In this work, we consider the properties of the two-term Machin-like formula and develop an algorithm for computing digits of π by using its rational approximation. In this approximation, both terms are constructed by using a representation of 1/π in the binary form. This approach provides the squared convergence in computing digits of π without any trigonometric functions and surd numbers. The Mathematica codes showing some examples are presented.

This document introduces methods and algorithms to characterize a series of values as patterns of repeating symbols or sequences selected with the criterion to maximize the information retrieved. The method converts a series of real-number values into texts. Then, it interprets the text from a point of view specified by several parameters. Located patterns are linked nested, with shorter sequences within longer ones. Texts are then synthesized and organized in a tree-like structure, substantially reducing entropy. The characterization of processes serves as the basis for constructing models to estimate likely projected values. Alternative ways to assess the multiscale complexity of unidimensional processes are applications of the results obtained here.

Despite several decades’ effort to detect and identify phytoplasmas (Tenericutes: Mollicutes) using PCR and Sanger sequencing focusing on diseased plants, knowledge of phytoplasma biodiversity and vector associations remains highly incomplete. To improve protocols for documenting phyto-plasma diversity and ecology, we used DNA extracted from phloem-feeding insects and com-pared traditional Sanger sequencing with a next-generation sequencing method, Anchored Hy-brid Enrichment (AHE) for detecting and characterizing phytoplasmas. Among 22 of 180 leaf-hopper samples that initially tested positive for phytoplasmas using qPCR, AHE yielded phyto-plasma 16Sr sequences for 20 (19 complete and 1 partial sequence) while Sanger sequencing yield-ed sequences for 16 (11 complete and 5 partial). AHE yielded phytoplasma sequences for an addi-tional 7 samples (3 complete and 4 partial) that did not meet the qPCR threshold for phytoplasma positivity or yielded non-phytoplasma sequences using Sanger sequencing. This suggests that AHE is more efficient for obtaining phytoplasma sequences. Twenty-three samples with sufficient data were classified into eight 16Sr subgroups (16SrI-B, I-F, I-AO, III-U, V-C, IX-J, XI-C, XXXVII-A), three new subgroups (designated as 16SrVI-L, XV-D, XI-G) and three possible new groups. Our results suggest that screening phloem-feeding insects using qPCR and AHE sequencing may be the most efficient method for discovering new phytoplasmas.

Ostreid herpesvirus 1 (OsHV-1) is one of the most economically important pathogens of Pacific oysters. Understanding the pathogenesis of this virus is critical to developing tools to control outbreaks on shellfish farms. OsHV-1 is genetically related to vertebrate herpesviruses, which have a lytic and a latent stage, with the latent stage capable of being reactivated to the lytic stage. Here, OsHV-1 latency in Pacific oysters was investigated in experimentally and naturally infected oysters. Lytic infection in one-year-old oysters injected with the Tomales Bay strain of OsHV-1 was detectable between 1 and 4 days post-infection (dpi) but was not detectable after 5 dpi. The infected oysters shed 1102 to 1104 DNA copies/ml during the 4-day acute phase. At 21 dpi, the recovered oysters were temperature and chemically stressed and were found to shed 1104 to 1105 DNA copies/ml over a period of 48 h. Lytic shedding was not detectable in two-year-old oysters injected similarly with the same strain of OsHV-1; however, the OsHV-1 genome was detectable by qPCR in the adductor muscle, gill, mantle, and hemocytes within the first 3 dpi, after which it became undetectable. No OsHV-1 was detectable in the adductor muscle, gill, or mantle from experimentally infected oysters on days 15 and 21 post-infection or from oysters sampled 9 months after surviving an OsHV-1 mortality event; however, OsHV-1 DNA could be detected in hemocytes of both experimentally infected oysters at 21 dpi and naturally infected oysters. In addition, lytic viral gene transcription was detectable in hemocytes of experimentally infected oysters between 1 and 21 dpi and in hemocytes of naturally infected oysters.

