Variable Region Research Articles

B Cells With Complementary B Cell Receptors Can Kill Each Other

ABSTRACTB cells differentiate from hematopoietic stem cells in the bone marrow (BM) and migrate as transitional cells to the spleen where final maturation takes place. Due to the enormous diversity in variable (V) regions of B cell receptors for antigen (BCR), B cells with complementary BCRs are likely to be generated. These could encounter each other in the BM or in secondary lymphoid organs. The outcome of such an event is unknown. To study this issue, we used two strains of gene‐modified mice whose B cells display complementary BCRs. B cells of one strain express an idiotype+ (Id+) BCR while B cells of the other strain display an anti‐idiotypic (αId) BCR. In vitro, B cells with complementary BCRs killed each other in a mechanism that required physical binding between BCR V‐regions. In contrast, killing was unilateral in vivo: αId B cells with a follicular (FO) B cell phenotype were expanded, while Id+ B cells with a marginal zone (MZ) phenotype became deleted. The results show that B cells with complementary BCRs can recognize and regulate each other in vivo. This mechanism should be taken into account in theories for idiotypic regulation of the immune system.

Chloroplast genome characteristic, comparative and phylogenetic analyses in Capsicum (Solanaceae)

BackgroundCapsicum (Solanaceae) is a globally important vegetable crop and is also used therapeutically in traditional medicine systems. However, little is known of the genetic variation within the commonly grown cultivars, the evolutionary relationships and differences in the chloroplast (cp.) genomes between Capsicum species remain unclear.ResultsThe cp. genomes of 32 Capsicum varieties in three species from 6 countries were investigated. The cp. genome of Capsicum was found to be ~ 156 kb in length and to contain 113 unique genes, of which 79 encoded proteins, 30 encoded transfer tRNAs, and 4 were for ribosomal RNAs. The 32 varieties that we chose for study represented 13 genotypes, containing a total of 608 indels, 83 SNPs, 47 SSRs and 281–306 repeat sequences. We then included several previously sequenced Capsicum cp. genomes, and found that the nine investigated species showed a number of differences in the characteristics of the four IR boundaries, and it was the non-coding regions that contained the most variable regions. We conducted a phylogenetic reconstruction using the cp. genomes of 43 representative species of Solanaceae, and the resulting phylogeny generally reflected the currently accepted classification, with the species of the pungent group having close relationship with one another.ConclusionsThis study provides a comprehensive analysis of Capsicum chloroplast genomes, revealing significant variations in IR boundaries and other genomic features. These findings enhance our understanding of Capsicum evolution and genetic diversity.

Advanced Bayesian Method for Timely Small-Scale Forest Loss Detection in the Brazilian Amazon and Cerrado with Sentinel-1 Time-Series

Abstract. The world’s forests are undergoing significant changes due to loss and degradation, emphasizing the need for Near Real-Time (NRT) monitoring to prevent further damage. Traditional monitoring methods using optical imagery are hindered by cloud coverage, while newer Synthetic Aperture Radar (SAR) systems, although operational in all weather conditions, face challenges such as sensitivity to soil moisture and the need for spatial filtering to reduce speckle effects. These limitations affect the detection of small-scale forest loss, especially in seasonally variable regions like dry forests and savannas. This paper presents a SAR-based forest disturbance detection method using Bayesian inference. Unlike traditional methods, this approach maintains the native resolution of the data by avoiding spatial filtering. Forest disturbance is modelled as a change-point detection problem within a non-filtered Sentinel-1 time series, where each new observation updates the probability of forest loss by leveraging prior information and a data model. This sequential adaptation ensures robustness against variations and trends, making it effective in monitoring disturbances across diverse forest types, including areas affected by seasonality. The proposed method was tested against other NRT monitoring systems for the year 2020, using small validation polygons (under 1 hectare) in the Brazilian Amazon and Cerrado savanna. Results demonstrate significant improvements in detecting small-scale disturbances and drastically reduced false alarm rates in both biomes. Notably, in the seasonality-sensitive Cerrado, our solution completely outperforms the leading and only existing optical technology.

