Higienização de ambiente em agroindústria familiar de embutidos cárneos / Environmental hygienization in family meat product

A higienização é um procedimento importante na indústria de alimentos e sua realização deve ocorrer rotineiramente para evitar que os alimentos sejam contaminados. Além disso, todos os manipuladores de alimentos devem receber treinamentos de modo a entender como ocorrem as contaminações e como evitá-las, para que não ocorra deterioração antecipada dos alimentos e para que não exponham os consumidores ao risco de doenças transmitidas por alimentos em caso de contaminação. Esta pesquisa avaliou o processo de higienização e sua eficiência em superfícies presentes em uma agroindústria da agricultura familiar produtora de embutidos cárneos. Apesar de ter instalações adequadas a agroindústria apresentava inadequações quanto aos produtos utilizados e a frequência inadequada para uma higienização eficiente. Foi realizada análise microbiológica das superfícies dos equipamentos para contagem de aeróbios mesófilos e notou-se uma elevada carga microbiana que indicou uma baixa eficiência no processo de higienização. Sugeriu-se melhorias na higiene ambiental associado à instrução dos colaboradores, para contribuir na promoção da qualidade dos produtos, aumento dos lucros e salvaguardando a saúde do consumidor.

Hygiene is an important procedure in the food industry, and its performance must occur routinely to prevent food from being contaminated. In addition, all food handlers must receive training in order to understand how contamination occurs and how to avoid it, so that there is no anticipated deterioration of food and that consumers are not exposed to the risk of foodborne diseases. in case of contamination by pathogenic microorganisms. Thus, this research evaluated the cleaning process and its efficiency on surfaces present in a family farming agroindustry that produces meat products, which despite having adequate facilities, had some difficulties such as product use and inadequate frequency for eficiente cleaning. After performing a microbiological analysis to count surface mesophilic aerobes, a high level of contamination was noted, relating to low efficiency in the cleaning process. Improvements in environmental hygiene are suggested, associated with the instruction of employees for the implementation of the Standard Operating Hygiene Procedure, promoting improvements in product quality, increasing profits and safeguarding consumer health.

X-ray-activated polymerization expanding the frontiers of deep-tissue hydrogel formation.

Photo-crosslinking polymerization stands as a fundamental pillar in the domains of chemistry, biology, and medicine. Yet, prevailing strategies heavily rely on ultraviolet/visible (UV/Vis) light to elicit in situ crosslinking. The inherent perils associated with UV radiation, namely the potential for DNA damage, coupled with the limited depth of tissue penetration exhibited by UV/Vis light, severely restrict the scope of photo-crosslinking within living organisms. Although near-infrared light has been explored as an external excitation source, enabling partial mitigation of these constraints, its penetration depth remains insufficient, particularly within bone tissues. In this study, we introduce an approach employing X-ray activation for deep-tissue hydrogel formation, surpassing all previous boundaries. Our approach harnesses a low-dose X-ray-activated persistent luminescent phosphor, triggering on demand in situ photo-crosslinking reactions and enabling the formation of hydrogels in male rats. A breakthrough of our method lies in its capability to penetrate deep even within thick bovine bone, demonstrating unmatched potential for bone penetration. By extending the reach of hydrogel formation within such formidable depths, our study represents an advancement in the field. This application of X-ray-activated polymerization enables precise and safe deep-tissue photo-crosslinking hydrogel formation, with profound implications for a multitude of disciplines.

Using sentinel-2 satellite images and machine learning algorithms to predict tropical pasture forage mass, crude protein, and fiber content.

Grasslands cover approximately 24% of the Earth's surface and are the main feed source for cattle and other ruminants. Sustainable and efficient grazing systems require regular monitoring of the quantity and nutritive value of pastures. This study demonstrates the potential of estimating pasture leaf forage mass (FM), crude protein (CP) and fiber content of tropical pastures using Sentinel-2 satellite images and machine learning algorithms. Field datasets and satellite images were assessed from an experimental area of Marandu palisade grass (Urochloa brizantha sny. Brachiaria brizantha) pastures, with or without nitrogen fertilization, and managed under continuous stocking during the pasture growing season from 2016 to 2020. Models based on support vector regression (SVR) and random forest (RF) machine-learning algorithms were developed using meteorological data, spectral reflectance, and vegetation indices (VI) as input features. In general, SVR slightly outperformed the RF models. The best predictive models to estimate FM were those with VI combined with meteorological data. For CP and fiber content, the best predictions were achieved using a combination of spectral bands and meteorological data, resulting in R2 of 0.66 and 0.57, and RMSPE of 0.03 and 0.04 g/g dry matter. Our results have promising potential to improve precision feeding technologies and decision support tools for efficient grazing management.

