A defined metabolic state in pre B cells governs B-cell development and is counterbalanced by Swiprosin-2/EFhd1.
B-cell development in the bone marrow comprises proliferative and resting phases in different niches. We asked whether B-cell metabolism relates to these changes. Compared to pro B and small pre B cells, large pre B cells revealed the highest glucose uptake and ROS but not mitochondrial mass, whereas small pre B cells exhibited the lowest mitochondrial membrane potential. Small pre B cells from Rag1-/-;33.C9 μ heavy chain knock-in mice revealed decreased glycolysis (ECAR) and mitochondrial spare capacity compared to pro B cells from Rag1-/- mice. We were interested in the step regulating this metabolic switch from pro to pre B cells and uncovered that Swiprosin-2/EFhd1, a Ca2+-binding protein of the inner mitochondrial membrane involved in Ca2+-induced mitoflashes, is expressed in pro B cells, but downregulated by surface pre B-cell receptor expression. Knockdown and knockout of EFhd1 in 38B9 pro B cells decreased the oxidative phosphorylation/glycolysis (OCR/ECAR) ratio by increasing glycolysis, glycolytic capacity and reserve. Prolonged expression of EFhd1 in EFhd1 transgenic mice beyond the pro B cell stage increased expression of the mitochondrial co-activator PGC-1α in primary pre B cells, but reduced mitochondrial ATP production at the pro to pre B cell transition in IL-7 cultures. Transgenic EFhd1 expression caused a B-cell intrinsic developmental disadvantage for pro and pre B cells. Hence, coordinated expression of EFhd1 in pro B cells and by the pre BCR regulates metabolic changes and pro/pre B-cell development.


Digital Identity: The effect of trust and reputation information on user judgement in the Sharing Economy.
The Sharing Economy (SE) is a growing ecosystem focusing on peer-to-peer enterprise. In the SE the information available to assist individuals (users) in making decisions focuses predominantly on community-generated trust and reputation information. However, how such information impacts user judgement is still being understood. To explore such effects, we constructed an artificial SE accommodation platform where we varied the elements related to hosts' digital identity, measuring users' perceptions and decisions to interact. Across three studies, we find that trust and reputation information increases not only the users' perceived trustworthiness, credibility, and sociability of hosts, but also the propensity to rent a private room in their home. This effect is seen when providing users both with complete profiles and profiles with partial user-selected information. Closer investigations reveal that three elements relating to the host's digital identity are sufficient to produce such positive perceptions and increased rental decisions, regardless of which three elements are presented. Our findings have relevant implications for human judgment and privacy in the SE, and question its current culture of ever increasing information-sharing.


Violations. How Nature Circumvents the Woodward-Hoffmann Rules and Promotes the Forbidden Conrotatory 4n + 2 Electron Electrocyclization of Prinzbach's Vinylogous Sesquifulvalene.
Woodward and Hoffmann, in their treatise on orbital symmetry in 1969, stated "Violations. There are none!" Prinzbach reported in 1978 that the electrocyclization of vinylogous sesquifulvalene occurs exclusively through the Woodward-Hoffmann orbital-symmetry-forbidden 14π-electron conrotatory pathway, despite the availability of a variety of orbital-symmetry-allowed processes. Prinzbach later demonstrated that an 18π-electron homologue exhibits the same forbidden behavior. And yet, the analogous vinylogous pentafulvalene and heptafulvalene both follow the orbital symmetry rules, each proceeding through its allowed conrotatory 12π and 16π process, respectively. We report the investigation of these reactions with ωB97X-D DFT. The physical origins of the flagrant Prinzbach violations of the Woodward-Hoffmann orbital symmetry selection rules have now been elucidated by these calculations in conjunction with extensive analyses and comparisons to electrocyclizations that obey the Woodward-Hoffmann rules. This remarkable reversal of the Rules (the 14π-electron-forbidden process is found to be 11 kcal/mol more energetically facile than the allowed process) occurs due to the high degree of polarization of this hydrocarbon, such that conrotatory electrocyclization of vinylogous sesquifulvalene behaves like a cyclopentadienide combining with a tropylium. These results are compared to other forbidden pericyclic processes driven by steric constraints and strain release or by diradical character of the reactants that facilitates the formation of diradical transition states for symmetry-forbidden reactions. We predict how strong donor-acceptor substitution can modify nodal properties to level the difference between allowed and forbidden electrocyclic reaction barriers, and we provide computational predictions of two such cases.


Tenancy, Marriage, and the Boll Weevil Infestation, 1892-1930.
In the early twentieth century, the cotton-growing regions of the U.S. South were dominated by families of tenant farmers. Tenant farming created opportunities and incentives for prospective tenants to marry at young ages. These opportunities and incentives especially affected African Americans, who had few alternatives to working as tenants. Using complete-count Census of Population data from 1900-1930 and Census of Agriculture data from 1889-1929, we find that increases in tenancy over time increased the prevalence of marriage among young African Americans. We then study how marriage was affected by one of the most notorious disruptions to southern agriculture at the turn of the century: the boll weevil infestation of 1892-1922. Using historical Department of Agriculture maps, we show that the boll weevil's arrival reduced the share of farms worked by tenants as well as the share of African Americans who married at young ages. When the boll weevil infestation altered African Americans' opportunities and incentives to marry, the share of African Americans who married young fell accordingly. Our results provide new evidence about the effect of economic and political institutions on demographic transformations.


Optical surface waves over metallo-dielectric nanostructures: Sommerfeld integrals revisited.
The asymptotic closed-form solution to the fundamental diffraction problem of a linear horizontal Hertzian dipole radiating over the metallo-dielectric interface is provided. For observation points just above the interface, we confirm that the total surface near-field is the sum of two components: a long-range surface plasmon polariton and a short-range radiative cylindrical wave. The relative phases, amplitudes and damping functions of each component are quantitatively elucidated through simple analytic expressions for the entire range of propagation: near and asymptotic. Validation of the analytic solution is performed by comparing the predictions of a dipolar model with recently published data.


SARS-CoV-2 Nonstructural Proteins 3 and 4 tune the Unfolded Protein Response.
Coronaviruses (CoV), including SARS-CoV-2, modulate host proteostasis through activation of stress-responsive signaling pathways such as the Unfolded Protein Response (UPR), which remedies misfolded protein accumulation by attenuating translation and increasing protein folding capacity. While CoV nonstructural proteins (nsps) are essential for infection, little is known about the role of nsps in modulating the UPR. We characterized the impact of SARS-CoV-2 nsp4, a key driver of replication, on the UPR using quantitative proteomics to sensitively detect pathway-wide upregulation of effector proteins. We find nsp4 preferentially activates the ATF6 and PERK branches of the UPR. Previously, we found an N-terminal truncation of nsp3 (nsp3.1) can suppress pharmacological ATF6 activation. To determine how nsp3.1 and nsp4 tune the UPR, their co-expression demonstrated that nsp3.1 suppresses nsp4-mediated PERK, but not ATF6 activation. Re-analysis of SARS-CoV-2 infection proteomics data revealed time-dependent activation of PERK targets early in infection, which subsequently fades. This temporal regulation suggests a role for nsp3 and nsp4 in tuning the PERK pathway to attenuate host translation beneficial for viral replication while avoiding later apoptotic signaling caused by chronic activation. This work furthers our understanding of CoV-host proteostasis interactions and highlights the power of proteomic methods for systems-level analysis of the UPR.


Human umbilical cord blood-derived mesenchymal stem cells upregulate myelin basic protein in shiverer mice.
Human umbilical cord blood is a rich source of pluripotent mesenchymal stem cells and possesses significant advantages over other stem cell sources such as the embryo and bone marrow. In the present study, we aimed to investigate the potential of human umbilical cord blood-derived pluripotent mesenchymal stem cells (hUCB) to myelinate the axons of shiverer mice brains. We also investigated the effect of hUCB treatment on regulation of myelin basic protein in vitro in PC-12 cells, which are normally not myelinated. The results of our study clearly demonstrated that hUCB survive and migrate in vivo and has the potential to myelinate shiverer mice brains. The expression level of myelin basic protein, a major component of the myelin sheath, has been significantly increased in vivo and in vitro as revealed by Western blot, reverse transcription (RT)-polymerase chain reaction, immunohistochemistry, immunocytochemistry, and fluorescent in situ hybridization results. Further, transmission electron microscopic images of hUCB-treated shiverer mice brains showed several layers of myelin around the axons compared with a thin and fragmented layer of myelin in untreated animals. Moreover, the frequency of shivering was diminished 1 month after hUCB treatment in shiverer mice. Our results strongly indicated that hUCB transplantation could be an effective means of treating demyelinating or hypomyelinating disorders.


Results from a community-based smoking cessation treatment program for LGBT smokers.
Little is known about lesbian, gay, bisexual, and transgender (LGBT) people's response to smoking cessation interventions. This descriptive study examined the benefits of a community-based, culturally tailored smoking cessation treatment program for LGBT smokers. A total of N = 198 LGBT individuals recruited from clinical practice and community outreach participated in group-based treatment. Sessions were based on the American Lung Association's "Freedom from Smoking Program" (ALA-FFS) and were tailored to LGBT smokers' needs. Seven-day smoking point prevalence abstinence served as the primary outcome. Participants (M age = 40.5) were mostly White (70.4%) and male (60.5%) and had at least a college degree (58.4%). Forty-four percent scored in the moderate range on the Fagerström Test for Nicotine Dependence pretreatment, and 42.4% completed treatment (≥75% sessions). Higher educational attainment and use of nicotine replacement therapy (NRT) were associated with treatment completion. Self-reported quit rates were 32.3% at posttreatment assessment. Treatment attendance (OR = 2.45), use of NRT (OR = 4.24), and lower nicotine dependency (OR = 0.73) were positively associated with quitting smoking. Results suggest the benefits of offering LGBT smokers culturally tailored smoking cessation treatments. Future research could improve outcomes by encouraging treatment attendance and promoting NRT uptake.