Durable improvements in atopic dermatitis in the head and neck and across other anatomic regions with rocatinlimab

In a randomized phase 2b trial (NCT03703102) for adult patients with moderate-to-severe atopic dermatitis (AD), treatment with the T cell rebalancing anti-OX40 receptor antibody rocatinlimab (AMG 451/KHK4083) led to significant improvements in clinical measurements versus placebo including whole-body Eczema Area and Severity Index (EASI) score. AD manifestations can impact variable anatomic regions, and involvement of the head and neck, a sensitive, hard-to-treat area, can negatively impact quality of life. In this post hoc analysis, we investigated response to rocatinlimab treatment across anatomic regions, including the head and neck. Least squares mean change from baseline to Week 56 in EASI score was analyzed by anatomic region (head and neck, trunk, upper extremities, or lower extremities) for patients with baseline moderate-to-severe AD in the respective anatomic region, using mixed models for repeated measures. Rocatinlimab groups were compared with placebo at Week 16. The proportion of patients achieving at least 75% reduction from baseline in EASI (EASI-75) was calculated. Probability of relapsing in EASI-75 during the off-treatment follow-up period (Weeks 36–56) was estimated using a Kaplan − Meier approach. At Week 16, decrease from baseline in mean EASI score was greater with all rocatinlimab regimens versus placebo across all anatomic regions for patients with baseline moderate-to-severe AD in the respective region (all P < 0.001). EASI scores continued to improve on treatment after Week 16 and were maintained during the off-treatment period across all regions. Among patients with baseline moderate-to-severe AD in the head and neck (n = 219; rocatinlimab, n = 174; placebo, n = 45), mean difference (rocatinlimab vs placebo) at Week 16 in LS mean percent change in head and neck EASI score ranged from − 30.4% to − 42.6% across treatment regimens. In patients who received rocatinlimab from the start of the trial, 47% − 71% achieved EASI-75 in the head and neck at Week 36. Among EASI-75 responders at Week 36, the probability of relapsing in EASI-75 in any region was low (< 25% in the head and neck) 20 weeks after treatment discontinuation until Week 56.Rocatinlimab treatment led to durable improvements in AD across multiple anatomic regions, including the sensitive head and neck region.Graphical abstract

Use of Affimer Technology for Inhibition of α2-antiplasmin and Enhancement of Fibrinolysis

Hypofibrinolysis is a documented abnormality in conditions with high risk of vascular occlusion. A key inhibitor of fibrinolysis is α2-antiplasmin (α2AP) and we hypothesise that the Affimer technology, comprising small conformational proteins with two nine amino acid variable regions, can be used to modulate α2AP activity and facilitate fibrinolysis.Using a phage display system, a library of Affimers was screened against α2AP. A total of 28 α2AP-specific Affimers were isolated of which one, termed Affimer A11, inhibited protein function and enhanced fibrinolysis. Affimer A11 displayed a monomeric form and consistently reduced lysis time of clots made from plasma samples of individuals with type 2 diabetes mellitus (n=15; from 150.8±100.9 to 109.8±104.8 mins) and those with cardiovascular disease (n=15; 117.6±40.6 to 79.7±33.3 mins); p<0.01 for both groups. The effects of A11 on fibrinolysis were maintained when clots were made from whole blood samples.Mechanistic studies demonstrated that A11 did not affect clot structure or interfere with incorporation of α2AP into fibrin networks but significantly enhanced plasmin activity and accelerated plasmin generation. Affimer A11 reduced α2AP binding to plasmin(ogen), while molecular modelling demonstrated interactions with α2AP in an area responsible for binding to plasminogen, explaining the effects on both plasmin activity and generation. Affimer A11, at 0.15-0.60 mg/ml, had the ability to bind 70-90% of plasma α2AP.In conclusion, we demonstrate that Affimers are viable tools for inhibiting α2AP function and facilitating fibrinolysis, making them potential future therapeutic agents to reduce thrombosis risk.

Genomic Vulnerability to Climate Change of an Australian Migratory Freshwater Fish, the Golden Perch (Macquaria ambigua).

