Intratumoral Heterogeneity and Longitudinal Changes in Gene Expression Predict Differential Drug Sensitivity in Newly Diagnosed and Recurrent Glioblastoma.
Background: Inevitable recurrence after radiochemotherapy is the major problem in the treatment of glioblastoma, the most prevalent type of adult brain malignancy. Glioblastomas are notorious for a high degree of intratumor heterogeneity manifest through a diversity of cell types and molecular patterns. The current paradigm of understanding glioblastoma recurrence is that cytotoxic therapy fails to target effectively glioma stem cells. Recent advances indicate that therapy-driven molecular evolution is a fundamental trait associated with glioblastoma recurrence. There is a growing body of evidence indicating that intratumor heterogeneity, longitudinal changes in molecular biomarkers and specific impacts of glioma stem cells need to be taken into consideration in order to increase the accuracy of molecular diagnostics still relying on readouts obtained from a single tumor specimen. Methods: This study integrates a multisampling strategy, longitudinal approach and complementary transcriptomic investigations in order to identify transcriptomic traits of recurrent glioblastoma in whole-tissue specimens of glioblastoma or glioblastoma stem cells. In this study, 128 tissue samples of 44 tumors including 23 first diagnosed, 19 recurrent and 2 secondary recurrent glioblastomas were analyzed along with 27 primary cultures of glioblastoma stem cells by RNA sequencing. A novel algorithm was used to quantify longitudinal changes in pathway activities and model efficacy of anti-cancer drugs based on gene expression data. Results: Our study reveals that intratumor heterogeneity of gene expression patterns is a fundamental characteristic of not only newly diagnosed but also recurrent glioblastomas. Evidence is provided that glioblastoma stem cells recapitulate intratumor heterogeneity, longitudinal transcriptomic changes and drug sensitivity patterns associated with the state of recurrence. Conclusions: Our results provide a transcriptional rationale for the lack of significant therapeutic benefit from temozolomide in patients with recurrent glioblastoma. Our findings imply that the spectrum of potentially effective drugs is likely to differ between newly diagnosed and recurrent glioblastomas and underscore the merits of glioblastoma stem cells as prognostic models for identifying alternative drugs and predicting drug response in recurrent glioblastoma. With the majority of recurrent glioblastomas being inoperable, glioblastoma stem cell models provide the means of compensating for the limited availability of recurrent glioblastoma specimens.


A previously unreported function of β(1)B integrin isoform in caspase-8-dependent integrin-mediated keratinocyte death.
Integrins regulate adhesive cell-matrix interactions and mediate survival signals. On the other hand, unligated or free cytoplasmic fragments of integrins induce apoptosis in many cell types (integrin-mediated death). We have previously shown that β(1) integrin expression protects keratinocyte stem cells from anoikis, whereas the role of the β(1)B integrin isoform has not been clarified. In this study we report that suspended keratinocytes undergo apoptosis through the activation of caspase-8, independently of the Fas/Fas ligand system. Indeed, anti-β(1) integrin-neutralizing antibodies induced apoptosis in short hairpin RNA Fas-associated death domain-treated cells. Moreover, before and during suspension, caspase-8 directly associated with β(1) integrin, which in turn internalized and progressively degraded, shedding the cytoplasmic domain. β(1)B was expressed only in the cytoplasm in a perinuclear manner and remained unaltered during suspension. At 24 hours, as β(1)A was located close to the nucleus, β(1)B colocalized with β(1)A and coimmunoprecipitated with caspase-8. Caspase-8 was activated earlier in β(1)B integrin-transfected keratinocytes, and these cells underwent a higher rate of apoptosis than mock cells. In contrast, caspase-8 was not activated in small interfering RNA (siRNA) β(1)B-transfected cells. These results indicate that when β(1)A is unligated, β(1)B is responsible for "integrin-mediated death" in human keratinocytes.


Chimeric RNA: DNA TracrRNA Improves Homology-Directed Repair In Vitro and In Vivo.
Nearly 90% of human pathogenic mutations are caused by small genetic variations, and methods to correct these errors efficiently are critically important. One way to make small DNA changes is providing a single-stranded oligo deoxynucleotide (ssODN) containing an alteration coupled with a targeted double-strand break (DSB) at the target locus in the genome. Coupling an ssODN donor with a CRISPR-Cas9-mediated DSB is one of the most streamlined approaches to introduce small changes. However, in many systems, this approach is inefficient and introduces imprecise repair at the genetic junctions. We herein report a technology that uses spatiotemporal localization of an ssODN with CRISPR-Cas9 to improve gene alteration. We show that by fusing an ssODN template to the trans-activating RNA (tracrRNA), we recover precise genetic alterations, with increased integration and precision in vitro and in vivo. Finally, we show that this technology can be used to enhance gene conversion with other gene editing tools such as transcription activator like effector nucleases.


Baculum morphology predicts reproductive success of male house mice under sexual selection.
Diversity in penile morphology is characterised by extraordinary variation in the size and shape of the baculum (penis bone) found in many mammals. Although functionally enigmatic, diversity in baculum form is hypothesised to result from sexual selection. According to this hypothesis, the baculum should influence the outcome of reproductive competition among males within promiscuous mating systems. However, a test of this key prediction is currently lacking. Here we show that baculum size explains significant variation in the reproductive success of male house mice under competitive conditions. After controlling for body size and other reproductive traits, the width (but not length) of the house mouse baculum predicts both the mean number of offspring sired per litter and total number of offspring sired. By providing the first evidence linking baculum morphology to male reproductive success, our results support the hypothesis that evolutionary diversity in baculum form is driven by sexual selection.


Detection of the survival motor neuron (SMN) genes by FISH: further evidence for a role for SMN2 in the modulation of disease severity in SMA patients.
Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder which presents with various clinical phenotypes ranging from severe to very mild. All forms are caused by the homozygous absence of the survival motor neuron ( SMN1 ) gene. SMN1 and a nearly identical copy ( SMN2 ) are located in a duplicated region at 5q13 and encode identical proteins. The genetic basis for the clinical variability of SMA remains unclear, but it has been suggested that the copy number of SMN2 could influence the disease severity. We have assessed the number of SMN2 genes in patients with different clinical phenotypes by fluorescence in situ hybridization (FISH) using as SMN probe a mixture of small specific DNA fragments. Gene copy number was established by FISH on interphase nuclei, but the presence of two SMN2 genes on the same chromosome could also be revealed by FISH on metaphase spreads. All patients had at least two SMN2 genes. We found two or three copies of SMN2 in severely affected type I patients, three copies in intermediately affected type II patients, generally four copies in mildly affected type III patients and four or eight copies in patients with very mild adult-onset SMA. No alterations of the genes were detected by Southern blot and sequence analysis, suggesting that all gene copies of SMN2 were intact. These data provide additional evidence that the SMN2 genes modulate the disease severity and suggest that knowledge of the gene copy number could be of some prognostic value.


'Pure' spindle cell variant of angiomatoid fibrous histiocytoma, lacking classic histologic features.
Angiomatoid fibrous histiocytoma (AFH) is a soft tissue tumor of intermediate biologic potential and uncertain differentiation that most frequently occurs in the superficial extremities of children and young adults. It is histologically typified by nodules of ovoid to spindle cells with pseudoangiomatoid spaces and a surrounding dense lymphoplasmacytic infiltrate, desmin expression in about 50%, and association with EWSR1-CREB1, EWSR1-ATF1 or FUS-ATF1 gene fusions. The diagnosis still poses a challenge because AFH may not display all classic features, can show a variety of unusual histologic findings and lacks a specific immunoprofile. We describe a case of 'pure' spindle cell AFH arising in the forearm musculature of a 19 year-old female, which harbored EWSR1-CREB1 fusion transcripts by reverse transcription-polymerase chain reaction. The neoplasm was composed entirely of highly cellular fascicles of spindled cells architecturally resembling spindle cell sarcoma, and lacked obvious pseudoangiomatoid spaces or a lymphoid cuff. This purely spindle cell variant adds to the significant morphologic spectrum of AFH, and emphasizes that even when occuring at a typical site, AFH may be difficult to recognize when showing non-classical morphology. This is of clinical relevance, as AFH with this morphology could be potentially misdiagnosed as a high-grade sarcoma, with the patient subject to more radical therapeutic approaches.


BioParts-A Biological Parts Search Portal and Updates to the ICE Parts Registry Software Platform.
Capturing, storing, and sharing biological DNA parts data are integral parts of synthetic biology research. Here, we detail updates to the ICE biological parts registry software platform that enable these processes, describe our implementation of the Web of Registries concept using ICE, and establish Bioparts, a search portal for biological parts available in the public domain. The Web of Registries enables standalone ICE installations to securely connect and form a distributed parts database. This distributed database allows users from one registry to query and access plasmid, strain, (DNA) part, plant seed, and protein entry types in other connected registries. Users can also transfer entries from one ICE registry to another or make them publicly accessible. Bioparts, the new search portal, combines the ease and convenience of modern web search engines with the capabilities of bioinformatics search tools such as BLAST. This portal, available at bioparts.org, allows anyone to search for publicly accessible biological part information (e.g., NCBI, iGEM, SynBioHub, Addgene), including parts publicly accessible through ICE Registries. Additionally, the portal offers a REST API that enables third-party applications and tools to access the portal's functionality programmatically.


Evaluation of bronchial asthma as risk factor for early childhood caries.
Asthma and dental caries are common chronic health conditions of childhood. The aim of this study was to determine if severity of bronchial asthma is a risk factor for early childhood caries. Asthmatic children aged 71 months or younger were examined for their plaque and caries status and were compared with controls matched for age, sex and socioeconomic status. The results showed that asthmatics as a group had a lower DMFT score than the control group. New asthma management and educational programs may be having a positive spillover effect to the dental arena.


[EEG-parameters dynamic during relaxation (frequencies analysis)].
The aim of this study was to analyze the wide frequency range of EEG-parameters to estimate a possible functional importance of different EEG-components to reach the relaxation state. Sixty five subjects 17 to 20 years old were involved in the investigation. The relaxation session with monopolar EEG-registration and autonomic parameters recording was conducted to each subject. Significant changes in all analyzed EEG-bands were observed during relaxation. Two types of changes of EEG-coherence were distinguished by wakefulness-relaxation shift of state, and correlation of the type of dynamic with LF/HF ratio were discovered.


