Trends and biases in African large carnivore population assessments: identifying priorities and opportunities from a systematic review of two decades of research.

African large carnivores have undergone significant range and population declines over recent decades. Although conservation planning and the management of threatened species requires accurate assessments of population status and monitoring of trends, there is evidence that biodiversity monitoring may not be evenly distributed or occurring where most needed. Here, we provide the first systematic review of African large carnivore population assessments published over the last two decades (2000-2020), to investigate trends in research effort and identify knowledge gaps. We used generalised linear models (GLMs) and generalised linear mixed models (GLMMs) to identify taxonomic and geographical biases, and investigated biases associated with land use type and author nationality. Research effort was significantly biased towards lion (Panthera leo) and against striped hyaena (Hyaena hyaena), despite the latter being the species with the widest continental range. African wild dog (Lycaon pictus) also exhibited a negative bias in research attention, although this was partly explained by its relatively restricted distribution. The number of country assessments for a species was significantly positively associated with its geographic range in that country. Population assessments were biased towards southern and eastern Africa, particularly South Africa and Kenya. Northern, western, and central Africa were generally under-represented. Most studies were carried out in photographic tourism protected areas under government management, while non-protected and trophy hunting areas received less attention. Outside South Africa, almost half of studies (41%) did not include authors from the study country, suggesting that significant opportunities exist for capacity building in range states. Overall, large parts of Africa remain under-represented in the literature, and opportunities exist for further research on most species and in most countries. We develop recommendations for actions aimed at overcoming the identified biases and provide researchers, practitioners, and policymakers with priorities to help inform future research and monitoring agendas.

Optimising the cost of roadkill surveys based on an analysis of carcass persistence.

Reliable estimates of wildlife mortality due to wildlife-vehicle collisions are key to understanding its impact on wildlife populations and developing strategies to prevent or reduce collisions. Standardised approaches for monitoring roadkill are needed to derive robust and unbiased estimates of mortality that are comparable across different study systems and ecological contexts. When designing surveys, there is a trade-off between survey frequency (and hence logistical effort and financial cost) and carcass detection. In this regard, carcass persistence (the period a carcass remains detectable before being removed by decomposition or scavengers) is important; the longer a carcass persists, the greater the likelihood it will be detected with lower survey effort by conducting more infrequent surveys. Using multi-taxon carcass data collected over a month of repeated driven surveys, combined with five covariates (species functional group, body weight, carcass position on road, carcass condition [either flattened or not after impact], and rainfall prior to each survey), we explored the drivers of carcass persistence with the overall aim of providing information to optimise the design of carcass surveys along linear infrastructure. Our methodological approach included a survival analysis to determine carcass persistence, linear regressions to test the effect of covariates, a subsampling analysis (using field data and a simulation exercise) to assess how the proportion of carcasses detected changes according to survey frequency, and an analysis to compare the costs of surveys based on study duration, transect length and survey frequency. Mean overall carcass persistence was 2.7 days and was significantly correlated with position on road and within-functional group body weight. There was no evidence for a significant effect of rainfall, while the effect of carcass condition was weakly non-significant. The proportion of carcasses detected decreased sharply when survey intervals were longer than three days. However, we showed that survey costs can be reduced by up to 80% by conducting non-daily surveys. Expanding on the call for a standardised methodology for roadkill surveys, we propose that carcass persistence be explicitly considered during survey design. By carefully considering the objectives of the survey and characteristics of the focal taxa, researchers can substantially reduce logistical costs. In addition, we developed an R Shiny web app that can be used by practitioners to compare survey costs across a variety of survey characteristics. This web app will allow practitioners to easily assess the trade-off between carcass detection and logistical effort.

ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells.

Management of infection with hepatitis B virus (HBV) remains a global health problem. Persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is responsible for modest curative efficacy of currently licensed drugs. Novel gene editing technologies, such as those based on CRISPR/Cas9, provide the means for permanently disabling cccDNA. However, efficient delivery of antiviral sequences to infected hepatocytes is challenging. A limiting factor is the large size of sequences encoding Cas9 from Streptococcus pyogenes, and resultant incompatibility with the popular single stranded adeno-associated viral vectors (ssAAVs). We thus explored the utility of ssAAVs for delivery of engineered CRISPR/Cas9 of Staphylococcus aureus (Sa), which is encoded by shorter DNA sequences. Short guide RNAs (sgRNAs) were designed with cognates in the S open reading frame of HBV and incorporated into AAVs that also encoded SaCas9. Intended targeted mutation of HBV DNA was observed after transduction of cells with the all-in-one vectors. Efficacy against HBV-infected hNTCP-HepG2 cells indicated that inactivation of cccDNA was successful. Analysis of likely off-target mutagenesis revealed no unintended sequence changes. Use of ssAAVs to deliver all components required to disable cccDNA by SaCas9 is novel and the technology has curative potential for HBV infection.

