Nonhuman Blood Research Articles

Droplet-Based Single-Cell 3' mRNA Sequencing of Marburg Virus-Infected Samples.

Single-cell technologies are continually evolving with emerging methods that are gradually uncovering the central DNA-RNA-protein dogma. Single-cell RNA sequencing is one arm of a multi-omic approach that achieves an astounding level of granularity to reveal the complexity of virus-host interactions at the transcriptomic level. Cell tropism, virus replication, pathogenesis, and gene expression changes mediated by the virus and the host's immune response to infection are just some areas of study that are gaining better clarity due to the high-resolution analysis afforded by the technology.We describe a single-cell sequencing protocol for Marburg virus infection in vivo using nonhuman primate blood and the 10× Chromium Next GEM single-cell genomics methodology. Working with pathogens of high consequence is logistically complicated, requiring containment in biosafety level (BSL)-4 laboratories and harsh inactivation procedures before samples can safely be removed to lower biosafety conditions. We provide procedural insight into sample isolation and processing conducted in BSL-4 and describe the requirements for safe sample removal without jeopardizing quality for down-stream sequencing and analysis in BSL-2 conditions. Characterization of complicated biological processes mediated by high-containment pathogens, typically restricted to analogous model systems, e.g., minigenome, can be achieved using live virus.

Ivermectin Inhibits Zika Virus Replication in Vitro But Does Not Prevent Zika Virus Infection in Rhesus Macaques (Macaca mulatta).

Zika virus (ZIKV) outbreaks occur sporadically in tropical and subtropical regions. At present, there are no licensed vaccines or specific treatments available for ZIKV. Ivermectin is approved for use in humans as an antiparasitic drug. In this study, we conducted invitro cell culture and invivo experiments in rhesus macaque hosts and Aedes aegypti vectors to investigate the potential of ivermectin as an inhibitor of ZIKV infection. In LLC-MK2 mammalian cells, ivermectin inhibited ZIKV growth invitro with 50% inhibitory concentration (IC50) values in the ranges of 7.4-21.3 µM and 4.0-11.6 µM for African and Asian genotypes, respectively. In C6/36 mosquito cells, ivermectin inhibited ZIKV growth invitro with IC50 values in the ranges of 10.1-17.4 µM and 8.0-15.6 µM for the African and Asian genotypes, respectively. Despite these invitro results, high-dose ivermectin prophylaxis (1.2 mg/kg for 3 consecutive days) failed to prevent ZIKV infection in rhesus macaque and did not alter ZIKV IgM antibody production. The secondary transfer of ivermectin from nonhuman primate blood to mosquito vectors at 3 days post-ZIKV inoculation and after the last dose of ivermectin administration showed no reduction in ZIKV replication in mosquitoes. However, mosquito survival rates were significantly (P <0.0001) lower after exposure to ivermectin, thereby potentially impacting ZIKV transmission through increased vector mortality. However, further investigation is needed to determine dosing regimens that may realize these effects invivo.

Analysis of the aging-related biomarker in a nonhuman primate model using multilayer omics

BackgroundAging is a prominent risk factor for diverse diseases; therefore, an in-depth understanding of its physiological mechanisms is required. Nonhuman primates, which share the closest genetic relationship with humans, serve as an ideal model for exploring the complex aging process. However, the potential of the nonhuman primate animal model in the screening of human aging markers is still not fully exploited. Multiomics analysis of nonhuman primate peripheral blood offers a promising approach to evaluate new therapies and biomarkers. This study explores aging-related biomarker through multilayer omics, including transcriptomics (mRNA, lncRNA, and circRNA) and proteomics (serum and serum-derived exosomes) in rhesus monkeys (Macaca mulatta).ResultsOur findings reveal that, unlike mRNAs and circRNAs, highly expressed lncRNAs are abundant during the key aging period and are associated with cancer pathways. Comparative analysis highlighted exosomal proteins contain more types of proteins than serum proteins, indicating that serum-derived exosomes primarily regulate aging through metabolic pathways. Finally, eight candidate aging biomarkers were identified, which may serve as blood-based indicators for detecting age-related brain changes.ConclusionsOur results provide a comprehensive understanding of nonhuman primate blood transcriptomes and proteomes, offering novel insights into the aging mechanisms for preventing or treating age-related diseases.