Genomic vulnerability is a measure of how much evolutionary change is required for a population to maintain optimal genotype-environment associations under projected climates. Aquatic species, and in particular migratory ectotherms, are largely underrepresented in studies of genomic vulnerability. Such species might be well equipped for tracking suitable habitat and spreading diversity that could promote adaptation to future climates. We characterised range-wide genomic diversity and genomic vulnerability in the migratory and fisheries-important golden perch (Macquaria ambigua) from Australia's expansive Murray-Darling Basin (MDB). The MDB has a steep hydroclimatic gradient and is one of the world's most variable regions in terms of climate and streamflow. Golden perch are threatened by fragmentation and obstruction of waterways, alteration of flow regimes, and a progressively hotter and drying climate. We gathered a genomic dataset of 1049 individuals from 186 MDB localities. Despite high range-wide gene flow, golden perch in the warmer, northern catchments had higher predicted vulnerability than those in the cooler, southern catchments. A new cross-validation approach showed that these predictions were insensitive to the exclusion of individual catchments. The results raise concern for populations at warm range edges, which may already be close to their thermal limits. However, a population with functional variants beneficial for climate adaptation found in the most arid and hydrologically variable catchment was predicted to be less vulnerable. Native fish management plans, such as captive breeding and stocking, should consider spatial variation in genomic vulnerability to improve conservation outcomes under climate change, even for dispersive species with high connectivity.

The SDSS-V Black Hole Mapper Reverberation Mapping Project: A Kinematically Variable Broad-line Region and Consequences for the Masses of Luminous Quasars

We present a velocity-resolved reverberation mapping analysis of the hypervariable quasar RM160 (SDSS J141041.25+531849.0) at z = 0.359 with 153 spectroscopic epochs of data representing a 10 yr baseline (2013–2023). We split the baseline into two regimes based on the 3× flux increase in the light curve: a “low state” phase during the years 2013–2019 and a “high state” phase during the years 2022–2023. The velocity-resolved lag profiles (VRLPs) indicate that gas with different kinematics dominates the line emission in different states. The Hβ VRLP begins with a signature of inflow onto the broad-line region (BLR) in the low state, while in the high state it is flatter with less signature of inflow. The Hα VRLP begins consistent with a virialized BLR in the low state, while in the high state shows a signature of inflow. The differences in the kinematics between the Balmer lines and between the low state and the high state suggests complex BLR dynamics. We find that the BLR radius and velocity (both FWHM and σ) do not obey a constant virial product throughout the monitoring period. We find that the BLR lags and continuum luminosity are correlated, consistent with rapid response of the BLR gas to the illuminating continuum. The BLR kinematic profile changes in unpredictable ways that are not related to continuum changes and reverberation lag. Our observations indicate that nonvirial kinematics can significantly contribute to observed line profiles, suggesting caution for black hole mass estimation in luminous and highly varying quasars like RM160.

Survival Distinctions for Cases Representing Immunologically Cold Tumors via Intrinsic Disorder Assessments for Blood-Sourced TRB Variable Regions

T cell receptor beta (TRB) sequences were recovered from the Cancer Genome Atlas Uveal Melanoma blood exome files. Intrinsic disorder scores for amino acid (AA) sequences of the entire TRB variable region were obtained and evaluated as potentially representative of overall survival (OS) distinctions, i.e., for cases representing the upper and lower 50th percentiles for intrinsic disorder scores. Analyses using four intrinsic disorder assessment tools indicated that a lower intrinsic disorder of the blood-sourced TRB variable regions, including continuous AA sequences of the V-gene segment, the complementarity-determining region-3, and the J-gene segment, was associated with a better OS probability (with log-rank p-values ranging from 0.002 to 0.014). We further determined that intrinsic disorder assessments could be used for OS stratification for a second, immunologically cold cancer: MYCN amplified neuroblastoma. Thus, intrinsic disorder assessments of blood-sourced, full TRB variable regions may provide a novel patient stratification approach for patients with immunologically cold cancers.

