Structural basis for heme detoxification by an ATP-binding cassette-type efflux pump in gram-positive pathogenic bacteria.

Bacterial pathogens acquire heme from the host hemoglobin as an iron nutrient for their virulence and proliferation in blood. Concurrently, they encounter cytotoxic-free heme that escapes the heme-acquisition process. To overcome this toxicity, many gram-positive bacteria employ an ATP-binding cassette heme-dedicated efflux pump, HrtBA in the cytoplasmic membranes. Although genetic analyses have suggested that HrtBA expels heme from the bacterial membranes, the molecular mechanism of heme efflux remains elusive due to the lack of protein studies. Here, we show the biochemical properties and crystal structures of Corynebacterium diphtheriae HrtBA, alone and in complex with heme or an ATP analog, and we reveal how HrtBA extracts heme from the membrane and releases it. HrtBA consists of two cytoplasmic HrtA ATPase subunits and two transmembrane HrtB permease subunits. A heme-binding site is formed in the HrtB dimer and is laterally accessible to heme in the outer leaflet of the membrane. The heme-binding site captures heme from the membrane using a glutamate residue of either subunit as an axial ligand and sequesters the heme within the rearranged transmembrane helix bundle. By ATP-driven HrtA dimerization, the heme-binding site is squeezed to extrude the bound heme. The mechanism sheds light on the detoxification of membrane-bound heme in this bacterium.

YRNA and tRNA fragments can differentiate benign from malignant canine mammary gland tumors.

Mammary gland tumors (MGT) are the most common tumors in sexually intact female dogs. The functional regulation of miRNAs, a type of noncoding RNAs (ncRNAs), in canine MGT has been extensively investigated. However, the expression of other ncRNAs, such as YRNAs and transfer RNA-derived fragments (tRFs) in canine MGT is unknown. We investigated ncRNAs other than miRNAs from our small RNA project (PRJNA716131) in different canine MGT histologic subtypes. This study included benign tumors (benign mixed tumor, complex adenoma) and malignant tumors (carcinoma in benign tumor and carcinoma with metastasis) samples. Aberrantly expressed ncRNAs were examined by comparisons among MGT subtypes. The relative expression trends were validated in canine MGT tissues, plasma, extracellular vesicles, and MGT cell lines using quantitative reverse transcription PCR. Three aberrantly expressed ncRNAs were identified by comparisons among MGT subtypes. YRNA and tRNA-Gly-GCC distinguished benign mixed tumor from other MGT histologic subtypes, while tRNA-Val differentiated complex adenoma, carcinoma in benign tumors, and carcinoma with metastasis. The ROC curve of the three ncRNAs showed they might be potential biomarkers to discriminate malignant from benign MGT. YRNA and tRFs expression levels were decreased in metastatic compared with primary canine MGT cell lines. To the best of our knowledge, this is the first investigation of YRNA and tRFs in canine MGT. The three identified ncRNAs may be biomarkers for differentiating MGT histologic subtypes. Suggested Reviewers: Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporatio.

Epidemiology and Risk Factors of Norovirus Infections Among Diarrhea Patients Admitted to Tertiary Care Hospitals in Bangladesh.

BACKGROUND: Norovirus is a major cause of endemic acute gastroenteritis (AGE) worldwide. We described the epidemiology, risk factors, and genotypic distribution of noroviruses among hospitalized patients of all ages in Bangladesh. METHODS: From March 2018 to October 2021, 1250 AGE case patients and controls (age, sex, season, and site matched) were enrolled at 10 hospitals. Demographic and clinical information was collected; real-time reverse-transcriptase polymerase chain reaction (RT-PCR) used to test stool specimens, and positive samples were genotyped. RESULTS: Norovirus was detected in 9% of cases (111 of 1250) and 15% (182 of 1250) of controls. Eighty-two percent of norovirus-positive cases were in children <5 years old. Norovirus-positive AGE hospitalizations occurred year-round, with peaks in April and October. Risk factors for norovirus included age <5 years (adjusted odds ratio, 3.1 [95% confidence interval, 1.9-5.2]) and exposure to a patient with AGE in the 10 days before enrollment (3.8 [1.9-7.2]). GII.3[P16] and GII.4 Sydney[P16] were the predominant genotypes. CONCLUSIONS: We highlight the burden of norovirus in hospital settings. Young age and recent exposure to a patient with AGE were risk factors for norovirus. A high prevalence of norovirus among controls might represent asymptomatic reinfections or prolonged shedding from a previous infection; carefully designed longitudinal studies are needed to improve our understanding of norovirus infections in Bangladesh.

