A DNA barcoding framework for taxonomic verification in the Darwin Tree of Life Project.

Biodiversity genomics research requires reliable organismal identification, which can be difficult based on morphology alone. DNA-based identification using DNA barcoding can provide confirmation of species identity and resolve taxonomic issues but is rarely used in studies generating reference genomes. Here, we describe the development and implementation of DNA barcoding for the Darwin Tree of Life Project (DToL), which aims to sequence and assemble high quality reference genomes for all eukaryotic species in Britain and Ireland. We present a standardised framework for DNA barcode sequencing and data interpretation that is then adapted for diverse organismal groups. DNA barcoding data from over 12,000 DToL specimens has identified up to 20% of samples requiring additional verification, with 2% of seed plants and 3.5% of animal specimens subsequently having their names changed. We also make recommendations for future developments using new sequencing approaches and streamlined bioinformatic approaches.

Identifying species based solely on their morphology can be difficult. DNA-based identification using DNA barcoding can aid species identification, but can be challenging to implement in biodiversity projects sampling diverse organismal groups. Here, we describe the development and implementation of DNA barcoding for the Darwin Tree of Life Project (DToL), which aims to sequence and assemble high quality reference genomes for all eukaryotic species in Britain and Ireland. We discuss how a standardised approach has been adapted by each partner to suit different organismal groups, show the efficacy of this approach for confirming species identities and resolving taxonomic issues, and make recommendations for future developments.

Umbilical cord blood T cells can be isolated and enriched by CD62L selection for use in 'off the shelf' chimeric antigen receptor T-cell therapies to widen transplant options.

Umbilical cord blood (UCB) T cells exhibit distinct naive ontogenetic profiles and may be an attractive source of starting cells for the production of chimeric antigen receptor (CAR) T cells. Pre-selection of UCB-T cells on the basis of CD62L expression was investigated as part of a machine-based manufacturing process, incorporating lentiviral transduction, CRISPR-Cas9 editing, T-cell expansion and depletion of residual TCReeeT cells. This provided stringent mitigation against the risk of graft versus host disease (GVHD), and was combined with simultaneous knockout of CD52 to enable persistence of edited T cells in combination with preparative lymphodepletion using Alemtuzumab. Under compliant manufacturing conditions, two cell banks were generated with high levels of CAR19 expression and minimal carriage of TCReeeT cells. Sufficient cells were cryopreserved in dose-banded aliquots at the end of each campaign to treat dozens of potential recipients. Molecular characterisation captured vector integration sites and CRISPR editing signatures and functional studies, including in vivo potency studies in humanised mice, confirmed antileukaemic activity comparable to peripheral blood-derived universal CAR19 T cells. Machine manufactured UCB derived T cells banks offer an alternative to autologous cell therapies and could help widen access to CAR T cells.

Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti.

Introduction: Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects' biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Methods: Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase and a plasmid-based method for expressing PhiC31 when injected into early embryos. Results: Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard. Discussion: Here we have expanded the toolbox for synthetic biology in Ae. aegypti. These methods can be easily transferred into other mosquito and even insect species by identifying appropriate promoter sequences. This advances the ability to manipulate these insects for fundamental studies, and for more applied approaches for pest control.

Driving down malaria transmission with engineered gene drives.

The last century has witnessed the introduction, establishment and expansion of mosquito-borne diseases into diverse new geographic ranges. Malaria is transmitted by female Anopheles mosquitoes. Despite making great strides over the past few decades in reducing the burden of malaria, transmission is now on the rise again, in part owing to the emergence of mosquito resistance to insecticides, antimalarial drug resistance and, more recently, the challenges of the COVID-19 pandemic, which resulted in the reduced implementation efficiency of various control programs. The utility of genetically engineered gene drive mosquitoes as tools to decrease the burden of malaria by controlling the disease-transmitting mosquitoes is being evaluated. To date, there has been remarkable progress in the development of CRISPR/Cas9-based homing endonuclease designs in malaria mosquitoes due to successful proof-of-principle and multigenerational experiments. In this review, we examine the lessons learnt from the development of current CRISPR/Cas9-based homing endonuclease gene drives, providing a framework for the development of gene drive systems for the targeted control of wild malaria-transmitting mosquito populations that overcome challenges such as with evolving drive-resistance. We also discuss the additional substantial works required to progress the development of gene drive systems from scientific discovery to further study and subsequent field application in endemic settings.

