Identification and validation of stable reference genes for expression profiling of target genes in diverse ovine tissues.

Quantitative PCR (qPCR) is a widely-used technique for quantifying the expression of target genes across various tissues, as well as under different pathological and physiological conditions. One of the challenges associated with this method is the need to identify optimal reference genes (RGs) that maintain consistent expression levels under diverse experimental settings, thereby ensuring accurate biological interpretation. In this study, we conducted a thorough analysis of 18 candidate RGs (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) across 10 ovine tissues (muscle, skin, kidney, liver, intestine, rumen, lung, testis, heart, and spleen) obtained from five individual sheep. We aimed to identify genes with stable expression across these tissues. A literature-based survey helped us shortlist candidate genes representing various functional classes from multiple livestock species. We employed four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), to rank these genes based on their stability. A consistent trend in the rankings was observed across these different algorithms. RefFinder was then used for a comprehensive ranking, integrating the outputs from the various methods. ACTB, PPIB, BACH1, and B2M emerged as the most stable RGs, while RPS9, RPS15, and PGK1 displayed variable expression. We validated our findings through qPCR analysis of four target genes (ACTN2, CRYAB, DLK1, and TRIM54) in the skin samples from two different sheep breeds. Based on these results, we recommend ACTB, PPIB, BACH1, and B2M as reliable internal control genes for qPCR experiments involving diverse ovine tissues.

Unraveling the genetic mechanisms governing the host response to bovine anaplasmosis.

Bovine anaplasmosis caused by Anaplasma marginale is a tick-borne disease of livestock with widespread prevalence and huge economic implications. In order to get new insights into modulation of host gene expression in response to natural infections of anaplasmosis, this study is the first attempt that compared the transcriptome profiles of peripheral blood mononuclear cells (PBMCs) of A. marginale infected and healthy crossbred cattle. Transcriptome analysis identified shared as well as unique functional pathways in the two groups. Translation and structural constituent of ribosome were the important terms for the genes abundantly expressed in the infected as well as healthy animals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes revealed that immunity and signal transduction related terms were enriched for the up-regulated genes in the infected animals. The over-represented pathways were cytokine-cytokine receptor interaction and signaling pathways involving chemokines, Interleukin 17 (IL17), Tumour Necrosis Factor (TNF), Nuclear Factor Kappa B (NFKB) etc. Interestingly, many genes previously associated with parasite-borne diseases such as amoebiasis, trypanosomiasis, toxoplasmosis, and leishmaniasis were profusely expressed in the dataset of the diseased animals. High expression was also evident for the genes for acute phase response proteins, anti-microbial peptides and many inflammatory cytokines. Role of cytokines in mediating communication between immune cells was the most conspicuous gene network identified through the Ingenuity Pathway Analysis. This study provides comprehensive information about the crosstalk of genes involved in host defense as well as parasite persistence in the host upon infection with A. marginale.

Validation of stable reference genes in peripheral blood mononuclear cells for expression studies involving vector-borne haemoparasitic diseases in bovines.

Normalization of gene expression data using appropriate reference genes is critical to diminish any technical bias in an experiment involving quantitative real-time PCR (qPCR). To the best of our knowledge, this is the first report offering a systematic assessment of 14 potential reference genes (RPLP0, ACTB, RPS28, YWHAZ, SDHA, PPIA, RPS9, RPS15, UXT, GAPDH, B2M, BACH1, HMBS, and PPIB) for the identification of the most stable normalizers for qPCR of target genes in peripheral blood mononuclear cells (PBMCs) of bovines for vector-borne haemoparasitic diseases such as anaplasmosis, babesiosis, theileriosis, and trypanosomiasis. A total of 38 blood samples were collected from healthy as well as diseased cattle and buffaloes representing different haemoparasitic diseases. RNA isolated from the PBMCs was subjected to qPCR for the 14 prospective internal control genes. The comprehensive ranking of the genes was accomplished by the RefFinder tool that integrates the results of three algorithms (geNorm, NormFinder, and BestKeeper) and the comparative CT method. RPS15, B2M, and GAPDH were ranked to be the most stable genes, whereas, PPIA and HMBS emerged to be the least suitable genes. Validation of the selected reference genes by the qPCR analysis of two immunity genes, ISG15 and GPX7 was congruent with the observations of this study. We recommend that a panel of three reference genes including RPS15, B2M, and GAPDH could prove useful in delineating the transcriptional landscape of PBMCs for vector-borne haemoparasitic diseases in bovines.

Expression profiling of cytokine genes in peripheral blood mononuclear cells from Anaplasma marginale infected and healthy cattle.