Recent advances in early pregnancy loss diagnosis in dairy cows: New approaches.

Early pregnancy loss is a primary cause of low reproductive rates in dairy cows, posing severe economic losses to dairy farming. The accurate diagnosis of dairy cows with early pregnancy loss allows for oestrus synchronization, shortening day open, and increasing the overall conception rate of the herd. Several techniques are available for detecting early pregnancy loss in dairy cows, including rectal ultrasound, circulating blood progesterone, and pregnancy-associated glycoproteins (PAGs). Yet, there is a need to improve on existing techniques and develop novel strategies to identify cows with early pregnancy loss accurately. This manuscript reviews the applications of rectal ultrasound, circulating blood progesterone concentration, and PAGs in the diagnosis of pregnancy loss in dairy cows. The manuscript also discusses the recent progress of new technologies, including colour Doppler ultrasound (CDUS), interferon tau-induced genes (ISGs), and exosomal miRNA in diagnosing pregnancy loss in dairy cows. This study will provide an option for producers to re-breed cows with pregnancy loss, thereby reducing the calving interval and economic costs. Meanwhile, this manuscript might also act as a reference for exploring more economical and precise diagnostic technologies for early pregnancy loss in dairy cows.

Development and application of one-step multiplex Real-Time PCR for detection of three main pathogens associated with bovine neonatal diarrhea.

Introduction: Neonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive. Methods: In this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+. Results: The established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect ~1 copy/µL for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/µL. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves' diarrhea in China. Discussion: The established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research. ​.

A highly divergent enteric calicivirus in a bovine calf in India.

A highly divergent bovine calicivirus was identified in an Indian calf with enteritis. The whole genome of this virus was sequenced, revealing distinct amino acid motifs in the polyprotein encoded by open reading frame 1 (ORF1) that are unique to caliciviruses. Phylogenetic analysis showed that it was related to members of the genus Nebovirus of the family Caliciviridae. Although it showed only 33.7-34.2% sequence identity in the VP1 protein to the nebovirus prototype strains, it showed 90.6% identity in VP1 to Kirklareli virus, a nebovirus detected in calves with enteritis in Turkey in 2012. An in-house-designed and optimized reverse transcription polymerase chain reaction (RT-PCR) assay was used to screen 120 archived bovine diarrhoeic fecal samples, 40 each from the Indian states of Uttar Pradesh, Haryana, and Himachal Pradesh, revealing frequent circulation of these divergent caliciviruses in the bovine population, with an overall positivity rate of 64.17% (77/120). This underscores the importance of conducting a comprehensive investigation of the prevalence of these divergent caliciviruses and assessing their associations with other pathogens responsible for enteritis in India.

The Difference of Milk-Derived Extracellular Vesicles from Cow Colostrum and Mature Milk on miRNAs Expression and Protecting Intestinal Epithelial Cells against Lipopolysaccharide Damage.