Water in Yorùbá (Southwest Nigeria) religious belief and practices

Among the Yorùbá, one of the three largest ethnic groups primarily based in southwest Nigeria, water holds several meanings which are usually demonstrated in its capacity as a regenerating and degenerating power. To draw upon its affirming quality and elude its caustic powers, the Yorùbá exhibit strong reverence for water and, therefore, give critical attention to it. This reverence manifests through various ritual observances and religious rites that may include the presentation of non-human life or blood or any other acceptable ritual material. In the literature, water is presented ordinarily in its fluid state or as a ‘staple’ consumed to sustain human, animal and plant life as well as satisfy all kinds of human needs. Among the Yorùbá, however, water is approached in a different light and beyond its mere physical state. Drawing on Yorùbá verbal texts such as odù ifá (divinatory poems or texts) and òwe (proverbs), oral interviews and secondary sources, I present a hermeneutic approach to the intrinsic capacity of water within the context of Yorùbá religious belief and practices. I also examine water as a numinous power in Yorùbá culture and myths. I argue that the Yorùbá people’s reverence for water informs the diverse religious and cultural practices that manifest across sites where sacred water bodies are situated and why verbal formulations have been developed to explain water’s ‘extraordinary’ powers.

Discrimination Between Human and Animal Blood Using Raman Spectroscopy and a Self-Reference Algorithm for Forensic Purposes: Method Expansion and Validation

Determining whether the origin of a bloodstain is human or non-human is important during a forensic investigation. In their pioneering work, Bian et al. introduced a self-reference peak algorithm for the analysis of the Raman spectra of bloodstains and demonstrated the great potential of this approach for differentiating between human and non-human blood. However, this work only used three non-human species in the creation of their original model. The current study expands the capability of a self-referencing algorithm to discriminate between human and 18 non-human species based on the Raman spectra of blood samples. The intensity ratios between the bands at 1003 and 1341 cm−1 of the samples’ Raman spectra were compared between species to determine whether a threshold existed that separates human samples from those of non-humans. The self-referencing algorithm was capable of correctly categorizing spectra averaged from donors of all 18 non-human species. The use of this algorithm is simple and requires little training or knowledge of statistics, which makes it accessible for forensic applications, compared to computationally difficult analysis methods. This technique using Raman spectroscopy is rapid, nondestructive, and highly accurate making it a promising tool for forensic applications.

Forensic DNA Analysis of Mixed Mosquito Blood Meals: STR Profiling for Human Identification

Mosquito vectors captured at a crime scene are forensically valuable since they feed on human blood, and hence, human DNA can be recovered to help identify the victim and/or the suspect. This study investigated the validity of obtaining the human short tandem repeats (STRs) profile from mixed blood meals of the mosquito, Culex pipiens L. (Diptera, Culicidae). Thus, mosquitoes were membrane-feed on blood from six different sources: a human male, a human female, mixed human male-female blood, mixed human male-mouse blood, mixed human female-mouse blood, and mixed human male-female-mouse blood. DNA was extracted from mosquito blood meals at 2 h intervals up to 72 h post-feeding to amplify 24 human STRs. Data showed that full DNA profiles could be obtained for up to 12 h post-feeding, regardless of the type of blood meal. Complete and partial DNA profiles were obtained up to 24 h and 36 h post-feeding, respectively. The frequencies of STR loci decreased over time after feeding on mixed blood until they became weakly detectable at 48 h post-feeding. This may indicate that a blood meal of human blood mixed with animal blood would contribute to maximizing DNA degradation and thus affects STR identification beyond 36 h post-feeding. These results confirm the feasibility of human DNA identification from mosquito blood meals, even if it is mixed with other types of non-human blood, for up to 36 h post-feeding. Therefore, blood-fed mosquitoes found at the crime scene are forensically valuable, as it is possible to obtain intact genetic profiles from their blood meals to identify a victim, a potential offender, and/or exclude a suspect.

