Direct Counting Research Articles

Transcriptomic gene signatures measure satellite cell activity in muscular dystrophies

The routine need for myonuclear turnover in skeletal muscle, together with more sporadic demands for hypertrophy and repair, are performed by resident muscle stem cells called satellite cells. Muscular dystrophies are characterized by muscle wasting, stimulating chronic repair/regeneration by satellite cells. Here, we derived and validated transcriptomic signatures for satellite cells, myoblasts/myocytes, and myonuclei using publicly available murine single cell RNA-Sequencing data. Our signatures distinguished disease from control in transcriptomic data from several muscular dystrophies including facioscapulohumeral muscular dystrophy (FSHD), Duchenne muscular dystrophy, and myotonic dystrophy type I. For FSHD, the expression of our gene signatures correlated with direct counts of satellite cells on muscle sections, as well as with increasing clinical and pathological severity. Thus, our gene signatures enable the investigation of myogenesis in bulk transcriptomic data from muscle biopsies. They also facilitate study of muscle regeneration in transcriptomic data from human muscle across health and disease.

Efficiency of moderately hypofractionated radiotherapy in NSCLC cell model.

The current standard of radiotherapy for inoperable locally advanced NSCLCs with single fraction doses of 2.0 Gy, results in poor outcomes. Several fractionation schedules have been explored that developed over the past decades to increasingly more hypofractionated treatments. Moderate hypofractionated radiotherapy, as an alternative treatment, has gained clinical importance due to shorter duration and higher patient convenience. However, clinical trials show controversial results, adding to the need for pre-clinical radiobiological studies of this schedule. We examined in comparative analysis the efficiency of moderate hypofractionation and normofractionation in four different NSCLC cell lines and fibroblasts using several molecular-biological approaches. Cells were daily irradiated with 24x2.75 Gy (moderate hypofractionation) or with 30x2 Gy (normofractionation), imitating the clinical situation. Proliferation and growth rate via direct counting of cell numbers, MTT assay and measurements of DNA-synthesizing cells (EdU assay), DNA repair efficiency via immunocytochemical staining of residual γH2AX/53BP1 foci and cell surviving via clonogenic assay (CSA) were experimentally evaluated. Overall, the four tumor cell lines and fibroblasts showed different sensitivity to both radiation regimes, indicating cell specificity of the effect. The absolute cell numbers and the CSA revealed significant differences between schedules (P < 0.0001 for all employed cell lines and both assays) with a stronger effect of moderate hypofractionation. Our results provide evidence for the similar effectiveness and toxicity of both regimes, with some favorable evidence towards a moderate hypofractionation. This indicates that increasing the dose per fraction may improve patient survival and therapy outcomes.

Long monitoring of Birds of Elssuki Area. Sinnar State, Sudan

This study aims to make a database of the birds in the area of Elssuki, Sinnar state - Sudan. The period of study spanning from 2008 to 2023 with a mix of methods used to identify bird species in many sites in the locality, these methods include road count, line transects, and direct count besides registering every strange, rare, or unusual single species seen in the area. All these methods are used by different researchers and applied in such studies in Sudan. The study revealed that the area is one of the important areas enriched of birds in Sinnar state of 19 orders 53 families. The total number of species is 129 species. It included all birds; water birds, tree birds, diving birds, dabbling birds, swimming birds, small waders, and passerines which the most. The study concluded that there is a need for comprehensive and regular studies and short and long-term monitoring to identify, classify, and establish a database for Sudan Birds Atlas.

High-speed railway infrastructure leads to species-specific changes and biotic homogenisation in surrounding bird community.

