A universal method for analyzing copolymer growth.

Polymers consisting of more than one type of monomer, known as copolymers, are vital to both living and synthetic systems. Copolymerization has been studied theoretically in a number of contexts, often by considering a Markov process in which monomers are added or removed from the growing tip of a long copolymer. To date, the analysis of the most general models of this class has necessitated simulation. We present a general method for analyzing such processes without resorting to simulation. Our method can be applied to models with an arbitrary network of sub-steps prior to addition or removal of a monomer, including non-equilibrium kinetic proofreading cycles. Moreover, the approach allows for a dependency of addition and removal reactions on the neighboring site in the copolymer and thermodynamically self-consistent models in which all steps are assumed to be microscopically reversible. Using our approach, thermodynamic quantities such as chemical work; kinetic quantities such as time taken to grow; and statistical quantities such as the distribution of monomer types in the growing copolymer can be directly derived either analytically or numerically from the model definition.

Giardia lamblia G6PD::6PGL Fused Protein Inhibitors Decrease Trophozoite Viability: A New Alternative against Giardiasis.

Treatments to combat giardiasis have been reported to have several drawbacks, partly due to the drug resistance and toxicity of current antiparasitic agents. These constraints have prompted many researchers to investigate new drugs that act against protozoan parasites. Enzyme inhibition is an important means of regulating pathogen metabolism and has recently been identified as a significant alternative target in the search for new treatments. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase (G6PD::6PGL) is a bifunctional enzyme involved in the pentose phosphate pathway (PPP) in Giardia lamblia (G. lamblia). The G. lamblia enzyme is unusual since, unlike the human enzyme, it is a fused enzyme. Here, we show, through inhibition assays, that an in-house chemical library of 120 compounds and four target compounds, named CNZ-7, CNZ-8, CMC-1, and FLP-2, are potent inhibitors of the G. lamblia G6PD::6PGL fused enzyme. With a constant (k2) of 2.3, 3.2, and 2.8 M−1 s−1, respectively, they provoke alterations in the secondary and tertiary protein structure and global stability. As a novel approach, target compounds show antigiardial activity, with IC50 values of 8.7, 15.2, 15.3, and 24.1 µM in trophozoites from G. lamblia. Moreover, these compounds show selectivity against G. lamblia, since, through counter-screening in Caco-2 and HT29 human cells, they were found to have low toxicity. This finding positions these compounds as a potential and attractive starting point for new antigiardial drugs.

Water mite assemblages reveal diverse genera, novel DNA barcodes and transitional periods of intermediate disturbance.

Water mites are important constituents of aquatic ecosystems, but their biodiversity is poorly understood. The goal of this study was to improve knowledge of water mite assemblages in the Detroit River through combined use of morphological and cytochrome oxidase I (COI) DNA barcode data and to elucidate seasonal water mite diversity. The diversity of water mites collected from Blue Heron Lagoon at Belle Isle, an island in the Detroit River, is described. Novel DNA barcodes for Albia, Hydrochoreutes, Madawaska, and Axonopsis are reported with a species level barcode for Lebertia. Novel DNA barcodes may represent the presence of previously undescribed variants or new species of several genera. The prevalence of water mites is higher in the summer, but a different pattern is observed for diversity. The diversity of water mites, by several measures, varies seasonally with lower diversity in summer and winter months and higher diversity during seasonal transitions. For these organisms, we interpret seasonal change as an intermediate disturbance resulting in increased biodiversity.

Nutritional Correlates of Human Oral Microbiome.

BACKGROUND: Despite many potential effects of the oral microbiome on oral and systemic health, scant information is available regarding the associations between diet and the oral microbiome. METHODS: Oral rinse DNA samples from 182 participants in a population-based case-control study for colorectal cancer were used to amplify a V3-V4 region of bacterial 16S rRNA gene. The amplicons were sequenced using Illumina MiSeq paired end chemistry on 2 runs, yielding approximately 33 million filtered reads that were assigned to bacterial classes. Relative abundances of each class and family as well microbial diversity/richness indices were correlated with selected dietary intakes from a food frequency questionnaire. RESULTS: Saturated fatty acids (SFAs) and vitamin C intakes were consistently correlated with alpha (within-subjects) diversity indexes in both richness and diversity. SFA intake was positively correlated with relative abundance of betaproteobacteria and fusobacteria. Vitamin C and other vitamins with correlated intakes-for example, the B vitamins and vitamin E-exhibited positive correlations with fusobacteria class, its family Leptotrichiaceae and a clostridia family Lachnospiraceae. In addition, glycemic load was positively correlated with Lactobacillaceae abundance. CONCLUSION: The observed associations in this study were modest. However, the results suggest that the effects of diets are likely to be habitat specific, and observations from the gut microbiome are not transferrable to the oral microbiome. Further studies are warranted, incorporating a range of host biomarkers, such as cytohistological, molecular, or biochemical measurements, in order to address biological consequences of these dietary intakes in human oral health.