Do community and autonomy moral violations elicit different emotions?
According to one important set of theories, different domains of immorality are linked to different discrete emotions-panculturally. Violations against the community elicit contempt, whereas violations against an individual elicit anger. To test this theory, American, Indian and Japanese participants (N = 480) indicated contempt and anger reactions (with verbal rating and face selection) to both the types of immorality. To remedy method problems in previous research, community and autonomy violations were created for the same story-frame, by varying the target to be either the community or an individual. Community and autonomy violations did not differ significantly in the emotion elicited: overall, both types of violations elicited more anger than contempt (and more negative emotion of any kind than positive emotion). By verbal rating, Americans and Indians reported more anger than contempt for both types of violation, whereas Japanese reported more contempt than anger for both types. By face selection, the three cultural groups selected anger more than contempt for both types of violation. The results speak against defining distinct domains of morality by their association with distinct emotions.


Cholesterol Sulfonation Enzyme, SULT2B1b, Modulates AR and Cell Growth Properties in Prostate Cancer.
Cholesterol accumulates in prostate lesions and has been linked to prostate cancer incidence and progression. However, how accumulated cholesterol contributes to prostate cancer development and progression is not completely understood. Cholesterol sulfate (CS), the primary sulfonation product of cholesterol sulfotransferase (SULT2B1b), accumulates in human prostate adenocarcinoma and precancerous prostatic intraepithelial neoplasia (PIN) lesions compared with normal regions of the same tissue sample. Given the enhanced accumulation of CS in these lesions, it was hypothesized that SULT2B1b-mediated production of CS provides a growth advantage to these cells. To address this, prostate cancer cells with RNAi-mediated knockdown (KD) of SULT2B1b were used to assess the impact on cell growth and survival. SULT2B1b is expressed and functional in a variety of prostate cells, and the data demonstrate that SULT2B1b KD, in LNCaP and other androgen-responsive (VCaP and C4-2) cells, results in decreased cell growth/viability and induces cell death. SULT2B1b KD also decreases androgen receptor (AR) activity and expression at mRNA and protein levels. While AR overexpression has no impact on SULT2B1b KD-mediated cell death, the addition of exogenous androgen is able to partially rescue the growth inhibition induced by SULT2B1b KD in LNCaP cells. These results suggest that SULT2B1b positively regulates the AR either through alterations in ligand availability or by interaction with critical coregulators that influence AR activity. These findings provide evidence that SULT2B1b is a novel regulator of AR activity and cell growth in prostate cancer and should be further investigated for therapeutic potential. Mol Cancer Res; 14(9); 776-86. ©2016 AACR.


Rapid and Efficient Generation of Transgene-Free iPSC from a Small Volume of Cryopreserved Blood.
Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.


Mouse models of telomere dysfunction phenocopy skeletal changes found in human age-related osteoporosis.
A major medical challenge in the elderly is osteoporosis and the high risk of fracture. Telomere dysfunction is a cause of cellular senescence and telomere shortening, which occurs with age in cells from most human tissues, including bone. Telomere defects contribute to the pathogenesis of two progeroid disorders characterized by premature osteoporosis, Werner syndrome and dyskeratosis congenital. It is hypothesized that telomere shortening contributes to bone aging. We evaluated the skeletal phenotypes of mice with disrupted telomere maintenance mechanisms as models for human bone aging, including mutants in Werner helicase (Wrn(-/-)), telomerase (Terc(-/-)) and Wrn(-/-)Terc(-/-) double mutants. Compared with young wild-type (WT) mice, micro-computerized tomography analysis revealed that young Terc(-/-) and Wrn(-/-)Terc(-/-) mice have decreased trabecular bone volume, trabecular number and trabecular thickness, as well as increased trabecular spacing. In cortical bone, young Terc(-/-) and Wrn(-/-)Terc(-/-) mice have increased cortical thinning, and increased porosity relative to age-matched WT mice. These trabecular and cortical changes were accelerated with age in Terc(-/-) and Wrn(-/-)Terc(-/-) mice compared with older WT mice. Histological quantification of osteoblasts in aged mice showed a similar number of osteoblasts in all genotypes; however, significant decreases in osteoid, mineralization surface, mineral apposition rate and bone formation rate in older Terc(-/-) and Wrn(-/-)Terc(-/-) bone suggest that osteoblast dysfunction is a prominent feature of precocious aging in these mice. Except in the Wrn(-/-) single mutant, osteoclast number did not increase in any genotype. Significant alterations in mechanical parameters (structure model index, degree of anistrophy and moment of inertia) of the Terc(-/-) and Wrn(-/-)Terc(-/-) femurs compared with WT mice were also observed. Young Wrn(-/-)Terc(-/-) mice had a statistically significant increase in bone-marrow fat content compared with young WT mice, which remained elevated in aged double mutants. Taken together, our results suggest that Terc(-/-) and Wrn(-/-)Terc(-/-) mutants recapitulate the human bone aging phenotype and are useful models for studying age-related osteoporosis.


Meaning and consequence of the coexistence of competitive hydrogen bond/salt forms on the dissociation orientation of non-covalent complexes.
Non-covalent complexes (NCC) between hexose monophosphates (HexP) and arginine (R) were analyzed using ESI MS and MS/MS in negative mode under different (hard, HC and soft, SC) desolvation conditions. High resolution mass spectrometry (HRMS) revealed the presence of different ionic species, namely, homo- and heteromultimers of R and HexP. Deprotonated heterodimers and corresponding sodiated species were enhanced under HC likely due to a decrease in available charge number associated with the reduction of H+/Na+ exchange. The quantum calculations showed that the formation of covalent systems is very little exothermic, therefore, such systems are disfavored. Desolvation dependent CID spectra of deprotonated [(HexP+R)‒H]- complexes demonstrated that they can exist within the hydrogen bond (HB) and salt bridge (SB) forms, yielding either NCC separation or covalent bond cleavages, respectively. Although HB forms are the main species, they cannot survive under HC; therefore, the minor SB forms became detectable. Energy-resolved mass spectrometry (ERMS) experiments revealed diagnostic fragment ions from both SB and HB forms, providing evidence that these isomeric forms are inconvertible. SB formation should result from the ionic interactions of highly acidic group of HexP with strongly basic guanidine group of arginine and thus requires an arginine zwitterion (ZW) form. This was confirmed by quantum calculations. Ion-ion interactions are significantly affected by the presence of sodium cation as demonstrated by the fragmentation patterns of sodiated complex species. Regarding CID data, only SB between protonated amino group of R and deprotonated phosphate group of HexP could be suggested, but the primary amine is not enough basic then, the SB must be fleeting. Nevertheless, the observation of the covalent bond cleavages suggests the presence of structures with a free negative charge able to induce fragmentations. Indeed, according to quantum calculations, solvated salt (SS) systems involving Na+/COO- salt solvated by neutral phosphate and negative charge on sugar ring are preferentially formed.


Carcinoma of the uterine cervix: a review of its pathology and commentary on the problem in Malaysians.
Since its recognition about 150 years ago, there has been much progress in the understanding of the pathogenesis, prevention, early detection and management of carcinoma of the uterine cervix. Important historical landmarks include the (1) recognition of pre-invasive and pre-clinical lesions, and the devise of various systems for reporting these lesions, (2) improvements in diagnostic techniques particularly colposcopy, (3) advent of therapeutic procedures (electrocoagulation, cryotherapy, laser therapy and loop electrosurgical excision), and (4) recognition of the aetiological relationship between the human papillomavirus and cervical neoplasia. The susceptibility of the cervical transformation zone to malignant change is now well recognised. The WHO classification system remains the one most commonly utilised for histological reporting of cervical cancers. In the recent 1994 update, cervical carcinoma is divided into 3 main categories: squamous cell carcinoma, adenocarcinoma and other epithelial tumours. Squamous cell carcinoma (60-80%) predominates among invasive cervical carcinoma. Recognised variants include verrucous, warty (condylomatous), papillary squamous (transitional) and lymphoepithelioma-like carcinoma. Adenocarcinoma (5-15% of invasive carcinomas) shows an increasing trend in young females. Like its squamous counterpart, preinvasive and microinvasive versions are known. Variants such as mucinous, endometrioid, clear cell, mesonephric, serous, villoglandular and minimal deviation carcinoma are now defined. Adenosquamous carcinoma (5-25%), adenoid-cystic, adenoid-basal, neuroendocrine and undifferentiated carcinomas constitute other epithelial tumours of the cervix. The management of invasive cervical carcinoma remains heavily dependent on its stage. The FIGO staging system remains the most widely used. The 1995 update provides more definite criteria in subdividing stage IA tumours by delimiting stromal invasion of stage IA1 lesions to a maximum depth of 3 mm and a horizontal axis of 7 mm. In Malaysia, an appreciation of the cervical carcinoma problem has to take into consideration the population at risk, its multi-ethnicity, its socio-economic and geographical diversities and the constraints of the health care system. Females form 48.9% of the Malaysian population. 52.9% of them are in the sexually active age group of 15-50 years, indicating a significant population at risk for cervical carcinoma. Cervical carcinoma was the third most common cause of death due to solid tumours among Malaysian females in 1995 following carcinoma of the breast and respiratory tract. East Malaysia is predominantly rural with many communities having limited modern facilities. Such areas imply a lower educational and socio-economic status, raising the worry of a population at higher risk for developing cervical carcinoma. The population: doctor for Malaysia of 2153:1 compares poorly with nearby Singapore. Besides a shortage of doctors, there is also an uneven distribution of doctors, resulting in a ratio in East Malaysia of > 4000:1. Although Malaysia does not have a national cervical cancer-screening programme, many action plans and cancer awareness campaigns have been launched throughout the years, which appear to have made an impact as evidenced by the decreasing mortality rates from cervical carcinoma. Another interesting feature of cervical carcinoma in Malaysia relates to its multiethnic population. In Malaysian Chinese and Malay females, the prevalence of cervical carcinoma ranks second to breast cancer whereas the pattern is reversed in Malaysian Indian females. Studies into its aetiology and pathogenesis are being undertaken and may shed more light on this matter.


