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Vascularized composite allotransplantation (VCA) is the most advanced reconstructive technique available to patients who suffer devastating burns to the limbs or face. However, VCA requires donor–recipient matching. Burn patients have been reported to experience sensitization, or the development of anti-human leukocyte antigen antibodies, during resuscitation and wound coverage, potentially precluding them from future VCA. This study sought to investigate the contributions of both blood and allograft to sensitization in burn patients. Four groups were compared: burn patients who received blood products and allograft (group 1), burn patients who received blood products only (group 2), trauma patients who received blood products only (group 3), and healthy volunteer controls (group 4). The average calculated panel-reactive antibody (indicating sensitization) was higher in group 1 compared to group 4 (P = .035). Additionally, the incidence of severe sensitization was higher in group 1 relative to the other groups (P = .049). When comparing groups of patients who had no sensitization, mild sensitization, moderate sensitization, and severe sensitization, there were no significant differences in age, sex, blood products received, total body surface area burned, or allograft used between groups, though severely sensitized patients tended to have greater total body surface area involvement and received more units of packed red blood cells and allograft (P = .079, P = .196, and P = .072, respectively). We therefore conclude that while burn patients who received allograft and blood demonstrated a higher incidence of anti-human leukocyte antigen sensitization relative to healthy controls, this difference cannot solely be attributed to either allograft use or transfusion.

Made available in DSpace on 2021-06-25T10:21:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 We analyzed the distribution and ecological relationships in the parasitic infracommunities of four species of the genus Hypostomus: Hypostomus regani, Hypostomus strigaticeps, Hypostomus hermanni, and Hypostomus ancistroides, collected in the Batalha River, State of São Paulo, Brazil. Twenty-four parasite taxa were identified among myxozoans, monogeneans, digeneans, nematodes, and hirudineans occurring in the gills, body surface, eyes, brain, and intestine. Monogeneans presented the highest species richness. The highest parasite prevalence was observed for ectoparasites in the Hypostomus spp. analyzed. Except for Phanerothecium sp., all parasites presented an aggregate pattern of distribution. In relation to parasitic diversity, H. regani presented higher mean values of diversity indexes, while H. hermanni presented higher values for the specific richness; equitability values were the same in all fish species analyzed. Infracommunities were most differentiated from one another (p < 0.05) between H. reganni and H. ancistroides. Standard length, body mass, and sex of Hypostomus spp. were significantly correlated (positively or negatively) with at least one parasite species. This study emphasizes the importance of the Batalha River as a source of local and regional biodiversity expanding the knowledge of ecological interactions between the parasite–host relationships of Siluriformes fish. Instituto de Biociências Universidade Estadual Paulista (UNESP) Pró-reitoria de Pesquisa e pós-graduação Centro Universitário do Sagrado Coração (UNISAGRADO) Programa de pós-graduação em Análise de Sistemas Ambientais Centro Universitário CESMAC Instituto de Biociências Universidade Estadual Paulista (UNESP)

Pore-forming proteins are found in prokaryotes, vertebrates, and invertebrates, and when involved in pathogenic processes they are classified as pore-forming toxins (PFTs). The use of gene engineering methods in combination with the information provided by the high-resolution crystal structures of the PFTs have allowed investigators to gain a deep understanding of their pore-forming mechanisms. In this chapter, we discuss how protein engineering has helped us and others to reveal the molecular mechanisms of pore formation by prokaryotic PFTs with an emphasis on our experiences with the cholesterol-dependent cytolysins (CDCs).

Introduction: Elevation in the inflammatory marker high sensitivity C-reactive protein (hsCRP) is associated with worse outcomes in patients with heart failure (HF). We aimed to determine if baseline or trajectory of hsCRP levels over time predict incident HF hospitalization. Methods: Jackson Heart Study (JHS) participants’ (n=4203 African Americans) hsCRP levels were measured over 3 visits (visit 1: 2000 to 2004; visit 2: 2005 to 2008; visit 3: 2009 to 2013). We assessed the association of a single hsCRP level measurement at baseline (visit 1) with incident HF hospitalization using Cox proportional hazard models. Furthermore, we assessed the association of trajectory of hsCRP over repeated measurements (visit 1-3) with incident HF using a joint model, which incorporates estimated hsCRP from a linear mixed effects model into a Cox hazards model to predict incident HF hospitalization while incorporating trajectory of hsCRP over visits. All hazard ratios (HR) are presented as an increase in hsCRP by 1 standard deviation on a Log 2 scale. Results: At baseline, mean age of participants was 55±13 years, 63.4% were women, and mean hsCRP level was 0.5±0.7 mg/dl. Over a median follow-up of 12 years, 353 (8.4%) participants were hospitalized with incident HF. After adjustment for covariates, baseline hsCRP was not associated with increased risk of incident HF hospitalization (Table, p>0.05). However, increases in hsCRP levels on follow-up were associated with a significantly increased risk of incident HF hospitalization (Table, p Conclusions: While an elevated hsCRP level at one time point may not be associated with incident HF, the increasing trajectory of change in hsCRP over time is predictive of increased risk for incident HF hospitalization in African Americans. These data support the role of increased inflammatory status in the development of heart failure.

Cartilage defects are common in the knee joint of active athletes and remain a problem as a strong risk factor for osteoarthritis. We hypothesized that landing during sport activities, implication for subfailure ACL loading, would generate greater contact pressures (CP) at the lateral knee compartment. The purpose of this study is to investigate tibiofemoral cartilage CP of athletes during landing. Tibiofemoral cartilage contact pressures (TCCP) under clinically relevant anterior cruciate ligament subfailure external loadings were predicted using four dynamic explicit finite element (FE) models (2 males and 2 females) of the knee. Bipedal landing from a jump for five cases of varying magnitudes of external loadings (knee abduction moment, internal tibial torque, and anterior tibial shear) followed by an impact load were simulated. Lateral TCCP from meniscus (area under meniscus) and from femur (area under femur) increased by up to 94% and %30 respectively when external loads were incorporated with impact load in all the models compared to impact-only case. In addition, FE model predicted higher CP in lateral compartment by up to 37% (11.87 MPa versus 8.67 MPa) and 52% (20.19 MPa versus 13.29 MPa) for 90% and 50% percentile models, respectively. For the same percentile populations, CPs were higher by up to 25% and 82% in smaller size models than larger size models. We showed that subfailure ACL loadings obtained from previously conducted in vivo study led to high pressures on the tibiofemoral cartilage. This knowledge is helpful in enhancing neuromuscular training for athletes to prevent cartilage damage.

The cholesterol-dependent cytolysins’ pore-forming mechanism relies on the ability to sense the completion of the oligomeric prepore structure and initiate the insertion of the β-barrel pore from the assembled prepore structure. These studies show that a conserved motif is an important component of the sensor that triggers the prepore-to-pore transition and that it is conserved in a large family of previously unidentified CDC-like proteins, the genes for which are present in a vast array of microbial species that span most terrestrial environments, as well as most animal and human microbiomes. These studies establish the foundation for future investigations that will probe the contribution of this large family of CDC-like proteins to microbial survival and human disease., The cholesterol-dependent cytolysins (CDCs) are bacterial, β-barrel, pore-forming toxins. A central enigma of the pore-forming mechanism is how completion of the prepore is sensed to initiate its conversion to the pore. We identified a motif that is conserved between the CDCs and a diverse family of nearly 300 uncharacterized proteins present in over 220 species that span at least 10 bacterial and 2 eukaryotic phyla. Except for this motif, these proteins exhibit little similarity to the CDCs at the primary structure level. Studies herein show this motif is a critical component of the sensor that initiates the prepore-to-pore transition in the CDCs. We further show by crystallography, single particle analysis, and biochemical studies of one of these CDC-like (CDCL) proteins from Elizabethkingia anophelis, a commensal of the malarial mosquito midgut, that a high degree of structural similarity exists between the CDC and CDCL monomer structures and both form large oligomeric pore complexes. Furthermore, the conserved motif in the E. anophelis CDCL crystal structure occupies a nearly identical position and makes similar contacts to those observed in the structure of the archetype CDC, perfringolysin O (PFO). This suggests a common function in the CDCs and CDCLs and may explain why only this motif is conserved in the CDCLs. Hence, these studies identify a critical component of the sensor involved in initiating the prepore-to-pore transition in the CDCs, which is conserved in a large and diverse group of distant relatives of the CDCs.