Abstract: The probability of existence or not of local extinctions of six species of carnivorous mammals was analyzed by ordering the species composition in nested subsets in 7 fragments of forest habitats in the Coastal Mountains in the Araucanía Region, in southern Chile. Nested Temperature Calculator (NTC) and BINMATNEST computer programs were used to process the results. The first software provided a temperature in the archipelago of 28.21 degree Celsius, whereas the second program showed a nesting temperature of 16.58 degree Celsius. The computer software used in this study suggest that there is no nesting pattern at the level species composition of carnivorous mammal assemblage in the evaluated area, which may be due to the habitat use characteristics of the different carnivorous mammals studied here.

：Sleep stage on the whole night is not steady. Sleepers generally pass through three to five cycles. In each cycle, there are occur four typical sleep stages, such as wake stage (WS), light stage (LS), deep sleep (DS), rapid eye movement sleep stage (REM). According to the natural routine, in this paper, we investigate the stage transition and analyze the feature of stage transition using the local cluster Algorithm (LCA). Two-cycle sleep model (TCSM) is proposed to automatically classify sleep stages using over-night continuous heart rate variability (HRV) data. The generated model is based on the characteristics of the nested cycle's sleep stage distribution and the transition probabilities of sleep stages. Experiments were conducted using a public data set including 400 healthy subjects (female 239, male 161) and the model’s classification accuracy was evaluated for four sleep stages: WS, LS, DS, REM. The experimental results showed that based on the TCSM model, the segmentation classification of pure sleep is 5.2% higher than that of the traditional method, and the accuracy of segmentation classification is 11.2% higher than the traditional sleep staging accuracy. The experimental performance is promising in terms of the accuracy, sensitivity, and specificity rates compared with the ones of the state-of-the-art methods. The study contributes to improve the quality of sleep monitoring in daily life using easy-to-wear HRV sensors.

Toxoplasmosis is a neglected anthropozoonosis caused by the obligate intracellular protozoan, Toxoplasma gondii. The role of non-human primates in the epidemiology of human toxoplasmosis is not clear. Acute and highly fatal cases of toxoplasmosis are frequently reported in new world monkeys and asymptomatic infections in old world monkeys. Here we report detection of latent natural T. gondii infection in olive baboons during a screening exercise to select animals for an experimental toxoplasmosis study. Archived serum samples from 32 olive baboons (Papio anubis), 23 wild caught and nine colony-born, were screened for T. gondii DNA using nested PCR. Eighteen (56.25%) samples were from females and fourteen (43.75%) from males. Toxoplasma gondii DNA was detected in 21 (65.6%) baboons. 13 females (40. 6%) and eight males (25%). Five baboons (24%) which tested positive were from the Institute of Primate Research colony but there was no statistical significance between them and the wild trapped (p=0.453). There was neither statistical significance (p=0.373) between sex and infection status nor between area of origin and infection status (p>0.05). These results indicate that olive baboons get infected with T. gondii in the wild and during captivity and may be significant reservoirs of human infections, especially where they may be trapped for bushmeat. We recommend a country-wide study to establish true prevalence of toxoplasmosis among non-human primates and identify associated parasite strains.