Phosphoproteome analysis of Drosophila melanogaster embryos.
Protein phosphorylation is a key regulatory event in most cellular processes and development. Mass spectrometry-based proteomics provides a framework for the large-scale identification and characterization of phosphorylation sites. Here, we used a well-established phosphopeptide enrichment and identification strategy including the combination of strong cation exchange chromatography, immobilized metal affinity chromatography, and high-accuracy mass spectrometry instrumentation to study phosphorylation in developing Drosophila embryos. In total, 13,720 different phosphorylation sites were discovered from 2702 proteins with an estimated false-discovery rate (FDR) of 0.63% at the peptide level. Because of the large size of the data set, both novel and known phosphorylation motifs were extracted using the Motif-X algorithm, including those representative of potential ordered phosphorylation events.


The impact of motivational therapy in the management of enuretic children.
The aim of this study is to determine the impact of a motivational therapy (MT) on the outcomes for individuals diagnosed with nocturnal enuresis (NE). We enrolled 158 patients with NE referred to the Service of Pediatrics, Campus Bio-Medico University Hospital of Rome, from January 2013 to September 2017. Of these, 21 were excluded because they didn't meet the inclusion criteria. The study was carried out in compliance with the Helsinki Declaration. A hundred and thirty seven enuretic patients [100 (72.9%) male and 37 (27.1%)] female patients with a median age of 8.8 years were included in the study. The patients were assigned to receive pharmacological therapy with desmopressin (dDAVP) (G1) (n=51), MT (G2) (n=33) and both of them (G3) (n=53). The three groups were homogeneous, with no significant differences in gender, age and family history of NE. In G1, 30/51 (58.82%) children achieved response vs. 1/33 (3.0%) children in G2 vs. 35/53 (66.04%) children in G3. About these results, The differences between G2 vs. G1 (p<0.01) and vs. G3 (p<0.01) were statistically significant while the difference between G1 vs G3 was not statistically significant (p=0.45). Our study underlines the importance of MT in the management of NE and highlights the safety of treatment and the positive effect of MT on the compliance and the adherence to pharmacological therapy. Considering the importance of the impact of such evidence on children's lifestyle, we expect that further study with a larger sample size may confirm our hypothesis.


An auditory multiclass brain-computer interface with natural stimuli: Usability evaluation with healthy participants and a motor impaired end user.
Brain-computer interfaces (BCIs) can serve as muscle independent communication aids. Persons, who are unable to control their eye muscles (e.g., in the completely locked-in state) or have severe visual impairments for other reasons, need BCI systems that do not rely on the visual modality. For this reason, BCIs that employ auditory stimuli were suggested. In this study, a multiclass BCI spelling system was implemented that uses animal voices with directional cues to code rows and columns of a letter matrix. To reveal possible training effects with the system, 11 healthy participants performed spelling tasks on 2 consecutive days. In a second step, the system was tested by a participant with amyotrophic lateral sclerosis (ALS) in two sessions. In the first session, healthy participants spelled with an average accuracy of 76% (3.29 bits/min) that increased to 90% (4.23 bits/min) on the second day. Spelling accuracy by the participant with ALS was 20% in the first and 47% in the second session. The results indicate a strong training effect for both the healthy participants and the participant with ALS. While healthy participants reached high accuracies in the first session and second session, accuracies for the participant with ALS were not sufficient for satisfactory communication in both sessions. More training sessions might be needed to improve spelling accuracies. The study demonstrated the feasibility of the auditory BCI with healthy users and stresses the importance of training with auditory multiclass BCIs, especially for potential end-users of BCI with disease.


Wnt-7a in feather morphogenesis: involvement of anterior-posterior asymmetry and proximal-distal elongation demonstrated with an in vitro reconstitution model.
How do vertebrate epithelial appendages form from the flat epithelia? Following the formation of feather placodes, the previously radially symmetrical primordia become anterior-posterior (A-P) asymmetrical and develop a proximo-distal (P-D) axis. Analysis of the molecular heterogeneity revealed a surprising parallel of molecular profiles in the A-P feather buds and the ventral-dorsal (V-D) Drosophila appendage imaginal discs. The functional significance was tested with an in vitro feather reconstitution model. Wnt-7a expression initiated all over the feather tract epithelium, intensifying as it became restricted first to the primordia domain, then to an accentuated ring pattern within the primordia border, and finally to the posterior bud. In contrast, sonic hedgehog expression was induced later as a dot within the primordia. RCAS was used to overexpress Wnt-7a in reconstituted feather explants derived from stage 29 dorsal skin to further test its function in feather formation. Control skin formed normal elongated, slender buds with A-P orientation, but Wnt-7a overexpression led to plateau-like skin appendages lacking an A-P axis. Feathers in the Wnt-7a overexpressing skin also had inhibited elongation of the P-D axes. This was not due to a lack of cell proliferation, which actually was increased although randomly distributed. While morphogenesis was perturbed, differentiation proceeded as indicated by the formation of barb ridges. Wnt-7a buds have reduced expression of anterior (Tenascin) bud markers. Middle (Notch-1) and posterior bud markers including Delta-1 and Serrate-1 were diffusely expressed. The results showed that ectopic Wnt-7a expression enhanced properties characteristic of the middle and posterior feather buds and suggest that P-D elongation of vertebrate skin appendages requires balanced interactions between the anterior and posterior buds.


Use of the meganuclease I-SceI of Saccharomyces cerevisiae to select for gene deletions in actinomycetes.
The search for new natural products is leading to the isolation of novel actinomycete species, many of which will ultimately require genetic analysis. Some of these isolates will likely exhibit low intrinsic frequencies of homologous recombination and fail to sporulate under laboratory conditions, exacerbating the construction of targeted gene deletions and replacements in genetically uncharacterised strains. To facilitate the genetic manipulation of such species, we have developed an efficient method to generate gene or gene cluster deletions in actinomycetes by homologous recombination that does not introduce any other changes to the targeted organism's genome. We have synthesised a codon optimised I-SceI gene for expression in actinomycetes that results in the production of the yeast I-SceI homing endonuclease which produces double strand breaks at a unique introduced 18 base pair recognition sequence. Only those genomes that undergo homologous recombination survive, providing a powerful selection for recombinants, approximately half of which possess the desired mutant genotype. To demonstrate the efficacy and efficiency of the system, we deleted part of the gene cluster for the red-pigmented undecylprodiginine complex of compounds in Streptomyces coelicolor M1141. We believe that the system we have developed will be broadly applicable across a wide range of actinomycetes.


Clinical Effects of Dry Needling Among Asymptomatic Individuals With Hamstring Tightness: A Randomized Controlled Trial.
Randomized controlled trial. The aim of this study was to determine the effects of dry needling on hamstring extensibility and functional performance tests among asymptomatic individuals with hamstring muscle tightness. Dry needling has been shown to increase range of motion in the upper quarter and may have similar effects in the lower quarter. 27 subjects with hamstring extensibility deficits were randomly assigned to side of treatment (dominant or nondominant) and group (blunt needling or dry needling). The first session included measurement of hamstring extensibility and performance on 4 unilateral hop tests, instruction in home hamstring stretching exercises and needling distal to the ischial tuberosity and midbellies of the medial and lateral hamstrings. A second session, 3-5 days following the first session, included outcome measures and a second needling intervention, and a third session, 4-6 weeks following the first session, included outcome measures only. A 2 × 3 × 2 ANOVA was used to statistically analyze the data. Hamstring extensibility showed a significant side × time interaction (P < .05). The single hop for distance, timed 6-meter hop, and the crossover hop test had a significant main effect of time (P < .05). The triple hop for distance showed a significant side × time × group interaction (P < .05). It does not appear dry needling results in increased extensibility beyond that of stretching alone in asymptomatic individuals. Our study findings suggest that dry needling may improve certain dimensions of functional performance, although no clear conclusion can be made. Intervention, level 2b.


Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells.
Recent evidence indicates there is a role for small membrane vesicles, including exosomes, as vehicles for intercellular communication. Exosomes secreted by most cell types can mediate transfer of proteins, mRNAs, and microRNAs, but their role in the transmission of infectious agents is less established. Recent studies have shown that hepatocyte-derived exosomes containing hepatitis C virus (HCV) RNA can activate innate immune cells, but the role of exosomes in the transmission of HCV between hepatocytes remains unknown. In this study, we investigated whether exosomes transfer HCV in the presence of neutralizing antibodies. Purified exosomes isolated from HCV-infected human hepatoma Huh7.5.1 cells were shown to contain full-length viral RNA, viral protein, and particles, as determined by RT-PCR, mass spectrometry, and transmission electron microscopy. Exosomes from HCV-infected cells were capable of transmitting infection to naive human hepatoma Huh7.5.1 cells and establishing a productive infection. Even with subgenomic replicons, lacking structural viral proteins, exosome-mediated transmission of HCV RNA was observed. Treatment with patient-derived IgGs showed a variable degree of neutralization of exosome-mediated infection compared with free virus. In conclusion, this study showed that hepatic exosomes can transmit productive HCV infection in vitro and are partially resistant to antibody neutralization. This discovery sheds light on neutralizing antibodies resistant to HCV transmission by exosomes as a potential immune evasion mechanism.


Speed cells in the medial entorhinal cortex.
Grid cells in the medial entorhinal cortex have spatial firing fields that repeat periodically in a hexagonal pattern. When animals move, activity is translated between grid cells in accordance with the animal's displacement in the environment. For this translation to occur, grid cells must have continuous access to information about instantaneous running speed. However, a powerful entorhinal speed signal has not been identified. Here we show that running speed is represented in the firing rate of a ubiquitous but functionally dedicated population of entorhinal neurons distinct from other cell populations of the local circuit, such as grid, head-direction and border cells. These 'speed cells' are characterized by a context-invariant positive, linear response to running speed, and share with grid cells a prospective bias of ∼50-80 ms. Our observations point to speed cells as a key component of the dynamic representation of self-location in the medial entorhinal cortex.