Radiation-associated secondary malignancies: a novel opportunity for applying immunotherapies.

Radiation is commonly used as a treatment intended to cure or palliate cancer patients. Despite remarkable advances in the precision of radiotherapy delivery, even the most advanced forms inevitably expose some healthy tissues surrounding the target site to radiation. On rare occasions, this results in the development of radiation-associated secondary malignancies (RASM). RASM are typically high-grade and carry a poorer prognosis than their non-radiated counterparts. RASM are characterized by a high mutation burden, increased T cell infiltration, and a microenvironment that bears unique inflammatory signatures of prior radiation, including increased expression of various cytokines (e.g., TGF-ß, TNF-α, IL4, and IL10). Interestingly, these cytokines have been shown to up-regulate the expression of PD-1 and/or PD-L1-an immune checkpoint receptor/ligand pair that is commonly targeted by immune checkpoint blocking immunotherapies. Here, we review the current understanding of the tumor-immune interactions in RASM, highlight the distinct clinical and molecular characteristics of RASM that may render them immunologically "hot," and propose a rationale for the formal testing of immune checkpoint blockade as a treatment approach for patients with RASM.

Protein, lipid, and chitin fractions from insects: Method of extraction, functional properties, and potential applications.

Edible insects are accepted as food and feed ingredients in many parts of the world. Insects account for more than 80% of animal kingdom providing rich biodiversity of protein and lipid profiles compared to conventional livestock. Insect biomasses contain an average of 35-62% protein, 3-57% lipid, and 3-12% chitin, and their nutritional values are widely recognized due to their presence, including minerals, and vitamins. While whole insects are consumed as eggs, larvae, pupae, or adults, there has been a recent uptick in interest to use fractions, e.g., protein, lipid, and chitin, as food and feed ingredients. To utilize these fractions in various food and feed preparations, a deeper understanding of the physicochemical as well as functional properties of the ingredients is required, which are generally impacted by extraction and preparation processes. Thus, the methods of extraction/purification are important to preserve the quality and functional properties of these ingredients. This paper discusses the extraction methods for insect protein, lipid, and chitin, their functional properties, and potential applications in food and feed applications.

Ultrasound-assisted extraction and modification of plant-based proteins: Impact on physicochemical, functional, and nutritional properties.

Ultrasonication is a green technology that has recently received an enormous research attention for extraction of plant-based proteins and tailoring the functionalities of these ingredients. Ultrasonication is generally used as a pretreatment method in the conventional protein solubilization protocols because it can break the cell matrix to improve the extractability. The rate of protein extraction and increase in the extraction yields depend on operating conditions such as sonic energy density, time of sonication, the substrate to slurry ratio, agitation, and so on. Ultrasonication is also applied to modify the physical, structural, and functional properties of protein-based ingredients, besides simultaneous extraction and modifications. Significant changes that occur in protein physical properties due to sonication include size reduction, rheology, electrical conductivity, and zeta (ζ) potential. These changes are due to cavitation-induced shear leading to changes in secondary and tertiary structures, including protein aggregation and cross-linking due to oxidation. Physical and structural changes affect the resulting ingredient functionality and nutritional quality of protein. Changes in the functional properties, especially hydrophobicity, solubility, emulsion, and foaming, depend on the extent of ultrasound energy applied to the protein. This study aims to review major ultrasound process parameters and conditions for extraction and modification of plant proteins and their impact on protein structural changes and resulting physicochemical, functional, and nutritional properties.

Reducing childhood malnutrition in Bangladesh: the importance of addressing socio-economic inequalities.