Despite the successes earned in cataloguing and finding the role of the most of molecular components in living matter, the “biochemical and molecular” perspective, popular in biology, medicine and genetics, is unable to give account for crucial topics as the faculty of living systems to “feel”, to “perceive” what a given stimulus implies (means, indeed) for their survival. Condensed matter physics too, if bounded to a local, short-range, and perturbative approach, fails dramatically. This is also due to the role commonly assigned to water – actually the main constituent of living matter – deemed for long time to be merely chemical (as “solvent” or a reactant/product). Nonetheless, today many evidences show how living matter can be right conceived as a super-structured coherent water-based matrix, suggesting that the characterization of bio(electro)chemical and physical processes undertaken at molecular level in living matter, would let us unable to answer a question like this: what allows an amoeba, moreover without any neurons, to “know” to get closer to a nutrient or escape away from a toxin? I propose that to pursue such a fundamental inquiry it’s necessary an essentially relational approach, that is: to consider the living being at its grounding as the outcome of a physical history of relationships where symmetry-breakings, dissipation and coherence yield the emergence of the living state of matter, conceivable only as a time-dependent open process, and not as a portraited “body”. The effective tools to build up such an approach may be retrieved in far-from-equilibrium thermodynamics (TD), symmetry-breakings and gauge-fields theory, science of complexity, within the framework of a Quantum Field Theory. Indeed, within a field-view of matter, and of water especially, as it has been developed by a Quantum Electrodynamic (QED) description of condensed matter, it’s possible to give account for a physical basis too such an epistemologically elusive, though crucial, feature of living systems (i.e.: perception and meaning). The emerging landscape allows some important meditations about adaptation, evolution, ecodynamics, and about different conceptions of complexity and “information” in living realm. Furthermore, some neuroscientific themes like consciousness, qualia and their links to artificial intelligence could be supplied with due insights.

Understanding the spatially varying effects of demographic factors on the spatio-temporal variation of intestinal parasites infections is important for public health intervention and monitoring. This paper presents a hierarchical Bayesian spatially varying coefficient model to evaluate the effects demographic factors on intestinal parasites morbidities in Ghana. The modeling relied on morbidity data collected by the District Health Information Management Systems. We developed Poisson and Poisson-gamma spatially varying coefficient Models. We used the demographic factors, unsafe drinking water, unsafe toilet and unsafe liquid waste disposal as model covariates. The models were fitted using the Integrated Nested Laplace Approximations (INLA). The overall risk of intestinal parasites infection was estimated to be 10.9 per 100 people with a wide spatial variation in the district-specific posterior risk estimates. Substantial spatial variation of increasing multiplicative effects of unsafe drinking water, unsafe toilet and unsafe liquid waste disposal occurs on the variation of intestinal parasites risk. The structured residual spatial variation widely dominates the unstructured component, suggesting that the unaccounted-for risk factors are spatially continuous in nature. The study concludes that both the spatial distribution of the posterior risk and the associated exceedance probability maps are essential for monitoring and control of intestinal parasites.

Urban sprawl is recognized as one of the most critical drivers disrupting the functional integrity of rural and natural landscapes. A systematic multiscale study has yet to be conducted to measure the Megalopolis of Mexico City's regional extent, comprising ca. 23 million inhabitants. This paper investigates land cover, land use, vegetation and soil changes at macro-regional, regional, local and site scales. Land cover/use data from the 1970s and 2018 were used as the primary input to analyze changes using remote sensing and geographical information systems. At the local scale, fieldwork data from the 1970s, 1990s, and 2020 served as the primary input and were subjected to multivariate analyses. At site scale, soils profiles and hydrological balance analyses at micro-basin level were conducted. Furthermore, social perception of changes was investigated among local residents/stakeholders using semi-structured interviews with four communities. Outcomes were validated to reach 85 percent accuracy in cartographic and 95 % statistical outputs. At the regional scale, land-use changes, especially human settlements, increased from about four to almost 40 % between 1970 and 2018 , with significant incursion into rural landscapes. At the local scale, analyses revealed significant decline in predominantly cold-humid vegetation types and conversion of agricultural fields into peri-urban landscapes. Site scale revealed the predominant importance of soils as the core landscape component for water infiltration and carbon sequestration. Nested multi-scale analyses revealed different disrupting processes which are hidden at one scale and evident at another. Poorly implemented, disconnected urban and environmental policies prevail as the main driving force, with implications for the weak local governance of rural communities. Regional integrated analyses are discussed in light of the relevance of urban resilience theory.