Effects of the central pair apparatus on microtubule sliding velocity in sea urchin sperm flagella.
To produce oscillatory bending movement in cilia and flagella, the activity of dynein arms must be regulated. The central-pair microtubules, located at the centre of the axoneme, are often thought to be involved in the regulation, but this has not been demonstrated definitively. In order to determine whether the central-pair apparatus are directly involved in the regulation of the dynein arm activity, we analyzed the movement of singlet microtubules that were brought into contact with dynein arms on bundles of doublets obtained by sliding disintegration of elastase-treated flagellar axonemes. An advantage of this new assay system was that we could distinguish the bundles that contained the central pair apparatus from those that did not, the former being clearly thicker than the latter. We found that microtubule sliding occurred along both the thinner and the thicker bundles, but its velocity differed between the two kinds of bundles in an ATP concentration dependent manner. At high ATP concentrations, such as 0.1 and 1 mM, the sliding velocity on the thinner bundles was significantly higher than that on the thicker bundles, while at lower ATP concentrations the sliding velocity did not change between the thinner and the thicker bundles. We observed similar bundle width-related differences in sliding velocity after removal of the outer arms. These results provide first evidence suggesting that the central pair and its associated structures may directly regulate the activity of the inner (and probably also the outer) arm dynein.


Neuronal calcium signaling, mitochondrial dysfunction, and Alzheimer's disease.
Alzheimer's disease (AD) is the most common neurodegenerative disorder among the aged worldwide. AD is characterized by extensive synaptic and neuronal loss that leads to impaired memory and cognitive decline. The cause of AD is not completely understood and no effective therapy has been developed. The accumulation of toxic amyloid-beta42 (Abeta42) peptide oligomers and aggregates in AD brain has been proposed to be primarily responsible for the pathology of the disease, an idea dubbed the 'amyloid hypothesis' of AD etiology. In addition to the increase in Abeta42 levels, disturbances in neuronal calcium (Ca2+) signaling and alterations in expression levels of Ca2+ signaling proteins have been observed in animal models of familial AD and in studies of postmortem brain samples from sporadic AD patients. Based on these data, the 'Ca2+ hypothesis of AD' has been proposed. In particular, familial AD has been linked with enhanced Ca2+ release from the endoplasmic reticulum and elevated cytosolic Ca2+ levels. The augmented cytosolic Ca2+ levels can trigger signaling cascades that affect synaptic stability and function and can be detrimental to neuronal health, such as activation of calcineurin and calpains. Here we review the latest results supporting the 'Ca2+ hypothesis' of AD pathogenesis. We further argue that over time, supranormal cytosolic Ca2+ signaling can impair mitochondrial function in AD neurons. We conclude that inhibitors and stabilizers of neuronal Ca2+ signaling and mitochondrial function may have therapeutic potential for AD treatment. We also discuss latest and planned AD therapeutic trials of agents targeting Ca2+ channels and mitochondria.


Structural insights into ISRIB, a memory-enhancing inhibitor of the integrated stress response.
The integrated stress response (ISR) regulates protein synthesis under conditions of stress. Phosphorylation of translation initiation factor eIF2 by stress-sensing kinases converts eIF2 from substrate to competitive inhibitor of its dedicated nucleotide exchange factor, eIF2B, arresting translation. A drug-like molecule called integrated stress response inhibitor (ISRIB) reverses the effects of eIF2 phosphorylation and restores translation by targeting eIF2B. When administered to mice, ISRIB enhances cognition and limits cognitive decline due to brain injury. To determine ISRIB's mechanism of action, we solved an atomic structure of ISRIB bound to the human eIF2B decamer. We found that ISRIB acts as a molecular staple, pinning together tetrameric subcomplexes of eIF2B along the assembly path to a fully active, decameric enzyme. In this Structural Snapshot, we discuss ISRIB's mechanism, its ability to rescue disease mutations in eIF2B and conservation of the enzyme and ISRIB-binding pocket.


Mucoepidermoid carcinoma of the parotid gland: the Mayo clinic experience.
To determine clinical and histopathologic features of mucoepidermoid carcinoma of the parotid gland, specifically, the relation of tumor stage and grade and treatment type with clinical outcome. Retrospective clinical and histopathologic review. Tertiary care medical center. From 1940 to 1994, 128 patients were treated at our institution for parotid mucoepidermoid carcinoma. Eighty-nine of these patients had their first treatment at our institution; these cases were chosen for retrospective clinical and histopathologic review. A head and neck pathologist independently reviewed the pathology specimens. Age, symptoms, stage, treatment type, tumor grade, pathological features, disease progression, and survival. Results of clinical staging were: T1 in 56 patients, T2 in 13, T3 in 1, T4 in 15, N0 in 85, N1 in 2, and N2 in 2. No patient had N3 or M1 disease. All patients underwent parotidectomy with or without neck dissection. Seven patients received postoperative radiotherapy. Tumor grade was low in 43 patients (48%), intermediate in 40 (45%), and high in 6 (7%). Only 5 patients had disease progression (local recurrence in 4, regional recurrence in 4, and distant recurrence in 2). At latest follow-up (mean follow-up, 14.7 years), 64 patients were alive without disease, 1 was alive with disease, 2 had died of mucoepidermoid carcinoma, and 22 had died of other causes. The Kaplan-Meier estimated cancer-specific survival rates at 5, 15, and 25 years were 98.8%, 97.4%, and 97.4%, respectively. In our study, tumor grade and stage appeared to be less important than previously described. With adequate parotidectomy and appropriate neck dissection, patients with mucoepidermoid carcinoma of the parotid gland appear to do well, with few recurrences.


Lack of association of hepatitis C virus load and genotype with risk of end-stage liver disease in patients with human immunodeficiency virus coinfection.
In hepatitis C virus (HCV) infection, virus load and the risk for HCV-related end-stage liver disease (ESLD) are increased among persons with human immunodeficiency virus (HIV) coinfection. To clarify these relationships, 42 hemophilic patients who developed ESLD and random samples from 164 hemophilic patients with HCV infection alone and 146 with HCV-HIV coinfection were tested for HCV load and genotype. HCV genotype was unrelated to HIV and age. In contrast, HCV load was higher with older age (P(trend)=.0001) and with HIV coinfection (6.2 vs. 5.9 log(10) genome equivalents/mL, P=.0001). During 16 years of follow-up of dually infected patients, ESLD risk was unrelated to HCV load overall (P(trend)=.64) or separately to HCV genotype 1 and genotypes 2 or 3 (P(trend)> or =.70). Irrespective of virus load, incidence of ESLD was marginally increased 2-fold (95% confidence interval, 0.8-5.6) with HCV genotype 1. Understanding the discordance between HCV load and ESLD, despite HIV's link to each of these, may help clarify the pathogenesis of HCV-related disease.


PyFolding: Open-Source Graphing, Simulation, and Analysis of the Biophysical Properties of Proteins.
For many years, curve-fitting software has been heavily utilized to fit simple models to various types of biophysical data. Although such software packages are easy to use for simple functions, they are often expensive and present substantial impediments to applying more complex models or for the analysis of large data sets. One field that is reliant on such data analysis is the thermodynamics and kinetics of protein folding. Over the past decade, increasingly sophisticated analytical models have been generated, but without simple tools to enable routine analysis. Consequently, users have needed to generate their own tools or otherwise find willing collaborators. Here we present PyFolding, a free, open-source, and extensible Python framework for graphing, analysis, and simulation of the biophysical properties of proteins. To demonstrate the utility of PyFolding, we have used it to analyze and model experimental protein folding and thermodynamic data. Examples include: 1) multiphase kinetic folding fitted to linked equations, 2) global fitting of multiple data sets, and 3) analysis of repeat protein thermodynamics with Ising model variants. Moreover, we demonstrate how PyFolding is easily extensible to novel functionality beyond applications in protein folding via the addition of new models. Example scripts to perform these and other operations are supplied with the software, and we encourage users to contribute notebooks and models to create a community resource. Finally, we show that PyFolding can be used in conjunction with Jupyter notebooks as an easy way to share methods and analysis for publication and among research teams.


[Prevalence and early determinants of common mental disorders in the 1982 birth cohort, Pelotas, Southern Brazil].
To estimate the prevalence of common mental disorders and assess its association with risk factors in a cohort of young adults. Cross-sectional study nested in a 1982 birth cohort study conducted in Pelotas, Southern Brazil. In 2004-5, 4,297 subjects were interviewed during home visits. Common mental disorders were assessed using the Self-Report Questionnaire. Risk factors included socioeconomic, demographic, perinatal, and environmental variables. The analysis was stratified by gender and crude and adjusted prevalence ratios were estimated by Poisson regression. The overall prevalence of common mental disorders was 28.0%; 32.8% and 23.5% in women and men, respectively. Men and women who were poor in 2004-5, regardless of their poor status in 1982, had nearly 1.5-fold increased risk for common mental disorders (p<0.001) when compared to those who have never been poor. Among women, being poor during childhood (p<0.001) and black/mixed skin color (p=0.002) increased the risk for mental disorders. Low birth weight and duration of breastfeeding were not associated to the risk of these disorders. Higher prevalence of common mental disorders among low-income groups and race-ethnic minorities suggests that social inequalities present at birth have a major impact on mental health, especially common mental disorders.


Monitoring dynamic interactions of tumor cells with tissue and immune cells in a lab-on-a-chip.
A complementary cell analysis method has been developed to assess the dynamic interactions of tumor cells with resident tissue and immune cells using optical light scattering and impedance sensing to shed light on tumor cell behavior. The combination of electroanalytical and optical biosensing technologies integrated in a lab-on-a-chip allows for continuous, label-free, and noninvasive probing of dynamic cell-to-cell interactions between adherent and nonadherent cocultures, thus providing real-time insights into tumor cell responses under physiologically relevant conditions. While the study of adherent cocultures is important for the understanding and suppression of metastatic invasion, the analysis of tumor cell interactions with nonadherent immune cells plays a vital role in cancer immunotherapy research. For the first time, the direct cell-to-cell interactions of tumor cells with bead-activated primary T cells were continuously assessed using an effector cell to target a cell ratio of 10:1.


