Methylene blue-mediated antimicrobial photodynamic therapy on chicken semen.

BACKGROUND: Artificial insemination is widely employed in poultry, but high degrees of bacterial contamination are often observed in semen because of its passage through the cloaca. Consequently, most semen extenders for birds have antibiotics that could aggravate bacterial resistance. METHODS: We evaluated the potential of antimicrobial photodynamic therapy (PDT) as an alternative to the use of antibiotics, and assessed whether changes in concentration and incubation time with methylene blue (MB), radiant exposure, and irradiance of light affect spermatozoa activity and bacteria in chicken semen. RESULTS: Incubation with MB (< 25 µM) did not alter sperm motility, regardless of the pre-irradiation time (PIT, 1 or 5 min). Following 1 min of PIT with MB at 10 µM, samples were irradiated for 30, 60, 120, and 180 s at irradiances of 44, 29, and 17 mW/ cm² (660 nm LedBox). MB and light alone did not interfere with the analyzed parameters. However, when both factors were associated, increases in light dose led to greater reductions in sperm parameters, regardless of the irradiance used. Besides, PDT conditions that were less harmful to spermatozoa were not able to significantly reduce bacterial colonies in chicken semen. CONCLUSIONS: A failure in MB selectivity could explain unsuccessful bacterial reduction following PDT. Further research involving other photosensitizers or conjugating molecules to MB to target microbial cells is needed for PDT application in poultry breeders.

Insights on sperm assays and cryopreservation in six Neotropical pit vipers.

Breeding snakes in captivity has become more and more relevant due not only to the growing interest on their venoms but also to the increasing number of endangered species worldwide. Unfortunately, studies on the formation of germplasm banks for these reptiles do not follow the same pace, and literature on sperm cryopreservation remains in its infancy when compared to other taxa. Herein, we first validated a sperm-egg binding assay (using chicken egg perivitelline membrane - EPM) and some nonfluorescent staining techniques for semen analysis of two pit viper genera (Bothrops and Crotalus), and then we investigated the protective effects of dimethylacetamide (DMA), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) at different concentrations (3, 6 and 12%) throughout the freezing process in five species of lancehead and one of rattlesnake. Our validation process showed high correlations among sperm functional tests (including sperm-binding to EPM) and motion parameters. A total of 166 fresh ejaculates were acquired from 233 collection attempts, and 63.9% of these samples exhibited minimal motility for freezing (≥20%). During cryopreservation we observed that post-thaw motility and quality was improved by higher levels of cryoprotectants (CPA), regardless the CPA type. Lower concentrations of CPA were less harmful to sperm motility and progressive motility following the equilibrium phase, but were ineffective in protecting these cells from the freeze-thaw cycle. Likewise, higher CPA concentrations increased post-thaw integrity of the acrosome and plasma membrane for most species, except for rattlesnakes in which only 12% DMSO produced better outcomes.

Insights on sperm assays and cryopreservation in six Neotropical pit vipers

Breeding snakes in captivity has become more and more relevant due not only to the growing interest on their venoms but also to the increasing number of endangered species worldwide. Unfortunately, studies on the formation of germplasm banks for these reptiles do not follow the same pace, and literature on sperm cryopreservation remains in its infancy when compared to other taxa. Herein, we first validated a sperm-egg binding assay (using chicken egg perivitelline membrane – EPM) and some nonfluorescent staining techniques for semen analysis of two pit viper genera (Bothrops and Crotalus), and then we investigated the protective effects of dimethylacetamide (DMA), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) at different concentrations (3, 6 and 12%) throughout the freezing process in five species of lancehead and one of rattlesnake. Our validation process showed high correlations among sperm functional tests (including sperm-binding to EPM) and motion parameters. A total of 166 fresh ejaculates were acquired from 233 collection attempts, and 63.9% of these samples exhibited minimal motility for freezing (≥20%). During cryopreservation we observed that post-thaw motility and quality was improved by higher levels of cryoprotectants (CPA), regardless the CPA type. Lower concentrations of CPA were less harmful to sperm motility and progressive motility following the equilibrium phase, but were ineffective in protecting these cells from the freeze-thaw cycle. Likewise, higher CPA concentrations increased post-thaw integrity of the acrosome and plasma membrane for most species, except for rattlesnakes in which only 12% DMSO produced better outcomes.

