Intercolony Comparisons of Gut Microbiome Composition From Lab Reared Eastern Subterranean Termites (Blattodea: Rhinotermitidae).

Termites are social insects living in colonies composed of worker, soldier, and reproductive castes. Termite hindguts are inhabited by all three domains of life- Eukarya (protists), Bacteria, and Archaea. These gut microorganisms are horizontally and vertically transferred by nestmates and reproductives, respectively. Prior evidence suggests that every colony potentially has a different gut microbiome that was transferred vertically and horizontally over time. However, we do not know if different colonies reared in the laboratory on the same diet will ultimately demonstrate similar microbial composition and structure. Therefore, we looked at gut bacteria in Eastern subterranean termite (Reticulitermes flavipes) colonies that were reared in the laboratory with identical diets and rearing conditions. Based on16S rRNA gene sequencing, the observed features, and Shannon's diversity were significantly different between the colonies while differences in Pielou evenness and Faith phylogenetic diversity were not statistically significant. In addition, the microbial community structures were significantly different between colonies. Based on ANCOM (Analysis of Composition of Microbiomes), the taxa Elizabethkingia (Bacteroidetes: Flavobacteriales) and Chryseobacterium (Bacteroidetes: Flavobacteriales) were differentially abundant between the colonies. These results suggest that providing the exact same diet and rearing environment for >2 yr cannot result in identical gut microbiomes between termite colonies.

High-content phenotypic screening identifies novel chemistries that disrupt mosquito activity and development.

New classes of chemistries are needed to control insecticide resistant populations of mosquitoes and prevent transmission of vector-borne diseases (VBDs). Organismal screens of chemical collections have played an important role in the search for new vector insecticides and the identification of active ingredients (AIs) that cause rapid mortality of mosquitoes. Advances in image-based screening offer an opportunity to identify chemistries that operate via novel biochemical modes and investigate the range of phenotypes exhibited by mosquitoes following exposure to lethal and sub-lethal chemical dose. An automated, high throughput phenotypic screen (HTS) employing high-content imaging of first instar (L1) Aedes aegypti larvae was developed to identify chemistries associated with mortality and atypical morphological phenotypes. A pilot screen of the Library of Pharmacologically Active Compounds (LOPAC1280) identified 92 chemistries that disrupted larval activity and development, including conventional insecticides and chemistries known to modulate G protein-coupled receptors (GPCRs) and other molecular targets in mammalian systems. Secondary assay series were used to evaluate a selection of chemistries for impacts on mosquito activity, survival and development. Ritodrine hydrochloride reduced mobility of larvae but had no observable effect on survival and development of mosquitoes. High doses of metergoline suppressed larval activity and sub-lethal dose resulted in pupal mortality. Assay data support the utility of phenotypic screening and diverse entomological end-points for discovery of novel insecticidal chemical scaffolds. The insecticide discovery process must consider how multi-modal efficacy spectra contribute to vector and VBD control.

Using trend arrows in continuous glucose monitoring systems for insulin adjustment in clinical practice: Brazilian Diabetes Society Position Statement.

This manuscript reports the Brazilian Diabetes Society Position Statement for insulin adjustments based on trend arrows observed in continuous glucose monitoring systems. The Brazilian Diabetes Society supports the utilization of trend arrows for insulin dose adjustments in patients with diabetes on basal-bolus insulin therapy, both with multiple daily insulin doses or insulin pumps without closed-loop features. For those on insulin pumps with predictive low-glucose suspend feature, we suggest that only upward trend arrows should be used for adjustments. In this paper, tables for insulin adjustment based on sensitivity factors are provided and strategies to optimize the use of trend arrows in clinical practice are discussed.

Switching from originator recombinant growth hormone (Genotropin™) to biosimilar (CRISCY™): Results from a 6-month, multicentric, non-inferiority, extension trial.

