Biological Characteristics Research Articles

Bioanalysis of antisense oligonucleotides: Techniques, challenges, regulatory considerations, and future perspectives

Antisense oligonucleotides (ASOs) are a powerful class of therapeutics designed to target RNA sequences and regulate gene expression, offering promising treatment avenues for various genetic disorders, neurodegenerative diseases, cancers, and viral infections. The bioanalysis of ASOs presents unique challenges due to their distinct chemical and biological characteristics, requiring specialized analytical methods and regulatory oversight. This review explores the latest methodologies in ASO bioanalysis, assesses the current regulatory environment, and considers future directions, particularly in the context of critical and emerging technologies (CETs) relevant to national security strategies. ASOs, classified under biotechnology, play a crucial role in gene expression modulation and protein regulation. By integrating insights from CETs, this review emphasizes the strategic significance of ASO bioanalysis in driving advancements in medical science and contributing to national security.

Biological characterisation and computational conformation dynamics of putative L-glutaminase YLaM identified from Bacillus licheniformis

ABSTRACT Glutaminases are a unique group of hydrolytic enzymes with potential applications in cancer therapy and the food industry. This study focused on the biological characterisation and computational conformation dynamics of YLaM, a potential L-glutaminase from a halothermotolerant Bacillus licheniformis. The research integrated experimental and computational methods to identify the YLaM sequence and predict its structural and functional properties. YLaM was amplified using a polymerase chain reaction, cloned into a pET-22b(+) vector, sequenced, and submitted to BankIt/NCBI. This experimental work was complemented by in silico analysis, including structural modelling via the I-TASSER web server, molecular docking using AutoDockTools 1.5.6, and molecular dynamic (MD) simulations using GROMACS v5.1.4. The study resulted in the development of a high-quality 3D model of YLaM through homology modelling and structural refinement, enabling a detailed exploration of its binding affinity with L-glutamine (L-Gln). By identifying active site residues using the homotetramer structure of human glutaminase, crucial interactions with L-Gln were validated, confirming its catalytic function. Finally, MD simulations confirmed the stability of the complex under physiological conditions. This combined molecular identification and functional simulations provide comprehensive insights into the physicochemical properties of the enzyme, offering valuable information for subsequent assessments, including recombinant production and biological impact evaluations.

Alkyl Pyridinol Compounds Exhibit Antimicrobial Effects against Gram-Positive Bacteria

Background/Objectives. The rise of antibiotic-resistant pathogens represents a significant global challenge in infectious disease control, which is amplified by the decline in the discovery of novel antibiotics. Staphylococcus aureus continues to be a highly significant pathogen, causing infections in multiple organs and tissues in both healthcare institutions and community settings. The bacterium has become increasingly resistant to all available antibiotics. Consequently, there is an urgent need for novel small molecules that inhibit the growth or impair the survival of bacterial pathogens. Given their large structural and chemical diversity, as well as often unique mechanisms of action, natural products represent an excellent avenue for the discovery and development of novel antimicrobial treatments. Anaephene A and B are two such naturally occurring compounds with significant antimicrobial activity against Gram-positive bacteria. Here, we report the rapid syntheses and biological characterization of five novel anaephene derivatives, which display low cytotoxicity against mammalian cells but potent antibacterial activity against various S. aureus strains, including methicillin-resistant S. aureus (MRSA) and the multi-drug-resistant community-acquired strain USA300LAC. Methods. A Sonogashira cross-coupling reaction served as the key step for the synthesis of the alkyl pyridinol products. Results/Conclusions. Using the compound JC-01-074, which displays bactericidal activity already at low concentrations (MIC: 16 μg/mL), we provide evidence that alkyl pyridinols target actively growing and biofilm-forming cells and show that these compounds cause disruption and deformation of the staphylococcal membrane, indicating a membrane-associated mechanism of action.