Assessment of the Concentration of Endogenous Factors Regulating Angiogenesis, VASH-1 and VEGF-A, in the Blood Serum of Patients with Neuroendocrine Neoplasms.

Neuroendocrine neoplasms (NENs) constitute about 2% of all malignant neoplasms, and the angiogenesis process in these tumors is still of a great interest. Vasohibin-1 (VASH-1) is an angiogenesis inhibitor, while vascular endothelial growth factor A (VEGF-A) is one of the main factors promoting vascular formation. The subject of this study was to assess serum concentration of these factors in patients with diagnosed NEN and in control group. Methods. The study group consisted of 120 patients with diagnosed NENs, while the control group consisted of 69 healthy volunteers. The concentrations of VASH-1 and VEGF-A in serum were tested using the ELISA. We also analyzed the association of the concentration of these factors with demographic data (e.g., age and gender), body mass index (BMI), primary tumor location, histological grade, metastasis, clinical staging, selected biochemical parameters and markers of NENs, and information on smoking habits. Results. The mean concentration of VASH-1 was 218.8 ± 359.8 pg/ml in the study group and 973.1 ± 1239.4 pg/ml in the control group, that difference was statistically significant (p < 0.05). In the NEN group, the highest concentration of VASH-1 was in patients with pancreatic NENs in relation to NENs with different location of the primary tumor (p < 0.05). Negative correlation was found between the concentration of VASH-1 and serotonin (r S = -0.19, p < 0.05). No statistically significant differences were observed for VEGF-A (p = 0.658). Conclusions. Patients with NENs showed lower serum level of VASH-1 in comparison to healthy volunteers. The highest level of VASH-1 was observed in tumors localized in pancreas. This might reflect the relevant function of VASH-1 in NENs and requires further evaluation to further knowledge of angiogenesis in NENs. Furthermore, the serum concentration of VEGF-A showed no statistical differences and probably does not have diagnostic value in this group of patients.

The levels of sICAM-1, sELAM-1, TNFα and sTNFR1 proteins in patients with colorectal adenocarcinoma in tumor and corresponding normal mucosa.

Colorectal cancer is a common malign disease of the gastrointestinal tract. The cancer survival rate depends on the stage of the disease at detection time. It is well known that several molecular mechanisms are involved in cancer and some molecules might affect or modulate cancerogenesis. The aim of the study was to assess the levels of sICAM-1, sELAM-1, TNFα and sTNFR1 protein in tumor and corresponding normal mucosa in a group of patients with colorectal adenocarcinoma and also associations of these parameters with demographic and clinical profiles of the patients. Tissue specimens were obtained during resection of neoplastic lesions. Protein levels were assayed in tissue homogenates by ELISA. The protein level of sICAM-1 in tumor was significantly increased in comparison to the corresponding normal mucosa (80.06 ng/mg vs 69.53 ng/mg, p=0.02). Furthermore, a significant positive correlation between sICAM-1 and sTNFR1 proteins levels in tumor (rs=0.58, p<0.001) and in corresponding normal mucosa (rs=0.48, p<0.001) was found. Also, significant correlations in corresponding normal mucosa were found between sELAM-1 and sICAM-1 (rs=0.58, p<0.001) and between sTNFR1 and sELAM-1 (rs=0.57, p<0.001). Significantly higher level of sTNFR1 in corresponding normal mucosa samples of patients with distant metastases was observed (p=0.04). Obtained results suggest that sICAM-1 protein could be considered as colorectal cancer marker. Furthermore, sTNFR1 also has the potential to become a good prognostic marker used during monitoring of the patients. Nevertheless, a further study in this area to confirm this correlation is required.

Comparison of Selected Protein Levels in Tumour and Surgical Margin in a Group of Patients with Oral Cavity Cancer.