OBJECTIVE: A previous 12-month comparative trial with Criscy™ (r-hGH Cristália), a biosimilar recombinant growth hormone, demonstrated equivalent efficacy and safety to Genotropin™. This extension trial evaluated the effects of switching patients treated with Genotropin™ to the biosimilar Criscy™ over an additional 6-month treatment period, comparing efficacy, safety, and immunogenicity parameters with patients remaining in the Criscy™ arm. DESIGN: This extension study included 11 research centers and 81 patients who participated in the CERES study (Czepielewski et al., 2019 [1]). Participants from the Genotropin™ arm (n = 39) had the drug replaced by Criscy™ and the remaining participants were kept in the Criscy™ arm (n = 42) for an additional 6-month period to evaluate immunogenicity, efficacy (growth rate, height SDS), and safety (laboratory tests, and adverse events). RESULTS: Before the switch, both Criscy™ and Genotropin groups were similar concerning demographics, and auxological measures: age, sex, height, height SDS, weight, and BMI. Height velocity (HV) after 18 months of treatment was 8.7 ± 1.56 cm/year for Criscy™ group and 8.9 ± 1.36 cm/year for Genotropin™ group in the ITT population (p = 0.43). The auxological parameters and IGF-1 and IGFBP-3 SDS were comparable between both groups of patients. No participants were excluded from the study due to adverse events. There were no clinical or statistical relevant differences between the treatment groups concerning frequency, distribution, intensity, and AEs outcome. Similarly, no new anti-r-hGH (ADA) cases among patients that switched from Genotropin™ to Criscy™ were reported. No neutralizing antibody (nAb) was detected in either group. CONCLUSIONS: This trial showed that switching from originator recombinant human growth hormone to Criscy™ had no impact on efficacy, safety, nor immunogenicity as compared to continued treatment with Criscy™. Growth rates and ADA incidence remained the same as seen before the switch.

Efficacy and safety of a biosimilar recombinant human growth hormone (r-hGH Cristalia) compared with reference r-hGH in children with growth hormone deficiency (CERES study): A randomized, multicentric, investigator-blind, phase 3 trial.

OBJECTIVE: The CERES study was a randomized, multicenter, investigator-blind trial aimed to evaluate the efficacy and safety of a recombinant human growth hormone (r-hGH) developed by Cristalia, as a biosimilar product, with analytical, functional and pharmacokinetics similarities comparable to Genotropin™, in children with growth hormone deficiency (GHD). DESIGN: A total of 135 naïve prepubertal children with GHD were recruited, of whom 97 were randomized in 14 Brazilian sites to received either r-hGH Cristalia (n = 49) or Genotropin™ (n = 48). Efficacy was evaluated considering the height standard deviation score (SDS) and growth velocity as auxological parameters, IGF-1 and IGFBP-3 were measured as pharmacodynamic parameters during 12 months treatment time. Safety was assessed by monitoring adverse events, immunogenicity, blood count with platelets, biochemical profile and hormonal levels particularly fasting glucose, insulin and HbA1C. RESULTS: The auxological parameters and IGF-1 and IGFBP-3 levels were comparable between both groups of patients. At end of study or the 12th month treatment, the means growth velocity was 9.7 cm/year and 9.5 cm/year, for r-hGH Cristalia and Genotropin™, respectively. The ANCOVA mean difference between the groups was 0.16 cm/year to Cristalia group (CI 95% = -0.72 to 1.03 cm/year). There was no difference in adherence among the treatment groups. The safety profile was comparable between groups. CONCLUSIONS: The clinical similarity between r-hGH and Genotropin™ was demonstrated within 12 month of treatment. On the basis of comparability of quality, safety, and efficacy to the reference product, r-hGH from Cristalia can be considered a cost-effective therapeutic option for patients with growth disorders.

RNA interference and functional characterization of a tergal gland alpha amylase in the German cockroach, Blattella germanica L.

German cockroach males possess tergal glands that secrete a combination of oligosaccharides, lipids and proteins. Four major proteins occur in the secretion, with one being the 63 kDa alpha-amylase Blattella germanica Tergal Gland protein-1 (BGTG-1). Denaturing and starch gel electrophoresis coupled with peptide sequencing verified amylase activity for the BGTG-1 protein. BGTG-1 gene expression profiles were determined by using quantitative real-time PCR to compare messenger RNA abundance among isolated tissues of males, females and gravid females. Differences in BGTG-1 gene expression occurred among male tissues, with tergal gland tissue showing the highest expression. Tissues of nongravid and gravid females had significantly lower expression in comparison with male tergal glands (gravid females lowest). RNA interference (RNAi) was used to silence BGTG-1 gene expression by injecting BGTG-1 homologous double-stranded RNA (dsRNA) into male cockroaches. Groups injected with BGTG-1 dsRNA showed ∼90% lower BGTG-1 gene and protein expression compared to controls, which correlated with lower amylase activity in colorimetric assays. However, behavioural assays comparing precopulatory behaviour and mating success between RNAi and control males did not reveal differences. These results connect amylase gene expression and activity in tergal gland tissue but suggest other factors, such as other tergal gland components, may contribute more strongly to mating success.

