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The copepod Calanus finmarchicusplays a crucial role in the north Atlantic food web. Its seasonal life cycle involves reproduction and development in surface waters before overwintering in diapause at depth. Although diapause has been studied for more than a century, the factors responsible for the initiation and termination of it are still unclear. Endogenous clocks have been identified as potent tools for photoperiod measurement and seasonal rhythmicity in many terrestrial species, but knowledge of these remains scarce in the marine realm. Focusing on the dominant CV copepodid stage, we sampled a population of C. finmarchicusfrom a Scottish sea loch to characterize population dynamics, several physiological parameters, and diel and seasonal expression rhythms of 35 genes representing different metabolic pathways, including the circadian clock machinery. This generated a detailed overview of the seasonal cycle of C. finmarchicusincluding the most extensive field dataset on circadian clock gene expression in a marine species to date. Gene expression patterns revealed distinct gene clusters upregulated at different phases of the copepod's seasonal cycle. While diel clock cycling was restricted to the active spring/summer phase, many clock genes exhibited the highest expression during diapause. Our results provide new insights into diapause on physiological and genetic levels. We suggest that photoperiod, in interaction with internal and external factors (lipid content, temperature, food availability) and the endogenous clock mechanism, plays an important role in the timing of diapause in C. finmarchicus.

The Arctic Ocean north of Svalbard has recently experienced large sea ice losses and the increasing prominence of Atlantic water (AW) advection. To investigate the impact of these ongoing changes on annual particle export, two moorings with sequential sediment traps were deployed in ice‐free and seasonally ice‐covered waters on the shelf north (NSv) and east (ESv) of Svalbard, collecting sinking particles nearly continuously from October 2017 to October 2018. Vertical export of particulate organic carbon (POC), total particulate matter (TPM), planktonic protists, chlorophyll a, and zooplankton fecal pellets were measured, and swimmers were quantified and identified. Combined with sensor data from the moorings, these time‐series measurements provided a first assessment of the factors influencing particle export in this region of the Arctic Ocean. Higher annual TPM and POC fluxes at the ice‐free NSv site were primarily driven by the advection of AW, higher grazing by large copepods, and a wind‐induced mixing event during winter. Higher diatom fluxes were observed during spring in the presence of sea ice at the ESv site. Along with sea ice cover, regional differences in AW advection and the seasonal presence of grazers played a prominent role in the biological carbon pump along the continental shelf off Svalbard. Recently, the area north of Svalbard has experienced the largest reduction of winter sea ice extent in the Arctic Ocean and an increasing influence of warm Atlantic water (AW) in surface waters. The consequences for the marine ecosystem remain unclear. In the present study, we investigated the fate of algal production and organic matter by measuring the amount and composition of the material sinking toward the seafloor. Using sediment traps with automatically rotating bottles, the seasonal variability in the quality and quantity of the organic matter sinking to ∼100 m was investigated at two sites with and without winter sea ice on the shelf north and east of Svalbard from 2017 to 2018. Our results suggest that the AW inflow along the shelf break induces a gradient with more zooplankton in the west and more ice in the east. More zooplankton in the west rework the organic matter through grazing, resulting in higher flux of carbon than in the east. Less zooplankton in the east potentially leaves more algal cells to sink ungrazed. As algal fluxes were higher in the presence of sea ice, a future with less sea ice may result in more reworked material sinking to the seafloor. Sea ice melt led to higher springtime and annual diatom fluxes at the seasonally ice‐covered siteAside from algal fluxes, annual particle fluxes were higher at the ice‐free siteAtlantic water advection, zooplankton grazing, wind‐induced mixing, and resuspension contributed to the higher fluxes at the ice‐free site Sea ice melt led to higher springtime and annual diatom fluxes at the seasonally ice‐covered site Aside from algal fluxes, annual particle fluxes were higher at the ice‐free site Atlantic water advection, zooplankton grazing, wind‐induced mixing, and resuspension contributed to the higher fluxes at the ice‐free site