The use of equivalent size distributions of natural phytoplankton assemblages for optical modeling.
The effective cell size is expected to be one of the principal causes of variability in the inherent optical properties (IOPs) of a phytoplankton population. However, establishing simple size descriptors is complicated by the typically complex particle size distributions of natural phytoplankton assemblages. This study compares the use of measured and equivalent particle size distributions on the modeled IOPs of a wide range of natural phytoplankton assemblages. It demonstrates that several equivalent size distributions, using simple parameterizations of complex size distributions based on the effective radius or diameter, are capable of modeling phytoplankton IOPs with sufficient accuracy for further use in marine bio-optical models. The results offered here are expected to be of use in bio-optical studies of phytoplankton dynamics e.g. harmful algal bloom oriented inverse reflectance models.


Case Report: Expanding the Phenotypic Spectrum of Timothy Syndrome Type 1: A Sporadic Case With a de novo CACNA1C Pathogenic Variant and Segmental Ileal Dilatation.
Background: Long QT syndactyly syndrome (long QT syndrome type 8), also known as Timothy Syndrome (TS) was first described in 1994 with still <50 case reported in the literature. The full spectrum of the syndrome is not yet known. Results: Here we report a girl who presented with new onset refractory seizures and an undiagnosed cause of intermittent abdominal distention. She also had syndactyly of her fingers and toes and was found to have prolonged QT. Upon further investigations she was found to have a de novo pathogenic variant in CACNA1C, along with Segmental Ileal Dilatation (SID), and subsequently diagnosed with Timothy syndrome. Conclusion: To our knowledge, the association of Timothy Syndrome with Segmental Ileal Dilatation, was not described before.


JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats.
Neuropathic pain, a debilitating pain condition, is a common consequence of damage to the nervous system. Optimal treatment of neuropathic pain is a major clinical challenge because the underlying mechanisms remain unclear and currently available treatments are frequently ineffective. Emerging lines of evidence indicate that peripheral nerve injury converts resting spinal cord glia into reactive cells that are required for the development and maintenance of neuropathic pain. However, the mechanisms underlying reactive astrogliosis after nerve injury are largely unknown. In the present study, we investigated cell proliferation, a critical process in reactive astrogliosis, and determined the temporally restricted proliferation of dorsal horn astrocytes in rats with spinal nerve injury, a well-known model of neuropathic pain. We found that nerve injury-induced astrocyte proliferation requires the Janus kinase-signal transducers and activators of transcription 3 signalling pathway. Nerve injury induced a marked signal transducers and activators of transcription 3 nuclear translocation, a primary index of signal transducers and activators of transcription 3 activation, in dorsal horn astrocytes. Intrathecally administering inhibitors of Janus kinase-signal transducers and activators of transcription 3 signalling to rats with nerve injury reduced the number of proliferating dorsal horn astrocytes and produced a recovery from established tactile allodynia, a cardinal symptom of neuropathic pain that is characterized by pain hypersensitivity evoked by innocuous stimuli. Moreover, recovery from tactile allodynia was also produced by direct suppression of dividing astrocytes by intrathecal administration of the cell cycle inhibitor flavopiridol to nerve-injured rats. Together, these results imply that the Janus kinase-signal transducers and activators of transcription 3 signalling pathway are critical transducers of astrocyte proliferation and maintenance of tactile allodynia and may be a therapeutic target for neuropathic pain.


Developing a Comprehensive Model of Intensive Care Unit Processes: Concept of Operations.
This study aimed to use a systems engineering approach to improve performance and stakeholder engagement in the intensive care unit to reduce several different patient harms. We developed a conceptual framework or concept of operations (ConOps) to analyze different types of harm that included 4 steps as follows: risk assessment, appropriate therapies, monitoring and feedback, as well as patient and family communications. This framework used a transdisciplinary approach to inventory the tasks and work flows required to eliminate 7 common types of harm experienced by patients in the intensive care unit. The inventory gathered both implicit and explicit information about how the system works or should work and converted the information into a detailed specification that clinicians could understand and use. Using the ConOps document, we created highly detailed work flow models to reduce harm and offer an example of its application to deep venous thrombosis. In the deep venous thrombosis model, we identified tasks that were synergistic across different types of harm. We will use a system of systems approach to integrate the variety of subsystems and coordinate processes across multiple types of harm to reduce the duplication of tasks. Through this process, we expect to improve efficiency and demonstrate synergistic interactions that ultimately can be applied across the spectrum of potential patient harms and patient locations. Engineering health care to be highly reliable will first require an understanding of the processes and work flows that comprise patient care. The ConOps strategy provided a framework for building complex systems to reduce patient harm.


CD4-Pseudomonas exotoxin hybrid proteins: modulation of potency and therapeutic window through structural design and characterization of cell internalization.
Replacing the Pseudomonas exotoxin A (PE) cell binding domain with the human immunodeficiency virus (HIV) gp120 binding domain from CD4 yields a hybrid toxin (CD4-PE) with potential therapeutic use in treating acquired immunodeficiency syndrome (AIDS). To find the most therapeutically potent combination of CD4 and PE four different hybrid toxins composed of one [CD4(122)] or two [CD4(181)] Ig-like CD4 domains and sequences of PE where the binding domain was partially [PE(392)] or completely [PE(364)] removed were constructed and expressed in Escherichia coli. The number of CD4 domains determined the binding affinity to gp120 and in cell viability assays the window between specific and nonspecific cytotoxicity. The length of PE determined the potency of the drug. The optimal hybrid toxin was composed of two Ig-like domains of CD4 and PE(392). Investigation of the internalization mechanism of CD4-PE revealed that the hybrid toxin binds to target cells and is endocytosed within one hour. However, more than 6 hours are required for maximum translation inhibition. In contrast to PE which is inhibited by ammonium chloride treatment, cell toxicity of CD4-PE is not affected by ammonium chloride. Further investigations showed that the acid-induced hydrophobicity change which is required for membrane translocation is also observed with CD4-PE but at significantly higher pH than with PE.


17p Deletion is associated with resistance of B-cell chronic lymphocytic leukemia cells to in vitro fludarabine-induced apoptosis.
We explored the relationship between the cytogenetic/biologic characteristics of B-chronic lymphocytic leukemia (B-CLL) cells and their tendency to undergo spontaneous or fludarabine-induced apoptosis in vitro. B cells from 36 B-CLL patients were incubated with or without fludarabine for 48 h. Apoptosis was determined by two assays: annexin V staining and DNA staining. Fluorescence in situ hybridization was used for detection of trisomy 12, 11q deletion, and 17p deletion. Bcl-2 and CD38 expressions were determined by flow cytometry. Five patients had 17p deletion, 6 had trisomy 12, and another 6 had 11q deletion. B-CLL cells with 17p deletion had significant resistance to apoptosis induced by fludarabine and a slight spontaneous resistance to apoptosis. Bcl-2 and CD38 were not associated with in vitro spontaneous and fludarabine-induced apoptosis. In conclusion, 17p deletion, which causes loss of p53 gene, is associated with resistance to fludarabine-induced apoptosis in vitro. New treatment modalities should be tried in B-CLL patients with 17p deletion.


CHESS improves cancer caregivers' burden and mood: results of an eHealth RCT.
Informal caregivers (family and friends) of people with cancer are often unprepared for their caregiving role, leading to increased burden or distress. Comprehensive Health Enhancement Support System (CHESS) is a Web-based lung cancer information, communication, and coaching system for caregivers. This randomized trial reports the impact on caregiver burden, disruptiveness, and mood of providing caregivers access to CHESS versus the Internet with a list of recommended lung cancer websites. A total of 285 informal caregivers of patients with advanced nonsmall cell lung cancer were randomly assigned to a comparison group that received Internet or a treatment group that received Internet and CHESS. Caregivers were provided a computer and Internet service if needed. Written surveys were completed at pretest and during the intervention period bimonthly for up to 24 months. Analyses of covariance (ANCOVAs) compared the intervention's effect on caregivers' disruptiveness and burden (CQOLI-C), and negative mood (combined Anxiety, Depression, and Anger scales of the POMS) at 6 months, controlling for blocking variables (site, caregiver's race, and relationship to patient) and the given outcome at pretest. Caregivers randomized to CHESS reported lower burden, t(84) = 2.36, p = .021, d = .39, and negative mood, t(86) = 2.82, p = .006, d = .44, than those in the Internet group. The effect on disruptiveness was not significant. Although caring for someone with a terminal illness will always exact a toll on caregivers, eHealth interventions like CHESS may improve caregivers' understanding and coping skills and, as a result, ease their burden and mood.


Surgical strategy for biventricular assist device implantation after previous coronary artery bypass grafting.
In an aging population, numerous patients who underwent previous coronary artery bypass grafting (CABG) are presenting with end-stage ischemic cardiomyopathy. Although redo CABG and cardiological interventions are possible treatment options, orthotopic heart transplantation remains an ultimate option for these patients. However, there is high morbidity and mortality on the waiting list, and mechanical circulatory support is a life-saving concept [Hetzer 2006; Taylor 2009].We developed a simplified and safe technique for implantation of a biventricular assist device as a redo in complex patients after previous CABG and end-stage heart failure.