Description of a multi-university education and collaborative care child psychiatry access program: New York State's CAP PC.

OBJECTIVE: Although, child mental health problems are widespread, few get adequate treatment, and there is a severe shortage of child psychiatrists. To address this public health need many states have adopted collaborative care programs to assist primary care to better assess and manage pediatric mental health concerns. This report adds to the small literature on collaborative care programs and describes one large program that covers most of New York state. PROGRAM DESCRIPTION: CAP PC, a component program of New York State's Office of Mental Health (OMH) Project TEACH, has provided education and consultation support to primary care providers covering most of New York state since 2010. The program is uniquely a five medical school collaboration with hubs at each that share one toll free number and work together to provide education and consultation support services to PCPs. METHODS: The program developed a clinical communications record to track information about all consultations which forms the basis of much of this report. 2-week surveys following consultations, annual surveys, and pre- and post-educational program evaluations have also been used to measure the success of the program. RESULTS: CAP PC has grown over the 6years of the program and has provided 8013 phone consultations to over 1500 PCPs. The program synergistically provided 17,523 CME credits of educational programming to 1200 PCPs. PCP users of the program report very high levels of satisfaction and self reported growth in confidence. CONCLUSIONS: CAP PC demonstrates that large-scale collaborative consultation models for primary care are feasible to implement, popular with PCPs, and can be sustained. The program supports increased access to child mental health services in primary care and provides child psychiatric expertise for patients who would otherwise have none.

Identification of the flotillin-1/2 heterocomplex as a target of autoantibodies in bona fide multiple sclerosis.

BACKGROUND: Autoantibodies, in particular those against aquaporin-4 and myelin-oligodendrocyte glycoprotein (MOG), aid as biomarkers in the differential diagnosis of demyelination. Here, we report on discovery of autoantibodies against flotillin in patients with multiple sclerosis (MS). METHODS: The target antigen was identified by histo-immunoprecipitation using the patients' sera and cryosections of rat or pig cerebellum combined with mass spectrometrical analysis. Correct identification was ascertained by indirect immunofluorescence and neutralization tests using the target antigens recombinantly expressed in HEK293 cells. RESULTS: Serum and CSF of the index patient produced a fine-granular IgG indirect immunofluorescence staining of the hippocampal and cerebellar molecular layers. Flotillin-1 and flotillin-2 were identified as target autoantigens. They also reacted with recombinant human flotillin-1/2 co-expressed in HEK293 cells, but not with the individual flotillins in fixed- and live-cell assays. Moreover, neutralization using flotillin-1/2, but not the single flotillins, abolished the tissue reactivity of patient serum. Screening of 521 patients, for whom anti-aquaporin-4 testing was requested and negative, revealed 8 additional patients with anti-flotillin-1/2 autoantibodies. All eight were negative for anti-MOG. Six patients ex post fulfilled the revised McDonald criteria for MS. Vice versa, screening of 538 MS sera revealed anti-flotillin-1/2 autoantibodies in eight patients. The autoantibodies were not found in a cohort of 67 patients with other neural autoantibody-associated syndromes and in 444 healthy blood donors. CONCLUSIONS: Autoantibodies against the flotillin-1/2 heterocomplex, a peripheral membrane protein that is involved in axon outgrowth and regeneration of the optic nerve, are present in 1-2% of patients with bona fide MS.

Silencing gut genes associated with the peritrophic matrix of Reticulitermes flavipes (Blattodea: Rhinotermitidae) increases susceptibility to termiticides.