The tellurium-based immunomodulator, AS101 ameliorates adjuvant-induced arthritis in rats.

Despite undeniable improvement in the management of rheumatoid arthritis (RA), the discovery of more effective, less toxic and, ideally, less immune suppressive drugs are much needed. In the current study, we set to explore the potential anti-rheumatic activity of the non-toxic, tellurium-based immunomodulator, AS101 in an experimental animal model of RA. The effect of AS101 was assessed on adjuvant-induced arthritis (AIA) rats. Clinical signs of arthritis were assessed. Histopathological examination was used to assess inflammation, synovial changes and tissue lesions. Very late antigen-4 (VLA-4)+ cellular infiltration was detected using immunohistochemical staining. Enzyme-linked immunosorbent assay (ELISA) was used to measure circulating anti-cyclic citrullinated-peptide autoantibody (ACPA) and real-time polymerase chain reaction (PCR) was used to measure the in-vitro effect of AS101 on interleukin (IL)-6 and IL-1ß expression in activated primary human fibroblasts. Prophylactic treatment with intraperitoneal AS101 reduced clinical arthritis scores in AIA rats (P < 0·01). AS101 abrogated the migration of active chronic inflammatory immune cells, particularly VLA-4+ cells, into joint cartilage and synovium, reduced the extent of joint damage and preserved joint architecture. Compared to phosphate-buffered saline (PBS)-treated AIA rats, histopathological inflammatory scores were significantly reduced (P < 0·05). Furthermore, AS101 resulted in a marked reduction of circulating ACPA in comparison to PBS-treated rats (P < 0·05). Importantly, AS101 significantly reduced mRNA levels of proinflammatory mediators such as IL-6 (P < 0·05) and IL-1ß (P < 0·01) in activated primary human fibroblasts. Taken together, we report the first demonstration of the anti-rheumatic/inflammatory activity of AS101 in experimental RA model, thereby supporting an alternative early therapeutic intervention and identifying a promising agent for therapeutic intervention.

IVIG ameliorate inflammation in collagen-induced arthritis: projection for IVIG therapy in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease that leads to joint destruction and disability. Despite a significant progress in administration of biological agents for RA patients, there is still a need for improved therapy. Intravenous immunoglobulins (IVIG), a pooled polyspecific immunoglobulin (Ig)G extracted from 5000 to 20 000 healthy subjects, showed beneficial therapeutic effect in patients with immune deficiency, sepsis and autoimmune diseases. The current study aimed to investigate the beneficial effect of treatment with IVIG in established collagen-induced arthritis in DBA/1j mice. Murine arthritis was induced in DBA/1j mice. Treatment with IVIG began when the disease was established. The clinical score was followed twice a week until day 48. The mice were bled for plasma and the paws were hematoxylin and eosin (H&E)-stained. Cytokine profile in the plasma was analyzed by Luminex technology and titers of circulating anti-collagen antibodies in the plasma was tested by enzyme-linked immunosorbent assay. Our results show that treatment with IVIG in murine significantly reduced the clinical arthritis score (P < 0·001). Moreover, mode of action showed that IVIG significantly reduced circulating levels of inflammatory cytokines [interferon (IFN)-γ, interleukin (IL)-1ß, IL-17, IL-6, tumor necrosis factor (TNF)-α, P < 0·001], inhibiting anti-collagen antibodies (P < 0·001) in the plasma of collagen-induced arthritis mice. Importantly, histopathological examination revealed that IVIG treatment prevented the migration of inflammatory immune cells into the cartilage and synovium, reduced the extent of joint damage and preserved joint architecture. Our results proved for the first time the valuable anti-inflammatory treatment of IVIG in experimental RA. We propose IVIG therapy for a subgroup of patients with rheumatologically related diseases.