Complete Chloroplast Genome of Crassula aquatica: Comparative Genomic Analysis and Phylogenetic Relationships

Background/Objectives: Crassula aquatica (L.) Schonl. is a very small annual plant growing along riverbanks. Chloroplast (cp) genomes, crucial for photosynthesis, are highly conserved and play a key role in understanding plant evolution. In this study, we conducted cp genome analysis of C. aquatica, aiming to elucidate its phylogenetic position and structural variations. We analyzed and described the features of the complete cp genome of C. aquatica and conducted comparative analysis with the cp genomes of closely related taxa. Rsults: The cp genome was 144,503 bp in length and exhibited the typical quadripartite structure, consisting of a large single-copy region (LSC; 77,993 bp), a small single-copy region (SSC; 16,784 bp), and two inverted repeats (24,863 bp). The cp genome of C. aquatica comprised 113 unique genes, including 79 protein-coding genes (PCGs), 30 tRNAs, and 4 rRNA genes. Comparative genomic analysis of 13 other Crassula species and six outgroups demonstrated highly conserved gene content and order among Crassula species. However, notable differences were observed, including the complete loss of the rpoC1 intron in C. aquatica and several closely related species, which may serve as a synapomorphic trait supporting the monophyly of the subgenus Disporocarpa. We analyzed the nucleotide diversity among 14 Crassula cp genomes and identified five highly variable regions (pi &gt; 0.08) in the IGS regions. Phylogenetic analysis based on 78 PCGs confirmed the monophyly of Crassula and its division into two subgenera: Crassula and Disporocarpa. Although the phylogenetic tree supported the subgeneric classification system, the sectional classification system requires reassessment. Conclusions: In this study, we conducted a comparative analysis of the cp genome of the genus Crassula. We inferred evolutionary trends within the Crassula cp genome and provided molecular evidence supporting the integration of the genus Tillaea into the genus Crassula. However, as this study does not represent all species within the genus Tillaea, further comprehensive phylogenetic analyses are requrired.

Comparative Chloroplast Genome Analysis in High-Yielding Pinus kesiya var. langbianensis

Pinus kesiya var. langbianensis, a species endemic to Yunnan, China, accounts for over 90% of Yunnan’s Pinus resin production. However, there is significant variation in resin yield among individuals, and molecular markers for identifying high-yielding individuals have yet to be developed. In this study, a comparative analysis of complete chloroplast genomes of P. kesiya var. langbianensis was conducted to perform a phylogenetic analysis and differentiate high-yielding individuals. Both high-yielding (HY) and low-yielding (LY) trees possess a typical quadripartite structure, with respective genome sizes of 119,812 bp and 119,780 bp. Each chloroplast genome contains 112 genes, including 72 protein-coding genes, 36 tRNAs, and 4 rRNAs. Furthermore, HY and LY trees contain 30 and 34 SSRs, respectively, with mononucleotide repeats being predominant; neither genome exhibited trinucleotide or pentanucleotide repeats. Six highly variable regions were identified: trnI-CAU-psbA, trnH-GUG-trnI-CAU, rpl16, rrn4.5-rrn5, petG-petL, and psaJ. Phylogenetic analysis based on 72 Pinus species revealed that HY and LY trees clustered separately, with the HY tree grouping with P. kesiya and the LY tree with P. yunnanensis. This study provides a theoretical foundation for the molecular identification of high-yield P. kesiya var. langbianensis individuals and enriches the understanding of its phylogenetic relationships.

Lactuca super-pangenome reduces bias towards reference genes in lettuce research

BackgroundBreeding of lettuce (Lactuca sativa L.), the most important leafy vegetable worldwide, for enhanced disease resistance and resilience relies on multiple wild relatives to provide the necessary genetic diversity. In this study, we constructed a super-pangenome based on four Lactuca species (representing the primary, secondary and tertiary gene pools) and comprising 474 accessions. We include 68 newly sequenced accessions to improve cultivar coverage and add important foundational breeding lines.ResultsWith the super-pangenome we find substantial presence/absence variation (PAV) and copy-number variation (CNV). Functional enrichment analyses of core and variable genes show that transcriptional regulators are conserved whereas disease resistance genes are variable. PAV-genome-wide association studies (GWAS) and CNV-GWAS are largely congruent with single-nucleotide polymorphism (SNP)-GWAS. Importantly, they also identify several major novel quantitative trait loci (QTL) for resistance against Bremia lactucae in variable regions not present in the reference lettuce genome. The usability of the super-pangenome is demonstrated by identifying the likely origin of non-reference resistance loci from the wild relatives Lactuca serriola, Lactuca saligna and Lactuca virosa.ConclusionsThe super-pangenome offers a broader view on the gene repertoire of lettuce, revealing relevant loci that are not in the reference genome(s). The provided methodology and data provide a strong basis for research into PAVs, CNVs and other variation underlying important biological traits of lettuce and other crops.

