A subset of chemosensory genes differs between two populations of a specialized leaf beetle after host plant shift

Due to its fundamental role in shaping host selection behavior, we have analyzed the chemosensory repertoire of Chrysomela lapponica. This specialized leaf beetle evolved distinct populations which shifted from the ancestral host plant, willow (Salix sp., Salicaceae), to birch (Betula rotundifolia, Betulaceae). We identified 114 chemosensory candidate genes in adult C. lapponica: 41 olfactory receptors (ORs), eight gustatory receptors, 17 ionotropic receptors, four sensory neuron membrane proteins, 32 odorant binding proteins (OBPs), and 12 chemosensory proteins (CSP) by RNA‐seq. Differential expression analyses in the antennae revealed significant upregulation of one minus‐C OBP (ClapOBP27) and one CSP (ClapCSP12) in the willow feeders. In contrast, one OR (ClapOR17), four minus‐C OBPs (ClapOBP02, 07, 13, 20), and one plus‐C OBP (ClapOBP32) were significantly upregulated in birch feeders. The differential expression pattern in the legs was more complex. To narrow down putative ligands acting as cues for host discrimination, the relative abundance and diversity of volatiles of the two host plant species were analyzed. In addition to salicylaldehyde (willow‐specific), both plant species differed mainly in their emission rate of terpenoids such as (E,E)‐α‐farnesene (high in willow) or 4,8‐dimethylnona‐1,3,7‐triene (high in birch). Qualitatively, the volatiles were similar between willow and birch leaves constituting an “olfactory bridge” for the beetles. Subsequent structural modeling of the three most differentially expressed OBPs and docking studies using 22 host volatiles indicated that ligands bind with varying affinity. We suggest that the evolution of particularly minus‐C OBPs and ORs in C. lapponica facilitated its host plant shift via chemosensation of the phytochemicals from birch as novel host plant.     

S. aureus Evades Macrophage Killing through NLRP3-Dependent Effects on Mitochondrial Trafficking

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Marmoset phylogenetics, conservation perspectives, and evolution of the mtDNA control region

Marmosets (genus Callithrix) are a diverse group of platyrrhine primates with 13-15 purported taxa, many of them considered endangered. Morphological analyses constitute most of the basis for recognition of these forms as distinct taxa. The purpose of this study was to provide a molecular view, based on mitochondrial control region sequences, of the evolutionary history of the marmosets, concomitant with a molecular phylogenetic perspective on species diversity within the group. An additional purpose was to provide the first comparative examination of a complete New World monkey control region sequence with those of other mammals. The phylogenetic analyses provide convincing support for a split between the Atlantic forest and Amazonian marmosets, with the inclusion of the pygmy marmoset (Cebuella pygmaea) at the base of the Amazonian clade. The earliest branch of the Atlantic forest group was C. aurita. In the Amazonian group, the analyses do not support the recognition of C. humeralifer and the recently described C mauesi as distinct taxa. They do, however, support a clear distinction between C. argentata and a strongly supported mixed clade of C. humeralifer and C. mauesi. In the Atlantic forest group, the phylogenetic tree suggests mixing between C. penicillata, C. kuhli, and possibly C. jacchus. Most of the sequence features characteristic of other mammal control regions were also evident in marmosets, with the exception that conserved sequence blocks (CSBs) 2 and 3 were not clearly identifiable. Tandem repeat units often associated with heteroplasmy in a variety of other mammals were not evident in the marmoset sequences.      

Assessment of viability and mitochondrial function of equine spermatozoa using double staining and flow cytometry