Made available in DSpace on 2020-12-12T01:28:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01. Added 1 bitstream(s) on 2021-07-15T14:37:52Z : No. of bitstreams: 1 S0001-37652020000300709.pdf: 399812 bytes, checksum: 69154f7a13469ad3becd92f812450d1c (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Eighty-one Hoplias malabaricus specimens were collected between February 2014 and June 2016. A total of 29 species of metazoan parasites were found, 13 of which were identified as monogeneans, seven were digenean species, seven of which were nematodes, and two of which were from the subclass Hirudinea. The highest prevalence values were presented by Contracaecum sp. and Tylodelphys sp. The highest mean abundance and mean intensity was recorded by Tylodelphys sp.; the values were 36.7 ± 61.8 and 55.65 ± 69.1, respectively. The abundance of the monogenean Urocleidoides cuiabai was found to be positively correlated with host weight. The abundance of Bucephalidae gen. sp. exhibited significant positive correlations with host weight and length. For Contracaecum sp., a significant negative correlation was found between its abundance and host length and weight. No significant differences between the diversity indexes (Margalef, Pielou and Shannon) of the parasites collected in the two points were found. The Sorensen similarity index, with a value of 0.82 between the two sampling points revealed that the parasitic diversity between them is similar. The findings from this study represent new records of occurrence of H. malabaricus, as well as of Urocleidoides margolisi, Scleroductus sp. and Helobdella sp. Universidade do Sagrado Coração/USC Pró-reitoria de Pesquisa e Pós-graduação Laboratório de Ictioparasitologia, Rua Irmã Arminda, 10-50, Jardim Brasil Universidade Estadual Paulista/UNESP Instituto de Biociências de Botucatu, Rua Prof. Dr. Antônio Celso Wagner Zanin, 250, Distrito de Rubião Júnior Universidade Estadual Paulista/UNESP Instituto de Biociências de Botucatu, Rua Prof. Dr. Antônio Celso Wagner Zanin, 250, Distrito de Rubião Júnior FAPESP: 2012/23655-0 FAPESP: 2015/17932-9

Zika virus remains a worldwide health threat, with outbreaks still occurring in the Americas. While mosquitos are the primary vector for the spread of the virus, sexual transmission of Zika virus is also a significant means of infection, especially in terms of passage from an infected to an uninfected partner. While sexual transmission has been documented in humans, and male-to-female transmission has been reported in mice, ours is the first study in nonhuman primates to demonstrate infection via vaginal deposition of Zika virus-infected semen. The latter is important since a recent publication indicated that human semen inhibited, in a laboratory setting, Zika virus infection of reproductive tissues. We also found that compared to the French Polynesian isolate, the Puerto Rican Zika virus isolate led to greater spread throughout the body, particularly in reproductive tissues. The American isolates of Zika virus appear to have acquired mutations that increase their efficacy., Zika virus (ZIKV) infection is now firmly linked to congenital Zika syndrome (CZS), including fetal microcephaly. While Aedes species of mosquito are the primary vector for ZIKV, sexual transmission of ZIKV is a significant route of infection. ZIKV has been documented in human, mouse, and nonhuman primate (NHP) semen. It is critical to establish NHP models of the vertical transfer of ZIKV that recapitulate human pathogenesis. We hypothesized that vaginal deposition of ZIKV-infected baboon semen would lead to maternal infection and vertical transfer in the olive baboon (Papio anubis). Epidemiological studies suggest an increased rate of CZS in the Americas compared to the original link to CZS in French Polynesia; therefore, we also compared the French Polynesian (FP) ZIKV isolate to the Puerto Rican (PR) isolate. Timed-pregnant baboons (n = 6) were inoculated via vaginal deposition of baboon semen containing 106 focus-forming units (FFU) of ZIKV (n = 3 for FP isolate H/PF/2013; n = 3 for PR isolate PRVABC59) at midgestation (86 to 95 days of gestation [dG]; term, 183 dG) on day 0 (all dams) and then at 7-day intervals through 3 weeks. Maternal blood, saliva, and cervicovaginal wash (CVW) samples were obtained. Animals were euthanized at 28 days (n = 5) or 39 days (n = 1) after the initial inoculation, and maternal/fetal tissues were collected. Viremia was achieved in 3/3 FP ZIKV-infected dams and 2/3 PR ZIKV-infected dams. ZIKV RNA was detected in CVW samples of 5/6 dams. ZIKV RNA was detected in lymph nodes but not the ovaries, uterus, cervix, or vagina in FP isolate-infected dams. ZIKV RNA was detected in lymph nodes (3/3), uterus (2/3), and vagina (2/3) in PR isolate-infected dams. Placenta, amniotic fluid, and fetal tissues were ZIKV RNA negative in the FP isolate-infected dams, whereas 2/3 PR isolate-infected dam placentas were ZIKV RNA positive. We conclude that ZIKV-infected semen is a means of ZIKV transmission during pregnancy in primates. The PR isolate appeared more capable of widespread dissemination to tissues, including reproductive tissues and placenta, than the FP isolate. IMPORTANCE Zika virus remains a worldwide health threat, with outbreaks still occurring in the Americas. While mosquitos are the primary vector for the spread of the virus, sexual transmission of Zika virus is also a significant means of infection, especially in terms of passage from an infected to an uninfected partner. While sexual transmission has been documented in humans, and male-to-female transmission has been reported in mice, ours is the first study in nonhuman primates to demonstrate infection via vaginal deposition of Zika virus-infected semen. The latter is important since a recent publication indicated that human semen inhibited, in a laboratory setting, Zika virus infection of reproductive tissues. We also found that compared to the French Polynesian isolate, the Puerto Rican Zika virus isolate led to greater spread throughout the body, particularly in reproductive tissues. The American isolates of Zika virus appear to have acquired mutations that increase their efficacy.

ZIKV infection is associated with pregnancy loss, fetal microcephaly and other malformations. While Aedes sp. of mosquito are the primary vector for ZIKV, sexual transmission of ZIKV is a significant route of infection. ZIKV has been documented in human, mouse and non-human primate (NHP) semen. It is critical to establish NHP models of vertical transfer of ZIKV that recapitulate human ZIKV pathogenesis. We hypothesized that vaginal deposition of ZIKV infected baboon semen would lead to maternal infection and vertical transfer in the olive baboon (Papio anubis). Timed pregnant baboons (n=6) were inoculated via vaginal deposition of baboon semen containing 106 ffu ZIKV (n=3, French Polynesian isolate:H/PF/2013, n=3 Puerto Rican isolate:PRVABC59) at mid-gestation (86-95 days gestation [dG]; term 183dG) on day (d) 0 (all dams), and then at 7 day intervals through three weeks. Maternal blood, saliva and cervico-vaginal washes were obtained at select days post-inoculation. Animals were euthanized at 28 days post initial inoculation (dpi; n=5) or 39 dpi (n=1) and maternal/fetal tissues collected. vRNA was quantified by qPCR. Viremia was achieved in 3/3 FP ZIKV infected dams and 2/3 PR ZIKV. ZIKV RNA was detected in cvw (5/6 dams;). ZIKV RNA was detected in lymph nodes, but not ovary, uterus, cervix or vagina in the FP ZIKV dams but was detected in uterus, vagina and lymph nodes. Placenta, amniotic fluid and all fetal tissues were ZIKV RNA negative in the FP infected dams whereas 2/3 PR infected dam placentas were ZIKV RNA positive. We conclude that ZIKV infected semen is a means of ZIKV transmission during pregnancy in primates. The PR isolate appeared more capable of wide spread dissemination to tissues, including placenta compared to the FP strain.IMPORTANCEDue to its established link to pregnancy loss, microcephaly and other major congenital anomalies, Zika virus (ZIKV) remains a worldwide health threat. Although mosquitoes are the primary means of ZIVK transmission, sexual transmission in human populations is well documented and provides a means for widespread dissemination of the virus. Differences in viremia, tissue distribution, immune responses and pregnancy outcome from sexually transmitted ZIKV compared to the subcutaneous route of infection are needed to better clinically manage ZIKV in pregnancy. Through our previous work, we have developed the olive baboon as a non-human primate model of ZIKV infection that is permissible to ZIKV infection via the subcutaneous route of inoculation and transfer of ZIKV to the fetus in pregnancy. The current study evaluated the course of ZIKV infection after vaginal inoculation of ZIKV in pregnant baboons at mid-gestation using baboon semen as the carrier and comparing two isolates of ZIKV, the French Polynesian isolate first associated with microcephaly and the Puerto Rican isolate, associated with an increased risk of microcephaly observed in the Americas.