Eurytemora carolleeae in the Laurentian Great Lakes revealed by phylogenetic and morphological analysis.

In the Laurentian Great Lakes, specimens of Eurytemora have been reported as E. affinis since its invasion in the late 1950s. During an intensive collection of aquatic invertebrates for morphological and molecular identification in Western Lake Erie in 2012-2013, several specimens of Eurytemora were collected. Analysis of these specimens identified them as the recently described species E. carolleeae Alekseev and Souissi 2011. This result led us to assess E. carolleeae's identifying features, geographic distribution and historical presence in the Laurentian Great Lakes in view of its recent description in 2011. Cytochrome oxidase I (COI) DNA sequences of Eurytemora specimens were identified as closer (2 - 4% different) to recently described E. carolleeae than to most Eurytemora affinis sequences (14% different). Eurytemora from other areas of the Great Lakes and from North American rivers as far west as South Dakota (Missouri River) and east to Delaware (Christina River) also keyed to E. carolleeae. Morphological analysis of archival specimens from 1962 and from all the Great Lakes were identified as E. carolleeae. Additionally, Eurytemora drawings in previous publications from studies in the Holarctic region were reassessed to determine if these specimens were E. carolleeae. Additional morphological characters that may distinguish the North American E. carolleeae from other taxa are also described. We conclude that E. carolleeae is the correct name for the species of Eurytemora that has inhabited the Great Lakes since its invasion, as established by both morphological and COI sequence comparisons to reference keys and sequence databases in present and archival specimens.

New species and a fascinating diversity of Chironomidae (Diptera, Insecta) in and around an overlooked urban vernal pool.

In this study, the biodiversity of Chironomidae was investigated in Palmer Park Pond A, an urban vernal pond in Detroit, Michigan, USA. This study is developed as part of our ongoing Public Environmental Outreach Program at the Detroit Exploration and Nature Center in Palmer Park. Twenty-one Chironomidae species were discovered in and on the adjacent riparian vegetation of this pond using molecular and morphological methods. Three species Bryophaenocladiuspalmerparcum Namayandeh & Hudson sp. nov., Limnophyesstagnum Namayandeh, Guerra & Ram sp. nov., and Rheocricotopus (s. s.) angustus Namayandeh & Hudson sp. nov. are new to science. Bryophaenocladiuspalmerparcum sp. nov. and L.stagnum sp. nov. are unusual Orthoclads, with B.palmerparcum sp. nov. possessing a setose, short, and wide anal point and L.stagnum sp. nov. lacking lanceolate setae on both sexes. Based on the shape of superior volsella, R.angustus sp. nov., belongs to the effusus group, which was also confirmed by DNA barcoding molecular analysis. In this study, a new faunistic record was also found for the Nearctic as well as four new faunistic records for the state of Michigan. Ephemeral aquatic habitats such as vernal pools are often overlooked or destroyed by urbanization activities, controlling vector species, creating groomed fields, and/or residential development. Therefore, finding these new species demonstrates the biodiversity value of vernal ponds as important habitats, further motivating us to preserve them.

Structural, biochemical and immunochemical characterization of an acidic phospholipase A2 from Lachesis acrochorda (Viperidae: Crotalinae) venom.