Clinical characteristics of 5 Chinese LQTS families and phenotype-genotype correlation.
In order to assess the clinical manifestations and electrocardiogram (ECG) characteristics of Chinese long QT syndrome (LQTS) patients and describe the phenotype-genotype correlation, the subjects from 5 congenital LQTS families underwent clinical detailed examination including resting body surface ECG. QT interval and transmural dispersion of repolarization (TDR) were manually measured. Five families were genotyped by linkage analysis (polymerase chain reacting-short tandem repeat, PCR-STR). The phenotype-genotype correlation was analyzed. Four families were LQT2, 1 family was LQT3. Twenty-eight gene carriers were (14 males and 14 females) identified from 5 families. The mean QTc and TDRc were 0.56 +/- 0.04 s (range 0.42 to 0.63) and 0.16 +/- 0.04 s (range 0.09 to 0.24) respectively. 35.7% (10/28) had normal to borderline QTc (< or = 0.460 s). There was significant difference in QTc and TDRc between the patients with symptomatic LQTS and those with asymptomatic LQTS, and there was significant difference in TDRc between the asymptomatic patients and normal people also. A history of cardiac events was present in 50% (14/28), including 9 with syncope, 2 with sudden death (SD) and occurred in the absence of beta-blocker. Three SDs occurred prior to the diagnosis of LQTS and had no ECG record. Two out of 5 SDs (40%) occurred as the first symptom. Typical LQT2 T wave pattern were found in 40% (6/15) of all affected members. The appearing-normal T wave was found in one LQT3 family. Low penetrance of QTc and symptoms resulted in diagnostic challenge. ECG patterns and repolarization parameters may be used to predict the genotype in most families. Genetic test is very important for identification of gene carriers.


Performance evaluation of 2-D adaptive prediction filters for detection of small objects in image data.
This work studies the performance of dimensional least mean square (TDLMS) adaptive filters as prewhitening filters for the detection of small objects in image data. The object of interest is assumed to have a very small spatial spread and is obscured by correlated clutter of much larger spatial extent. The correlated clutter is predicted and subtracted from the input signal, leaving components of the spatially small signal in the residual output. The receiver operating characteristics of a detection system augmented by a TDLMS prewhitening filter are plotted using Monte-Carlo techniques. It is shown that such a detector has better operating characteristics than a conventional matched filter in the presence of correlated clutter. For very low signal-to-background ratios, TDLMS-based detection systems show a considerable reduction in the number of false alarms. The output energy in both the residual and prediction channels of such filters is shown to be dependent on the correlation length of the various components in the input signal. False alarm reduction and detection gains obtained by using this detection scheme on thermal infrared sensor data with known object positions is presented.


Prediction of the in planta Phakopsora pachyrhizi secretome and potential effector families.
Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, can cause losses greater than 80%. Despite its economic importance, there is no soybean cultivar with durable ASR resistance. In addition, the P. pachyrhizi genome is not yet available. However, the availability of other rust genomes, as well as the development of sample enrichment strategies and bioinformatics tools, has improved our knowledge of the ASR secretome and its potential effectors. In this context, we used a combination of laser capture microdissection (LCM), RNAseq and a bioinformatics pipeline to identify a total of 36 350 P. pachyrhizi contigs expressed in planta and a predicted secretome of 851 proteins. Some of the predicted secreted proteins had characteristics of candidate effectors: small size, cysteine rich, do not contain PFAM domains (except those associated with pathogenicity) and strongly expressed in planta. A comparative analysis of the predicted secreted proteins present in Pucciniales species identified new members of soybean rust and new Pucciniales- or P. pachyrhizi-specific families (tribes). Members of some families were strongly up-regulated during early infection, starting with initial infection through haustorium formation. Effector candidates selected from two of these families were able to suppress immunity in transient assays, and were localized in the plant cytoplasm and nuclei. These experiments support our bioinformatics predictions and show that these families contain members that have functions consistent with P. pachyrhizi effectors.


Immunohistochemical expression of Bcl-2 and Ki-67 in oral lichen planus and leukoplakia with different degrees of dysplasia.
Oral lichen planus (OLP) is a chronic inflammatory disease of unknown cause. Malignant transformation in OLP lesions may be favored by changes in the expression of proteins that regulate cell proliferation and apoptosis. This study aimed to investigate these issues by immunohistochemical staining for Bcl-2 and Ki-67 and by correlating histopathological findings in samples from lesions of OLP and leukoplakia with epithelial dysplasia. Data for patients with OLP or leukoplakia with moderate or severe epithelial dysplasia recorded during 2006-2011 were retrospectively reviewed. The study samples represented 37 subjects with OLP (n = 14), leukoplakia with moderate (n = 8) or severe (n = 6) epithelial dysplasia, and normal buccal mucosa (controls, n = 9). New sections were subjected to histological examination and immunohistochemistry for Bcl-2 and Ki-67 in the basal layer, suprabasal layer, and inflammatory infiltrate, respectively. All basal layer sections stained either negative or positive in <10% of cells for Bcl-2 in OLP (92.9% and 7.1%, respectively) and control (77.8% and 22.2%, respectively) samples. In leukoplakia, 85.7% of sections indicated positivity in <10% of cells, and 14.3% indicated positivity in 10-26% of cells. Most OLP (42.9%) and leukoplakia (64.3%) sections stained positive for Ki-67 in >50% of cells. All suprabasal sections stained either negative or positive in <10% of cells for Bcl-2 in OLP (92.9% and 7.1%, respectively), leukoplakia (42.9% and 57.1%, respectively), and control (88.9% and 11.1%, respectively) samples. Suprabasal staining for Ki-67 was negative or positive in <10% of cells in OLP (14.3% and 85.7%, respectively), leukoplakia (7.1% and 92.9%, respectively), and controls (88.9% and 11.1%, respectively). Staining for Bcl-2 in inflammatory infiltrate in OLP was positive in 92.9% of sections. Expression of Bcl-2 may play a dual role in tumor development and progression. Increased cell proliferation in the epithelium may present a predisposition to cancer in OLP. The expression of Ki-67 can be considered as an adjunct marker for proliferative activity in lesions with malignant potential. The prognostic value of these immunomarkers in the evaluation of precancerous oral lesions requires further investigation.


Attenuating RNA Viruses with Expanded Genetic Codes to Evoke Adjustable Immune Response in PylRS-tRNACUAPyl Transgenic Mice.
Ribonucleic acid (RNA) viruses pose heavy burdens on public-health systems. Synthetic biology holds great potential for artificially controlling their replication, a strategy that could be used to attenuate infectious viruses but is still in the exploratory stage. Herein, we used the genetic-code expansion technique to convert Enterovirus 71 (EV71), a prototypical RNA virus, into a controllable EV71 strain carrying the unnatural amino acid (UAA) Nε-2-azidoethyloxycarbonyl-L-lysine (NAEK), which we termed an EV71-NAEK virus. After NAEK supplementation, EV71-NAEK could recapitulate an authentic NAEK time- and dose-dependent infection in vitro, which could serve as a novel method to manipulate virulent viruses in conventional laboratories. We further validated the prophylactic effect of EV71-NAEK in two mouse models. In susceptible parent mice, vaccination with EV71-NAEK elicited a strong immune response and protected their neonatal offspring from lethal challenges similar to that of commercial vaccines. Meanwhile, in transgenic mice harboring a PylRS-tRNACUAPyl pair, substantial elements of genetic-code expansion technology, EV71-NAEK evoked an adjustable neutralizing-antibody response in a strictly external NAEK dose-dependent manner. These findings suggested that EV71-NAEK could be the basis of a feasible immunization program for populations with different levels of immunity. Moreover, we expanded the strategy to generate controllable coxsackieviruses for conceptual verification. In combination, these results could underlie a competent strategy for attenuating viruses and priming the immune system via artificial control, which might be a promising direction for the development of amenable vaccine candidates and be broadly applied to other RNA viruses.


[Hormonal control of liver gluconeogenesis].
The stimulation of gluconeogenesis by glucagon results from a concerted mechanism involving: 1) the stimulation of pyruvate transport and carboxylation in mitochondria; 2) the cyclic AMP dependent phosphorylation and inactivation of pyruvate kinase resulting in a re-routing of phosphoenolpyruvate towards glucose; 3) the inhibition of phosphofructokinase and the stimulation of fructose bisphosphatase resulting from the disappearance of fructose-2,6-bisphosphate. Catecholamines and vasopressin stimulate gluconeogenesis in starvation whereas in the fed state they promote glycogenolysis together with glycolysis.


Difference density quality (DDQ): a method to assess the global and local correctness of macromolecular crystal structures.
Methods for the evaluation of the accuracy of crystal structures of proteins and nucleic acids are of general importance for structure-function studies as well as for biotechnological and biomedical research based upon three-dimensional structures of biomacromolecules. The structure-validation program DDQ (difference-density quality) has been developed to complement existing validation procedures. The DDQ method is based on the information present in a difference electron-density map calculated with the water molecules deliberately omitted from the structure-factor calculation. The quality of a crystal structure is reflected in this difference map by (i) the height of solvent peaks occurring at physical chemically reasonable positions with respect to protein and ligand atoms and (ii) the number and height of positive and negative 'shift' peaks next to protein atoms. The higher the solvent peaks and the lower the shift peaks, the better the structure is likely to be. Moreover, extraneous positive density due to an incomplete molecular model is also monitored, since this is another indicator of imperfections in the structure. Automated analysis of these types of features in difference electron densities is used to quantify the local as well as global accuracy of a structure. In the case of proteins, the DDQ structure-validation method is found to be very sensitive to small local errors, to omitted atoms and also to global errors in crystal structure determinations.


The future of antiviral immunotoxins.
There is a constant need for new therapeutic interventions in a wide range of infectious diseases. Over the past few years, the immunotoxins have entered the stage as promising antiviral treatments. Immunotoxins have been extensively explored in cancer treatment and have achieved FDA approval in several cases. Indeed, the design of new anticancer immunotoxins is a rapidly developing field. However, at present, several immunotoxins have been developed targeting a variety of different viruses with high specificity and efficacy. Rather than blocking a viral or cellular pathway needed for virus replication and dissemination, immunotoxins exert their effect by killing and eradicating the pool of infected cells. By targeting a virus-encoded target molecule, it is possible to obtain superior selectivity and drastically limit the side effects, which is an immunotoxin-related challenge that has hindered the success of immunotoxins in cancer treatment. Therefore, it seems beneficial to use immunotoxins for the treatment of virus infections. One recent example showed that targeting of virus-encoded 7 transmembrane (7TM) receptors by immunotoxins could be a future strategy for designing ultraspecific antiviral treatment, ensuring efficient internalization and hence efficient eradication of the pool of infected cells, both in vitro and in vivo. In this review, we provide an overview of the mechanisms of action of immunotoxins and highlight the advantages of immunotoxins as future anti-viral therapies.


