Obesity Differs from Diabetes Mellitus in Antibody and T Cell Responses Post COVID-19 Recovery.

Obesity and type 2 diabetes (DM) are risk factors for severe COVID-19 outcomes, which disproportionately affect South Asian populations. This study aims to investigate the humoral and cellular immune responses to SARS-CoV-2 in adult COVID-19 survivors with obesity and DM in Bangladesh. In this cross-sectional study, SARS-CoV-2-specific antibody and T cell responses were investigated in 63 healthy and 75 PCR-confirmed COVID-19 recovered individuals in Bangladesh, during the pre-vaccination first wave of the COVID-19 pandemic in 2020. In COVID-19 survivors, SARS-CoV-2 infection induced robust antibody and T cell responses, which correlated with disease severity. After adjusting for age, sex, DM status, disease severity, and time since onset of symptoms, obesity was associated with decreased neutralising antibody titers, and increased SARS-CoV-2 spike-specific IFN-γ response along with increased proliferation and IL-2 production by CD8+ T cells. In contrast, DM was not associated with SARS-CoV-2-specific antibody and T cell responses after adjustment for obesity and other confounders. Obesity is associated with lower neutralising antibody levels and higher T cell responses to SARS-CoV-2 post COVID-19 recovery, while antibody or T cell responses remain unaltered in DM.

Exploration of genes associated with induction of the viable but non-culturable state of Campylobacter jejuni.

Campylobacter jejuni is known to enter a viable but non-culturable (VBNC) state when exposed to environmental stresses. Microarray and quantitative real-time polymerase chain reaction (qPCR) analyses were performed to elucidate the genes related to the induction of the VBNC state. The C. jejuni NCTC11168 strain was cultured under low-temperature or high-osmotic stress conditions to induce the VBNC state. mRNA expression in the VBNC state was investigated using microarray analysis, and the gene encoding peptidoglycan-associated lipoprotein, Pal, was selected as the internal control gene using qPCR analysis and software. The three genes showing particularly large increases in mRNA expression, cj1500, cj1254, and cj1040, were involved in respiration, DNA repair, and transporters, respectively. However, formate dehydrogenase encoded by cj1500 showed decreased activity in the VBNC state. Taken together, C. jejuni actively changed its mRNA expression during induction of the VBNC state, and protein activities did not always match the mRNA expression levels.

Rapid analysis of untreated food samples by gel loading tip spray ionization mass spectrometry.

Rapid, efficient, versatile, easy-to-use, and non-expensive analytical approaches are globally demanded for food analysis. Many ambient ionization approaches based on electrospray ionization (ESI) have been developed recently for the rapid molecular characterization of food products. However, those approaches mainly suffer from insufficient signal duration for comprehensive chemical characterization by tandem MS analysis. Here, a commercially available disposable gel loading tip is used as a low-cost emitter for the direct ionization of untreated food samples. The most important advantages of our approach include high stability, and durability of the signal (> 10 min), low cost (ca. 0.1 USD per run), low sample and solvent consumption, prevention of tip clogging and discharge, operational simplicity, and potential for automation. Quantitative analysis of sulfapyridine, HMF (hydroxymethylfurfural), and chloramphenicol in real sample shows the limit-of-detection 0.1 µg mL-1, 0.005 µg mL-1, 0.01 µg mL-1; the linearity range 0.1-5 µg mL-1, 0.005-0.25 µg mL-1, 0.01-1 µg mL-1; and the linear fits R2 ≥ 0.980, 0.991, 0.986. Moreover, we show that tip-ESI can also afford sequential molecular ionization of untreated viscous samples, which is difficult to achieve by conventional ESI. We conclude that tip-ESI-MS is a versatile analytical approach for the rapid chemical analysis of untreated food samples.

Floral volatiles identification and molecular differentiation of Osmanthus fragrans by neutral desorption extractive atmospheric pressure chemical ionization mass spectrometry.