Metathesis Mastery: Crafting Linear Triquinane Frameworks for Natural Product Synthesis

AbstractOrganic and medicinal chemists have shown great interest in developing simple approaches for creating linear triquinane structures, as they are crucial for synthesizing both natural and synthetic products. Linear triquinane shows various biological characteristics like being commonly used for antitumor, antibacterial, and antimalarial purposes, making it a popular subject of study among synthetic chemists. These come from different sources like marine organisms, plants, and fungi and make up a significant group of sesquiterpenoids. These can be classified as cis‐syn‐cis or cis‐anti‐cis based on the stereochemistry at the ring junction, with cis‐anti‐cis being the most common type and exhibiting significant biological activity. In this review, we summarized different methods using a combination of photothermal and olefin metathesis strategy to construct a triquinane framework by utilizing both traditional heat and microwave radiation. Some commonly used synthetic methods, including metathesis protocol and key reactions like Grignard reaction and Diels–Alder reaction, were utilized together with ring‐closing, ring‐opining, and ring‐rearrangement metathesis to create the linear triquinane core for use in total synthesis of natural products. Additionally, the use of photothermal olefin metathesis along with Hosomi‐Sakurai reaction conditions allows for the creation of intricate polyquinanes, which can be used to produce complex natural compounds and for designing unique molecules theoretically.

Sandy loam soil maintains better physicochemical parameters and more abundant beneficial microbiomes than clay soil in Stevia rebaudiana cultivation

Depending on the texture of soil, different physicochemical and microbiological parameters are characterized, and these characteristics are influenced by crop cultivation. Stevia, a popular zero-calorie sweetener crop, is widely cultivated around the world on various soil textures. Sandy loam and clay soil show great differences in physicochemical and biological parameters and are often used for Stevia cultivation. To understand the effects of Stevia cultivation on soil physicochemical and biological features, we investigated the changes of physicochemical and microbiological parameters in sandy loam and clay soil following Stevia cultivation. This study was carried out through different physiological and biochemical assays and microbiomic analysis. The results indicated that the sandy loam soil had significantly lower pH and higher nutrient content in the rhizosphere and bulk soils after the Stevia cultivation. The sandy loam soil maintained higher bacterial diversity and richness than the clay soil after Stevia harvest. Beneficial bacteria such as Dongia, SWB02, Chryseolinea, Bryobacter and Devosia were enriched in the sandy loam soil; however, bacteria such as RB41, Haliangium and Ramlibacter, which are unfavorable for nutrient accumulation, predominated in clay soil. Redundancy analysis indicated that the variation in the composition of bacterial community was mainly driven by soil pH, organic matter, total nitrogen, available phosphorus, and microbial biomass phosphorus. This study provides a deeper understanding of physicochemical and microbiological changes in different soil textures after Stevia cultivation and guidance on fertilizer management for Stevia rotational cultivation.

Biological activity and chemical characteristics studies of new oligomannose produced by Erwinia gerundensis

Natural polymers have attracted considerable attention in recent decades among scientists due to their potential therapeutic uses, particularly as antimicrobial and antitumor agents. In this research, novel EPSs were extracted from garlic rhizosphere bacteria. The antibacterial and antitumor activities of the polymer were evaluated through biological assays. The antibacterial activity was tested against gram-positive microorganisms (such as Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus) and gram-negative organisms (such as Shigella sonnei and Escherichia coli). The most significant inhibition zone was observed with Listeria monocytogenes and S. typhi, measuring 35 mm, while the most miniature antibacterial effect was seen with Staphylococcus aureus at 23.67 mm.Furthermore, the inhibitory effect of the crude polymer was assessed using a broth medium with two strains of E. coli and Bacillus cereus. Electron microscope images displayed varying degrees of damage to bacterial cells in the treated broth. The antitumor activity was determined using the MTT test on colon carcinoma cells (HCT-116), hepatocellular carcinoma cells (HepG-2), and CaCO2 (intestinal carcinoma cells), with IC50 values of 188.86±6.17 µg/mL, 221.66±8.02 µg/mL, and 203.65±7.43 µg/mL, respectively, after 48 h. The bacteria responsible for polymer production were isolated from garlic plant rhizospheres and identified as Erwinia gerundensis CCASU-2024–69 through 16S rRNA sequencing. FTIR and NMR techniques determined the crude EPS's main components and functional groups, including carbonyl, carboxylic, methylene, and silanol. GC–MS analysis revealed 34 bioactive compounds, while HPLC analysis indicated that the EPS was a hetero-monosaccharide consisting of d-xylose, d-glucose, l-arabinose, ribose, and d-mannose. This research study represents the initial exploration into the exopolysaccharide derived from Erwinia gerundensis. To assess the interaction between the exopolysaccharide and the active sites of Bacillus cereus and E. coli, molecular docking experiments were conducted using five monosaccharides: d-xylose, d-glucose, l-arabinose, ribose, and d-mannose. The data obtained from the molecular docking analysis strongly correlates with the findings from biological studies.Furthermore, these highly active compounds exhibit a favorable proposed ADMET profile. This particular exopolysaccharide shows potential as a natural antibiotic and holds promise in treating gastrointestinal cancer. A comprehensive assessment of laboratory animals is essential before its potential use as a prebiotic in nutrition.