Eggplant or brinjal is one of the most consumed and important nutrient-dense tropical solanaceous vegetable crops grown worldwide. Little leaf is a disease especially widespread in brinjal in India where it is associated with the presence of phytoplasmas. To clarify the epidemiology of this disease and verify its seed transmission potential, seedlings and their progeny derived from symptomatic mother plants field-collected in Dharwad district of Karnataka State, India, were sowed under insect proof conditions in greenhouse. The resulting first- and second-generation seedlings obtained were tested at different times after germination by DNA extraction and amplification of 16S rRNA gene of phytoplasmas. Tissues from field collected symptomatic brinjal plants were also tested following the same procedure. The amplicons obtained were subjected to restriction fragment length polymorphism (RFLP) analysis and sequencing for phytoplasma identification. The detected phytoplasmas belonged to 16SrI, 16SrII, 16SrIII, 16SrV, 16SrVI, and 16SrXII groups. Several fruits produced from the first-generation seedlings showed germination of the seeds inside the fruit and resulted to be phytoplasma positive. This demonstrates the seed transmission of phytoplasmas in eggplants for two subsequent generations. This finding highlights the risk of additional source of infection represented by asymptomatic and infected seedlings under field conditions and contribute to explain the epidemic spread of phytoplasmas in brinjal cultivations in India and in the other eggplant growing areas of the world.

Drug induced liver injury (DILI) remains one of the challenges in the safety profile of both authorized drugs and candidate drugs and predicting hepatotoxicity from the chemical structure of a substance remains a challenge worth pursuing, being also coherent with the current tendency for replacing non-clinical tests with in vitro or in silico alternatives. In 2016 a group of researchers from FDA published an improved annotated list of drugs with respect to their DILI risk, constituting “the largest reference drug list ranked by the risk for developing drug-induced liver injury in humans”, DILIrank. This paper is one of the few attempting to predict liver toxicity using the DILIrank dataset. Molecular descriptors were computed with the Dragon 7.0 software, and a variety of feature selection and machine learning algorithms were implemented in the R computing environment. Nested (double) cross-validation was used to externally validate the models selected. A number of 78 models with reasonable performance have been selected and stacked through several approaches, including the building of multiple meta-models. The performance of the stacked models was slightly superior to other models published. The models were applied in a virtual screening exercise on over 100,000 compounds from the ZINC database and about 20% of them were predicted to be non-hepatotoxic.

Extracting entities from large volumes of chicken epidemic texts is crucial for knowledge sharing, integration, and application. However, Named Entity Recognition (NER) encounters significant challenges in this domain, particularly due to the prevalence of nested entities and domain-specific named entities, coupled with a scarcity of labeled data. To address these challenges, we compiled a corpus from 50 books on chicken diseases, covering 28 different disease types. Utilizing this corpus, we developed a nested NER model, MFGFF-BiLSTM-EGP. This model integrates the Multiple Fine-Grained Feature Fusion (MFGFF) module with a BiLSTM neural network and employs an Efficient Global Pointer (EGP) for predicts the entity location encoding. In MFGFF module we designed three encoders, character encoder, word encoder and sentence encoder, this design effectively captures fine-grained features and improves the recognition accuracy of nested entities. Experimental results show that the model performs robustly, with F1 scores of 91.98%, 73.32%, and 82.54% on the CDNER, CMeEE V2, and CLUENER datasets, respectively, outperforming other commonly used NER models. Specifically, on the CDNER dataset, the model achieved an F1 score of 79.68% for nested entity recognition. This research not only advances the development of knowledge graph and intelligent question-answering system for chicken diseases but also provides a viable solution for extracting disease information that can be applied to other livestock species.

This paper shows that by applying the Nyquist-Shannon sampling theorem, the spatial and temporal resolution of a simulation can be no more than half the resolution of the simulating reality. This has significant implications for not only the values of the observables in the simulation but also its physical laws. This progressive halving of nested simulations coupled with the minimum resolution compatible with the production of a simulation also sets a limit to the nestedness of a lineage of simulations (it is by no means infinite). The limit of nestedness is then used to calculate the probability that we are living in a simulation assuming a single base reality. This will be shown to be significantly lower than popular expectation. A Kardashev-like scale, with three variants, is also developed to gauge the technological advancement of a civilization in relation to the extent to which it can extract information from space and time.