RATIONALE: Floral volatiles are commonly present only at trace amounts and can be degraded or lost during vapor collection, which is often challenging from the analytical standpoint. Osmanthus fragrans Lour. is a widely cultivated plant known for the highly distinct fragrance of its flowers. The identification of specific volatile organic compounds (VOCs) and molecular differentiation of O. fragrans without any chemical pretreatment and VOC collection are important. METHODS: Twenty-eight VOCs released by the flowers from ten different cultivars of O. fragrans were identified using neutral desorption extractive atmospheric pressure chemical ionization mass spectrometry (ND-EAPCI-MS) without any chemical pretreatment or VOC collection. Chemical identification was performed by high-resolution MSn analysis and whenever possible was confirmed by the analysis of standards. RESULTS: According to our literature search, nine of the identified VOCs, 3-buten-2-one, cyclohexadiene, 2-methylfuran, 3-allylcyclohexene, cuminyl alcohol, hotrienol oxide, epoxy-linalool oxide, N-(2-hydroxyethyl) octanamide, and 3-hydroxy-dihydro-ß-ionone, have not been reported in O. fragrans in earlier studies. Confident differentiation between ten different cultivars of O. fragrans was achieved by the principal component analysis of the mass spectrometric results. CONCLUSIONS: The results of our ND-EAPCI-MS analysis substantially increase our knowledge about the chemistry of the O. fragrans floral fragrance and demonstrate the power of this technique for direct molecular profiling for plant recognition or in biotechnological applications.

Realizing nano electrospray ionization using disposable pipette tips under super atmospheric pressure.

Nano electrospray with a solution flow rate below 100 nL min(-1) is usually performed using a metallized pulled glass capillary with an inner diameter tip of <10 µm. In this paper, we demonstrated that it was possible to achieve a stable solution flow rate of ~10 nL min(-1) for an aqueous solution using a disposable plastic pipette tip with an inner diameter of 100 µm as an ESI emitter. This pipette tip (gel loading tip) was originally designed for the loading of sample onto the gel in gel electrophoresis. The inner and outer diameters were much larger than those of a nanoESI emitter, but the low solution flow rate could be sustained by conducting the electrospray under a super-atmospheric gas pressure. The high pressure condition ensured the absence of electrical discharge which was essential for stable electrospray of aqueous solution. ESI with this low cost tip was equally as sensitive as the standard nanoESI emitter and capable of producing a long lasting signal from a small amount of sample liquid. Due to a small initial droplet size under the low flow rate regime, ESI with the present method exhibited the property of nanoESI where the ion suppression of the hydrophilic analyte by the hydrophobic compound present in the solution was greatly reduced. Furthermore, owing to its larger inner diameter, the tip relatively easily avoided the clogging problem often encountered when dealing with "dirty" samples when compared with a nanoESI emitter made from a fine glass capillary.

Development of high-pressure probe electrospray ionization for aqueous solution.

RATIONALE: Probe electrospray ionization (PESI) is a recently developed method that uses a sharp solid needle as electrospray emitter and the sample is loaded to the needle tip by repetitive movement of the needle probe. This method has been previously used for the analysis of sample with high salt concentration and real-world samples without sample pretreatment. Although PESI is also applicable to aqueous solution, the ion signal stability and reproducibility were not satisfactory due to the spontaneous occurrence of corona discharge taking place on the metallic needle tip. METHODS: In the previous version of PESI, the ionization was performed under an open environment, thus ambient condition such as humidity and the presence of environmental contaminant also affected the performance of PESI. In this paper, we report a modified version of PESI in which the electrospray and the desolvation of charged droplets are performed inside an enclosed chamber which is pressurized to >1 atm with dry air. RESULTS: Under a super atmospheric condition, the dielectric strength of the ambient gas was enhanced and stable electrospray could to be initiated without the occurrence of corona discharge even for liquid sample with high surface tension such as pure water. A comparative study of atmospheric-pressure PESI and high-pressure PESI (HP-PESI) had been performed to demonstrate the improvement of PESI in sensitivity and signal stability for the detection analytes in aqueous solution. CONCLUSIONS: HP-PESI offers a higher sensitivity and signal stability over PESI due to the absence of gaseous breakdown, better desolvation, and higher ion sampling rate by the mass spectrometer.

