Biogenic Synthesis, Purification, and Chemical Characterization of Anti-inflammatory Resolvins Derived from Docosapentaenoic Acid (DPAn-6)

Enzymatically oxygenated derivatives of the ω-3 fatty acids cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) and cis-5,8,11,14,17-eicosapentaenoic acid, known as resolvins, have potent inflammation resolution activity (Serhan, C. N., Clish, C. B., Brannon, J., Colgan, S. P., Chiang, N., and Gronert, K. (2000) J. Exp. Med. 192, 1197–1204; Hong, S., Gronert, K., Devchand, P. R., Moussignac, R., and Serhan, C. N. (2003) J. Biol. Chem. 278, 14677–14687). Our objective was to determine whether similar derivatives are enzymatically synthesized from other C-22 fatty acids and whether these molecules possess inflammation resolution properties. The reaction of DHA, DPAn-3, and DPAn-6 with 5-, 12-, and 15-lipoxygenases produced oxylipins, which were identified and characterized by liquid chromatography coupled with tandem mass-spectrometry. DPAn-6 and DPAn-3 proved to be good substrates for 15-lipoxygenase. 15-Lipoxygenase proved to be the most efficient enzyme of the three tested for conversion of long chain polyunsaturated fatty acids to corresponding oxylipins. Since DPAn-6 is a major component of Martek DHA-S™ oil, we focused our attention on reaction products obtained from the DPAn-6 and 15-lipoxygenase reaction. (17S)-hydroxy-DPAn-6 and (10,17S)-dihydroxy-DPAn-6 were the main products of this reaction. These compounds were purified by preparatory high performance liquid chromatography techniques and further characterized by NMR, UV spectrophotometry, and tandem mass spectrometry. We tested both compounds in two animal models of acute inflammation and demonstrated that both compounds are potent anti-inflammatory agents that are active on local intravenous as well as oral administration. These oxygenated DPAn-6 compounds can thus be categorized as a new class of DPAn-6-derived resolvins.Enzymatically formed oxygenation products of C-20 and C-22 long chain polyunsaturated fatty acids (LC-PUFAs),⁴ have important biological roles in inflammation, allergies, and blood clotting and are thus believed to have therapeutic potential in several chronic immune diseases (1–10) Several biologically important products of cis-5,8,11,14-eicosatetraenoic acid/arachidonic acid (ARA), cis-5,8,11,14,17-eicosapentaenoic acid (EPA), and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) have been described (4, 11, 12). Proinflammatory oxylipins, such as leukotrienes and some prostaglandins, are derived from ARA, an ω-6 fatty acid. Interestingly, the same fatty acid also serves as a precursor to anti-inflammatory or proresolution molecules like lipoxins (13, 14). Stable analogues of lipoxins are being developed as drugs for asthma and other inflammatory airway diseases (15, 16). Oxylipins derived from ω-3 fatty acids, such as DHA and EPA, known as resolvins, are primarily anti-inflammatory in nature (17). EPA acts as a precursor to the E-series resolvins that have shown potential in the treatment of colitis, arthritis, and periodontitis (18–20). The resolvins of the D-series derived from DHA are useful as neuroprotective agents. 10,17-Dihydroxy-4,7,11,13,15,19-docosahexaenoic acid (10,17-HDHA) or neuroprotectin D1 is a resolvin that is formed endogenously in the human brain and eye and is believed to exert its protective effect against cell injury-induced oxidative stress (21–23).The main enzymes responsible for the production of these oxygenated LC-PUFA products are primarily lipoxygenases and, in addition, cyclo-oxygenases and cytochromes P450. These enzymes produce oxylipins via transcellular activity, often involving multiple cell types (24). This activity mainly results in mono-, di-, and tri-hydroxylation products of fatty acids that have varying potencies, depending on the exact structure of the compound. Lipoxygenases are non-heme, iron-containing dioxygenases that catalyze the regioselective and enantioselective oxidation of polyunsaturated fatty acids containing one or more cis,cis-1,4-pentadienoic moieties to give the corresponding hydroperoxy derivatives (25, 26). We thus considered that, in addition to DHA and EPA, other C-22 PUFAs containing such methylene interrupted double bonds may also be substrates for lipoxygenases and that resulting products may have anti-inflammatory activity similar to DHA-derived resolvins. DPAn-6 (cis-4,7,10,13,16-docosapentaenoic acid) is present in algal oils, and recent studies have demonstrated that this fatty acid has anti-inflammatory activities in vitro and, in conjunction with DHA, also has anti-inflammatory activity in vivo.⁵ Also, it has been suggested that a combination of DHA and DPAn-6 could be a beneficial natural therapy in neuroinflammatory conditions like Alzheimer disease. Specifically, in a 3×Tg-AD mouse model of Alzheimer disease, DPAn-6 was shown to reduce levels of early stage phospho-Tau epitopes, which in turn correlated with a reduction in phosphorylated c-Jun N-terminal kinase, a putative Tau kinase (27). Although the precise mechanism of action of DPAn-6 in these inflammatory milieus is not known, it suggests a possible role for oxylipin products of DPAn-6 in resolution of inflammation. Also, another LC-PUFA, DPAn-3 (cis-7,10,13,16,19-docosapentaenoic acid) usually present along with DHA and EPA in marine oils is known to be a potent inhibitor of platelet aggregation (28–30). In addition, this LC-PUFA has a potent inhibitory effect on angiogenesis through the suppression of VEGFR-2 (vascular endothelial-cell growth factor receptor 2) expression. Angiogenesis is known to contribute to tumor growth, inflammation, and microangiopathy, again pointing to the possibility that anti-inflammatory activity of DPAn-3 might be mediated through resolvin-like products as in the case of DHA and EPA (31).The purpose of this research was to determine whether oxylipins are formed from the C-22 LC-PUFAs, DPAn-6 and DPAn-3, by lipoxygenase activity; to compare them to products formed from DHA; to chemically characterize products; to purify key oxylipin products from the DPAn-6/15-lipoxygenase reaction; and to test whether these compounds have resolvin-like anti-inflammatory activity. This research also sets the stage for preparation and isolation of a wide range of other C-22 oxylipins that could be evaluated as potential anti-inflammatory compounds.     