Decrease in 14-3-3η protein levels is correlated with improvement in disease activity in patients with rheumatoid arthritis treated with Tofacitinib.

14-3-3η protein is a proinflammatory mediator that may represent a novel diagnostic and prognostic biomarker for rheumatoid arthritis (RA). We assessed the correlation between changes in serum 14-3-3η levels and changes in clinical disease activity measures in RA patients treated with Tofacitinib (TOF). Paired serum samples from 35 patients with RA were obtained at baseline and 5 months after the initiation of treatment with TOF. The levels of 14-3-3η were measured by JOINT stat 14-3-3η ELISA test kits (Augurex Life Sciences Corp.). The cut-off was defined as 0.19 ng/ml. 14-3-3η positivity was found in 57% of the patients at baseline and in 37% of the patients after 5 months of treatment. Mean ± SD baseline 14-3-3η levels [4.92 ± 8.86 ng/ml] were significantly higher (p < 0.005) than 14-3-3η levels following treatment [1.97 ± 4.59 ng/ml]. A statistically significant improvement (p < 0.001) of CDAI, SDAI, DAS4ESR and DAS4CRP was achieved after 5 month of treatment. Decrease in 14-3-3η protein levels was highly correlated with improvement in DAS4ESR (r = 0.50, p < 0.01), DAS4CRP (r = 0.46, p < 0.01) and ESR (r = 0.36, p = 0.03) and moderately correlated with improvement in CDAI (r = 0.32, p = 0.065) and SDAI (r = 0.33, p = 0.051). The correlation between decrease in 14-3-3η levels and improvement in DAS4ESR remained significant in a partial correlation analysis controlling for ESR (r = 0.39, p = 0.02). This study demonstrates that in RA patients who were treated with TOF, decrease in 14-3-3η levels is correlated with improvement in clinical disease activity parameters. The 14-3-3η protein may serve as an objective biomarker for monitoring of TOF therapy response.

Return to sport after acute acromioclavicular stabilization: a randomized control of double-suture-button system versus clavicular hook plate compared to uninjured shoulder sport athletes.

PURPOSE: Traumatic high-grade acromioclavicular joint (ACJ) separations can be surgically stabilized by numerous anatomic and non-anatomic procedures. The return to sport (Maffe et al. in Am J Sports Med 23:93-98, 1995] and remaining sport-associated impairments after acute ACJ stabilization has not yet been investigated. METHODS: 73 consecutive athletes with acute high-grade ACJ separation were prospectively assigned into two groups (64.4% randomized, 35.6% intention-to-treat): open clavicular hook plate (cHP) implantation (GI) or arthroscopically assisted double double-suture-button (dDSB) implantation (GII). Patients were analyzed using shoulder sport-specific measurement tools for sport ability (ASOSS), sport activity (SSAS), and numerical analog scales: NASpain during sport, NASshoulder function in sport, and NASre-achievement of sport level. Four points of examination were established: preoperative evaluation (FU0) and first postoperative follow-up (FU1) at 6 months; FU2 at 12 months; and FU3 at 24 months after surgery. The control group (GIII) consisted of 140 healthy athletes without anamnesis of prior macro-injury or surgery. RESULTS: After surgical stabilization, 29 of 35 athletes in GI (82.9%; 38.6 ± 9.9 years) and 32 of 38 in GII (82.9%; 38.6 ± 9.9 years) were followed up for 24 months (FU3) (loss 17.8%). All operated athletes showed significantly increased scores compared to FU0 (p < 0.05). Compared to GI, GII showed significantly superior outcome data for sporting ability as well as for NASre-achievement of sport level (p < 0.05). While GII re-achieved GIII-comparable SSAS and ASOSS levels, GI remained at a significantly inferior level. Athletes after ACJ injury of Rockwood grade IV/V and overhead athletes benefited significantly from the dDSB procedure. CONCLUSION: The dDSB procedure enabled significantly superior sport-specific outcomes compared to the cHP procedure. Athletes after dDSB surgeries re-achieved the sporting ability and the sport activity levels of healthy athletes, whereas athletes after cHP implantation remained at significantly inferior levels. The more extensive dDSB procedure and the more restrictive rehabilitation are recommended for treatment of acute high-grade ACJ separations of functionally high-demanding athletes. LEVEL OF EVIDENCE: I.