Introduction Skin graft survival relies on imbibition, inosculation, and revascularization from the wound bed. When a wound bed is poorly vascularized, as in the case of exposed fascia, tendon or bone, skin grafting may be delayed until the wound bed improves. We propose that topical nutrient supplementation may be able to increase take of skin grafts applied over an avascular wound bed. Methods Twenty full-thickness 5cm-diameter wounds were created on the dorsum of anesthetized swine and a dermal substitute (0.4mm, 0.8mm, 1.2mm, or 1.6mm thick) was placed on each wound. Negative pressure therapy with and without intermittent (3x per day) saline instillation was applied. Wounds were analyzed using a non-contact 3D camera at day 7 and day 14. Results Dermal substitutes of 0.8mm, 1.2mm, and 1.6mm thicknesses inhibited graft take significantly (p< 0.01, p=0.02, p< 0.01, respectively) for all wounds treated with wound vac alone. Addition of the normal saline instill showed a significant improvement in graft take (p=0.03) over wound vac alone for the wounds treated with the 0.8mm dermal substitute. Wounds covered with 1.2mm and 1.6mm dermal substitute continued to show significantly decreased graft take (p=0.03 and p=0.02, respectively). Wounds with 0.4mm dermal substitute showed similar graft take to control for both the wound vac and wound vac + instill treatments. Conclusions Dermal substitutes ≥0.8mm create a successful model of an avascular wound bed. Vac + instill treatment overcame the impedance of an avascular wound bed only for the 0.8mm dermal substitute thickness. This thickness of dermal substitute creates an ideal avascular wound bed model from which to conduct further studies incorporating topical nutrients instilled directly onto skin grafts placed onto avascular wound beds. Applicability of Research to Practice Single-stage skin grafting procedures onto avascular wound beds may become feasible with topical nutrient supplementation providing the environment to maintain graft survival until the wound bed is able to support the skin graft.

The cholesterol-dependent cytolysins (CDCs) are the archetype for the superfamily of oligomeric pore-forming proteins that includes the membrane attack complex/perforin (MACPF) family of immune defense proteins and the stonefish venom toxins (SNTX). The CDC/MACPF/SNTX family exhibits a common protein fold, which forms a membrane-spanning β-barrel pore. We show that changing the relative stability of an extensive intramolecular interface within this fold, which is necessarily disrupted to form the large β-barrel pore, dramatically alters the kinetic and temperature-dependent properties of CDC pore formation. These studies show that the CDCs and other members of the CDC/MACPF/SNTX superfamily have the capacity to significantly alter their pore-forming properties to function under widely different environmental conditions encountered by these species., The cholesterol-dependent cytolysin (CDC) genes are present in bacterial species that span terrestrial, vertebrate, and invertebrate niches, which suggests that they have evolved to function under widely different environmental conditions. Using a combination of biophysical and crystallographic approaches, we reveal that the relative stability of an intramolecular interface in the archetype CDC perfringolysin O (PFO) plays a central role in regulating its pore-forming properties. The disruption of this interface allows the formation of the membrane spanning β-barrel pore in all CDCs. We show here that the relative strength of the stabilizing forces at this interface directly impacts the energy barrier posed by the transition state for pore formation, as reflected in the Arrhenius activation energy (Ea) for pore formation. This change directly impacts the kinetics and temperature dependence of pore formation. We further show that the interface structure in a CDC from a terrestrial species enables it to function efficiently across a wide range of temperatures by minimizing changes in the strength of the transition state barrier to pore formation. These studies establish a paradigm that CDCs, and possibly other β-barrel pore-forming proteins/toxins, can evolve significantly different pore-forming properties by altering the stability of this transitional interface, which impacts the kinetic parameters and temperature dependence of pore formation.

OBJECTIVE: This study aims to evaluate differences in the midtrimester cervicovaginal microbiota between women who developed puerperal infections at term and those who did not, and whether obesity modulates this relationship. METHODS: Previously, cervicovaginal swabs were collected at 21 to 25 weeks gestation (stored at −80°C). Samples were identified from Black women with normal vaginal flora (Nugent score: 0–2) delivering term singletons. Patients were in one of four equally sized groups (total n = 120) characterized by absence or presence of puerperal infection and maternal obesity. Samples were thawed, DNA extracted, and polymerase chain reaction with primers targeting the 16S rDNA V4 region was used to prepare an amplicon library sequenced and analyzed using Quantitative Insights into Microbial Ecology (QIIME) suite. Microbiota differences were assessed using permutation-based anodis over three β-diversity measures; Kruskal-Wallis test was used for taxa level analysis. RESULTS: After quality control measures, 113 samples were analyzed. Overall, there was significant clustering by puerperal infection (p = 0.03), but not by obesity (p > 0.05). Detailed taxa level analysis revealed approximately 66% less Proteobacteria phylum and 400% more BVAB1 genera in the second-trimester microbiota of women who had puerperal infections at term (p < 0.05). CONCLUSION: Women who develop puerperal infections at term have a significantly altered midtrimester cervicovaginal microbiome with less Proteobacteria and greater BVAB1. This finding may represent a potential method to identify women at an increased risk of puerperal infection.

We studied rooting damage during five-years of feral swine control at Avon Park Air Force Range, a significant botanical biodiversity hotspot in peninsular Florida with many globally imperiled plant species and communities. While control reduced swine abundance, remaining animals consistently rooted the 49 studied sites in both middle-dry season (MDS) and late-dry season (LDS) each year. At each study site, we measured rooting with sub-meter accuracy. Neither total nor proportional area rooted differed in either season, across study years, or among plant community types: herbaceous seepage slopes, wet pine savannas, wet grasslands. The proportion of sites with damage during MDS was at least 25 % less than pre-control baseline. During LDS, the proportion of sites with damage increased over years but remained below the initial 2 years’ MDS results. Fresh rooting frequency (rooting

We sought to compare the skin microbiota above and below the panniculus of third-trimester pregnant women with class III obesity.We collected swabs from the anterior panniculus and from the intertriginous area under the panniculus of women with class III obesity (body mass index ≥40 kg/m(2)) carrying a live singleton at ≥28 weeks. DNA was extracted. PCR with primers targeting the 16S rDNA V4 region were used to prepare an amplicon library. PCR products were sequenced (Illumina MiSeq platform). The 16S rDNA sequences were processed, integrated, analyzed and reported using QIIME and in-house developed scripts. Taxonomy was assigned using RDP Classifier (threshold 0.8) against the Greengenes 16S rDNA database.Twenty women were enrolled. One sample pair was excluded for low sequence depth. Using permutation testing, there was significantly less bacterial diversity in the samples above compared to below the panniculus (beta diversity using weighted UniFrac metrics; p 0.001).In pregnant women with class III obesity, the skin microbiota in the subpannicular fold differs from and is more diverse than that on the anterior panniculus.

The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and β), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.