Penicillinase plasmid DNA from Staphylococcus aureus.
A penicillinase plasmid from Staphylococcus aureus and three of its derivatives, all previously identified as extrachromosomal genetic elements, have been isolated in high yield as circular duplex DNA molecules. The wild-type plasmid was found by contour-length measurements of electron micrographs to have a molecular weight of 18.6 x 10(6) daltons. Two plasmids with deletions encompassing six and eight of the eleven known plasmid cistrons had molecular weights of 16.4 x 10(6) and 15.3 x 10(6) daltons, respectively. This information was used to establish approximate physical distances for the genetic map. A high-frequency transducing element also derived from the plasmid had a molecular weight of approximately 24 x 10(6) daltons. Although each plasmid preparation appeared homogeneous by ultracentrifugal analysis, electron micrographs always revealed the presence of a low percentage of complex oligomeric forms, particularly circular and catenated dimers.


Benchmarking risk-adjusted complications and mortality: a case study on outcomes assessment.
This article illustrates a process by which hospitals can assess their outcomes performance on rate-related complications and mortality using readily available administrative data. This approach allows hospitals to demonstrate the value of their clinical services to managed care organizations and further investigate services that may be experiencing adverse outcomes. The analysis compares outcome performance to national norms and benchmarks, as well as to local competitors.


Microbialites and global environmental change across the Permian-Triassic boundary: a synthesis.
Permian-Triassic boundary microbialites (PTBMs) are thin (0.05-15 m) carbonates formed after the end-Permian mass extinction. They comprise Renalcis-group calcimicrobes, microbially mediated micrite, presumed inorganic micrite, calcite cement (some may be microbially influenced) and shelly faunas. PTBMs are abundant in low-latitude shallow-marine carbonate shelves in central Tethyan continents but are rare in higher latitudes, likely inhibited by clastic supply on Pangaea margins. PTBMs occupied broadly similar environments to Late Permian reefs in Tethys, but extended into deeper waters. Late Permian reefs are also rich in microbes (and cements), so post-extinction seawater carbonate saturation was likely similar to the Late Permian. However, PTBMs lack widespread abundant inorganic carbonate cement fans, so a previous interpretation that anoxic bicarbonate-rich water upwelled to rapidly increase carbonate saturation of shallow seawater, post-extinction, is problematic. Preliminary pyrite framboid evidence shows anoxia in PTBM facies, but interbedded shelly faunas indicate oxygenated water, perhaps there was short-term pulsing of normally saturated anoxic water from the oxygen-minimum zone to surface waters. In Tethys, PTBMs show geographic variations: (i) in south China, PTBMs are mostly thrombolites in open shelf settings, largely recrystallised, with remnant structure of Renalcis-group calcimicrobes; (ii) in south Turkey, in shallow waters, stromatolites and thrombolites, lacking calcimicrobes, are interbedded, likely depth-controlled; and (iii) in the Middle East, especially Iran, stromatolites and thrombolites (calcimicrobes uncommon) occur in different sites on open shelves, where controls are unclear. Thus, PTBMs were under more complex control than previously portrayed, with local facies control playing a significant role in their structure and composition.


Cerebellar connections to the dorsomedial and posterior nuclei of the hypothalamus in the rat.
The stimulation or ablation of cerebellar structures has produced a variety of visceral responses, indicating a cerebellar role in visceral functions. Studies using anterograde and retrograde tracing methods have revealed connections between the hypothalamus and cerebellar structures. The aim of this study is to investigate the cerebellar connections of the dorsomedial (DMH) and posterior hypothalamic nuclei using retrograde axonal transport of horseradish peroxidase (HRP). In the present study, micro-injection of HRP restricted within the borders of the DMH showed that the projections of this nucleus are not uniform throughout its extent. The posterior DMH receives projections from the cerebellum, whereas the anterior DMH does not. These projections were from the (greatest to least concentration) lateral (dentate), anterior interposed (emboliform), and medial (fastigial) cerebellar nuclei. In addition, both the anterior and posterior DMH receive projections from various areas of the brainstem which confirms earlier studies and provides detailed descriptions. This study also demonstrates the distribution of labelled neurons to cerebellar and brainstem nuclei following HRP injection into the posterior hypothalamic nucleus. It provides clear evidence for a direct cerebellar nuclei-posterior DMH and cerebellar nuclei-posterior hypothalamic nucleus connections. We suggest that the brainstem reticular nuclei and other connections, such as the solitary, trigeminal and vestibular nuclei, of both DMH and posterior hypothalamus may contribute to the indirect cerebellohypothalamic connections. These observations offer a new perspective on the question of how the cerebellum may influence autonomic activity.


Role of the galactose lectin of Entamoeba histolytica in adherence-dependent killing of mammalian cells.
Entamoeba histolytica extracellular killing of host cells is contact dependent. Adherence to human colonic epithelial cells and mucins is mediated by a galactose-specific lectin. The effect on cytotoxicity of a panel of monoclonal antibodies (MAb) directed against the galactose lectin was tested. As expected, those MAb which inhibited adherence also decreased cytotoxicity. However, one antilectin MAb blocked cytotoxicity after adherence had occurred, indicating that the lectin has a role in cell killing that is distinct from its adherence function.


Altered Plant and Nodule Development and Protein S-Nitrosylation in Lotus japonicus Mutants Deficient in S-Nitrosoglutathione Reductases.
Nitric oxide (NO) is a crucial signaling molecule that conveys its bioactivity mainly through protein S-nitrosylation. This is a reversible post-translational modification (PTM) that may affect protein function. S-nitrosoglutathione (GSNO) is a cellular NO reservoir and NO donor in protein S-nitrosylation. The enzyme S-nitrosoglutathione reductase (GSNOR) degrades GSNO, thereby regulating indirectly signaling cascades associated with this PTM. Here, the two GSNORs of the legume Lotus japonicus, LjGSNOR1 and LjGSNOR2, have been functionally characterized. The LjGSNOR1 gene is very active in leaves and roots, whereas LjGSNOR2 is highly expressed in nodules. The enzyme activities are regulated in vitro by redox-based PTMs. Reducing conditions and hydrogen sulfide-mediated cysteine persulfidation induced both activities, whereas cysteine oxidation or glutathionylation inhibited them. Ljgsnor1 knockout mutants contained higher levels of S-nitrosothiols. Affinity chromatography and subsequent shotgun proteomics allowed us to identify 19 proteins that are differentially S-nitrosylated in the mutant and the wild-type. These include proteins involved in biotic stress, protein degradation, antioxidant protection and photosynthesis. We propose that, in the mutant plants, deregulated protein S-nitrosylation contributes to developmental alterations, such as growth inhibition, impaired nodulation and delayed flowering and fruiting. Our results highlight the importance of GSNOR function in legume biology.


Examining the effects of turnover intentions on organizational citizenship behaviors and deviance behaviors: A psychological contract approach.
Although turnover intentions are considered the most proximal antecedent of organizational exit, there is often temporal separation between thinking about leaving and actual exit. Using field data from 2 diverse samples of working adults, we explore a causal model of the effects of turnover intentions on employee behavior while they remain with the organization, focusing specifically on organizational citizenship behaviors (OCBs) and deviance behaviors (DBs). Utilizing expectancy theory as an explanatory framework, we argue that turnover intentions result in high levels of transactional contract orientation and low levels of relational contract orientation, which in turn lead to a decrease in the incidence of OCBs and an increase in the incidence of DBs. We first used a pilot study to investigate the direction of causality between turnover intentions and psychological contract orientations. Then, in Study 1, we tested our mediated model using a sample of employees from a large drug retailing chain. In Study 2, we expanded our model by arguing that the mediated effects are much stronger when the organization is deemed responsible for potential exit. We then tested our full model using a sample of employees from a large state-owned telecommunications corporation in China. Across both studies, results were generally consistent and supportive of our hypotheses. We discuss the implications of our findings for future theory, research, and practice regarding the management of both the turnover process and discretionary behaviors at work. (PsycINFO Database Record


A simplified cloning strategy for the generation of an endothelial cell selective recombinant adenovirus vector.
Specifically targeting adenoviral vectors to particular cell/tissue types can be achieved by genetically modifying the adenovirus fiber protein. Two common strategies are: (1) directly modifying the fiber gene in the adenovirus genome and (2) in trans supply of the modified fiber. The former however, suffers from difficulties in directly manipulating large adenoviral genomic DNA. Although the latter allows easy manipulation of the small fiber gene, our studies show that the in trans supplement of the modified fiber causes incomplete fiber assimilation in the virus. Thus an alternate cloning strategy was devised to facilitate the insertion of cell-targeting sequences into the HI loop of a CAR binding-ablated fiber gene in the Ad5 genomic backbone. Our approach retains the advantage of easily modifying the fiber with the additional benefit of genetic re-insertion into the Ad genomic backbone to ensure complete modified fiber incorporation. Using this strategy, an endothelial cell binding peptide sequence (Asn-Gly-Arg) was introduced into the Ad fiber and showed that the generated Ad vector displayed selective transduction of endothelial cells both in vitro and in vivo compared to the conventional vector. Furthermore, this Ad vector cloning strategy can be adapted to introduce other peptide sequences to target other cell types.


Transcriptional repressor HipB regulates the multiple promoters in Escherichia coli.
HipB is a DNA-binding protein in Escherichia coli and negatively regulates its own promoter by binding to the palindromic sequences [TATCCN8GGATA (N represents any nucleotides)] on the hipBA promoter. For such sequences, bioinformatic analysis revealed that there are a total of 39 palindromic sequences (TATCCN(x)GGATA: N is any nucleotides and x is the number of nucleotides from 1 to 30) in the promoter regions of 33 genes on the E. coli genome. Notably, eutH and fadH have two and three TATCCN(x)GGATA palindromic sequences located in their promoters, respectively. Another significant finding was that a palindromic sequence was also identified in the promoter region of hipAB locus, known to be involved in the RelA-dependent persister cell formation in bacteria. Here, we demonstrated that HipB binds to the palindromic structures in the eutH, fadH, as well as the relA promoter regions and represses their expressions. We further demonstrated that HipA enhances the repression of the relA promoter activity by HipB. This effect was not observed with D291A HipA mutant which was previously shown to lack an ability to interact with HipB, indicating that HipA enhances the HipB's repressor activity through direct interaction with HipB.