Viperids of the genus Lachesis, also known as bushmasters, are capable of injecting great amounts of venom that cause severe envenomation incidents. Since phospholipases type A2 are mainly involved in edema and myonecrosis within the snakebite sites, in this work, the isolation, amino acid sequence and biochemical characterization of the first phospholipase type A2 from the venom of Lachesis acrochorda, named Lacro_PLA2, is described. Lacro_PLA2 is an acidic aspartic 49 calcium-dependent phospholipase A2 with 93% similarity to the L. stenophrys phospholipase. Lacro_PLA2 has a molecular mass of 13,969.7 Da and an experimental isoelectric point around 5.3. A combination of N-terminal Edman degradation and MS/MS spectrometry analyses revealed that Lacro_PLA2 contains 122 residues including 14 cysteines that form 7 disulfide bridges. A predicted 3D model shows a high resemblance to other viperid phospholipases. Nevertheless, immunochemical and phospholipase neutralization tests revealed a notorious level of immunorecognition of the isolated protein by two polyclonal antibodies from viperids from different genus, which suggest that Lacro_PLA2 resembles more to bothropic phospholipases. Lacro_PLA2 also showed significantly high edema activity when was injected into mice; so, it could be an alternative antigen in the development of antibodies against toxins of this group of viperids, seeking to improve commercial polyclonal antivenoms.

Immunological Cross-Reactivity and Preclinical Assessment of a Colombian Anticoral Antivenom against the Venoms of Three Micrurus Species.

Snakebite accident treatment requires the administration of antivenoms that provide efficacy and effectiveness against several snake venoms of the same genus or family. The low number of immunogenic components in venom mixtures that allow the production of antivenoms consequently gives them partial neutralization and a suboptimal pharmacological response. This study evaluates the immunorecognition and neutralizing efficacy of the polyvalent anticoral antivenom from the Instituto Nacional de Salud (INS) of Colombia against the heterologous endemic venoms of Micrurus medemi, and M. sangilensis, and M. helleri by assessing immunoreactivity through affinity chromatography, ELISA, Western blot, and neutralization capability. Immunorecognition towards the venoms of M. medemi and M. sangilensis showed values of 62% and 68% of the protein composition according to the immunoaffinity matrix, respectively. The analysis by Western blot depicted the highest recognition patterns for M. medemi, followed by M. sangilensis, and finally by M. helleri. These findings suggest that the venom compositions are closely related and exhibit similar recognition by the antivenom. According to enzyme immunoassays, M. helleri requires a higher amount of antivenom to achieve recognition than the others. Besides reinforcing the evaluation of INS antivenom capability, this work recommends the use of M. helleri in the production of Colombian antisera.

Alaskacladius gen. nov., (Diptera: Chironomidae), a unique new orthoclad from Alaska.

We describe a new genus Alaskacladius gen. nov., based on the adult stages collected from Alaska, USA, and British Columbia, Canada. Molecular and morphological assessment of adult specimens supports the presence of a new genus. Alaskacladius gen. nov., is related to the genera Doithrix Sther & Sublette, 1983; Georthocladius Strenzke, 1941; Parachaetocladius Wlker, 1959; and Pseudorthocladius Goetghebuer, 1932. Based on the molecular analysis result and intergeneric K2P distance obtained from Cytochrome Oxidase I (COI) genes, Alaskacladius is closest and forms a sister group with Doithrix.

Wastewater Monitoring for Infectious Disease: Intentional Relationships between Academia, the Private Sector, and Local Health Departments for Public Health Preparedness.

The public health emergency caused by the COVID-19 pandemic stimulated stakeholders from diverse disciplines and institutions to establish new collaborations to produce informed public health responses to the disease. Wastewater-based epidemiology for COVID-19 grew quickly during the pandemic and required the rapid implementation of such collaborations. The objective of this article is to describe the challenges and results of new relationships developed in Detroit, MI, USA among a medical school and an engineering college at an academic institution (Wayne State University), the local health department (Detroit Health Department), and an environmental services company (LimnoTech) to utilize markers of the COVID-19 virus, SARS-CoV-2, in wastewater for the goal of managing COVID-19 outbreaks. Our collaborative team resolved questions related to sewershed selection, communication of results, and public health responses and addressed technical challenges that included ground-truthing the sewer maps, overcoming supply chain issues, improving the speed and sensitivity of measurements, and training new personnel to deal with a new disease under pandemic conditions. Recognition of our complementary roles and clear communication among the partners enabled city-wide wastewater data to inform public health responses within a few months of the availability of funding in 2020, and to make improvements in sensitivity and understanding to be made as the pandemic progressed and evolved. As a result, the outbreaks of COVID-19 in Detroit in fall and winter 2021-2022 (corresponding to Delta and Omicron variant outbreaks) were tracked in 20 sewersheds. Data comparing community- and hospital-associated sewersheds indicate a one- to two-week advance warning in the community of subsequent peaks in viral markers in hospital sewersheds. The new institutional relationships impelled by the pandemic provide a good basis for continuing collaborations to utilize wastewater-based human and pathogen data for improving the public health in the future.