Linear infrastructure networks, including railways, are undergoing rapid development in order to connect distant urban areas. Particularly, High-Speed Railways are increasingly seen as a viable alternative to domestic flights in many countries. However, this development of linear infrastructures is known to affect the surrounding faunal communities due to the changes in the landscape and operation of said linear infrastructures. Both positive and negative effects of linear infrastructures on adjacent faunal communities have been reported. In this study, we determined the influence of the High-Speed Railway infrastructure on the bird community that surrounds it. Birds were surveyed by using both linear transect and direct counting methods, both in the area directly adjacent to the railway infrastructure and 500m away from it in a period of two years of surveys. A total of 16114 individuals belonging to 71 species were recorded. The presence of the High-Speed Railway caused species-specific changes in the bird communities that surround it, causing the attraction of some species and the rejection of others. Furthermore, we show that the presence of the infrastructure altered the natural species turnover as the landscape changes by attracting the same bird species regardless of changes in the landscape, and filtering out others. We propose that further work in mitigation and development plans should focus on species-specific measures to assess the risk bird communities are exposed to.

Observation on identification of birds and reptile in Taya - Basonda – Al galabat National Park, Gedaref State - Sudan

Birds are significant members of many ecosystems. They play a vital role incontrolling pests, acting as pollinators, and maintaining ecology while reptiles are important both aspredator and prey species. This study was conducted in Taya - Basonda - Al galabat National Park in March and December 2019, Gedaref state - Sudan. It aims to identify reptiles and birds and determine their diversity and richness in Taya - Basonda - Al galabat National Park.The direct count method and inventory were used to count birds and reptiles through observations across the park in March and December. Identification tools were used. The result revealed that, Seventy-two birds and sixteen reptiles species belonging to 35 and 9 families respectively were observed and identified out of 2915 and 1025 bird’s individuals in December and March respectively, and 290 and 155 reptile’s individuals in December and March respectively. The highest richness of birds was recorded in December (69 species) while in March (44 species), and highest number of reptiles recorded in December (16 species) compared to 11 species in March. The highest Shannon diversity index (H') was reported in Taya (3.23) in March compared to Basonda and Al galabat. The park faces major threats such as human settlement, expansion of agricultural schemes, hunting, fishing, grazing of livestock and fire. The study recommended preservation of natural habitat for birds in particular and wildlife in general by stopping all human activities in the park and setting laws and firm enforcement against violators.

Indirect Questioning Technique Related to Sensitive Quantitative Variables with Options for Direct, Randomized and Item Count Responses

Randomized Response (RR) Technique (RRT) pioneered by Warner (1965) is a useful tool to elicit responses on sensitive characteristics, such as induced abortions, drug abuse, drunken driving, total amount of counterfeit notes of a particular denomination held by individuals in the population, etc. There exists a huge literature on Randomized Response (RR) devices for estimation of finite population mean of quantitative variables, sensitive in nature mostly based on Eichhorn and Hayre (1983). Device-I and Device-II vide Chaudhuri and Christofides (2013) allow estimation of population mean of sensitive quantitative variables using sample chosen by a general sampling design. On the other hand, Item Count Technique (ICT), described elaborately in Chaudhuri and Christofides (2013), is an alternative to RRT for respondents who do not choose to provide RRs. While some respondents may find a variable as sensitive, others may find it innocuous enough to provide a direct response (DR) about his/her true value. In such a case, Optional Randomized Response (ORR) Technique (ORRT) with options for DR and RR was introduced by Chaudhuri and Mukherjee (1985). Pal (2007) proposed an ORR device which offers choices for RR and ICT to the respondents for giving their answers. A new ORRT with options for DR, RR and ICT was provided by Shaw and Pal (2021) for eliciting indirect responses on sensitive characteristics. As this device relates to estimation of population proportion of sensitive characteristics, an attempt has been made to extend it for sensitive quantitative variables. Further, to take care of individuals’ varying choices for DR, RR and ICT and to protect the privacy of the respondents’ choices, this paper develops an ORR device allowing the respondents chosen by a general sampling design, to choose any one of the three options according to their choices. International Journal of Statistical Sciences, Vol.24(1), March, 2024, pp 155-170

From solution to structure: empowering inclusive cryo-EM with a pre-characterization pipeline for biological samples.