Intestinal epithelial cells (IECs) play crucial roles in forming an essential barrier, providing host defense against pathogens and regulating nutrients absorption. Milk-derived extracellular vesicles (EVs) within its miRNAs are capable of modulating the recipient cell function. However, the differences between colostrum and mature milk EVs and their biological function in attenuating intestinal epithelial cell injury remain poorly understood. Thus, we carried out the present study to characterize the difference between colostrum and mature milk-derived miRNA of EVs and the effect of colostrum and mature milk EVs on the proliferation, apoptosis, proinflammatory cytokines and intestinal epithelial barrier related genes in IEC-6 induced by LPS. Differential expression of 329 miRNAs was identified between colostrum and mature milk EVs, with 185 miRNAs being downregulated and 144 upregulated. In addition, colostrum contains a greater number and protein concentration of EVs than mature milk. Furthermore, compared to control, EVs derived from colostrum significantly inhibited the expression of apoptosis- (Bax, p53, and caspase-3) and proinflammatory-related genes (TNFα, IL6, and IL1ß). EVs derived from mature milk did not affect expression of apoptosis-related genes (Bax, p53, bcl2, and caspase-3). The EVs derived from mature milk significantly inhibited the expression of proinflammatory-related genes (TNFα and IL6). Western blot analysis also indicated that colostrum and mature milk EVs significantly decreased the apoptosis of IEC-6 cells. The EdU assay results showed that colostrum and mature milk EVs significantly increased the proliferation of IEC-6 cells. The expression of intestinal barrier-related genes (TJP1, CLDN1, OCLN, CDX2, MUC2, and IGF1R) was significantly promoted in IEC-6 cells after colostrum and mature milk EVs addition. Importantly, colostrum and mature milk EVs significantly relieved the LPS-induced inhibition of proliferation and intestinal barrier-related genes expression and attenuated apoptosis and proinflammatory responses induced by LPS in IEC-6 cells. Flow cytometry and Western blot analysis also indicated that colostrum and mature milk EVs significantly affect the apoptosis of IEC-6 cells induced by LPS. The results also indicated that EVs derived from colostrum had better effects on inhibiting the apoptosis- and proinflammatory cytokines-related genes expression. However, the EVs derived from mature milk exhibited beneficial effects on intestinal epithelial barrier protection. The present study will provide a better understanding of the role of EVs derived from colostrum and milk in dairy cows with different responses in the regulation of intestinal cells function, and also presents new evidence for the change of EVs cargos during various stages of lactation.

Effects of Regulating Hippo and Wnt on the Development and Fate Differentiation of Bovine Embryo.

The improvement of in vitro embryo development is a gateway to enhance the output of assisted reproductive technologies. The Wnt and Hippo signaling pathways are crucial for the early development of bovine embryos. This study investigated the development of bovine embryos under the influence of a Hippo signaling agonist (LPA) and a Wnt signaling inhibitor (DKK1). In this current study, embryos produced in vitro were cultured in media supplemented with LPA and DKK1. We comprehensively analyzed the impact of LPA and DKK1 on various developmental parameters of the bovine embryo, such as blastocyst formation, differential cell counts, YAP fluorescence intensity and apoptosis rate. Furthermore, single-cell RNA sequencing (scRNA-seq) was employed to elucidate the in vitro embryonic development. Our results revealed that LPA and DKK1 improved the blastocyst developmental potential, total cells, trophectoderm (TE) cells and YAP fluorescence intensity and decreased the apoptosis rate of bovine embryos. A total of 1203 genes exhibited differential expression between the control and LPA/DKK1-treated (LD) groups, with 577 genes upregulated and 626 genes downregulated. KEGG pathway analysis revealed significant enrichment of differentially expressed genes (DEGs) associated with TGF-beta signaling, Wnt signaling, apoptosis, Hippo signaling and other critical developmental pathways. Our study shows the role of LPA and DKK1 in embryonic differentiation and embryo establishment of pregnancy. These findings should be helpful for further unraveling the precise contributions of the Hippo and Wnt pathways in bovine trophoblast formation, thus advancing our comprehension of early bovine embryo development.

Phenotypic Characteristics, Antimicrobial Susceptibility and Virulence Genotype Features of Trueperella pyogenes Associated with Endometritis of Dairy Cows.

Trueperella pyogenes can cause various infections in the organs and tissues of different livestock (including pigs, cows, goats, and sheep), including mastitis, endometritis, pneumonia, or abscesses. Moreover, diseases induced by T. pyogenes cause significant economic losses in animal husbandry. In recent large-scale investigations, T. pyogenes has been identified as one of the main pathogens causing endometritis in lactating cows. However, the main treatment for the above-mentioned diseases is still currently antibiotic therapy. Understanding the impact of endometritis associated with T. pyogenes on the fertility of cows can help optimize antibiotic treatment for uterine diseases, thereby strategically concentrating the use of antimicrobials on the most severe cases. Therefore, it is particularly important to continuously monitor the prevalence of T. pyogenes and test its drug resistance. This study compared the uterine microbiota of healthy cows and endometritis cows in different cattle farms, investigated the prevalence of T. pyogenes, evaluated the genetic characteristics and population structure of isolated strains, and determined the virulence genes and drug resistance characteristics of T. pyogenes. An amount of 186 dairy cows were involved in this study and 23 T. pyogenes strains were isolated and identified from the uterine lavage fluid of dairy cows with or without endometritis.

Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 µM KAE and compared to native ejaculates (negative control-CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control-CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 µM KAE (p < 0.01). At the same time, supplementation with 25 µM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 µM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.

Consumption of Tap Water and Sociodemographic-Associated Characteristics: A Nationwide Cross-Sectional Study.

Safe water is a global public health concern amid increasing scarcity and pollution. Bottled water production and consumption contribute to these problems. This study examines tap water consumption in Italy, assessing associated sociodemographic factors and related health outcomes such as obesity and self-perceived health status. Data from the Italian National Statistics Institute's "Aspects of daily life" survey (N = 45,597) were analyzed. Covariates included education, age, gender, economic status, region, concerns about waste and climate change, consumption of carbonated drinks excluding water, alcohol consumption, consumption of vegetables, consumption of snacks, body mass index, and self-perceived health status. Bivariate analyses and mixed-effect logistic regression models explored the associations. People who drink tap water made up 19,674, with a higher prevalence in people aged 45 to 59 old, people with a graduate/post-graduate degree diploma, with optimal economic resources, people concerned about waste production and climate change, and those coming from the north-east regions of Italy. Underweight people showed a higher prevalence of TW consumption as well as those who less than occasionally drank carbonated drinks, drank alcohol, consumed vegetables more than once a day and snacks less than once a week, dairy products more than once a day, sweet less than once a week, cured meat less than once a week, and chicken meat less than once a week, those with no consumption of sheep meat, consumption of beef meat less than once a week and consumption of pork meat less than once a week, and those with a satisfactory level of perceived health status. Regressions showed that all other age classes are less likely to drink tap water than people younger than 20 years old. The category with "inadequate" economic resources is more likely to consume tap water. Low educational classes show a low likelihood of consuming tap water as well as islands. A concern about waste production and climate change is associated with an increased likelihood of consuming tap water. Tap water consumption was negatively associated with obesity but not with a satisfactory self-perceived health status. Insights from this study can inform public health strategies.

Contribution of Beef to Key Nutrient Intakes and Nutrient Adequacy in Pregnant and Lactating Women: NHANES 2011-2018 Analysis.

Beef is an important source of high-quality protein and several micronutrients, including iron, zinc, and B-vitamins. The objective was to assess the association of beef intake with nutrient intake and adequacy among pregnant and lactating women using 24-h dietary recall data. Usual intakes from foods were determined with the National Cancer Institute (NCI) method and % population below Estimated Average Requirement (EAR) or above Adequate Intake (AI) were estimated. A high proportion of pregnant and lactating women had inadequate intakes for vitamin D (94%), vitamin E (82%), vitamin C (52%), and vitamin A (50%), magnesium (35%), folate (31%), zinc (25%), and vitamin B6 (22%); only 4% and 35% met AI for choline and potassium, respectively. About 67% of pregnant and lactating women were beef consumers, consuming 49 g beef/day. Beef consumers had higher intakes (p < 0.05) of energy, protein, calcium, iron, phosphorus, selenium, sodium, zinc, thiamin, riboflavin, and niacin, and a higher proportion (p < 0.05) met nutrient recommendations for protein, calcium, iron, zinc, thiamin, riboflavin, niacin, vitamin B6, and vitamin B12 compared to non-consumers. In conclusion, pregnant and lactating women generally have inadequate nutrient intakes from their diets. Beef consumers have higher intakes and adequacy for certain nutrients, many of which are inherently available in beef or in foods eaten with beef.

Establishment of the HPLC fluorescence detection method for plasma trace ivermectin and its pharmacokinetics in Bactrian camel.

