Multitracer: a Java-based tool for anatomic delineation of grayscale volumetric images.
A Java-based tool for delineating anatomic boundaries in 8- and 16- bit grayscale volumetric images is described. Modern features implemented by the tool include the ability to simultaneously view the current cursor position and the previously delineated boundaries on three orthogonal planes, the ability to magnify images during delineation using high-quality interpolation, the ability to encode and save boundaries with subvoxel resolution, and the ability to utilize coregistered images interchangeably during delineation. Additional features facilitate use of the tool in a multiuser, multiplatform environment and provide support for the documentation of anatomic delineation protocols. In addition to providing direct estimates of structure volumes, areas, and lengths, the tool allows contoured boundaries to be exported for more sophisticated analyses. The tool also provides support for manual editing of image volumes to remove confounding structures and for manual correction of image volumes that have been inaccurately edited. In addition to its research utility, the tool also has potential value in education, allowing students to interact with volumetric data and structural boundaries in three dimensions.


Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase α.
Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase α (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.


Distribution of the 72-kd heat-shock protein as a function of transient focal cerebral ischemia in rats.
The significance and physiological implications of the expression of the 72-kd heat-shock protein in ischemic tissue are unknown. To enhance our understanding of the relation between ischemic cell damage and 72-kd heat-shock protein expression, we evaluated the cellular expression and the anatomic distribution of 72-kd heat-shock protein in conjunction with the morphological analysis of rat brain, as a function of the duration of a single arterial occlusion. Adult Wistar rats were subjected to graded transient middle cerebral artery occlusion (for a duration of 10, 20, 30, 60, 90, and 120 minutes and sham; n = 4 per group). Forty-eight hours after reopening the artery, brain tissue sections were analyzed to determine the extent of neuronal damage (hematoxylin and eosin staining), the extent of astrocytic reactivity (immunohistochemistry, using anti-glial fibrillary acidic protein), and the distribution of 72-kd heat-shock protein (immunohistochemistry, using a monoclonal antibody to 72-kd heat-shock protein). We found that 72-kd heat-shock protein was sequentially expressed in morphologically intact neurons, microglia, and endothelial cells with increasing duration of ischemia; 72-kd heat-shock protein immunoreactivity was not detected in astrocytes. The duration of ischemia required to evoke a 72-kd heat-shock protein response in neurons was dependent on the anatomic site and followed a pattern of increasing neuronal sensitivity to ischemic cell damage with duration of ischemia: 72-kd heat-shock protein and neuronal damage were sequentially detected in the caudate putamen, globus pallidus, cerebral cortex, amygdala, and hippocampus with increasing duration of ischemia. With ischemia of long duration (greater than or equal to 90 minutes), neurons expressing 72-kd heat-shock protein were localized to a zone peripheral to the severely damaged ischemic core. These studies suggest that 1) the expression of 72-kd heat-shock protein in neurons precedes the development of ischemic cellular alterations detectable by conventional hematoxylin and eosin light microscopy methods; 2) there is a hierarchy of cell types and anatomic sites that express 72-kd heat-shock protein, and this hierarchy reflects cellular and anatomic vulnerability to ischemic cell damage; and 3) 72-kd heat-shock protein induction in neurons bordering a necrotic ischemic core may be the morphological equivalent of the ischemic penumbra.


Epsilon-aminocaproic acid inhibits the activity of factor VIII inhibitors in patients with severe haemophilia A in vivo and in vitro.
Haemophilia patients with inhibitors pose a formidable challenge for patient management. This is particularly problematic in developing countries, where porcine factor VIII, FEIBA, factor VIIa or immunoadsorption column are generally unavailable or unaffordable. Under these circumstances, any effective modality of affordable treatment is welcome. We investigated, both in vivo and in vitro, the effect of epsilon-aminocaproic acid (EACA) on the inhibitory activity of factor VIII inhibitor. It was found that in vitro EACA (final concentration 1.25-5 mg/ml) substantially inhibited the activity of the inhibitors, while the same concentration of EACA had no effect on other immunological reactions like red cell agglutination and immunofluorescence. The inhibitory action of EACA on factor VIII inhibitor was also confirmed in an improvised antigen-binding ELISA system. Further, the inhibitory activity of EACA was confirmed in 2 patients, in whom the inhibitory activity persisted for 15 min following infusion of EACA (100 mg/kg over 10 min). EACA was found to be even more effective in local wound application in patients of haemophilia A with inhibitors. EACA at the concentration cited did not act as an inhibitor of factor VIII inhibitor through occupancy of lysine binding sites. The inhibitory activity of EACA on factor VIII inhibitor was equally seen with recombinant factor VIII also; hence this action cannot be explained by its antifibrinolytic activity.


Sampling strategies for phlebotomine sand flies (Diptera: Psychodidae) in Europe.
The distribution of phlebotomine sand flies is widely reported to be changing in Europe. This can be attributed to either the discovery of sand flies in areas where they were previously overlooked (generally following an outbreak of leishmaniasis or other sand fly-related disease) or to true expansion of their range as a result of climatic or environmental changes. Routine surveillance for phlebotomines in Europe is localized, and often one of the challenges for entomologists working in non-leishmaniasis endemic countries is the lack of knowledge on how to conduct, plan and execute sampling for phlebotomines, or how to adapt on-going sampling strategies for other haematophagous diptera. This review brings together published and unpublished expert knowledge on sampling strategies for European phlebotomines of public health concern in order to provide practical advice on: how to conduct surveys; the collection and interpretation of field data; suitable techniques for the preservation of specimens obtained by different sampling methods; molecular techniques used for species identification; and the pathogens associated with sand flies and their detection methods.


Three-Quarters Adrenalectomy for Infantile-Onset Cushing Syndrome due to Bilateral Adrenal Hyperplasia in McCune-Albright Syndrome.
Bilateral adrenalectomy is performed in cases with infantile-onset Cushing syndrome due to bilateral adrenal hyperplasia in McCune-Albright syndrome (MAS) because severe Cushing syndrome with heart failure and liver dysfunction can have a lethal outcome. This procedure can completely ameliorate hypercortisolism, although lifetime steroid replacement therapy and steps to prevent adrenal crisis are necessary. Recently, the efficacy of unilateral adrenalectomy has been reported in adult cases of bilateral macronodular adrenal hyperplasia, but there is no consensus regarding the appropriate surgical treatment for bilateral adrenal hyperplasia in MAS. A 6-month-old girl presented with café-au-lait spots, short stature, central obesity, a moon face, and hypertension. Endocrinological tests and imaging studies led to the diagnosis of ACTH-independent Cushing syndrome due to bilateral adrenal hyperplasia induced by MAS. "Three-quarters adrenalectomy", namely right-sided total adrenalectomy and left-sided half adrenalectomy, was carried out. An activating mutation of the GNAS1 gene (p.Arg201Cys) was identified in the adrenal tissues. Since the operation, our patient has been in a state of clinical remission for more than 2 years. Our original surgical intervention, three-quarters adrenalectomy, may be a new treatment option for Cushing syndrome associated with MAS.


Asthma-like symptoms: is it always a pulmonary issue?
Double aortic arch is a rare congenital and complete vascular ring around trachea and esophagus. It is usually diagnosed during infancy. The symptoms are generally related to respiratory and gastroesophageal tracts. A 20-year-old female patient was referred to our outpatient clinic for persistent dry cough. She had a history of an episode of inhalation of food bolus as an infant and recurrent bronchitis, anorexia and allergic bronchial asthma since the childhood. Since the beginning, an intrathoracic obstruction was suspected at pulmonary function tests. After 1 month of complete asthma treatment, the cough was unchanged and the spirometry confirmed the presence of an intrathoracic obstruction. Then, she underwent a chest CT with contrast medium, a contrast transthoracic echocardiography, a fiberbronchoscopy and an esophageal radiography with contrast medium. The final diagnosis was made and a double aortic arch was found. A careful observation of the flow/volume curve should always be guaranteed and the presence of congenital vascular anomalies should be suspected in case of difficult-to-treat asthma.


The ACA's pediatric essential health benefit has resulted in a state-by-state patchwork of coverage with exclusions.
The Affordable Care Act (ACA) establishes essential health benefits as the coverage standard for health plans sold in the individual and small-group markets for all fifty states and the District of Columbia, including the health insurance Marketplaces. "Pediatric services" is one of the required classes of coverage under the ACA. However, other than oral health and vision care, neither the act nor the regulations for implementing it define what these services should be. We investigated how state benchmark plans-the base plan chosen in each state as the standard or benchmark of coverage in that state under ACA rules-address pediatric coverage in plans governed by the essential health benefits standard. Our review of summaries of all the state benchmark plans found that no state specified a distinct pediatric services benefit class. Furthermore, although benchmark plans explicitly included multiple pediatric conditions, many plans also specifically excluded services for children with special health care needs. The Department of Health and Human Services has made a commitment in the essential health benefits regulations to review its approach for the 2016 plan year. Thus, our findings have implications for future regulations regarding the essential health benefits standard for pediatric services.