Prevalence of HIV Viral Load Suppression Among Psychiatric Inpatients with Comorbid Substance Use Disorders.

A consistently suppressed viral load enables HIV (+) patients to live longer, healthier lives and reduces the probability of transmitting the virus. Since the prevalence of HIV is four times higher among those with psychiatric disorders than in the general population, it is likely that this group would also have greater difficulty remaining in care and achieving viral suppression. A secondary data analysis utilizing screening data from the Preventing AIDS Through Health (PATH) for Triples (PFT) Study were examined to assess HIV load suppression among 254 psychiatric inpatients with comorbid substance use disorders in Philadelphia. Viral load results from the past 12 months were obtained from medical records for 63 inpatients identified as HIV (+). The sample was predominately African American (76%), male (56%), and the average age was 43 years. Psychiatric disorders included depression (64%), schizophrenia (21%), and bipolar disorder (13%) with patients reporting use of alcohol (73%), cocaine (64%), cannabis (29%) and opioids (16%) prior to admission. Among this high risk sample of HIV (+) patients, about one-half (52%) achieved viral suppression, with recent opioid users six times more likely to have a detectable viral load than non-opioid users (OR 6.0; CI 1.1-31.7, p = .035). The 52% viral load suppression rate among psychiatric inpatient was higher than expected, given that the CDC's national suppression rate among those diagnosed with HIV in the general population is 58%. However, individuals with mental illness and substance use disorders require constant surveillance, monitoring, and supportive services to achieve viral suppression. Many of those who were virally suppressed were engaged in Philadelphia's extensive treatment network, whereas those who were detectable and enrolled in the PFT intervention were often homeless with unstable psychiatric symptoms and current substance use disorders, particularly opioid abuse.

Tuftsin-phosphorylcholine (TPC) equally effective to methylprednisolone in ameliorating lupus nephritis in a mice model.

The role of helminth treatment in autoimmune diseases is growing constantly. Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with challenging treatment options. Tuftsin-phosphorylcholine (TPC) is a novel helminth-based compound that modulates the host immune network. This study was conducted to evaluate the potential value of TPC in ameliorating lupus nephritis in a murine model and specifically to compare the efficacy of TPC to the existing first-line therapy for SLE: corticosteroids (methylprednisolone). Lupus-prone NZBxW/F1 mice were treated with TPC (5 µg/mouse), methylprednisolone (MP; 5 mg/body weight) or phosphate-buffered saline (PBS) (control) three times per week once glomerulonephritis, defined as proteinuria of grade > 100 mg/dl, was established. Levels of anti-dsDNA autoantibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), splenic cytokines were measured in vitro and the kidney microscopy was analysed following staining. TPC and MP treatments improved lupus nephritis significantly and prolonged survival in NZBxW/F1 mice. TPC-treated mice showed a significantly decreased level of proteinuria (P < 0·001) and anti-dsDNA antibodies (P < 0·001) compared to PBS-treated mice. Moreover, TPC and MP inhibited the production of the proinflammatory cytokines interferon IFN-γ, interleukin IL-1ß and IL-6 (P < 0·001) and enhanced expression of the anti-inflammatory cytokine IL-10 (P < 0·001). Finally, microscopy analysis of the kidneys demonstrated that TPC-treated mice maintained normal structure equally to MP-treated mice. These data indicate that the small molecule named TPC hinders lupus development in genetically lupus-prone mice equally to methylprednisolone in most of the cases. Hence, TCP may be employed as a therapeutic potential for lupus nephritis.