High pressure nanoelectrospray ionization mass spectrometry for analysis of aqueous solutions.

Nanoelectrospray ionization (nanoESI) with a very fine emitter and nanoliter solution flow rate is known to be suitable for aqueous solutions. However, under atmospheric pressure, its stability with aqueous solutions is not always guaranteed particularly in the negative ion mode where corona and arc discharge tend to occur more easily. Electrical discharge can be quenched to a certain extent by adding electron scavenging gases like SF6 or CO2 to the ion source. The onset potential that is required to induce the discharge also increases with an increase of gas pressure. Recently, we have reported on a series of high pressure electrospray ion sources that were stable in both positive and negative ion modes using air or N2 as the working gas. In this paper, we compare the performance of nanoelectrospray under atmospheric pressure and super-atmospheric pressure for the analysis of samples in aqueous solution. The comparative study was performed using the same ion source chamber that could be pressurized up to 6 bar. The pressure in the first pumping stage of the mass spectrometer was kept constant when the ion source pressure was changed by using an additional pump with variable pumping speed. High pressure nanoESI optimized at 2-3 bar demonstrated a 3-5 times improvement in ion signal intensity compared to atmospheric pressure nanoESI, and the signal stability was significantly improved particularly in the negative mode.

Assessing of the use of proteins A, G, and chimeric protein AG to detect marine mammal immunoglobulins.

In recent years, there has been an increase in infectious diseases in marine mammals, including brucellosis, infections of morbillivirus, herpesvirus, and poxvirus. Several serological diagnostic methods, including enzyme-linked immunosorbent assays, immunofluorescence assays (ELISA), and western blotting, have been used to detect antibodies against pathogens in marine mammals. However, options for commercial secondary antibodies used to detect antibodies in marine mammals are limited; therefore, the use of proteins A, G, or chimeric protein AG may provide a suitable alternative. This study aimed to assess the use of proteins A, G, and chimeric protein AG to detect marine mammal immunoglobulins. Currently, there are no comparative studies on the use of proteins A, G, and chimeric protein AG for the detection of immunoglobulins in marine mammals. In this study, we used ten pinnipeds' species (Baikal seal, California sea lion, harbor seal, northern fur seal, ringed seal, South American fur seal, South American sea lion, spotted seal, Steller sea lion, and walrus) and five cetacean species (beluga whale, bottlenose dolphin, harbor porpoise, killer whale, and Pacific white-sided dolphin) and compare binding ability to proteins A, G, or chimeric protein AG by ELISA. The results revealed that the immunoglobulins from pinniped and cetacean species reacted more strongly to protein A than protein G. In addition, the immunoglobulins of pinnipeds and cetaceans showed a strong binding ability to chimeric protein AG. These results suggest that proteins A, G, and chimeric protein AG would be used to help further develop serological assays.

Research Note: Detection of Campylobacter spp. in chicken meat using culture methods and quantitative PCR with propidium monoazide.

Globally, Campylobacter spp. are prominent causative agents of food-borne gastroenteritis. These pathogens are commonly detected using conventional culture methods; however, culture methods are unable to detect viable but nonculturable (VBNC) bacteria. Currently, the detection rate of Campylobacter spp. on chicken meat does not correlate with the seasonal peak of human campylobacteriosis. We hypothesized that this may be due to the presence of undetectable VBNC Campylobacter spp. Therefore, we previously established a quantitative PCR assay using propidium monoazide (PMA-qPCR), which can detect viable Campylobacter cells. In this study, PMA-qPCR was conducted to detect viable Campylobacter spp. in chicken meat, and the detection rates of PMA-qPCR and the culture method throughout all 4 seasons were compared. A total of 105 chicken meat samples (whole legs, breast fillets, and livers) were screened for the presence of Campylobacter spp. using both PMA-qPCR and the conventional culture method. The detection rates of the 2 methods did not differ significantly; however, the positive and negative samples were not always consistent. Detection rates in March were significantly lower compared to months with the highest detection rates. These results suggest that, to increase the detection rate of Campylobacter spp., the 2 methods should be used in parallel. In this study, PMA-qPCR could not detect VBNC Campylobacter spp. effectively in C. jejuni-spiked chicken meat. Further studies using improved viability-qPCR should be performed to describe the impact of the VBNC state of Campylobacter spp. on the detection of this bacterium in chicken meat.