A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD.
Amyotrophic Lateral Sclerosis (ALS) is a fatal and progressive neurodegenerative disorder with identified genetic causes representing a significant minority of all cases. A GGGGCC hexanucleotide repeat expansion (HRE) mutation within the C9ORF72 gene has recently been identified as the most frequent known cause of ALS. The expansion leads to partial heterochromatinization of the locus, yet mutant RNAs and dipeptide repeat proteins (DPRs) are still produced in sufficient quantities to confer neurotoxicity. The levels of these toxic HRE products positively correlate with cellular toxicity and phenotypic severity across multiple disease models. Moreover, the degree of epigenetic repression inversely correlates with some facets of clinical presentation in C9-ALS patients. Recently, bacterial artificial chromosomes (BAC) have been used to generate transgenic mice that harbor the HRE mutation, complementing other relevant model systems such as patient-derived induced pluripotent stem cells (iPSCs). While epigenetic features of the HRE have been investigated in various model systems and post-mortem tissues, epigenetic dysregulation at the expanded locus in C9-BAC mice remains unexplored. Here, we sought to determine whether clinically relevant epigenetic perturbations caused by the HRE are mirrored in a C9-BAC mouse model. We used complementary DNA methylation assessment and immunoprecipitation methods to demonstrate that epigenetic aberrations caused by the HRE, such as DNA and histone methylation, are recapitulated in the C9-BAC mice. Strikingly, we found that cytosine hypermethylation within the promoter region of the human transgene occurred in a subset of C9-BAC mice similar to what is observed in patient populations. Moreover, we show that partial heterochromatinization of the C9 HRE occurs during the first weeks of the mouse lifespan, indicating age-dependent epigenetic repression. Using iPSC neurons, we found that preventing R-loop formation did not impede heterochromatinization of the HRE. Taken together, these observations provide further insight into mechanism and developmental time-course of epigenetic perturbations conferred by the C9ORF72 HRE. Finally, we suggest that epigenetic repression of the C9ORF72 HRE and nearby gene promoter could impede or delay motor neuron degeneration in C9-BAC mouse models of ALS/FTD.


A study exploring attributes and nature of the retracted literature on mental disorders.
This study was aimed at assessing the retracted medical literature on mental disorders. Another aim was to test the hypothesis that the weak research infrastructure in certain countries and the rising pressure to publish in Asia due to the progress of science in that continent may have contributed to the increase in the number of retractions. A bibliometric search was carried out using the PubMed database. The data were analysed using SPSS version 21. The retraction rate for articles on mental disorders (number of retracted articles per 100,000 published articles on mental disorders) varied from a low of 3.56 (for 2005) to a high of 49.25 (for 2012). Of the 38 articles for which the reasons for retraction could be accessed, 10 (26.31%) were retracted for fraud. Overall, 0.0138% of all articles on the biomedical sciences were retracted. Of the articles on mental disorders, 0.0095% were retracted. There was a disproportionately greater number of retractions in the case of articles originating from low- and middle-income countries than high-income countries. Similarly, there was a disproportionately greater number of retractions in the case of articles originating in Asian countries than non-Asian countries.


Lowering cortisol and CVD risk in postmenopausal women: a pilot study using the Transcendental Meditation program.
Unlike younger women, the risk of cardiovascular disease in older women matches or exceeds that of men. Excessive cortisol may play a role in this increased risk. Here we explore the possibility that the Transcendental Meditation (TM) program may reduce the cortisol response to a metabolic stressor as a way of reducing disease risk in older women. Data from 16 women who were long-term practitioners of transcendental meditation (mean = 23 y) were compared with data from 14 control women matched for age (mean = 75 y, range = 65-92 y). Data on demographics, disease symptoms, and psychological variables were collected, and cortisol response to a metabolic stressor (75 g of glucose, orally) was examined in saliva and urine. Pre-glucose levels of salivary cortisol were identical for the two groups. Post-glucose cortisol rose faster in the controls and was significantly higher than that in the TM women (P < 1 3 10(-4)). Urinary excretion of cortisol during this period was 3 times higher in controls than in the TM women (2.4 +/- 0.17 and 0.83 +/- 0.10 microg/h, respectively; P = 2 x 10(-4)). In addition, the number of months practicing transcendental meditation was inversely correlated with CVD risk factors. Lower cortisol response to metabolic challenge may reflect improved endocrine regulation relevant to the disease-preventing effects of transcendental meditation in older women.


Cannabinoids modulate voltage sensitive potassium A-current in hippocampal neurons via a cAMP-dependent process.
Previous studies have shown that cannabinoid receptor analogs increase voltage-dependent potassium A-current (IA) in cultured hippocampal cells. Because cannabinoid receptors inhibit adenylate cyclase, the present study explored whether cAMP played a role in mediating this effect on IA. The specific issue of whether cannabinoid receptor modulation of voltage-dependent IA acts via a cAMP-dependent process was investigated. The cAMP analog, 8-bromo-cAMP, as well as the adenylate cyclase stimulant forskolin, produced concentration-dependent shifts in IA that were opposite those produced by cannabinoid receptor ligands. Moreover, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also produced a marked negative shift in the steady-state voltage dependence of IA and increased the effect of forskolin on IA. As shown in previous studies, the cannabinoid agonist WIN 55,212-2 increased IA via a decrease in steady-state voltage-dependent inactivation of IA. WIN 55,212-2 also reversed the effects of forskolin on IA. The electrophysiological studies were paralleled by direct assays of cAMP in these cells, where cannabinoids inhibited forskolin-stimulated cAMP by 50% in a pertussis toxin-sensitive manner. The results confirmed that pertussis toxin-sensitive cannabinoid receptor-mediated changes in IA were probably the result of inhibition of adenylate cyclase. The findings are discussed in terms of modulation of IA conductance properties via cannabinoid receptor-mediated inhibition of cAMP levels within the cell.


A bio-inspired polymeric gradient refractive index (GRIN) human eye lens.
A synthetic polymeric lens was designed and fabricated based on a bio-inspired, "Age=5" human eye lens design by utilizing a nanolayered polymer film-based technique. The internal refractive index distribution of an anterior and posterior GRIN lens were characterized and confirmed against design by µATR-FTIR. 3D surface topography of the fabricated aspheric anterior and posterior lenses was measured by placido-cone topography and exhibited confirmation of the desired aspheric surface shape. Furthermore, the wavefronts of aspheric posterior GRIN and PMMA lenses were measured and simulated by interferometry and Zemax software, respectively. Their results show that the gradient index distribution reduces the overall wavefront error as compared a homogenous PMMA lens of an identical geometry. Finally, the anterior and posterior GRIN lenses were assembled into a bio-inspired GRIN human eye lens through which a clear imaging was possible.


The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine-activated killer cells.
Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Ralphabetagamma and IL-2Rbetagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. For the first time, we show that a chemically synthesized fragment of the IL-2 sequence can fold into a molecule mimicking the quaternary structure of a hemopoietin. Indeed, peptide p1-30 (containing amino acids 1-30, covering the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rbeta, whereas shorter versions of the peptide lack helical structure and are inactive. We also demonstrate that this neocytokine interacts with a previously undescribed dimeric form of IL-2Rbeta. In agreement with its binding to IL-2Rbeta, p1-30 activates Shc and p56(lck) but unlike IL-2, fails to activate Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription 5 (STAT5). Unexpectedly, we also show that p1-30 activates Tyk2, thus suggesting that IL-2Rbeta may bind to different Jaks depending on its oligomerization. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8(low) lymphocytes and natural killer cells, which constitutively express IL-2Rbeta. A significant interferon gamma production is also detected after p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys), which is likely unable to induce vascular leak syndrome, remains capable of generating LAK cells, like the original p1-30 peptide. Altogether, our data suggest that p1-30 has therapeutic potential.


[General anesthesia after failed spinal block for emergency surgery in a patient with mucopolysaccharidosis: case report.].
Mucopolysaccharidosis are a group of heterogenous disorders secondary to the deficiency of a lysosomal enzyme, with accumulation of acid mucopolysaccharides in the tissues. It carries a 20% perioperative mortality rate due mainly to difficulties on handling the airways. The objective of this report was to present the anesthetic approach of a patient with mucopolysaccharidosis who presented in the operating room for emergency surgery. A 15 year-old male patient with a diagnosis of mucopolysaccharidosis was admitted to the operating room for treatment of an incarcerated umbilical hernia. After monitoring, a subarachnoid block with hyperbaric bupivacaine was performed with the patient in the sitting position. After identifying failure of the spinal block it was changed to general anesthesia. Anesthetic induction consisted of intravenous clonidine, fentanyl and propofol, followed by endotracheal intubation without intercurrences. After a 45-minute surgery, the patient was extubated and transferred to the postanesthetic care unit where he remained under observation for 12 hours, being discharged to the regular ward without intercurrences. In the case presented here we did not have any difficulties handling the airways. However, this is the main problem in this group of patients and it is up to the anesthesiologist to choose the better technique to guarantee adequate handling of the airways.


A new technique for determining the refractive index of ices at cryogenic temperatures.
A reflection-absorption optical (RAO) spectrometer, operating across the ultra-violet/visible (UV/visible) wavelength region, has been developed that allows simultaneous measurements of optical properties and thickness of thin solid films at cryogenic temperatures in ultrahigh vacuum. The RAO spectrometer enables such measurements to be made after ice deposition, as opposed to most current approaches which make measurements during deposition. This allows changes in the optical properties and in the thickness of the film to be determined subsequent to thermal, photon or charged particle processing. This is not possible with current techniques. A data analysis method is presented that allows the wavelength dependent n and k values for ices to be extracted from the reflection-absorption spectra. The validity of this analysis method is shown using model data from the literature. New data are presented for the reflection UV/visible spectra of amorphous and crystalline single component ices of benzene, methyl formate and water adsorbed on a graphite surface. These data show that, for benzene and methyl formate, the crystalline ice has a larger refractive index than amorphous ice, reflecting changes in the electronic environment occurring in the ice during crystallisation. For water, the refractive index does not vary with ice phase.