OBJECTIVE: To obtain projections of the prevalence of childhood malnutrition indicators up to 2030 and to analyse the changes of wealth-based inequality in malnutrition indicators and the degree of contribution of socio-economic determinants to the inequities in malnutrition indicators in Bangladesh. Additionally, to identify the risk factors of childhood malnutrition. DESIGN: Cross-sectional study. A Bayesian linear regression model was used to estimate trends and projections of malnutrition. For equity analysis, slope index, relative index and decomposition in concentration index were used. Multilevel logistic models were used to identify risk factors of malnutrition. SETTING: Household surveys in Bangladesh from 1996 to 2014. PARTICIPANTS: Children under the age of 5 years. RESULTS: A decreasing trend was observed for all malnutrition indices. In 1990, predicted prevalence of stunting, wasting and underweight was 55·0, 15·9 and 61·8 %, respectively. By 2030, prevalence is projected to reduce to 28·8 % for stunting, 12·3 % for wasting and 17·4 % for underweight. Prevalence of stunting, wasting and underweight were 34·3, 6·9 and 32·8 percentage points lower in the richest households than the poorest households. Contribution of the wealth index to child malnutrition increased over time and the largest contribution of pro-poor inequity was explained by wealth index. Being an underweight mother, parents with a lower level of education and poorer households were the key risk factors for stunting and underweight. CONCLUSIONS: Our findings show an evidence-based need for targeted interventions to improve education and household income-generating activities among poor households to reduce inequalities and reduce the burden of child malnutrition in Bangladesh.

Go clean label: replacement of commercial dough strengtheners with hard red spring wheat flour in bread formulations.

There is growing interest in the application of natural ingredients to replace chemical dough improvers in bread formulations in order to meet consumer demands of clean label products. The goal of this study was to evaluate the dough quality and baking quality of hard red spring (HRS) wheat flour blends to replace commercial dough improvers. Hard red winter (HRW) wheat flour is commonly used in bread and diner roll formulations. In this study, doughs were prepared by adding 10%, 20%, 30%, and 40% of HRS wheat flour to HRW wheat base-flour to compare the dough quality and baking quality relative to different levels of commercial improvers. Additional to commercial HRS flour, two commonly grown HRS wheat varieties (Glenn and Linkert) were included in the study. All of the HRS wheat flour blends had significantly (p < 0.05) higher farinograph stability and extensograph resistance at 135 min than doughs containing most of the commercial additives. Bread flour with 40% Glenn and 40% Linkert showed the highest loaf volumes of 920 cm3 and 950 cm3, respectively with the firmness of 1553.50 and 1525.50 mN, respectively. Baking quality of HRS wheat flour blends also showed significant (p < 0.05) correlation with dough rheology but commercial additives did not have the correlations. Therefore, HRS wheat flour may be used as a replacement for dough improvers, as it had better dough and bread properties compared to commercial additives and provides a great alternative for "clean-label" bread products.

Hybrid graphene metasurface for near-infrared absorbers.

We experimentally demonstrated an amorphous graphene-based metasurface yielding near-infrared super absorber characteristic. The structure is obtained by alternatively combining magnetron-sputtering deposition and graphene transfer coating fabrication techniques. The thickness constraint of the physical vapor-deposited amorphous metallic layer is unlocked and as a result, the as-fabricated graphene-based metasurface absorber achieves near-perfect absorption in the near-infrared region with an ultra-broad spectral bandwidth of 3.0 µm. Our experimental characterization and theoretical analysis further point out that the strong light-matter interaction observed is caused by localized surface plasmon resonance of the metal film's particle-like surface morphology. In addition to the enhanced light absorption characteristics, such an amorphous metasurface can be used for surface-enhanced Raman scattering applications. Meanwhile, the proposed graphene-based metasurface relies solely on CMOS-compatible, low cost and large-area processing, which can be flexibly scaled up for mass production.

Micro RNA Transcriptome Profile in Canine Oral Melanoma.