Identification of Suitable Internal Control miRNAs in Bovine Milk Small Extracellular Vesicles for Normalization in Quantitative Real-Time Polymerase Chain Reaction.

This study aimed to identify a suitable RNA extraction kit and stable internal control microRNA (miRNA) in bovine milk small extracellular vesicles (sEVs) for a quantitative polymerase chain reaction (qPCR) analysis. Two RNA extraction kits, miRNeasy Micro Kit, and Maxwell RSC miRNA Tissue Kit, were compared and evaluated using bovine milk sEVs via qPCR analysis. Five miRNAs, bta-miR-29a, bta-miR-200a, bta-miR-26b, hsa-miR-27b-3p, and hsa-miR-30b-5p, were selected by microarray analyses, and their cycle threshold (Ct) values were further evaluated mathematically using geNorm, NormFinder, BestKeeper, and ∆Ct algorithms. The results revealed that both the miRNeasy Micro Kit and Maxwell RSC miRNA Tissue Kit are useful for the efficient recovery of RNA from bovine milk sEVs. According to the final stability ranking analyzed by RefFinder, hsa-miR-27b-3p and bta-miR-29a can be used as suitable internal control miRNAs in bovine milk sEVs. The study also indicated that using a suitable internal control miRNA may improve the reliability and accuracy of the qPCR analysis for normalization in bovine milk sEVs. To the best of our knowledge, this is the first study to uncover the suitable internal control miRNAs in bovine milk sEVs.

Enhanced ultrasonic spray ionization for direct mass spectrometry analysis of aqueous solution and complex samples using a single-orifice piezoelectric atomizer.

An efficient and superior soft ionization approach for direct mass spectrometry analysis of a variety of samples such as aqueous solution, raw biological sample and proteins, was developed based on commercially available piezoelectric atomizers. A single conical orifice (5 µm in diameter) was created on the atomizer, which resulted in generation of uniform fine droplets and long-duration of MS signal. The two electrodes of piezoelectric atomizer were connected to the two sides of ceramic ring which was insulated from the metallic substrate. The unique design allowed an additional high voltage input towards the spray reagents, which facilitated direct analysis of more complex samples without sample pre-treatment, such as biological samples (tomato tissue). The ionization was driven by an extremely low electrical power (3.5 V rechargeable battery) yet providing an efficient and superior soft ionization. The method displayed a better thermal and pH stability than nano electrospray ionization (nanoESI) and electrospray ionization (ESI) on direct analysis of Vitamin B and protein aqueous solutions. Quantitative analysis of Vitamin B and Rhodamine B aqueous solutions was also investigated, showing a good linearity (R2 > 0.99). In addition, our results suggested that compared with ESI and nanoESI, the method not only could be used for direct analysis of intact protein, but also provide more information concerning the association between intact protein and the subunits.

Characterization of mRNA Signature in Milk Small Extracellular Vesicles from Cattle Infected with Bovine Leukemia Virus.