Development of a 40+ color full spectrum flow cytometry panel for in-depth immunophenotyping of chief cell subsets in non-human primate peripheral blood mononuclear cells

Abstract Leveraging full-spectrum technology, we designed and optimized a 40+ color flow cytometry panel for the analysis of non-human primate peripheral blood mononuclear cells (PBMCs). Non-human primate models of disease allow for the closest approximation of human outcomes to vaccination, disease, and treatment due to similarities in physiological host responses, which increase predictive accuracy. This first panel of its kind achieves comprehensive profiling of immune subpopulations by the inclusion of surface, intracellular, and cytokine markers representative of differentiation, activation, functionality, and proliferation to deeply interrogate the immune response to infectious disease and vaccination. The optimized panel is additionally appropriate for the discovery of new immune phenotypes important for protection against pathogens. After initial optimization for use with cynomolgus macaque (Macaca fascicularis) and rhesus macaque (Macaca mulatta) PBMCs, the panel was further adapted for use with African green monkey (Chlorocebus aethiops) PBMCs to allow for cross-comparison between three frequently implemented non-human primate models of infectious disease. This study was supported by the US Army Medical Research Acquisition Activity contract number W81XWH1910027 to TWG and Department of Health and Human Services, National Institutes of Health grant number UC7AI094660 for BSL-4 operations support of the Galveston National Laboratory.

Development of surface‐enhanced Raman spectroscopy evidence swabs using a silver nanoparticle biosynthesis for the detection of animal blood

AbstractIdentifying the type of body fluid recovered from crime scenes before DNA typing may be critical to qualify its relevancy to the case. Presumptive tests are often used first to detect blood traces; however, they require confirmation, are destructive, and are susceptible to false positives. This study aims to develop a method of collection and identification for nonhuman blood using biosynthesized SERS swabs as a more accurate, specific, and nondestructive detection technique. Silver nanoparticles were synthesized directly on nylon swabs using a biosynthesis reaction and curcumin as the reducing agent to form the nanoparticles. The detection of bovine blood was investigated by analyzing decreasing volumes of blood swabbed with the surface‐enhanced Raman spectroscopy (SERS) swabs. Although swabbing 30 and 20 μl of blood resulted in the most resolved spectra, swabbing 10 μl of blood still produced identifiable peaks. Wetted swabs were used to analyze dried bovine bloodstains on cotton fabrics and glass slides: The spectra collected when swabbing the bloodstain on the glass sides had a higher signal‐to‐noise ratio than on fabrics. The SERS swabs were subsequently used to collect and analyze horse and sheep blood. Although Raman bands characteristic of blood were identifiable for each specie, showing that the SERS swabs allowed the successful detection of the three animal blood species, the spectra from the three species could not be differentiated. This study demonstrates the efficiency of the biosynthesis to grow nanoparticles on the swabs and shows their viability to be used as SERS substrates to collect and identify animal blood.

Livestock and rodents within an endemic focus of Visceral Leishmaniasis are not reservoir hosts for Leishmania donovani.

Leishmaniasis on the Indian subcontinent is thought to have an anthroponotic transmission cycle. There is no direct evidence that a mammalian host other than humans can be infected with Leishmania donovani and transmit infection to the sand fly vector. The aim of the present study was to evaluate the impact of sand fly feeding on other domestic species and provide clinical evidence regarding possible non-human reservoirs through experimental sand fly feeding on cows, water buffalo goats and rodents. We performed xenodiagnosis using colonized Phlebotomus argentipes sand flies to feed on animals residing in villages with active Leishmania transmission based on current human cases. Xenodiagnoses on mammals within the endemic area were performed and blood-fed flies were analyzed for the presence of Leishmania via qPCR 48hrs after feeding. Blood samples were also collected from these mammals for qPCR and serology. Although we found evidence of Leishmania infection within some domestic mammals, they were not infectious to vector sand flies. Monitoring infection in sand flies and non-human blood meal sources in endemic villages leads to scientific proof of exposure and parasitemia in resident mammals. Lack of infectiousness of these domestic mammals to vector sand flies indicates that they likely play no role, or a very limited role in Leishmania donovani transmission to people in Bihar. Therefore, a surveillance system in the peri-/post-elimination phase of visceral leishmaniasis (VL) must monitor absence of transmission. Continued surveillance of domestic mammals in outbreak villages is necessary to ensure that a non-human reservoir is not established, including domestic mammals not present in this study, specifically dogs.