Community-based colorectal cancer intervention in underserved Korean Americans

Background: Despite evidence of a decline in both incidence and prevalence of colorectal cancer nationwide, it remains the second most commonly diagnosed cancer and the third highest cause of mortality among Asian Americans, including Korean Americans. This community-based and theoretically guided study evaluated a culturally appropriate intervention program that included a bilingual cancer educational program among Korean Americans including information on CRC risks, counseling to address psychosocial and access barriers, and patient navigation assistance. Methods: A two-group quasi-experimental design with baseline and post-intervention assessment and a 12-month follow-up on screening was used in the study. Korean Americans (N = 167) were enrolled from six Korean churches. The intervention group received culturally appropriate intervention program addressing accessibility and psychosocial barriers, and navigation assistance for screening. The control group received general health education that included cancer-related health issues and screening. Results: There was a significant difference (p < 0.05) between the post-intervention and control groups in awareness of CRC risk factors. There was also a significant improvement in the pre–post across HBM measures in the intervention group for perceived susceptibility (p < 0.05) and benefits and barriers to screening (p < 0.001). At baseline, 13% of participants in the intervention group and 10% in control group reported having had a CRC cancer screening test in the previous year. At the 12-month post-intervention follow-up, 77.4% of participants in the intervention group had obtained screening compared to 10.8% in the control group. Conclusion: While health disparities result from numerous factors, a culturally appropriate and church-based intervention can be highly effective in increasing knowledge of and access to, and in reducing barriers to CRC screening among underserved Koreans.     