CBL mutations in chronic myelomonocytic leukemia often occur in the RING domain with multiple subclones per patient: Implications for targeting

Chronic myelomonocytic leukemia (CMML) is a rare blood cancer of older adults (3 in every 1,000,000 persons) characterized by poor survival and lacking effective mutation-specific therapy. Mutations in the ubiquitin ligase Cbl occur frequently in CMML and share biological and molecular features with a clonal disease occurring in children, juvenile myelomonocytic leukemia (JMML). Here we analyzed the clinical presentations, molecular features and immunophenotype of CMML patients with CBL mutations enrolled in a prospective Phase II clinical trial stratified according to molecular markers. Clinically, CBL mutations were associated with increased bone marrow blasts at diagnosis, leukocytosis and splenomegaly, similar to patients harboring NRAS or KRAS mutations. Interestingly, 64% of patients presented with more than one CBL variant implying a complex subclonal architecture, often with co-occurrence of TET2 mutations. We found CBL mutations in CMML frequently clustered in the RING domain in contrast to JMML, where mutations frequently involve the linker helix region (P<0.0001). According to our comparative alignment of available X-ray structures, mutations in the linker helix region such as Y371E give rise to conformational differences that could be exploited by targeted therapy approaches. Furthermore, we noted an increased percentage of CMML CD34+ stem and progenitor cells expressing CD116 and CD131 in all CBL mutant cases and increased CD116 receptor density compared to healthy controls, similar to CMML overall. In summary, our data demonstrate that CBL mutations are associated with distinct molecular and clinical features in CMML and are potentially targetable with CD116-directed immunotherapy.

Multiomics and single‐cell sequencings reveal the specific biological characteristics of low Ki‐67 triple‐negative breast cancer

Multiomics and single‐cell sequencings reveal the specific biological characteristics of low Ki‐67 triple‐negative breast cancer

Corrosion Resistance and In Vitro Biological Properties of TiO2 on MAO-Coated AZ31 Magnesium Alloy via ALD

The surface corrosion of magnesium alloys is effectively addressed currently by the creation of a micro-arc oxidation (MAO) ceramic layer. However, oxide film porousness restricts magnesium alloy use. Thus, this work used atomic layer deposition (ALD) to create a TiO2 coating on MAO-coated AZ31B magnesium alloy to plug micropores and increase corrosion resistance and biological characteristics. The samples were analyzed using SEM, EDS, XPS, and XRD to determine their surface appearance, chemical content, and microstructure. Micro-arc oxidation produced a 20 μm oxide coating. The TiO2 film reached 47.41 nm after 400 atomic layer deposition cycles. All corroded samples were tested for corrosion resistance using electrochemical and hydrogen evolution methods and examined for surface morphology. In vitro cell experiments examined biocompatibility. The results indicate that the TiO2 layer sealed the MAO coating’s micro-pores and micro-cracks, enhanced corrosion resistance, and preserved surface morphology following corrosion. The TiO2/MAO composite coating is more biocompatible than the substrate and MAO coating. This research proposes coating AZ31B magnesium alloy for bio-remediation to increase corrosion resistance and biocompatibility.

Development of new pyrazoles as class I HDAC inhibitors: Synthesis, molecular modeling, and biological characterization in leukemia cells.

Class I histone deacetylases (HDACs) are considered promising targets in current cancer research. To obtain subtype-selective and potent HDAC inhibitors, we used the aminobenzamide scaffold as the zinc-binding group and prepared new derivatives with a pyrazole ring as the linking group. The synthesized compounds were analyzed in vitro using an enzymatic assay against HDAC1, -2, and -3. Compounds 12b, 15b, and 15i were found to be potent HDAC1 inhibitors, also in comparison to the reference compounds entinostat and tacedinaline, with IC50 values of 0.93, 0.22, and 0.68 μM, respectively. The best compounds were measured for their cellular effect and target engagement in acute myeloid leukemia (AML) cells. In addition, we studied the interaction of the compounds with HDAC subtypes using docking and molecular dynamic simulations. In summary, we have developed a new chemotype of HDAC1 inhibitors that can be used for further structure-based optimization.