Socio-cultural and service delivery dimensions of maternal mortality in rural central India: a qualitative exploration using a human rights lens.
Despite the avoidable nature of maternal mortality, unacceptably high numbers of maternal deaths occur in developing countries. Considering its preventability, maternal mortality is being increasingly recognised as a human rights issue. Integration of a human rights perspective in maternal health programmes could contribute positively in eliminating avertable maternal deaths. This study was conducted to explore socio-cultural and service delivery-related dimensions of maternal deaths in rural central India using a human rights lens. Social autopsies were conducted for 22 maternal deaths during 2011 in Khargone district in central India. The data were analysed using thematic analysis. The factors associated with maternal deaths were classified by using the 'three delays' framework and were examined by using a human rights lens. All 22 women tried to access medical assistance, but various factors delayed their access to appropriate care. The underestimation of the severity of complications by family members, gender inequity, and perceptions of low-quality delivery services delayed decisions to seek care. Transportation problems and care seeking at multiple facilities delayed reaching appropriate health facilities. Negligence by health staff and unavailability of blood and emergency obstetric care services delayed receiving adequate care after reaching a health facility. The study highlighted various socio-cultural and service delivery-related factors which are violating women's human rights and resulting in maternal deaths in rural central India. This study highlights that, despite the health system's conscious effort to improve maternal health, normative elements of a human rights approach to maternal health (i.e. availability, accessibility, acceptability, and quality of maternal health services) were not upheld. The data and analysis suggest that the deceased women and their relatives were unable to claim their entitlements and that the duty bearers were not successful in meeting their obligations. Based on the findings of our study, we conclude that to prevent maternal deaths, further concentrated efforts are required for better community education, women's empowerment, and health systems strengthening to provide appropriate and timely services, including emergency obstetric care, with good quality.


Social outcomes in the Childhood Cancer Survivor Study cohort.
Difficulties with negotiating and achieving desired social outcomes in life may be exacerbated by the experience of childhood cancer, including adverse effects from therapies used to achieve a cure. This review of previous publications from the Childhood Cancer Survivor Study (CCSS) and other relevant literature provides insight into the prevalence of, and risk factors for, poor educational attainment, less than optimal employment status, and interpersonal relationship issues among long-term survivors of childhood cancer. The impacts of emotional health and physical disability on social outcomes are also examined. Study results suggest that childhood cancer survivors generally have similar high school graduation rates, but are more likely to require special education services than sibling comparison groups. Survivors are slightly less likely than expected to attend college, and are more likely to be unemployed and not married as young adults. Cancers and treatments that result in impairment to the CNS, particularly brain tumors, or that impact sensory functioning, such as hearing loss, are associated with greater risk for undesirable social outcomes, as are emotional health problems and physical disability. This review of relevant data from CCSS and other studies provides information on risk factors for social problems into adulthood. A greater understanding of the long-term social impacts from the diagnosis and treatment of childhood cancer is critically important for developing targeted interventions to prevent or ameliorate adverse psychosocial effects.


Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB.
In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400-2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair-the attenuation of DNA-end processing and the initiation of homology search by RecA-thereby helping to ensure that genomic integrity is maintained during DSB repair.


Choroidal thickness in Behcet's uveitis: an enhanced depth imaging-optical coherence tomography and its association with angiographic changes.
To evaluate the change in subfoveal choroidal thickness between active and quiescent phases of Behcet's posterior uveitis and compare this with the healthy population using enhanced depth imaging optical coherence tomography (EDI-OCT). Thirty eyes from 30 patients with Behcet's posterior uveitis (mean age, 47.03 ± 11.01 years) were retrospectively enrolled in the study. Their subfoveal choroidal thickness was measured using EDI-OCT in the active and quiescent phases of Behcet's uveitis, and compared with the age, sex, and spherical equivalent-matched healthy population. Changes in retinal vascular leakage on fluorescein angiography (FA) were correlated with the changes in subfoveal choroidal thickness. Mean subfoveal choroidal thickness in the acute phase of Behcet's uveitis was significantly greater than that in the quiescent phase (398.77 ± 155.59 μm versus 356.72 ± 141.09 μm; P = 0.004). Subfoveal choroidal thickness in the quiescent phase was also significantly greater than that of the healthy population (259.96 ± 65.16 μm; P < 0.0001). There was a statistically significant association between the change in subfoveal choroidal thickness and the change in vascular leakage revealed by FA (ρ = 0.381, P = 0.046). Subfoveal choroidal thickness in the uninvolved fellow eyes of patients with unilateral Behcet's uveitis was also evaluated and it was significantly greater than that of the healthy population (n = 13 eyes; P = 0.001) CONCLUSIONS: This study found choroidal thickening during the active phase of Behcet's posterior uveitis. Subfoveal choroidal thickness during the quiescent phase was also significantly greater than in normal eyes. The degree of reduction in choroidal thickening was significantly correlated with improvement in retinal vascular leakage as revealed by FA.


Flowing lattices of bubbles as tunable, self-assembled diffraction gratings.
We demonstrate tunable, fluidic, two-dimensional diffraction gratings based on a microfluidic platform comprising a flow-focusing bubble generator and flowing, regular lattices of bubbles formed by dynamic self-assembly. The structure of these lattices can be tuned with switching times of less than ten seconds by changing the pressures and rates of flow applied to the device. These diffraction gratings exhibit high stability (over hours of operation if properly designed and operated). For our devices, we achieved tunable ranges in pitch from 12 to 51 microm, corresponding to first-order diffraction angles from 3.2 degrees to 0.7 degrees for light with a wavelength of 632 nm.


Jaw lift causes less laryngeal interference during lightwand-guided intubation than combined jaw and tongue traction applied by single operator.
Lightwand-guided intubation is a semi-blind technique that takes advantage of the anterior location of the trachea in relation to the oesophagus. Fibreoptic evaluation of lightwand-guided intubation has revealed a possibility of laryngeal interference and epiglottic distortion. Jaw lift, tongue traction or a combination of both have been used to assist in lightwand-guided intubation. This study fibreoptically evaluates lightwand-guided intubation using jaw lift and combined jaw and tongue traction. Eighty four patients with normal airway undergoing general anaesthesia were studied. This randomised, double blinded, cross over study was done in two phases. First phase - after achieving adequate depth of anaesthesia, a fibrescope was advanced nasally, and lightwand-guided intubation was carried out under direct fibreoptic visualisation with the aid of either jaw lift or combined jaw and tongue traction. Second phase - Extubation followed by reintubation using the other manoeuvre. Interference with laryngeal structures during intubation and position of the epiglottis at the end of intubation were noted. Epiglottic distortion (deviated to one side/infolded into trachea) was observed in 6 patients with jaw lift and 17 patients with combined jaw and tongue traction (P=0.003). Laryngeal interference was significantly higher (P=0.012) with combined manoeuvre (30/78) than with jaw lift alone (9/81). Although lightwand-guided intubation can be performed quickly and easily, interference with laryngeal structures and distortion of the epiglottis can occur. Jaw lift manoeuvre causes less laryngeal interference than combined jaw and tongue traction applied by a single operator.


Thymine functionalised porphyrins, synthesis and heteromolecular surface-based self-assembly.
The synthesis and surface-based self-assembly of thymine-functionalised porphyrins is described. Reaction of 1-formylphenyl-3-benzoyl-thymine with suitable pyrollic species leads to the formation of tetra-(phenylthymine)porphyrin (tetra-TP) or mono-thymine-tri-(3,5-di-tert-butylphenyl)porphyrin (mono-TP). Single crystal X-ray diffraction studies demonstrate the self-association of mono-TP in the solid state through thymine···thymine hydrogen-bonding interactions but in solution this interaction (Kd = 6.1 ± 3.0 M-1) is relatively weak in comparison to the heteromolecular interaction between mono-TP and 9-propyladenine (K = 91.8 ± 20.5 M-1). STM studies of the tetratopic hydrogen-bonding tecton, tetra-TP, deposited on an HOPG substrate reveal the formation of an almost perfectly square self-assembled lattice through thymine···thymine hydrogen-bonding. Co-deposition of tetra-TP with 9-propyladenine leads to the adoption of preferable thymine···adenine interactions leading to the formation of a heteromolecular tetra-TP···9-propyladenine hydrogen bonded array including both Watson-Crick thymine···adenine interactions and adenine···adenine hydrogen-bonding. The studies demonstrate a pathway for the self-assembly of tetratopic hydrogen-bonding tectons and the use of preferential heteromolecular thymine···adenine interactions for the disruption of the homomolecular tetra-TP array. Studies of the self-assembly of tetra-TP and 9-propyladenine demonstrate a strong dependence on overall concentration and molar ratio of components indicating the importance of kinetic effects in surface self-assembly processes.


Protein dynamics at Eph receptor-ligand interfaces as revealed by crystallography, NMR and MD simulations.
The role of dynamics in protein functions including signal transduction is just starting to be deciphered. Eph receptors with 16 members divided into A- and B- subclasses are respectively activated by 9 A- and B-ephrin ligands. EphA4 is the only receptor capable of binding to all 9 ephrins and small molecules with overlapped interfaces. We first determined the structures of the EphA4 ligand binding domain (LBD) in two crystals of P1 space group. Noticeably, 8 EphA4 molecules were found in one asymmetric unit and consequently from two crystals we obtained 16 structures, which show significant conformational variations over the functionally critical A-C, D-E, G-H and J-K loops. The 16 new structures, together with previous 9 ones, can be categorized into two groups: closed and open forms which resemble the uncomplexed and complexed structures of the EphA4 LBD respectively. To assess whether the conformational diversity over the loops primarily results from the intrinsic dynamics, we initiated 30-ns molecular dynamics (MD) simulations for both closed and open forms. The results indicate that the loops do have much higher intrinsic dynamics, which is further unravelled by NMR H/D exchange experiments. During simulations, the open form has the RMS deviations slightly larger than those of the closed one, suggesting the open form may be less stable in the absence of external contacts. Furthermore, no obvious exchange between two forms is observed within 30 ns, implying that they are dynamically separated. Our study provides the first experimental and computational result revealing that the intrinsic dynamics are most likely underlying the conformational diversity observed for the EphA4 LBD loops mediating the binding affinity and specificity. Interestingly, the open conformation of the EphA4 LBD is slightly unstable in the absence of it natural ligand ephrins, implying that the conformational transition from the closed to open has to be driven by the high-affinity interaction with ephrins because the weak interaction with small molecule was found to be insufficient to trigger the transition. Our results therefore highlight the key role of protein dynamics in Eph-ephrin signalling and would benefit future design of agonists/antagonists targeting Eph receptors.