Tracking data from nine free-roaming Cheetahs (Acinonyx jubatus) collared in the Thabazimbi area, Limpopo Province, South Africa.

BACKGROUND: In partnership with the University of Pretoria, the Endangered Wildlife Trust's Carnivore Conservation Programme collared six male and three female free-roaming Cheetahs (Acinonyx jubatus) in the Thabazimbi area in Limpopo Province, South Africa. This study was undertaken to determine the spatial ecology of free-roaming Cheetahs that occur outside of formal protected areas on private ranchland, where they frequently come into conflict with, and are sometimes killed by, private landowners. The data were collected between September 2003 and November 2008, resulting in a total of 3165 location points (65 points from VHF collars and 3100 from GPS collars) for nine individual Cheetahs. NEW INFORMATION: This dataset provides distribution information about this Vulnerable species occurring outside of protected areas within South Africa. The dataset has been published to the Global Biodiversity Information Facility (www.GBIF.org) and provides the largest dataset on Cheetahs thus far, and, although it is spatially limited to a relatively small region on the African continent, it is the first study of its kind within South Africa. Also of significance is that the fate of 6 of the 9 collared Cheetahs is known, all except one of which died of anthropogenic causes.

The African Crane Database (1978-2014): Records of three threatened crane species (Family: Gruidae) from southern and eastern Africa.

BACKGROUND: The International Crane Foundation (ICF) / Endangered Wildlife Trust's (EWT) African Crane Conservation Programme has recorded 26 403 crane sightings in its database from 1978 to 2014. This sightings collection is currently ongoing and records are continuously added to the database by the EWT field staff, ICF/EWT Partnership staff, various partner organizations and private individuals. The dataset has two peak collection periods: 1994-1996 and 2008-2012. The dataset collection spans five African countries: Kenya, Rwanda, South Africa, Uganda and Zambia; 98% of the data were collected in South Africa. Georeferencing of the dataset was verified before publication of the data. The dataset contains data on three African crane species: Blue Crane Anthropoides paradiseus, Grey Crowned Crane Balearica regulorum and Wattled Crane Bugeranus carunculatus. The Blue and Wattled Cranes are classified by the IUCN Red List of Threatened Species as Vulnerable and the Grey Crowned Crane as Endangered. NEW INFORMATION: This is the single most comprehensive dataset published on African Crane species that adds new information about the distribution of these three threatened species. We hope this will further aid conservation authorities to monitor and protect these species. The dataset continues to grow and especially to expand in geographic coverage into new countries in Africa and new sites within countries. The dataset can be freely accessed through the Global Biodiversity Information Facility data portal.

Improved antiviral efficacy using TALEN-mediated homology directed recombination to introduce artificial primary miRNAs into DNA of hepatitis B virus.

Chronic infection with hepatitis B virus (HBV) remains an important global health problem. Currently licensed therapies have modest curative efficacy, which is as a result of their transient effects and limited action on the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Gene editing with artificial HBV-specific endonucleases and use of artificial activators of the RNA interference pathway have shown anti-HBV therapeutic promise. Although results from these gene therapies are encouraging, maximizing durable antiviral effects is important. To address this goal, a strategy that entails combining gene editing with homology-directed DNA recombination (HDR), to introduce HBV-silencing artificial primary microRNAs (pri-miRs) into HBV DNA targets, is reported here. Previously described transcription activator-like effector nucleases (TALENs) that target the core and surface sequences of HBV were used to introduce double stranded breaks in the viral DNA. Simultaneous administration of donor sequences encoding artificial promoterless anti-HBV pri-miRs, with flanking arms that were homologous to sequences adjoining the TALENs' targets, augmented antiviral efficacy. Analysis showed targeted integration and the length of the flanking homologous arms of donor DNA had a minimal effect on antiviral efficiency. These results support the notion that gene editing and silencing may be combined to effect improved inhibition of HBV gene expression.

CRISPR-Cas: Revolutionising genome engineering.