A remarkable new species of the genus Digitivalva Gaedike, 1970 (Lepidoptera, Glyphipterigidae, Acrolepiinae) from Japan

A new species of the genus Digitivalva Gaedike, 1970 from Japan, D. bella Zhang, Yagi & Hirowatari, sp. nov., is described. The host plant of this species is identified as Carpesium divaricatum Sieb. & Zucc. (Asteraceae). Larvae of this species pupate beneath the host tissue; this habit is for the first time reported for the genus Digitivalva in Japan. Adult specimens with male and female genitalia and pupae are illustrated and described in detail. Although the wing markings differ, the new species is considered closely related to D. arnicella (Heyden, 1863) from Europe, with which it shares similar biological and morphological features.

Genetic history and biological adaptive landscape of the Tujia people inferred from shared haplotypes and alleles

BackgroundHigh-quality genomic datasets from under-representative populations are essential for population genetic analysis and medical relevance. Although the Tujia are the most populous ethnic minority in southwestern China, previous genetic studies have been fragmented and only partially reveal their genetic diversity landscape. The understanding of their fine-scale genetic structure and potentially differentiated biological adaptive features remains nascent.ObjectivesThis study aims to explore the demographic history and genetic architecture related to the natural selection of the Tujia people, focusing on a meta-Tujia population from the central regions of the Yangtze River Basin.ResultsPopulation genetic analyses conducted on the meta-Tujia people indicate that they occupy an intermediate position in the East Asian North-South genetic cline. A close genetic affinity was identified between the Tujia people and neighboring Sinitic-speaking populations. Admixture models suggest that the Tujia can be modeled as a mixture of northern and southern ancestries. Estimates of f3/f4 statistics confirmed the presence of ancestral links to ancient Yellow River Basin millet farmers and the BaBanQinCen-related groups. Furthermore, population-specific natural selection signatures were explored, revealing highly differentiated functional variants between the Tujia and southern indigenous populations, including genes associated with hair morphology (e.g., EDAR) and skin pigmentation (e.g., SLC24A5). Additionally, both shared and unique selection signatures were identified among ethnically diverse but geographically adjacent populations, highlighting their extensive admixture and the biological adaptations introduced by this admixture.ConclusionsThe study unveils significant population movements and genetic admixture among the Tujia and other ethno-linguistically diverse East Asian groups, elucidating the differentiated adaptation processes across geographically diverse populations from the current genetic landscape.

Clinical-Biological Particularities and Clinical Forms of Metabolic Liver Steatosis in a Group of Romanian Patients

Formerly named Non-alcoholic fatty liver disease (NAFLD) is now known as Metabolic associated steatotic liver disease (MASLD) and is frequent pathology in daily clinical practice. The paper present a study of 125 romanian patients with MASLD with anlysis of important data on the characteristics of disease in our geographical area, with highlight on some interesting correlations between clinical and biological features and the particularities of the disease: correlations between clinical aspects and laboratory examinations (imaging, biochemical), frequency and type of risk factors, clinical aspects and forms of liver steatosis, associated diseases, biochemical characteristics.

Design, Biological Characterization, and Discovery of Novel Cyclohexenyl Derivatives as Kinesin KIF18A Inhibitors for the Treatment of Ovarian Cancer

Design, Biological Characterization, and Discovery of Novel Cyclohexenyl Derivatives as Kinesin KIF18A Inhibitors for the Treatment of Ovarian Cancer

Inoculation with Jeotgalicoccus sp. improves nutritional quality and biological value of Eruca sativa by enhancing amino acid and phenolic metabolism and increasing mineral uptake, unsaturated fatty acids, vitamins, and antioxidants