We evaluate the nested sum $\sum_{a_{n - 1} = c}^{a_n } {\sum_{a_{n - 2} = c}^{a_{n - 1} } { \cdots \sum_{a_0 = c}^{a_1 } {x^{a_0 } } } }$ where $a_n$ and $c$ are any integers and $x$ is a real or complex variable. Consequently, we evaluate multiple sums involving the terms of a general second order sequence, the Horadam sequence (W_j (a, b; p, q)), defined for all non-negative integers j by the recurrence relation W₀ = a, W₁ = b; W_j = pW_j−1 − qW_j−2 (j≥2); where a, b, p and q are arbitrary complex numbers, with p ≠ 0, q ≠ 0.

5G low-earth orbit satellite communications offer enhanced wireless coverage beyond the reach of 5G terrestrial networks, with important implications, particularly for automated and connected vehicles. Such wireless automotive mass-market applications demand well-designed compact user equipment antenna terminals offering non-terrestrial jointly with terrestrial communications. The antenna should be low-profile, conformal, and meet specific parameter values for gain and operational frequency bandwidth, tailored to the intended applications, in line with the aesthetic design requirements of passenger cars. This work presents an original concept for a dual-band nested circularly polarized automotive user terminal that operates at the S-band frequencies around 3.5 GHz and Ka-band frequencies around 28 GHz, namely within the 5G new-radio bands n78 and n257, respectively. The proposed terminal is designed to be integrated into the plastic components of a passenger vehicle. The arrays consist of 2×2 aperture-coupled corner-truncated microstrip slot patch antenna elements for the n78 band and of 4×4 single-layer edge-truncated microstrip circular slot patch antenna elements for the n257 band. The embedded arrays offer, across the two bands, respectively 9.9 and 13.7 dBi measured realized gain and 3-dB axial ratio bandwidths of 100 and 1500 MHz for the n78 and n257 bands along the broadside direction. Detailed link budget calculations anticipate uplink data rates of 21 and 6 Mbit/s, respectively, deeming it suitable for various automotive mobility and internet-of-things applications.

Formal Calculation uses an auxiliary form to calculate various nested sums and provides results in three forms. In addition to computation, it is also a powerful tool for analysis, allowing one to study various numbers in a unified way. This article contains many results of two types of Stirling numbers, associated Stirling numbers, and Eulerian numbers, making a great generalization of Euler polynomials, Wilson's theorem, and Wolstenholme's theorem, showing that they are just special cases. Formal Calculation provides a novel method for obtaining combinatorial identities and analyzing q-binomial.This article has obtained a large number of results in q-analogues, including inversion formulas for q-binomial coefficients. This article also introduces a theorem on symmetry.

We address security and privacy problems for digital devices and biometrics from an information-theoretic optimality perspective, where a secret key is generated for authentication, identification, message encryption/decryption, or secure computations. A physical unclonable function (PUF) is a promising solution for local security in digital devices and this review gives the most relevant summary for information theorists, coding theorists, and signal processing community members who are interested in optimal PUF constructions. Low-complexity signal processing methods such as transform coding that are developed to make the information-theoretic analysis tractable are discussed. The optimal trade-offs between the secret-key, privacy-leakage, and storage rates for multiple PUF measurements are given. Proposed optimal code constructions that jointly design the vector quantizer and error-correction code parameters are listed. These constructions include modern and algebraic codes such as polar codes and convolutional codes, both of which can achieve small block-error probabilities at short block lengths, corresponding to a small number of PUF circuits. Open problems in the PUF literature from a signal processing, information theory, coding theory, and hardware complexity perspectives and their combinations are listed to stimulate further advancements in the research on local privacy and security.