Evaluation of Virulence in Cynomolgus Macaques Using a Virus Preparation Enriched for the Extracellular Form of Monkeypox Virus.

The 2022 global human monkeypox outbreak emphasizes the importance of maintaining poxvirus research, including enriching a basic understanding of animal models for developing and advancing therapeutics and vaccines. Intravenous administration of monkeypox virus in macaques is arguably one of the best animal models for evaluating the efficacy of medical countermeasures. Here we addressed one criticism of the model, a requirement for a high-titer administration of virus, as well as improving our understanding of monkeypox virus pathogenesis. To do so, we infected macaques with a challenge dose containing a characterized inoculum enriched for the extracellular form of monkeypox virus. Although there were some differences between diseases caused by the enriched preparation compared with a relatively similar unpurified preparation, we were unable to reduce the viral input with the enriched preparation and maintain severe disease. We found that inherent factors contained within the serum of nonhuman primate blood affect the stability of the monkeypox extracellular virions. As a first step to study a role of the extracellular form in transmission, we also showed the presence of this form in the oropharyngeal swabs from nonhuman primates exposed to monkeypox virus.

Tick Intrastadial Feeding and Its Role on IgE Production in the Murine Model of Alpha-gal Syndrome: The Tick "Transmission" Hypothesis.

Recent studies have provided strong evidence indicating that lone star tick bites are a cause of AGS (alpha-gal syndrome, also known as red meat allergy RMA) in humans. AGS is characterized by an increase in IgE antibody production against galactose-alpha-1,3-galactose (aGal), which is a common glycan found in mammalian tissue, except in Old World monkeys and humans. The main causative factor of AGS, the lone star tick (Amblyomma americanum), is broadly distributed throughout the east and midwest of the United States and is a vector of a wide range of human and animal pathogens. Our earlier glycomics study of the salivary glands of partially fed male and female ticks revealed relatively high levels of aGal epitopes. In this study, we found that partially fed males of A. americanum on bovine blood, which engage in multiple intrastadial feedings, carry a large amount of aGal in the salivary glands. In our current work, we aimed to test whether ticks mediate the transmission of the aGal sensitizer acquired from nonhuman blood to humans in the intrastadial host switch (referred to as the “transmission” hypothesis). To test this hypothesis, we used an alpha-galactosyltransferase knockout mutant mouse (aGT-KO) model system infested with ticks that were unfed or partially fed on bovine blood. Based on the levels of total IgE and specific IgG and IgE antibodies against aGal after tick feedings, aGT-KO mice significantly responded to tick feeding and injection of aGal (Galα1-3Galβ1-4GlcNAc) conjugated to human serum albumin or mouse serum albumin (aGal-HSA or aGal-MSA) by increasing total IgE and aGal-specific IgE levels compared to those in C57BL/6 control mice. All of the treatments of aGT-KO mice involving the feeding of partially fed and unfed ticks functioned as sensitizers that increased the levels of specific IgE against aGal, with large individual variations. The data in this study do not support the “transmission” component of AGS, although they confirmed that aGT-KO mice can be used as a model for RMA studies.

Spectra-based blood species discrimination by machine learning: Between human and non-human

With the fast development of data-processing technologies, more and more machine learning algorithms and a large volume of data were involved in spectra analysis. This study aims to validate the spectra’ capability of discriminating blood species between human and non-human with a large volume of data and investigate the predictive ability of the binary classification model for unseen animal species. This study used eight machine learning algorithms and six balancing re-sampling strategies to train the binary classifier on 8431 samples from 14 species, including human. The hold-out experiment was conducted to evaluate the trained model’s predictive ability for unseen animal species. The optimal binary classifier between human and non-human produced a testing accuracy of 98.9% and Youden’s index of 0.976. This model was trained with the linear discriminant analysis with the re-sampling strategy of cluster centroids. Nine out of thirteen species produced sensitivities greater than 0.9 on the hold-out data for the hold-out experiment. This result indicates that the human and non-human blood classification model trained with a limited number of animal species could correctly recognize many unseen animal species as non-human and suggests careful data selection for the model building.