Plant growth-promoting bacteria (PGPB) are considered a promising tool for triggering the synthesis of bioactive compounds in plants and to produce healthy foods. This study aimed to demonstrate the impact of PGPB on the growth, accumulation of primary and secondary metabolites, biological activities, and nutritional qualities of Eruca sativa (arugula), a key leafy vegetable worldwide. To this end, Jeotgalicoccus sp. (JW0823), was isolated and identified by using partial 16S rDNA-based identification and phylogenetic analysis. The findings revealed that JW0823 significantly boosted plant biomass production by about 45% (P<0.05) and enhanced pigment contents by 47.5% to 83.8%. JW0823-treated plants showed remarkable improvements in their proximate composition and vitamin contents, with vitamin E levels increasing by 161.5%. JW0823 induced the accumulation of bioactive metabolites including antioxidants, vitamins, unsaturated fatty acids, and essential amino acids, thereby improving the nutritional qualities of treated plants. An increase in the amounts of amino acids was recorded, with isoleucine showing the highest increase of 270.2%. This was accompanied by increased activity of the key enzymes involved in amino acid biosynthesis, including glutamine synthase, dihydrodipicolinate synthase, cystathionine γ-synthase, and phenylalanine ammonia-lyase enzymes. Consequently, the total antioxidant and antidiabetic activities of the inoculated plants were enhanced. Additionally, JW0823 improved antimicrobial activity against several pathogenic microorganisms. Overall, the JW0823 treatment is a highly promising method for enhancing the health-promoting properties and biological characteristics of E. sativa, making it a valuable tool for improving the quality of this important leafy vegetable.

A New Approach for CRISPR/Cas9 Editing and Selection of Pathogen-Resistant Plant Cells of Wine Grape cv. ‘Merlot’

Grape is one of the most economically significant berry crops. Owing to the biological characteristics of grapes, such as the long juvenile period (5–8 years), high degree of genome heterozygosity, and the frequent occurrence of inbreeding depression, homozygosity during crossbreeding leads to loss of varietal characteristics and viability. CRISPR/Cas editing has become the tool of choice for improving elite technical grape varieties. This study provides the first evidence of a decrease in the total fraction of phenolic compounds and an increase in the concentration of peroxide compounds in grape callus cells upon the addition of chitosan to the culture medium. These previously unreported metabolic features of the grape response to chitosan have been described and used for the first time to increase the probability of selecting plant cells with MLO7 knockout characterised by an oxidative burst in response to the presence of a pathogen modulated by chitosan in the high-metabolite black grape variety ‘Merlot’. This was achieved by using a CRISPR/Cas9 editing vector construction with the peroxide sensor HyPer as a reporter. This research represents the first CRISPR/Cas9 editing of ‘Merlot’, one of the most economically important elite technical grape varieties.

Estimation of the ecological integrity of the Guadiana River using Partial Least Squares Path Modelling and simulation scenarios

Ecological integrity is fundamental to human life and ecosystems, so its assessment and management are crucial. This concept assesses ecosystem health by examining physico-chemical and biological characteristics, riparian vegetation and macroinvertebrate communities. In recent decades, water resources have undergone significant changes due to various factors that have contributed to the physical, chemical and biological pollution of water. To address this problem, a specific model has been developed using the Partial Least Squares Path Modelling methodology to analyse and quantify the main factors affecting the ecological integrity of the Spanish part of the Guadiana River (Spain). The variables analysed at the different sampling points in the catchment include forest cover, anthropogenic pressure, water quality and biological integrity. Water quality and biological integrity, in turn, constitute the concept of ecological integrity. The model predicts 60.3 % of the physico-chemical water quality and 56.6 % of the biological integrity, showing that ¨Forest cover¨ negatively impacts water quality (W = −0.476) by reducing pollution, while ¨Anthropogenic Pressure¨ positively impacts it (W = 0.680) by increasing pollution. Based on the modelling, three future scenarios were designed, from the lowest to the highest pressure considering changes in riparian forest quality based on QBR and changes in the number of reservoirs: a favourable scenario with high riparian forest quality and no reservoirs; an intermediate scenario with good riparian forest quality and no change in the number of reservoirs; and an unfavourable scenario, characterised by very poor riparian forest quality and an increase in the number of reservoirs. In this context, the importance of the conservation and enhancement of riparian vegetation as a nature-based solution is highlighted, as well as the pressure generated by industrial activity and agricultural practices on the ecological integrity of the study area. The favourable scenario, with very good quality riparian vegetation, improves water quality by up to 85 %, positively impacting the ecological integrity of the river. In contrast, the unfavourable scenario, with extremely degraded riparian forest, would decrease water quality by up to 62 %, negatively affecting ecological integrity. Modelling and future scenarios is an essential tool in the decision-making process to improve environmental governance and water security. In addition, the PLS-PM methodology allows the identification and quantification of relationships between complex variables, providing a solid basis for the design of effective environmental management strategies.