This cross‐sectional study investigated performances of two consecutive cohorts of second year dental students on completion of a conventional didactic course and two succeeding cohorts of second year dental students on a recently introduced problem‐based learning (PBL) course at The University of Liverpool School of Dentistry. A 40 part true/false questionnaire tested recall of factual knowledge in anatomy, biochemistry, oral biology and physiology. The results showed no significant difference in the total scores when negatively marked between the conventional and PBL course groups but higher total scores in the PBL groups when positively marked. Performances in anatomy, oral biology and physiology did not differ between the groups when negatively marked but the scores of the conventional course groups in biochemistry were significantly lower than the others. With positive marking biochemistry scores were not significantly different but all other subjects were significantly higher in the PBL course groups. The PBL course groups offered fewer blank responses than the conventional course groups but the ratio of correct to incorrect responses, for both definite and intelligent guess responses, were similar in these groups. The compositional profile of the study groups was similar with respect to educational background but the PBL course groups included more females and more older students. The results of this study show that the overall knowledge recall in the basic sciences by dental students on PBL or conventional didactic courses does not differ and may be helpful to those considering the introduction of PBL into the curriculum.

We have expressed G1-G2 mutants with amino acid changes at the DIPEN(341) downward arrow(342)FFGVG and ITEGE(373) downward arrow(374)ARGSV cleavage sites, in order to investigate the relationship between matrix metalloproteinase (MMP) and aggrecanase activities in the interglobular domain (IGD) of aggrecan. The mutation DIPEN(341) to DIGSA(341) partially blocked cleavage by MMP-13 and MMP-8 at the MMP site, while the mutation (342)FFGVG to (342)GTRVG completely blocked cleavage at this site by MMP-1, -2, -3, -7, -8, -9, -13, -14. Each of the MMP cleavage site mutants, including a four-amino acid deletion mutant lacking residues ENFF(343), were efficiently cleaved by aggrecanase, suggesting that the primary sequence at the MMP site had no effect on aggrecanase activity in the IGD. The mutation (374)ARGSV to (374)NVYSV completely blocked cleavage at the aggrecanase site by aggrecanase, MMP-8 and atrolysin C but had no effect on the ability of MMP-8 and MMP-13 to cleave at the Asn(341) downward arrowPhe bond. Susceptibility to atrolysin C cleavage at the MMP site was conferred in the DIGSA(341) mutant but absent in the wild-type, (342)GTRVG, (374)NVYSV, and deletion mutants. To further explore the relationship between MMP and aggrecanase activities, sequential digest experiments were done in which MMP degradation products were subsequently digested with aggrecanase and vice versa. Aggrecanase-derived G1 domains with ITEGE(373) C termini were viable substrates for MMPs; however, MMP-derived G2 fragments were resistant to cleavage by aggrecanase. A 10-mer peptide FVDIPENFFG, which is a substrate analogue for the MMP cleavage site, inhibited aggrecanase cleavage at the Glu(373) downward arrowAla bond. This study demonstrates that MMPs and aggrecanase have unique substrate recognition in the IGD of aggrecan and suggests that sequences at the C terminus of the DIPEN(341) G1 domain may be important for regulating aggrecanase cleavage.

This cross‐sectional study aimed to assess and compare, by performances in a questionnaire, the level of knowledge of basic medical sciences in 6th‐form school pupils studying science subjects as entrance requirements to University and in 2nd, 3rd and 4th‐year undergraduate dental students. A 40‐part multiple response, true/false questionnaire, testing recall of factual knowledge in anatomy, biochemistry, physiology and oral biology, was used as the method of assessment. The results suggested that this simple format was an acceptable and useful method of assessment of the knowledge level of the study groups. The difference in scores of knowledge, expected to be higher in 2nd‐year students compared to 6th form groups, was greatest in anatomy and oral biology, less in biochemistry and, unexpectedly, was not apparent in physiology. A difference in performance on the knowledge questionnaire was observed between 4th and 2nd year dental students, attributable primarily to decreased scores for 4th year dental students in biochemistry and, to a lesser extent, anatomy. The results obtained with this standardised test of factual knowledge recall may be of value to teachers compiling medical sciences courses for dental undergraduates and to those planning and evaluating new curricula with a less didactic approach.

