Repurposing cancer drugs, batimastat and marimastat, to inhibit the activity of a group I metalloprotease from the venom of the Western Diamondback rattlesnake, Crotalus atrox

Snakebite envenomation causes over 140,000 deaths every year predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with an incredibly complex pathophysiology due to the vast number of unique toxins/proteins found in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a group I metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity was completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 M, while it is partially potentiated by calcium chloride. Molecular docking studies demonstrate that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites

Mechanisms underpinning the permanent muscle damage induced by snake venom metalloprotease

Snakebite is a major neglected tropical health issue that affects over 5 million people worldwide resulting in around 1.8 million envenomations and 100,000 deaths each year. Snakebite envenomation also causes innumerable morbidities specifically loss of limbs as a result of excessive tissue/muscle damage. Snake venom metalloproteases (SVMPs) are a predominant component of viper venoms, and are involved in the degradation of basement membrane proteins (particularly collagen) surrounding the tissues around the bite site. Although their collagenolytic properties have been established, the molecular mechanisms through which SVMPs induce permanent muscle damage are poorly understood. Here, we demonstrate the purification and characterisation of an SVMP from a viper (Crotalus atrox) venom. Mass spectrometry analysis confirmed that this protein is most likely to be a group III metalloprotease (showing high similarity to VAP2A) and has been referred to as CAMP (Crotalus atrox metalloprotease). CAMP displays both collagenolytic and fibrinogenolytic activities and inhibits CRP-XL-induced platelet aggregation. To determine its effects on muscle damage, CAMP was administered into the tibialis anterior muscle of mice and its actions were compared with cardiotoxin I (a three-finger toxin) from an elapid snake (Naja pallida) venom. Extensive immunohistochemistry analyses revealed that CAMP significantly damages skeletal muscles by attacking the collagen scaffold and other important basement membrane proteins, and prevents their regeneration through disrupting the functions of satellite cells. In contrast, cardiotoxin I destroys skeletal muscle by damaging the plasma membrane, but does not impact regeneration due to its inability to affect the extracellular matrix. Overall, this study provides novel insights into the mechanisms through which SVMPs induce permanent muscle damage

Impact of Naja nigricollis Venom on the Production of Methaemoglobin

Snakebite envenomation is an affliction currently estimated to be killing upwards of 100,000 people annually. Snakebite is associated with a diverse pathophysiology due to the magnitude of variation in venom composition that is observed worldwide. The haemolytic (i.e., lysis of red blood cells) actions of snake venoms are well documented, although the direct impact of venoms on haemoglobin is not fully understood. Here we report on the varied ability of a multitude of snake venoms to oxidise haemoglobin into methaemoglobin. Moreover, our results demonstrate that the venom of an elapid, the black necked spitting cobra, Naja nigricollis, oxidises oxyhaemoglobin (Fe2+) into methaemoglobin (Fe3+) in a time- and concentration-dependent manner that is unparalleled within the 47 viper and elapid venoms evaluated. The treatment of venom with a reducing agent, dithiothreitol (DTT) is observed to potentiate this effect at higher concentrations, and the use of denatured venom demonstrates that this effect is dependent upon the heat-sensitive proteinaceous elements of the venom. Together, our results suggest that Naja nigricollis venom appears to promote methaemoglobin production to a degree that is rare within the Elapidae family, and this activity appears to be independent of proteolytic activities of venom components on haemoglobin