The peritrophic matrix (PM) is a noncellular structure that lines the gut of most insects. Because of its close involvement in digestive processes and its role as a barrier against pathogens and toxins, the PM is an attractive target for pest management strategies. The objectives of this study were to (1) reduce the expression of a chitin synthase gene (Reticulitermes flavipes chitin synthase B, RfCHSB), a putative peritrophin [R. flavipes Protein with Peritrophin-A domain 1, (RfPPAD1)] and a confirmed peritrophin [R. flavipes Protein with Peritrophin-A domain 2 (RfPPAD2)] in R. flavipes by means of RNA interference, and (2) to evaluate the susceptibility of R. flavipes to termiticides and a bacterial pathogen, after silencing the target genes. Force feeding termites with 55 and 100 ng of long double-stranded RNAs (dsRNAs), targeting RfCHSB and RfPPAD2, respectively, resulted in the highest levels of transcript suppression. RfCHSB expression was reduced by 70%, whereas the transcript level of RfPPAD2 was decreased by 90%. Force feeding 100 ng/termite of a long RfPPAD1 dsRNA reduced the expression of the transcript by 30%. Challenging termites with imidacloprid, chlorantraniliprole and noviflumuron, after silencing RfCHSB, significantly increased termite mortality. Force feeding termites a dsRNA cocktail, targeting RfCHSB, RfPPAD1 and RfPPAD2, caused the highest significant increase in termite mortality after challenging the insects with imidacloprid. These results demonstrate the viability of the R. flavipes PM as a target in termite pest management.

[High versus moderate intense running exercise - effects on cardiometabolic risk-factors in untrained males]. / Hoch- versus moderat-intensive Laufbelastung - Einfluss auf kardio-metabolische Risikogrößen bei untrainierten Männern.

Introduction | The philosophy on how to improve cardiometabolic risk factors most efficiently by endurance exercise is still controversial. To determine the effect of high-intensity (interval) training (HI[I]T) vs. moderate-intensity continuous exercise (MICE) training on cardiometabolic risk factors we conducted a 16-week crossover randomized controlled trial. Methods | 81 healthy untrained middle aged men were randomly assigned to a HI(I)T-group and a control-group that started the MICE running program after their control status. HI(I)T consisted of running exercise around or above the individual anaerobic threshold (≈ 80- 100 % HRmax); MICE focused on continuous running exercise at ≈ 65-77.5 % HRmax. Both protocols were comparable with respect to energy consumption. Study endpoints were cardiorespiratory fitness (VO2max), left ventricular mass index (LVMI), metabolic syndrome Z-score (MetS-Z-score), intima-media-thickness (IMT) and body composition. Results | VO2max-changes in this overweighed male cohort significantly (p=0.002) differ between HIIT (14.7 ± 9.3 %, p=0.001) and MICE (7.9 ± 7.4 %,p=0.001). LVMI, as determined via magnetic resonance imaging, significantly increased in both exercise groups (HIIT: 8.5 ± 5.4 %, p=0.001 vs. MICE: 5.3 ± 4.0 %, p=0.001), however the change was significantly more pronounced (p=0.005) in the HIIT-group. MetS-Z-score (HIIT: -2.06 ± 1.31, p=0.001 vs. MICE: -1.60 ± 1.77, p=0.001) and IMT (4.6 ± 5.9 % p=0.011 vs. 4.4 ± 8.1 %, p=0.019) did not show significant group-differences. Reductions of fat mass (-4.9 ± 9.0 %, p=0.010 vs. -9.5 ± 9.4, p=0.001) were significantly higher among the MICE-participants (p=0.034), however, the same was true (p=0.008) for lean body mass (0.5 ± 2.3 %, p=0.381 vs. -1.3 ± 2.0 %, p=0.003). Conclusion | In summary high-intensity interval training tends to impact cardiometabolic health more favorable compared with a moderate-intensity continuous endurance exercise protocol.

Dose reduction in computed tomography of the chest: image quality of iterative reconstructions at a 50% radiation dose compared to filtered back projection at a 100% radiation dose.