Oral cavity cancer belongs to head-and-neck squamous cell carcinoma group. The purpose of the study was to assess the levels of certain proteins in a tumour and surgical margin in a group of patients with oral cavity cancer. The levels of DAPK1, MGMT, CDH1, SFRP1, SFRP2, RORA, TIMP3, p16, APC and RASSF1 proteins were measured by ELISA in tissue homogenates. The protein levels of DAPK1, MGMT, CDH1, SFRP2 and RASSF1 were significantly higher in tumour tissue than in the margin, contrary to TIMP3 which was lower in the tumour itself. DAPK1 level in the tumour was significantly higher in females than in males, the MGMT and p16 levels were lower in the tumours with lymph node metastasis (N1 + N2) than in N0 samples. The CDH1 expression was higher in a group with smoking habits, whereas TIMP3 was lower in this group. Changes in the levels of proteins in tumour and surgical margin may be either reflective of tumour occurrence and development, or they might be also responsible for the progress and reoccurrence of the disease. Levels of the studied proteins might be good prognostic factors; however, further studies are required.

Ticks (Ixodida) from the collection of the Natural History Department, Museum of Upper Silesia in Bytom, Poland - A contribution to knowledge on tick fauna and the first record of Hyalomma marginatum presence in Poland.

There is no doubt that museum collections provide a wide variety of information on ticks. The tick collection at the Natural History Department of the Museum of Upper Silesia in Bytom consists only of 37 specimens as the department is focused mainly on building collections of insects and birds. However, this does not mean that such collection cannot contribute to our knowledge about these arthropods. The most valuable results of studies on the museum's tick collection concerned Polish fauna. There are specimens of I. ricinus dating back as far as 1930-1948, which are the first known records of the presence of this tick in the Upper Silesia. Two specimens collected in copula in 1941 might be the earliest record of the mating behaviour of this species in Poland. The most important result was the detection of 2 cases of H. marginatum presence in Poland, which by far are the oldest documented cases of its presence in this country.

Seasonal changes in the fatty acid profile of the tick Ixodes ricinus (Acari, Ixodidae).

Fatty acids (FAs) from nymphs, females and males of Ixodes ricinus were analysed by gas chromatography/mass spectrometry. Ticks were collected from May to October 2013. The most abundant FAs were 18:1, 18:0, 16:0 and 18:2 which are also dominant FAs of insects. Adults contained higher concentrations of FAs in general than nymphs because they contain more fat body and probably a thicker layer of epicuticular lipids. Larger quantities of FAs > 20 carbon atoms in the carboxylic chain were present in females, which generally show higher content of lipids essential for oogenesis, whereas there were similar amounts of 14-18 in both sexes. In September and October, ticks contained large concentrations of the majority of FAs except for 18:1, the most abundant one in ticks collected from May through August. Thus, most FAs, especially those with more than 20 C atoms, tend to increase at lower temperatures.

Prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from southern Poland.

In 2008-2011 ticks were collected from southern Poland. Out of 6336 individuals collected and identified as Ixodes ricinus, 768 (2 larvae, 84 nymphs, 417 females, 265 males) were included in molecular study. The aim of this study was to investigate the prevalence and types of genospecies of Borrelia burgdorferi sensu lato in ticks. The polymerase chain reaction (PCR) was applied to detect the presence of pathogens in ticks. Subsequently the amplified DNA was digested with TasI enzyme. The infection rate was 15% (116) of examined ticks. PCR-RFLP analysis allowed distinguishing three genospecies of B. burgdorferi s.l.: B. burgdorferi sensu stricto, B. afzelii, and B. garinii. RFLP analyses of 116 positive samples revealed 96 (83%) monoinfections and 13 (11%) coinfections, whereas unidentified genospecies were present in 7 (6%) of positive samples. In the case of monoinfections, B. burgdorferi s.s. was the predominant species of pathogen in infected ticks - 61.4%. Other genospecies: B. garinii and B. afzelii were detected in 22.9% and 15.6% of the samples, respectively. To sum up, 15 % of ticks were infected by B. burgdorferi s.l which increases the risk of human infections in the recreational areas of southern Poland. Furthermore, there is a need to increase public awareness and implement more preventive measures concerning Lyme disease.

