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Tanner crab (Chionoecetes bairdi) is an economically important species that is threatened by ocean warming and bitter crab disease, which is caused by an endoparasitic dinoflagellate, Hematodinium. Little is known about disease transmission or its link to host mortality, or how ocean warming will affect pathogenicity or host susceptibility. To provide a transcriptomic resource for the Tanner crab, we generated a suite of RNA-seq libraries encompassing pooled hemolymph samples from crab displaying differing infection statuses and maintained at different temperatures (ambient (7.5˚C), elevated (10˚C), or decreased (4˚C)). After assembling a transcriptome and performing a multifactor differential gene expression analysis, we found genes influenced by temperature in relation to infection and detected some of those genes over time at the individual level using RNA-seq data from one crab. Biological processes associated with those genes include lipid storage, transcription, response to oxidative stress, cell adhesion, and morphogenesis. Alteration in lipid storage and transcription provide insight into how temperature impacts energy allocation in Hematodinium infected crabs. Alteration in expression patterns in genes associated with morphogenesis could suggest that hemocytes were changing morphology and/or type in response to temperature. This project provides insight into how Hematodinium infection could influence crab physiology as oceans warm., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.

Chemokine receptors belong to the large family of 7-transmembrane (7TM) G-protein-coupled receptors. These receptors are targeted and activated by a variety of different ligands, indicating that activation is a result of similar molecular mechanisms but not necessarily similar modes of ligand binding. Attempts to unravel the activation mechanism of 7TM receptors have led to the conclusion that activation involves movements of the transmembrane segments VI and VII in particular, as recently gathered in the Global Toggle Switch Model. However, to understand the activation mechanism completely, more research has to be done in this field. Chemokine receptors are interesting tools in this matter. First, the chemokine system has a high degree of promiscuity that allows several chemokines to target one receptor in different ways, as well as a single chemokine ligand to target several receptors in different ways. Second, the endogenous ligands are large proteins that mainly activate their cognate receptors by interacting with various extracellular-located receptor regions. It is, however, also possible to introduce agonism of simple ligands like metal ions. Thus, the chemokine system offers the possibility to test and compare the activation profiles of several chemically diverse ligands. This also brings up the interesting discussion of allosterism, because small molecules in the chemokine field often interact with allosteric receptor sites.

7 transmembrane-spanning (7TM) chemokine receptors having multiple endogenous ligands offer special opportunities to understand the molecular basis for allosteric mechanisms. Thus, CC-chemokine receptor 1 (CCR1) binds CC-chemokine 3 and 5 (CCL3 and CCL5) with K(d) values of 7.3 and 0.16 nm, respectively, as determined in homologous competition binding assays. However, CCL5 appears to have a >10,000-fold lower affinity in competition against (125)I-CCL3. Mutational mapping revealed that CCL3 and CCL5 both are strongly affected by systematic truncations of the N-terminal extension, whereas only CCL5 and not CCL3 activation is affected by substitutions in the main ligand binding pocket including the conserved GluVII:06 anchor point. A series of metal ion chelator complexes were found to act as full agonists on CCR1 and to be critically affected by the same substitutions in the main ligand binding pocket as CCL5 but not by mutations in the extracellular domain. In agreement with the overlapping binding sites, the small non-peptide agonists displaced radiolabeled CCL5 with high affinity. Interestingly, the same compounds acted as allosteric enhancers of the binding of CCL3, with which they did not overlap in binding site, leading to an increased B(max) and affinity of this chemokine mainly due to an increased association rate. It is concluded that a small molecule agonist through binding deep in the main ligand binding pocket can act as an allosteric enhancer for one endogenous chemokine and at the same time as a competitive blocker of the binding of another endogenous chemokine.

Most nonpeptide antagonists for CC-chemokine receptors share a common pharmacophore with a centrally located, positively charged amine that interacts with the highly conserved glutamic acid (Glu) located in position 6 of transmembrane helix VII (VII:06). We present a novel CCR8 nonpeptide agonist, 8-[3-(2-methoxyphenoxy)benzyl]-1-phenethyl-1,3,8-triaza-spiro[4.5]decan-4-one (LMD-009), that also contains a centrally located, positively charged amine. LMD-009 selectively stimulated CCR8 among the 20 identified human chemokine receptors. It mediated chemotaxis, inositol phosphate accumulation, and calcium release with high potencies (EC50 from 11 to 87 nM) and with efficacies similar to that of the endogenous agonist CCL1, and it competed for 125I-CCL1 binding with an affinity of 66 nM. A series of 29 mutations targeting 25 amino acids broadly distributed in the minor and major ligand-binding pockets of CCR8 uncovered that the binding of LMD-009 and of four analogs [2-(1-(3-(2-methoxyphenoxy)benzyl)-4-hydroxypiperidin-4-yl)benzoic acid (LMD-584), N-ethyl-2-4-methoxybenzenesulfonamide (LMD-902), N-(1-(3-(2-methoxyphenoxy)benzyl)piperidin-4-yl)-2-phenyl-4-(pyrrolidin-1yl)butanamide (LMD-268), and N-(1-(3-(2-methoxyphenoxy)benzyl)piperidin-4-yl)-1,2,3,4-tetrahydro-2-oxoquinoline-4-carboxamide (LMD-174)] included several key-residues for nonpeptide antagonists targeting CCR1, -2, and -5. It is noteworthy that a decrease in potency of nearly 1000-fold was observed for all five compounds for the Ala substitution of the anchor-point GluVII:06 (Glu(286)) and a gain-of-function of 19-fold was observed for LMD-009 (but not the four other analogs) for the Ala substitution of PheVI:16 (Phe(254)). These structural hallmarks were particularly important in the generation of a model of the molecular mechanism of action for LMD-009. In conclusion, we present the first molecular mapping of the interaction of a nonpeptide agonist with a chemokine receptor and show that the binding pocket of LMD-009 and of analogs overlaps considerably with the binding pockets of CC-chemokine receptor nonpeptide antagonists in general.