Solution structure and characterisation of the human pyruvate dehydrogenase complex core assembly.
Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a central pentagonal dodecahedral core consisting of two subunit types (E2 and E3BP) to which peripheral enzymes (E1 and E3) bind tightly but non-covalently. Currently, there are two conflicting models of PDC (E2+E3BP) core organisation: the 'addition' model (60+12) and the 'substitution' model (48+12). Here we present the first ever low-resolution structures of human recombinant full-length PDC core (rE2/E3BP), truncated PDC core (tE2/E3BP) and native bovine heart PDC core (bE2/E3BP) obtained by small-angle X-ray scattering and small-angle neutron scattering. These structures, corroborated by negative-stain and cryo electron microscopy data, clearly reveal open pentagonal core faces, favouring the 'substitution' model of core organisation. The native and recombinant core structures are all similar to the truncated bacterial E2 core crystal structure obtained previously. Cryo-electron microscopy reconstructions of rE2/E3BP and rE2/E3BP:E3 directly confirm that the core has open pentagonal faces, agree with scattering-derived models and show density extending outwards from their surfaces, which is much more structurally ordered in the presence of E3. Additionally, analytical ultracentrifugation characterisation of rE2/E3BP, rE2 (full-length recombinant E2-only) and tE2/E3BP supports the substitution model. Superimposition of the small-angle neutron scattering tE2/E3BP and truncated bacterial E2 crystal structures demonstrates conservation of the overall pentagonal dodecahedral morphology, despite evolutionary diversity. In addition, unfolding studies using circular dichroism and tryptophan fluorescence spectroscopy show that the rE2/E3BP is less stable than its rE2 counterpart, indicative of a role for E3BP in core destabilisation. The architectural complexity and lower stability of the E2/E3BP core may be of benefit to mammals, where sophisticated fine-tuning is required for cores with optimal catalytic and regulatory efficiencies.


Mannose trimming is required for delivery of a glycoprotein from EDEM1 to XTP3-B and to late endoplasmic reticulum-associated degradation steps.
Although the trimming of α1,2-mannose residues from precursor N-linked oligosaccharides is an essential step in the delivery of misfolded glycoproteins to endoplasmic reticulum (ER)-associated degradation (ERAD), the exact role of this trimming is unclear. EDEM1 was initially suggested to bind N-glycans after mannose trimming, a role presently ascribed to the lectins OS9 and XTP3-B, because of their in vitro affinities for trimmed oligosaccharides. We have shown before that ER mannosidase I (ERManI) is required for the trimming and concentrates together with the ERAD substrate and ERAD machinery in the pericentriolar ER-derived quality control compartment (ERQC). Inhibition of mannose trimming prevents substrate accumulation in the ERQC. Here, we show that the mannosidase inhibitor kifunensine or ERManI knockdown do not affect binding of an ERAD substrate glycoprotein to EDEM1. In contrast, substrate association with XTP3-B and with the E3 ubiquitin ligases HRD1 and SCF(Fbs2) was inhibited. Consistently, whereas the ERAD substrate partially colocalized upon proteasomal inhibition with EDEM1, HRD1, and Fbs2 at the ERQC, colocalization was repressed by mannosidase inhibition in the case of the E3 ligases but not for EDEM1. Interestingly, association and colocalization of the substrate with Derlin-1 was independent of mannose trimming. The HRD1 adaptor protein SEL1L had been suggested to play a role in N-glycan-dependent substrate delivery to OS9 and XTP3-B. However, substrate association with XTP3-B was still dependent on mannose trimming upon SEL1L knockdown. Our results suggest that mannose trimming enables delivery of a substrate glycoprotein from EDEM1 to late ERAD steps through association with XTP3-B.


MicroRNA-206 functions as a pleiotropic modulator of cell proliferation, invasion and lymphangiogenesis in pancreatic adenocarcinoma by targeting ANXA2 and KRAS genes.
Recent advances in cancer biology have emerged important roles for microRNAs (miRNAs) in regulating tumor responses. However, their function in mediating intercellular communication within the tumor microenvironment is thus far poorly explored. Here, we found miR-206 to be abrogated in human pancreatic ductal adenocarcinoma (PDAC) specimens and cell lines. We show that miR-206 directly targets the oncogenes KRAS and annexin a2 (ANXA2), thereby acting as tumor suppressor in PDAC cells by blocking cell cycle progression, cell proliferation, migration and invasion. Importantly, we identified miR-206 as a negative regulator of oncogenic KRAS-induced nuclear factor-κB transcriptional activity, resulting in a concomitant reduction of the expression and secretion of pro-angiogenic and pro-inflammatory factors including the cytokine interleukin-8, the chemokines (C-X-C motif) ligand 1 and (C-C motif) ligand 2, and the granulocyte macrophage colony-stimulating factor. We further show that miR-206 abrogates the expression and secretion of the potent pro-lymphangiogenic factor vascular endothelial growth factor C in pancreatic cancer cells through an NF-κB-independent mechanism. By using in vitro and in vivo approaches, we reveal that re-expression of miR-206 in PDAC cells is sufficient to inhibit tumor blood and lymphatic vessel formation, thus leading to a significant delay of tumor growth and progression. Taken together, our study sheds light onto the role of miR-206 as a pleiotropic modulator of different hallmarks of cancer, and as such raising the intriguing possibility that miR-206 may be an attractive candidate for miRNA-based anticancer therapies.


Risk- and response-adapted strategies for the management of Hodgkin lymphoma.
Therapy for Hodgkin lymphoma (HL) is associated with excellent long-term survival rates, of 80% of more. Extended follow up has described late treatment-related toxicities, principally secondary malignancies, cardiovascular disease and infertility. Given the young age of many patients, there is a desire to offer a more personalised approach, correlated to individual tumour biology that enables treatment de-escalation in low risk patients to reduce toxicity, and treatment intensification in high risk patients to reduce treatment failure. Contemporary therapeutic strategies have involved risk assessment based on staging and clinical factors. The use of functional imaging with 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) as a predictive tool to identify early non-responders has been well validated and outperforms the risk stratifying International Prognostic Score (IPS). HL has particularly high FDG-avidity (97-100%), with FDG-PET scanning reflecting metabolic activity and acting as a surrogate biomarker for chemosensitivity. International consensus on the methods of reporting and interpreting FDG-PET scans has enabled their use to be standardised and reproducible. Given that primary therapy fails for 15-20% of patients, the use of combined FDG-PET and computerised tomography (FDG-PET/CT) to provide a response-adapted strategy to guide management is under investigation in numerous prospective clinical trials. They aim to determine whether early response scanning can be used to directly modulate subsequent therapy, through intensifying or abbreviating chemotherapy regimens and/or omitting radiotherapy. Integrated multi-modality imaging and advanced conformal planning techniques have led to the emergence of radiotherapy strategies such as involved-node radiation (INRT) that aim to optimise treatment volumes and maintain efficacy whilst lowering toxicity. Study groups have incorporated these modalities in trial designs to assess whether a PET-directed, individualised approach can become the new standard of care.


What happens to lost nets: a multi-country analysis of reasons for LLIN attrition using 14 household surveys in four countries.
While significant focus has been given to net distribution, little is known about what is done with nets that leave a household, either to be used by others or when they are discarded. To better understand the magnitude of sharing LLIN between households and patterns of discarding LLIN, the present study pools data from 14 post-campaign surveys to draw larger conclusions about the fate of nets that leave households. Data from 14 sub-national post-campaign surveys conducted in Ghana, Senegal, Nigeria (10 states), and Uganda between 2009 and 2012 were pooled. Survey design and data collection methods were similar across surveys. The timing of surveys ranged from 2-16 months following their respective mass LLIN distributions. Among the 14 surveys a total of 14,196 households reported owning 25,447 nets of any kind, of which 23,955 (94%) were LLINs. In addition, a total of 4,102 nets were reported to have left the households in the sample: 63% were discarded, and 34% were given away. Only 255 of the discarded nets were reported used for other purposes, representing less than 1% of the total sample of nets. The majority (62.5%) of nets given away were given to or taken by relatives, while 31.1% were given to non-relatives. Campaign nets were almost six times (OR 5.95, 4.25-8.32, p < 0.0001) more likely to be given away than non-campaign nets lost during the same period. Nets were primarily given away within the first few months after distribution. The overall rate of net redistribution was 5% of all nets. Intra-household re-allocation of nets does occur, but was sensitive to current household net ownership and the time elapsed since mass distribution. These factors can be addressed programmatically to further facilitate reallocation within a given community. Secondly, the overwhelming majority of nets were used for malaria prevention. Of the repurposed nets (<1% overall), the majority were already considered too torn, indicating they had already served out their useful life for malaria prevention. National programmes and donor agencies should remain confident that overall, their investments in LLIN are being appropriately used.