Risk of exposure to ticks (Ixodidae) and the prevalence of tick-borne encephalitis virus (TBEV) in ticks in Southern Poland.

The article presents the results of the first study on seasonal activity of ticks and prevalence of tick-borne encephalitis virus (TBEV) in nymphs from the Silesian Province (Southern Poland). Previous studies on the prevalence of TBEV in ticks in Poland have been conducted mostly in northern and eastern regions, but none in the Silesian Province itself. The aims of this study were to analyse the seasonal variation in tick populations and compare TBEV prevalence in nymphs from different geographical locations in the Silesia. A total of 5160 questing Ixodes ricinus ticks were collected by the flagging method from 23 localities in southern Poland in 2010. Micro-climatic parameters (air temperature and humidity) were measured in order to estimate their influence on tick population. The highest tick activity was recorded in spring and was positively correlated with relative air humidity (RH). TBEV in the Silesian Province was analysed in 1750 nymphs and an overall prevalence was 0.11% (2 pools out of 175 analysed). The results of this study show that TBEV pool prevalence in nymphs is low in accordance with the low number of TBE cases reported within the region.

Selected mechanisms of molecular resistance of Candida albicans to azole drugs.

A phenomenon of increasing resistance of Candida spp. to azoles has been observed for several years now. One of the mechanisms of lack of sensitivity to azoles is associated with CDR1, CDR2, MRD1 genes (their products are active transport pumps conditioning drug efflux from pathogen's cell), and ERG11 gene (encoding lanosterol 14α-demethylase). Test material was 120 strains of Candida albicans (60 resistant and 60 susceptible to azole drugs) obtained from clinical samples. The first stage of experiment assessed the expression of CDR1, CDR2, MDR1 and ERG11 genes by Q-PCR. The impact of ERG11 gene's mutations on the expression of this gene was analysed. The final stage of the experiment assessed the level of genome methylation of Candida albicans strains. An increase in the expression of CDR2, MDR1 and ERG11 was observed in azole-resistant strains of Candida albicans in comparison to strains sensitive to this class of drugs. Furthermore, 19 changes in the sequence of ERG11 were detected in tested strains. Four of the discovered mutations: T495A, A530C, G622A and A945C led to the following amino acid substitutions: D116E, K128T, V159I and E266D, respectively. It has also been found that statistically five mutations: T462C, G1309A, C216T, C1257T and A945C affected the expression of ERG11. The applied method of assessing the level of methylation of Candida albicans genome did not confirm its role in the development of resistance to azoles. The results indicate however, that resistance of Candida albicans strains to azole drugs is multifactorial.

Detection of protozoans Babesia microti and Toxoplasma gondii and their co-existence in ticks (Acari: Ixodida) collected in Tarnogórski district (Upper Silesia, Poland).

INTRODUCTION: Ticks (Acari: Ixodida) are vectors of many pathogens i. a: Babesia microti [Bm] and Toxoplasma gondii [Tg]. In Poland, Ixodes ricinus [Ir] is the main vector of both pathogens. This tick species and pathogens transmitted by them are a significant threat to human and animal health. OBJECTIVES OF THE STUDY: Detection of the protozoans Bm and Tg in ticks collected in the Tarnogórski district area. MATERIAL AND METHODS: The ticks were collected from vegetation and pets in the spring period of their activity on the territory of the Tarnogórski district. The parasites were preserved in 70% ethanol. DNA was isolated by ammonia method. Bm was detected by nested-PCR using specific primers for the 18S rRNA sequence. To detect the Tg a commercial kit was used. The PCR products were separated on 2% ethidium bromide stained agarose gels and visualised under UV light. RESULTS: It was showed that all collected ticks belonged to the species Ir. Bm was detected in 50.87% and Tg in 64.91% of all examined ticks. Co-existence of these both protozoans in 36.84% of total examined ticks was noted. CONCLUSIONS: The study showed a high risk of exposure to Bm and Tg in the studied area. Ticks Ir play an important role in the transmission of Bm in this region. Demonstrating a high percentage of ticks collected from animals infected with Tg may indicate their important role in the transmission of this pathogen, but it requires a further studies.