Viral infections complicated by a bacterial infection are typically referred to as coinfections or superinfections. Streptococcus pyogenes, the group A streptococcus (GAS), is not the most common bacteria associated with influenza A virus (IAV) superinfections but did cause significant mortality during the 2009 influenza pandemic even though all isolates are susceptible to penicillin. One approach to improve the outcome of these infections is to use passive immunization targeting GAS. To test this idea, we assessed the efficacy of passive immunotherapy using antisera against either the streptococcal M protein or streptolysin O (SLO) in a murine model of IAV-GAS superinfection. Prophylactic treatment of mice with antiserum to either SLO or the M protein decreased morbidity compared to mice treated with non-immune sera; however, neither significantly decreased mortality. Therapeutic use of antisera to SLO decreased morbidity compared to mice treated with non-immune sera but neither antisera significantly reduced mortality. Overall, the results suggest that further development of antibodies targeting the M protein or SLO may be a useful adjunct in the treatment of invasive GAS diseases, including IAV-GAS superinfections, which may be particularly important during influenza pandemics.

Here we conduct a qualitative and quantitative analysis of ectoparasites and endoparasites of Astyanax altiparanae (Garutti & Britski, 2000), popularly known as lambari-do-rabo-amarelo collected from the Batalha River, State of São Paulo, Brazil. Of the 67 specimens of A. altiparanae, 52 were parasitized by at least one parasitic metazoan species. The following metazoan parasites were found: Monogenea, Digenea and Nematoda. The monogenetic Diaphorocleidus sp. was the predominant species, presenting a higher frequency of dominance, prevalence and mean abundance. However, the monogenean Trinigyrus sp. was the one with the highest mean intensity value. All parasites showed aggregate distribution pattern. The parasitic community of A. altiparanae was characterized by low diversity, low richness and low uniformity. The digenetic Rhipidocotyle santanaensis is recorded for the first time in A. altiparanae, in the same way, this parasite is being registered for the first time in the Batalha River. Resumo Foi realizada uma análise qualitativa e quantitativa de ectoparasitas e endoparasitas de Astyanax altiparanae (Garutti & Britski, 2000), popularmente conhecido como lambari-do-rabo-amarelo coletados no rio Batalha, estado de São Paulo, Brasil. Dos 67 espécimes de A. altiparanae, 52 estavam parasitados por pelo menos uma espécie de metazoário parasito. Foram encontrados os seguintes metazoários parasitos: Monogenea, Digenea e Nematoda. O monogenético Diaphorocleidus sp. foi a espécie predominante, apresentando maior frequência de dominância, prevalência e abundância média. Porém o monogenético Trinigyrus sp. foi o que apresentou maior valor de intensidade média. Todos os parasitos mostraram padrão de distribuição agregado. A comunidade parasitária de A. altiparanae foi caracterizada pela baixa diversidade, baixa riqueza e baixa uniformidade. Registra-se pela primeira vez o digenético Rhipidocotyle santanaensis em A. altiparanae, da mesma forma, este parasito está sendo registrado pela primeira vez no rio Batalha.

This study aims to evaluate vaginal microbiota differences by bacterial vaginosis (BV), birth timing, and race, and to estimate parameters to power future vaginal microbiome studies.Previously, vaginal swabs were collected at 21 to 25 weeks (stored at -80°C), and vaginal smears evaluated for BV (Nugent criteria). In a blinded fashion, 40 samples were selected, creating 8 equal-sized groups stratified by race (black/white), BV (present/absent), and birth timing (preterm/term). Samples were thawed, DNA extracted, and prepared. Polymerase chain reaction (PCR) with primers targeting the 16S rDNA V4 region was used to prepare an amplicon library. PCR products were sequenced and analyzed using quantitative insight into microbial ecology; taxonomy was assigned using ribosomal database program classifier (threshold 0.8) against the modified Greengenes database.After quality control, 97,720 sequences (mean) per sample, single-end 250 base-reads, were analyzed. BV samples had greater microbiota diversity (p 0.05)-with BVAB1, Prevotella, and unclassified genus, Bifidobacteriaceae family (all p 0.001) more abundant; there was minimal content of Gardnerella or Mobiluncus. Microbiota did not differ by race or birth timing, but there was an association between certain microbial clusters and preterm birth (p = 0.07). To evaluate this difference, 159 patients per group are needed.There are differences in the vaginal microbiota between patients with and without BV. Larger studies should assess the relationship between microbiota composition and preterm birth.

The mechanism by which the cholesterol-dependent cytolysins (CDCs) assemble their giant β-barrel pore in cholesterol-rich membranes has been the subject of intense study in the past two decades. A combination of structural, biophysical, and biochemical analyses has revealed deep insights into the series of complex and highly choreographed secondary and tertiary structural transitions that the CDCs undergo to assemble their β-barrel pore in eukaryotic membranes. Our knowledge of the molecular details of these dramatic structural changes in CDCs has transformed our understanding of how giant pore complexes are assembled and has been critical to our understanding of the mechanisms of other important classes of pore-forming toxins and proteins across the kingdoms of life. Finally, there are tantalizing hints that the CDC pore-forming mechanism is more sophisticated than previously imagined and that some CDCs are employed in pore-independent processes.

Pore-forming proteins (PFPs) encompass a broad family of proteins that are used for virulence or immune defense. Members of the cholesterol-dependent cytolysins (CDCs) and membrane attack complex/perforin (MACPF) family of PFPs form large β-barrel pores in the membrane. The CDC/MACPF proteins contain a characteristic four-stranded β-sheet that is flanked by two α-helical bundles, which unfold to form two transmembrane β-hairpins. Apicomplexan eukaryotic parasites express CDC/MACPFs termed perforin-like proteins (PLPs). Here we review recent studies that provide key insights into the assembly and regulation of the Apicomplexan PLP (ApiMACPF) molecular pore-forming mechanisms, which are necessary for the osmotically driven rupture of the parasitophorous vacuole and host cell membrane, and cell traversal by these parasites.

The majority of cholesterol-dependent cytolysins (CDCs) utilize cholesterol as a membrane receptor, whereas a small number are restricted to the GPI-anchored protein CD59 for initial membrane recognition. Two cholesterol-binding CDCs, perfringolysin O (PFO) and streptolysin O (SLO), were found to exhibit strikingly different binding properties to cholesterol-rich natural and synthetic membranes. The structural basis for this difference was mapped to one of the loops (L3) in the membrane binding interface that help anchor the toxin monomers to the membrane after receptor (cholesterol) binding by the membrane insertion of its amino acid side chains. A single point mutation in this loop conferred the binding properties of SLO to PFO and vice versa. Our studies strongly suggest that changing the side chain structure of this loop alters its equilibrium between membrane-inserted and uninserted states, thereby affecting the overall binding affinity and total bound toxin. Previous studies have shown that the lipid environment of cholesterol has a dramatic effect on binding and activity. Combining this data with the results of our current studies on L3 suggests that the structure of this loop has evolved in the different CDCs to preferentially direct binding to cholesterol in different lipid environments. Finally, the efficiency of β-barrel pore formation was inversely correlated with the increased binding and affinity of the PFO L3 mutant, suggesting that selection of a compatible lipid environment impacts the efficiency of membrane insertion of the β-barrel pore.

The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as theta toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250-300 angstrom in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis.

Septic cardiomyopathy is a severe complication among some patients who develop group A streptococcal toxic shock syndrome. Despite the importance of cardiac dysfunction in determining prognosis, very little is known about mechanisms that reduce cardiac output in association with streptococcal infection. Here, we investigated the effects of streptococcal extracellular toxins on mechanical contractility of electrically paced primary murine cardiomyocytes. Our data demonstrate that streptolysin O (SLO) is the major streptococcal toxin responsible for cardiomyocyte contractile dysfunction. Streptolysin O dose-dependently affected cardiac myocyte function in discrete stages. Exposure to SLO caused a failure of cardiac cells to respond to electrical pacing, followed by spontaneous dysregulated contractions and augmented strength of contraction. Central to these SLO-mediated effects is a marked influx of calcium into the cytosol through SLO-mediated pores in the cytoplasmic membrane. Such calcium mobilization in response to SLO correlated temporally with hypercontractility and unpaced contractions. During continued exposure to SLO, cardiomyocytes exhibited periods of reversion to normal electrical pacing suggestive of membrane lesion repair and restoration of calcium handling. Together, these observations are consistent with the clinical observation that septic cardiomyopathy is a reversible condition in patients who survive streptococcal toxic shock syndrome. These data provide strong evidence that streptococcal exotoxins, specifically SLO, can directly impact cardiac mechanical function.