Should celecoxib be contraindicated in patients who are allergic to sulfonamides? Revisiting the meaning of 'sulfa' allergy.
Celecoxib, a selective cyclo-oxygenase-2 inhibitor, is a diaryl-substituted pyrazole derivative containing a sulfonamide substituent. Because of this structural component, celecoxib is contraindicated for use in patients who have demonstrated allergic reactions to sulfonamides. However, there is a lack of data demonstrating cross-reactivity among sulfonamide medications. A sulfonamide is any compound with an SO2NH2 moiety. The major difference between sulfonamide antimicrobials and other sulfonamide-containing medications such as furosemide, thiazide diuretics and celecoxib, is that sulfonamide antimicrobials contain an aromatic amine group at the N4 position. This allows for division of the sulfonamides into 2 groups: aromatic amines (i.e., sulfonamide antimicrobials) and nonaromatic amines. In addition, sulfonamide antimicrobials contain a substituted ring at the N1-position; this group is not found with nonaromatic amine-containing sulfonamides. Adverse reactions to sulfonamide antimicrobials include type I, or immunoglobulin (Ig) E-mediated reactions, hypersensitivity syndrome reactions, and severe skin reactions such as toxic epidermal necrolysis. The aromatic amine portion of the sulfonamide antimicrobial is considered to be critical in the development of latter 2 reactions. In susceptible individuals, the hydroxylamine metabolite is unable to be detoxified leading to a cascade of cytotoxic and immunological events that eventually results in the adverse reaction. Since celecoxib does not contain the aromatic amine, adverse reactions such as hypersensitivity syndrome reactions and toxic epidermal necrolysis would not be expected to occur at the same frequency as they do with sulfonamide antimicrobials. Similarly, for IgE-mediated reactions, the N1-substituent and not the sulphonamide moiety is important in determining specificity to antibodies. Celecoxib and other nonaromatic amine-containing sulfonamide medications do not contain the N1-substituent. Cross-reactivity among the various sulfonamide-containing medications has also not been substantiated by published case reports. In fact, conflicting information exists in the literature. Reports showing lack of cross-reactivity balance the few case reports suggesting cross-reactivity. Cross-reactivity between sulfonamide medications should be based on scientific data, including chemistry, metabolism, immune responses and clinical data. Based on the current information, there is no documentation for cross-reactivity between sulfonamide antimicrobials and other sulfonamide medications, such as celecoxib.


Essential function for the kinase TAK1 in innate and adaptive immune responses.
Transforming growth factor-beta-activated kinase 1 (TAK1) has been linked to interleukin 1 receptor and tumor necrosis factor receptor signaling. Here we generated mouse strains with conditional expression of a Map3k7 allele encoding part of TAK1. TAK1-deficient embryonic fibroblasts demonstrated loss of responses to interleukin 1beta and tumor necrosis factor. Studies of mice with B cell-specific TAK1 deficiency showed that TAK1 was indispensable for cellular responses to Toll-like receptor ligands, CD40 and B cell receptor crosslinking. In addition, antigen-induced immune responses were considerably impaired in mice with B cell-specific TAK1 deficiency. TAK1-deficient cells failed to activate transcription factor NF-kappaB and mitogen-activated protein kinases in response to interleukin 1beta, tumor necrosis factor and Toll-like receptor ligands. However, TAK1-deficient B cells were able to activate NF-kappaB but not the kinase Jnk in response to B cell receptor stimulation. These results collectively indicate that TAK1 is key in the cellular response to a variety of stimuli.


Refinement of docked protein-ligand and protein-DNA structures using low frequency normal mode amplitude optimization.
Prediction of structural changes resulting from complex formation, both in ligands and receptors, is an important and unsolved problem in structural biology. In this work, we use all-atom normal modes calculated with the Elastic Network Model as a basis set to model structural flexibility during formation of macromolecular complexes and refine the non-bonded intermolecular energy between the two partners (protein-ligand or protein-DNA) along 5-10 of the lowest frequency normal mode directions. The method handles motions unrelated to the docking transparently by first applying the modes that improve non-bonded energy most and optionally restraining amplitudes; in addition, the method can correct small errors in the ligand position when the first six rigid-body modes are switched on. For a test set of six protein receptors that show an open-to-close transition when binding small ligands, our refinement scheme reduces the protein coordinate cRMS by 0.3-3.2 A. For two test cases of DNA structures interacting with proteins, the program correctly refines the docked B-DNA starting form into the expected bent DNA, reducing the DNA cRMS from 8.4 to 4.8 A and from 8.7 to 5.4 A, respectively. A public web server implementation of the refinement method is available at http://lorentz.immstr.pasteur.fr.


c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont.
Both microbial and host genetic factors contribute to the pathogenesis of autoimmune diseases. There is accumulating evidence that microbial species that potentiate chronic inflammation, as in inflammatory bowel disease, often also colonize healthy individuals. These microorganisms, including the Helicobacter species, can induce pathogenic T cells and are collectively referred to as pathobionts. However, how such T cells are constrained in healthy individuals is not yet understood. Here we report that host tolerance to a potentially pathogenic bacterium, Helicobacter hepaticus, is mediated by the induction of RORγt+FOXP3+ regulatory T (iTreg) cells that selectively restrain pro-inflammatory T helper 17 (TH17) cells and whose function is dependent on the transcription factor c-MAF. Whereas colonization of wild-type mice by H. hepaticus promoted differentiation of RORγt-expressing microorganism-specific iTreg cells in the large intestine, in disease-susceptible IL-10-deficient mice, there was instead expansion of colitogenic TH17 cells. Inactivation of c-MAF in the Treg cell compartment impaired differentiation and function, including IL-10 production, of bacteria-specific iTreg cells, and resulted in the accumulation of H. hepaticus-specific inflammatory TH17 cells and spontaneous colitis. By contrast, RORγt inactivation in Treg cells had only a minor effect on the bacteria-specific Treg and TH17 cell balance, and did not result in inflammation. Our results suggest that pathobiont-dependent inflammatory bowel disease is driven by microbiota-reactive T cells that have escaped this c-MAF-dependent mechanism of iTreg-TH17 homeostasis.


Perceived suprathreshold depth under conditions that elevate the stereothreshold.
Previous studies considered the possibility that individuals with impaired stereoacuity can be identified by estimating the perceived depth of a target with a suprathreshold retinal image disparity. These studies showed that perceived suprathreshold depth is reduced when the image presented to one eye is blurred, but they did not address whether a similar reduction of perceived depth occurs when the stereothreshold is elevated using other manipulations. Stereothresholds were measured in six adult observers for a pair of bright 1-degree vertical lines during normal viewing and under five conditions that elevated the stereothreshold: monocular dioptric blur, monocular glare, binocular luminance reduction, monocular luminance reduction, and imposed disjunctive image motion. The observers subsequently matched the perceived depth of degraded targets presented with crossed or uncrossed disparities corresponding to two, four, and six times the elevated stereothreshold for each stimulus condition. The image manipulations used elevated the stereothreshold by a factor of 3.7 to 5.5 times. For targets with suprathreshold disparities, monocular blur, monocular luminance reduction, and disjunctive image motion resulted in a significant decrease in perceived depth. However, the magnitude of perceived suprathreshold depth was unaffected when monocular glare was introduced or the binocular luminance of the stereotargets was reduced. Not all conditions that increase the stereothreshold reduce the perceived depth of targets with suprathreshold disparities. Observers who have poor stereopsis therefore may or may not exhibit an associated reduction of perceived suprathreshold depth.


The impact of systems redesign on staff, patient, and financial outcomes.
The purpose of this study was to measure the impact of a change initiative using the strategies of inpatient bed consolidation and patient population reaggregation on staff, patient, and financial outcomes. Bed consolidation and patient population reaggregation are extensively used strategies in the hospital industry. However, the state of science as it relates to these two strategies is limited, with no studies measuring the effect of bed consolidation, patient reaggregation, or both in isolation of multiple other concurrent care model changes. An exploratory, single case, longitudinal field study design with embedded levels of analysis was used for the study. The impact associated with the reaggregation and consolidation of the acute inpatient system of care was evaluated at the organization, the acute inpatient nursing unit, and the individual nursing staff member levels. This article reports the outcomes found at both the nursing unit level and the staff member level on three general medicine nursing units. Baseline nursing unit-level data are reported from fiscal year 1994-1995 and the first half of fiscal year 1995-1996 (July 1995 through December 1995); data from January 1996 through June 1996 reflect the planning phase of the project. Outcomes related to the change process are reported from the time period January 1996 through June 1997. Staff perceptions of multiple variables were measured at five different data points over a 2-year period. Both quantitative and qualitative data were collected. Unit-level cost and hours per patient day (HPPD) increased during or immediately after the merger of major patient populations on two of the study units; no significant variation was found in medication errors or patient falls. The quantitative and qualitative data analysis of the nursing staff member surveys revealed a nursing staff that was dissatisfied with many aspects of their job, worried about job security, had low morale, and had many concerns about the quality of care provided to patients. This study suggests that consolidation and reaggregation strategies may cause an increase in costs and HPPDs, at least on a short-term basis. Also, these strategies may have a significant effect on the morale and job satisfaction of unit-level nursing. In light of these findings, on-going testing of the efficacy of consolidation and patient reaggregation strategies in improving quality and cost outcomes is essential.