PURPOSE: The aim of this study was to evaluate the potential of iterative reconstruction (IR) in chest computed tomography (CT) to reduce radiation exposure. The qualitative and quantitative image quality of standard reconstructions with filtered back projection (FBP) and half dose (HD) chest CT data reconstructed with FBP and IR was assessed. MATERIALS AND METHODS: 52 consecutive patients underwent contrast-enhanced chest CT on a dual-source CT system at 120 kV and automatic exposure control. The tube current was equally split on both tube detector systems. For the HD datasets, only data from one tube detector system was utilized. Thus, FD and HD data was available for each patient with a single scan. Three datasets were reconstructed from the raw data: standard full dose (FD) images applying FBP which served as a reference, HD images applying FBP and IR. Objective image quality analysis was performed by measuring the image noise in tissue and air. The subjective image quality was evaluated by 2 radiologists according to European guidelines. Additional assessment of artifacts, lesion conspicuity and edge sharpness was performed. RESULTS: Image noise did not differ significantly between HD-IR and FD-FBP (p = 0.254) but increased substantially in HD-FBP (p < 0.001). No statistically significant differences were found for the reproduction of anatomical and pathological structures between HD-IR and FD-FBP, subsegmental bronchi and bronchioli. The image quality of HD-FBP was rated inferior because of increased noise. CONCLUSION: A 50 % dose reduction in contrast-enhanced chest CT is feasible without a loss of diagnostic confidence if IR is used for image data reconstruction. Iterative reconstruction is another powerful tool to reduce radiation exposure and can be combined with other dose-saving techniques. KEY POINTS: • Iterative reconstructions allow for image noise and artifact reduction.• Comparable image data can thus be attained even at 50 % radiation dose.• Diagnostic confidence remains unaffected.

Tissue characteristics in tendon-to-bone healing change after rotator cuff repair using botulinumneurotoxin A for temporary paralysis of the supraspinatus muscle in rats.

We hypothesized that botulinumneurotoxin A (BoNtA) positively influences tissue characteristics at the re-insertion site when used as an adjuvant prior to rotator cuff repair. One hundred and sixty Sprague-Dawley rats were randomly assigned to either a BoNtA or saline-injected control group. BoNtA or saline solution was injected into the supraspinatus muscle one week prior to repair of an artificially created supraspinatus tendon defect. Post-operatively, one subgroup was immobilized using a cast on the operated shoulder while the other had immediate mobilization. Histologically, the fibrocartilage transition zone was more prominent and better organized in the BoNtA groups when compared to the saline control group. In the immediately mobilized BoNtA groups significantly more collagen 2 at the insertion was detected than in the control groups (p<0.05). Fiber orientation of all BoNtA groups was better organized and more perpendicular to the epiphysis compared with control groups. Tendon stiffness differed significantly (p<0.05) between casted BoNtA and casted saline groups. Tendon viscoelasticity was significantly higher (p<0.05) in the immobilized saline groups no matter if repaired with increased or normal repair load. The results of this study suggest that reduction of load at the healing tendon-to-bone interface leads to improved repair tissue properties.

Comparative metatranscriptomic signatures of wood and paper feeding in the gut of the termite Reticulitermes flavipes (Isoptera: Rhinotermitidae).

Termites are highly eusocial insects that thrive on recalcitrant materials like wood and soil and thus play important roles in global carbon recycling and also in damaging wooden structures. Termites, such as Reticulitermes flavipes (Rhinotermitidae), owe their success to their ability to extract nutrients from lignocellulose (a major component of wood) with the help of gut-dwelling symbionts. With the aim to gain new insights into this enzymatic process we provided R. flavipes with a complex lignocellulose (wood) or pure cellulose (paper) diet and followed the resulting differential gene expression on a custom oligonucleotide-microarray platform. We identified a set of expressed sequence tags (ESTs) with differential abundance between the two diet treatments and demonstrated the source (host/symbiont) of these genes, providing novel information on termite nutritional symbiosis. Our results reveal: (1) the majority of responsive wood- and paper-abundant ESTs are from host and symbionts, respectively; (2) distinct pathways are associated with lignocellulose and cellulose feeding in both host and symbionts; and (3) sets of diet-responsive ESTs encode putative digestive and wood-related detoxification enzymes. Thus, this study illuminates the dynamics of termite nutritional symbiosis and reveals a pool of genes as potential targets for termite control and functional studies of termite-symbiont interactions.