Passive swab versus grab sampling for detection of SARS-CoV-2 markers in wastewater.

Early detection of the COVID-19 virus, SARS-CoV-2, is key to mitigating the spread of new outbreaks. Data from individual testing is increasingly difficult to obtain as people conduct non-reported home tests, defer tests due to logistics or attitudes, or ignore testing altogether. Wastewater based epidemiology is an alternative method for surveilling a community while maintaining individual anonymity; however, a problem is that SARS-CoV-2 markers in wastewater vary throughout the day. Collecting grab samples at a single time may miss marker presence, while autosampling throughout a day is technically challenging and expensive. This study investigates a passive sampling method that would be expected to accumulate greater amounts of viral material from sewers over a period of time. Tampons were tested as passive swab sampling devices from which viral markers could be eluted with a Tween-20 surfactant wash. Six sewersheds in Detroit were sampled 16-22 times by paired swab (4 h immersion before retrieval) and grab methods over a five-month period and enumerated for N1 and N2 SARS-CoV-2 markers using ddPCR. Swabs detected SARS-CoV-2 markers significantly more frequently (P < 0.001) than grab samples, averaging two to three-fold more copies of SARS-CoV-2 markers than their paired grab samples (p < 0.0001) in the assayed volume (10 mL) of wastewater or swab eluate. No significant difference was observed in the recovery of a spiked-in control (Phi6), indicating that the improved sensitivity is not due to improvements in nucleic acid recovery or reduction of PCR inhibition. The outcomes of swab-based sampling varied significantly between sites, with swab samples providing the greatest improvements in counts for smaller sewersheds that otherwise tend to have greater variation in grab sample counts. Swab-sampling with tampons provides significant advantages in detection of SARS-CoV-2 wastewater markers and are expected to provide earlier detection of new outbreaks than grab samples, with consequent public health benefits.

Phenolic Compounds from Wild Plant and In Vitro Cultures of Ageratina pichichensis and Evaluation of Their Antioxidant Activity.

Ageratina pichichensis, is commonly used in traditional Mexican medicine. In vitro cultures were established from wild plant (WP) seeds, obtaining in vitro plant (IP), callus culture (CC), and cell suspension culture (CSC) with the objective to determine total phenol content (TPC) and flavonoids (TFC), as well as their antioxidant activity by DPPH, ABTS and TBARS assays, added to the compound's identification and quantification by HPLC, from methanol extracts obtained by sonication. CC showed significantly higher TPC and TFC than WP and IP, while CSC produced 2.0-2.7 times more TFC than WP, and IP produced only 14.16% TPC and 38.8% TFC compared with WP. There were identified compounds such as epicatechin (EPI), caffeic acid (CfA), and p-coumaric acid (pCA) in in vitro cultures that were not found in WP. The quantitative analysis shows gallic acid (GA) as the least abundant compound in samples, whereas CSC produced significantly more EPI and CfA than CC. Despite these results, in vitro cultures show lower antioxidant activity than WP, for DPPH and TBARS WP > CSC > CC > IP and ABTS WP > CSC = CC > IP. Overall, A. pichichensis WP and in vitro cultures produce phenolic compounds with antioxidant activity, especially CC and CSC, which are shown to be a biotechnological alternative for obtaining bioactive compounds.

Unveiling the Venom Composition of the Colombian Coral Snakes Micrurus helleri, M. medemi, and M. sangilensis.