Functional heterogeneity of the UpaH autotransporter protein from uropathogenic Escherichia coli.
Uropathogenic Escherichia coli (UPEC) is responsible for the majority of urinary tract infections (UTI). To cause a UTI, UPEC must adhere to the epithelial cells of the urinary tract and overcome the shear flow forces of urine. This function is mediated primarily by fimbrial adhesins, which mediate specific attachment to host cell receptors. Another group of adhesins that contributes to UPEC-mediated UTI is autotransporter (AT) proteins. AT proteins possess a range of virulence properties, such as adherence, aggregation, invasion, and biofilm formation. One recently characterized AT protein of UPEC is UpaH, a large adhesin-involved-in-diffuse-adherence (AIDA-I)-type AT protein that contributes to biofilm formation and bladder colonization. In this study we characterized a series of naturally occurring variants of UpaH. We demonstrate that extensive sequence variation exists within the passenger-encoding domain of UpaH variants from different UPEC strains. This sequence variation is associated with functional heterogeneity with respect to the ability of UpaH to mediate biofilm formation. In contrast, all of the UpaH variants examined retained a conserved ability to mediate binding to extracellular matrix (ECM) proteins. Bioinformatic analysis of the UpaH passenger domain identified a conserved region (UpaH(CR)) and a hydrophobic region (UpaH(HR)). Deletion of these domains reduced biofilm formation but not the binding to ECM proteins. Despite variation in the upaH sequence, the transcription of upaH was repressed by a conserved mechanism involving the global regulator H-NS, and mutation of the hns gene relieved this repression. Overall, our findings shed new light on the regulation and functions of the UpaH AT protein.


Factors associated with Leishmania chagasi infection in domestic dogs from Teresina, State of Piauí, Brazil.
Many studies have evaluated risk factors for human visceral leishmaniasis, but few have focused on the infection among dogs. The objective of this study was to assess the association between peridomestic socioeconomic and environmental factors and the presence of dogs seropositive for Leishmania chagasi in the City of Teresina, Brazil. This case-control study was based on the results of a routine seroepidemiological survey among domestic dogs carried out in 2007. Serological tests were performed by means of indirect immunofluorescence antibody test. All dwellings in which at least one seropositive dog was detected were considered cases, and controls were a random sample of dwellings in which only seronegative dogs were identified. Associations between variables were expressed as odds ratios (OR) and their respective 95% confidence intervals (95%CI) estimated using multivariate logistic regression. Dwellings with a history of dogs removed by the visceral leishmaniasis control program in the last 12 months had five-fold higher odds of having at least one seropositive dog as compared with dwellings having no history of dog removal (OR = 5.19; 95%CI = 3.20-8.42). Dwellings with cats had 58% increased odds of dog infection as compared with those having no cats (OR = 1.58; 95%CI = 1.01-2.47). Identification of factors associated with canine visceral leishmaniasis might be used for the delimitation of areas of higher risk for human visceral leishmaniasis, since infection in dogs generally precedes the appearance of human cases.


Variation in Aedes aegypti mRNA populations related to strain, sex, and development.
Translation products were used to monitor changes in gene expression between different strains and developmental stages of Aedes aegypti. Total RNA was isolated from fourth stadium larvae, male and female pupae, and male and female adults collected at fixed time intervals following pupation and ecdysis. Differences in RNA populations were assessed by in vitro translation followed by one- and two-dimensional polyacrylamide gel electrophoresis and fluorography. Variations in gene expression between sexes and during development were examined in Liverpool (LVP) and Rockefeller (RKF) Ae. aegypti strains. Sex-related differences consisted primarily of differing lengths of expression for certain polypeptides, although two-dimensional electrophoresis revealed changes in intensity of a 44-kD polypeptide with a pI of 7.9 between males and females. Fourth stadium larvae, pupae, and adults expressed different translation products, which probably correlated with developmental differences. Strain-related differences were observed between LVP and RKF.


Peculiar macrophagous adaptations in a new Cretaceous pliosaurid.
During the Middle and Late Jurassic, pliosaurid plesiosaurs evolved gigantic body size and a series of craniodental adaptations that have been linked to the occupation of an apex predator niche. Cretaceous pliosaurids (i.e. Brachaucheninae) depart from this morphology, being slightly smaller and lacking the macrophagous adaptations seen in earlier forms. However, the fossil record of Early Cretaceous pliosaurids is poor, concealing the evolution and ecological diversity of the group. Here, we report a new pliosaurid from the Late Hauterivian (Early Cretaceous) of Russia. Phylogenetic analyses using reduced consensus methods recover it as the basalmost brachauchenine. This pliosaurid is smaller than other derived pliosaurids, has tooth alveoli clustered in pairs and possesses trihedral teeth with complex serrated carinae. Maximum-likelihood ancestral state reconstruction suggests early brachauchenines retained trihedral teeth from their ancestors, but modified this feature in a unique way, convergent with macrophagous archosaurs or sphenacodontoids. Our findings indicate that Early Cretaceous marine reptile teeth with serrated carinae cannot be unequivocally assigned to metriorhynchoid crocodylomorphs. Furthermore, they extend the known diversity of dental adaptations seen in Sauropterygia, the longest lived clade of marine tetrapods.


Test-retest reliability of functional connectivity networks during naturalistic fMRI paradigms.
Functional connectivity analysis has become a powerful tool for probing the human brain function and its breakdown in neuropsychiatry disorders. So far, most studies adopted resting-state paradigm to examine functional connectivity networks in the brain, thanks to its low demand and high tolerance that are essential for clinical studies. However, the test-retest reliability of resting-state connectivity measures is moderate, potentially due to its low behavioral constraint. On the other hand, naturalistic neuroimaging paradigms, an emerging approach for cognitive neuroscience with high ecological validity, could potentially improve the reliability of functional connectivity measures. To test this hypothesis, we characterized the test-retest reliability of functional connectivity measures during a natural viewing condition, and benchmarked it against resting-state connectivity measures acquired within the same functional magnetic resonance imaging (fMRI) session. We found that the reliability of connectivity and graph theoretical measures of brain networks is significantly improved during natural viewing conditions over resting-state conditions, with an average increase of almost 50% across various connectivity measures. Not only sensory networks for audio-visual processing become more reliable, higher order brain networks, such as default mode and attention networks, but also appear to show higher reliability during natural viewing. Our results support the use of natural viewing paradigms in estimating functional connectivity of brain networks, and have important implications for clinical application of fMRI. Hum Brain Mapp 38:2226-2241, 2017. © 2017 Wiley Periodicals, Inc.


Recovering 3D human pose from monocular images.
We describe a learning-based method for recovering 3D human body pose from single images and monocular image sequences. Our approach requires neither an explicit body model nor prior labeling of body parts in the image. Instead, it recovers pose by direct nonlinear regression against shape descriptor vectors extracted automatically from image silhouettes. For robustness against local silhouette segmentation errors, silhouette shape is encoded by histogram-of-shape-contexts descriptors. We evaluate several different regression methods: ridge regression, Relevance Vector Machine (RVM) regression, and Support Vector Machine (SVM) regression over both linear and kernel bases. The RVMs provide much sparser regressors without compromising performance, and kernel bases give a small but worthwhile improvement in performance. The loss of depth and limb labeling information often makes the recovery of 3D pose from single silhouettes ambiguous. To handle this, the method is embedded in a novel regressive tracking framework, using dynamics from the previous state estimate together with a learned regression value to disambiguate the pose. We show that the resulting system tracks long sequences stably. For realism and good generalization over a wide range of viewpoints, we train the regressors on images resynthesized from real human motion capture data. The method is demonstrated for several representations of full body pose, both quantitatively on independent but similar test data and qualitatively on real image sequences. Mean angular errors of 4-6 degrees are obtained for a variety of walking motions.


An essential role for the Escherichia coli DnaK protein in starvation-induced thermotolerance, H2O2 resistance, and reductive division.
During a 3-day period, glucose starvation of wild-type Escherichia coli produced thermotolerant, H2O2-resistant, small cells with a round morphology. These cells contained elevated levels of the DnaK protein, adjusted either for total protein or on a per-cell basis. Immunoprecipitation of [35S]methionine-labeled protein produced by such starving cells demonstrated that DnaK underwent continuous synthesis but at decreasing rates throughout this time. Glucose resupplementation of starving cells resulted in rapid loss of thermotolerance, H2O2 resistance, and the elevated DnaK levels. A dnaK deletion mutant, but not an otherwise isogenic wild-type strain, failed to develop starvation-induced thermotolerance or H2O2 resistance. The filamentous phenotype associated with DnaK deficiency was suppressed by cultivation in a defined glucose medium. When starved for glucose, the nonfilamentous and rod-shaped dnaK mutant strain failed to convert into the small spherical form typical of starving wild-type cells. The dnaK mutant retained the ability to develop adaptive H2O2 resistance during growth but not adaptive resistance to heat. Complementation of DnaK deficiency by using Ptac-regulated dnaK+ and dnaK+J+ expression plasmids confirmed a specific role for the DnaK molecular chaperone in these starvation-induced phenotypes.


Translational behaviour-genetic studies of alcohol: are we there yet?
In biomedical research, one key stage of translating basic science knowledge to clinical practice is the reconciliation of phenotypes employed for laboratory animal studies with those important for the clinical condition. Alcohol dependence (AD) is a prototypic complex genetic trait. There is a long history of behaviour-genetic studies of AD in both human subjects and various genetic animal models. This review assesses the state of the art in our understanding of the genetic contributions to AD. In particular, it primarily focuses on the phenotypes studied in mouse genetic animal models, comparing them to the aspects of the human condition they are intended to target. It identifies several features of AD where genetic animal models have been particularly useful, and tries to identify understudied areas where there is good promise for further genetic animal model work.


Surgeons, surgery, and immunomodulation.
With the definition over the past 15 years of the altered immune state of surgical patients as a result of disease itself and surgical therapy, there have been multiple approaches to the modulation of immune status in experimental or clinical situations, but with conflicting or unhelpful results. The variable that has never been assessed is the significance of the surgeon as an immunomodulator. The expediency and the quality of the surgical act in a variety of surgical diseases have a positive effect on the immune system. Indeed, the data indicate that correction of shock, drainage of infection, excision or drainage of necrotic material, restoration of body composition, and solid basic care all have a positive influence on patients' immune responses. An immunomodulator might get credit if the role of surgical care is not properly assessed. A framework for the study of immunomodulators with the integration of clinical behavior is outlined.