Made available in DSpace on 2018-12-11T16:44:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Pollution in aquatic ecosystems due to negative human activities remains a problem in both freshwater and marine environments and is an ongoing subject of research. Several studies have shown that some fish parasites can be used as a tool for biomonitoring because they demonstrate higher metal accumulation capacity compared to their host tissues. However, compared to acanthocephalans, information regarding the absorption mechanisms and accumulation rates in nematodes is relatively limited. Here, we evaluated the potential of larvae Contracaecum sp. (L3) as indicators of contamination by metals by analyzing thirteen element concentrations: magnesium (Mg), aluminum (Al), titanium (Ti), chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), cadmium (Cd), barium (Ba), and lead (Pb) in the parasites and host Acestrorhynchus lacustris, using inductively coupled plasma mass spectrometry. Twelve of the thirteen analyzed elements were detected in at least 2-fold higher concentrations (e.g. Ni) and were up to approximately 50-fold higher (e.g. Pb) in parasites than in host tissues, including elements known for their high toxicity (As, Cd, Pb) and those considered to be essential (Cu and Zn). Our results suggest that Contracaecum sp. larvae can be used as bioindicators of metal contamination because even in early stages of development, numerous essential and non-essential elements were accumulated, making this system a useful tool for monitoring polluted environments. Laboratório de Ictioparasitologia Central de Laboratórios de Ciência e Tecnologia Ambiental Pró-reitoria de Pesquisa e Pós-graduação Universidade do Sagrado Coração (USC), Rua Irmã Arminda, 10-50 Instituto de Biociências de Botucatu Universidade Estadual Paulista “Julio de Mesquita Filho” (UNESP) Instituto de Física Universidade de São Paulo (USP) Instituto de Biociências de Botucatu Universidade Estadual Paulista “Julio de Mesquita Filho” (UNESP) FAPESP: 2012/23655-0

Made available in DSpace on 2018-11-26T17:42:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01. Added 1 bitstream(s) on 2019-10-09T18:27:07Z : No. of bitstreams: 1 S0001-37652017000502317.pdf: 638413 bytes, checksum: 87ca090c193f714c5d961c6554b5e6bd (MD5) The parasites are indicative of several biological aspects of their hosts, including their diet, migration, recruitment and phylogeny, but they can also be direct indicators of the environmental quality. Hoplosternum littorale is a fish that has a high commercial value in some South American countries. The fishes were collected from January to July 2012, in two locations of the Peixe River in the municipality of Anhembi, State of Sao Paulo, Brazil, which had different environmental and limnological characteristics (river mouth and Lagoa Cabeca de Boi). An amount of 30 fish specimens were captured in each location for the analysis of their parasitic fauna. Nine species of metazoan parasites were collected. Both Brillouin's diversity index and Margalef's richness index were greater in the Lagoa Cabeca de Boi. The ratio of heteroxenous and monoxenous parasite species richness (S-H/S-M) was higher in the Lagoa Cabeca de Boi. The results of the physicochemical analysis of the water showed that the mouth of the Peixe River can be considered an area that suffers more from anthropogenic actions than the Lagoa da Cabeca de Boi. Proteocephalus sp., Gyrodactylus sp., Scleroductus sp. and Capillaria (Neocapillaria) pterophylli are being registered for the first time in this host. Univ Estadual Paulista, Inst Biociencias Botucatu, Dept Parasitol, Rua Prof Dr Antonio Celso Wagner Zanin S-N, BR-18618689 Botucatu, SP, Brazil Univ Sagrado Coracao, Cent Ciencia & Tecnol Ambiental, Rua Irma Arminda 1050, BR-17011160 Bauru, SP, Brazil Univ Estadual Paulista, Inst Biociencias Botucatu, Dept Parasitol, Rua Prof Dr Antonio Celso Wagner Zanin S-N, BR-18618689 Botucatu, SP, Brazil

Netrin and its receptor, Frazzled, dictate the strength of synaptic connections in the giant fiber system (GFS) ofDrosophila melanogasterby regulating gap junction localization in the presynaptic terminal. InNetrinmutant animals, the synaptic coupling between a giant interneuron and the “jump” motor neuron was weakened and dye coupling between these two neurons was severely compromised or absent. In cases in whichNetrinmutants displayed apparently normal synaptic anatomy, half of the specimens exhibited physiologically defective synapses and dye coupling between the giant fiber (GF) and the motor neuron was reduced or eliminated, suggesting that gap junctions were disrupted in theNetrinmutants. When we examined the gap junctions with antibodies to Shaking-B (ShakB) Innexin, they were significantly decreased or absent in the presynaptic terminal of the mutant GF. Frazzled loss of function mutants exhibited similar defects in synaptic transmission, dye coupling, and gap junction localization. These data are the first to show that Netrin and Frazzled regulate the placement of gap junctions presynaptically at a synapse.

Cholesterol-dependent cytolysins (CDCs) are a large family of bacterial toxins that exhibit a dependence on the presence of membrane cholesterol in forming large pores in cell membranes. Significant changes in the three-dimensional structure of these toxins are necessary to convert the soluble monomeric protein into a membrane pore. We have determined the crystal structure of the archetypical member of the CDC family, streptolysin O (SLO), a virulence factor from Streptococcus pyogenes. The overall fold is similar to previously reported CDC structures, although the C-terminal domain is in a different orientation with respect to the rest of the molecule. Surprisingly, a signature stretch of CDC sequence called the undecapeptide motif, a key region involved in membrane recognition, adopts a very different structure in SLO to that of the well-characterized CDC perfringolysin O (PFO), although the sequences in this region are identical. An analysis reveals that, in PFO, there are complementary interactions between the motif and the rest of domain 4 that are lost in SLO. Molecular dynamics simulations suggest that the loss of a salt bridge in SLO and a cation-pi interaction are determining factors in the extended conformation of the motif, which in turn appears to result in a greater flexibility of the neighboring L1 loop that houses a cholesterol-sensing motif. These differences may explain the differing abilities of SLO and PFO to efficiently penetrate target cell membranes in the first step of toxin insertion into the membrane.

MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly linked with diseased states, including hepatitis C virus (HCV) infection. Herein, we demonstrate that HCV replication induces the expression of miR-27 in cell culture and in vivo HCV infectious models. Overexpression of the HCV proteins core and NS4B independently activates miR-27 expression. Furthermore, we establish that miR-27 overexpression in hepatocytes results in larger and more abundant lipid droplets, as observed by coherent anti-Stokes Raman scattering (CARS) microscopy. This hepatic lipid droplet accumulation coincides with miR-27b's repression of peroxisome proliferator-activated receptor (PPAR)-α and angiopoietin-like protein 3 (ANGPTL3), known regulators of triglyceride homeostasis. We further demonstrate that treatment with a PPAR-α agonist, bezafibrate, is able to reverse the miR-27b-induced lipid accumulation in Huh7 cells. This miR-27b-mediated repression of PPAR-α signaling represents a novel mechanism of HCV-induced hepatic steatosis. This link was further demonstrated in vivo through the correlation between miR-27b expression levels and hepatic lipid accumulation in HCV-infected SCID-beige/Alb-uPa mice. Conclusion: Collectively, our results highlight HCV's up-regulation of miR-27 expression as a novel mechanism contributing to the development of hepatic steatosis. (Hepatology 2014;58:98–108)

Many viruses including the hepatitis C virus (HCV) induce changes to the infected host cell metabolism that include the up-regulation of lipogenesis to create a favorable environment for the virus to propagate. The enzyme acetyl-CoA carboxylase (ACC) polymerizes to form a supramolecular complex that catalyzes the rate-limiting step of de novo lipogenesis. The small molecule natural product Soraphen A (SorA) acts as a nanomolar inhibitor of acetyl-CoA carboxylase activity through disruption of the formation of long highly active ACC polymers from less active ACC dimers. We have shown that SorA inhibits HCV replication in HCV cell culture models expressing subgenomic and full-length replicons (IC50 = 5 nM) as well as a cell culture adapted virus. Using coherent anti-Stokes Raman scattering (CARS) microscopy, we have shown that SorA lowers the total cellular lipid volume in hepatoma cells, consistent with a reduction in de novo lipogenesis. Furthermore, SorA treatment was found to depolymerize the ACC complexes into less active dimers. Taken together, our results suggest that SorA treatment reverses HCV-induced lipid accumulation and demonstrate that SorA is a valuable probe to study the roles of ACC polymerization and enzymatic activity in viral pathogenesis.