A recombinant human aggrecan G1-G2 fragment comprising amino acids Val(1)-Arg(656) has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M(r) 90,000-95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. Neoepitope antibodies recognizing the N-terminal F(342)FGVG or C-terminal DIPEN(341) sequences were used to confirm MMP cleavage at the Asn(341) downward arrow Phe bond, while neoepitope antibodies recognizing the N-terminal A(374)RGSV or C-terminal ITEGE(373) sequences were used to confirm aggrecanase cleavage at the Glu(373) downward arrow Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1alpha or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.

The evidence that the moon has a profound effect on the timing of reproductive activities of marine animals is compelling. Some moon phase related spawning events are revealed by the constant phase relationship between the timing of “once per year” spawning events and the lunar phase as in the highly synchronised breeding of the palolo worm Palola viridisand the Japanese crinoid Comanthus japonicusIn other cases there is a repeated lunar cycle of reproductive activity and again the marine worms provide many good examples. The breeding of the palolo worm involves the highly synchronised release of what are in effect detached sexual satellites and the timing of this has annual (solar year), lunar, daily and tidal rhythm components. In a similar way, the onset of sexual maturation and participation in the nuptial dance of Platynereis dumerillihas strong lunar components. Sexual reproduction is the culmination of a process of sexual maturation that takes many months for completion and the mechanisms by which moon phase relationships are imposed on this process must have been selected for by mechanisms relating to reproductive success. The polychaetes provide excellent models for investigation of both the selective advantage and the physiological processes involved in reproductive synchrony. We have recently shown that the spawning of the lugworm Arenicola marinahas lunar components and we conclude that an interaction between solar and lunar signals is widespread in the timing of reproduction in marine animals. Carl Hauenschild was the first to demonstrate the existence of a free-running circa-lunar rhythm in marine animals using captive populations of Platynereis dumerilliHis experiments also provided clear evidence for the influence of moonlight (light at night) as the zeitgeber for this rhythm. This implies a high level of sensitivity to light, and the operation of appropriate endogenous biological rhythms. Using Nereis virenswe have demonstrated a high level of sensitivity to low intensity solar light signals in relation to rhythmic processes with the properties similar to the circadian and photoperiodic mechanisms of terrestrial organisms. The spawning of N. virensis also believed to have lunar components reflecting the complex influences of the sun and the moon in the marine domain, where influences of the moon and sun are more equal. We suggest that evolution of biological rhythmicity that can be entrained to either lunar or solar systems arose in the marine domain. Recent advances in understanding the molecular components of the circadian clock system suggest that a search for a common molecular mechanism for both lunar and solar related biological rhythms in marine organisms might be very fruitful.

Proteoglycan was extracted from alveolar and basal bone of New Zealand White rabbits using a sequential extraction procedure. Proteoglycans not associated with the bone mineral represented 1% of the total organic matrix whereas proteoglycans associated with the mineral represented 20% of the alveolar organic matrix and 12% of the basal organic matrix. Chondroitin-4-sulphate and keratan sulphate were identified in both alveolar and basal bone following protease treatment of the proteoglycan extracts and enzymic digestion with chondroitinase AC, ABC and keratanase. Differences were observed in the percentage of each glycosaminoglycan (GAG) in the total organic matrix. In alveolar bone samples, keratan sulphate and chondroitin-4-sulphate is present in equal proportions. In basal bone chondroitin-4-sulphate represents approximately half the value found in alveolar bone and keratan sulphate about a quarter. The extracts were examined by gel filtration on Sepharose 4B under associative conditions. The 280 nm absorbance profiles of proteoglycan from alveolar and basal bone were essentially similar with three main peaks evident, including molecular weight material in excess of 2 × 106. The bulk of the bone GAG appeared in the medium molecular weight range with trace amounts in lower molecular weight fractions.