Pharmacological screening technologies for venom peptide discovery.
Venomous animals occupy one of the most successful evolutionary niches and occur on nearly every continent. They deliver venoms via biting and stinging apparatuses with the aim to rapidly incapacitate prey and deter predators. This has led to the evolution of venom components that act at a number of biological targets - including ion channels, G-protein coupled receptors, transporters and enzymes - with exquisite selectivity and potency, making venom-derived components attractive pharmacological tool compounds and drug leads. In recent years, plate-based pharmacological screening approaches have been introduced to accelerate venom-derived drug discovery. A range of assays are amenable to this purpose, including high-throughput electrophysiology, fluorescence-based functional and binding assays. However, despite these technological advances, the traditional activity-guided fractionation approach is time-consuming and resource-intensive. The combination of screening techniques suitable for miniaturization with sequence-based discovery approaches - supported by advanced proteomics, mass spectrometry, chromatography as well as synthesis and expression techniques - promises to further improve venom peptide discovery. Here, we discuss practical aspects of establishing a pipeline for venom peptide drug discovery with a particular emphasis on pharmacology and pharmacological screening approaches. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'


Incident Type 2 Myocardial Infarction in a Cohort of Patients Undergoing Coronary or Peripheral Arterial Angiography.
Despite growing recognition of type 2 myocardial infarction (T2MI; related to supply/demand mismatch), little is known about its risk factors or its association with outcome. A single-center cohort of patients undergoing coronary or peripheral angiography with or without intervention was prospectively enrolled and followed for incident type 1 and T2MI, and major adverse cardiovascular events (MACE, a composite of all-cause death, nonfatal myocardial infarction [MI], heart failure, stroke, transient ischemic attack, peripheral arterial complication, and cardiac arrhythmia), as well. T2MI was adjudicated using criteria from the Third Universal Definition of MI. Baseline characteristics, blood samples, and angiography information were obtained. Major end points subsequent to first MI were assessed using landmark analyses to compare the rates of first events only where everyone with a prior history of any MACE before MI were censored and adjusted for follow-up times. Cox proportional hazard models were used for time-to-event analyses with age and sex forced into all models and additional covariates evaluated by using the stepwise option for the selection. One thousand two hundred fifty-one patients were enrolled and followed for a median of 3.4 years. Of these patients, 152 (12.2%) had T2MI during follow-up; T2MI was frequently recurrent. Multivariable predictors of T2MI were older age, lower systolic blood pressure, history of coronary artery disease, heart failure, chronic obstructive pulmonary disease, diabetes mellitus, nitrate use, and elevated concentrations of glucose, N-terminal pro-B type natriuretic peptide, and cystatin C. Patients with T2MI had higher rates of subsequent adverse events than those without T2MI (per 100 person-years: MACE, 53.7 versus 21.1, P<0.001; all-cause death, 23.3 versus 3.3, P<0.001; cardiovascular death, 17.5 versus 2.6, P<0.001; heart failure events, 22.4 versus 7.4, P<0.001); these rates are similar to those seen in patients with type 1 MI. Incident diagnosis of T2MI strongly predicted risk for subsequent MACE (adjusted hazard ratio, 1.90; 95% confidence interval, 1.46-2.48; P<0.001), all-cause death (adjusted hazard ratio, 2.96; 95% confidence interval, 2.01-4.36; P<0.001), and cardiovascular death (adjusted hazard ratio, 2.16; 95% confidence interval, 1.36-3.43; P=0.001). T2MI is common and associated with poor prognosis. Studies evaluating treatment strategies for management of T2MI are needed. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00842868.


Behavioural treatment for chronic low-back pain.
Behavioural treatment, commonly used in the treatment of chronic low-back pain (CLBP), is primarily focused at reducing disability through the modification of environmental contingencies and cognitive processes. In general, three behavioural treatment approaches are distinguished: operant, cognitive and respondent. To determine if behavioural therapy is more effective than reference treatments for CLBP, and which type of behavioural treatment is most effective. We searched the CENTRAL, MEDLINE, EMBASE, and PsycLIT databases up to October 2003. References of identified randomised trials and relevant systematic reviews were screened. Only randomised trials on behavioural treatment for non-specific CLBP were included. Two reviewers independently assessed the methodological quality and extracted the data. The magnitude of effect was assessed by computing a pooled effect size for post-treatment and long-term results for each comparison, for each domain (i.e., behavioural outcomes, overall improvement, back pain specific and generic functional status, return to work, and pain intensity) using the random effects model. Seven studies (33%) were considered high quality. Comparing behavioural treatment to waiting list control (WLC) revealed strong evidence (4 trials, 134 people) in favour of a combined respondent-cognitive therapy for a medium positive effect on pain, and moderate evidence (2 trials, 39 people) in favour of progressive relaxation for a large positive effect on pain and behavioural outcomes (short-term only). When comparing operant treatment to WLC no significant differences could be detected on general functional status (strong evidence: 2 trials, 87 people) or on behavioural outcomes (moderate evidence; 3 trials, 153 people) (short-term only). There is limited evidence (1 trial, 98 people) that a graded activity program in an industrial setting is more effective than usual care for early return to work and reduced long-term sick leave. There is limited evidence (1 trail, 39 people) that there are no differences between behavioural treatment and exercises. Finally, there is moderate evidence (6 trials, 210 people) that there are no significant differences in short-term and long-term effectiveness when behavioural components are added to usual treatment programs for CLBP (i.e. physiotherapy, back education) on pain, generic functional status and behavioural outcomes. Combined respondent-cognitive therapy and progressive relaxation therapy are more effective than WLC on short-term pain relief. However, it is unknown whether these results sustain in the long term. No significant differences could be detected between behavioural treatment and exercise therapy. Whether clinicians should refer patients with CLBP to behavioural treatment programs or to active conservative treatment cannot be concluded from this review.


A Chemically Defined, Xeno- and Blood-Free Culture Medium Sustains Increased Production of Small Extracellular Vesicles From Mesenchymal Stem Cells.
Cell therapy is witnessing a notable shift toward cell-free treatments based on paracrine factors, in particular, towards small extracellular vesicles (sEV), that mimic the functional effect of the parental cells. While numerous sEV-based applications are currently in advanced preclinical stages, their promised translation depends on overcoming the manufacturing hurdles posed by the large-scale production of purified sEV. Unquestionably, the culture medium used with the parental cells plays a key role in the sEV's secretion rate and content. An essential requisite is the use of a serum-, xeno-, and blood-free medium to meet the regulatory entity requirements of clinical-grade sEV's production. Here, we evaluated OxiumTMEXO, a regulatory complying medium, with respect to production capacity and conservation of the EV's characteristics and functionality and the parental cell's phenotype and viability. A comparative study was established with standard DMEM and a commercially available culture medium developed specifically for sEV production. Under similar conditions, OxiumTMEXO displayed a three-fold increase of sEV secretion, with an enrichment of particles ranging between 51 and 200 nm. These results were obtained through direct quantification from the conditioned medium to avoid the isolation method's interference and variability and were compared to the two culture media under evaluation. The higher yield obtained was consistent with several harvest time points (2, 4, and 6 days) and different cell sources, incluiding umbilical cord-, menstrual blood-derived mesenchymal stromal cells and fibroblasts. Additionally, the stem cell phenotype and viability of the parental cell remained unchanged. Furthermore, OxiumTMEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The in vitro internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV in vivo indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumTMEXO medium sustains an efficient and robust production of large quantities of sEV, conserving the classic functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of EVs for the development of cell-free therapies.


An Af9 cis-element directly targets Dot1a to mediate transcriptional repression of the αENaC gene.
The epithelial Na(+) channel subunit-α (αENaC) of the distal nephron is essential for salt balance. We previously demonstrated that the histone methyltransferase Dot1a and its protein partner Af9 basally repress αENaC transcription in mouse inner medullary collecting duct type 3 (mIMCD3) cells and link aldosterone-elicited chromatin modifications to αENaC transcriptional activation. Af9 DNA-binding activity has never been demonstrated, and whether and where Af9 binds to the αENaC promoter to target Dot1a are unknown. The present study sought to identify functional Af9 cis-element(s) in the -57/+439 "R3" subregion of αENaC, the principal site for Dot1a-Af9 interaction, in mIMCD3 cells. We also exploited connecting tubule/collecting duct-specific Dot1l-deficient mice (Dot1l(AC)) to determine the impact of Dot1l inactivation on renal αENaC expression in vivo. mIMCD3 cell lines expressing αENaC promoter-reporter constructs harboring deletion of +74/+107 demonstrated greatly reduced association of Af9 and Dot1a by ChIP/qPCR. Aldosterone treatment resulted in further decrements in Af9 and Dot1a association with the αENaC promoter. Gel shift and antibody competition assays using wild-type and mutant oligomers revealed Af9-containing +78/+92 αENaC DNA-protein complexes in nuclear extracts of mIMCD3 cells. Mutation of the +78/+92 element resulted in higher basal αENaC promoter activity and impaired Dot1a-mediated inhibition in trans-repression assays. In agreement, mice with connecting tubule/collecting duct-specific knockout of Dot1l exhibited greater αENaC mRNA levels in kidney compared with control. Thus, we conclude that +78/+92 of αENaC represents the primary Af9 binding site involved in recruiting Dot1a to repress basal and aldosterone-sensitive αENaC transcription and that Dot1l inactivation promotes αENaC mRNA expression by eliminating Dot1a-mediated repression.