MicroRNAs (miRNAs) dysregulation contribute the cancer pathogenesis. However, the miRNA profile of canine oral melanoma (COM), one of the frequent malignant melanoma in dogs is still unrevealed. The aim of this study is to reveal the miRNA profile in canine oral melanoma. MiRNAs profile of oral tissues from normal healthy dogs and COM patients were compared by next-generation sequencing. Along with tumour suppressor miRNAs, we report 30 oncogenic miRNAs in COM. The expressions of miRNAs were further confirmed by quantitative real-time PCR (qPCR). Pathway analysis showed that deregulated miRNAs impact on cancer and signalling pathways. Three oncogenic miRNAs targets (miR-450b, 301a, and 223) from human study also were down-regulated in COM and had a significant negative correlation with their respective miRNA. Furthermore, we found that miR-450b expression is higher in metastatic cells and regulated MMP9 expression through a PAX9-BMP4-MMP9 axis. In silico analysis indicated that miR-126, miR-20b, and miR-106a regulated the highest numbers of differentially expressed transcription factors with respect to human melanoma. Chromosomal enrichment analysis revealed the X chromosome was enriched with oncogenic miRNAs. We comprehensively analyzed the miRNA's profile in COM which will be a useful resource for developing therapeutic interventions in both species.

Effects of processing techniques on drying characteristics, physicochemical properties and functional compounds of green and red chilli (Capsicum annum L.) powder.

This study was aimed to investigate the effect of processing techniques on the characteristics of green and red chilli powder. Four samples, such as pretreated green chilli paste (PTGP), pretreated green chilli longitudinal slit (PTGL), pretreated whole red chilli (PTWR) and untreated green chilli paste (UTGP), were prepared and dried at 60 °C in a cabinet dryer. The pretreatment was blanching in acetic acid solution and soaking immediately in a combined solution of Na2S2O5 and CaCl2. Pretreated samples took a shorter drying time than the untreated sample in reducing moisture content from 86.31 to 8%. Pretreatment before drying resulted in retaining total chlorophyll (~ 86%), phenolic compounds (~ 32%), green color, and pungency of chilli. Analysis result indicated that more than 60% retention of ß-carotene was found while retention of ascorbic acid was comparable. Conclusively, this research reveals a good nutritional profile in cabinet dried green chilli powder, which may open the scope for commercial production.

Structural basis for binding and transfer of heme in bacterial heme-acquisition systems.

Periplasmic heme-binding proteins (PBPs) in Gram-negative bacteria are components of the heme acquisition system. These proteins shuttle heme across the periplasmic space from outer membrane receptors to ATP-binding cassette (ABC) heme importers located in the inner-membrane. In the present study, we characterized the structures of PBPs found in the pathogen Burkholderia cenocepacia (BhuT) and in the thermophile Roseiflexus sp. RS-1 (RhuT) in the heme-free and heme-bound forms. The conserved motif, in which a well-conserved Tyr interacts with the nearby Arg coordinates on heme iron, was observed in both PBPs. The heme was recognized by its surroundings in a variety of manners including hydrophobic interactions and hydrogen bonds, which was confirmed by isothermal titration calorimetry. Furthermore, this study of 3 forms of BhuT allowed the first structural comparison and showed that the heme-binding cleft of BhuT adopts an "open" state in the heme-free and 2-heme-bound forms, and a "closed" state in the one-heme-bound form with unique conformational changes. Such a conformational change might adjust the interaction of the heme(s) with the residues in PBP and facilitate the transfer of the heme into the translocation channel of the importer.

An evaluation of automated chest radiography reading software for tuberculosis screening among public- and private-sector patients.

Computer-aided reading (CAR) of medical images is becoming increasingly common, but few studies exist for CAR in tuberculosis (TB). We designed a prospective study evaluating CAR for chest radiography (CXR) as a triage tool before Xpert MTB/RIF (Xpert).Consecutively enrolled adults in Dhaka, Bangladesh, with TB symptoms received CXR and Xpert. Each image was scored by CAR and graded by a radiologist. We compared CAR with the radiologist for sensitivity and specificity, area under the receiver operating characteristic curve (AUC), and calculated the potential Xpert tests saved.A total of 18 036 individuals were enrolled. TB prevalence by Xpert was 15%. The radiologist graded 49% of CXRs as abnormal, resulting in 91% sensitivity and 58% specificity. At a similar sensitivity, CAR had a lower specificity (41%), saving fewer (36%) Xpert tests. The AUC for CAR was 0.74 (95% CI 0.73-0.75). CAR performance declined with increasing age. The radiologist grading was superior across all sub-analyses.Using CAR can save Xpert tests, but the radiologist's specificity was superior. Differentiated CAR thresholds may be required for different populations. Access to, and costs of, human readers must be considered when deciding to use CAR software. More studies are needed to evaluate CAR using different screening approaches.

