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.

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.

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.

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.

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.

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.

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.

Heavy chain variable region. Multiple gene segments encode anti-4-(hydroxy-3-nitro-phenyl)acetyl idiotypic antibodies.

The hapten (4-hydroxy-3-nitrophenyl)acetyl (NP), when conjugated to carrier proteins, elicits a characteristic idiotypic response (NPb) in C57BL/6 mice. The response can be divided serologically into two distinct NPb-positive groups of antibodies. The first group consists of four crossreacting subgroups (I-IV), the second of two subgroups (V, VI). Some antibodies of subgroups I and II have been shown to express the unmutated heavy chain variable region (VH) germline gene 186.2. Antibodies of subgroups V and VI crossreact extensively with the NPa-positive antibodies of BALB/c mice. We sequenced heavy chain complementary DNA from eight hybridomas producing anti-NP antibodies. Six of these belong to subgroups V and VI, and two were NPa-positive hybridomas of BALB/c origin. All sequences were homologous to each other, and differed by approximately 80 basepairs from the 186.2 C57BL/6 germline VH gene. From our sequence and Southern blot analyses we suggest: (a) the NPb idiotypic response is the product of several VH germline genes, (b) some of these genes are very homologous to the gene coding for the BALB/c NPa idiotype, and might represent the C57BL/6 allelic forms of this gene, (c) the diversity regions of NPb and NPa-positive antibodies are diverse in length and amino acid composition, except for the first residue, which is always tyrosine, (d) all four heavy chain joining region gene segments are expressed without mutation. We discuss our data in terms of diversity in the germline VH gene repertoire, as well as diversity created by gene segment-joining events and somatic mutation.

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.

Rebound insomnia: a new clinical syndrome.

Rebound insomnia followed the withdrawal of three benzodiazepine hypnotic drugs, each of which had been administered in a single nightly dose for only short-term periods. The intense worsening of sleep is attributed to the short duration of the action of these drugs. A hypothesis involving benzodiazepine receptors in the brain is proposed in which there is a delay or lag in replacement of endogenous benzodiazepine-like molecules after the abrupt withdrawal of exogenous drugs.

Cigarette smoking associated with sleep difficulty.

A group of 50 smokers experienced greater sleep difficulty than a group of 50 nonsmokers matched by age and sex. The two groups did not differ in personality patterns or drug consumption. Also, sleep patterns significantly improved in a group of eight chronic smokers when they abstained from cigarette smoking. These findings are consistent with reports on the stimulant effects of nicotine.

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.

Simultaneous stimulation of slow-wave sleep and growth hormone secretion by gamma-hydroxybutyrate in normal young Men.

The aim of this study was to investigate, in normal young men, whether gamma-hydroxybutyrate (GHB), a reliable stimulant of slow-wave (SW) sleep in normal subjects, would simultaneously enhance sleep related growth hormone (GH) secretion. Eight healthy young men participated each in four experiments involving bedtime oral administration of placebo, 2.5, 3.0, and 3.5 g of GHB. Polygraphic sleep recordings were performed every night, and blood samples were obtained at 15-min intervals from 2000 to 0800. GHB effects were mainly observed during the first 2 h after sleep onset. There was a doubling of GH secretion, resulting from an increase of the amplitude and the duration of the first GH pulse after sleep onset. This stimulation of GH secretion was significantly correlated to a simultaneous increase in the amount of sleep stage IV. Abrupt but transient elevations of prolactin and cortisol were also observed, but did not appear to be associated with the concomitant stimulation of SW sleep. Thyrotropin and melatonin profiles were not altered by GHB administration. These data suggest that pharmacological agents that reliably stimulate SW sleep, such as GHB, may represent a novel class of powerful GH secretagogues.

Mixed chimerism and tolerance without whole body irradiation in a large animal model.

Mixed hematopoietic chimerism may provide a treatment for patients with nonmalignant hematologic diseases, and may tolerize patients to organ allografts without requiring chronic immunosuppression. However, the toxicity of the usual conditioning regimens has limited the clinical applicability of this approach. These regimens generally include some level of whole body irradiation (WBI), which is thought to facilitate engraftment either by making room for donor hematopoietic stem cells or by providing sufficient host immunosuppression to enable donor cells to engraft. Here, we have established mixed chimerism across both minor and major histocompatibility barriers in swine, by using high doses of peripheral blood stem cells in the absence of WBI. After mixed chimerism was established, swine leukocyte antigen-matched (SLA-matched) donor skin grafts were tolerated and maintained for a prolonged period, whereas third-party SLA-matched skin was rejected promptly. Donor-matched kidney allografts were also accepted without additional immunosuppression. Because of its low toxicity, this approach has potential for a wide range of clinical applications. Our data may indicate that niches for engrafting stem cells are filled by mass action and that WBI, which serves to empty some of these niches, can be omitted if the donor inoculum is sufficiently large and if adequate host T-cell depletion is achieved before transplant.

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.

Two hexamerin genes from the termite Reticulitermes flavipes: Sequence, expression, and proposed functions in caste regulation.

Previous molecular studies on the termite Reticulitermes flavipes have revealed that two hexamerin proteins serve an important status quo role in the regulation of juvenile hormone (JH)-dependent caste differentiation. Here, we report sequence data and other experimental evidence suggesting how these two hexamerins function in achieving caste regulation. The two hexamerin genes, named Hex-1 and Hex-2, encode highly unique sequence features relative to the 100+ other known insect hexamerins. These features include a long hydrophobic tail and prenylation motif in Hex-1, and a long hydrophilic insertion plus several putative protease cleavage sites in Hex-2. Both hexamerin genes are primarily expressed in fat body tissue, but only Hex-2 expression is substantially induced by JH. SDS-PAGE showed that the hexamerin proteins constitute a major proportion of total soluble termite protein. Also, although each protein occurs in both the membrane and soluble protein fractions, Hex-2 has stronger membrane affinity. Anti-JH antiserum specifically recognizes hemolymph-soluble Hex-1 protein, supporting that the unique prenylation site in Hex-1 facilitates covalent JH binding to the primary amino acid chain. Finally, increased ratios of Hex-2 to Hex-1 transcription occur in caste phenotypes and developmental stages that differentiate in response to rising JH titers. Two main conclusions can be taken from these studies. First, elevated ratios of Hex-2 to Hex-1 expression are associated with caste phenotypes that differentiate in response to rising JH titers (i.e., workers, presoldiers and soldiers). Second, due to their unique structural features and other observed characteristics, our findings support the hypothesis that the two hexamerins participate in the regulation of caste-differentiation by modulating JH availability.