Development and Pilot of a Checklist for Management of Acute Liver Failure in the Intensive Care Unit.
Acute liver failure (ALF) is an ideal condition for use of a checklist. Our aims were to develop a checklist for the management of ALF in the intensive care unit (ICU) and assess the usability of the checklist among multiple providers. The initial checklist was developed from published guidelines and expert opinion. The checklist underwent pilot testing at 11 academic liver transplant centers in the US and Canada. An anonymous, written survey was used to assess the usability and quality of the checklist. Written comments were used to improve the checklist following the pilot testing period. We received 81 surveys involving the management of 116 patients during the pilot testing period. The overall quality of the checklist was judged to be above average to excellent by 94% of users. On a 5-point Likert scale, the majority of survey respondents agreed or agreed strongly with the following checklist characteristics: the checklist was easy to read (99% agreed/agreed strongly), easy to use (97%), items are categorized logically (98%), time to complete the checklist did not interfere with delivery of appropriate and safe patient care (94%) and was not excessively burdensome (92%), the checklist allowed the user the freedom to use his or her clinical judgment (80%), it is a useful tool in the management of acute liver failure (98%). Web-based and mobile apps were developed for use of the checklist at the point of care. The checklist for the management of ALF in the ICU was shown in this pilot study to be easy to use, helpful and accepted by a wide variety of practitioners at multiple sites in the US and Canada.


Generation of an expandable intermediate mesoderm restricted progenitor cell line from human pluripotent stem cells.
The field of tissue engineering entered a new era with the development of human pluripotent stem cells (hPSCs), which are capable of unlimited expansion whilst retaining the potential to differentiate into all mature cell populations. However, these cells harbor significant risks, including tumor formation upon transplantation. One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential. Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties. This cell population, referred to as intermediate mesodermal progenitor (IMP) cells, is capable of unlimited expansion, lacks tumor formation potential, and, upon appropriate stimulation, readily acquires properties of a sub-population of kidney cells. Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate.


Human ING1 proteins differentially regulate histone acetylation.
ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33(ING1b), but not of p47(ING1a), induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47(ING1a) inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33(ING1b) affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT.ING1.PCNA protein complexes.


Modifying the Phenotypic Frailty Model in Predicting Risk of Major Osteoporotic Fracture in the Elderly.
The phenotypic frailty (PF) model (including slow walking, low physical activity, exhaustion, weakness, and unintentional weight loss) has been widely used to quantify the degree of frailty and predict risks of adverse health outcomes for the elderly. However, evidence has shown that not all the components included in the PF model contribute equally, and low predictive accuracy of the PF model has been reported in predicting risks of outcomes. We aimed to improve predictive accuracy of the PF model in risk of major osteoporotic fracture (MOF) in the elderly by modifying its weighting of individual components. Data from the Global Longitudinal Study of Osteoporosis in Women (GLOW) 3-year Hamilton cohort were used for this study. We used the multivariable Cox regression model to identify the updated weighting for components in the original PF model. The goodness of fit and discrimination were assessed for model performances. There were 3985 women included for analyses (mean age: 69.4 years). In the modified PF model, the updated weighting was 3 points for slowness and weakness, 2 points for weight loss, 1 point for poor endurance and exhaustion, and 1 point for low physical activity, respectively. The modified PF model could capture and categorize the future risk of MOF more accurately than the original model. Significant relationship between risks of MOF, falls, and death and the modified PF model was found. Compared with the original model, the modified PF model was a better fit to the data and with improved predictive accuracy. Based on a simple and practical rescoring and recategorizing algorithm, the modified PF model could predict risks of adverse outcomes more accurately than the original model, reflecting a cost-effective way. More evidence is needed to validate the modified PF model and support its application in geriatric practice.


Predictive Systems Toxicology.
In this review we address to what extent computational techniques can augment our ability to predict toxicity. The first section provides a brief history of empirical observations on toxicity dating back to the dawn of Sumerian civilization. Interestingly, the concept of dose emerged very early on, leading up to the modern emphasis on kinetic properties, which in turn encodes the insight that toxicity is not solely a property of a compound but instead depends on the interaction with the host organism. The next logical step is the current conception of evaluating drugs from a personalized medicine point of view. We review recent work on integrating what could be referred to as classical pharmacokinetic analysis with emerging systems biology approaches incorporating multiple omics data. These systems approaches employ advanced statistical analytical data processing complemented with machine learning techniques and use both pharmacokinetic and omics data. We find that such integrated approaches not only provide improved predictions of toxicity but also enable mechanistic interpretations of the molecular mechanisms underpinning toxicity and drug resistance. We conclude the chapter by discussing some of the main challenges, such as how to balance the inherent tension between the predicitive capacity of models, which in practice amounts to constraining the number of features in the models versus allowing for rich mechanistic interpretability, i.e., equipping models with numerous molecular features. This challenge also requires patient-specific predictions on toxicity, which in turn requires proper stratification of patients as regards how they respond, with or without adverse toxic effects. In summary, the transformation of the ancient concept of dose is currently successfully operationalized using rich integrative data encoded in patient-specific models.


A Comparison of the effectiveness of Mammographic Film-Screen and Standard Film-Screen in the Detection of Small Bone Fractures.
The use of mammography film-screen is limited in general radiography. The purpose of this study was to compare the effectiveness of mammographic film-screen and standard film-screen systems in the detection of small bone fractures. Radiographs were taken from patients' extremities and neck areas using mammography film-screen and standard film-screen (n=57 each). Fourteen other radiographs were taken from other views (predominantly oblique views), making a total number of 128 radiographs. Paired radiographs, taken from the same areas, were compared by two radiologists in terms of image visual sharpness, presence of bony fractures, and soft tissue injuries. The surface dose received by patients in the two systems was also compared. The radiographs taken by mammography film-screen had a statistically better visual sharpness compared to those taken by the standard film-screen system. However, there was no statistically significant difference between the diagnostic accuracy of the two systems. Mammography film-screen was able to detect only one out of 57 lesions, whereas standard film-screen system did not detec any lesion. The surface dose received by patients in mammography film-screen was higher than that in standard film-screen system. The findings of the present study suggest that mammography film-screen may be recommended as a diagnostic tool for the detection of small fractures of tinny parts of body such as fingers, hand or foot. They also suggest that mammography film-screen has no advantage over standard film-screen for radiography of thick body parts such as neck and knee.


Difficulties with neurological prognostication in a young woman with delayed-onset generalised status myoclonus after cardiac arrest due to acute severe asthma.
Neurological prognostication in cardiac arrest survivors is difficult, especially when the primary etiology is respiratory arrest. Prognostic factors designed to have zero false-positive rates to robustly confirm poor outcome are usually inadequate to rule out poor outcomes (i.e., high specificity and low sensitivity). One of the least understood prognosticators is generalised status myoclonus (GSM), with case reports confusing GSM, isolated myoclonic jerks and post-hypoxic intention myoclonus (Lance Adams syndrome [LAS]). With several prognostic indicators (including status myoclonus) having been validated in the pre-hypothermia era, their current relevance is debatable. New modalities such as brain magnetic resonance imaging (MRI) and continuous electroencephalography are being evaluated. We describe here a pregnant woman resuscitated from a cardiac arrest due to acute severe asthma, and an inability to reach a consensus based on published guidelines, with a brief overview of myoclonus, LAS and the role of MRI brain in assisting prognostication.


Spin-Holstein Models in Trapped-Ion Systems.
In this work, we highlight how trapped-ion quantum systems can be used to study generalized Holstein models, and benchmark expensive numerical calculations. We study a particular spin-Holstein model that can be implemented with arrays of ions confined by individual microtraps, and that is closely related to the Holstein model of condensed matter physics, used to describe electron-phonon interactions. In contrast to earlier proposals, we focus on simulating many-electron systems and inspect the competition between charge-density wave order, fermion pairing, and phase separation. In our numerical study, we employ a combination of complementary approaches, based on non-Gaussian variational ansatz states and matrix product states, respectively. We demonstrate that this hybrid approach outperforms standard density-matrix renormalization group calculations.


Engineered phages for electronics.
Phages are traditionally widely studied in biology and chemistry. In recent years, engineered phages have attracted significant attentions for functionalization or construction of electronic devices, due to their specific binding, catalytic, nucleating or electronic properties. To apply the engineered phages in electronics, these are a number of interesting questions: how to engineer phages for electronics? How are the engineered phages characterized? How to assemble materials with engineered phages? How are the engineered phages micro or nanopatterned? What are the strategies to construct electronics devices with engineered phages? This review will highlight the early attempts to address these questions and explore the fundamental and practical aspects of engineered phages in electronics, including the approaches for selection or expression of specific peptides on phage coat proteins, characterization of engineered phages in electronics, assembly of electronic materials, patterning of engineered phages, and construction of electronic devices. It provides the methodologies and opens up ex-cit-ing op-por-tu-ni-ties for the development of a variety of new electronic materials and devices based on engineered phages for future applications.


Descriptive analysis of individual and community factors among African American youths in urban public housing.
African American adolescents are disproportionately represented in urban public housing developments. These neighborhoods are generally characterized by high rates of poverty, crime, violence, and disorganization. Although evidence is emerging on youths in these communities, little is known about their depressive symptoms, perceived efficacy, or frequency of substance use and sex-risk behavior. Further, even less is known about their exposure to community and household violence, their parents' behavior, or their sense of connection to their communities. Using a sample of 782 African American adolescents living in public housing neighborhoods located in four large U.S. cities, this article attempts to rectify the observed gap in knowledge by presenting a descriptive overview of their self-reported depressive symptoms; self-efficacy; frequencies of delinquent and sexual-risk behavior; and alcohol, tobacco, and other drug use. The self-reported ratings of their parents' behavior as well as their exposure to community and household violence are presented. Analytic procedures include descriptive statistics and mean comparisons between genders and across research cities. Results suggest several differences between genders and across research sites. However, results are not very different from national data. Implications for social work practice are discussed.