The diagnostic value of 14-3-3η protein levels in patients with rheumatoid arthritis.

14-3-3η may represent a useful diagnostic biomarker for rheumatoid arthritis (RA). We assessed the prevalence and serum levels of 14-3-3η in patients with RA and in patients with other rheumatic diseases. Serum levels of 14-3-3η were measured in 96 patients with RA, in 101 patients with other rheumatic diseases, and in 66 healthy subjects. All of the sera samples were evaluated by JOINT stat 14-3-3η ELISA test kits (Augurex Life Sciences Corp.). Median (IQR) 14-3-3η levels were significantly higher in the early RA group [0.25 ng/ml (0.075-3.11)] and in patients with established RA [0.15 ng/ml (0.08-1.26)] than in healthy subjects [0 ng/ml (0-0)] and disease controls: SLE [0.01 ng/ml (0-0.055)], AS [0.05 ng/ml (0-0.255)], and PsA [0.01 ng/ml (0-0.065)]. The prevalence of 14-3-3η positivity in patients with early RA was 58%, significantly higher than that in disease controls and healthy subjects (p < 0.001). In patients with established RA, this prevalence was 43%, and it was significantly higher than that in patients with other rheumatic diseases and healthy subjects (p < 0.05), excluding the AS group (p = 0.054). In the early RA cohort, the positivity for 14-3-3η, RF, and anti-CCP was 58%, 67%, and 71%, respectively. Eighty-two percent of the patients in this cohort were positive for at least one of these biomarkers. The concentration of 14-3-3η protein may be used to distinguish between patients with early RA and patients with other rheumatic diseases.

Analysis of genetic diversity of Hyptis pectinata (L.) Poit. plants using ISSR markers.

Hyptis pectinata, popularly known as 'sambacaitá' or 'canudinho', is a medicinal and aromatic species widely used in the Brazilian Northeast. In Sergipe, the excessive extraction of natural resources may reduce the genetic variability of native plants. Thus, molecular markers have frequently been applied to the characterization of genetic diversity as the basis for germplasm conservation and breeding programs. The objective of the present study was to evaluate the genetic diversity of H. pectinata plants collected in different municipalities of the State of Sergipe using ISSR molecular markers. Thirty-four primers were tested, nine of which were selected for providing reproducible and analyzable amplification products, resulting in 67 polymorphic bands. The expected heterozygosity ranged from 0.32 to 0.45, with a mean of 0.39. Polymorphism information content was of 0.49, which classifies the markers as moderately informative. A dendrogram was constructed using unweighted pair group method with arithmetic mean, forming three clusters: Cluster I (79 plants); Cluster II (4 plants); and Cluster III (2 plants). Jaccard's similarity coefficients ranged from 0.06 to 0.98. The plants SAM-117 and SAM-119 presented greater similarity. Conversely, SAM-107 and SAM-171 were the most genetically distant. In general, H. pectinata plants collected in the State of Sergipe presented low to moderate genetic diversity.

Assessment of genetic diversity of a native population of Eplingiella fruticosa: a plant with therapeutic potential.

Eplingiella fruticosa (Salzm. ex Benth.) Harley & J.F.B. Pastore, ex Hyptis fruticosa Salzm. ex Benth. is an aromatic and medicinal plant of the family Lamiaceae, found mainly in regions with intense anthropic activity. Information on the genetic diversity of this species is scarce. However, it can be assessed using molecular markers that identify the level of diversity among phenotypically identical individuals. The present study aimed to characterize the genetic diversity of a native population of E. fruticosa from the State of Sergipe using ISSR molecular markers. Samples of 100 plants were collected in 11 municipalities of the State of Sergipe and analyzed using eight ISSR primers, resulting in 72 informative bands. The cluster analysis obtained using the neighbor joining method resulted in three groups: Group I consisted of 50 plants, mainly from the municipalities of Areia Branca, Estância, Japaratuba, Moita Bonita, Pirambu, and Salgado; Group II was formed by 21 plants, with nine representatives from the municipality of Itaporanga d'Ajuda and 13 representatives from other municipalities; Group III was composed by 29 plants, being represented mainly by the municipalities of Malhada dos Bois and São Cristóvão. The smallest genetic distance occurred between plants EPF94 and EPF96 (0.250), and the greatest distance occurred between plants EPF50 and EPF96 (0.9778). The Shannon index had a mean value of 0.42, and diversity was considered moderate. Heterozygosity had a mean value of 0.267 and was considered low. Polymorphic information content (0.253) was considered moderately informative. Genetic diversity of E. fruticosa plants was intermediate, and the results of the present study can assist in the conservation and use of the genetic resources of this species.