Assessment of biorational larvicides and botanical oils against Culex quinquefasciatus Say (Diptera: Culicidae) larvae in laboratory conditions.

Mosquitoes are known vectors that transmit deadly diseases to millions of people across the globe. The reliance on synthetic insecticides has been the sole way to combat mosquito vectors for decades. In recent years, the extensive use of conventional insecticides in mosquito suppression has led to significant pesticide resistance and serious human health hazards. In this light, investigating the potential application of biorational compounds for vector management has drawn significant attention. We, hereby, evaluated the efficacy of three microbial derivative biorational insecticides, abamectin, spinosad, and buprofezin, and two botanical oils, neem (Azadirachta indica A. Juss) and karanja oil (Pongamia pinnata Linn.) against the Culex quinquefasciatus under laboratory conditions. The fourth-instar C. quinquefasciatus larvae were exposed to different concentrations of the selected larvicides and lethality was estimated based on LC50 and LT50 with Probit analysis. All larvicides showed concentration-dependent significant effects on survival and demonstrated larvicidal activity against C. quinquefasciatus larvae. However, abamectin exerted the highest toxicity (LC50 = 10.36 ppm), exhibited statistically significant effects on C. quinquefasciatus larval mortality, followed by spinosad (LC50 = 21.32 ppm) and buprofezin (LC50 = 56.34 ppm). Abamectin caused larval mortality ranged from 30.00 to 53.33 % and 53.00-70.00 % at 06 and 07 h after treatment (HAT), respectively. In the case of botanicals, karanja oil (LC50 = 216.61 ppm) was more lethal (more than 1.5 times) and had a shorter lethal time than neem oil (LC50 = 330.93 ppm) and showed a classic pattern of relationship between concentrations and mortality over time. Overall, the present study highlighted the potential of deploying new generation biorational pesticides and botanicals in mosquito vector control programs.

Antimicrobial Activity of Polycaprolactone Nanofiber Coated with Lavender and Neem Oil Nanoemulsions against Airborne Bacteria.

The development of efficient, eco-friendly antimicrobial agents for air purification and disinfection addresses public health issues connected to preventing airborne pathogens. Herein, the antimicrobial activity of a nanoemulsion (control, 5%, 10%, and 15%) containing neem and lavender oils with polycaprolactone (PCL) was investigated against airborne bacteria, including Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. Various parameters such as the physicochemical properties of the nanoemulsion, pH, droplet size, the polydispersity index (PDI), the minimum inhibitory concentration (MIC), the minimum bacterial concentration (MBC), and the color measurement of the emulsion have been evaluated and optimized. Our results showed that the antimicrobial activity of PCL combined with neem and lavender oil was found to be the highest MIC and MBC against all tested bacteria. The droplet sizes for lavender oil are 21.86-115.15 nm, the droplet sizes for neem oil are 23.92-119.15 nm, and their combination is 25.97-50.22 nm. The range of pH and viscosity of nanoemulsions of various concentrations was found to be 5.8 to 6.6 pH and 0.372 to 2.101 cP. This study highlights the potential of nanotechnology in harnessing the antimicrobial properties of natural essential oils, paving the way for innovative and sustainable solutions in the fight against bacterial contamination.

Hypoxia-related Y RNA fragments as a novel potential biomarker for distinguishing metastatic oral melanoma from non-metastatic oral melanoma in dogs.