This study aimed to characterize the mRNA signature of milk small extracellular vesicles (sEVs) from BLV-infected cattle. A total of 23 mRNAs, which showed greater abundance in milk sEVs from BLV-infected cattle compared to those from BLV-uninfected (control) cattle, were identified through microarray analyses conducted in our previous study. To assess the significance of these differences in mRNA abundance, milk was collected from six control cattle and twenty-six cattle infected with BLV. The infected cattle were categorized into two distinct groups based on their proviral loads: a group of eight cattle with low proviral loads (LPVL), characterized by <10,000 copies per 105 white blood cells (WBC), and a group of eighteen cattle with high proviral loads (HPVL), marked by ≥10,000 copies per 105 WBC. The qPCR analysis quantified 7 out of 23 mRNAs, including BoLA, CALB1, IL33, ITGB2, MYOF, TGFBR1, and TMEM156, in the milk sEVs from control cattle, LPVL cattle, and HPVL cattle. Significantly, the average relative expression of CALB1 mRNA in milk sEVs was higher in LPVL cattle compared to HPVL cattle and control cattle (p < 0.05), while it was relatively lower in HPVL cattle compared to LPVL cattle and control cattle (p > 0.05). Likewise, the average relative expression of TMEM156 mRNA in milk sEVs was significantly higher in LPVL cattle compared to HPVL cattle (p < 0.05), and relatively lower in HPVL cattle compared to LPVL cattle and control cattle (p > 0.05). The results indicate distinct patterns of CALB1 and TMEM156 mRNA levels in milk sEVs, with higher levels observed in LPVL cattle and lower levels in HPVL cattle. The current study could provide essential information to comprehend the complexities during the progression of BLV infection and direct the exploration of mRNA biomarkers for monitoring the clinical stage of BLV infection.

Two-Round Treatment With Propidium Monoazide Completely Inhibits the Detection of Dead Campylobacter spp. Cells by Quantitative PCR.

Campylobacter spp. are known as important foodborne gastroenteric pathogens worldwide. Campylobacter spp. can exist in a viable but non-culturable (VBNC) state under unsuitable environmental conditions, which is undetectable by conventional culture methods. Quantitative polymerase chain reaction (qPCR) can be used to detect VBNC Campylobacter spp.; however, both viable and dead bacteria are detected during qPCR and are indistinguishable. Propidium monoazide (PMA), which can only enter dead bacterial cells through a damaged cell wall/cell membrane, binds to DNA and inhibits qPCR. PMA treatment has been performed along with qPCR (PMA-qPCR) to detect viable bacteria. However, the efficacy of detection inhibition differed among studies, and PMA can potentially enter living cells after changes in cell membrane permeability. In this study, we optimized the PMA treatment method by conducting it before qPCR. Two-round PMA treatment completely inhibited the qPCR signals from dead cells, whereas single-round PMA treatment failed to facilitate this. An optimized PMA-qPCR method was developed using commercial chicken meat, and VBNC Campylobacter spp., which are undetectable using conventional culture-based methods, were successfully detected. In conclusion, this study presents a novel, efficient PMA treatment method for the detection of viable Campylobacter spp., including VBNC Campylobacter spp., in chicken meat. We believe that this method will aid the reliable risk assessment of commercial chicken meat.

A New Enzyme-linked Immunosorbent Assay for Serological Diagnosis of Seal Parapoxvirus Infection in Marine Mammals.

Introduction: Seal parapoxvirus (SPPV) infection has been reported among pinnipeds in aquaria in Japan; however, its seroprevalence is unknown. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed for serological diagnosis of SPPV infection. Material and Methods: The gene encoding the major envelope protein of SPPV was cloned into the eukaryotic expression vector pAcGFP1-N1, which encodes the green fluorescence protein (GFP), thereby producing a fusion protein (Env-GFP). Parental and cloned vector DNA was independently transfected into cultured seal cells for the expression of GFP and Env-GFP. The wells of an ELISA plate were coated with either GFP- or Env-GFP-transfected cell lysates. The light absorbance of each serum sample was adjusted by subtracting the absorbance of GFP-coated wells from that of Env-GFP-coated wells. Sera from two spotted seals (Phoca largha), six beluga whales (Delphinapterus leucas), three Pacific white-sided dolphins (Lagenorhynchus obliquidens), and ten bottlenose dolphins (Tursiops truncatus) from an aquarium in Japan were examined using the ELISA. Results: Positive reactions were not observed, except in one preserved sample collected ten years ago from a naturally SPPV-infected spotted seal. Conclusion: The established ELISA could be useful in screening marine mammal sera for anti-SPPV antibodies.

Cellular infiltration, cytokines, and histopathology of skin lesions associated with different clinical forms and stages of naturally occurring lumpy skin disease in cattle.