Laboratory review: the role of gait analysis in seniors' mobility and fall prevention.
Walking is a complex motor task generally performed automatically by healthy adults. Yet, by the elderly, walking is often no longer performed automatically. Older adults require more attention for motor control while walking than younger adults. Falls, often with serious consequences, can be the result. Gait impairments are one of the biggest risk factors for falls. Several studies have identified changes in certain gait parameters as independent predictors of fall risk. Such gait changes are often too discrete to be detected by clinical observation alone. At the Basel Mobility Center, we employ the GAITRite electronic walkway system for spatial-temporal gait analysis. Although we have a large range of indications for gait analyses and several areas of clinical research, our focus is on the association between gait and cognition. Gait analysis with walking as a single-task condition alone is often insufficient to reveal underlying gait disorders present during normal, everyday activities. We use a dual-task paradigm, walking while simultaneously performing a second cognitive task, to assess the effects of divided attention on motor performance and gait control. Objective quantification of such clinically relevant gait changes is necessary to determine fall risk. Early detection of gait disorders and fall risk permits early intervention and, in the best-case scenario, fall prevention. We and others have shown that rhythmic movement training such as Jaques-Dalcroze eurhythmics, tai chi and social dancing can improve gait regularity and automaticity, thus increasing gait safety and reducing fall risk.


Physical and mental health impacts of COVID-19 on healthcare workers: a scoping review.
Coronavirus disease (COVID-19) pandemic has spread to 198 countries, with approximately 2.4 million confirmed cases and 150,000 deaths globally as of April 18. Frontline healthcare workers (HCWs) face a substantially higher risk of infection and death due to excessive COVID-19 exposure. This review aimed at summarizing the evidence of the physical and mental health impacts of COVID-19 pandemic on health-care workers (HCWs). We used the Arksey O'Malley framework to conduct a scoping review. A systematic literature search was conducted using two databases: PubMed and Google Scholar. We found 154 studies, and out of which 10 met our criteria. We collected information on the date of publication, first author's country, the title of the article, study design, study population, intervention and outcome, and key findings, and divided all research articles into two domains: physical and mental health impact. We reviewed a total of 154 articles from PubMed (126) and Google Scholar (28), of which 58 were found to be duplicate articles and were excluded. Of the remaining 96 articles, 82 were excluded after screening for eligibility, and 4 articles did not have available full texts. Ten full-text articles were reviewed and included in this study. Our findings identified the following risk factors for COVID-19-related health impact: working in a high-risk department, diagnosed family member, inadequate hand hygiene, suboptimal hand hygiene before and after contact with patients, improper PPE use, close contact with patients (≥ 12 times/day), long daily contact hours (≥ 15 h), and unprotected exposure. The most common symptoms identified amongst HCWs were fever (85%), cough (70%), and weakness (70%). Prolonged PPE usage led to cutaneous manifestations and skin damage (97%), with the nasal bridge (83%) most commonly affected site. HCWs experienced high levels of depression, anxiety, insomnia, and distress. Female HCWs and nurses were disproportionately affected. The frontline healthcare workers are at risk of physical and mental consequences directly as the result of providing care to patients with COVID-19. Even though there are few intervention studies, early data suggest implementation strategies to reduce the chances of infections, shorter shift lengths, and mechanisms for mental health support could reduce the morbidity and mortality amongst HCWs.


Frequency modulation of entorhinal cortex neuronal activity drives distinct frequency-dependent states of brain-wide dynamics.
Human neuroimaging studies have shown that, during cognitive processing, the brain undergoes dynamic transitions between multiple, frequency-tuned states of activity. Although different states may emerge from distinct sources of neural activity, it remains unclear whether single-area neuronal spiking can also drive multiple dynamic states. In mice, we ask whether frequency modulation of the entorhinal cortex activity causes dynamic states to emerge and whether these states respond to distinct stimulation frequencies. Using hidden Markov modeling, we perform unsupervised detection of transient states in mouse brain-wide fMRI fluctuations induced via optogenetic frequency modulation of excitatory neurons. We unveil the existence of multiple, frequency-dependent dynamic states, invisible through standard static fMRI analyses. These states are linked to different anatomical circuits and disrupted in a frequency-dependent fashion in a transgenic model of cognitive disease directly related to entorhinal cortex dysfunction. These findings provide cross-scale insight into basic neuronal mechanisms that may underpin flexibility in brain-wide dynamics.


Syndrome produced by lesions of the amygdala in monkeys (Macaca mulatta).
Behavioral effects of subtotal amygdaloid lesions were investigated in an attempt to dissociate some of the abnormalities seen after total amygdalectomy. Twelve monkeys received bilateral stereotaxic lesions centered in the basolateral amygdala, lateral amygdala, dorsal amygdala, or the temporal white matter lying adjacent to the lateral amygdala. These monkeys were compared with others with control operations. The control monkeys then received total amygdaloid lesions (AMX). The AMX monkeys exhibited the typical amygdaloid syndrome of hypoemotionality, meat eating, coprophagia, and excessive exploration. In contrast, the monkeys with subtotal amygdaloid lesions would not eat meat or feces, though they were more willing than control monkeys to investigate inanimate objects. Although minor changes in affect were observed, the extreme emotional changes seen after total amygdalectomy were found only in the monkey with the largest subtotal lesion. Only those animals that were hypoemotional showed a deficit in learning successive reversals of an object discrimination. This close association suggests that both the hypoemotionality and the successive reversal deficit arise from the same underlying dysfunction.


B cell receptor-mediated uptake of CD1d-restricted antigen augments antibody responses by recruiting invariant NKT cell help in vivo.
Highly regulated activation of B cells is required for the production of specific antibodies necessary to provide protection from pathogen infection. This process is initiated by specific recognition of antigen through the B cell receptor (BCR), leading to early intracellular signaling followed by the late recruitment of T cell help. In this study we demonstrate that specific BCR uptake of CD1d-restricted antigens represents an effective means of enhancing invariant natural killer T (iNKT)-dependent B cell responses in vivo. This mechanism is effective over a wide range of antigen affinities but depends on exceeding a tightly regulated avidity threshold necessary for BCR-mediated internalization and CD1d-dependent presentation of particulate antigenic lipid. Subsequently, iNKT cells provide the help required for stimulating B cell proliferation and differentiation. iNKT-stimulated B cells develop within extrafollicular foci and mediate the production of high titers of specific IgM and early class-switched antibodies. Thus, we have demonstrated that in response to particulate antigenic lipids iNKT cells are recruited for the assistance of B cell activation, resulting in the enhancement of specific antibody responses. We propose that such a mechanism may operate to potentiate adaptive immune responses against pathogens in vivo.


How should laboratories communicate with primary care? Obtaining general practitioners' views.
Recognising the importance of communication with our primary care colleagues, focus groups were held with GPs to determine how they perceived the current lines of communication with their local microbiology laboratory and the PHLS, and how they could be improved. Focus groups were held in Plymouth, Gloucester, Bristol and Hereford. Between four and 10 GPs and/or PCG Board members attended each workshop. The modes of communication i.e. websites, face-to-face contact, laboratory reporting, telephone advice, newsletters, guidance and surveillance were discussed. Microbiology websites should be user friendly, with clear labelling as to whom the page is directed. They should contain locally relevant data, antibiotic guidance and information leaflets. Despite great variation in laboratory reporting protocols GPs were mostly happy with reports received. Results, especially serology, should contain a clear conclusion and could refer to a website for further information. Electronic reporting was enthusiastically awaited. All GPs felt they had excellent access to telephone advice. GPs would value data and guidance on their use of diagnostic tests. These workshops highlight the variation in laboratory reporting protocols that should be addressed. Website development for GPs should include locally relevant data. GPs would value details of their laboratory use and costs.


NAction! How Can Neuraminidase-Based Immunity Contribute to Better Influenza Virus Vaccines?
Neuraminidase is one of the two surface glycoproteins of influenza A and B viruses. It has enzymatic activity that cleaves terminal sialic acid from glycans, and that activity is essential at several points in the virus life cycle. While neuraminidase is a major target for influenza antivirals, it is largely ignored in vaccine development. Current inactivated influenza virus vaccines might contain neuraminidase, but the antigen quantity and quality are varied and not standardized. While there are data that show a protective role of anti-neuraminidase immunity, many questions remain unanswered. These questions, among others, concern the targeted epitopes or antigenic sites, the potential for antigenic drift, and, connected to that, the breadth of protection, differences in induction of immune responses by vaccination versus infection, mechanisms of protection, the role of mucosal antineuraminidase antibodies, stability, and the immunogenicity of neuraminidase in vaccine formulations. Reagents for analysis of neuraminidase-based immunity are scarce, and assays are not widely used for clinical studies evaluating vaccines. However, efforts to better understand neuraminidase-based immunity have been made recently. A neuraminidase focus group, NAction!, was formed at a Centers of Excellence for Influenza Research and Surveillance meeting at the National Institutes of Health in Bethesda, MD, to promote research that helps to understand neuraminidase-based immunity and how it can contribute to the design of better and broadly protective influenza virus vaccines. Here, we review open questions and knowledge gaps that have been identified by this group and discuss how the gaps can be addressed, with the ultimate goal of designing better influenza virus vaccines.