Policy and practice for preventing skin cancer in children.
This paper identifies what is known and what is not known about the link between sun exposure and skin cancer and what can be done to reduce sun exposure in children. There is evidence for the use of sun protective clothing and of the effectiveness of sunscreens for some, but not all, forms of skin cancer. While there is some evidence to support interventions in schools, there is insufficient evidence to support other interventions aimed at children or their caregivers. There is no clear means of assessing the "trade-off" between the benefits and harms of sunlight exposure, and the impact of sun protection strategies on other health promotion messages aimed at children is unknown. Reliance on educational interventions in schools may benefit from a broader approach that includes more emphasis on protective clothing and structural changes in the school day. Sun protection messages should be linked with other health promotion messages targeting children.


Non-Invasive Genetic Mark-Recapture as a Means to Study Population Sizes and Marking Behaviour of the Elusive Eurasian Otter (Lutra lutra).
Quantifying population status is a key objective in many ecological studies, but is often difficult to achieve for cryptic or elusive species. Here, non-invasive genetic capture-mark-recapture (CMR) methods have become a very important tool to estimate population parameters, such as population size and sex ratio. The Eurasian otter (Lutra lutra) is such an elusive species of management concern and is increasingly studied using faecal-based genetic sampling. For unbiased sex ratios or population size estimates, the marking behaviour of otters has to be taken into account. Using 2132 otter faeces of a wild otter population in Upper Lusatia (Saxony, Germany) collected over six years (2006-2012), we studied the marking behaviour and applied closed population CMR models accounting for genetic misidentification to estimate population sizes and sex ratios. We detected a sex difference in the marking behaviour of otters with jelly samples being more often defecated by males and placed actively exposed on frequently used marking sites. Since jelly samples are of higher DNA quality, it is important to not only concentrate on this kind of samples or marking sites and to invest in sufficiently high numbers of repetitions of non-jelly samples to ensure an unbiased sex ratio. Furthermore, otters seemed to increase marking intensity due to the handling of their spraints, hence accounting for this behavioural response could be important. We provided the first precise population size estimate with confidence intervals for Upper Lusatia (for 2012: N = 20 ± 2.1, 95% CI = 16-25) and showed that spraint densities are not a reliable index for abundances. We further demonstrated that when minks live in sympatry with otters and have comparably high densities, a non-negligible number of supposed otter samples are actually of mink origin. This could severely bias results of otter monitoring if samples are not genetically identified.


Epidemic non-A non-B hepatitis in urban Karachi, Pakistan.
An outbreak of icteric non-A non-B (NANB) hepatitis occurred in a residential community of urban Karachi, Pakistan, from August 1986 through October 1986. Of the 114 cases reported from this community during the 1986 calendar year, a clustering of 85 cases was seen during the above period. Twenty-seven percent of 226 households and 9% of 1,250 individuals were affected. Five persons were hospitalized and 1 death occurred in a young pregnant woman. Cases occurred predominantly in the less than or equal to 29-year-old age group (72%), with a male:female ratio of 1.8:1. Thirty-four cases occurred singly within households, while in 28 households multiple cases were seen. Analysis of the epidemic curve and intervals of onset of multiple cases within households suggested prolonged common source exposure rather than secondary person-to-person transmission. No single water source was implicated but a contaminated municipal supply was presumed. Information collected from several other communities and from a university hepatitis reference laboratory suggested that the outbreak was part of a larger urban epidemic of NANB hepatitis. Based upon this investigation and data from recently published reports, it is concluded that NANB hepatitis is endemic in Pakistan.


Relevant microclimate for determining the development rate of malaria mosquitoes and possible implications of climate change.
The relationship between mosquito development and temperature is one of the keys to understanding the current and future dynamics and distribution of vector-borne diseases such as malaria. Many process-based models use mean air temperature to estimate larval development times, and hence adult vector densities and/or malaria risk. Water temperatures in three different-sized water pools, as well as the adjacent air temperature in lowland and highland sites in western Kenya were monitored. Both air and water temperatures were fed into a widely-applied temperature-dependent development model for Anopheles gambiae immatures, and subsequently their impact on predicted vector abundance was assessed. Mean water temperature in typical mosquito breeding sites was 4-6 degrees C higher than the mean temperature of the adjacent air, resulting in larval development rates, and hence population growth rates, that are much higher than predicted based on air temperature. On the other hand, due to the non-linearities in the relationship between temperature and larval development rate, together with a marginal buffering in the increase in water temperature compared with air temperature, the relative increases in larval development rates predicted due to climate change are substantially less. Existing models will tend to underestimate mosquito population growth under current conditions, and may overestimate relative increases in population growth under future climate change. These results highlight the need for better integration of biological and environmental information at the scale relevant to mosquito biology.


Characterization of the DNA-dependent GTPase activity of T4 gene 41 protein, an essential component of the T4 bacteriophage DNA replication apparatus.
The product specified by T4 bacteriophage gene 41 is known from genetic analyses to be essential for phage DNA replication in vivo. Correspondingly, the purified gene 41 protein is an essential component of an efficient in vitro DNA replication system reconstructed from seven purified T4 replication proteins; it is required both for the synthesis of short RNA primers (in conjunction with the T4 gene 61 protein) and for the rapid unwinding of the double-helical DNA template at a replication fork. The purified gene 41 protein exhibits a DNA-dependent GTPase (and ATPase) activity. In this report, we have used this associated GTPase activity as a biochemical probe for the analysis of the interactions between DNA and the 41 protein. Our results suggest that, upon binding GTP, the 41 protein monomer is induced to form a dimer, which can them form a tight complex with single-stranded DNA. Driven by the repeated hydrolysis of GTP molecules, the 41 protein dimer appears to run rapidly along the bound DNA chain. Studies with the synthetic GTP analogue, GTP gamma S, suggest that GTP hydrolysis is required for this 41 protein movement, but that it is not essential for the function of the 41 protein in RNA primer synthesis. In sum, our observations suggest that a 41 protein dimer runs along the lagging strand template at a DNA replication fork; from this position, it functions as a DNA helicase and simultaneously interacts with the T4 gene 61 protein to make the pentaribonucleotide primers which initiate Okazaki pieces at specific primer initiation sites.


Magnetic resonance phase contrast velocity mapping for flow quantification in irregular heart rhythms using radial k-space ultrashort echo time imaging.
Phase contrast velocity mapping sequences utilising ultrashort echo time (UTE) radial k-space sequences have been used to reduce intravoxel dephasing at high velocities. We evaluated the accuracy of the UTE flow sequence for mitral regurgitation (MR) quantification, including patients with atrial fibrillation. Forty patients underwent cardiac MRI for indirect MR quantification by assessment of aortic flow using a UTE phase contrast sequence (TE 0.65 ms) combined with left ventricular stroke volume. Retrospective ECG-gating was used in sinus rhythm (30 patients), prospective ECG-triggering in atrial fibrillation (10). MR was also quantified by a standard phase contrast sequence (TE 2.85 ms, standard flow method) and by comparing stroke volumes (volumetric method). UTE flow-derived MR measurement showed modest agreement in sinus rhythm (95% limits of agreement: ±38.2 ml; ±29.8%) and atrial fibrillation (±33.7 ml; ±30.3%) compared to standard flow assessment. There was little systematic bias in sinus rhythm (mean offset -4.4 ml /-3.5% compared to standard flow assessment), but a slight bias towards greater regurgitation in atrial fibrillation (+15.2 ml /+14.0%). There were wider limits of agreement between the UTE flow method and volumetric method than between the regular flow method and the volumetric method in sinus rhythm (±48.4 ml; ±36.4%; mean offset: -12.2 ml /-9.0%) and similar limits of agreement in atrial fibrillation (±29.6 ml; 25.8%; +12.0 ml /+10.3%). UTE flow imaging is inferior to conventional flow techniques for MR assessment in patients with sinus rhythm as well as atrial fibrillation. However, the number of atrial fibrillation patients in this initial study is small.


Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics.
14-3-3 proteins play a pivotal role in controlling cell proliferation and survival, two commonly dysregulated hallmarks of cancers. 14-3-3 protein expression is enhanced in many human cancers and correlates with more aggressive tumors and poor prognosis, suggesting a role for 14-3-3 proteins in tumorigenesis and/or progression. We showed previously that the dimeric state of 14-3-3 proteins is regulated by the lipid sphingosine, a physiological inducer of apoptosis. As the functions of 14-3-3 proteins are dependent on their dimeric state, this sphingosine-mediated 14-3-3 regulation provides a possible means to target dimeric 14-3-3 for therapeutic effect. However, sphingosine mimics are needed that are not susceptible to sphingolipid metabolism. We show here the identification and optimization of sphingosine mimetics that render dimeric 14-3-3 susceptible to phosphorylation at a site buried in the dimer interface and induce mitochondrial-mediated apoptosis. Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells. Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice. Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery.