Evaluating the Renal Safety of Investigational New Drugs: Where Should We Be Going?

We the authors work in the US Food and Drug Administration (FDA) review division responsible for the therapeutic agents for primary renal disease. We also field consultative inquiries regarding off-target adverse renal effects of drugs intended to treat other diseases. We do neither basic science research on renal diseases nor clinical studies of new drugs, but we are professional spectators of both. We offer here our thoughts on the challenge of identifying renal safety signals in the preclinical space and in the earliest phases of clinical development.

Genetic diversity of native populations of Croton tetradenius Baill. using ISSR markers.

Brazil has about 300 Croton species in different types of vegetation. Croton tetradenius Baill., which is endemic to the Northeast region and predominant in the Caatinga vegetation, stands out among the several species of this genus. Considering the importance of knowing the genetic variability of a species, the objective of this study was to analyze the genetic diversity of the genotypes of natural populations of C. tetradenius in the State of Sergipe, using ISSR molecular markers. Forty individuals were sampled in four natural populations of the State of Sergipe, Brazil. Thirteen primers were used for DNA amplification using ISSR-PCR, totaling 77 amplified fragments, of which 94.8% were polymorphic. Results of the cluster analysis obtained by the Jaccard's similarity index, using the UPGMA method, resulted in the formation of six distinct clusters. Analysis of molecular variance (AMOVA), used to estimate the genetic variability among populations, revealed significant genetic variance (P < 0.01) between and within the studied populations, and most of the genetic diversity was found (87%) within populations. According to the Jaccard's similarity index, none of the studied plants was genetically identical. CTE210 and CTE305 presented high similarity index (0.76), while CTE105 presented low similarity index (<0.16) with all related individuals. ISSR markers were efficient and allowed the formation of a molecular profile, and had sufficient polymorphism to estimate the genetic variability between the accessions of the studied populations.

Evaluation of swimming performance for fish passage of longnose dace Rhinichthys cataractae using an experimental flume.

The swimming performance of longnose dace Rhinichthys cataractae, the most widely distributed minnow (Cyprinidae) in North America, was assessed in relation to potential passage barriers. The study estimated passage success, maximum ascent distances and maximum sprint speed in an open-channel flume over a range of water velocities and temperatures (10·7, 15·3 and 19·3° C). Rhinichthys cataractae had high passage success (95%) in a 9·2 m flume section at mean test velocities of 39 and 64 cm s-1 , but success rate dropped to 66% at 78 cm s-1 . Only 20% of fish were able to ascend a 2·7 m section with a mean velocity of 122 cm s-1 . Rhinichthys cataractae actively selected low-velocity pathways located along the bottom and corners of the flume at all test velocities and adopted position-holding behaviour at higher water velocities. Mean volitional sprint speed was 174 cm s-1 when fish volitionally sprinted in areas of high water velocities. Swimming performance generally increased with water temperature and fish length. Based on these results, fishways with mean velocities <64 cm s-1 should allow passage of most R. cataractae. Water velocities >100 cm s-1 within structures should be limited to short distance (<1 m) and structures with velocities ≥158 cm s-1 would probably represent movement barriers. Study results highlighted the advantages of evaluating a multitude of swimming performance metrics in an open-channel flume, which can simulate the hydraulic features of fishways and allow for behavioural observations that can facilitate the design of effective passage structures.