Helicobacter pylori DprA alleviates restriction barrier for incoming DNA.
Helicobacter pylori is a Gram-negative bacterium that colonizes human stomach and causes gastric inflammation. The species is naturally competent and displays remarkable diversity. The presence of a large number of restriction-modification (R-M) systems in this bacterium creates a barrier against natural transformation by foreign DNA. Yet, mechanisms that protect incoming double-stranded DNA (dsDNA) from restriction enzymes are not well understood. A DNA-binding protein, DNA Processing Protein A (DprA) has been shown to facilitate natural transformation of several Gram-positive and Gram-negative bacteria by protecting incoming single-stranded DNA (ssDNA) and promoting RecA loading on it. However, in this study, we report that H. pylori DprA (HpDprA) binds not only ssDNA but also dsDNA thereby conferring protection to both from various exonucleases and Type II restriction enzymes. Here, we observed a stimulatory role of HpDprA in DNA methylation through physical interaction with methyltransferases. Thus, HpDprA displayed dual functional interaction with H. pylori R-M systems by not only inhibiting the restriction enzymes but also stimulating methyltransferases. These results indicate that HpDprA could be one of the factors that modulate the R-M barrier during inter-strain natural transformation in H. pylori.


Differential diagnosis of retinal vasculitis.
Retinal vaculitis is a sight-threatening inflammatory eye condition that involves the retinal vessels. Detection of retinal vasculitis is made clinically, and confirmed with the help of fundus fluorescein angiography. Active vascular disease is characterized by exudates around retinal vessels resulting in white sheathing or cuffing of the affected vessels. In this review, a practical approach to the diagnosis of retinal vasculitis is discussed based on ophthalmoscopic and fundus fluorescein angiographic findings.


Systems analysis of seed filling in Arabidopsis: using general linear modeling to assess concordance of transcript and protein expression.
Previous systems analyses in plants have focused on a single developmental stage or time point, although it is often important to additionally consider time-index changes. During seed development a cascade of events occurs within a relatively brief time scale. We have collected protein and transcript expression data from five sequential stages of Arabidopsis (Arabidopsis thaliana) seed development encompassing the period of reserve polymer accumulation. Protein expression profiling employed two-dimensional gel electrophoresis coupled with tandem mass spectrometry, while transcript profiling used oligonucleotide microarrays. Analyses in biological triplicate yielded robust expression information for 523 proteins and 22,746 genes across the five developmental stages, and established 319 protein/transcript pairs for subsequent pattern analysis. General linear modeling was used to evaluate the protein/transcript expression patterns. Overall, application of this statistical assessment technique showed concurrence for a slight majority (56%) of expression pairs. Many specific examples of discordant protein/transcript expression patterns were detected, suggesting that this approach will be useful in revealing examples of posttranscriptional regulation.


Dark accumulation of downstream glycolytic intermediates initiates robust photosynthesis in cyanobacteria.
Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin-Benson-Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies have shown that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid dynamic changes (on a time scale of seconds) in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enabled estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicated that downstream glycolytic intermediates, including phosphoglycerate and phosphoenolpyruvate, accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant decreases in the initial oxygen evolution rate were observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathways. Accordingly, the degree of decrease in the initial oxygen evolution rate was proportional to the accumulated pool size of glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.


An overview of mobile applications (apps) to support the coronavirus disease 2019 response in India.
The potential benefits of mobile health (mHealth) initiatives to manage the coronavirus disease 2019 (COVID-19) pandemic have been explored. The Government of India, State governments, and healthcare organizations have developed various mobile apps for the containment of COVID-19. This study was aimed to systematically review COVID-19 related mobile apps and highlight gaps to inform the development of future mHealth initiatives. Google Play and the Apple app stores were searched using the terms 'COVID-19', 'coronavirus', 'pandemic', and 'epidemic' in the first week of April 2020. A list of COVID-19-specific functions was compiled based on the review of the selected apps, the literature on epidemic surveillance, and national and international media reports. The World Health Organization guideline on Digital Health Interventions was used to classify the app functions under the categories of the general public, health workers, health system managers, and data services. The search yielded 346 potential COVID-19 apps, of which 50 met the inclusion criteria. Dissemination of untargeted COVID-19-related information on preventative strategies and monitoring the movements of quarantined individuals was the function of 27 (54%) and 19 (32%) apps, respectively. Eight (16%) apps had a contact tracing and hotspot identification function. Our study highlights the current emphasis on the development of self-testing, quarantine monitoring, and contact tracing apps. India's response to COVID-19 can be strengthened by developing comprehensive mHealth solutions for frontline healthcare workers, rapid response teams and public health authorities. Among this unprecedented global health emergency, the Governments must ensure the necessary but least intrusive measures for disease surveillance.


Synthetic mucus barrier arrays as a nanoparticle formulation screening platform.
A mucus gel layer lines the luminal surface of tissues throughout the body to protect them from infectious agents and particulates. As a result, nanoparticle drug delivery systems delivered to these sites may become trapped in mucus and subsequently cleared before they can reach target cells. As such, optimizing the properties of nanoparticle delivery vehicles, such as their surface chemistry and size, is essential to improving their penetration through the mucus barrier. In previous work, we developed a mucin-based hydrogel that has viscoelastic properties like that of native mucus which can be further tailored to mimic specific mucosal tissues and disease states. Using this biomimetic hydrogel system, a 3D-printed array containing synthetic mucus barriers was created that is compatible with a 96-well plate enabling its use as a high-throughput screening platform for nanoparticle drug delivery applications. To validate this system, we evaluated several established design parameters to determine their impact on nanoparticle penetration through synthetic mucus barriers. Consistent with the literature, we found nanoparticles of smaller size and coated with a protective PEG layer more efficiently penetrated through synthetic mucus barriers. In addition, we evaluated a mucolytic (tris (2-carboxyethyl) phosphine, TCEP) for use as a permeation enhancer for mucosal drug delivery. In comparison to N-acetyl cysteine (NAC), we found TCEP significantly improved nanoparticle penetration through a disease-like synthetic mucus barrier. Overall, our results establish a new high-throughput screening approach using synthetic mucus barrier arrays to identify promising nanoparticle formulation strategies for drug delivery to mucosal tissues.


Evaluation of the Completeness of ALS Case Ascertainment in the US National ALS Registry: Application of the Capture-Recapture Method.
The Centers for Disease Control and Prevention (CDC) National Amyotrophic Lateral Sclerosis (ALS) Registry is the first national registry for a chronic neurologic disease in the USA and uses a combination of case-finding methods including administrative healthcare data and patient self-registration. We applied capture-recapture methodology to estimate the completeness of the Registry for ascertaining patients with ALS for the first full year and the fourth year of the Registry (2011, 2014). The Registry uses the combination of two national administrative claims databases (Medicare and Veterans Affairs) with a self-register option at the registry portal. We conducted descriptive analyses of the demographic and clinical characteristics of the ALS cases identified by each of the sources and estimated the completeness of case ascertainment for each of the three ALS Registry sources individually, pairwise, and in all combinations. Case-finding completeness was 54% in 2011 and improved to 56% in 2014. A smaller proportion of ALS patients under age 65 were ascertained than those 65 or older, and ascertainment was also lower for nonwhite than white patients. The uncorrected ALS prevalence was 4.3/100,000 in 2011 (in 2014, 5.0/100,000), but after correction for underascertainment, annual prevalence in 2011 was 7.9/100,000 (95% CI: 7.6-8.2) (in 2014 was 8.9/100,000 [95% CI: 8.7-9.2]). Our findings indicate that administrative healthcare databases are a very efficient method for identifying the majority of ALS prevalent cases in the National ALS Registry and that the inclusion of a web registry portal for patients to self-register is important to ensure a more representative population for estimating ALS prevalence. Nonetheless, more than 40% of ALS cases were not ascertained by the Registry, with individuals younger than age 65 and people of color underrepresented. Recommendations are provided for additional methods that can be considered to improve the completeness of case ascertainment.


Long-term outcomes of gastrografin in small bowel obstruction.
The gastrografin (GG) challenge is a diagnostic and therapeutic tool used to treat patients with small bowel obstruction (SBO); however, long-term data on SBO recurrence after the GG challenge remain limited. We hypothesized that patients treated with GG would have the same long-term recurrence as those treated before the implementation of the GG challenge protocol. Patients ≥18 years who were treated for SBO between July 2009 and December 2012 were identified. We excluded patients with contraindications to the GG challenge (i.e., signs of strangulation), patients having SBO within 6-wk of previous abdominal or pelvic surgery and patients with malignant SBO. All patients had been followed a minimum of 1 y or until death. Kaplan-Meier method and Cox regression models were used to describe the time-dependent outcomes. A total of 202 patients were identified of whom 114 (56%) received the challenge. Mean patients age was 66 y (range, 19-99 y) with 110 being female (54%). A total of 184 patients (91%) were followed minimum of 1 year or death (18 patients lost to follow-up). Median follow-up of living patients was 3 y (range, 1-5 y). During follow-up, 50 patients (25%) experienced SBO recurrences, and 24 (12%) had exploration for SBO recurrence. The 3-year cumulative rate of SBO recurrence in patients who received the GG was 30% (95% confidence interval [CI], 21%-42%) compared to 27% (95% CI, 18%-38%) for those who did not (P = 0.4). The 3-year cumulative rate of exploration for SBO recurrence in patients who received the GG was 15% (95% CI, 8%-26%) compared to 12 % (95% CI, 6%-22%) for those who did not (P = 0.6). The GG challenge is a clinically useful tool in treating SBO patients with comparable long-term recurrence rates compared to traditional management of SBO.