In addressing the challenges faced by laboratories and universities with limited (or no) cryo-electron microscopy (cryo-EM) infrastructure, the ESRF, in collaboration with the Grenoble Institute for Structural Biology (IBS), has implemented the cryo-EM Solution-to-Structure (SOS) pipeline. This inclusive process, spanning grid preparation to high-resolution data collection, covers single-particle analysis and cryo-electron tomography (cryo-ET). Accessible through a rolling access route, proposals undergo scientific merit and technical feasibility evaluations. Stringent feasibility criteria demand robust evidence of sample homogeneity. Two distinct entry points are offered: users can either submit purified protein samples for comprehensive processing or initiate the pipeline with already vitrified cryo-EM grids. The SOS pipeline integrates negative stain imaging (exclusive to protein samples) as a first quality step, followed by cryo-EM grid preparation, grid screening and preliminary data collection for single-particle analysis, or only the first two steps for cryo-ET. In both cases, if the screening steps are successfully completed, high-resolution data collection will be carried out using a Titan Krios microscope equipped with a latest-generation direct electron counting detector coupled to an energy filter. The SOS pipeline thus emerges as a comprehensive and efficient solution, further democratizing access to cryo-EM research.

Abstract 7551: Combined epigenetic therapy and immune checkpoint blockade drive reshaping of the tumor microenvironment of platinum resistant ovarian cancer

Abstract Introduction: Immunotherapy has demonstrated modest effectiveness for patients with platinum-resistant ovarian cancer, which is in part related to the immunosuppressive tumor microenvironment (TME) of ovarian cancers. Epigenetic modulators in combination with immune checkpoint inhibitors (ICIs) may represent a potential way to sensitize ovarian cancer to immunotherapy by TME reprogramming. Methods: We evaluated the effect of epigenetic modulation in combination with ICI by comprehensive transcriptomic analyses of serially biopsied platinum-resistant/refractory ovarian cancer. Leveraging serial tumor samples from 30 patients that received oral azacitidine and pembrolizumab in the TRIO026 phase II clinical trial (NCT02900560), we performed bulk RNA sequencing and direct digital counting of 770 target genes using molecular barcodes (nCounter, NanoString) for 72 serial tumor samples, prior to treatment initiation and 6 weeks on-therapy. RNA sequencing of ~200 million reads for each sample was used for differential gene expression and gene set enrichment analysis (GSEA) and cell type deconvolution. Target gene expression data was normalized and analyzed for differential gene and pathway expression. Results: Differential gene expression analyses revealed an upregulation of inflammatory and cytolytic genes (IFNG, GZMA and GZMH; FDR-adjusted p=0.0184) as well as the co-inhibitory molecule CTLA-4 (FDR-adjusted p=0.0184). GSEA revealed an enrichment of IFNG response, natural killer cell mediated cytotoxicity, neutrophil degranulation, inflammatory response, and antigen processing/presentation related gene sets on-therapy (FDR-adjusted p &amp;lt; 0.001). Target gene expression analyses orthogonally confirmed these findings, which were also concordant with pathway scoring analyses showing an increased score for immune inflammatory pathways such as INFG signaling (Wilcoxon rank sum test p=0.01). Immune cell subset deconvolution suggested TME reshaping driven by a greater density of intra-tumoral T cells on-therapy (Wilcoxon rank sum test p=0.012). Higher IFNG, CXCL13, CXCR5 expression, CD8 T cell and NK cell density were associated with longer time on treatment, both at baseline and at the on-therapy timepoint. GSEA revealed an upregulation of interferon gamma, adaptive immunity, antigen presentation as well as conserved immune response gene sets on-therapy, for the patients attaining longer time on-treatment. Conclusion: Combination epigenetic and immunotherapy induces an inflammatory response and reshaping of the tumor microenvironment that may enhance their clinical efficacy, highlighting the therapeutic potential of utilizing epigenetic modulators as a way to sensitize platinum-resistant ovarian cancer to immune checkpoint inhibition. Citation Format: Blair V. Landon, Guneet Kaleka, Archana Balan, Julia L. Boland, Christopher Cherry, Gavin Pereira, Cynthia Zahnow, Boris Winterhoff, Stephen Baylin, Victor E. Velculescu, Gottfried E. Konecny, John A. Glaspy, Valsamo Anagnostou. Combined epigenetic therapy and immune checkpoint blockade drive reshaping of the tumor microenvironment of platinum resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7551.