Surface enhanced Raman scattering specificity for detection and identification of dried bloodstains

Surface enhanced Raman spectroscopy (SERS) provides highly specific vibrational signatures identifying dried blood for a variety of forensic applications. SERS spectra on Au nanoparticle substrates excited at 785 nm are found to identify dried stains of human and nonhuman blood from seven animals, and distinguish stains due to menstrual and peripheral blood. In addition, the unique SERS bloodstain spectrum is distinct from the SERS spectra of thirty red-brown stains of potential household substances that could be visually mistaken for bloodstains and from food stains that have been shown to give positive results with presumptive colorimetric blood tests. Finally, a SERS swab procedure has been developed and demonstrates that the substrates that a blood sample dried on does not offer any Raman or fluorescence interference for the SERS identification of dried blood. Such bloodstains on porous and nonporous materials are all identical and exclusively due to the heme moiety of hemoglobin. Optimized selection of the extraction solvent is found to control the chemical composition of molecular components appearing in the SERS spectrum of complex, multicomponent biological mixtures, such as body fluids.

Proteomic Analysis of Non-human Primate Peripheral Blood Mononuclear Cells During Burkholderia mallei Infection Reveals a Role of Ezrin in Glanders Pathogenesis.

Burkholderia mallei, the causative agent of glanders, is a gram-negative intracellular bacterium. Depending on different routes of infection, the disease is manifested by pneumonia, septicemia, and chronic infections of the skin. B. mallei poses a serious biological threat due to its ability to infect via aerosol route, resistance to multiple antibiotics and to date there are no US Food and Drug Administration (FDA) approved vaccines available. Induction of innate immunity, inflammatory cytokines and chemokines following B. mallei infection, have been observed in in vitro and small rodent models; however, a global characterization of host responses has never been systematically investigated using a non-human primate (NHP) model. Here, using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified alterations in expression levels of host proteins in peripheral blood mononuclear cells (PBMCs) originating from naïve rhesus macaques (Macaca mulatta), African green monkeys (Chlorocebus sabaeus), and cynomolgus macaques (Macaca fascicularis) exposed to aerosolized B. mallei. Gene ontology (GO) analysis identified several statistically significant overrepresented biological annotations including complement and coagulation cascade, nucleoside metabolic process, vesicle-mediated transport, intracellular signal transduction and cytoskeletal protein binding. By integrating an LC-MS/MS derived proteomics dataset with a previously published B. mallei host-pathogen interaction dataset, a statistically significant predictive protein-protein interaction (PPI) network was constructed. Pharmacological perturbation of one component of the PPI network, specifically ezrin, reduced B. mallei mediated interleukin-1β (IL-1β). On the contrary, the expression of IL-1β receptor antagonist (IL-1Ra) was upregulated upon pretreatment with the ezrin inhibitor. Taken together, inflammasome activation as demonstrated by IL-1β production and the homeostasis of inflammatory response is critical during the pathogenesis of glanders. Furthermore, the topology of the network reflects the underlying molecular mechanism of B. mallei infections in the NHP model.

Vector Competence of Aedes aegypti Populations from Entebbe, Uganda, for Zika Virus.

We evaluated the vector competence of three strains of Aedes aegypti formosus from Entebbe, Uganda, for Zika virus (ZIKV). The aim was to find out if these strains were competent or incompetent vectors for ZIKV, to explain the lack of ZIKV outbreaks in the city of Entebbe. We observed transmission rates ranging from 33% to 78%; however, these rates were not statistically significantly different, suggesting that there were no real differences among the strains. Nonetheless, this showed that populations of Ae. aegypti formosus in Entebbe are competent vectors for ZIKV. The reason why there is no detectable transmission of ZIKV to humans in Entebbe is currently unknown. The lack of detectable transmission despite Ae. aegypti formosus competence for ZIKV suggests that other parameters, such as preference for nonhuman blood, may be limiting its ability to serve as a vector.