Hypoxia may promote tumor progression, and hypoxically altered noncoding RNA (ncRNA) expression may play a role in metastasis. Canine oral melanoma (COM) frequently metastasizes, and ncRNA expression under hypoxia may be clinically significant. We aimed to elucidate ncRNA fragments whose expression is altered by hypoxia in COM-derived primary KMeC and metastatic LMeC cell lines using next-generation sequencing to validate these results in qRT-PCR, and then compare expression between metastatic and non-metastatic COM. The NGS analysis and subsequent qRT-PCR validation were performed using hypoxic and normoxic KMeC and LMeC cells, and clinical samples [tumor tissue, plasma, and plasma-derived extracellular vesicles] obtained from dogs with metastatic or non-metastatic melanoma were analyzed with qRT-PCR. Y RNA was significantly decreased in metastatic LMeC cells versus primary KMeC cells in hypoxic and normoxic conditions. The expression of Y RNA was decreased in dogs with metastatic melanoma versus those with non-metastatic melanoma for all clinical sample types, reflecting the pattern found with hypoxia. Receiver operating characteristic analysis demonstrated that Y RNA level is a promising biomarker for discriminating metastatic from non-metastatic melanoma in plasma [area under the curve (AUC) = 0.993, p < 0.0001] and plasma-derived extracellular vesicles (AUC = 0.981, p = 0.0002). Overall, Y RNA may be more resistant to hypoxic stress in the metastatic than the non-metastatic state for COM. However, further investigation is required to elucidate the biological functions of Y RNA under hypoxic conditions.

Disparities and Determinants of Testing for Early Detection of Cervical Cancer among Nepalese Women: Evidence from a Population-Based Survey.

BACKGROUND: Cervical cancer presents a considerable challenge in South Asia, notably in Nepal, where screening remains limited. Past research in Nepal lacked national representation and a thorough exploration of factors influencing cervical cancer screening, such as educational and socioeconomic disparities. This study aims to measure these gaps and identify associated factors in testing for early detection of cervical cancer among Nepalese women. METHODS: Data from the 2019 Nepal Noncommunicable Disease Risk Factors survey (World Health Organization STEPwise approach to noncommunicable risk factor surveillance), involving 2,332 women aged 30 to 69 years, were used. Respondents were asked if they had undergone cervical cancer testing through visual inspection with acetic acid, Pap smear, or human papillomavirus test ever or in the past 5 years. The slope index of inequality (SII) and relative concentration index were used to measure socioeconomic and education-based disparities in cervical cancer test uptake. RESULTS: Only 7.1% [95% confidence interval (CI): 5.1-9.9] Nepalese women had ever undergone cervical cancer testing, whereas 5.1% (95% CI: 3.4-7.5) tested within the last 5 years. The ever uptake of cervical cancer testing was 5.1 percentage points higher (SII: 5.1, 95% CI: -0.1 to 10.2) among women from the richest compared with the poorest households. Education-based disparities were particularly pronounced, with a 13.9 percentage point difference between highly educated urban residents and their uneducated counterparts (SII: 13.9, 95% CI: 5.8-21.9). CONCLUSIONS: Less than one in ten women in Nepal had a cervical cancer testing, primarily favoring higher educated and wealthier individuals. IMPACT: Targeted early detection and cervical cancer screening interventions are necessary to address these disparities and improve access and uptake.

Prediction and assessment of deleterious and disease causing nonsynonymous single nucleotide polymorphisms (nsSNPs) in human FOXP4 gene: An in - silico study.

In humans, FOXP gene family is involved in embryonic development and cancer progression. The FOXP4 (Forkhead box protein P4) gene belongs to this FOXP gene family. FOXP4 gene plays a crucial role in oncogenesis. Single nucleotide polymorphisms are biological markers and common determinants of human diseases. Mutations can largely affect the function of the corresponding protein. Therefore, the molecular mechanism of nsSNPs in the FOXP4 gene needs to be elucidated. Initially, the SNPs of the FOXP4 gene were extracted from the dbSNP database and a total of 23124 SNPs was found, where 555 nonsynonymous, 20525 intronic, 1114 noncoding transcript, 334 synonymous were obtained and the rest were unspecified. Then, a series of bioinformatics tools (SIFT, PolyPhen2, SNAP2, PhD SNP, PANTHER, I-Mutant2.0, MUpro, GOR IV, ConSurf, NetSurfP 2.0, HOPE, DynaMut2, GeneMANIA, STRING and Schrodinger) were used to explore the effect of nsSNPs on FOXP4 protein function and structural stability. First, 555 nsSNPs were analyzed using SIFT, of which 57 were found as deleterious. Following, PolyPhen2, SNAP2, PhD SNP and PANTHER analyses, 10 nsSNPs (rs372762294, rs141899153, rs142575732, rs376938850, rs367607523, rs112517943, rs140387832, rs373949416, rs373949416 and rs376160648) were common and observed as deleterious, damaging and diseases associated. Following that, using I-Mutant2.0 and MUpro servers, 7 nsSNPs were found to be the most unstable. GOR IV predicted that these seven nsSNPs affect protein structure by altering the protein contents of alpha helixes, extended strands, and random coils. Following DynaMut2, 5 nsSNPs showed a decrease in the ΔΔG value compared with the wild-type and were found to be responsible for destabilizing the corresponding protein. GeneMANIA and STRING network revealed interaction of FOXP4 with other genes. Finally, molecular dynamics simulation analysis revealed consistent fluctuation in RMSD and RMSF values, Rg and hydrogen bonds in the mutant proteins compared with WT, which might alter the functional and structural stability of the corresponding protein. As a result, the aforementioned integrated comprehensive bioinformatic analyses provide insight into how various nsSNPs of the FOXP4 gene change the structural and functional properties of the corresponding protein, potentially proceeding with the pathophysiology of human diseases.