The effect of using nano and microbubbles as aeration strategies on the nitrification process, microbial community composition, and growth of Penaeus vannamei in a super-intensive biofloc system

The aim of this study was to evaluate the effect of using nano and microbubbles as aeration strategies on the nitrification process, microbial community composition of water and biofilm, microbial flocs proximate composition, and growth of Penaeus vannamei in a pilot scale super-intensive system with biofloc technology. A grow-out (stocking density: 450 shrimp m−3) was carried out for 74 days in a greenhouse with nine 35 m3 tanks, using the following treatments: T1: nanobubbles, T2: microbubbles, T3: mixed (nano + microbubbles). The nanobubbles were generated by nozzle-type air injectors and the microbubbles were generated by microperforated hoses. Artificial substrates, with an area equivalent to 200% of the lateral surface, were used into the experimental units. Molasses was used as an organic carbon source at a carbon:nitrogen ratio of 15:1. The main morphotypes of bacteria in the water and in the biofilm were analyzed using direct counting and a profile of the bacterial composition of the water was performed using metagenomic analysis. Control of total ammonia nitrogen (TAN) was better in treatments T2 and T3 than in T1. At the end of the experiment, treatment T3 showed a higher total abundance of bacteria in the water than T1 and T2. Bacillus abundance in the biofilm was higher in the T3 treatment than in T1 and T2. The relative abundance of nitrifying bacteria was <1% in all treatments. Treatment T1 had a higher relative abundance of nitrite-oxidizing bacteria than the others. However, the T3 treatment had a better distribution of nitrifying bacteria species. T3 treatment showed a higher diversity and evenness of operational taxonomic units. The microbial flocs in the T2 had a higher crude protein content than in the T1. These results indicate that the use of a mixed aeration system promotes a higher load of microorganisms, which has proven to improve water quality and can influence shrimp growth. The shrimp final weight was higher in T1 (12.57 g) and T3 (12.54 g) than in T2 (11.70 g), survival was higher in T3 (97.17%) than in T1 (80.33%), and yield was higher in T3 (4.61 Kg m−3) than in T1 (4.06 Kg m−3) and T2 (4.21 Kg m−3). The treatment that used nano and microbubble as aeration strategy (T3) proved to be the best as it provided better TAN control, higher abundance of bacillus bacteria in the biofilm, higher diversity and evenness of bacteria, and even higher shrimp growth and survival.

Development and validation of a DNA-based multi-species biomonitoring toolkit using a high-throughput qPCR platform: A case study of Irish shellfish species.

Biomonitoring of marine life has been enhanced in recent years by the integration of innovative DNA-based approaches, which offer advantages over more laborious techniques (e.g. microscopy). However, trade-offs between throughput, sensitivity and quantitative measurements must be made when choosing between the prevailing molecular methodologies (i.e. metabarcoding or qPCR/dPCR). Thus, the aim of the present study was to demonstrate the utility of a microfluidic-enabled high-throughput quantitative PCR platform (HTqPCR) for the rapid and cost-effective development and validation of a DNA-based multi-species biomonitoring toolkit, using larvae of 23 commercially targeted bivalve and crustacean species as a case study. The workflow was divided into three main phases: definition of (off-) target taxa and establishment of reference databases (PHASE 1); selection/development and assessment of molecular assays (PHASE 2); and protocol optimization and field validation (PHASE 3). 42 assays were eventually chosen and validated. Genetic signal not only showed good correlation with direct visual counts by microscopy but also showed the ability to provide quantitative data at the highest taxonomic resolution (species level) in a time- and cost-effective fashion. This study developed a biomonitoring toolkit, demonstrating the considerable advantages of this state-of-the-art technology in boosting the developmental testing and application of panels of molecular assays for the monitoring and management of natural resources. Once developed, this approach provides a cost and time-effective alternative compared to other multi-species approaches (e.g. metabarcoding). In addition, it is transferable to a wide range of species and will aid future monitoring programmes.