Split-thickness skin grafting (STSG) is the current gold standard for treatment of extensive burn and traumatic skin injuries. However, STSG is limited by donor-site morbidity and availability, and often leads to scarring and wound contracture. Furthermore, these thin grafts lack dermal elements such as nerves and adnexa which are important in recapitulating normal skin function. Methods of fractional skin replacement either as minced STSGs or microscopic skin tissue columns have been proposed, though these techniques have not been fully characterized and lack evidence of regenerated adnexal structures. Here, we describe an alternative method of fractional skin replacement using full-thickness skin micrografts containing deep dermal components and intact adnexa. Full-thickness wounds measuring 3 cm in diameter and 2 cm apart were created on adult female Yorkshire swine. Full-thickness skin tissue columns (FTSTCs) 1.5 mm in diameter with intact adnexa and subcutaneous tissue were obtained using a suction-assisted device. Explant culture was initiated to demonstrate the capacity of FTSTCs to act as reservoirs of viable and proliferative epidermal and dermal cells. FTSTCs were applied directly to excisional wounds at three different expansion ratios (1:16, 1:40, 1:100) in fibrin sealant. Biopsies were collected at defined time points postwounding and processed for histology and immunohistochemistry. Wounds grafted with FTSTCs showed enhanced reepithelialization and epidermal differentiation over untreated control wounds in a dosage dependent manner. Adnexal structures such as hair follicles and sweat glands were only evident in FTSTC-treated wounds. Furthermore, whereas ungrafted wounds were marked by extensive infiltration of α-Smooth Muscle Actin+ (α-SMA+ ) myofibroblasts at POD 60, α-SMA expression was sparse and largely limited to perivascular cells in FTSTC-treated wounds. The number of Ki67+ cells was also greatly reduced in FTSTC-treated wounds. Transplantation of FTSTCs containing intact adnexa improved wound healing parameters in porcine full-thickness wounds and may have implications for the treatment of large, traumatic wounds.

During development, a coordinated and integrated series of events must be accomplished in order to generate functional neural circuits. Axons must navigate toward target cells, build synaptic connections, and terminate outgrowth. The PHR proteins (consisting of mammalian Phr1/MYCBP2, Drosophila Highwire and C. elegans RPM-1) function in each of these events in development. Here, we review PHR function across species, as well as the myriad of signaling pathways PHR proteins regulate. These findings collectively suggest that the PHR proteins are intracellular signaling hubs, a concept we explore in depth. Consistent with prominent developmental functions, genetic links have begun to emerge between PHR signaling networks and neurodevelopmental disorders, such as autism, schizophrenia and intellectual disability. Finally, we discuss the recent and important finding that PHR proteins regulate axon degeneration, which has further heightened interest in this fascinating group of molecules.

Cholesterol-dependent cytolysins (CDCs) are a family of pore-forming toxins that punch holes in the outer membrane of eukaryotic cells. Cholesterol serves as the receptor, but a subclass of CDCs first binds to human CD59. Here we describe the crystal structures of vaginolysin and intermedilysin complexed to CD59. These studies, together with small-angle X-ray scattering, reveal that CD59 binds to each at different, though overlapping, sites, consistent with molecular dynamics simulations and binding studies. The CDC consensus undecapeptide motif, which for the CD59-responsive CDCs has a proline instead of a tryptophan in the motif, adopts a strikingly different conformation between the structures; our data suggest that the proline acts as a selectivity switch to ensure CD59-dependent CDCs bind their protein receptor first in preference to cholesterol. The structural data suggest a detailed model of how these water-soluble toxins assemble as prepores on the cell surface.

To compare rates of preterm birth and pregnancy-related hypertension in women with and without human papillomavirus (HPV) infection.We performed a retrospective cohort study of all women delivered at our institution in 2013 who had cervical cancer screening test results within 3 years before delivery. Patients were excluded if they had prior procedure(s) for cervical dysplasia other than biopsy. There were two primary outcomes: preterm birth (less than 37 weeks of gestation) and pregnancy-related hypertension (gestational hypertension, preeclampsia, or eclampsia). Multivariable logistic regression was performed to adjust for confounders including demographic variables, diabetes, prior preterm birth, chronic hypertension, and other genital infections. Assuming a 10% prevalence of HPV, a rate of 12% in the HPV-negative group for both preterm birth and pregnancy-related hypertension, α of 0.05, and β of 0.2, we needed 2,207 patients to detect a 60% increase in the rate of either outcome in the HPV-positive group.A total of 3,958 patients delivered in 2013, of whom 2,321 met eligibility criteria, 242 (10.4%) of whom were HPV-positive and 2,079 (89.2%) of whom were HPV-negative. In multivariate analyses, the rate of preterm birth was not significantly different between HPV-positive and HPV-negative women (16.5% compared with 12.2%, adjusted odds ratio [OR] 1.3, 95% confidence interval [CI] 0.9-1.9); rates of pregnancy-related hypertension also were not significantly different between HPV-positive and HPV-negative women (17.0% compared with 16.4%, adjusted OR 1.0, 95% CI 0.7-1.5).Maternal HPV infection is not an independent risk factor for preterm birth or pregnancy-related hypertension.

The ATP-binding cassette (ABC) transporter multidrug resistance protein 1 (MRP1/ ABCC1 ) is responsible for the cellular export of a chemically diverse array of xenobiotics and endogenous compounds. Arsenic, a human carcinogen, is a high-affinity MRP1 substrate as arsenic triglutathione [As(GS)3]. In this study, marked differences in As(GS)3 transport kinetics were observed between MRP1-enriched membrane vesicles prepared from human embryonic kidney 293 (HEK) ( Km 3.8 µ M and Vmax 307 pmol/mg per minute) and HeLa ( Km 0.32 µ M and Vmax 42 pmol/mg per minute) cells. Mutant MRP1 lacking N -linked glycosylation [Asn19/23/1006Gln; sugar-free (SF)-MRP1] expressed in either HEK293 or HeLa cells had low Km and Vmax values for As(GS)3, similar to HeLa wild-type (WT) MRP1. When prepared in the presence of phosphatase inhibitors, both WT- and SF-MRP1-enriched membrane vesicles had a high Km value for As(GS)3 (3–6 µ M), regardless of the cell line. Kinetic parameters of As(GS)3 for HEK-Asn19/23Gln-MRP1 were similar to those of HeLa/HEK-SF-MRP1 and HeLa-WT-MRP1, whereas those of single glycosylation mutants were like those of HEK-WT-MRP1. Mutation of 19 potential MRP1 phosphorylation sites revealed that HEK-Tyr920Phe/Ser921Ala-MRP1 transported As(GS)3 like HeLa-WT-MRP1, whereas individual HEK-Tyr920Phe- and -Ser921Ala-MRP1 mutants were similar to HEK-WT-MRP1. Together, these results suggest that Asn19/Asn23 glycosylation and Tyr920/Ser921 phosphorylation are responsible for altering the kinetics of MRP1-mediated As(GS)3 transport. The kinetics of As(GS)3 transport by HEK-Asn19/23Gln/Tyr920Glu/Ser921Glu were similar to HEK-WT-MRP1, indicating that the phosphorylation-mimicking substitutions abrogated the influence of Asn19/23Gln glycosylation. Overall, these data suggest that cross-talk between MRP1 glycosylation and phosphorylation occurs and that phosphorylation of Tyr920 and Ser921 can switch MRP1 to a lower-affinity, higher-capacity As(GS)3 transporter, allowing arsenic detoxification over a broad concentration range.