Little is known of the biochemical composition and functional features of the venoms of poorly known Colombian coral snakes. Here, we provide a preliminary characterization of the venom of two Colombian endemic coral snake species, Micrurus medemi and M. sangilensis, as well as Colombian populations of M. helleri. Electrophoresis and RP-HPLC techniques were used to identify venom components, and assays were conducted to detect enzyme activities, including phospholipase A2, hyaluronidase, and protease activities. The median lethal dose was determined using murine models. Cytotoxic activities in primary cultures from hippocampal neurons and cancer cell lines were evaluated. The venom profiles revealed similarities in electrophoretic separation among proteins under 20 kDa. The differences in chromatographic profiles were significant, mainly between the fractions containing medium-/large-sized and hydrophobic proteins; this was corroborated by a proteomic analysis which showed the expected composition of neurotoxins from the PLA2 (~38%) and 3FTx (~17%) families; however, a considerable quantity of metalloproteinases (~12%) was detected. PLA2 activity and protease activity were higher in M. helleri venom according to qualitative and quantitative assays. M. medemi venom had the highest lethality. All venoms decreased cell viability when tested on tumoral cell cultures, and M. helleri venom had the highest activity in neuronal primary culture. These preliminary studies shed light on the venoms of understudied coral snakes and broaden the range of sources that could be used for subsequent investigations of components with applications to specific diseases. Our findings also have implications for the clinical manifestations of snake envenoming and improvements in its medical management.

Chemical characterization of yellow-orange and purple varieties of Opuntia ficus-indica fruits and thermal stability of their betalains.

Betalains are plant pigments with biological properties and can be used instead of synthetic colorants to confer color and functional properties to foods. The objective of this work was to carry out the chemical characterization of two varieties of prickly pear of Opuntia ficus-indica, one of yellow-orange coloration (Mandarina) and the other of purple coloration (Vigor), through measurements of chemical parameters and color in pulp, antioxidant activity, total phenolic compounds, and betalain content. Considering the thermolability of betalains and their potential applications in food, the thermal stability and activation energy of betacyanins from Vigor variety and betaxanthins from the Mandarina variety were also evaluated and compared with those from beetroot, the main source of betalains. Results for chemical characterization agreed with previous prickly pear reports of other regions, while the thermal degradation kinetics of betalains showed a first-order degradation pattern with respect to time and temperature treatment. Betacyanins from Vigor prickly pear showed similar thermal stability to those from beetroot, which was reflected in similar values of activation energy, while betaxanthins from Mandarina prickly pear showed a higher stability, and therefore a higher activation energy, than those from beetroot. Based on the results, the prickly pear varieties used in this study can be considered as a good source of betalains with potential applications in food and, in addition, the methodology for the evaluation of thermostability can be used to compare the stability of betalains from different sources in a temperature range of 50-90°C. PRACTICAL APPLICATION: The varieties of prickly pear used in this study can be considered a good source of red-purple and yellow-orange easily extractable pigments. In addition, we report a methodology that can be used for the evaluation of the thermal stability of these pigments and to compare this stability between different plant sources. Gaining knowledge on betalain thermal stability will make it possible to propose specific applications, for example, in processed foods requiring different pigment stabilities.

Variation in the diversity of bacterial communities and potential sources of fecal contamination of beaches in the Huron to Erie corridor.

Understanding the diversity of bacteria and E.coli levels at beaches is important for managing health risks. This study compared temporal changes of the bacterial communities of Belle Isle Beach (Detroit, MI) and Sand Point Beach (Windsor, ONT), both located near the Lake St. Clair origin of the Detroit River. Water samples collected 4 days/week for 12 weeks in summer, were subjected to 16S rRNA analysis of amplicon sequencing and E. coli enumeration. Bacterial communities changed over time, as determined by cluster dendrogram analysis, exhibiting different communities in July and August than in June and different communities at the two beaches. After June, alpha diversity decreased and relative abundance of Enterobacter (Gammaproteobacteria) increased at Sand Point; whereas, Belle Isle maintained its alpha diversity and dominance by Betaproteobacteria and Actinobacteria. Contamination at both beaches is dominated by birds (23% to 50% of samples), while only ∼10% had evidence of human-associated bacteria. High E. coli at both beaches was often associated with precipitation. Nearshore sampling counts were higher than waist-deep sampling counts. Despite the dynamic changes in bacterial communities between the two beaches, this analysis based on 16S rRNA amplicon sequencing is able to provide information about bacterial types associated with high E. coli levels and to use bacterial sequences to more precisely determine sources and health relevance of contaminants.

Sensitive detection of SARS-CoV-2 molecular markers in urban community sewersheds using automated viral RNA purification and digital droplet PCR.