An objective double-staining method was developed to evaluate viability and mitochondrial function of stallion spermatozoa using flow cytometry. Sperm viability was assessed by propidium iodide (PI) exclusion, and mitochondrial function was measured by the intensity of rhodamine 123 (R123) fluorescence. Flow cytometry estimates of sperm viability measured by PI were equivalent (P > 0.05) to estimates made using Hoechst 33258 stain and fluorescent microscopy (% dead: 25 +/- 2.4 vs 21.5 +/- 3.5). The use of both PI and R123 was validated by addition of various proportions of freeze-shocked (membrane damaged) cells to viable spermatozoa. There was a high correlation (r(2) = 0.996) between increased PI positivestained (dead) cells and the number of membrane-damaged spermatozoa added (% dead: 29 +/- 0.4, 44 +/- 1.4, 58 +/- 0.9, 75 +/- 0.7 and 91+/- 0.25 vs 0, 25, 50, 75 and 100% damaged cells, respectively). Optimal mitochondrial activity (OMA), as assessed by R123 uptake, was also reduced proportionally (r(2) = 0.976) by the percentage of membrane-damaged cells added (% OMA: 48 +/- 0.6, 37 +/- 1.7, 29 +/- 0.5, 16 +/- 1, 3.8 +/- 1.3 vs 0, 25, 50, 75 and 100% damaged cells, respectively). The mitochondrial inhibitors rotenone and monensin significantly depressed optimal mitochondrial activity (P < 0.001), and there was a significant positive correlation (r(2) = 0.959) between the dose of inhibitors added and the population of sperm cells exhibiting minimal R123 staining (4 -/+ 0.9, 12 -/+ 1.6, 14 -/+ 0.1 and 28 -/+ 2% for treatments with 0, 0.5, 1 and 2 x 10(-5) M rotenone and 0, 0.5, 1, and 2 x 10(-4) M monensin, respectively). Finally, it was shown that treatments containing identical proportions of membrane-damaged cells yielded similar results in terms of viability and mitochondrial activity, irrespective of whether the staining procedure was single or double (P > 0.05). The results of the double-staining method revealed that the percentage of spermatozoa with optimally functioning mitochondria was significantly correlated with the percentage of viable (PI negative) sperm cells (r(2) = 0.998). Flow cytometric analyses using this staining procedure provides reliable and rapid (10,000 cells/min) qualitative assessment of stallion semen.     

Effects of culturing bovine oocytes either singly or in groups on development to blastocysts

In vitro maturation, fertilization and culture (IVM/IVF/IVC) of cattle oocytes from individual cows requires adapting existing culture protocols so that small numbers of oocytes can be cultured. The culture of single oocytes is desirable for correlating the relationship between follicular properties with oocyte developmental competence or for facilitating ovum pick-up procedures. In Experiment 1 we compared group and single culture under cell-free conditions on embryo development; significantly higher (P<0.001) rates of cleavage (66.4 vs 47.6%) and blastocyst formation (7.5 vs 0.5%) were observed in the group cultured oocytes. In Experiment 2 we compared group and single oocyte co-culture with granulosa cells. Although there was no effect of oocyte number on the percentage cleaving (73.1 vs 66.6%), there were significantly higher blastocyst yields (37.4 vs 10.1%) and blastocyst cell numbers (91.6 vs 66.2) in group-cultured oocytes. In Experiment 3 we examined the effect of group size (1, 5, 10, 20 and 40 oocytes) in a co-culture system using granulosa cell monolayers. The results show a difference in cleavage rates between the single cultured oocytes (66.8%) and each group of cultured oocytes, with the highest cleavage rate (81.5%) obtained in the 20-oocyte group. The blastocyst yield from both cleaved and total oocytes showed that group culture of 20 or 40 oocytes resulted in the highest number of blastocysts (32.5%), with smaller group sizes yielding significantly (P<0.05) fewer blastocysts. In Experiment 4 we examined the effects of co-culture on the development of single vs group-cultured oocytes. The results showed no significant difference (P>0.05) in the cleavage rate between single and group culture systems. No blastocysts were formed with single oocytes cultured without monolayers, while the blastocyst formation rate for those co-cultured with granulosa cells was 12.4%. Blastocyst formation was significantly higher (P < 0.006) in group co-culture on monolayers (24.2 vs 8.5%). These data indicate that oocytes cultured in groups are developmentally more competent and suggest that for optimum development oocytes need some undefined paracrine activity that is absent from the culture medium in addition to coculture with granulosa cells, which enhances development to the blastocyst stage of both group and singly cultured oocytes.     

Phylogenetic Analysis of Heterothallic Neurospora Species

We examined the phylogenetic relationships among five heterothallic species of Neurospora using restriction fragment polymorphisms derived from cosmid probes and sequence data from the upstream regions of two genes, al-1 and frq. Distance, maximum likelihood, and parsimony trees derived from the data support the hypothesis that strains assigned to N. sitophila, N. discreta, and N. tetrasperma form respective monophyletic groups. Strains assigned to N. intermedia and N. crassa, however, did not form two respective monophyletic groups, consistent with a previous suggestion based on analysis of mitochondrial DNAs that N. crassa and N. intermedia may be incompletely resolved sister taxa. Trees derived from restriction fragments and the al-1 sequence position N. tetrasperma as the sister species of N. sitophila. None of the trees produced by our data supported a previous analysis of sequences in the region of the mating type idiomorph that grouped N. crassa and N. sitophila as sister taxa, as well as N. intermedia and N. tetrasperma as sister taxa. Moreover, sequences from al-1, frq, and the mating-type region produced different trees when analyzed separately. The lack of consensus obtained with different sequences could result from the sorting of ancestral polymorphism during speciation or gene flow across species boundaries, or both.     