The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that are produced, secreted and contribute to the pathogenesis of many species of Gram-positive bacteria. The assembly of the CDC pore-forming complex has been under intense study for the past 20years. These studies have revealed a molecular mechanism of pore formation that exhibits many novel features. The CDCs form large β-barrel pore complexes that are assembled from 35 to 40 soluble CDC monomers. Pore formation is dependent on the presence of membrane cholesterol, which functions as the receptor for most CDCs. Cholesterol binding initiates significant secondary and tertiary structural changes in the monomers, which lead to the assembly of a large membrane embedded β-barrel pore complex. This review will focus on the molecular mechanism of assembly of the CDC membrane pore complex and how these studies have led to insights into the mechanism of pore formation for other pore-forming proteins. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Sixty specimens of singing catfish Trachelyopterus striatulus (Steindachner, 1877) (Siluriformes: Auchenipteridae) collected from Guandu River (22º 48' 32" S and 43º 37' 35" W), in the state of Rio de Janeiro, Brazil from October 2006 to March 2009, were necropsied to study their parasites. From the 60 specimens of T. striatulus examined 57 were parasitised by at least one parasite species. The majority of the parasite specimens collected were monogeneans followed by Nematoda, Digenea and Hirudinea. Cosmetocleithrum sp. was the numerically predominant species with highest prevalence and abundance. The parasites of T. striatulus showed the typical pattern of aggregated distribution. No parasite species showed significant correlation between the body total length of the host and their abundance. The mean parasite species richness was not correlated with the host's total body length and sex. Values of the Brillouin index of diversity had a mean of H = 0.083 ± 0.136.

CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.

The recognition and binding of cholesterol is an important feature of many eukaryotic, viral, and prokaryotic proteins, but the molecular details of such interactions are understood only for a few proteins. The pore-forming cholesterol-dependent cytolysins (CDCs) contribute to the pathogenic mechanisms of a large number of Gram-positive bacteria. Cholesterol dependence of the CDC mechanism is a hallmark of these toxins, yet the identity of the CDC cholesterol recognition motif has remained elusive. A detailed analysis of membrane interactive structures at the tip of perfringolysin O (PFO) domain 4 reveals that a threonine-leucine pair mediates CDC recognition of and binding to membrane cholesterol. This motif is conserved in all known CDCs and conservative changes in its sequence or order are not well tolerated. Thus, the Thr-Leu pair constitutes a common structural basis for mediating CDC-cholesterol recognition and binding, and defines a unique paradigm for membrane cholesterol recognition by surface-binding proteins.

Here we have simultaneously characterized the influence of inhibitors of peroxisome proliferator-activated receptor alpha (PPARalpha) and the mevalonate pathway on hepatocyte lipid metabolism and the subcellular localization of hepatitis C virus (HCV) RNA using two-photon fluorescence (TPF) and coherent anti-Stokes Raman scattering (CARS) microscopy. Using this approach, we demonstrate that modulators of PPARalpha signaling rapidly cause the dispersion of HCV RNA from replication sites and simultaneously induce lipid storage and increases in lipid droplet size. We demonstrate that reductions in the levels of cholesterol resulting from inhibition of the mevalonate pathway upregulates triglyceride levels. We also show that the rate of dispersion of HCV RNA is very rapid when using a PPARalpha antagonist. This occurs with a faster rate to that of direct inhibition of 3-hydroxy-3-methyglutaryl CoA reductase (HMG-CoA reductase) using lovastatin in living cells, demonstrating the potential therapeutic value of modulating host cell pathways as part of a strategy to eliminate chronic HCV infection.

Intermedilysin (ILY) is an unusual member of the family of cholesterol-dependent cytolysins because it binds to human CD59 (hCD59) rather than directly to cholesterol-rich membranes. Binding of ILY to hCD59 initiates a series of conformational changes within the toxin that result in the conversion of the soluble monomer into an oligomeric membrane-embedded pore complex. In this study the association of ILY with its membrane receptor has been examined throughout the assembly and formation of the pore complex. Using ILY mutants trapped at various stages of pore assembly, we show ILY remains engaged with hCD59 throughout the assembly of the prepore oligomer, but it disengages from the receptor upon the conversion to the pore complex. We further show that the assembly intermediates increase the sensitivity of the host cell to lysis by its complement membrane attack complex, apparently by blocking the hCD59-binding site for complement proteins C8α and C9.

Clostridium septicum alpha-toxin is a β-barrel pore-forming cytolysin that is functionally similar to aerolysin. Residues important in receptor binding, oligomerization, and pore formation have been identified; however, little is known about the activity of the toxin in an infection, although it is essential for disease. We have now shown that deletion of a small portion of the transmembrane domain, so that the toxin is no longer able to form pores, completely abrogates its ability to contribute to disease, as does replacement of the sole cysteine residue with leucine. However, although previous biochemical and cytotoxicity assays clearly indicated that mutations in residues important in oligomerization, binding, and prepore conversion greatly reduced activity or rendered the toxin inactive, once the mutated toxins were overexpressed by the natural host in the context of an infection it was found they were able to cause disease in a mouse model of myonecrosis. These results highlight the importance of testing the activity of virulence determinants in the normal host background and in an infectious disease context and provide unequivocal evidence that it is the ability of alpha-toxin to form a pore that confers its toxicity in vivo.

The signaling mechanisms that allow the conversion of a growth cone into a mature and stable synapse are yet to be completely understood. Ubiquitination plays key regulatory roles in synaptic development and may be involved in this process. Previous studies identified theDrosophilaubiquitin conjugasebendless(ben) to be important for central synapse formation, but the precise role it plays has not been elucidated. Our studies indicate that Ben plays a pivotal role in synaptic growth and maturation. We have determined that an incipient synapse is present with a high penetrance inbenmutants, suggesting that Ben is required for a developmental step after target recognition. We used cell-autonomous rescue experiments to show that Ben has a presynaptic role in synapse growth. We then harnessed the TARGET system to transiently express UAS (upstream activating sequence)–benin abenmutant background and identified a well defined critical period for Ben function in establishing a full-grown, mature synaptic terminal. We demonstrate that the protein must be present at a time point before but not during the actual growth process. We also provide phenotypic evidence demonstrating that Ben is not a part of the signal transduction pathway involving the well characterized ubiquitin ligasehighwire. We conclude that Bendless functions as a novel developmental switch that permits the transition from axonal growth and incipient synapse formation to synaptic growth and maturation in the CNS.