Biological Characteristics of the Cytochrome P 450 Family and the Mechanism of Terpinolene Metabolism in Hyalomma asiaticum (Acari: Ixodidae)

The CYP450 enzyme is a superfamily enzyme ubiquitously found in nearly all organisms, playing a vital role in the metabolism of both endogenous and exogenous compounds, and in biosynthesis. Unfortunately, an understanding of its classification, functions, expression characteristics, and other biological traits in Hyalomma asiaticum, a vector for Crimean–Congo Hemorrhagic Fever, as well as of the genes implicated in its natural product metabolism, is lacking. Towards this end, this study has identified 120 H. asiaticum CYP450 genes via transcriptome data in the face of a joint genome threat from terpinolene. The proteins these genes encode are of higher molecular weight, devoid of a signal peptide, and composed of unstable hydrophobic proteins principally containing 1–3 variable transmembrane regions. Phylogenetic evolution classifies these H. asiaticum CYP450 genes into four subfamilies. These genes all encompass complete CYP450 conserved domains, and five specific conserved motifs, albeit with different expression levels. GO and KEGG annotation findings suggest a widespread distribution of these CYP450 genes in many physiological systems, predominantly facilitating lipid metabolism, terpenoid compound metabolism, and polyketone compound metabolism, as well as cofactor and vitamin metabolism at a cellular level. Molecular docking results reveal a hydrophobic interaction between the ARG-103, ARG-104, LEU-106, PHE-109, and ILE-119 amino acid residues in CYP3A8, which is primarily expressed in the fat body, and terpinolene, with a notably up-regulated expression, with affinity = −5.6 kcal/mol. The conservation of these five key amino acid residues varies across 12 tick species, implying differences in terpinolene metabolism efficacy among various tick species. This study thereby fills an existing knowledge gap regarding the biological characteristics of H. asiaticum CYP450 genes and paves the way for further research into the functions of these particular genes.

Immunoglobulin secretion influences the composition of chicken caecal microbiota

The chicken caecum is colonised by hundreds of different bacterial species. Which of these are targeted by immunoglobulins and how immunoglobulin expression shapes chicken caecal microbiota has been addressed in this study. Using cell sorting followed by sequencing of V3/V4 variable region of 16S rRNA, bacterial species with increased or decreased immunoglobulin coating were determined. Next, we determined also caecal microbiota composition in immunoglobulin knockout chickens. We found that immunoglobulin coating was common and major taxa were coated with immunoglobulins. Similarly, more taxa required immunoglobulin production for caecum colonisation compared to those which became abundant in immunoglobulin-deficient chickens. Taxa with low immunoglobulin coating such as Lactobacillus, Blautia, [Eubacterium] hallii, Megamonas, Fusobacterium and Desulfovibrio all encode S-layer proteins which may reduce interactions with immunoglobulins. Although there were taxa which overgrew in Ig-deficient chickens (e.g. Akkermansia) indicating immunoglobulin production acted to exclude them from the chicken caecum, in most of the cases, immunoglobulin production more likely contributed to fixing the desired microbiota in the chicken caecum.

An integrative taxonomic revision of the Chaerophyllum hirsutum complex (Apiaceae) using morphological and molecular markers