L-deprenyl (selegiline) exerts anticonvulsant effects against different seizure types in mice.
L-Deprenyl (selegiline), a selective inhibitor of monoamine oxidase type B, has recently been shown to exert anticonvulsant and antiepileptogenic effects in the kindling model of partial (focal) epilepsy. In the present study, we examined if L-deprenyl exerts anticonvulsant effects in standard rodent models of generalized seizures. In addition to anticonvulsant activity, behavioral effects induced by L-deprenyl were monitored closely. To assess the stereoselectivity of anticonvulsant and behavioral effects of deprenyl, the D-enantiomer was included in the studies. Furthermore, the antiepileptic drug phenobarbital was used for comparison. The following tests were performed in mice: 1) the threshold for tonic electroconvulsions; 2) the maximal electroshock seizure test with fixed supramaximal (suprathreshold) stimulation; 3) the threshold for myoclonic, clonic and tonic seizures in response to i.v. infusion of pentylenetetrazole (PTZ); 4) the s.c. PTZ seizure test, with a fixed dose of PTZ (80 microgram/kg) for seizure induction; 5) the rotarod and chimney tests for determination of motor impairment. Furthermore, animals were observed in cage and open field for stereotyped behavior and other behavioral abnormalities. L-Deprenyl, tested at doses of 1 to 40 microgram/kg i.p., significantly increased myoclonic and clonic PTZ thresholds and the threshold for tonic electroconvulsions, whereas D-deprenyl was either ineffective or exhibited a lower anticonvulsant potency than L-deprenyl. Both drugs were ineffective in the maximal electroshock seizure and s.c. PTZ seizure tests. In contrast to the higher anticonvulsant potency of L-deprenyl in seizure threshold tests, D-deprenyl was more potent than L-deprenyl to induce behavioral abnormalities, such as hyperlocomotion. The data indicate that L-deprenyl exerts anticonvulsant activity against different seizure types. This anticonvulsant activity and the previously reported neuroprotective and cognition-enhancing action of L-deprenyl offer a unique combination of drug effects which might be of clinical benefit in patients with epilepsy.


Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton.
By immunogold labeling, we demonstrate that "millipede-like" structures seen previously in mammalian cell cytoskeletons after removal of actin by treatment with gelsolin are composed of the cores of vimentin IFs with sidearms containing plectin. These plectin sidearms connect IFs to microtubules, the actin-based cytoskeleton and possibly membrane components. Plectin binding to microtubules was significantly increased in cells from transgenic mice lacking IFs and was reversed by microinjection of exogenous vimentin. These results suggest the existence of a pool of plectin which preferentially associates with IFs but may also be competed for by microtubules. The association of IFs with microtubules did not show a preference for Glu-tubulin. Nor did it depend upon the presence of MAP4 since plectin links were retained after specific immunodepletion of MAP4. The association of IFs with stress fibers survived actin depletion by gelsolin suggesting that myosin II minifilaments or components closely associated with them may play a role as plectin targets. Our results provide direct structural evidence for the hypothesis that plectin cross-links elements of the cytoskeleton thus leading to integration of the cytoplasm.


Assessment of the Readability of Genetic Counseling Patient Letters.
Patient letters are a powerful tool that genetic counselors use to communicate with their patients. Patient letters are often sent to provide information on a new diagnosis, reiterate test results, and to serve as a permanent record of the visit. Patient letters, however, are only helpful if the patients can understand them. More than 50 % of the US population reads below a 9th grade reading level and over one-third of the population has low health literacy skills. In this study we evaluate the readability of genetic counseling patient letters by assessing reading level, image use, and terminology use. One hundred forty-nine genetic counselors participated in the survey and of these, 79 submitted a sample patient letter. Analyses of the letters revealed a mean reading level of 10.93. On average, 6 genetic terms were included in each letter, and only 25 % of these terms were defined. Analyses of survey responses revealed over 75 % of the genetic counselors did not include images in their patient letters. These results indicate there is room for improvement in order to make genetic counseling patient letters more accessible to the general population.


Severe experimental folate deficiency in a human subject - a longitudinal investigation of red-cell folate immunoassay errors as megaloblastic anaemia develops.
The few published studies comparing results between commercial red-cell folate immunoassays have found significant differences. None have provided longitudinal data during the development of megaloblastic anaemia from severe folate deficiency. The objective was to produce longitudinal data, comparing results between three commercial immunoassays for red-cell folate, generated by means of severe experimental folate deficiency. This 58 year old male, initially replete in folate, used a folate-deficient diet to severely deplete himself of folate until overt megaloblastic anaemia developed. The Siemens Advia Centaur, Roche Elecsys 2010 and Beckman UniCel DxI 800 Access immunoassay systems were used, by different clinical pathology laboratories, to perform weekly assays for red-cell folate throughout the depletion stage. The results were analysed graphically four ways: comparison with lines of equality; number of standard deviations difference against the means; number of standard deviations difference over time; variation over time. There were very significant differences, varying with time and folate concentration, between the results reported by the three laboratories. The differences were greatest, up to 17 standard deviations, between the Siemens Advia Centaur and each of the other two systems. Of the 85 results comparing the Siemens Advia Centaur and the Roche Elecsys 2010, two were within the 99.9% confidence interval. Of the 91 results comparing the Siemens Advia Centaur and the Beckman UniCel DxI 800 Access, 22 were within the 99.9% confidence interval. Of the 83 results comparing the Beckman UniCel DxI 800 Access and the Roche Elecsys 2010, 37 were within the 99.9% confidence interval. Comparative longitudinal data from clinical pathology laboratories, produced during experimental folate deficiency, have exposed very significant differences in results between commercial red-cell folate immunoassays. One immunoassay, the Roche Elecsys 2010, failed to detect overt megaloblastic anaemia of severe folate deficiency.


Intravitreal anti-VEGF therapy for macular radiation retinopathy: a 10-year study.
To report long-term experience with intravitreal anti-vascular endothelial growth factor treatment for radiation maculopathy. From 2005-2015, 120 consecutive patients underwent intravitreal anti-VEGF therapy for radiation maculopathy. Inclusion criteria included a diagnosis of uveal melanoma treated with plaque radiotherapy and subsequent macular radiation vasculopathy (exudate, retinal hemorrhage, intraretinal microangiopathy, neovascularization, edema). Anti-VEGF therapy involved continuous injections in 4- to 12-week intervals with doses of 1.25 mg/0.05 mL, 2.0 mg/0.08 mL, 2.5 mg/0.1 mL, or 3.0 mg/0.12 mL of bevacizumab as well as 0.5 mg/0.05 mL or 2.0 mg/0.05 mL of ranibizumab. Goals were maintenance of visual acuity and normative macular anatomy. Safety and tolerability (retinal detachment, hemorrhage, infection), visual acuity, central foveal thickness on optical coherence tomography imaging, and clinical features of radiation maculopathy were analyzed. Progressive reductions in macular edema, hemorrhages, exudates, cotton-wool spots, and microangiopathy were noted. At last follow-up, 80% remained within 2 lines of their initial visual acuity or better, with a mean treatment interval of 38 months (range 6-108 months). Kaplan-Meier analysis of the probability of remaining within 2 lines of initial visual acuity was 69% at 5 years and 38% at 8 years of anti-VEGF therapy. Discontinuation of therapy was rare. Relatively few acute or long-term side effects were noted, allowing for good long-term patient accrual. Continuous intravitreal anti-VEGF therapy in patients with radiation maculopathy was well-tolerated and preserved vision. In most cases, reductions or resolution of retinal hemorrhages, cotton-wool spots, and retinal edema were noted for up to 10 years.


Exploring the variation in implementation of a COPD disease management programme and its impact on health outcomes: a post hoc analysis of the RECODE cluster randomised trial.
This study aims to (1) examine the variation in implementation of a 2-year chronic obstructive pulmonary disease (COPD) management programme called RECODE, (2) analyse the facilitators and barriers to implementation and (3) investigate the influence of this variation on health outcomes. Implementation variation among the 20 primary-care teams was measured directly using a self-developed scale and indirectly through the level of care integration as measured with the Patient Assessment of Chronic Illness Care (PACIC) and the Assessment of Chronic Illness Care (ACIC). Interviews were held to obtain detailed information regarding the facilitators and barriers to implementation. Multilevel models were used to investigate the association between variation in implementation and change in outcomes. The teams implemented, on average, eight of the 19 interventions, and the specific package of interventions varied widely. Important barriers and facilitators of implementation were (in)sufficient motivation of healthcare provider and patient, the high starting level of COPD care, the small size of the COPD population per team, the mild COPD population, practicalities of the information and communication technology (ICT) system, and hurdles in reimbursement. Level of implementation as measured with our own scale and the ACIC was not associated with health outcomes. A higher level of implementation measured with the PACIC was positively associated with improved self-management capabilities, but this association was not found for other outcomes. There was a wide variety in the implementation of RECODE, associated with barriers at individual, social, organisational and societal level. There was little association between extent of implementation and health outcomes.


Discovery-based protein expression profiling identifies distinct subgroups and pathways in leiomyosarcomas.
Soft tissue sarcomas (STS) are malignant tumors of mesenchymal origin. A substantial portion of these tumors exhibits complex karyotypes and lack characterized chromosomal aberrations. Owing to such properties, both histopathologic and molecular classification of these tumors has been a significant challenge. This study examines the protein expression of a large number of human STS, including subtype heterogeneity, using two-dimensional gel proteomics. In addition, detailed proteome profiles of a subset of pleomorphic STS specimens using an in-depth mass-spectrometry approach identified subgroups within the leiomyosarcomas with distinct protein expression patterns. Pathways analysis indicates that key biologic nodes like apoptosis, cytoskeleton remodeling, and telomere regulation are differentially regulated among these subgroups. Finally, investigating the similarities between protein expression of leiomyosarcomas and undifferentiated pleomorphic sarcomas (UPS) revealed similar protein expression profiles for these tumors, in comparison with pleomorphic leiomyosarcomas. These results suggest that UPS tumors share a similar lineage as leiomyosarcomas and are likely to originate from different stages of differentiation from mesenchymal stem cells to smooth muscle cells.


Comparative genomics of the dormancy regulons in mycobacteria.
In response to stresses, Mycobacterium cells become dormant. This process is regulated by the DosR transcription factor. In Mycobacterium tuberculosis, the dormancy regulon is well characterized and contains the dosR gene itself and dosS and dosT genes encoding DosR kinases, nitroreductases (acg; Rv3131), diacylglycerol acyltransferase (DGAT) (Rv3130c), and many universal stress proteins (USPs). In this study, we apply comparative genomic analysis to characterize the DosR regulons in nine Mycobacterium genomes, Rhodococcus sp. RHA1, Nocardia farcinica, and Saccharopolyspora erythraea. The regulons are highly labile, containing eight core gene groups (regulators, kinases, USPs, DGATs, nitroreductases, ferredoxins, heat shock proteins, and the orthologs of the predicted kinase [Rv2004c] from M. tuberculosis) and 10 additional genes with more restricted taxonomic distribution that are mostly involved in anaerobic respiration. The largest regulon is observed in M. marinum and the smallest in M. abscessus. Analysis of large gene families encoding USPs, nitroreductases, and DGATs demonstrates a mosaic distribution of regulated and nonregulated members, suggesting frequent acquisition and loss of DosR-binding sites.