The viridans streptococci, which include Streptococcus mitis, Streptococcus mutans, Streptococcus salivarius, and Streptococcus sanguis, are normally found as members of the normal flora of the oropharyx. These organisms can escape this niche, however, and cause a variety of diseases that include infective endocarditis, bacteremia, and septicemia. S. mitis is one of the more common isolates, and its antibiotic resistance is increasing, making treatment of its infections difficult (1–4). S. mitis was recently associated with a large outbreak of a toxic shock-like syndrome that affected thousands of individuals in China (5). S. mitis is typically not considered to be toxigenic, and it is characterized by an α-hemolytic phenotype. Jeffries et al. (6), however, recently showed that some S. mitis strains express a cholesterol-dependent cytolysin (CDC), designated mitilysin (MLY), which is nearly identical to Streptococcus pneumoniae pneumolysin. Another potential S. mitis pathogenesis factor is human platelet aggregation factor (Sm-hPAF)1 that was purified from S. mitis isolated from a patient with Kawasaki disease (7). This protein was shown to aggregate human platelets and was designated S. mitis human platelet aggregation factor or Sm-hPAF. More recently, Ohkuni et al. (8) showed that antibody titers to a Sm-hPAF-derived peptide were significantly elevated in children with Kawasaki disease, a disease that is often associated with platelet aggregation and coronary artery thrombosis. The peptide sequence reported by Ohkuni et al. (8) was derived from the sequence for a Sm-hPAF gene from S. mitis Nm-65 recently reported in GenBank (accession number {"type":"entrez-nucleotide","attrs":{"text":"AB051299.1","term_id":"84579713","term_text":"AB051299.1"}}AB051299.1). The sequence of Sm-hPAF encodes a predicted CDC structure that is most closely related to inter-medilysin (ILY), the CDC from Streptococcus intermedius (9). The DNA-derived primary structure of Sm-hPAF exhibits an extra amino-terminal domain of about 162 amino acids. Based on the DNA-derived primary structure, this domain is related to the European eel Anguilla anguilla agglutinin (AAA), a fucose-binding lectin that preferentially binds glycans containing fucosylated structures found in the blood group antigens A, B, H, and Lewis a (Lea) (10, 11). Herein we describe the properties of purified recombinant Sm-hPAF (referred to as lectinolysin or LLY hereafter) from S. mitis SK597. These studies show that LLY is a pore-forming CDC with a functional glycan-binding domain. LLY does not trigger platelet aggregation but does form pores in platelet membranes. The isolated fucose-binding lectin domain of LLY exhibits specificity for glycans containing the Lewis y (Ley) and b (Leb) antigens. The lectin domain of LLY appears to modulate the pore-forming activity of LLY in a glycan-dependent manner after LLY binds to the cell surface.

Estuaries play an important role as nurseries and migration corridors for Chinook salmon and other fishes. The invasive New Zealand mudsnail, Potamopyrgus antipodarum (Gray, 1843), has been noted in the Columbia River Estuary and other estuaries in the western USA, yet no studies have addressed the estuarine impacts of this invader. Our data show P. antipodarum is currently found in five peripheral bays and many tributaries of the Columbia River Estuary, where it can constitute a major portion of the benthic invertebrate biomass and where it co-occurs with native amphipod species. We review the history of the P. antipodarum invasion in the Columbia River Estuary and discuss potential impacts on estuarine food webs. We also report the first occurrence of P. antipodarum in the diet of juvenile Chinook salmon from the Columbia River Estuary. Although present in Chinook diets at very low frequencies, our observations of P. antipodarum in Chinook gut contents may represent early stages of food web change due to the establishment of dense estuarine snail populations. Additional research is needed to determine the effects of P. antipodarum on benthic resources, native benthic invertebrates, and benthic predators. We encourage biologists working in western USA estuaries to be alert to the possibility of encountering P. antipodarum in benthic habitats and predator diets.

Conditional targeted cell ablation is a powerful approach for investigating the pathogenesis of human diseases and in vivo cellular functions. Intermedilysin (ILY) is a cytolytic pore-forming toxin secreted by Streptococcus intermedius that lyses human cells exclusively, owing to its receptor specificity for human CD59. We generated two transgenic mouse strains that express human CD59 either on erythrocytes (strain ThCD59(RBC)) or on endothelia (strain ThCD59(END)). Intravenous injection of ILY in ThCD59(RBC) mice induced acute intravascular hemolysis, leading to reduced nitric oxide bioavailability, increased platelet activation and rapid death. In ThCD59(END) mice, ILY induced rapid endothelial damage, leading to acute death and disseminated intravascular coagulation. Additionally, we show that human serum contains ILY-specific neutralizing antibodies not found in any other animal species. Together, these results suggest that this new rapid conditional targeted ILY-mediated cell ablation technique can be used in combination with any available transgenic expression system to study the physiologic role of specific cell populations.

The pore-forming mechanism of the cholesterol-dependent cytolysins (CDCs) exhibits an absolute requirement for membrane cholesterol. The structural elements of the CDCs that mediate this interaction are not well understood. Three short hydrophobic loops (L1–L3) and a highly conserved undecapeptide sequence at the tip of domain 4 of the CDC structure are known to anchor the CDC to the membrane. It has been thought that the undecapeptide directly mediates the interaction of the CDCs with a cholesterol-rich cell surface. Herein we show that the L1–L3 loops, not the undecapeptide, are responsible for mediating the specific interaction of the CDCs with cholesterol-rich membranes. The membrane insertion of the undecapeptide was uncoupled from membrane binding by the covalent modification of the undecapeptide cysteine thiol. Modification of the cysteine prevented prepore to pore conversion, but did not affect membrane binding, thus demonstrating that undecapeptide membrane insertion follows that of the L1–L3 loops. These studies provide an example of a structural motif that specifically mediates the interaction of a bacterial toxin with a cholesterol-rich membrane.

The largest central synapse in adult Drosophila is a mixed electro-chemical synapse whose gap junctions require the product of the shaking-B (shak-B) gene. Shak-B2 mutant flies lack gap junctions at this synapse, which is between the giant fibre (GF) and the tergotrochanteral motor neuron (TTMn), but it still exhibits a long latency response upon GF stimulation. We have targeted the expression of the light chain of tetanus toxin to the GF, to block chemical transmission, in shak-B2 flies. The long latency response in the tergotrochanteral muscle (TTM) was abolished indicating that the chemical component of the synapse mediates this response. Attenuation of GAL4-mediated labelling by a cha-GAL80 transgene, reveals the GF to be cholinergic. We have used a temperature-sensitive allele of the choline acetyltransferase gene (chats2) to block cholinergic synapses in adult flies and this also abolished the long latency response in shak-B2 flies. Taken together the data provide evidence that both components of this mixed synapse are functional and that the chemical neurotransmitter between the GF and the TTMn is acetylcholine. Our findings show that the two components of this synapse can be separated to allow further studies into the mechanisms by which mixed synapses are built and function.

Three short hydrophobic loops and a conserved undecapeptide at the tip of domain 4 (D4) of the cholesterol-dependent cytolysins (CDCs) mediate the binding of the CDC monomers to cholesterol-rich cell membranes. But intermedilysin (ILY), from Streptococcus intermedius, does not bind to cholesterol-rich membranes unless they contain the human protein CD59. This observation suggested that the D4 loops, which include loops L1–L3 and the undecapeptide, of ILY were no longer required for its cell binding. However, we show here that membrane insertion of the D4 loops is required for the cytolysis by ILY. Receptor binding triggers changes in the structure of ILY that are necessary for oligomerization, but membrane insertion of the D4 loops is critical for oligomer assembly and pore formation. Defects that prevent membrane insertion of the undecapeptide also block assembly of the prepore oligomer, while defects in the membrane insertion of the L1–L3 loops prevent the conversion of the prepore oligomer to the pore complex. These studies reveal that pore formation by ILY, and probably other CDCs, is affected by an intricate and coupled sequence of interactions between domain 4 and the membrane.

Immune regulation of cellular metabolism can be responsible for successful responses to invading pathogens. Viruses alter their hosts' cellular metabolism to facilitate infection. Conversely, the innate antiviral responses of mammalian cells target these metabolic pathways to restrict viral propagation. We identified miR-130b and miR-185 as hepatic microRNAs (miRNAs) whose expression is stimulated by 25-hydroxycholesterol (25-HC), an antiviral oxysterol secreted by interferon-stimulated macrophages and dendritic cells, during hepatitis C virus (HCV) infection. However, 25-HC only directly stimulated miR-185 expression, whereas HCV regulated miR-130b expression. Independently, miR-130b and miR-185 inhibited HCV infection. In particular, miR-185 significantly restricted host metabolic pathways crucial to the HCV life cycle. Interestingly, HCV infection decreased miR-185 and miR-130b levels to promote lipid accumulation and counteract 25-HC's antiviral effect. Furthermore, miR-185 can inhibit other viruses through the regulation of immunometabolic pathways. These data establish these microRNAs as a key link between innate defenses and metabolism in the liver.