Our study examines how dengue incidence is associated with spatial (demographic and socioeconomic) alongside temporal (environmental) factors at multiple scales. We used the incidence of dengue in Ibagué, Colombia, from 2013 to 2018, to examine the associations with climate, socioeconomic and demographic factors from the census and satellite imagery at various levels of local spatial aggregation, including Manzanas, Secciones, Sectores, and Comunas. Our findings show a significant effect of spatial variables at finer levels of aggregation, showing varying degrees of correlation with dengue incidence. Temporal variables such as temperature and precipitation displayed consistent patterns across all spatial levels, with notable variations in Relative Risk (RR). Our study employs Geographically Weighted Regression (GWR) to identify relevant socioeconomic and demographic predictors. Then, these predictors were integrated into hierarchical models implemented in Integrated Nested Laplace Approximation (INLA) at each spatial level to assess spatiotemporal interactions. We comprehensively analyzed the three distinct models developed for each level: spatial, temporal, and spatiotemporal. A comparative evaluation of the models reveals that while higher aggregation levels often yield a better overall model fit, finer levels offer more detailed insights into the localized impacts of socioeconomic and demographic variables on dengue incidence. Our results underscore the importance of considering macro and micro-level factors in epidemiological modeling and highlight the potential for targeted public health interventions based on localized risk factor analysis. Notably, the intermediate level Secciones emerged as the most informative, balancing spatial heterogeneity and case distribution density, thereby providing a robust framework for understanding the spatial determinants of dengue.

HMG-CoA reductase is an enzyme that regulates the initial stage of cholesterol synthesis and its inhibitors are widely used in the treatment of cardiovascular diseases. Methods: We have created a set of quantitative structure-activity relationship (QSAR) models for human HMG-CoA reductase inhibitors using nested cross-validation as the primary validation method. To develop the QSAR models, we employed various machine learning regression algorithms, feature selection methods, and fingerprints or descriptor datasets. Results: We built and evaluated a total of 300 models, selecting 21 that demonstrated good performance (coefficient of determination, R2 ≥ 0.70 or concordance correlation coefficient, CCC ≥ 0.85). Six of these top-performing models met both performance criteria and were used to construct five ensemble models. We identified the descriptors most important in explaining HMG-CoA inhibition for each of the six best-performing models. We used the top models to search through over 220,000 chemical compounds from a large database (ZINC 15) for potential new inhibitors. Only a small fraction (237 out of approximately 220,000 compounds) had reliable predictions with mean pIC50 values ≥8 (IC50 values ≤10 nM). Our svm-based ensemble model predicted IC50 values <10 nM for roughly 0.08% of the screened compounds. We have also illustrated the potential applications of these QSAR models in understanding the cholesterol-lowering activities of herbal extracts, such as those reported for an extract prepared from the Iris × germanica rhizome. Conclusions: Our QSAR models can accurately predict human HMG-CoA reductase inhibitors, having the potential to accelerate the discovery of novel cholesterol-lowering agents and may also be applied to understand the mechanisms underlying the reported cholesterol-lowering activities of herbal extracts.

We lack a unified theory of aging that spans multiple scientific disciplines, including life history theory, evolution, cellular and molecular biology, and medicine. This gap has hindered both our ability to effectively target and treat age-related diseases in humans and our understanding of the vast variation in aging rates across species. This paper builds on the Blueprint Theory of Aging, which suggests that biological pathways can be ‘dis’-activated outside their intended context, leading to pathological pathways (patho-pathways) that drive cellular and tissue ‘dys’-function and age-related ‘dis’-eases, ultimately limiting both healthspan and lifespan. We propose that evolution operates through complex trade-offs within interconnected networks (network constraints), optimizing beneficial functions while minimizing the costs of misactivated patho-pathways. These optimizations are shaped by a species' environment and are driven by a central evolutionary dilemma: prioritize immediate reproduction or reduce current fecundity in favour of future reproductive success. Evolution adjusts network constraints along a continuum, balancing early-life reproduction with lifetime reproductive output. When environmental pressures result in lower juvenile survival than adult survival, the optimal strategy shifts toward maximizing total progeny over a lifetime. This strategy, in turn, determines the final trade-offs selected, the structure of network constraints, and ultimately, the rate of aging for that species. Understanding these evolutionary calculations not only explains species-specific aging patterns but also sheds light on the wide variability in aging rates across nature. Finally, we reveal hidden evolutionary connections between mechanisms such as Antagonistic Pleiotropy and Mutational Accumulation, deepening our insight into the evolutionary dynamics underlying aging.