In this study, changes in expression profiles of genes encoding 14 cytokines (IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL12A, IL12B, IL16, IFNA, IFNB, TGFB1, and TNFA) were investigated amongst six Anaplasma marginale infected and six healthy crossbred cattle. Health status of the animals was determined based on clinical signs, blood smear examination and molecular detection using A. marginale-specific primers. Total RNA was isolated from the peripheral blood mononuclear cells of the infected animals as well as the healthy controls, which was further reverse transcribed to cDNA. Primers for real time PCR were designed using Primer3 software and the results were analyzed by the 2-ΔΔCt method with RPS15 and GAPDH as the reference genes. The expression levels of IL1A, IL1B, IL6, IL10, IL12A, IL12B, and TNFA varied significantly between the two groups, with higher expression in the infected cattle. The transcript abundance of IL4, IL16, and TGFB1 did not vary between the diseased and healthy animals. The expression of IL2 and IL8 was higher in the healthy animals, but the results were non-significant. Taken together, this study provides evidence for difference in expression of cytokine genes in response to anaplasmosis in crossbred cattle.

Comparative expression profiling of cytokine genes in Theileria annulata-infected and healthy cattle.

Theileriosis is one of the top ten economically important diseases in cattle in India. Cytokines are considered important mediators and regulators of the immune response to an infection. In the present study, the gene expression profiles of fourteen cytokines (IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL12A, IL12B, IL16, TGFB1, TNFA, IFNA and IFNB) were compared in Theileria annulata-infected and healthy crossbred cattle. Blood samples were obtained from the District Disease Diagnostic Laboratory, Karnal. The presence/absence of T. annulata infection in the animals was determined on the basis of blood smear examination and molecular detection through PCR using the genus-specific primers. Total RNA was extracted from peripheral blood mononuclear cells, which was further reverse transcribed to cDNA. Primer3 software was employed to design the primers for Real-Time qPCR. The results were examined using 2-∆∆Ct method with RPS15 and GAPDH as the reference genes. The expression of IL1B, IL6, IL8, IL10, IL12A, IL12B, TNFA, IFNA and IFNB was significantly higher, whereas the expression of IL2 was lower in the infected animals. The transcript levels of IL1A and TGFB1 were also higher in the diseased animals, but the results were non-significant. This study profiles the expression kinetics of various pro-inflammatory and anti-inflammatory cytokine genes in response to bovine theileriosis.

Outcome Analysis of En bloc Resection and Reconstruction by Non-vascularized Proximal Fibular Autograft in Giant Cell Tumor of Distal Radius supplemented with Brachytherapy.

Introduction: Giant cell tumors of the bone are aggressive and potentially malignant lesions. Juxtaarticular giant cell tumors of the lower end radius are common and is a challenge for reconstruction after tumor excision. Several reconstructive procedures like vascularized and non-vascularized fibular graft, osteoarticular allograft, ceramic prosthesis, and megapros thesis are in use for substitution of the defect in the distal radius following resection. Here, we have analyzed the results of aggressive benign Giant cell tumor of the distal radius treated by en bloc excision and reconstruction using autogenous non-vascularized fibular graft along with brachytherapy. Material and Methods: Eleven patients with either Campanacci Grade II or III histologically proven giant cell tumors of lower end radius were treated with en bloc excision and reconstruction with ipsilateral non-vascularized proximal fibular autograft. Host graft junction was fixed with low contact dynamic compression plate (LC-DCP) in all cases. Fixation of the head of the fibula with carpal bones and distal end of the ulna, if not resected, using K-wires at graft host junction was done. Brachytherapy was given in all 11 cases. Routine radiographs and clinical assessments regarding pain, instability, recurrence, hand grip strength, and functional status were done using Mayo modified wrist score at regular intervals. Result: The follow-up ranged from 12 to 15 months. At last follow-up, the average combined range of motion was 76.1%. The average union time was 19 weeks. Out of 11 patients, two patients had good results, five patients had fair results, and four patient had poor results. There was no case of graft fracture, metastasis, death, local recurrence, or significant donor site morbidity. Conclusion: En bloc resection of giant cell tumors of the lower end radius is a widely accepted method. Reconstruction with non-vascularized fibular graft and internal fixation with LC-DCP along with brachytherapy minimizes the problem and gives satisfactory functional results with no recurrence.

Correlation between Chondromalacia Patella and Patellofemoral Factors in Middle-Age Population: A Clinical, Functional, and Radiological Analysis.

Background Despite higher incidence of patellofemoral pain (PFP) and consequently morbidity, the understanding about PF factors leading to PF arthritis is way lacking. Material and Methods A prospective study of first 80 patients who were diagnosed with chondromalacia patella (CMP) on magnetic resonance imaging (MRI) divided into early and late CMP groups were evaluated clinically, radiologically, and in terms of functional outcome. Results : Quadriceps angle, Clark's test, and Insall-Salvati ratio results were nonsignificant despite greater values were observed in late CMP group, whereas trochlear morphology results (sulcus angle: 153:138 degrees and sulcus depth 3.9:5.4 mm) and clinical scores were significant in late CMP group (Kujala's score: 61:78, whereas PF pain score: 43:25). Type-C patellar morphology was found in greater number in late CMP cohort. Conclusion Trochlear and patellar morphologies along with clinical scores play a key role in understanding of the CMP.