Non-Human Primate Blood-Brain Barrier and In Vitro Brain Endothelium: From Transcriptome to the Establishment of a New Model.

The non-human primate (NHP)-brain endothelium constitutes an essential alternative to human in the prediction of molecule trafficking across the blood–brain barrier (BBB). This study presents a comparison between the NHP transcriptome of freshly isolated brain microcapillaries and in vitro-selected brain endothelial cells (BECs), focusing on important BBB features, namely tight junctions, receptors mediating transcytosis (RMT), ABC and SLC transporters, given its relevance as an alternative model for the molecule trafficking prediction across the BBB and identification of new brain-specific transport mechanisms. In vitro BECs conserved most of the BBB key elements for barrier integrity and control of molecular trafficking. The function of RMT via the transferrin receptor (TFRC) was characterized in this NHP-BBB model, where both human transferrin and anti-hTFRC antibody showed increased apical-to-basolateral passage in comparison to control molecules. In parallel, eventual BBB-related regional differences were Investig.igated in seven-day in vitro-selected BECs from five brain structures: brainstem, cerebellum, cortex, hippocampus, and striatum. Our analysis retrieved few differences in the brain endothelium across brain regions, suggesting a rather homogeneous BBB function across the brain parenchyma. The presently established NHP-derived BBB model closely mimics the physiological BBB, thus representing a ready-to-use tool for assessment of the penetration of biotherapeutics into the human CNS.

Forensic Human and Non-human Blood Discrimination using Mitochondrial 12S rRNA gene

Background: The blood samples of spots typing regards most important step in forensic analysis. Determining the blood affiliation as human on non-human is not enough as a forensic tool leading to the truth . The mitochondrial 12S rRNA gene are suitable for taxonomy and species identification, especially for the discrimination of even closely related species. The current study aimed to showed the validity of designed 12S rRNA species specific primer pairs for human and nonhuman animal blood typing as a forensic tool. Methodology: Seventy two blood samples were collected from Homo sapiens, Ovis aries, Capra hircus and Bos taurus (18 blood samples from each). Five milliliter were withdrawn and placed in EDTAtube and stored in refrigerator for further processing. Four sets of species specific primer pairs targeting mitochondrial 12S rRNA were designed and checked. PCR and sequencing were performed and sequences were analyzed and register in GenBank. Results: the results revealed that, the amplicon of 12S rRNA gene of Homo sapiens were 800bp, Ovis aries were 560bp, Capra hircus were 460bp, and Bos taurus were 600bp The first confirmatory test for validity and specificity of designed primer pairs is no amplification for intraspecies primer pairs (i.e. using homo12S rRNA primer pairs to amplify Ovis aries, Capra hircus, and Bos taurus 12S rRNA will not give product and vice versa). The second confirmatory test is the results of sequencing. The identity percentage and alignment of sequences results of amplified 12S rRNA gene of homo sapiens, Ovis aries, Capra hircus and Bos taurus revealed that, the similarity percentage ranged from 98.03% to 100%.Conclusion: The current study conclude that, validity and accuracy of designed 12S rRNA species specific primer pairs for human and nonhuman animal blood typing as a forensic tool and there is no intraspecies cross amplification.