BACKGROUND AND OBJECTIVE: Ivermectin (IVM), a widely used veterinary anthelmintic, lacks recommended doses for Bactrian camels. This study aims to establish its pharmacokinetics in Bactrian camels, comparing with other livestock. METHODS: A method for high-performance liquid chromatography fluorescence detection of IVM in plasma was developed. RESULTS: IVM exhibited linear scaling (y = 0.6946x + 0.0088, R2 = 0.9988) within 0.025-5 ng/mL, with a lower limit of quantification of 25.00 pg/mL, high recovery (>70%) and low RSD (<7%). In Bactrian camels, IVM injection showed a low Cmax, extended Tmax and apparent secondary absorption compared to cattle and sheep. CONCLUSIONS: Slow absorption and widespread distribution were observed, with peak concentration and area under the curve correlating positively with the dose. This study provides insights into IVM pharmacokinetics in Bactrian camels, informing dose determination and highlighting potential metabolic differences compared to other livestock.

Interaction of blood calcium with milk yield, energy metabolites, subclinical mastitis, and reproductive performance in crossbred dairy cows.

Limited literature is available on the consequences of postpartum low blood calcium (Ca) concentration in crossbred cows. The research aimed to investigate the correlation between postpartum serum Ca levels and various parameters, including milk yield, serum energy metabolites, milk somatic cell count, and reproductive factors in crossbred cows. Following parturition, a total of 45 potential high-yielding F2 (HF × Sahiwal; Genotype: 75:25) dairy cows were enrolled . These cows were categorized based on plasma calcium concentrations into three groups: a low calcium group (Ca-L) with a calcium concentration of <5 mg/dL, a medium calcium group (Ca-M) with a calcium concentration ranging from 5 to 8.5 mg/dL, and a high calcium group (Ca-H) with a calcium concentration exceeding 8.5 mg/dL. The study parameters were measured over an 8-week period. The results indicated that overall milk yield and blood glucose were significantly higher in the Ca-H group compared to Ca-M and Ca-L (p < .01). Blood cholesterol was significantly higher in Ca-M (p < .01), while blood triglyceride was significantly lower in both Ca-M and Ca-H. Overall, blood cortisol did not show a significant change between these groups (p < .01); however, progesterone levels were higher (p < .01) in Ca-M and Ca-H cows. Furthermore, somatic cell count (SCC) significantly (p < .01) decreased in cows with Ca-H compared to Ca-L. Additionally, postpartum oestrous interval and interestrus interval decreased significantly (p < .01) in Ca-M and Ca-H compared to Ca-L. These findings suggest that cows with blood calcium levels exceeding 8.5 mg/dL exhibited significantly higher milk yield, blood metabolite levels, a lower likelihood of subclinical mastitis, and earlier reproductive activity after calving.

Nucleic acid-extracted torula yeast from the paper industry as a protein feed for ruminants: A comparison with soybean meal.

We compared nucleic acid-extracted torula yeast (NTY) with soybean meal (SBM) to evaluate NTY as a potential protein feed for ruminants in a metabolic trial using four castrated male goats. NTY was replaced isonitrogenously with SBM at a 25% crude protein (CP) level on a dry matter (DM) basis. NTY has 55% CP and 74% total digestive nutrients on DM. Absorbed N was lower on the NTY diet, but since the urinary N excretion was lower on the NTY diet, no significant between-diet difference in retained N was observed. The efficiency of N utilization (retained N/absorbed N) was significantly higher on the NTY diet. The Lys and Met contents (presumed limiting amino acids for dairy cattle) were higher in NTY than SBM, which may be why N utilization efficiency was higher for the NTY diet. Ruminal ammonia-N and blood serum N were lower on the NTY diet, suggesting that NTY has more rumen undegradable protein than SBM. There was no significant between-diet difference in the visceral disorder indicators or antioxidant activities. Our results indicate that NTY is a safe protein feed with a high CP ratio and high-quality amino acid profile for ruminants that is equivalent to SBM.

Differential responses of rumen and fecal fermentation and microbiota of Liaoning cashmere goats after 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester supplementation.

