies in 3 distinct but interrelated domains: the underlying pathophysiology of INPH, the diagnosis of INPH, and the identification of likely shunt-responders. This review focuses on these 3 areas and their relation to surgical treatment outcomes. Despite the limitations of published outcome studies and limitations in our understanding of INPH pathophysiology, shunting is a safe and effective means of achieving meaningful clinical improvement in most patients with INPH.


Nanocomposites Based on Biodegradable Polymers.
In the present review paper, our main results on nanocomposites based on biodegradable polymers (on a time scale from 2010 to 2018) are reported. We mainly focused our attention on commercial biodegradable polymers, which we mixed with different nanofillers and/or additives with the final aim of developing new materials with tunable specific properties. A wide list of nanofillers have been considered according to their shape, properties, and functionalization routes, and the results have been discussed looking at their roles on the basis of different adopted processing routes (solvent-based or melt-mixing processes). Two main application fields of nanocomposite based on biodegradable polymers have been considered: the specific interaction with stem cells in the regenerative medicine applications or as antimicrobial materials and the active role of selected nanofillers in food packaging applications have been critically revised, with the main aim of providing an overview of the authors' contribution to the state of the art in the field of biodegradable polymeric nanocomposites.


Antibiotic resistance mitigation: the development of alternative general strategies.
Antibiotic resistance has become one of the major, deadly threats to public health worldwide. This paper highlights several recent works, which may initiate the development of comprehensive approaches to mitigate antibiotic resistance. The new strategies demonstrate efficiency and efficacy, with very little probability of inducing drug resistance, paving the way for further breakthroughs in drug discovery for infection control.