The isolation, PCR amplification, and descriptive statistics of six microsatellite loci are described for the Dungeness crab, Cancer magister. Also reported is the inheritance of these loci in two families obtained from artificial crosses in the laboratory. All six loci conform to expectations under Mendelian inheritance and there is no evidence for linkage between any of the loci. Allelic size ranges for three of the loci are relatively large, ranging from 135-357 bp between the smallest and largest allele detected at that locus. At two of these loci upper allelic drop out (non-amplification of the larger allele in a heterozygous individual) can be problematic for scoring. Results from cross-species amplification in nine congeners are summarized. These loci will be valuable in studies requiring high-resolution genetic markers in Dungeness crabs and related species.

Disease-specific registries have many important applications in epidemiologic, clinical and health services research. Since 1989 the Department of Veterans Affairs has maintained a national HIV registry. VA's HIV registry is national in scope, it contains longitudinal data and detailed resource utilization and clinical information. To describe the structure, function, and limitations of VA's national HIV registry, and to test its accuracy and completeness. The VA's national HIV registry contains data that are electronically extracted from VA's computerized comprehensive clinical and administrative databases, called Veterans Integrated Health Systems Technology and Architecture (VISTA). We examined the number of AIDS patients and the number of new patients identified to the registry, by year, through December 1996. We verified data elements against information obtained from the medical records at five VA sites. By December 1996, 40,000 HIV-infected patients had been identified to the registry. We encountered missing data and problems with data classification. Missing data occurred for some elements related to the computer programming that creates the registry (e.g., pharmacy files), and for other elements because manual entry is required (e.g., ethnicity). Lack of a standardized data classification system was a problem, especially for the pharmacy and laboratory files. In using VA's national HIV registry we have learned important lessons, which, if taken into account in the future, could lead to the creation of model disease-specific registries.

Objective: A Phase II, open-label, randomized, parallel-arm, multicentre trial to compare the antiviral activity and safety of two formulations of saquinavir (SQV), soft gelatin (SQV-SGC) and hard gelatin (SQV-HGC) capsules, in combination with two nucleoside reverse transcriptase inhibitors (NRTI), in antiretroviral-naive, HIV-1-infected individuals., Participants: A total of 171 people of > or = 13 years, with plasma HIV-1 RNA levels > or = 5000 copies/ml, who had received no protease inhibitor therapy, < or = 4 weeks NRTI therapy and no antiretroviral treatment within 28 days of screening. Eighty-one people were randomized to the SQV-HGC group and 90 to the SQV-SGC group. A total of 148 patients completed 16 weeks of therapy., Intervention: Therapy for 16 weeks with either SQV-SGC 1200 mg or SQV-HGC 600 mg, both three times a day, in combination with two NRTI., Results: Using an on-treatment analysis, patients taking SQV-SGC had a larger reduction in plasma HIV-1 RNA than those taking SQV-HGC (-2.0 versus -1.6 log10 copies/ml). Eighty per cent of those on SQV-SGC had < 400 copies HIV RNA/ml, compared with 43% in the SQV-HGC group (P = 0.001). A statistically significant difference in the area under the curve (AUC) values between the SQV-SGC and SQV-HGC arms (-1.7 versus -1.5 log10 copies/ml, respectively; P = 0.0054) was observed when withdrawals prior to week 12, major protocol violators and patients with < 75% compliance were excluded from the analysis; however, the difference between the values for the intent-to-treat population was not significant (P = 0.1929). Adverse events (mostly mild) included diarrhoea and nausea., Conclusions: SQV-SGC was generally well tolerated and gave significantly more potent suppression of plasma HIV-1 RNA in antiretroviral-naive patients than SQVHGC.