Anti-HMGCR antibodies demonstrate high diagnostic value in the diagnosis of immune-mediated necrotizing myopathy following statin exposure.

Anti-HMGCR antibodies represent a characteristic serological feature of statin-exposed and statin-unexposed patients with immune-mediated necrotizing myopathy (IMNM). We assessed anti-HMGCR antibodies in patients with suspected IMNM following statin exposure and patients with other autoimmune rheumatic diseases. We evaluated the presence of anti-HMGCR autoantibodies in sera samples from 13 statin-exposed patients who were suspected of having IMNM, 38 patients with different inflammatory and autoimmune rheumatic diseases and 29 healthy subjects. The autoantibodies were evaluated by two assays: a new chemiluminescence QUANTA Flash HMGCR kit utilizing BIO-FLASH system and QUANTA Lite® HMGCR ELISA kit. Twelve samples from patients with suspicion for IMNM were found positive for anti-HMGCR antibodies by both assays. Only one of the 13 samples that were found positive by ELISA was negative by CIA. A very good qualitative correlation (κ = 0.95; 95 % CI 0.85-1.0) and quantitative agreement (Spearman's rho 0.87; P value < 0.0001; 95 % CI 0.62-0.96) were found between these two assays. All samples from healthy subjects and from the disease-controlled patient cohort were negative for anti-HMGCR antibodies. In comparison with ELISA results, the CIA exhibited high sensitivity and specificity values of 92.3 and 100 %, respectively. Receiver operating characteristic analysis for CIA and ELISA yielded area under the curve values of 0.99. The presence of anti-HMGCR antibodies may be a useful biomarker of IMNM in statin-exposed patients. There is a good correlation between the two anti-HMGCR antibody assays evaluated in the present study.

Cross-reactivity between annexin A2 and Beta-2-glycoprotein I in animal models of antiphospholipid syndrome.

Antiphospholipid syndrome (APS) affects coagulation and the brain by autoimmune mechanisms. The major antigen in APS is beta-2-glycoprotein I (ß2-GPI) is known to complex with annexin A2 (ANXA2), and antibodies to ANXA2 have been described in APS. We measured these antibodies in mice with experimental APS (eAPS) induced by immunization with ß2-GPI. Sera of these mice reacted significantly with recombinant ANXA2 by enzyme-linked immunosorbent assay (ELISA) and the eAPS mice had significantly high levels of immunoglobulin G (IgG) in the brain by immunoblot assays compared to adjuvant immunized controls. Immunoprecipitation performed by mixing eAPS brain tissue with protein-G beads resulted in identification of two autoantigens unique to the eAPS group, one of which was ANXA2. In order to study more directly and methodically the specific role of anti-ANXA2 antibodies in APS, we immunized mice with ß2-GPI which contained no ANXA2 or with ANXA2 and measured antibodies to these proteins. Levels of antibodies to ANXA2 measured by ELISA were 0.72 ± 0.007 arbitrary units (a.u), 0.24 ± 0.03 and 0.02 ± 0.01 a.u for sera from ANXA2, ß2-GPI and control mice, respectively (p < 0.0001 and p = 0.037 for the comparison of the ANXA2 and ß2-GPI groups to the controls). Purified IgG from ß2-GPI sera did not show cross-binding with ANXA2. Antibodies to ß2-GPI and phospholipids were found in the ß2-GPI immunized group only. The present study suggests an immune response to the ß2-GPI-ANXA2 complex in eAPS and provides a novel ANXA2 immunization model which will serve to study the role of ANXA2 antibodies in of APS.

Analysis of genetic diversity of a native population of Myrcia lundiana Kiaersk. plants using ISSR markers.