60 kD Ro and nRNP A Frequently Initiate Human Lupus Autoimmunity

Systemic lupus erythematosus (SLE) is a clinically heterogeneous, humoral autoimmune disorder. The unifying feature among SLE patients is the production of large quantities of autoantibodies. Serum samples from 129 patients collected before the onset of SLE and while in the United States military were evaluated for early pre-clinical serologic events. The first available positive serum sample frequently already contained multiple autoantibody specificities (65%). However, in 34 SLE patients the earliest pre-clinical serum sample positive for any detectable common autoantibody bound only a single autoantigen, most commonly 60 kD Ro (29%), nRNP A (24%), anti-phospholipids (18%) or rheumatoid factor (15%). We identified several recurrent patterns of autoantibody onset using these pre-diagnostic samples. In the serum samples available, anti-nRNP A appeared before or simultaneously with anti-nRNP 70 K in 96% of the patients who had both autoantibodies at diagnosis. Anti-60 kD Ro antibodies appeared before or simultaneously with anti-La (98%) or anti-52 kD Ro (95%). The autoantibody response in SLE patients begins simply, often binding a single specific autoantigen years before disease onset, followed by epitope spreading to additional autoantigenic specificities that are accrued in recurring patterns.     

An ATM/Chk2-mediated DNA damage-responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells

Epstein-Barr virus (EBV), an oncogenic herpesvirus that causes human malignancies, infects and immortalizes primary human B cells in?vitro into indefinitely proliferating lymphoblastoid cell lines, which represent a model for EBV-induced tumorigenesis. The immortalization efficiency is very low, suggesting that an innate tumor suppressor mechanism is operative. We identify the DNA damage response (DDR) as?a major component of the underlying tumor suppressor mechanism. EBV-induced DDR activation was not due to lytic viral replication, nor did the DDR marks colocalize with latent episomes. Rather, a transient period of EBV-induced hyperproliferation correlated with DDR activation. Inhibition of the DDR kinases ATM and Chk2 markedly increased transformation efficiency of primary B cells. Further, the viral latent oncoprotein EBNA3C was required to attenuate the EBV-induced DDR. We propose that heightened oncogenic activity in early cell divisions activates a growth-suppressive DDR that is attenuated by viral latency products to induce cell immortalization.     

UniEuk: Time to Speak a Common Language in Protistology!

Universal taxonomic frameworks have been critical tools to structure the fields of botany, zoology, mycology, and bacteriology as well as their large research communities. Animals, plants, and fungi have relatively solid, stable morpho‐taxonomies built over the last three centuries, while bacteria have been classified for the last three decades under a coherent molecular taxonomic framework. By contrast, no such common language exists for microbial eukaryotes, even though environmental ‘‐omics’ surveys suggest that protists make up most of the organismal and genetic complexity of our planet's ecosystems! With the current deluge of eukaryotic meta‐omics data, we urgently need to build up a universal eukaryotic taxonomy bridging the protist ‐omics age to the fragile, centuries‐old body of classical knowledge that has effectively linked protist taxa to morphological, physiological, and ecological information. UniEuk is an open, inclusive, community‐based and expert‐driven international initiative to build a flexible, adaptive universal taxonomic framework for eukaryotes. It unites three complementary modules, EukRef, EukBank, and EukMap, which use phylogenetic markers, environmental metabarcoding surveys, and expert knowledge to inform the taxonomic framework. The UniEuk taxonomy is directly implemented in the European Nucleotide Archive at EMBL‐EBI, ensuring its broad use and long‐term preservation as a reference taxonomy for eukaryotes.     

Traction forces of neutrophils migrating on compliant substrates

Proper functioning of the innate immune response depends on migration of circulating neutrophils into tissues at sites of infection and inflammation. Migration of highly motile, amoeboid cells such as neutrophils has significant physiological relevance, yet the traction forces that drive neutrophil motion in response to chemical cues are not well characterized. To better understand the relationship between chemotactic signals and the organization of forces in motile neutrophils, force measurements were made on hydrogel surfaces under well-defined chemotactic gradients created with a microfluidic device. Two parameters, the mean chemoattractant concentration (C_M) and the gradient magnitude (Δc/Δx) were varied. Cells experiencing a large gradient with C_M near the chemotactic receptor K_D displayed strong punctate centers of uropodial contractile force and strong directional motion on stiff (12 kPa) surfaces. Under conditions of ideal chemotaxis—cells in strong gradients with mean chemoattractant near the receptor K_D and on stiffer substrates—there is a correlation between the magnitude of force generation and directional motion as measured by the chemotactic index. However, on soft materials or under weaker chemotactic conditions, directional motion is uncorrelated with the magnitude of traction force. Inhibition of either β₂ integrins or Rho-associated kinase, a kinase downstream from RhoA, greatly reduced rearward traction forces and directional motion, although some vestigial lamellipodium-driven motility remained. In summary, neutrophils display a diverse repertoire of methods for organizing their internal machinery to generate directional motion.     