True volumetric counting of CD34+ cells using flow cytometry

While the single-platform flow cytometric CD34+ cell counting method is the preferred choice to predict the yield of mobilized peripheral blood stem cells, most flow cytometers lack the ability of hematology counter analyzers to perform volumetric counting. However, one of the problems using reference microbeads is the vanishing counting bead phenomenon. This phenomenon results in a drop in microbeads concentration and reduces the total and relative number of beads in calibration procedures. In the last years, flow cytometers including a volumetric system to quantify cells have been developed and may represent a promising alternative to enumerate CD34+ cells avoiding the use of beads.In this study we have used a direct true volumetric counting of CD34+ cells under continuous flow pump to overcome potential drawbacks with impact in rare cell analysis. To confirm this hypothesis, we have compared the results of CD34+ cell enumeration using non-volumetric vs. volumetric systems with FC500 (Beckman Coulter) and Attune NxT (ThermoFisher) flow cytometers, respectively, in mobilized peripheral blood samples.No statistically significant differences were observed between measurements of CD34+ cells using beads, when the FC500 and Attune NxT absolute counting values were compared, or when CD34+ counts were compared on the Attune NxT, either using or not using beads. Linear regressions to study the relationship between volumetric and non-volumetric CD34+ counts confirmed the accuracy of each method. Bland-Altman test showed agreement between both methods.Our data showed that CD34+ cell enumeration using a volumetric system is comparable with current counting systems. This method represents an alternative with the advantage of the simplification of sample preparation and the reduction of the analysis subjectivity.

The CDK Inhibitor Dinaciclib Improves Cisplatin Response in Nonseminomatous Testicular Cancer: A Preclinical Study.

Most patients with testicular germ cell tumors (GCTs) are treated with cisplatin (CP)-based chemotherapy. However, some of them may develop CP resistance and therefore represent a clinical challenge. Cyclin-dependent kinase 5 (CDK5) is involved in chemotherapy resistance in different types of cancer. Here, we investigated the possible role of CDK5 and other CDKs targeted by dinaciclib in nonseminoma cell models (both CP-sensitive and CP-resistant), evaluating the potential of the CDK inhibitor dinaciclib as a single/combined agent for the treatment of advanced/metastatic testicular cancer (TC). The effects of dinaciclib and CP on sensitive and resistant NT2/D1 and NCCIT cell viability and proliferation were evaluated using MTT assays and direct count methods. Flow cytometry cell-cycle analysis was performed. The protein expression was assessed via Western blotting. The in vivo experiments were conducted in zebrafish embryos xenografted with TC cells. Among all the CDKs analyzed, CDK5 protein expression was significantly higher in CP-resistant models. Dinaciclib reduced the cell viability and proliferation in each cell model, inducing changes in cell-cycle distribution. In drug combination experiments, dinaciclib enhances the CP effect both in vitro and in the zebrafish model. Dinaciclib, when combined with CP, could be useful for improving nonseminoma TC response to CP.

A review of the status, threats and management priorities of a remnant population of Indus River dolphins in the Beas River, India

Abstract The Indus River dolphin (Platanista minor) is a severely threatened species of freshwater dolphin that occurs only in the lower Indus River system of Pakistan and India. The dolphin's range has declined by 80% since the 1870s, and total species abundance is estimated as approximately 2000 individuals. In 2007, a remnant population of Indus dolphins was discovered above Harike Barrage, in the Beas River in India, 600 km away from all other individuals of its species. This paper provides an overview of the conservation status of Indus dolphins in the Beas River, details the threats they face, and suggests priorities for their conservation and management. Between 2011 and 2022, 40 dolphin direct count surveys were conducted. Indus dolphins occur only in the lower third of the Beas River and reported counts have been from one to eight individuals. The data do not indicate an increase in abundance, and instead suggest a potential decrease; however, sightings of calves continue to be reported annually indicating reproduction is still taking place. Threats to the Beas River dolphin population include accidental entanglement in fishing gear, pollution, escapement downstream of Harike barrage, altered and depleted river flow regimes' and the effects of a very small population size. Urgent conservation measures are essential if this small, but important satellite population is to persist. Recommended management actions include the complete removal of fishing nets from dolphin habitat, reducing pollution, ensuring adequate river discharge to sustain aquatic ecology including dolphins, evaluating and monitoring dolphin movement through Harike barrage and into canals, and engaging riverside communities to protect dolphins. In addition, the possibility of conservation translocations to supplement this population with individuals from larger healthy populations elsewhere in the range of the species should be explored.