We report a new synthesis method for multiple walled nested thin film nanostructures by combining hydrothermal growth methods with Atomic Layer Deposition (ALD) thin film technology and sacrificial films thereby increasing the surface-to-volume ratio to improve the sensing performance of novel ZnO gas sensors. Single crystal ZnO nanorods serve as the core of the nanostructure assembly and were synthesized hydrothermally on fine-grained ALD ZnO seed films. Subsequently the ZnO core nanotubes were coated with alternating sacrificial co-axial 3-D wraparound ALD Al2O3 films and ALD ZnO films. Basically, an ALD 3-D wrap-around Al2O3 sacrificial layer was coating the center nanorod to realize a nested coaxial ZnO thin film nanotube. To increase the surface-to-volume ratio of the nested multiple film nanostructure both front and backside of the nested coaxial ZnO films must be exposed by selectively removing the intermittent Al2O3 sacrificial films. The selective removal of the sacrificial films exposes the front and backside of the free-standing ZnO films for interacting with target gases during sensing operation while steadily increasing the surface-to-volume ratio. The sensing response of the novel ZnO gas sensor architecture with nested nanotubes achieved a maximum 150% enhancement at low temperature compared to a conventional ZnO nanorod sensor.

Our goal was to determine the cellular immune response (CIR) among a random sample of the Borriana COVID-19 cohort (Spain) to identify its associated factors and their relationship with infection, reinfection and sequelae. We conducted a nested case-control study using a randomly selected sample of 225 individuals age 18 and older, including 36 individual naïve to SARS-CoV-2 infection, and 189 infected patients. We employed flow cytometry-based immunoassays for intracellular cytokine staining, utilizing Wuhan and BA.2 antigens and chemoluminescence microparticle immunoassay for detection SARS-CoV-2 antibodies. Logistic regression models were used. A total of 215 (95.6%) participants exhibited T-cell response (TCR) to at least one antigen. Positive responses of CD4+ and CD8+ T cells were 89.8% and 85.3% respectively. No difference in CIR was found for naïve and infected patients. Patients who experienced sequelae exhibited higher CIR than those without. A positive correlation was observed between TCR and Anti-Spike IgG levels. Factors positive associated with TCR included A-blood group, number of SARS-CoV-2 vaccine doses received, and Anti-N IgM; factors inversely related were the time elapsed since the last vaccine dose or infection, and B-blood group. These findings contribute valuable insights into the nuance immune landscape shaped by SARS-CoV-2 infection and vaccination.

Rodents are known to be reservoirs of Toxoplasma gondii and keep the parasite circulation in the environment. We conducted biomonitoring to assess the role of sylvatic rodents in maintaining T. gondii and to analyse the prevalence and seroprevalence of the parasite in seven wild rodent species. Rodents were collected in our study sites (woodland and open grasslands) located in northeastern Poland and dissected. We collected brain, spleen, blood and serum samples. We applied both molecular (PCR assay, nested-PCR assay) and serological (ELISA and agglutination tests) methods to indicate the best approach for application in the biomonitoring of T. gondii in small mammals. We screened samples from 95 individuals sing PCR assays and found no T. gondii DNA. The agglutination test showed no signal. We found antibodies against T. gondii in 6 sera samples out of 74 analysed (seroprevalence = 8.11% [4.0-17.1]). Our results confirm that rodents participate in the life cycle of T. gondii as reservoirs of this parasite in the sylvatic environment. However, biomonitoring should be performed with the ELISA tests to search for T. gondii antigens, rather than a molecular approach only.

As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein along with certain mutations in other regions within the S protein as specific variants in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.