16a-METHYL CORTICOSTEROIDS—A New Series of Anti-Inflammatory Compounds; Clinical Appraisal of Their Antirheumatic Potencies

Four new derivatives of hydrocortisone, each containing in common a methyl grouping at the 16a-carbon position of the steroid molecule, have been synthesized and are being studied in human subjects. The compounds are 16a-methyl 9a-fluoroprednisolone (MK-125: hexadecadrol), 16a-methyl 9a-fluorohydrocortisone (MK-126), 16a-methylprednisolone (MK-110), and 16a-methylhydrocortisone (MK-117). Biologic tests in animals have indicated that these analogues exhibit, in varying degrees, striking alterations of several physiologic properties, including enhanced anti-inflammatory activity unassociated with corresponding disturbance of electrolyte metabolism.In the present study preliminary observations of the effects of the four new compounds were made in patients with rheumatoid arthritis. Clinical estimates of the antirheumatic potencies of the compounds, as compared with prednisolone, were accomplished by determining the milligram dosages required to maintain similar degrees of improvement of active rheumatoid manifestations. The approximate antirheumatic potencies of the compounds, on an average, were gauged as follows: for 16a-methyl 9a-fluoroprednisolone, about seven times greater than prednisolone; for 16a-methyl 9a-fluorohydrocortisone, about three times greater; for 16a-methylprednisolone, approximately one-third greater; and for 16a-methylhydrocortisone, about 70 per cent that of prednisolone. In the dosage used, none of the compounds promoted discernible salt and water retention.These observations would indicate that 16a-methyl 9a-fluoroprednisolone (hexadecadrol) possesses greater anti-inflammatory potency per milligram than any steroid yet produced. The therapeutic efficiency of the compound on longterm administration is being studied.     

Outcomes among children and adults at risk of severe dengue in Sri Lanka: Opportunity for outpatient case management in countries with high disease burden

BackgroundHealthcare systems in dengue-endemic countries are often overburdened due to the high number of patients hospitalized according to dengue management guidelines. We systematically evaluated clinical outcomes in a large cohort of patients hospitalized with acute dengue to support triaging of patients to ambulatory versus inpatient management in the future.Methods/Principal findingsFrom June 2017- December 2018, we conducted surveillance among children and adults with fever within the prior 7 days who were hospitalized at the largest tertiary-care (1,800 bed) hospital in the Southern Province, Sri Lanka. Patients who developed platelet count ≤100,000/μL (threshold for hospital admission in Sri Lanka) and who met at least two clinical criteria consistent with dengue were eligible for enrollment. We confirmed acute dengue by testing sera collected at enrollment for dengue NS1 antigen or IgM antibodies. We defined primary outcomes as per the 1997 and 2009 World Health Organization (WHO) classification criteria: dengue hemorrhagic fever (DHF; WHO 1997), dengue shock syndrome (DSS; WHO 1997), and severe dengue (WHO 2009). Overall, 1064 patients were confirmed as having acute dengue: 318 (17.4%) by NS1 rapid antigen testing and 746 (40.7%) by IgM antibody testing. Of these 1064 patients, 994 (93.4%) were adults ≥18 years and 704 (66.2%) were male. The majority (56, 80%) of children and more than half of adults (544, 54.7%) developed DHF during hospitalization, while 6 (8.6%) children and 22 (2.2%) adults developed DSS. Overall, 10 (14.3%) children and 113 (11.4%) adults developed severe dengue. A total of 2 (0.2%) patients died during hospitalization.ConclusionsOne-half of patients hospitalized with acute dengue progressed to develop DHF and a very small number developed DSS or severe dengue. Developing an algorithm for triaging patients to ambulatory versus inpatient management should be the future goal to optimize utilization of healthcare resources in dengue-endemic countries.