Myrcia lundiana Kiaersk. is a tree of the family Myrtaceae found in tropical and subtropical areas of the southern hemisphere that produces essential oil. The aim of this study was to characterize the genetic diversity of M. lundiana plants from a native population of Parque Nacional de Itabaiana, using inter-simple sequence repeat molecular markers. Thirty-five primers were tested, 20 of which were polymorphic, resulting in 135 polymorphic and informative bands. Results of the cluster analysis, obtained using the unweighted pair group method with arithmetic mean, grouped plants into three clusters: Cluster I - MLU001, MLU002, MLU003, MLU004, MLU005, MLU006, MLU018, MLU019, MLU020, MLU021, MLU022; MLU008, MLU011, MLU012, MLU014, MLU015, MLU017, MLU026, and MLU028; Cluster II - MLU007, MLU009, MLU010, MLU013, and MLU016; and Cluster III - MLU023, MLU024, MLU025, and MLU027. Jaccard similarity coefficients for pair-wise comparisons of plants ranged between 0.15 and 0.87. MLU014 and MLU015 presented low genetic diversity, with a similarity index of 0.87. Conversely, MLU007 and MLU019 presented high diversity, with a similarity index of 0.15. According to the structure analysis, three distinct clusters were formed. Genetic diversity of M. lundiana plants was intermediate, and expansion of its genetic diversity is necessary. MLU026 and MLU028 are the most suitable for selection in breeding programs, since they clearly represent all of the diversity present in these plants. Moreover, these results provide important information on the existing genetic variability, highlighting the importance of Parque Nacional de Itabaiana for the conservation of this species.

Genetic diversity analysis of Varronia curassavica Jacq. accessions using ISSR markers.

Varronia curassavica Jacq. is a medicinal and aromatic plant from Brazil with significant economic importance. Studies on genetic diversity in active germplasm banks (AGB) are essential for conservation and breeding programs. The aim of this study was to analyze the genetic diversity of V. curassavica accessions of the AGB of Medicinal and Aromatic Plants of the Federal University of Sergipe (UFS), using inter-simple sequence repeat molecular markers. Twenty-four primers were tested, and 14 were polymorphic and informative, resulting in 149 bands with 97.98% polymorphism. The UPGMA dendrogram divided the accessions into Clusters I and II. Jaccard similarity coefficients for pair-wise comparisons of accessions ranged between 0.24 and 0.78. The pairs of accessions VCUR-001/VCUR-503, VCUR-001/VCUR-504, and VCUR-104/VCUR-501 showed relatively low similarity (0.24), and the pair of accessions VCUR-402/VCUR- 403 showed medium similarity (0.78). Twenty-eight accessions were divided into three distinct clusters, according to the STRUCTURE analysis. The genetic diversity of V. curassavica in the AGB of UFS is low to medium, and it requires expansion. Accession VCUR-802 is the most suitable for selection in breeding program of this species, since it clearly represents all of the diversity present in the AGB.

Genetic diversity of Lippia sidoides Cham. and L. gracilis Schauer germplasm.

The conservation of plants in germplasm banks ensures the characterization and availability of these resources for future generations. The present study used DNA markers to obtain genetic information about germplasm collections of Lippia sidoides and L. gracilis, which are maintained in an Active Germplasm Bank (AGB). Genetic variability of samples in the AGB was assessed using 12 combinations of amplified fragment length polymorphism (AFLP) primers (EcoRI/MseI). Twenty simple sequence repeat primers designed for L. alba were tested to determine their transferability in L. sidoides and L. gracilis. The AFLP markers generated 789 markers. The assessed loci exhibited a moderate Shannon diversity index (I = 0.42) in both species, suggesting that the conserved accessions possess an intermediate level of genetic diversity. Twelve microsatellite loci amplified satisfactorily, and nine loci were polymorphic in each species. A total of 23, 22, and 36 alleles, with an average of 2.5, 2.4, and 3.27 alleles per locus were identified for L. sidoides and L. gracilis accessions in the AGB, and Lippia sp sampled plants, respectively. Analyses of genetic structure permitted the identification of three different groups using both sets of markers, of which two were representative of L. sidoides. The information generated in this study may help to create, expand, and maintain collections of these species and may assist in genetic-breeding programs.