Leptin and pubertal development.
Sexual development after birth in rodents, nonhuman primates, and humans is driven by the gonadotropin-releasing hormone (GnRH) pulse generator. During the neonatal period in primates, pulsatile GnRH discharge from the medial basal hypothalamus drives an active period of pituitary gonadotropin and gonadal hormone secretion. During the transition from the neonatal to the juvenile period, however, the activity of the GnRH pulse generator is restrained or arrested and gonadotropin and gonadal hormone secretion enters a quiescent period that continues until the onset of puberty. As puberty approaches the GnRH pulse generator is reactivated, resulting in enhanced gonadotropin secretion, accelerated growth, maturation of the gonads, and the achievement of sexual competence. Rodents do not appear to exhibit a developmental phase analogous to the quiescent juvenile period in primates when the GnRH pulse generator is held in check. Instead, progressive maturational changes in the pattern of GnRH pulsatility appear to drive sexual development in rodents. The role that leptin plays in sexual development has not been fully defined, but the balance of current evidence appears to support the idea that, in both rodents and primates, leptin plays a permissive rather than a causal role in timing this process. When body energy reserves rise above a critical level, blood leptin increases to a threshold concentration signaling to the central nervous system that the body can support sexual function. Puberty can apparently occur over a wide range of concentrations above this critical leptin threshold. Leptin does not appear to act as a trigger to time the initiation of puberty but, instead, once leptin reaches this threshold pubertal development may proceed if, and only if, other critical control mechanisms are operational.


Human NK cell-mediated cytotoxicity triggered by CD86 and Gal alpha 1,3-Gal is inhibited in genetically modified porcine cells.
Delayed xenograft rejection is a major hurdle that needs to be addressed to prolong graft survival in pig-to-primate xenotransplantation. NK cell activation has been implicated in delayed xenograft rejection. Both Ab-dependent and independent mechanisms are responsible for the high susceptibility of porcine cells to human NK cell-mediated cytotoxicity. Previous reports demonstrated a role of Galalpha1,3-Gal Ag in triggering the Ab-independent responses. We hypothesize that expression of CD80 and/or CD86 on porcine cells may also play a role in NK cell activation as human NK cells express a variant of CD28. Our initial analysis showed that porcine endothelial cells and fibroblasts express CD86, but not CD80. Genetic engineering of these cells to express hCD152-hCD59, a chimeric molecule designed to block CD86 in cis, was accompanied by a reduction in susceptibility to human NK cell-mediated cytotoxicity. The use of a specific anti-porcine CD86-blocking Ab and the NK92 and YTS cell lines further confirmed the involvement of CD86 in triggering NK cell-mediated lysis of porcine cells. Maximal protection was achieved when hCD152-hCD59 was expressed in H transferase-transgenic cells, which show reduced Galalpha1,3-Gal expression. In this work, we describe two mechanisms of human NK cell-mediated rejection of porcine cells and demonstrate that genetically modified cells resist Ab-independent NK cell-mediated cytotoxicity.


An electron microscopic study of Babesia microti invading erythrocytes.
Intracellular sporozoan parasites invade the host cell through the invagination of the plasma membrane of the host and a vacuole is formed which accommodates the entering parasite. The vacuole may disappear and the invaginated membrane of the host then becomes closely apposed to that of the parasite's own membrane. As a result the parasite is covered by two membranes. Members of the class Piroplasmea differ from other Sporozoa in that their trophozoites are covered by a single membrane. By screening numerous sections of intraerythrocytic Babesia microti belonging to the class Piroplasmea, it was found that merozoites of Babesia enter the erythrocytes of hamsters in the same way as those of the other Sporozoa. When a merozoite touches the red blood cell with its anterior end it becomes attached to the membrane of the host, which starts to invaginate and a parasitophorous vacuole is formed. The vacuolar space disappears rapidly and the membrane of the vacuole and that of the parasite become closely adjacent. At this stage the parasite is surrounded by two plasma membranes. The outer membrane derived from the invaginated host membrane disintegrates quickly and the parasite is left with a single membrane throughout its life span.


A membrane is born: origin of the autophagosomal compartment.
Autophagy is one of the major catabolic processes present in eukaryotic cells, conserved through evolution, by which damaged or superfluous organelles are degraded in response to different stimuli. A hallmark of the autophagic pathway is the formation of double or multiple layered membranes that engulf the material to be finally degraded in the lysosomes. Despite enormous advances in the last few years to understand the autophagic process at the molecular level, the origin of the sequestering membrane has remained elusive for more than forty years and it is still a matter of debate. In this review we have summarized recent experimental evidence indicating that more than one membrane source may exist. Even though de novo formation or assembly of the isolation membrane has been proposed, recent data points to the participation of specific organelles in the biogenesis of the sequestering membrane.


Evaluation of the use of transbronchial biopsy in patients with clinical suspicion of interstitial lung disease.
To study the clinical, radiological, and histopathological patterns of transbronchial biopsy (TBB) used in order to confirm the diagnosis in patients with clinical suspicion of interstitial lung disease (ILD) treated at a tertiary-care university hospital. We reviewed the medical records, radiology reports, and reports of transbronchial biopsies from all patients with suspected ILD who underwent TBB between January of 1999 and December of 2006 at the Hospital das Clínicas de Botucatu, located in the city of Botucatu, Brazil. The study included 56 patients. Of those, 11 (19.6%) had a definitive diagnosis of idiopathic pulmonary fibrosis (IPF), the rate of which was significantly higher in the patients in which ILD was a possible diagnosis in comparison with those in which ILD was the prime suspect (p = 0.011), demonstrating the contribution of TBB to the diagnostic confirmation of these diseases. The histopathological examination of the biopsies revealed that 27.3% of the patients with IPF showed a pattern of organizing pneumonia, which suggests greater disease severity. The most common histological pattern was the indeterminate pattern, reflecting the peripheral characteristic of IPF. However, the fibrosis pattern showed high specificity and high negative predictive value. For CT scan patterns suggestive of IPF, the ROC curve showed that the best relationship between sensitivity and specificity occurred when five radiological alterations were present. Honeycombing was found to be strongly suggestive of IPF (p = 0.01). For ILDs, chest CT should always be performed, and TBB should be used in specific situations, according to the suspicion and distribution of lesions.


Burden of non-communicable disease studies in Europe: a systematic review of data sources and methodological choices.
Assessment of disability-adjusted life years (DALYs) resulting from non-communicable diseases (NCDs) requires specific calculation methods and input data. The aims of this study were to (i) identify existing NCD burden of disease (BoD) activities in Europe; (ii) collate information on data sources for mortality and morbidity; and (iii) provide an overview of NCD-specific methods for calculating NCD DALYs. NCD BoD studies were systematically searched in international electronic literature databases and in grey literature. We included all BoD studies that used the DALY metric to quantify the health impact of one or more NCDs in countries belonging to the European Region. A total of 163 BoD studies were retained: 96 (59%) were single-country or sub-national studies and 67 (41%) considered more than one country. Of the single-country studies, 29 (30%) consisted of secondary analyses using existing Global Burden of Disease (GBD) results. Mortality data were mainly derived (49%) from vital statistics. Morbidity data were frequently (40%) drawn from routine administrative and survey datasets, including disease registries and hospital discharge databases. The majority (60%) of national BoD studies reported mortality corrections. Multimorbidity adjustments were performed in 18% of national BoD studies. The number of national NCD BoD assessments across Europe increased over time, driven by an increase in BoD studies that consisted of secondary data analysis of GBD study findings. Ambiguity in reporting the use of NCD-specific BoD methods underlines the need for reporting guidelines of BoD studies to enhance the transparency of NCD BoD estimates across Europe.


Comparative activity of telithromycin against macrolide-resistant isolates of Streptococcus pneumoniae: results of two years of the PROTEKT surveillance study.
The increase in resistance to macrolides has been linked with increasing use of these agents as empirical therapy for community-acquired respiratory tract infections (CARTIs). As part of the ongoing PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) surveillance program, over 7600 Streptococcus pneumoniae isolates were collected worldwide from 1999-2001 and evaluated for macrolide resistance. Globally, macrolide resistance was 31-33%, with considerable inter-country variation (<10-88%) and particularly high prevalence in the Far East (>71%). In Europe, France had the most resistant isolates (>53%). The highest rates of macrolide resistance were seen in 0-2 year olds. Co-resistance to clindamycin (64%) and all beta-lactams (14-79%) was seen among macrolide-resistant isolates, but >99% of these isolates were susceptible to telithromycin, vancomycin and linezolid. There was considerable variation in the prevalence of erm(B) (56-64%) and mef(A) (30-35%): erm(B) was prevalent in Europe and mef(A) in North America. Globally, 5-7% isolates carried both mechanisms (erm(B)+mef(A)); of these, 47-65% were from South Korea. These double resistance isolates were >90% resistant to the beta-lactams, except amoxicillin-clavulanate. Clindamycin was active against >98% mef(A) but poorly active against erm(B) and erm(B)+mef(A) isolates. Telithromycin, vancomycin and linezolid were highly active (>99.5%) across all three genotypes. In vitro, telithromycin, vancomycin and linezolid are highly active against antibiotic-resistant strains of S. pneumoniae. Telithromycin may be a useful therapeutic alternative to macrolides for the treatment of CARTIs.


Commercially available pedometers: considerations for accurate step counting.
Many commercially available pedometers undercount, especially at slower speeds. We examined the effects of age, obesity, and self-selected walking speed on pedometer accuracy. We also compared the accuracy of piezoelectric and spring-levered pedometers at slow walking speeds. Study 1: 259 subjects walked on a motorized treadmill at two self-selected walking speeds. Steps were counted using a spring-levered pedometer. Study 2: 32 subjects walked on a motorized treadmill at slow walking (1.0-2.6 MPH) speeds. Steps were counted using spring-levered and piezoelectric pedometers. Study 1: self-selected walking speed and pedometer accuracy decreased with increasing age, weight, and body mass index (BMI). Accuracy was 71% below 2.0 MPH, 74-91% between 2.0 and 3.0 MPH, and 96% above 3.0 MPH. Decreased accuracy was best predicted by increasing age. Study 2: between 1.8 and 2.0 MPH, the accuracy of the piezoelectric pedometer (>97%) exceeded that of the spring-levered pedometers (52-95%). Even at 1.0 MPH, accuracy of the piezoelectric pedometer (56.4 +/- 33.8%) was superior to the spring-levered pedometers (7-20%). Accuracy of all pedometers tested exceeded 96% at speeds 3.0 MPH, but decreased at slower walking speeds. In individuals that naturally ambulate at slower walking speeds (e.g., elderly), we recommend the use of more sensitive (e.g., piezoelectric) pedometers.