Patient-specific analysis of displacement forces acting on fenestrated stent grafts for endovascular aneurysm repair

Treatment options for abdominal aortic aneurysm (AAA) include highly invasive open surgical repair or minimally invasive endovascular aneurysm repair (EVAR). Despite being minimally invasive, some patients are not suitable for EVAR due to hostile AAA morphology. Fenestrated-EVAR (F-EVAR) was introduced to address these limitations of standard EVAR, where AAA is treated using a Fenestrated Stent Graft (FSG). In order to assess durability of F-EVAR, displacement forces acting on FSGs were analysed in this study, based on patient-specific geometries reconstructed from computed tomography (CT) scans. The magnitude and direction of the resultant displacement forces acting on the FSG were numerically computed using computational fluid dynamics (CFD) with a rigid wall assumption. Although displacement force arises from blood pressure and friction due to blood flow, numerical simulations elucidated that net blood pressure is the dominant contributor to the overall displacement force; as a result, time dependence of the resultant displacement force followed pressure waveform very closely. The magnitude of peak displacement force varied from 1.9 N to 14.3 N with a median of 7.0 N. A strong positive correlation was found between inlet cross-sectional area (CSA), anterior/posterior (A/P) angle and the peak displacement force i.e. as inlet CSA or A/P angle increases, the magnitude of resultant displacement increases. This study manifests that while loads exerted by the pulsatile flow dictates the cyclic variation of the displacement force, its magnitude depends not only on blood pressure but also the FSG morphology, with the latter determining the direction of the displacement force.     

Evaluating the Clinical and Physiological Effects of Long Term Ultraviolet B Radiation on Guinea Pigs (Cavia porcellus)

Vitamin D is an important hormone in vertebrates. Most animals acquire this hormone through their diet, secondary to exposure to ultraviolet B (UVB) radiation, or a combination thereof. The objectives for this research were to evaluate the clinical and physiologic effects of artificial UVB light supplementation on guinea pigs (Cavia porcellus) and to evaluate the long-term safety of artificial UVB light supplementation over the course of six months. Twelve juvenile acromelanic Hartley guinea pigs were randomly assigned to one of two treatment groups: Group A was exposed to 12 hours of artificial UVB radiation daily and Group B received only ambient fluorescent light for 12 hours daily. Animals in both groups were offered the same diet and housed under the same conditions. Blood samples were collected every three weeks to measure blood chemistry values, parathyroid hormone, ionized calcium, and serum 25-hydroxyvitamin D₃ (25-OHD₃) levels. Serial ophthalmologic examinations, computed tomography scans, and dual energy x-ray absorptiometry scans were performed during the course of the study. At the end of the study the animals were euthanized and necropsied. Mean ± SD serum 25-OHD₃ concentrations differed significantly in the guinea pigs (p<0.0001) between the UVB supplementation group (101.49±21.81 nmol/L) and the control group (36.33±24.42 nmol/L). An increased corneal thickness in both eyes was also found in the UVB supplementation compared to the control group (right eye [OD]: p<0.0001; left eye [OS]: p<0.0001). There were no apparent negative clinical or pathologic side effects noted between the groups. This study found that exposing guinea pigs to UVB radiation long term significantly increased their circulating serum 25-OHD₃ levels, and that this increase was sustainable over time. Providing guinea pigs exposure to UVB may be an important husbandry consideration that is not currently recommended.