Lumpy skin disease (LSD) caused by the Capripoxvirus LSD virus which infects cattle, leading to a serious disease characterized by fever and the eruption of skin nodules all over the surface of the body. Our understanding of the pathogenesis of this disease is still incomplete, particularly the immunopathological alterations occurring in the skin nodules of infected animals. Therefore, we collected skin nodules from naturally infected cattle with different forms of the disease, both in the early stage of clinical infection and after disease progression. The skin samples were examined both histopathologically and immunohistochemically using a variety of antibodies targeting immune cellular markers and cytokines. As a result, the dermatohistopathology revealed orthokeratotic hyperkeratosis, vasculitis, epidermal microvesicles, and cellules claveleuses of Borrel in the early stage of infection, with the severity of the lesions correlating with the severity of the clinical disease. Meanwhile, late-stage samples had epidermal hyperkeratosis as well as dermal lymphocytic and histiocytic infiltrations. The predominant cellular infiltrates in the cutaneous lesions of early-stage LSD samples were interferon (IFN)-γ+ cells and CD4+ T lymphocytes with few macrophage lineage cells. However, in the late-stage samples, numerous Iba-1+ macrophages, with few IFN-γ+ cells and CD4+ T lymphocytes, were detected. Our findings indicate that IFN-γ+ cells, CD4+ T lymphocytes, and macrophages play a key role in the immunity against natural LSD virus infection and imply that cutaneous vasculopathy associated with LSD virus infection is an immune-mediated lesion. The current study contributes to our understanding of the pathogenesis of LSD.

Comprehensive Proteomic Analysis Revealed a Large Number of Newly Identified Proteins in the Small Extracellular Vesicles of Milk from Late-Stage Lactating Cows.

Bovine milk contains small extracellular vesicles (sEVs) that provide proteins, miRNAs, mRNAs, DNAs, and lipids to target cells and play a role in intracellular communications. Previous studies have characterized proteins in milk sEVs from early- and mid-stage lactation. However, the proteins in milk sEVs from late-stage lactation are mostly unexplored. The aim of this study was to determine the proteomic profile of milk sEVs from late-stage lactating cows. A comprehensive nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) approach was carried out to reveal the proteins in milk sEVs. Additionally, bioinformatics analysis was carried out to interpret the molecular signatures of newly identified proteins in milk sEVs from three late-stage lactating cows. NanoLC-MS/MS analysis revealed a total of 2225 proteins in milk sEVs from cows. Notably, after comparing these identified proteins with previously deposited datasets of proteins in bovine milk sEVs, 429 proteins were detected as newly identified. Bioinformatic analysis indicated that these newly identified proteins in milk sEVs were engaged in a diverse range of molecular phenomena relevant to mammary gland physiology, milk production, immunity, and immune response. These findings suggest that the newly identified proteins could expand the inventory application of molecular cargos, nutritional status, and immune modulation of sEVs in milk during the late-stage lactation.

Proteomic profiling of milk small extracellular vesicles from bovine leukemia virus-infected cattle.

Milk small extracellular vesicles (sEV) contain proteins that provide potential information of host physiology and immunology. Bovine leukemia virus (BLV) is an oncogenic virus that causes progressive B-cell lymphosarcoma in cattle. In this study, we aimed to explore the proteomic profile of milk sEV from BLV-infected cattle compared with those from uninfected cattle. Milk sEV were isolated from three BLV-infected and three uninfected cattle. Proteomic analysis was performed by using a comprehensive nanoLC-MS/MS method. Furthermore, gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to evaluate the candidates for uniquely or differentially expressed proteins in milk sEV from BLV-infected cattle. Proteomic analysis revealed a total of 1330 common proteins in milk sEV among BLV-infected cattle, whereas 118 proteins were uniquely expressed compared with those from uninfected cattle. Twenty-six proteins in milk sEV were differentially expressed proteins more than two-fold significant difference (p < 0.05) in BLV-infected cattle. GO and KEGG analyses indicated that the candidates for uniquely or differentially expressed proteins in milk sEV had been involved in diverse biological activities including metabolic processes, cellular processes, respond to stimulus, binding, catalytic activities, cancer pathways, focal adhesion, and so on. Taken together, the present findings provided a novel insight into the proteomes of milk sEV from BLV-infected cattle.