Wastewater based epidemiology (WBE) has emerged as a strategy to identify, locate, and manage outbreaks of COVID-19, and thereby possibly prevent surges in cases, which overwhelm local to global health care networks. The WBE process is based on assaying municipal wastewater for molecular markers of the SARS-CoV-2 virus. Standard processes for purifying viral RNA from municipal wastewater are often time-consuming and require the handling of large quantities of wastewater, negatively affecting throughput, timely reporting, and safety. We demonstrate here an automated, faster system to purify viral RNA from smaller volumes of wastewater but with increased sensitivity for detection of SARS-CoV-2 markers. We document the effectiveness of this new approach by way of comparison to the PEG/NaCl/Qiagen method prescribed by the State of Michigan for SARS-CoV-2 wastewater monitoring and show its application to several Detroit sewersheds. Specifically, compared to the PEG/NaCl/Qiagen method, viral RNA purification using the PerkinElmer Chemagic™ 360 lowered handling time, decreased the amount of wastewater required by ten-fold, increased the amount of RNA isolated per µl of final elution product by approximately five-fold, and effectively removed ddPCR inhibitors from most sewershed samples. For detection of markers on the borderline of viral detectability, we found that use of the Chemagic™ 360 enabled the measurement of viral markers in a significant number of samples for which the result with the PEG/NaCl/Qiagen method was below the level of detectability. The improvement in detectability of the viral markers might be particularly important for early warning to public health authorities at the beginning of an outbreak. Applied to sewersheds in Detroit, the technique enabled more sensitive detection of SARS-CoV-2 markers with good correlation between wastewater signals and COVID-19 cases in the sewersheds. We also discuss advantages and disadvantages of several automated RNA purification systems, made by Promega, PerkinElmer, and ThermoFisher.

Chemical structure of three basic Asp-49 phospholipases A2 isolated from Crotalus molossus nigrescens venom with cytotoxic activity against cancer cells.

Snake venoms are complex mixtures of molecules with several biological activities. Among these molecules, the enzymes with phospholipase A2 activity have been extensively studied in the venoms from snakes because of their importance in the envenomation process and symptoms. The Mexican rattlesnake Crotalus molossus nigrescens is widely distributed in the Mexican plateau. Unlike other crotalids, its venom components have been poorly studied. Here, we characterized the phospholipase activity of one fraction isolated from the venom of this snake and we determined the cytotoxic and neurotoxic effects on brain tumor cells and neuronal primary cultures, respectively. After reverse phase chromatography, we obtained a fraction which was analyzed by mass spectrometry showing higher activity than that from a PLA2 from bee venom used as control. This fraction was enriched with three basic Asp49 phospholipases with molecular masses of 12.5, 13.9 and 14.2 kDa. Their complete amino acid sequences were determined, and their predicted tertiary structures were generated using the model building softwares I-tasser and Chimera. Viability assays revealed that the fraction showed cytotoxic activity against brain tumor cells (C6, RG2 and Daoy) with IC50 values ranging between 10 and 100 ng/ml, whereas an IC50 > 100 ng/ml was exerted in rat primary astrocytes. These findings might be relevant in oncological medicine due to their potential as anticancer agents and low neurotoxic effects compared to conventional drugs.

Molecular diet studies of water mites reveal prey biodiversity.

Water mites are diverse aquatic invertebrates that provide potentially important ecosystem and economic services as bioindicators and mosquito biocontrol; however, little is known about water mite digestive physiology, including their diet in nature. Water mites, much like their spider relatives, liquefy their prey upon consumption. This results in the absence of morphologically identifiable prey in water mite mid-gut. Previous studies have reported associations in the field of water mites with presumed prey and laboratory observations of water mites feeding on specific organisms offered for ingestion; however, the present work aims to determine what water mites have ingested in nature based on molecular studies of gut contents from freshly collected organisms from the field. To elucidate water mite prey, we used next-generation sequencing to detect diverse cytochrome oxidase I DNA barcode sequences of putative prey in the guts of 54 specimens comprising two species of Lebertia and a few specimens of Arrenurus (2) and Limnesia (1). To our knowledge this is the first molecular study of the diets of water mites as they feed in nature. While the presence of chironomid DNA confirmed previous observations of midge larvae as part of the diets of Lebertia, we also found the DNA of diverse organisms in all four species of water mites, including the DNA of mosquitoes in 6 specimens of Lebertia and a large number of previously unknown prey, especially from oligochaete worms. These studies thereby reveal a greater diversity of prey and a potentially broader significance than previously appreciated for water mites in aquatic food webs. Molecular studies like this can detect water mite predators of mosquito larvae and add knowledge of water mite predatory contributions to freshwater food webs.