The risk of exposure to Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Babesia sp. and co-infections in Ixodes ricinus ticks on the territory of Niepolomice forest (southern Poland).

Niepolomice Forest is located about 20 kilometers east of Cracow (Malopolska province, southern Poland). Its natural and touristic values, as well as wide range of hosts occurring within indicate this to be an area of high risk of exposure to Ixodes ricinus and tick-borne diseases it transfers. I. ricinus is a common species in Poland and Europe. Its seasonal activity begins in Poland in the early spring, and ends with late autumn. A total number of 129 specimens of I. ricinus was collected by flagging in Niepolomice Forest. DNA was isolated by ammonia method from 30 randomly-selected individuals. PCR was used to detect tick-borne pathogens with primers specific for Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and Babesia sp. Molecular studies confirmed the presence of all three pathogens in I. ricinus. A. phagocytophilum was found in 76.7%, Babesia sp., 60%, B. burgdorferi s. l., in 3.3% of studied ticks. A. phagocytophilum co-infection with Babesia sp., was found in 46.7% of the specimens. A co-infection of all three tested pathogens was recorded in one case (3.3%). In Poland the problem of tick-borne diseases is a growing issue, therefore people residing in southern Polish touristic areas should be informed about the prevention and protection against ticks.

The first record and occurrence of the ornate cow tick Dermacentor reticulatus (Fabricius, 1794) in south-western Poland.

The following report concerns the first record and occurrence of an ornate cow tick (Dermacentor reticulatus) in the Silesian province. One female and one male were collected from a dog in the veterinary clinic in Racib6rz, while in the vicinity of Zywiec one female tick was found also on a dog. Ticks collected in Racib6rz are most probably part of the local fauna, while the specimen from Zywiec may have come from the Warsaw area, the source of the host. These findings confirm changes of European distribution recorded lately for this species and the role of humans in this process. However, they need to be supported by more thorough research involving collection by flagging and/or other hosts such as wild animals.

Prevalence of tick borne encephalitis virus in tick nymphs in relation to climatic factors on the southern coast of Norway.

BACKGROUND: Tick-borne encephalitis (TBE) is among the most important vector borne diseases of humans in Europe and is currently identified as a major health problem in many countries. TBE endemic zones have expanded over the past two decades, as well as the number of reported cases within endemic areas. Multiple factors are ascribed for the increased incidence of TBE, including climatic change. The number of TBE cases has also increased in Norway over the past decade, and the human cases cluster along the southern coast of Norway. In Norway the distribution and prevalence of TBE virus (TBEV) in tick populations is largely unknown. The objectives of this study were to estimate the TBEV prevalence in Ixodes ricinus from seven locations and to assess the relationship between the TBEV prevalence and site-specific climatic variables. METHODS: A total of 5630 questing nymphs were collected and analyzed in pools of ten. All pools were screened with an in-house real-time RT-PCR, and the positive pools were pyrosequenced. Two methods, minimum infection rate (MIR) and a frequentist method (EPP) for pooled prevalence estimations were calculated and compared. Climatic data were descriptively compared to the corresponding EPP of each location in order to explain variations in TBEV prevalence. RESULTS: The seven foci of TBEV had an estimated overall prevalence (EPP) in pools of nymphs combined, of 0.53% with 95% CI (0.35-0.75), with point prevalence ranging between 0.11%-1.22%. The sites with the highest point prevalences were within the municipalities which had the highest numbers of registered TBE cases. The results indicate that the location with highest point prevalence had the highest relative mean humidity and lowest mean saturation deficit and vice versa for the lowest EPP. CONCLUSION: Our study confirms the existence of TBEV endemic foci in Norway. These results are of importance to increase the awareness of TBEV infections in Norway and could be used for public information and recommendations of TBE vaccination. EPP is the method of choice for pooled prevalence calculations, since it provides estimated prevalences with confidence intervals. Our findings emphasise the possible importance of microclimatic conditions regarding the TBEV prevalence in ticks.