Putative Internal Control Genes in Bovine Milk Small Extracellular Vesicles Suitable for Normalization in Quantitative Real Time-Polymerase Chain Reaction.

Bovine milk small extracellular vesicles (sEVs) contain many biologically important molecules, including mRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for quantifying mRNA in tissues and cells. However, the use, selection, and stability of suitable putative internal control genes in bovine milk sEVs for normalization in qRT-PCR have not yet been identified. Thus, the aim of the present study was to determine suitable putative internal control genes in milk sEVs for the normalization of qRT-PCR data. Milk sEVs were isolated from six healthy Holstein-Friesian cattle, followed by RNA extraction and cDNA synthesis. In total, 17 mRNAs were selected for investigation and quantification using qRT-PCR; they were further evaluated using geNorm, NormFinder, BestKeeper, and ∆CT algorithms to identify those that were highly stable putative internal control genes in milk sEVs. The final ranking of suitable putative internal control genes was determined using RefFinder. The mRNAs from TUB, ACTB, DGKZ, ETFDH, YWHAZ, STATH, DCAF11, and EGFLAM were detected in milk sEVs from six cattle by qRT-PCR. RefFinder demonstrated that TUB, ETFDH, and ACTB were highly stable in milk sEVs, and thus suitable for normalization of qRT-PCR data. The present study suggests that the use of these genes as putative internal control genes may further enhance the robustness of qRT-PCR in bovine milk sEVs. Since these putative internal control genes apply to healthy bovines, it would be helpful to include that the genes were stable in sEVs under "normal or healthy conditions".

Data on proteomic analysis of milk extracellular vesicles from bovine leukemia virus-infected cattle.

Milk extracellular vesicles (EVs) are nanoparticles that contain proteins, mRNAs, microRNAs, DNAs, and lipids that involved in several biological functions. Milk EVs provide proteins that could represent relevant novel biomarkers for monitoring of different diseases such as breast cancer and mastitis in humans and animals, respectively. Bovine leukemia virus (BLV) is an oncogenic virus that causes progressive B-cell lymphosarcoma in cattle. Here, we aimed to identify proteins in milk EVs from BLV-infected cattle compared with those from uninfcetd cattle. Proteomic analysis was performed by using a comprehensive nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) approach. Identified proteins were analyzed by using a proteomic software, Scaffold-Data Independent Acquisition (Scaffold-DIA).

mRNA Profile in Milk Extracellular Vesicles from Bovine Leukemia Virus-Infected Cattle.

Milk extracellular vesicles (EVs) form an excellent source of mRNAs, microRNAs (miRNAs), proteins, and lipids that represent the physiological and pathological status of the host. Recent studies have reported milk EVs as novel biomarkers for many infectious diseases in both humans and animals. For example, miRNAs in milk EVs from cattle were used for early detection of bacterial infection in the mammary gland. Based on these findings, we hypothesized that mRNAs in milk EVs are suitable for gaining a better understanding of the pathogenesis of bovine leukemia virus (BLV) infection and prognosis of the clinical stage in cattle. For that purpose, milk EVs were isolated from BLV-infected and uninfected cattle, and mRNAs were investigated using microarray analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed mainly focusing on the differentially expressed genes (DEGs) in milk EVs from BLV-infected cattle. GO and KEGG analyses suggested the DEGs in milk EVs from BLV-infected cattle had involved in diverse molecular functions, biological processes, and distinct disease-related pathways. The present study suggested that BLV infection causes profound effects on host cellular activity, changing the mRNA expression profile in milk EVs obtained from BLV-infected cattle. Overall, our results suggested that the mRNA profile in milk EVs to be a key factor for monitoring the clinical stage of BLV infection. This is the first report of mRNA profiling of milk EVs obtained from BLV-infected cattle.