Dynamic contrast-enhanced ultrasound (DCE-US) for easy and rapid evaluation of hepatocellular carcinoma compared to dynamic contrast-enhanced computed tomography (DCE-CT)--a pilot study.

PURPOSE: To check the feasibility of the easy quantification of tumor vascularization derived from dynamic contrast-enhanced ultrasound (DCE-US) in comparison to dynamic contrast-enhanced computed tomography (DCE-CT) in patients with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: 19 patients with cirrhosis and histologically proven HCC prospectively underwent CEUS (SonoVue) and CT (Imeron400). Following CEUS, the software ImageJ was used for the easy quantification of the echogenicity in HCC lesions and tumor-free liver parenchyma. For DCE-CT we used the software Hepacare and created arterial enhancement fraction color maps of the whole liver and HCC lesions. RESULTS: Unifocal/multifocal HCCs were detected in 12/7 (US) and 10/9 patients (CT) and biopsied nodules were defined as a reference lesion with a median of 40 mm (US) and 42 mm (CT). CEUS showed HCC-typical hyper-/hypoenhancement in the arterial/late phase in 16/19 reference lesions, while all reference lesions showed an HCC-typical vascular pattern in CT. With DCE-US, quantitative assessment could not be performed in 3/19 patients due to respiratory motion or insufficient image quality. 13/16 reference lesions showed an HCC-typical vascular pattern. Quantitative assessment was possible with DCE-CT in all patients and all reference nodules showed HCC-typical values of the arterial enhancement fraction. There was no statistical difference between CEUS, DCE-US and DCE-CT in the quantitative assessment of contrast enhancement. CONCLUSION: The quantitative evaluation of DCE-US was feasible in HCC without a statistical difference with respect to DCE-CT. Further studies with a larger study population including small nodules ≤ 2 cm are needed to assess whether this technique is helpful in routine ultrasound.

Diagnosis of prostate cancer in patients with persistently elevated PSA and tumor-negative biopsy in ambulatory care: performance of MR imaging in a multi-reader environment.

PURPOSE: False-negative results are obtained in approx. 20 % of prostate cancer (PCa) patients (pts) at initial systematic transrectal biopsy (Bx), in particular when digital rectal examination (DRE) or transrectal ultrasound (TRUS) is negative. The aim of this study was to assess whether MR endorectal imaging of the prostate in a multi-reader ambulatory care setting may assist in patient selection for re-biopsy. MATERIALS AND METHODS: 115 consecutive pts with persistent PSA elevation, negative Bx, DRE and TRUS were examined using T2w axial and coronal and T1w axial sequences for tumor diagnosis. MR images were prospectively read as tumor-suspicious or tumor-negative by the MR radiologist on duty. Additionally, a retrospective readout of a prostate MR expert and an abdominal imaging fellowship-trained radiologist was performed to evaluate the effect of the reader's experience on tumor detection. Imaging findings were compared to the results of the repeat Bx (61 pts) or the clinical course of at least two years. RESULTS: For the prospective reading, the sensitivity of MRI was 83 %, the specificity was 69 %, the PPV was 33 % and the NPV was 96 %. ROC analysis revealed a significantly better performance of the prostate MR imaging expert compared to the abdominal imaging radiologist (area under ROC 0.88 vs. 0.66, p < 0.001). Based on the prospective reading, a pre-test probability for PCa of 17.4 % as in our study can be reduced to 5 % when obtaining a tumor-negative result in MRI. CONCLUSION: MR imaging in a multi-reader ambulatory care setting assists in patient selection for re-biopsy. Reducing the post-test probability for PCa to 5 % allows for further follow-up instead of re-biopsy in MR tumor-negative patients. Specific training and experience improve tumor detection in prostate MR imaging.

Characterization of five CYP4 genes from Asian citrus psyllid and their expression levels in Candidatus Liberibacter asiaticus-infected and uninfected psyllids.