The 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester (HMBi), a rumen protective methionine, has been extensively studied in dairy cows and beef cattle and has been shown to regulate gastrointestinal microbiota and improve production performance. However, knowledge of the application of HMBi on cashmere goats and the simultaneous study of rumen and hindgut microbiota is still limited. In this study, HMBi supplementation increased the concentration of total serum protein, the production of microbial protein in the rumen and feces, as well as butyrate production in the feces. The results of PCoA and PERMANOVA showed no significant difference between the rumen microbiota, but there was a dramatic difference between the fecal microbiota of the two groups of Cashmere goats after the HMBi supplementation. Specifically, in the rumen, HMBi significantly increased the relative abundance of some fiber-degrading bacteria (such as Fibrobacter) compared with the CON group. In the feces, as well as a similar effect as in the rumen (increasing the relative abundance of some fiber-degrading bacteria, such as Lachnospiraceae FCS020 group and ASV32), HMBi diets also increased the proliferation of butyrate-producing bacteria (including Oscillospiraceae UCG-005 and Christensenellaceae R-7 group). Overall, these results demonstrated that HMBi could regulate the rumen and fecal microbial composition of Liaoning cashmere goats and benefit the host.

HOXA9 gene inhibits proliferation and differentiation and promotes apoptosis of bovine preadipocytes.

BACKGROUND: Hox gene family is an important transcription factor that regulates cell process, and plays a role in the process of adipocytes differentiation and fat deposition. Previous transcriptome sequencing studies have indicated that the Homeobox A9 gene (HOXA9) is a candidate gene for regulating the process of bovine lipid metabolism, but the function and specific mechanism of action remain unclear. Therefore, this study aims to explore the role of HOXA9 in the proliferation, differentiation and apoptosis of bovine preadipocytes through gain-of-function and lose-of-function. RESULT: It found HOXA9 highly expressed in bovine adipose tissue, and its expression level changed significantly during adipocytes differentiation process. It gave a hint that HOXA9 may be involved in the process of bovine lipid metabolism. The results of HOXA9 gain-of-function experiments indicated that HOXA9 appeared to act as a negative regulator not only in the differentiation but also in the proliferation of bovine preadipocytes, which is mainly reflected that overexpression of HOXA9 down-regulate the mRNA and protein expression level of PPARγ, CEBPα and FABP4 (P < 0.05). The mRNA expression level of CDK1, CDK2, PCNA, CCNA2, CCNB1, CCND1 and CCNE2, as well as the protein expression of CDK2 also significantly decreased. The decrease of lipid droplets content was the main characteristic of the phenotype (P < 0.01), which further supported the evidence that HOXA9 was a negative regulator of preadipocytes differentiation. The decrease of cell proliferation rate and EdU positive rate, as well as the limitation of transition of preadipocytes from G0/G1 phase to S phase also provided evidence for the inhibition of proliferation. Apart from this above, we noted an interesting phenomenon that overexpression of HOXA9 showed in a significant upregulation of both mRNA and protein level of apoptosis markers, accompanied by a significant increase in cell apoptosis rate. These data led us not to refute the fact that HOXA9 played an active regulatory role in apoptosis. HOXA9 loss-of-function experiments, however, yielded the opposite results. Considering that HOXA9 acts as a transcription factor, we predicted its target genes. Dual luciferase reporter assay system indicated that overexpression of HOXA9 inhibits activity of PCNA promoter. CONCLUSION: Taken together, we demonstrated for the first time that HOXA9 played a role as a negative regulatory factor in the differentiation and proliferation of preadipocytes, but played a positive regulatory role in apoptosis, and it may play a regulatory role by targeting PCNA. This study provides basic data for further exploring the regulatory network of intramuscular fat deposition in bovine.

Investigating direct current potentials that affect native protein conformation during trapped ion mobility spectrometry-mass spectrometry.

Trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOFMS) has emerged as a tool to study protein conformational states. In TIMS, gas-phase ions are guided across the IM stages by applying direct current (DC) potentials (D1-6), which, however, might induce changes in protein structures through collisional activation. To define conditions for native protein analysis, we evaluated the influence of these DC potentials using the metalloenzyme bovine carbonic anhydrase (BCA) as primary test compound. The variation of DC potentials did not change BCA-ion charge and heme content but affected (relative) charge-state intensities and adduct retention. Constructed extracted-ion mobilograms and corresponding collisional cross-section (CCS) profiles gave useful insights in (alterations of) protein conformational state. For BCA, the D3 and D6 potential (which are applied between the deflection transfer and funnel 1 [F1] and the accumulation exit and the start of the ramp, respectively) had most profound effects, showing multimodal CCS distributions at higher potentials indicating gradual unfolding. The other DC potentials only marginally altered the CCS profiles of BCA. To allow for more general conclusions, five additional proteins of diverse molecular weight and conformational stability were analyzed, and for the main protein charge states, CCS profiles were constructed. Principal component analysis (PCA) of the obtained data showed that D1 and D3 exhibit the highest degree of correlation with the ratio of folded and unfolded protein (F/U) as extracted from the mobilograms obtained per set D potential. The correlation of D6 with F/U and protein charge were similar, and D2, D4, and D5 showed an inverse correlation with F/U but were correlated with protein charge. Although DC boundary values for induced conformational changes appeared protein dependent, a set of DC values could be determined, which assured native analysis of most proteins.

The Identification of a Novel Nucleomodulin MbovP467 of Mycoplasmopsis bovis and Its Potential Contribution in Pathogenesis.

Mycoplasmopsis bovis is a causative agent of crucial diseases in both dairy and beef cattle leading to substantial economic losses. However, limited control measures for M. bovis-related diseases exist due to a lack of understanding about the virulence factors of this pathogen, a common challenge in mycoplasma research. Consequently, this study aimed to characterize a novel nucleomodulin as a virulence-related factor of M. bovis. Employing bioinformatic tools, we initially predicted MbovP467 to be a secreted protein with a nuclear localization signal based on SignalP scores and the cNLS (Nuclear Localization Signal) Mapper, respectively. Subsequently, the MbovP467 gene was synthesized and cloned into a pEGFP plasmid with EGFP labeling to obtain a recombinant plasmid (rpEGFP-MbovP467) and then was also cloned in pET-30a with a consideration for an Escherichia coli codon bias and expressed and purified for the production of polyclonal antibodies against the recombinant MbovP467 protein. Confocal microscopy and a Western blotting assay confirmed the nuclear location of MbovP467 in bovine macrophages (BoMacs). RNA-seq data revealed 220 up-regulated and 20 down-regulated genes in the rpEGFP-MbovP467-treated BoMac group compared to the control group (pEGFP). A GO- and KEGG-enrichment analysis identified associations with inflammatory responses, G protein-coupled receptor signaling pathways, nuclear receptor activity, sequence-specific DNA binding, the regulation of cell proliferation, IL-8, apoptotic processes, cell growth and death, the TNF signaling pathway, the NF-κB signaling pathway, pathways in cancer, and protein families of signaling and cellular processes among the differentially expressed up-regulated mRNAs. Further experiments, investigating cell viability and the inflammatory response, demonstrated that MbovP467 reduces BoMac cell viability and induces the mRNA expression of IL-1ß, IL-6, IL-8, TNF-α, and apoptosis in BoMac cells. Further, MbovP467 increased the promoter activity of TNF-α. In conclusion, this study identified a new nucleomodulin, MbovP467, for M. bovis, which might have an important role in M. bovis pathogenesis.

Specific features of a scaffolding antibody light chain.

The antigen-binding sites in conventional antibodies are formed by hypervariable complementarity-determining regions (CDRs) from both heavy chains (HCs) and light chains (LCs). A deviation from this paradigm is found in a subset of bovine antibodies that bind antigens via an ultra-long CDR. The HCs bearing ultra-long CDRs pair with a restricted set of highly conserved LCs that convey stability to the antibody. Despite the importance of these LCs, their specific features remained unknown. Here, we show that the conserved bovine LC found in antibodies with ultra-long CDRs exhibits a distinct combination of favorable physicochemical properties such as good secretion from mammalian cells, strong dimerization, high stability, and resistance to aggregation. These physicochemical traits of the LCs arise from a combination of the specific sequences in the germline CDRs and a lambda LC framework. In addition to understanding the molecular architecture of antibodies with ultra-long CDRs, our findings reveal fundamental insights into LC characteristics that can guide the design of antibodies with improved properties.