Background and aims – Chaerophyllum hirsutum represents a complex of taxa with varying treatments and ranks across floras. Using both morphometric and molecular markers, we assessed the robustness of C. hirsutum, C. elegans, C. villarsii, and C. villarsii var. cicutariiforme. Material and methods – Ten morphometric variables and two ratios were calculated. Based on the sequencing of six plastomes, the rps16 intron was selected as the more variable region and sequenced on a broader sampling. Additionally, we also sequenced the nrDNA internal transcribed spacer 2 (ITS2) using Illumina technology to obtain intra-individual allelic diversity. Key results – Morphologically, the most easily differentiated taxon was C. elegans, especially using the number of subterminal umbels. The distinction between C. hirsutum and C. villarsii was rather clinal, but is mainly based on the degree of carpophore division. Finally, C. villarsii var. cicutariiforme was less easily distinguishable from the three others, but partly using the carpophore length and the total length of basal leaf blade. The cpDNArps16 clearly distinguished C. elegans from the three other taxa of the complex, which rather showed a geographical pattern of cpDNA diversity. The nrDNA ITS2 partially distinguished C. villarsii from the other taxa, without distinction of C. elegans. Conclusions – The present study supports the species differentiation of C. elegans based on both morphology and chloroplast genome. Furthermore, C. villarsii var. villarsii and C. villarsii var. cicutariiforme could potentially be recognized as distinct varieties within C. hirsutum. This will need to be confirmed by future studies using a larger sampling size and more comprehensive markers, covering a broader portion of the nuclear genome.

Comparative and phylogenetic analysis of the chloroplast genomes of four commonly used medicinal cultivars of Chrysanthemums morifolium

‘Boju’ and ‘Huaiju’ are cultivars of the Chrysanthemum (Chrysanthemum morifolium Ramat.) in the family Asteraceae, valued for their medicinal, tea, and ornamental properties, and valued by individuals. However, the yield and quality of medicinal chrysanthemums are limited by the characteristics of the germplasm resources, including the identification at the varieties and cultivation levels. Currently, research characterizing the chloroplast genomes of medicinal Chrysanthemum flowers is relatively limited. This study conducted chloroplast whole-genome sequencing on two cultivars of Chrysanthemum, ‘Boju’ and ‘Huaiju’, and compared them with the previously published chloroplast genomes of ‘Hangbaiju’ and ‘Gongju’. The study analyzed the chloroplast genome structures of these four medicinal Chrysanthemums, identifying mutation hotspots and clarifying their phylogenetic relationships. The chloroplast genome sizes of four medicinal Chrysanthemum cultivation products ranged from 151,057 to 151,109 bp, with GC content ranging from 37.45% to 37.76%. A total of 134 genes were identified, including 89 protein-coding genes, 37 ribosomal RNA genes, and 8 transfer RNA genes. Comparative genomic analysis revealed 159 large repeat sequences, 276 simple sequence repeats, 1 gene, and 8 intergenic regions identified as highly variable regions. Nucleotide diversity (Pi) values were high (≥ 0.004) for the petN-psbM, trnR-UCU-trnT-GGU, trnT-GGU-psbD, ndhC-trnV-UCA, ycf1, ndhI-ndhG, trnL-UGA-rpl32, rpl32-ndhF, and ndhF-ycf1 fragments, aiding in variety identification. Phylogenetic analysis revealed consistent results between maximum likelihood and Bayesian inference trees, showing that the four medicinal Chrysanthemum cultivars, along with their wild counterparts and related species, evolved as a monophyletic group, forming a sister clade to Artemisia and Ajania. Among the six Chrysanthemum species, the wild Chrysanthemum diverged first (Posterior probability = 1, bootstrap = 1,000), followed by Ajania, while C. indicum and C. morifolium clustered together (Bootstrap = 100), indicating their closest genetic relationship. The chloroplast whole-genome data and characteristic information provided in this study can be used for variety identification, genetic conservation, and phylogenetic analysis within the family Asteraceae.

Distributed Autonomous Economic Control Strategy for the AC/DC Interconnected Microgrid Considering the Regulation Boundary of the Consensus Variable

ABSTRACTThe autonomous economic control for the islanded AC/DC interconnected microgrid has various modes due to the restriction of the feasible regions of inner and inter‐microgrid resources. To properly handle the cooperative relationship between resources with different regulation characteristics and realize the frequency, voltage stability, and economic operation of the AC/DC interconnected microgrid, a distributed control strategy considering the regulation boundary of the consensus variable is proposed. Firstly, the influence of the variable feasible regions on the consensus control objective is analyzed, and the fundamental principle of the consensus control algorithm considering the variable regulation boundary is expounded. Secondly, the trilevel control strategy consisting of the local equipment control and inner and inter‐microgrid cooperative control is constructed. The local equipment control adopts droop control, and the cooperative control adopts consensus control to realize the elimination of the frequency and voltage deviation and the economic dispatch of active power within and between microgrids. Finally, the AC/DC interconnected microgrid model is built based on PSCAD/EMTDC, and the simulation results verify the effectiveness of the proposed control strategy.

Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells.

Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the DNA of the isolated cells, rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of IGV gene subgroup-specific primers recognizing nearly all IGV genes together with primers for the J genes. By sequence analysis of IGV region genes from distinct cells, the clonal relationship of the B-lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D and J gene usage. Moreover, the presence and pattern of somatic IGV gene mutations give valuable insight into the differentiation stage of the B cells.

Generation of canine neutralizing antibodies against canine parvovirus by single B cell antibody technology.

Canine parvovirus (CPV) is a significant threat to canines and is widely distributed worldwide. While vaccination is currently the most effective preventive measure, existing vaccines are not able to offer comprehensive and dependable protection against CPV infection. Hence, there is a need to explore alternative or complementary strategies to tackle this problem. In this study, we present an approach for the efficient screening of canine antibodies targeting CPV using a single B cell antibody technique. We sorted single IgM- IgG+ CPV+ B cells from canine peripheral blood mononuclear cells using fluorescence-activated cell sorting (FACS) and obtained the variable region genes of heavy and light chains (VH and VL) by nested PCR amplification. Canine monoclonal antibodies were expressed in HEK293 cells, and a total of 60 antibodies were obtained, five of which demonstrated neutralizing activity against CPV. Those findings demonstrate the effectiveness of the method for obtaining canine monoclonal antibodies, which in turn aids in the identification and screening of neutralizing antibodies against various canine pathogens.

Microstructural correlates of olfactory dysfunction in Parkinson's Disease: a systematic review of diffusion MRI studies.

Olfactory dysfunction (OD) is a non-motor symptom of Parkinson's Disease, affecting 75-95% of the patients. This symptom usually emerges before the clinical diagnosis, and patients with OD present with more severe forms of PD and need higher doses of therapy. It remains unknown whether OD is just a mere non-motor symptom or if it is a part of a series of pathological changes in different brain regions of the affected patients. We performed a systematic review to find the microstructural correlates of OD in people with PD. The systematic search in PubMed, Scopus, Embase, and Web of Science yielded ten eligible studies. Assessments in most included studies were inconclusive. However, we found variable brain regions and tracts associated with OD. The most repeated areas included the primary olfactory cortex, gyrus rectus, inferior fronto-occipital fasciculus, inferior longitudinal fasciculus, corticospinal tract, uncinate fasciculus, cingulum, and cerebellar peduncle. Despite some limitations, we pointed out the microstructural correlates of OD, which were also present in areas other than the olfactory system. These findings imply that OD might be a manifestation of an unknown, greater pathology in the brain of patients with PD.

A multispecific antibody against SARS-CoV-2 prevents immune escape in vitro and confers prophylactic protection in vivo.

Despite effective countermeasures, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists worldwide because of its ability to diversify and evade human immunity. This evasion stems from amino acid substitutions, particularly in the receptor binding domain (RBD) of the spike protein that confers resistance to vaccine-induced antibodies and antibody therapeutics. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different RBD sites into multispecific antibodies. Here, we describe multispecific antibodies, including a trivalent trispecific antibody that potently neutralized diverse SARS-CoV-2 variants and prevented virus escape more effectively than single antibodies or mixtures of the parental antibodies. Despite being generated before the appearance of Omicron, this trispecific antibody neutralized all major Omicron variants through BA.4/BA.5 at nanomolar concentrations. Negative stain electron microscopy suggested that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated binding across more than one spike protein. Moreover, a tetravalent trispecific antibody containing the same variable regions as the trivalent trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2, and BA.5 challenge, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. These results demonstrated that multispecific antibodies have the potential to provide broad SARS-CoV-2 coverage, decrease the likelihood of escape, simplify treatment, and provide a strategy for antibody therapies that could help eliminate pandemic spread for this and other pathogens.