HPA1-29w Genotyping and the Foundation for the Platelet Apheresis Registry in Jiangsu Province of China by MassARRAY Spectrometry

Introduction: This study aimed to investigate the allele frequencies of the human platelet antigens (HPA) HPA-1-29w system in Jiangsu (China) and establish the platelet apheresis registry in blood donors. Methods: HPA genotyping was performed using the MassARRAY iPLEX® platform. Allele and genotype frequencies were estimated by direct counting and tested for Hardy-Weinberg equilibrium. The transfusion mismatch probability was calculated for every HPA specificity. Results: The HPA allele frequencies in the Jiangsu Han population of HPA-1b, -2b, -3b, -4b, -5b, -6b, -11b, -15b, and -21b were 0.0055, 0.0530, 0.4116, 0.0015, 0.0155, 0.0162, 0.0003, 0.4683, and 0.0070, respectively, in which a heterozygote of HPA-11a/b was first detected in China. Only allele a was detected for HPA-7-10w,-12-14w,-16-20w, and -22-29w quasi-systems. The highest mismatch rate of HPA genes in 1,640 platelet donors was the HPA-15 system, followed by the HPA-3 system with a rate of 37.4% and 36.71%, respectively. Conclusion: China’s largest-scale platelet registry of HPA-1-29w has been explored. The MassARRAY platform may help found the platelet apheresis registry which would be useful to provide matching platelets and lead to a more accurate, effective, and safe transfusion for patients with platelet therapy.

Investigation of Genetic Polymorphisms of CSN1S1 and BLG Genes in Norduz sheep by PCR-RFLP method

This study was undertaken to examine genetic polymorphisms of Alpha-S1 casein (CSN1S1) and β-Lactoglobulin (BLG) genes in Norduz sheep by PCR-RFLP method. In the study, 10 ml blood samples were taken from 102 heads of Norduz sheep into tubes with EDTA. DNA isolation from blood samples was done with ready-made commercial kits. The target DNA region of alpha-s1 casein and beta-lactoglobulin genes was amplified by PCR technique. The obtained PCR patterns were digested with MboII and RsaI restriction enzymes to determine the genotypes of Alpha-S1 casein and β-lactoglobulin genes, respectively. The cut fragments were run on 2% agarose gel electrophoresis and genotypes were determined under UV light. Statistical analysis, after calculating allele and genotype frequencies by direct gene counting method, the distribution of observed and expected genotypic frequencies was determined according to the Hardy-Weinberg Equilibrium, and whether it was compatible with the χ2 test. β-Lactoglobulin AA, AB and BB genotype frequencies were found to be 17.6%, 69.6 and 12.7%, respectively. It was determined that the β-Lactoglobulin gene was not in Hardy-Weinberg Equilibrium. AlphaS1-Casein AA, AC and CC genotype frequencies were determined as 0.0, 2.9 and 97.1%, respectively. The Alpha-S1 casein gene was determined to be in Hardy-Weinberg equilibrium. It was determined that Norduz sheep showed polymorphism in terms of Alpha-S1 Casein and β-Lactoglobulin genes. In conclusion, it was determined that the A allele of the β-Lactoglobulin gene and the AB genotype were more common, the C allele and the CC genotype of the Alpha-S1 casein gene were more common, and the AA genotype was absent in Norduz sheep. It was concluded that it would be useful to investigate the economic effects of genetic polymorphisms of Alpha-S1 casein and β-Lactoglobulin genes on various yield traits in larger Norduz sheep populations.