Oral and intestinal bacterial exotoxins: Potential linked to carcinogenesis.

Growing evidence suggests that imbalances in resident microbes (dysbiosis) can promote chronic inflammation, immune-subversion, and production of carcinogenic metabolites, thus leading to neoplasia. Yet, evidence to support a direct link of individual bacteria species to human sporadic cancer is still limited. This chapter focuses on several emerging bacterial toxins that have recently been characterized for their potential oncogenic properties toward human orodigestive cancer and the presence of which in human tissue samples has been documented. These include cytolethal distending toxins produced by various members of gamma and epsilon Proteobacteria, Dentilisin from mammalian oral Treponema, Pasteurella multocida toxin, two Fusobacterial toxins, FadA and Fap2, Bacteroides fragilis toxin, colibactin, cytotoxic necrotizing factors and α-hemolysin from Escherichia coli, and Salmonella enterica AvrA. It was clear that these bacterial toxins have biological activities to induce several hallmarks of cancer. Some toxins directly interact with DNA or chromosomes leading to their breakdowns, causing mutations and genome instability, and others modulate cell proliferation, replication and death and facilitate immune evasion and tumor invasion, prying specific oncogene and tumor suppressor pathways, such as p53 and ß-catenin/Wnt. In addition, most bacterial toxins control tumor-promoting inflammation in complex and diverse mechanisms. Despite growing laboratory evidence to support oncogenic potential of selected bacterial toxins, we need more direct evidence from human studies and mechanistic data from physiologically relevant experimental animal models, which can reflect chronic infection in vivo, as well as take bacterial-bacterial interactions among microbiome into consideration.

In vivo intervertebral disc regeneration using stem cell-derived chondroprogenitors.

OBJECT: There is currently no biologic therapy to repair or restore a degenerated intervertebral disc. A potential solution may rest with embryonic stem cells (ESCs), which have a potential to grow indefinitely and differentiate into a variety of cell types in vitro. Prior studies have shown that ESCs can be encouraged to differentiate toward specific cell lineages by culture in selective media and specific growth environment. Among these lineages, there are cells capable of potentially producing nucleus pulposus (NP) in vivo. In this investigation, the authors studied ESCderived chondroprogenitors implanted into a degenerated disc in a rabbit. For this purpose, a rabbit model of disc degeneration was developed. METHODS: A percutaneous animal model of disc degeneration was developed by needle puncture of healthy intact discs in 16 New Zealand white rabbits. Series of spine MR imaging studies were obtained before disc puncture and after 2, 6, and 8 weeks. Prior to implantation, murine ESCs were cultured with cis-retinoic acid, transforming growth factor beta, ascorbic acid, and insulin-like growth factor to induce differentiation toward a chondrocyte lineage. After confirmation by MR imaging, degenerated disc levels were injected with chondrogenic derivatives of ESCs expressing green fluorescent protein. At 8 weeks post-ESC implantation, the animals were killed and the intervertebral discs were harvested and analyzed using H & E staining, confocal fluorescent microscopy, and immunohistochemical analysis. Three intervertebral disc groups were analyzed in 16 rabbits, as follows: 1) Group A, control: naïve, nonpunctured discs (32 discs, levels L4-5 and L5-6); 2) Group B, experimental control: punctured disc (16 discs, level L2-3); and 3) Group C, experimental: punctured disc followed by implantation of chondroprogenitor cells (16 discs, level L3-4). RESULTS: The MR imaging studies confirmed intervertebral disc degeneration at needle-punctured segments starting at approximately 2 weeks. Postmortem H & E histological analysis of Group A discs showed mature chondrocytes and no notochordal cells. Group B discs displayed an intact anulus fibrosus and generalized disorganization within fibrous tissue of NP. Group C discs showed islands of notochordal cell growth. Immunofluorescent staining for notochordal cells was negative for Groups A and B but revealed viable notochordal-type cells within experimental Group C discs, which had been implanted with ESC derivatives. Notably, no inflammatory response was noted in Group C discs. CONCLUSIONS: This study illustrates a reproducible percutaneous model for studying disc degeneration. New notochordal cell populations were seen in degenerated discs injected with ESCs. The lack of immune response to a xenograft of mouse cells in an immunocompetent rabbit model may suggest an as yet unrecognized immunoprivileged site within the intervertebral disc space.