Using a specific enzyme-linked immunosorbent assay, plasminogen activator inhibitor-2 (PAI-2) was quantitated in cultures of human monocytes. Lipopolysaccharide (LPS) increased both extracellular and cell-associated PAI-2 levels, as well as PAI-2 mRNA measured by Northern analysis. Both the lymphokine, interleukin-4 (IL-4) (greater than or equal to 10 pmol/L), and the glucocorticoid, dexamethasone (100 nmol/L), inhibited PAI-2 formation and PAI-2 mRNA induction. Another lymphokine, interferon-gamma (IFN-gamma) (100 U/mL), as for IL-4 alone, did not stimulate PAI-2 formation; however, in contrast to IL-4, IFN- gamma did not reverse the LPS effect but could potentiate it. The suppression of PAI-2 formation by IL-4 and glucocorticoid in stimulated human monocytes extends the list of monocyte products whose synthesis can be downregulated in these cells by the two agents. The findings could have relevance to the control by monocytes/macrophages of connective tissue resorption, including that of fibrin, at sites of inflammation.

Native and recombinant neutrophil collagenase (MMP-8) was shown to cleave at the E373-A374 ‘aggrecanase’ site in the interglobular domain of aggrecan. The time course of digestion in vitro showed that MMP-8 cleaved initially at N341-F342, the predominant metalloproteinase site, before cleaving at the E373-A374 site. A synthetic peptide, IPENFFG, inhibited cleavage at E373-A374 but not N341-F342 in vitro, indicating that the E373-A374 sequence was a less preferred site for MMP-8 cleavage than N341-F342. IPENFFG also inhibited release of A374 RGSVI fragments from cartilage in explant culture, suggesting that a metalloproteinase cleaved at the aggrecanase site in situ. The possibility remains that ‘aggrecanase’ may be a metalloproteinase in cartilage.

We have developed a monoclonal antibody AF-28 that specifically recognizes a neo-epitope on polypeptides with N-terminal FFGVG ... sequences. This sequence is found at the N-terminus of aggrecan fragments that have been digested with matrix metalloproteinases (MMPs). By immunoblotting, monoclonal antibody AF-28 specifically detected G2 fragments derived from an aggrecan G1-G2 substrate digested with stromelysin, collagenase, gelatinase and matrilysin, but failed to detect G2 fragments obtained from elastase, trypsin or cathepsin B digests. Undigested G1-G2 was not detected. In addition, AF-28 antibody detected fragments derived from whole aggrecan and this detection did not require prior treatment with chondroitinase or keratanase. Competition experiments confirmed that peptides containing internal ... FFGVG ... sequences were not detected by the antibody, while native MMP-digested aggrecan fragments and a synthetic 32-mer peptide with FFGVG ... N-termini were equally competitive on a molar basis. An FFGVG 5-mer, and an FGVGGEEDI9-mer which lacked the N-terminal phenylalanine residue, were 50 times and 230 times respectively less competitive than the FFGVG ... 32-mer. Two fragments from the interglobular domain, F342-F373 and F342-D441, that are predicted products of G1-G2 digestion by neutrophil collagenase but have not previously been detected, could be detected with AF-28. The epitope recognized by AF-28 was also detected in human synovial fluids by Western blot analysis. A broad band of 100-200 kDa was detected in some patients and a dominant band of 40-60 kDa was found in two patients. The size of this small fragment corresponds with that seen for the porcine F342-E373 product and may represent the natural physiological product of aggrecan cleaved in vivo at both the MMP site (... DIPEN341 decreases F342FGVG ...) and the aggrecanase site (... ITEGE373 decreases A374RGSVI ...).

The actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan G1-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and typsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.

The cyclic olefins (I) and (VI) react with NBS and 2‐propynol (II), forming the β‐bromo‐α‐propargyl ethers (III) or (VII) which are cyclized upon treatment with sodium borohydride in the presence of cobaloxime to give the tetrahydrofuran derivatives (IV) or (VIII).