First Report of Phenotype Prevalence of Five Major Rh Blood Group Antigens in the Blood Donor Population of Peshawar, Pakistan

Objective: The current study investigated the prevalence of five major Rh blood group antigens in the Peshawar region of Khyber Pakhtunkhwa province, Pakistan.&#x0D; Methodology: This was a cross-sectional study, conducted at the Peshawar Regional Blood Centre from February to April 2023. A total of 13,148 blood donor samples were analyzed for Rh D, C, c, E, and e antigens using monoclonal reagents. The frequencies were calculated by direct counting on a spreadsheet programme and the Statistical Package for Social Sciences (SPSS for Windows version 25.0. Armonk, NY, USA) was used to conduct a meta-analysis for comparison of results with national and international studies.&#x0D; Results: The findings of the current study revealed that 92.17% of donors were RhD positive. The e antigen showed the highest frequency of 98.31%, followed by D antigen and the C antigen (92.17% and 78.37%, respectively). Comparative analyses with earlier Pakistani studies and international data indicated significant variations in antigen frequencies, potentially attributed to the diverse ethnic population and geography of the region.&#x0D; Conclusion: The findings emphasize the importance of local antigen frequency data for efficient blood transfusion strategies, especially for thalassaemia patients requiring frequent transfusions.&#x0D; Key words: Rhesus, Blood Group, Phenotype

INVASIVE INSECT SPECIES CYDALIMA PERSPECTALIS (WALKER, 1859): GLOBAL DISTRIBUTION, INVASION IN UKRAINE AND DAMAGE IN CHERNIVTSI REGION

Invasions of species into ecosystems cause their transformation and, in certain cases, their degradation. The study was carried out to determine the extent of the invasion of Cydalima perspectalis (Walker, 1859). For this purpose, the following aspects were analyzed: distribution of the species in different countries of the worldі, history of invasion of the pest in Ukraine, degree of damage to plantations of different box (Buxus L.) species and colony density in certain areas. The chronology of the distribution of C. perspectalis was studied using literature sources and international databases: Global Biodiversity Information Facility (GBIF), Delivering Alien Invasive Species Inventories for Europe (DAISIE), European and Mediterranean Plant Protection Organization (EPPO). The information on the history of the species' invasion in Ukraine and in particular in the Chernivtsi region was found in the literature, using the database of the National Biodiversity Information Network (UkrBIN), through questionnaires and personal observations using the route method (counting the number of C. perspectalis on a certain route). The degree of damage to Buxus sp. shrubs was determined visually using a scale. The density of C. perspectalis in each area was assessed by total counts and direct counts in situ with a parallel assessment of habitat size. It was found that the expansion of C. perspectalis beyond its native range began in 2006 from southwestern Germany. In 15 years, the species has occupied the territories of about 40 mainland and island countries of the North American and European continents. Since 2014, after the invasion of the Zakarpattia region, C. perspectalis has begun to spread throughout the Ukrainian territories. Currently, the secondary range of the species is represented by 14 regions. The presence of C. perspectalis has been detected in 16 settlements of Chernivtsi region, where the degree of damage to box shrubs is mostly severe. The density of caterpillars on most bushes is up to 100 individuals/m2, which indicates the high damage of the species. Key words: Cydalima perspectalis (Walker, 1859), invasive species, damage, expansion.

Earthquake impacts on a protected pinniped in New Zealand

Abstract The impacts of natural disasters on marine mammals are poorly understood and difficult to study, which can hamper management responses following such events. This study provides the first distribution and abundance assessment of New Zealand fur seal (NZFS: Arctocephalus forsteri) colonies around Kaikōura, New Zealand, since a 7.8 magnitude earthquake in November 2016 caused substantial changes to both the local marine and terrestrial environments, and led to the reconstruction of a major highway that runs adjacent to NZFS colonies. Mark–recapture and direct counts in the 2022–2023 breeding season estimated pup production for NZFS breeding colonies along the Kaikōura coast. Using established multipliers, pup estimates were used to provide the first comprehensive population estimate for Kaikōura's NZFS population since the earthquake. Three new colonies and three new subcolonies were assessed and recorded, additional to reassessments of two established colonies. Overall, Kaikōura's NZFS population has grown and spread post‐earthquake, with an upper total population estimate of between 21,560 and 28,327 animals in the 2022–2023 breeding season. Some sites, such as Lynch's Reef, appear to have benefited from earthquake‐induced coastal uplift, with pup production increasing. Contrastingly, the estimated 2,401 (±99) pups produced at Ōhau Point in 2023 is similar to pre‐earthquake estimates. This indicates that the earthquake has disrupted previously documented growth at this site. The distribution of NZFS breeding at Ōhau Point has also changed substantially since the last pre‐earthquake assessment. From these findings, alterations to the Ōhau Point New Zealand Fur Seal Sanctuary and similar protections at other locations on the Kaikōura coast are suggested, as greater numbers of NZFSs are now accessible to human interaction and disturbance. The results demonstrate both how natural disasters and subsequent infrastructure modifications can impact coastal species and how conservation measures may need to be amended accordingly.