Otter qPCR Data at SAFE

<b>Description: </b><p>All data collected during my project at the SAFE project, March-April 2017. This document contains metadata that should summarise all data collected.</p><p><b>Project: </b>This dataset was collected as part of the following SAFE research project: <a href="https://www.safeproject.net/projects/project_view/166"><b>Using environmental DNA (eDNA) as a tool for monitoring the biodiversity of tropical otter species</b></a></p><p><b>XML metadata: </b>GEMINI compliant metadata for this dataset is available <a href="https://www.safeproject.net/datasets/xml_metadata?id=13">here</a></p><p><b>Data worksheets: </b>There are 2 data worksheets in this dataset:</p><ol><li><p><b>eDNA</b> (Worksheet eDNA)</p><p>Dimensions: 66 rows by 33 columns</p><p>Description: eDNA' contains all data related to the qPCR elements of this project</p><p>Fields: </p><ul><li><b>Date</b>: Date Collected (Field type: Date)</li><li><b>Session</b>: Morning or Evening (Field type: Categorical)</li><li><b>Site</b>: Catchment Site; indicates metres upriver from SAFE Project hydrology datalogger (Field type: ID)</li><li><b>Code</b>: Unique Code given to each samplea and subsample (1a, 1b, 1c…) (Field type: ID)</li><li><b>Location</b>: Riparian transect sample collected from (Field type: Location)</li><li><b>Time_Start</b>: Time arrived at site (Field type: Time)</li><li><b>Time_Finish</b>: Time left site (Field type: Time)</li><li><b>eDNA_Start</b>: Sample collection starting time (Field type: Time)</li><li><b>eDNA_Stop</b>: Sample collection ending time (Field type: Time)</li><li><b>T_w</b>: Water Temperature at time of sample (Field type: Numeric)</li><li><b>T_a</b>: Air Temperature at time of sample (Field type: Numeric)</li><li><b>pH</b>: pH of surface water (Field type: Numeric)</li><li><b>R_H</b>: Relative humidty at site (Field type: Numeric)</li><li><b>Lux</b>: Lux score at time of sample (Field type: Numeric)</li><li><b>Precip</b>: Precipitation Present (Yes or No) (Field type: Categorical)</li><li><b>Shade</b>: Presence of shade during collection (Field type: Categorical)</li><li><b>Leaf_Litter</b>: Presence of leaf litter during collection (Field type: Categorical)</li><li><b>Substrate</b>: Type of substrate (Field type: Categorical)</li><li><b>Time1</b>: Time taken for "bottle" to travel River_dist; used to calculate flow rate (Field type: Numeric)</li><li><b>Time2</b>: Time taken for "bottle" to travel River_dist; used to calculate flow rate (Field type: Numeric)</li><li><b>Time3</b>: Time taken for "bottle" to travel River_dist; used to calculate flow rate (Field type: Numeric)</li><li><b>TimeAv</b>: Average time taken for "bottle" to travel River_dist; used to calculate flow rate (Field type: Numeric)</li><li><b>Depth1</b>: Depth of sample (Field type: Numeric)</li><li><b>Depth2</b>: Depth of sample (Field type: Numeric)</li><li><b>Depth3</b>: Depth of sample (Field type: Numeric)</li><li><b>DepthAv</b>: Average_depth of sample (Field type: Numeric)</li><li><b>River_Dist</b>: Distance "bottle" travelled at sample site ; used to calculate flow rate (Field type: Numeric)</li><li><b>Flow</b>: Calculated flow (speed = distance/ time) (Field type: Numeric)</li><li><b>Presence</b>: Presence of otters detected (to a level deemed above background noise on qPCR) (Field type: Categorical)</li><li><b>N. Wells Pos</b>: Presence of otters detected (to a level deemed above background noise on qPCR) (Field type: Abundance)</li><li><b>Positive NTC</b>: Number of positive negative controls out of 12 (Field type: Numeric)</li><li><b>Notes</b>: Any important notes during sampling (Field type: Comments)</li></ul><br></li><li><p><b>Traditional</b> (Worksheet Traditional)</p><p>Dimensions: 122 rows by 12 columns</p><p>Description: Traditional' contains data collected using traditional surveys</p><p>Fields: </p><ul><li><b>Location</b>: Riparian transect sample collected from (Field type: Location)</li><li><b>Session</b>: Morning or Evening (Field type: Categorical)</li><li><b>Date</b>: Date of sampling (Field type: Date)</li><li><b>Time</b>: Time of sampling (Field type: Time)</li><li><b>Site</b>: Catchment Site; indicates metres upriver from SAFE Project hydrology datalogger (Field type: ID)</li><li><b>Otter</b>: Otter observed at site? 0=no, 1=yes (Field type: Abundance)</li><li><b>Spraint</b>: Otter faeces at site? 0=no, 1=yes (Field type: Abundance)</li><li><b>Den</b>: Den present at site? 0=no, 1=yes (Field type: Abundance)</li><li><b>Footprints</b>: Footprints at site? 0=no, 1=yes (Field type: Abundance)</li><li><b>Remains</b>: Remains of prey present? 0=no, 1=yes (Field type: Abundance)</li><li><b>Notes </b>: Any additional notes deemed important (Field type: Comments)</li></ul><br></li></ol><p><b>Date range: </b>2017-03-14 to 2017-04-11</p><p><b>Latitudinal extent: </b>4.6508 to 4.7255</p><p><b>Longitudinal extent: </b>117.5765 to 117.5980</p><p><b>Taxonomic coverage: </b><br> All taxon names are validated against the GBIF backbone taxonomy. If a dataset uses a synonym, the accepted usage is shown followed by the dataset usage in brackets. Taxa that cannot be validated, including new species and other unknown taxa, morphospecies, functional groups and taxonomic levels not used in the GBIF backbone are shown in square brackets.</p><div>Animalia<br> - Chordata<br> -  - Mammalia<br> -  -  - Carnivora<br> -  -  -  - Mustelidae<br></div><p></p

SOX9 has both conserved and novel roles in marsupial sexual differentiation

In addition to an essential role in chondrogenesis, SOX9 is a highly conserved and integral part of the testis determining pathway in human and mouse. To determine whether SOX9 is involved in sex determination in noneutherian mammals we cloned a marsupial orthologue and studied its expression. The tammar wallaby SOX9 gene proved to be highly conserved, and maps to a region of the tammar genome syntenic to human chromosome 17. Marsupial SOX9 transcripts were detected by RT-PCR in the developing limb buds and both the developing ovary and testis from the first sign of gonadal development through to adulthood. Northern blot, in situ hybridisation, and immunohistochemical analyses showed that SOX9 reaches high levels of expression in the developing testis, where it is confined to the Sertoli cell nuclei, and the brain. This is similar to the expression pattern seen in human and mouse embryos and is consistent with a conserved role for SOX9 in vertebrate brain, skeletal, and gonadal development. In addition, SOX9 was expressed in the developing scrotum and mammary gland primordium regions of the tammar up to the time of birth. SOX9 protein was also detected in the developing Wolffian duct epithelium in the male mesonephros. These previously undescribed locations of SOX9 expression suggest that SOX9 may play additional roles in the differentiation of the marsupial reproductive system

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present