Developing digital interventions for people living with serious mental illness: perspectives from three mHealth studies.
The rapidly expanding field of mobile health (mHealth) seeks to harness increasingly affordable and ubiquitous mobile digital technologies including smartphones, tablets, apps and wearable devices to enhance clinical care. Accumulating evidence suggests that mHealth interventions are increasingly being adopted and valued by people living with serious mental illnesses such as schizophrenia and bipolar disorder, as a means of better understanding and managing their condition. We draw on experiences from three geographically and methodologically distinct mHealth studies to provide a pragmatic overview of the key challenges and considerations relating to the process of developing digital interventions for this population.


High risk of adult-onset asthma and work-related wheeze in farmers despite low prevalence of asthma in young farmers.
To investigate risk factors for asthma in farmers. A questionnaire was sent to all farms (n = 2499) and to 2900 controls aged 19-65 years from the general population in the county of Uppsala. Sixty per cent of the farms (1514 men and 248 women) and 64% of the controls (900 men, 943 women) responded. Only 13% of the male farmers had heredity for allergy compared to 24% of the controls, and fewer farmers were smokers. After adjusting for confounders, male farmers had a significantly lower prevalence of doctor-diagnosed asthma and nocturnal breathlessness than the controls (OR 0.75, 95%CI 0.57-0.98 and OR 0.61, 95%CI 0.44-0.84), but a significantly higher prevalence of work-related wheeze (OR 1.74, 95%CI 1.30-2.35). The risk for asthma increased with age in the farmers. Of male farmers with asthma, 70% had developed asthma after the age of 21, in contrast with only 30% of the asthmatic controls. The young female farmers had a high prevalence of asthma. Male farmers have an increased risk of work-related wheeze and adult-onset asthma increasing with age despite a lower prevalence of asthma during childhood and young age than in the general population.


Rehabilitation of Dentofacial Asymmetry Secondary to Unilateral Temporomandibular Joint Ankylosis with Dual Distraction and Fixed Orthodontics - Stability at Three-year Follow-up.
Optimal rehabilitation of asymmetric dentofacial deformity secondary to unilateral temporomandibular joint (TMJ) ankylosis is often a challenge. The purpose of this case series is to present an insight into esthetic, occlusal and functional rehabilitation of two patients with varying degree of asymmetric Class II dentofacial deformities secondary to long-standing unilateral TMJ ankylosis. The patients were treated with one-stage surgical protocol employing simultaneous dual distraction technique along with interpositional arthroplasty. Dual distraction technique entailed the simultaneous use of two distractors which allowed for proper control of proximal condylar segment during the course of distraction and lowering the risk of ankylosis recurrence. Thereafter, comprehensive fixed orthodontic mechanotherapy involving the use of temporary anchorage devices was instituted to align and level the compensated dentition. Post-treatment records showed significant improvements in skeletal disharmony and functional stability with good functional occlusion. At the three-year follow-up, the morphological and functionally acceptable results were reasonably well-maintained, with no signs of relapse. Through the two cases reported here, we would like to highlight that one-stage concurrent arthroplasty and dual distraction technique is a safe, stable, and reliable approach for surgical and functional rehabilitation of an adult asymmetric dentofacial deformity secondary to unilateral TMJ ankylosis. Meticulously executed comprehensive orthodontic manipulations involving use of acrylic bite-blocks, elastic traction, and temporary skeletal anchorage device play a crucial role in enhancing the final occlusal outcomes.


Predicting Monthly Community-Level Radon Concentrations with Spatial Random Forest in the Northeastern and Midwestern United States.
In 1987, the United States Environmental Protection Agency recommended installing a mitigation system when the indoor concentration of radon, a well-known carcinogenic radioactive gas, is at or above 148 Bq/m3. In response, tens of millions of short-term radon measurements have been conducted in residential buildings over the past three decades either for disclosure or to initially evaluate the need for mitigation. These measurements, however, are currently underutilized to assess population radon exposure in epidemiological studies. Based on two relatively small radon surveys, Lawrence Berkeley National Laboratory developed a state-of-the-art national radon model. However, this model only provides coarse and invariant radon estimations, which limits the ability of epidemiological studies to accurately investigate the health effects of radon, particularly the effects of acute exposure. This study involved obtaining over 2.8 million historical short-term radon measurements from independent laboratories. With the use of these measurements, an innovative spatial random forest (SRF) model was developed based on geological, architectural, socioeconomical, and meteorological predictors. The model was used to estimate monthly community-level radon concentrations for ZIP Code Tabulation Areas (ZCTAs) in the northeastern and midwestern regions of the United States from 2001 to 2020. Via cross-validation, we found that our ZCTA-level predictions were highly correlated with observations. The prediction errors declined quickly as the number of radon measurements in a ZCTA increased. When ≥15 measurements existed, the mean absolute error was 24.6 Bq/m3, or 26.5% of the observed concentrations (R2 = 0.70). Our study demonstrates the potential of the large amount of historical short-term radon measurements that have been obtained to accurately estimate longitudinal ZCTA-level radon exposures at unprecedented levels of resolutions and accuracy.


Increase in endogenous glucose production with SGLT2 inhibition is attenuated in individuals who underwent kidney transplantation and bilateral native nephrectomy.
The glucosuria induced by sodium-glucose cotransporter 2 (SGLT2) inhibition stimulates endogenous (hepatic) glucose production (EGP), blunting the decline in HbA1c. We hypothesised that, in response to glucosuria, a renal signal is generated and stimulates EGP. To examine the effect of acute administration of SGLT2 inhibitors on EGP, we studied non-diabetic individuals who had undergone renal transplant with and without removal of native kidneys. This was a parallel, randomised, double-blind, placebo-controlled, single-centre study, designed to evaluate the effect of a single dose of dapagliflozin or placebo on EGP determined by stable-tracer technique. We recruited non-diabetic individuals who were 30-65 years old, with a BMI of 25-35 kg/m2 and stable body weight (±2 kg) over the preceding 3 months, and HbA1c <42 mmol/mol (6.0%). Participants had undergone renal transplant with and without removal of native kidneys and were on a stable dose of immunosuppressive medications. Participants received a single dose of dapagliflozin 10 mg or placebo on two separate days, at a 5- to 14-day interval, according to randomisation performed by our hospital pharmacy, which provided dapagliflozin and matching placebo, packaged in bulk bottles that were sequentially numbered. Both participants and investigators were blinded to group assignment. Twenty non-diabetic renal transplant patients (ten with residual native kidneys, ten with bilateral nephrectomy) participated in the study. Dapagliflozin induced greater glucosuria in individuals with residual native kidneys vs nephrectomised individuals (8.6 ± 1.1 vs 5.5 ± 0.5 g/6 h; p = 0.02; data not shown). During the 6 h study period, plasma glucose decreased only slightly and similarly in both groups, with no difference compared with placebo (data not shown). Following administration of placebo, there was a progressive time-related decline in EGP that was similar in both nephrectomised individuals and individuals with residual native kidneys. Following dapagliflozin administration, EGP declined in both groups, but the differences between the decrement in EGP with dapagliflozin and placebo in the group with bilateral nephrectomy (Δ = 1.11 ± 0.72 μmol min-1 kg-1) was significantly lower (p = 0.03) than in the residual native kidney group (Δ = 2.56 ± 0.33 μmol min-1 kg-1). In the population treated with dapagliflozin, urinary glucose excretion was correlated with EGP (r = 0.34, p < 0.05). Plasma insulin, C-peptide, glucagon, prehepatic insulin:glucagon ratio, lactate, alanine and pyruvate concentrations were similar following placebo and dapagliflozin treatment. β-Hydroxybutyrate increased with dapagliflozin treatment in the residual native kidney group, while a small increase was observed only at 360 min in the nephrectomy group. Plasma adrenaline (epinephrine) did not change after dapagliflozin and placebo treatment in either group. Following dapagliflozin administration, plasma noradrenaline (norepinephrine) increased slightly in the residual native kidney group and decreased in the nephrectomy group. In nephrectomised individuals, the hepatic compensatory response to acute SGLT2 inhibitor-induced glucosuria was attenuated, as compared with individuals with residual native kidneys, suggesting that SGLT2 inhibitor-mediated stimulation of hepatic glucose production via efferent renal nerves occurs in an attempt to compensate for the urinary glucose loss (i.e. a renal-hepatic axis). ClinicalTrials.gov NCT03168295 FUNDING: This protocol was supported by Qatar National Research Fund (QNRF) Award No. NPRP 8-311-3-062 and NIH grant DK024092-38. Graphical abstract.


Genetically Proxied Sarcopenia-Related Muscle Traits and Depression: Evidence from the FinnGen Cohort.
Sarcopenia and depression are common and often coexist in the elderly. This study aims to determine the impact of sarcopenia-related muscle traits on depression. A two-sample Mendelian randomization (MR) study was performed on the summary-level data from the FinnGen cohort to estimate the causal association of appendicular lean mass (ALM), walking pace, or low hand grip strength with depression. Additionally, multivariable MR (MVMR) was performed to assess the dependence of each muscle trait in the causality and adjust the effect of body mass index (BMI). Supplementary backward MR analyses were performed to estimate the effect of depression on sarcopenia-related muscle traits. Univariable MR analyses demonstrated that there were causal associations of ALM (odds ratio [OR]: 0.94; 95% confidence interval [CI]: 0.88-0.99), walking pace (OR: 0.53; 95% CI: 0.32-0.88), and low hand grip strength (OR: 1.20; 95% CI: 1.05-1.38) with depression. MVMR analyses showed that ALM was the only trait that had a significant causal relationship with depression (OR: 0.91; 95% CI: 0.85-0.98) after accounting for the other two muscle traits. Moreover, the independent association of ALM with depression remained (OR: 0.92; 95% CI: 0.85-0.99) after being adjusted by BMI. The backward MR analyses showed no causal associations of depression with any sarcopenia-related muscle traits. Low muscle mass independently increases the risk of depression. This study determined the muscle-related risk factors of depression, which may help establish the causality between sarcopenia and depression and provide evidence-based recommendations for improving mental health in the elderly.