Gut Microbiota, a Potential New Target for Chinese Herbal Medicines in Treating Diabetes Mellitus.
The gut microbiota, as an important factor affecting host health, plays a significant role in the occurrence and development of diabetes mellitus (DM), and the mechanism may be related to excessive endotoxins, altered short-chain fatty acids (SCFAs), and disordered bile acid metabolism. Traditional Chinese medicine (TCM) has a long history of treating DM, but its mechanism is not very clear. Recent research has suggested that Chinese herbal medicine can improve glucose metabolism by remodeling the gut microbiota, which opens new avenues for further research on hypoglycemic mechanisms. This review presents the recent progress of Chinese herbs, herbal extracts, and herbal compound preparations in treating DM through regulating the gut microbiota and summarizes the main mechanisms involved, namely, anti-inflammatory and antioxidative effects, protecting the intestinal barrier and inhibiting lipotoxicity. In addition, some suggestions for improvement are also proposed.


Genetic and Genomic Analyses Reveal Boundaries between Species Closely Related to Cryptococcus Pathogens.
Speciation is a central mechanism of biological diversification. While speciation is well studied in plants and animals, in comparison, relatively little is known about speciation in fungi. One fungal model is the Cryptococcus genus, which is best known for the pathogenic Cryptococcus neoformans/Cryptococcus gattii species complex that causes >200,000 new human infections annually. Elucidation of how these species evolved into important human-pathogenic species remains challenging and can be advanced by studying the most closely related nonpathogenic species, Cryptococcus amylolentus and Tsuchiyaea wingfieldii However, these species have only four known isolates, and available data were insufficient to determine species boundaries within this group. By analyzing full-length chromosome assemblies, we reappraised the phylogenetic relationships of the four available strains, confirmed the genetic separation of C. amylolentus and T. wingfieldii (now Cryptococcus wingfieldii), and revealed an additional cryptic species, for which the name Cryptococcus floricola is proposed. The genomes of the three species are ∼6% divergent and exhibit significant chromosomal rearrangements, including inversions and a reciprocal translocation that involved intercentromeric ectopic recombination, which together likely impose significant barriers to genetic exchange. Using genetic crosses, we show that while C. wingfieldii cannot interbreed with any of the other strains, C. floricola can still undergo sexual reproduction with C. amylolentus However, most of the resulting spores were inviable or sterile or showed reduced recombination during meiosis, indicating that intrinsic postzygotic barriers had been established. Our study and genomic data will foster additional studies addressing fungal speciation and transitions between nonpathogenic and pathogenic Cryptococcus lineages.IMPORTANCE The evolutionary drivers of speciation are critical to our understanding of how new pathogens arise from nonpathogenic lineages and adapt to new environments. Here we focus on the Cryptococcus amylolentus species complex, a nonpathogenic fungal lineage closely related to the human-pathogenic Cryptococcus neoformans/Cryptococcus gattii complex. Using genetic and genomic analyses, we reexamined the species boundaries of four available isolates within the C. amylolentus complex and revealed three genetically isolated species. Their genomes are ∼6% divergent and exhibit chromosome rearrangements, including translocations and small-scale inversions. Although two of the species (C. amylolentus and newly described C. floricola) were still able to interbreed, the resulting hybrid progeny were usually inviable or sterile, indicating that barriers to reproduction had already been established. These results advance our understanding of speciation in fungi and highlight the power of genomics in assisting our ability to correctly identify and discriminate fungal species.


Optimal surgical approach for esophageal cancer in the era of minimally invasive esophagectomy and neoadjuvant therapy.
The optimal surgical technique for the potentially curative treatment of patients with esophageal cancer is still under debate. The transhiatal esophagectomy (THE) with limited lymphadenectomy mainly focuses on a decrease of postoperative morbidity and mortality by preventing a formal thoracotomy. The transthoracic esophagectomy (TTE) with extended two-field lymphadenectomy attempts to improve the radicality of the resection and thus to increase locoregional tumor control, but is associated with increased postoperative morbidity. The recent introduction of different minimally invasive techniques probably decreases postoperative morbidity following TTE, with reduction of especially pulmonary complications, but high-quality evidence is still limited. It is widely agreed that extended lymphadenectomy as performed during TTE provides the benefit of more accurate staging, but its effect on improvement of survival is still debated. The literature on this topic is contradictory and the choice of surgical approach is primarily driven by personal opinions and institutional preferences. Moreover, the available evidence is mainly based on patients who underwent surgery alone without neoadjuvant therapy. Results of recent studies suggest that neoadjuvant chemoradiotherapy abolishes any possibly positive effect of extended lymphadenectomy as performed during TTE on survival, but this effect should be confirmed in future research. This review gives an overview and reflects the authors' personal view on the role of TTE and THE in the treatment of potentially curative treatment of patients with locally advanced esophageal cancer in the era of minimally invasive esophagectomy and neoadjuvant treatment and outlines future research perspectives.


A statistical method for estimating wood thermal diffusivity and probe geometry using in situ heat response curves from sap flow measurements.
The heat pulse method is widely used to measure water flux through plants; it works by using the speed at which a heat pulse is propagated through the system to infer the velocity of water through a porous medium. No systematic, non-destructive calibration procedure exists to determine the site-specific parameters necessary for calculating sap velocity, e.g., wood thermal diffusivity and probe spacing. Such parameter calibration is crucial to obtain the correct transpiration flux density from the sap flow measurements at the plant scale and subsequently to upscale tree-level water fluxes to canopy and landscape scales. The purpose of this study is to present a statistical framework for sampling and simultaneously estimating the tree's thermal diffusivity and probe spacing from in situ heat response curves collected by the implanted probes of a heat ratio measurement device. Conditioned on the time traces of wood temperature following a heat pulse, the parameters are inferred using a Bayesian inversion technique, based on the Markov chain Monte Carlo sampling method. The primary advantage of the proposed methodology is that it does not require knowledge of probe spacing or any further intrusive sampling of sapwood. The Bayesian framework also enables direct quantification of uncertainty in estimated sap flow velocity. Experiments using synthetic data show that repeated tests using the same apparatus are essential for obtaining reliable and accurate solutions. When applied to field conditions, these tests can be obtained in different seasons and can be automated using the existing data logging system. Empirical factors are introduced to account for the influence of non-ideal probe geometry on the estimation of heat pulse velocity, and are estimated in this study as well. The proposed methodology may be tested for its applicability to realistic field conditions, with an ultimate goal of calibrating heat ratio sap flow systems in practical applications.


Susceptibility loci for adiposity phenotypes on 8p, 9p, and 16q in American Samoa and Samoa.
Obesity is a complex phenotype affected by genetic and environmental influences such as sociocultural factors and individual behaviors. Previously, we performed two separate genome-wide investigations for adiposity-related traits (BMI, percentage body fat (%BF), abdominal circumference (ABDCIR), and serum leptin and serum adiponectin levels) in families from American Samoa and in families from Samoa. The two polities have a common evolutionary history but have lately been influenced by variations in economic development, leading to differences in income and wealth and in dietary and physical activity patterns. We now present a genome-wide linkage scan of the combined samples from the two polities. We adjust for environmental covariates, including polity of residence, education, cigarette smoking, and farm work, and use variance component methods to calculate univariate and bivariate multipoint lod scores. We identified a region on 9p22 with genome-wide significant linkage for the bivariate phenotypes ABDCIR-%BF (1-d.f. lod 3.30) and BMI-%BF (1-d.f. lod 3.31) and two regions with genome-wide suggestive linkage on 8p12 and 16q23 for adiponectin (lod 2.74) and the bivariate phenotype leptin-ABDCIR (1-d.f. lod 3.17), respectively. These three regions have previously been reported to be linked to adiposity-related phenotypes in independent studies. However, the differences in results between this study and our previous polity-specific studies suggest that environmental effects are of different importance in the samples. These results strongly encourage further genetic studies of adiposity-related phenotypes where extended sets of carefully measured environmental factors are taken into account.


Burden of Multiple Chronic Conditions in Delaware, 2011-2014.
The objective of this study was to use data from the Behavioral Risk Factor Surveillance System (BRFSS) to examine the prevalence of multiple chronic conditions (MCC) by select sociodemographic groups and determine the prevalence of most common MCC dyads and triads among Delaware adults. Combined data for 2011 through 2014 from BRFSS (n = 18,052) were analyzed to determine prevalence of MCC. Delaware adults were categorized as having 0, 1, 2, or 3 or more of the following diagnosed chronic conditions: angina, arthritis, asthma, cancer, chronic kidney disease, chronic obstructive pulmonary disease, diabetes, high blood pressure, high cholesterol, myocardial infarction (heart attack), obesity, or stroke. More than 65% of Delaware adults had at least 1 of the 12 selected chronic conditions. Furthermore, 36.8% of Delaware adults had MCC. The arthritis/obesity dyad and the arthritis/high blood pressure/high cholesterol triad were the 2 most prevalent MCC combinations. The findings of this study contribute information to the field of MCC research.


The role of genetic diversity in cancer.
The role of genetic heterogeneity within neoplasms is increasingly recognized as important for understanding the dynamics of cancer progression, cancer stem cells, and therapeutic resistance, and there is interest in intratumoral heterogeneity measurements as potential biomarkers for risk stratification. In this issue of the JCI, Park et al. characterize this genetic diversity in carcinoma in situ and in invasive regions from 3 types of human breast cancers and lay the groundwork for translation of these measures to the clinic.