Biological and genomic characterization of a polyvalent phage PSH-1 against multidrug-resistant Salmonella Enteritidis

BackgroundBacteriophage has been renewed attention as a new antibacterial agent due to the limitations of antibiotic treatment. Bacteriophages are generally thought to be highly host specific and even strain specific, but a small number of polyvalent bacteriophages have been found to infect bacteria of different genera.ResultsIn this study, a virulent lytic bacteriophage (named Salmonella phage PSH-1) of Salmonella Enteritidis was isolated from the sewage samples of a large-scale pig farm, PSH-1 demonstrated lytic activity against four multidrug-resistant Salmonella Enteritidis isolates and Escherichia coli, and then its biological characteristics, genome and bacteriostatic ability were investigated. The results showed that the initial titer of PSH-1 was 1.15 × 1010 PFU/mL and the optimal multiplicity of infection (MOI) was 0.01, PSH-1 has stable activity in the range of pH 3.0–11.0. One-step growth curve showed that its latent period was 20 min, burst time was 80 min, and the burst was 495 particles. The whole-genome sequencing results revealed phage PSH-1 had a linear dsDNA with 48,466 bp length. The G/C content was 45.33%. Non-coding RNA genes and virulence factors were not found. Eighty- five open reading frames (ORFs) were identified after online annotation. By tests, the use of phage could succeed in controlling the artificial Salmonella contamination in milk at a range of temperatures.ConclusionsThis study reports a novel Salmonella Enteritidis phage PSH-1, which has a robust lytic ability, no virulence factors, and good stability. The characterization and genomic analysis of PSH-1 will develop our understanding of phage biology and diversity and provide a potential arsenal for controlling of salmonellosis.

Chromosome-level genome assembly of cotton thrips Thrips tabaci (Thysanoptera: Thripidae)

Cotton thrip, Thrips tabaci is a major polyphagous pest widely distributed on a variety of crops around the world, causing huge economic losses to agricultural production. Due to its biological and genomic characteristics, this pest can reproduce quickly and develop resistance to various pesticides in a very short time. However, the lack of high-quality reference genomes has hindered deeper gene function exploration and slows down the development of new management strategies. Here, we assembled a high-quality genome of T. tabaci at the chromosome level for the first time by using Illumina, PacBio long reads, and Hi-C technologies. The 329.59 Mb genome was obtained from 320 contigs, with a contig N50 of 1.53 Mb, and 94.21% of the assembly was anchored to 18 chromosomes. In total, 17,816 protein-coding genes were annotated, and 96.78% of BUSCO genes were fully represented. In conclusion, this high-quality genome provides a valuable genetic basis for our understanding of the biology of T. tabaci and contributes to the development of management strategies for cotton thrip.

Histological differences related to autophagy in the minor salivary gland between primary and secondary types of Sjögren’s syndrome

Some forms of Sjögren’s syndrome (SS) follow a clinical course accompanied by systemic symptoms caused by lymphocyte infiltration and proliferation in the liver, kidneys, and other organs. To better understand the clinical outcomes of SS, here we used minor salivary gland tissues from patients and examine their molecular, biological, and pathological characteristics. A retrospective study was performed, combining clinical data and formalin-fixed paraffin-embedded (FFPE) samples from female patients over 60 years of age who underwent biopsies at Okayama University Hospital. We employed direct digital RNA counting with nCounter® and multiplex immunofluorescence analysis with a PhenoCycler™ on the labial gland biopsies. We compared FFPE samples from SS patients who presented with other connective tissue diseases (secondary SS) with those from stable SS patients with symptoms restricted to the exocrine glands (primary SS). Secondary SS tissues showed enhanced epithelial damage and lymphocytic infiltration accompanied by elevated expression of autophagy marker genes in the immune cells of the labial glands. The close intercellular distance between helper T cells and B cells positive for autophagy-associated molecules suggests accelerated autophagy in these lymphocytes and potential B cell activation by helper T cells. These findings indicate that examination of FFPE samples from labial gland biopsies can be an effective tool for evaluating molecular histological differences between secondary and primary SS through multiplexed analysis of gene expression and tissue imaging.