Anatomical and behavioral aspects of reproduction in female Dungeness crab, Cancer magister, were investigated. Female crabs were collected over a two-year period and external indicators of reproductive condition were recorded. A subset of crabs was retained for macroscopic and histological examination of the reproductive tract. In addition, males and females were held in the laboratory for mating observations, thus providing females of known mating history for dissection. The spermatheca is of the "ventral type," i.e., the vagina and the oviduct open into the spermatheca in close proximity to each other. A novel organ that often contains sperm, the bursa, is separate and distinct from the spermatheca in both position and origin and is reported for the first time for a brachyuran crab. The bursa is located just inside the vulva, distal to the spermatheca, and opens into the vagina. The sperm plug of C. magister, described here for the first time, is unique in form among cancrids. The sperm plug is placed in the vagina by the first male to mate with a soft-shelled female and prevents subsequent access to the spermatheca. However, the sperm plug does not occlude the vulva nor prevent subsequent copulation; ejaculates from subsequent matings are deposited in the bursa. Hypotheses of the possible function of the bursa are discussed.

Following a 4-year controlled trial comparing early and later zidovudine treatment, we conducted an additional 3-year follow-up. Of the original 338 patients, 275 participated. Clinical outcome measures were AIDS and death. In the early therapy group (n = 170), 67 patients progressed to AIDS compared with 85 in the later therapy group (n = 168); the relative risk (RR) comparing early with later therapy was 0.72% (95% confidence interval [CI] 0.52-0.99; p = 0.044). The early therapy group had 74 deaths compared with 73 in the later therapy (RR = 0.98; 95% CI, 0.71-1.36; p = 0.91). The early group had a peak CD4+ count increase at 1-2 months and a delay of 1 year before CD4+ counts fell below baseline. For patients who received zidovudine for more than the median duration (20.3 months) before their first AIDS diagnosis, the RR for death was 2.08 (95% CI, 1.36-3.19, p = 0.001). Additional factors independently associated with poor prognosis following AIDS were a CD4+ count of < 100 cells/mm3 and increased severity of the first AIDS diagnosis, whereas use of another antiretroviral agent was associated with improved survival. We conclude that early zidovudine therapy delays progression to AIDS but does not affect survival. Patients who progress to AIDS while on prolonged zidovudine monotherapy many benefit from a change to other antiretroviral therapy(ies).

Human immunodeficiency virus (HIV)-infected individuals often demonstrate neuropsychiatric impairment; however, it is unclear how brain metabolism may be altered in such patients. We used in vivo phosphorus 31 magnetic resonance spectroscopy to noninvasively assess brain energy and phospholipid metabolism by measuring brain concentrations of adenosine triphosphate (ATP), phosphocreatine (PCr), and inorganic phosphate (Pi), as well as phospholipid compounds and intracellular pH. In study 1, 17 HIV-seropositive men with varying degrees of neuropsychiatric impairment and six control subjects were studied. Localized spectra were obtained from a heterogeneous 5 x 5 x 5-cm volume of interest (VOI). Patients with HIV infection had a significantly lower ATP/Pi ratio and a trend for a lower PCr/Pi ratio than did the control group. In addition, the ATP/Pi and PCr/Pi ratios were both significantly negatively correlated with overall severity of neuropsychiatric impairment. In study 2, three HIV-seropositive men with neuropsychiatric impairment were compared with 11 HIV-seronegative men. Localized phosphorus 31 magnetic resonance spectra were obtained from two relatively homogeneous VOIs: (1) a predominantly white matter VOI, and (2) a predominantly subcortical gray matter VOI. The three HIV-infected patients demonstrated significantly decreased ATP and PCr concentrations in the white matter VOI. These results suggest that HIV infection of the brain may impair brain cellular oxidative metabolism and that the degree of metabolic compromise may be related to the severity of neuropsychiatric impairment.

The bisphosphonate whole body retention test (WBR) has been used to estimate bone mineralization rate (bone turnover). Bisphosphonates given i.v. are taken up by bone or excreted in urine. The aim of the present investigation was to test the efficacy of WBR in estimating bone mineralization rate (m) and to evaluate the influence of renal function (Clcr) and bone mass (forearm bone mineral content; BMC) on WBR. The 24-h retention of 3.7 MBq 99mTc-HMBP (1-hydroxymethylene-1,1-bisphosphonate) (Osteoscan) given i.v. was measured by a medium sensitive whole body counter in thirty-one patients with hyperparathyroidism (n = 14), hyperthyroidism (n = 8) or hypothyroidism (n = 9) (group 1) and in seventy-six females with postmenopausal spinal crush fracture osteoporosis (group 2). In the same individuals m was calculated from a 7-day 47Ca-kinetic study using the expanding calcium pool model. Multiple regression analysis of WBR vs. m and Clcr in group 1 disclosed that WBR correlated positively to m [rp = 0.49, P less than 0.01 (rp = partial correlation coefficient)] and inversely to Clcr (rp = -0.44, P less than 0.02). Inclusion of BMC in the analysis did not reveal any significant partial correlation between WBR and BMC (rp = -0.33, 0.05 less than P less than 0.10). In group 2 WBR correlated inversely to Clcr (rp = -0.48, P less than 0.001) but showed no significant relation to m (rp = 0.10, NS).(ABSTRACT TRUNCATED AT 250 WORDS)