The ability to permanently alter or repair the human genome has been the subject of a number of science fiction films, but with the recent advent of several customisable sequence-specific endonuclease technologies, genome engineering looks set to become a clinical reality in the near future. This article discusses recent advancements in the technology called 'clustered regularly interspaced palindromic repeat (CRISPR)-associated genes' (CRISPR-Cas), the potential of CRISPR-Cas to revolutionise molecular medicine, and the ethical and regulatory hurdles facing its application.

The role of long non-coding RNAs in hepatitis B virus-related hepatocellular carcinoma.

Chronic infection with hepatitis B virus (HBV) is a major risk for development of hepatocellular carcinoma (HCC), which is the fifth most common cancer and a leading global cause of mortality. Long noncoding RNAs (lncRNAs) are regulators of complex biological processes and their functional disruption is implicated in the etiology of many cancers including HCC. Several lncRNAs have been shown to have oncogenic or tumor suppressive roles and have recently become the focus of intense investigation. However, the contributions of lncRNAs to HBV-related HCC remain to be fully elucidated. In this review we concentrate on the functional roles of various lncRNAs in HBV-associated HCC. Their involvement in viral replication, the specific association of certain lncRNAs with HBV-related HCC, potential utility as therapeutic targets and diagnostic markers are discussed.

Progress and prospects of engineered sequence-specific DNA modulating technologies for the management of liver diseases.

Liver diseases are one of the leading causes of mortality in the world. The hepatic illnesses, which include inherited metabolic disorders, hemophilias and viral hepatitides, are complex and currently difficult to treat. The maturation of gene therapy has heralded new avenues for developing effective intervention for these diseases. DNA modification using gene therapy is now possible and available technology may be exploited to achieve long term therapeutic benefit. The ability to edit DNA sequences specifically is of paramount importance to advance gene therapy for application to liver diseases. Recent development of technologies that allow for this has resulted in rapid advancement of gene therapy to treat several chronic illnesses. Improvements in application of derivatives of zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), homing endonucleases (HEs) and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated (Cas) systems have been particularly important. These sequence-specific technologies may be used to modify genes permanently and also to alter gene transcription for therapeutic purposes. This review describes progress in development of ZFPs, TALEs, HEs and CRISPR/Cas for application to treating liver diseases.

The recA operon: A novel stress response gene cluster in Bacteroides fragilis.

Bacteroides fragilis, an opportunistic pathogen of humans, is a leading cause of bacteraemias and anaerobic abscesses which are often treated with metronidazole, a drug which damages DNA. This study investigated the responses of the B. fragilis recA three gene operon to the stress experienced during metronidazole treatment and exposure to reactive oxygen species simulating those generated by the host immune system during infection. A transcriptionally regulated response was observed using quantitative RT-PCR after metronidazole and hydrogen peroxide treatment, with all three genes being upregulated under stress conditions. In vivo and in vitro analysis of the functional role of the second gene of the operon was done using heterologous complementation and protein expression (in Escherichia coli), with subsequent biochemical assay. This gene encoded a functional bacterioferritin co-migratory protein (BCP) which was thiol-specific and had antioxidant properties, including protection of the glutamine synthetase III enzyme. This in vitro data supports the hypothesis that the genes of the operon may be involved in protection of the bacteria from the oxidative burst during tissue invasion and may play a significant role in bacterial survival and metronidazole resistance during treatment of B. fragilis infections.

Bacteroides fragilis RecA protein overexpression causes resistance to metronidazole.

Bacteroides fragilis is a human gut commensal and an opportunistic pathogen causing anaerobic abscesses and bacteraemias which are treated with metronidazole (Mtz), a DNA damaging agent. This study examined the role of the DNA repair protein, RecA, in maintaining endogenous DNA stability and its contribution to resistance to Mtz and other DNA damaging agents. RT-PCR of B. fragilis genomic DNA showed that the recA gene was co-transcribed as an operon together with two upstream genes, putatively involved in repairing oxygen damage. A B. fragilis recA mutant was generated using targeted gene inactivation. Fluorescence microscopy using DAPI staining revealed increased numbers of mutant cells with reduced intact double-stranded DNA. Alkaline gel electrophoresis of the recA mutant DNA showed increased amounts of strand breaks under normal growth conditions, and the recA mutant also showed less spontaneous mutagenesis relative to the wild type strain. The recA mutant was sensitive to Mtz, ultraviolet light and hydrogen peroxide. A B. fragilis strain overexpressing the RecA protein exhibited increased resistance to Mtz compared to the wild type. This is the first study to show that overexpression of a DNA repair protein in B. fragilis increases Mtz resistance. This represents a novel drug resistance mechanism in this bacterium.