Discrimination the Gender in the Criminal Evidence at Crime Scene

Background: An ideal method for gender identification would be accurate, simple, and cheap,enabling its use in most laboratories. In addition, it should also be able as much as possible to be used for all animals as well as tissues and/or cells. For chromosomal analysis, we need viable cells that are able to divide, and if this is not possible, these methods cannot be used. On the other hand, genetic methods are reliable and do not need living cells, and it is easy to obtain DNA for these studies even in very ancient and nonviable tissues. These methods are therefore the most accepted ones The current study aimed to Determine the gender of a given DNA samples by designed vaulable specific-PCR tool. Methodology: Thirty eight ( ninteen from each male and female) human blood samples were collected using EDTA-Na2 tubes for direct DNA extraction after taken the agreement of the volunteers to give the blood sample . via expert .The blood sample mixed well by rotation of EDTA tube. two sets of species specific primer pairs targeting Homo sapiens (AMLE X and AMLE Y) genes. were designed and checked. PCR and sequencing were performed and sequences were analyzed and register in GenBank. Results: the results revealed that, the amplicon of AMLE X gene of Homo sapiens were 1164bp and AMLE Y were 744bp Conclusion: The current study conclude that, validity and accuracy of designed 12S rRNA species specific primer pairs for human and nonhuman animal blood typing as a forensic tool and there is no intraspecies cross amplification.

Novel Expression of GABAA Receptors on Resistance Arteries That Modulate Myogenic Tone

The clinical administration of GABAergic medications leads to hypotension which has classically been attributed to the modulation of neuronal activity in the central and peripheral nervous systems. However, certain types of peripheral smooth muscle cells have been shown to express GABA_A receptors, which modulate smooth muscle tone, by the activation of these chloride channels on smooth muscle cell plasma membranes. Limited prior studies demonstrate that non-human large-caliber capacitance blood vessels mounted on a wire myograph are responsive to GABA_A ligands. We questioned whether GABA_A receptors are expressed in human resistance arteries and whether they modulate myogenic tone. We demonstrate the novel expression of GABA_A subunits on vascular smooth muscle from small-caliber human omental and mouse tail resistance arteries. We show that GABA_A receptors modulate both plasma membrane potential and calcium responses in primary cultured cells from human resistance arteries. Lastly, we demonstrate functional physiologic modulation of myogenic tone via GABA_A receptor activation in human and mouse arteries. Together, these studies demonstrate a previously unrecognized role for GABA_A receptors in the modulation of myogenic tone in mouse and human resistance arteries.

Validation of presumptive tests for non-human blood using Kastle Meyer and Hemastix reagents

Kastle Meyer and Hemastix reagents are presumptive tests commonly used in forensic casework for the detection of blood, and their suitability has been reviewed in numerous publications. However, studies to date have focused on the validation of these tests on human blood alone, and no published work has looked at the sensitivity, specificity and effect on DNA analysis when using these reagents to presumptively test for animal blood. The aim of this study was to validate the two reagents for use with animal blood, and compare their performance in order to choose the best test based on the circumstances in wildlife crime investigation.The sensitivity, specificity, stability and robustness of the methods were assessed by experiments with dilutions of animal blood (from 1:4 to 1:65536) using direct and indirect (rub) tests, potential interfering substances, blood sources from different species and aged blood. The effects of the two reagents on subsequent DNA analysis were also investigated.During the direct tests, Kastle Meyer showed a higher sensitivity, detecting blood down to a dilution of 1:16,384, one order of magnitude lower than Hemastix. However during the rub test, Hemastix showed a higher sensitivity, detecting blood down to a dilution of 1:64 on porous materials while Kastle Meyer was positive only down to a dilution of 1:16. Moreover, when using the same swab for presumptive testing and DNA extraction, Hemastix testing allowed amplification of a sufficient amount of DNA for species identification at its limit of sensitivity on porous materials (1:64) while Kastle Meyer inhibited most amplification of DNA at its less sensitive limit of 1:16 dilution. On the other hand, Hemastix showed a much lower specificity, producing false positive results when exposed to tomato, potato, rust, avian uric acid, bleach and sink rot, while Kastle Meyer only produced a faint positive reaction from potato. Both tests performed equally well detecting fresh blood of different animal species. The stability test gave comparable results among the tests except for aged fish blood stains, where the Kastle Meyer test performed poorly.Owing to its ease of use, higher sensitivity, and lack of interference with downstream DNA analysis, and despite its reduced specificity compared to Kastle Meyer, the Hemastix method is more appropriate for use in wildlife crime investigations. Positive results would always be confirmed with DNA analysis and the low interference of the reagent will allow the use of a single swab for presumptive testing and DNA sampling.