Upregulation and functional roles of miR-450b in canine oral melanoma.

Canine oral melanoma (COM) is a common and highly aggressive disease with the potential to model human melanomas. Dysregulated microRNAs represent an interesting line of research for COM because they are implicated in tumor progression. One example is miR-450b, which has been investigated for its molecular mechanisms and biological functions in multiple human cancers, but not human or canine melanoma. Here, we aimed to investigate miR-450b as a potential diagnostic biomarker of COM and its functional roles in metastatic and non-metastatic forms of the disease. We investigated the expression of miR-450b and its target mRNA genes in clinical (tumor tissue and plasma) samples and metastatic and primary-tumor cell lines. Knockdown and overexpression experiments were performed to determine the influence of miR-450b on cell proliferation, migration, colony formation, and apoptosis. miR-450b was significantly upregulated in COM and differentiated between metastatic and non-metastatic tumors, and its potential as a biomarker of metastatic and non-metastatic COM was further confirmed in ROC analysis. miR-450b knockdown promoted cell proliferation, migration, and clonogenicity and inhibited apoptosis, whereas its overexpression yielded the reverse pattern. miR-450b directly binds 3' UTR of PAX9 mRNA and modulates its function leading to BMP4 downregulation and MMP9 upregulation at the transcript level. Furthermore, we surmised that miR-450b activates the Wnt signaling pathway based on gene ontology and enrichment analyses. We concluded that miR-450b has the potential as a diagnostic biomarker and could be a target candidate for COM treatment.

Elevated expression of miR-301a and its functional roles in canine oral melanoma.

miR-301a is one of numerous dysregulated microRNAs (miRNAs) in canine oral melanoma (COM), one of which is miR-301a (upregulated). Its biological role has been described in various human cancer types, including malignant melanoma, but not in COM. Accordingly, in this study, we investigated miR-301a expression in COM in greater detail to ascertain whether it could serve as a diagnostic biomarker, elucidate its functional roles in this cancer, and predict the possible pathways by which it exerts its effects. Relative expression of miR-301a was investigated in clinical oral tissue and plasma samples and COM cell (KMeC and LMeC) lines using qRT-PCR. Knockdown of miR-301a was also validated for KMeC and LMeC cells using qRT-PCR. We performed CCK-8 assays to assess cell proliferation, monolayer wound-healing, and transwell migration assays to assess cell migration, a colony-formation assay to assess clonogenicity, a TUNEL assay and flow cytometry to assess apoptosis-related effects, and gene enrichment analyses to predict possible related pathways. miR-301a was markedly upregulated in COM oral tissue and plasma clinically, suggesting its potential as a diagnostic biomarker for COM diagnosis. In vitro assays demonstrated that miR-301 significantly inhibited apoptosis in COM cells while promoting cell migration, proliferation, and clonogenicity. We also predicted that miR-301 exerts cancer-promoting effects through the Wnt signalling pathway for COM. Our findings suggest that miR-301a is a COM oncomiR that regulates several oncogenic phenotypes with the potential to be a diagnostic biomarker.