Roost characteristics of Indian flying fox along urban noise gradient: A case study in Sallaghari Forest, Kathmandu Valley, Nepal

The Indian flying fox, Pteropus medius is a fruit bat that contributes significantly to seed dispersal and pollination services. These bats face serious threats from the loss of roosting trees due to urbanization and deforestation. This study aimed to assess the roost characteristics of Indian flying foxes and the effects of anthropogenic noise on roost tier selection in an urban forest patch. We carried out population surveys of Indian flying foxes in the Sallaghari Forest in Kathmandu Valley, Nepal by direct roost count method from July to November 2023. Further, we quantified the intensities of anthropogenic noise at every five-meter interval along the transects from the forest edge towards the main forest and recorded the minimum roosting heights at each point. Indian flying foxes were found to be roosting on trees belonging to six species. Eucalyptus sp. was the most abundant roost tree (50%) in the forest whereas the highest relative abundance of roosting Indian flying foxes was on Populus ciliata (40.1%). The regression analysis revealed that tree DBH and height and the combined interaction of height and canopy cover significantly increase the abundance of Indian flying foxes. The noise intensity at ground level and roosting height of Indian flying foxes showed a significant strong positive relationship indicating their roosting at higher tier under intense noise. We recommend measures for reducing anthropogenic noises and conserving large and tall trees to ensure the roosting habitat of Indian flying foxes in urban setups.

Metagenomic insights into isolable bacterial communities and antimicrobial resistance in airborne dust from pig farms.

This study aims to investigate bacterial communities and antimicrobial resistance (AMR) in airborne dust from pig farms. Airborne dust, pig feces and feed were collected from nine pig farms in Thailand. Airborne dust samples were collected from upwind and downwind (25 meters from pig house), and inside (in the middle of the pig house) of the selected pig house. Pig feces and feed samples were individually collected from the pen floor and feed trough from the same pig house where airborne dust was collected. A direct total bacteria count on each sampling plate was conducted and averaged. The ESKAPE pathogens together with Escherichia coli, Salmonella, and Streptococcus were examined. A total of 163 bacterial isolates were collected and tested for MICs. Pooled bacteria from the inside airborne dust samples were analyzed using Metagenomic Sequencing. The highest bacterial concentration (1.9-11.2 × 103 CFU/m3) was found inside pig houses. Staphylococcus (n = 37) and Enterococcus (n = 36) were most frequent bacterial species. Salmonella (n = 3) were exclusively isolated from feed and feces. Target bacteria showed a variety of resistance phenotypes, and the same bacterial species with the same resistance phenotype were found in airborne dust, feed and fecal from each farm. Metagenomic Sequencing analysis revealed 1,652 bacterial species across all pig farms, of which the predominant bacterial phylum was Bacillota. One hundred fifty-nine AMR genes of 12 different antibiotic classes were identified, with aminoglycoside resistance genes (24%) being the most prevalent. A total of 251 different plasmids were discovered, and the same plasmid was detected in multiple farms. In conclusion, the phenotypic and metagenomic results demonstrated that airborne dust from pig farms contained a diverse array of bacterial species and genes encoding resistance to a range of clinically important antimicrobial agents, indicating the significant role in the spread of AMR bacterial pathogens with potential hazards to human health. Policy measurements to address AMR in airborne dust from livestock farms are mandatory.