Toward a unified framework for research on instructions and other messages: An introduction to the special issue on the power of instructions.
Instructions are known to have a profound impact on human behavior. Nevertheless, research on the effects of instructions is relatively scarce and scattered across different areas of research in psychology and neuroscience. The current issue of this journal contains six papers that review research on instructions in different research areas. In this introduction to the special issue, we provide the outline of a framework that focuses on five components that can be varied in research on this topic (sender, message, receiver, context, and outcome). The framework brings order to the boundless potential variability in research on the effects of messages (i.e., it has heuristic value) and highlights that past research explored only a tiny fraction of what is possible (i.e., it has predictive value). Moreover, it reveals that research in different areas tends to examine different instantiations of the five components. The latter observation implies that much can be gained from closer interactions between researchers from different areas.


Different versions of Pseudomonas syringae pv. tomato DC3000 exist due to the activity of an effector transposon.
SUMMARY The plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000 is a key model organism to study plant-pathogen interactions. We realized that two versions of this strain, which carry plasmids of different sizes, exist in our strain collections. The difference was located to a 9.4-kb deletion within the larger of the two endogenous plasmids encompassing the partitioning genes parA and parB and a putative mobile element encoding the type III effector hopAM1-2 (formerly avrPpiB2). Both plasmid variants are lost in similar frequency, indicating that the partitioning genes are not essential for stability of the plasmid. In addition, the deletion derivative strain DC3001 exhibited the same virulence towards Arabidopsis as strain DC3000. The deletion site in DC3001 is located immediately adjacent to a putative transposon that carries the effector hopX1 (formerly avrPphE), suggesting that the deletion originated from an aberrant transposition event of this element. By tagging the hopX1 transposon with an antibiotic resistance cassette on a suicide plasmid it was shown that the element is functional and produces a target site duplication of 5 bp. The plasmid also integrated into the chromosome in several cases, possibly mediated by one-ended transposition of the hopX1 transposon. This is the first report that describes an active effector-transposon. Comparison of DC3000 strains from several sources revealed that strains exist with differences in the endogenous plasmid composition.


Delayed identification of pediatric abuse-related fractures.
Because physicians may have difficulty distinguishing accidental fractures from those that are caused by abuse, abusive fractures may be at risk for delayed recognition; therefore, the primary objective of this study was to determine how frequently abusive fractures were missed by physicians during previous examinations. A secondary objective was to determine clinical predictors that are associated with unrecognized abuse. Children who were younger than 3 years and presented to a large academic children's hospital from January 1993 to December 2007 and received a diagnosis of abusive fractures by a multidisciplinary child protective team were included in this retrospective review. The main outcome measures included the proportion of children who had abusive fractures and had at least 1 previous physician visit with diagnosis of abuse not identified and predictors that were independently associated with missed abuse. Of 258 patients with abusive fractures, 54 (20.9%) had at least 1 previous physician visit at which abuse was missed. The median time to correct diagnosis from the first visit was 8 days (minimum: 1; maximum: 160). Independent predictors of missed abuse were male gender, extremity versus axially located fracture, and presentation to a primary care setting versus pediatric emergency department or to a general versus pediatric emergency department. One fifth of children with abuse-related fractures are missed during the initial medical visit. In particular, boys who present to a primary care or a general emergency department setting with an extremity fracture are at a particularly high risk for delayed diagnosis.


Ventromedial prefrontal cortex activity and rapid eye movement sleep are associated with subsequent fear expression in human subjects.
In humans, activity patterns in the ventromedial prefrontal cortex (vmPFC) have been found to be predictive of subsequent fear memory consolidation. Pioneering work in rodents has further shown that vmPFC-amygdala theta synchronization is correlated with fear memory consolidation. We aimed to evaluate whether vmPFC activity during fear conditioning is (1) correlated with fear expression the subsequent day and whether (2) this relationship is mediated by rapid eye movement (REM) sleep. We analyzed data from 17 young healthy subjects undergoing a fear conditioning task, followed by a fear extinction task 24 h later, both recorded with simultaneous skin conductance response (SCR) and functional magnetic resonance imaging measurements, with a polysomnographically recorded night sleep in between. Our results showed a correlation between vmPFC activity during fear conditioning and subsequent REM sleep amount, as well as between REM sleep amount and SCR to the conditioned stimulus 24 h later. Moreover, we observed a significant correlation between vmPFC activity during fear conditioning and SCR responses during extinction, which was no longer significant after controlling for REM sleep amount. vmPFC activity during fear conditioning was further correlated with sleep latency. Interestingly, hippocampus activity during fear conditioning was correlated with stage 2 and stage 4 sleep amount. Our results provide preliminary evidence that the relationship between REM sleep and fear conditioning and extinction observed in rodents can be modeled in healthy human subjects, highlighting an interrelated set of potentially relevant trait markers.


Cosmetic face, neck, and brow lifts with local anesthesia.
The sections on the face, neck, and brow include descriptions of facelift, neck lift, and open brow lift techniques, anesthesia, treatment goals, procedural approaches, complications, management, preoperative and postoperative care, rehabilitation, recovery, and outcomes. The approach to facial rejuvenation the midface and periorbital area is detailed. These operations are often and easily performed entirely with the use of local anesthesia and mild oral sedation. There are very high satisfaction rates.


A First Step Toward the Operationalization of the Learned Non-Use Phenomenon: A Delphi Study.
The negative discrepancy between residual functional capacity and reduced use of the contralesional hand, frequently observed after a brain lesion, has been termed Learned Non-Use (LNU) and is thought to depend on the interaction of neuronal mechanisms during recovery and learning-dependent mechanisms. Albeit the LNU phenomenon is generally accepted to exist, currently, no transdisciplinary definition exists. Furthermore, although therapeutic approaches are implemented in clinical practice targeting LNU, no standardized diagnostic routine is described in the available literature. Our objective was to reach consensus regarding a definition as well as synthesize knowledge about the current diagnostic procedures. We used a structured group communication following the Delphi method among clinical and scientific experts in the field, knowledge from both, the work with patient populations and with animal models. Consensus was reached regarding a transdisciplinary definition of the LNU phenomenon. Furthermore, the mode and strategy of the diagnostic process, as well as the sources of information and outcome parameters relevant for the clinical decision making, were described with a wide range showing the current lack of a consistent universal diagnostic approach. The need for the development of a structured diagnostic procedure and its implementation into clinical practice is emphasized. Moreover, it exists a striking gap between the prevailing hypotheses regarding the mechanisms underlying the LNU phenomenon and the actual evidence. Therefore, basic research is needed to bridge between bedside and bench and eventually improve clinical decision making and further development of interventional strategies beyond the field of stroke rehabilitation.


Identification and characterization of outer membrane vesicle-associated proteins in Salmonella enterica serovar Typhimurium.
Salmonella enterica serovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enable Salmonella to survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influenced Salmonella survival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into how Salmonella utilizes OMVs to adapt to environmental changes.


Intron-tagged epitope: a tool for facile detection and purification of proteins expressed in Agrobacterium-transformed plant cells.
Epitope tagging provides a useful tool for immunological detection and cellular localization of proteins in vivo. Using T-DNA-mediated transformation, the detection of epitope-tagged proteins in planta is currently feasible only in transgenic plants, because an artificial expression of cDNA and gene constructs driven by plant promoters in bacteria obscures an early detection of epitope-tagged proteins in Agrobacterium-infected plant cells. We have developed a method for labelling plant coding sequences with intron-tagged epitope-coding domains that are not processed in Agrobacterium. Here we show that the expression of HA-epitope-tagged constructs encoding beta-glucuronidase and S-phase kinase-associated (AtSKP1/ASK1) proteins can be specifically and exclusively detected in cultured Arabidopsis cells as early as five days after Agrobacterium infection. This epitope-tagging approach offers an unlimited source of transformed material for purification and localization of proteins expressed individually or simultaneously in Agrobacterium-transformed plant cells.


Gene expression in the lung of p53 mutant mice exposed to cigarette smoke.
We showed previously that p53 mutations play a role in cigarette smoke-related carcinogenesis not only in humans but also in A/J mice. In fact, (UL53-3 x A/J)F(1) mice, carrying a dominant-negative germ-line p53 mutation, responded to exposure to environmental cigarette smoke more efficiently than their wild-type (wt) littermate controls in terms of molecular alterations, cytogenetic damage, and lung tumor yield. To clarify the mechanisms involved, we analyzed by cDNA array the expression of 1,185 cancer-related genes in the lung of the same mice. Neither environmental cigarette smoke nor the p53 status affected the expression of the p53 gene, but the p53 mutation strikingly increased the basal levels of p53 nuclear protein in the lung. Environmental cigarette smoke increased p53 protein levels in wt mice only. The p53 mutation enhanced the expression of positive cell cycle regulators in sham-exposed mice, which suggests a physiologic protective role of p53. In environmental cigarette smoke-exposed mice, the p53 mutation resulted in a lack of induction of proapoptotic genes and in overexpression of genes involved in cell proliferation, signal transduction, angiogenesis, inflammation, and immune response. Mutant mice and wt mice reacted to environmental cigarette smoke in a similar manner regarding genes involved in metabolism of xenobiotics, multidrug resistance, and protein repair. Irrespective of the p53 status, environmental cigarette smoke poorly affected the expression of oncogenes, tumor suppressor genes, and DNA repair genes. Taken together, these findings may explain the increased susceptibility of p53 mutant mice to smoke-related alterations of intermediate biomarkers and lung carcinogenesis.