Previously, we reported that Candidatus Liberibacter asiaticus (Las)-infected Diaphorina citri are characterized by lower levels of cytochrome P450 monooxygenases than uninfected counterparts. In the present study, we investigated expression levels of family 4 cytochrome P450 (CYP4) genes in Las-infected and uninfected D.citri adults. Five novel CYP4 genes (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1 and CYP4G70) were identified. Four of the five CYP4 genes were expressed at significantly higher levels in uninfected than Las-infected males, whereas only one was expressed at significantly higher levels in uninfected than Las-infected females. These results suggest that levels of cytochrome P450 monooxygenases in D.citri may be linked to expression levels of these CYP4 genes. Expression of all five CYP4 genes was induced by exposure of D.citri to imidacloprid, suggesting their possible involvement in metabolism of this toxin. Higher expression of the five CYP4 genes was found in nymphs than adults, which is congruent with previous results indicating higher levels of cytochrome P450 monooxygenases in nymphs than adults. These five CYP4 genes may be promising candidates for RNA-interference to silence overexpression of genes associated with insecticide resistance in D.citri. These newly identified genes may also serve as DNA-based screening markers for cytochrome P450-mediated insecticide resistance in field populations of D.citri.

The BAR domain protein PICK1 regulates cell recognition and morphogenesis by interacting with Neph proteins.

Neph proteins are evolutionarily conserved membrane proteins of the immunoglobulin superfamily that control the formation of specific intercellular contacts. Cell recognition through these proteins is essential in diverse cellular contexts such as patterning of the compound eye in Drosophila melanogaster, neuronal connectivity in Caenorhabditis elegans, and the formation of the kidney filtration barrier in mammals. Here we identify the PDZ and BAR domain protein PICK1 (protein interacting with C-kinase 1) as a Neph-interacting protein. Binding required dimerization of PICK1, was dependent on PDZ domain protein interactions, and mediated stabilization of Neph1 at the plasma membrane. Moreover, protein kinase C (PKCα) activity facilitated the interaction through releasing Neph proteins from their binding to the multidomain scaffolding protein zonula occludens 1 (ZO-1), another PDZ domain protein. In Drosophila, the Neph homologue Roughest is essential for sorting of interommatidial precursor cells and patterning of the compound eye. RNA interference-mediated knockdown of PICK1 in the Drosophila eye imaginal disc caused a Roughest destabilization at the plasma membrane and a phenotype that resembled rst mutation. These data indicate that Neph proteins and PICK1 synergistically regulate cell recognition and contact formation.

Phenol-oxidizing laccases from the termite gut.

cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma - mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (∼4 copper molecules per laccase protein molecule and ∼3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion.

HLA-B*9553: a novel HLA allele shown in three generations of a family.

Human leukocyte antigen (HLA)-B*9553, a novel HLA-B allele, was identified in a volunteer hematopoietic stem cell donor. HLA-B*9553 differs from the closely related allele HLA-B*1518 in one single nucleotide substitution resulting in an amino acid substitution.

Characterization of BGTG-1, a tergal gland-secreted alpha-amylase, from the German cockroach, Blattella germanica (L.).

The protein fraction of the German cockroach, Blattella germanica (L.), tergal gland secretion was examined. SDS-PAGE separation of proteins present in B. germanica tergal gland secretion revealed a tergal gland-secreted protein, BGTG-1, at approximately 63 kDa. BGTG-1 first appeared in tergal gland secretion at 2 days postimaginal moult and the amount of protein observed increased through day 5. A 2051 bp cDNA sequence, bgtg-1, was obtained by RACE polymerase chain reaction and contains a 1494 bp ORF encoding a predicted protein of 498 amino acids. In a Northern hybridization experiment using total RNA from B. germanica tergal gland tissue, a (32)P-labelled bgtg-1 probe hybridized to an RNA approximately 2000 bp and confirmed the 2051 bp cDNA size obtained by RACE PCR. Using the BLASTx sequence similarity search tool, the top match to the bgtg-1 ORF was found to be an alpha-amylase from Drosophila kikkawai (e-value = 1 x 10(-178)). Alignment of the bgtg-1 deduced protein sequence with alpha-amylases from fruit fly, Drosophila melanogaster, honey bee, Apis mellifera (L.) and yellow mealworm, Tenebrio molitor (L.), revealed conserved residues throughout the ORF and sequence identities ranging from 58.4 to 58.2%. Using a gel-based assay, degradation of starch by native BGTG-1 was demonstrated in vitro and we propose that BGTG-1 may be involved in processing phagostimulatory sugars present in B. germanica tergal gland secretion.