Benign fibrous histiocytoma of the lower lip.

The fibrous histiocytoma is a soft-tissue neoplasm of the biphasic cell population of fibroblasts and histiocytes that affects the dermis and the subcutaneous tissue. The objective of this article is to report a case of benign fibrous histiocytoma (BFH) of the lower lip in a 32-year-old female patient with a chief complaint of swelling in the lower lip for the past 1 month. With diagnostic clinical hypothesis of fibrous hyperplasia, fibrous histiocytoma and mucocele, an excisional biopsy was performed. The histopathological examination revealed a nonencapsulated proliferation of spindle cells with some giant multinucleated cells in the periphery of the lesion. Multinucleated giant cells and lymphocytes were noted throughout the lesion. Immunohistochemical reactions were performed, staining only CD68 in the multinucleated giant cells. According to these characteristics, the final diagnosis was BFH.

Correlation of acromial morphology in association with rotator cuff tear: a retrospective study.

PURPOSE: There have been many studies that have attempted to correlate radiographic acromial characteristics with rotator cuff tears, but the results have not been conclusive. Rotator cuff tears (RCT) are the common aetiology of shoulder pain. We assessed the association of rotator cuff tears with commonly used radiographic parameters of acromial morphology and their different radiographic characteristics. MATERIAL AND METHODS: From a retrospective study of 98 patients, we characterised acromial type and measured acromial thickness (AT), critical shoulder angle (CSA), lateral acromial angle (LAA), acromiohumeral distance (AHD), and acromion index (AI) on a 1.5T MRI, from 68 patients with partial or full-thickness supraspinatus tendon tears and 30 controls without tears. RESULTS: Out the 68 patients with rotator cuff tear, supraspinatus was the most commonly affected tendon, with 86% (59) cases showing abnormalities. The average age of the patients was 45.11 ± 21.45 years with male dominance (80%). Partial tears of rotator cuff were more common than complete tears. Forty-eight cases showed partial tears in supraspinatus as compared to 11 cases of complete tears. The acromial type did not show any correlation with any particular cuff lesion. The AT and AI of controls were significantly smaller than cuff-tear patients. The LAA of cuff-tear patients was significantly different from that of control patients. The impingement patients demonstrated a significantly greater acromial thickness, larger CSA, decreased AHD, and decreased LAA than their control counterparts. CONCLUSIONS: A higher prevalence of rotator cuff tears and impingement associated with low lateral acromial angle, larger CSA and decreased AHD was observed. AT and AI have a direct correlation with rotator cuff tear.

Role of Computed Tomography (CT) in Localisation and Characterisation of Suprahyoid Neck Masses.

BACKGROUND: Suprahyoid neck lesions are difficult to assess only by means of clinical inspection and therefore imaging techniques are required to precisely evaluate suprahyoid neck spaces. The aim of this study was to evaluate the distinctive role of computed tomography in the assessment of anatomical source and pathological type of masses involving the suprahyoid neck spaces. MATERIAL/METHODS: Sixty patients presenting with suprahyoid neck masses underwent computed tomography of the neck. The CT findings were correlated with histopathological findings and a final diagnosis was made. RESULTS: Overall, male preponderance was seen except in the case of parotid space lesions where female predominance was seen. The most common aetiology was squamous cell carcinoma and the majority of cases (30%) were seen in patients aged 41-50 years. The majority of lesions were found in the pharyngeal mucosal space (n=16) with squamous cell carcinoma being the most common pathology. In the parotid space, pleomorphic adenoma and in the prestyloid parapharyngeal space, squamous cell carcinoma were the most common lesions, respectively. In the retropharyngeal space, an equal incidence of malignant and inflammatory aetiologies was seen. Abscesses were the most common lesions in the prevertebral space. The pleomorphic adenoma was the most common benign tumour and was also the second most common tumour in the suprahyoid neck spaces. CT had an excellent correlation with histopathological findings with sensitivity of 96.4%, specificity of 100%, positive predictive value of 100% and a negative predictive value of 91.67%. CONCLUSIONS: Computed tomography definitely has a major role to play in the evaluation of suprahyoid neck masses as it has an excellent correlation with post-operative histopathological diagnosis.

Neglected hip dislocation: an unusual presentation, its management and review of the literature.

Neglected traumatic posterior hip dislocation is not an uncommon condition seen in developing countries. Its treatment is fraught with great difficulties and controversies. Here we present a 31-year-old female patient presented to our hospital 2 years after sustaining an accident. She had gone through multiple treatments for her dislocated hip without any success. After arrival to our hospital, patient managed by constrained total hip replacement (THR). Her Harris hip score improved from 48 (preoperative) to 81 (postoperative). On follow-up she had a good range of movement of the hip with no pain, deformity and limb length discrepancy. Constrained THR provides promising results in neglected hip dislocation.

Giant-cell tumour of proximal radius in a 50-year-old female with wrist drop: a rare case report.

Giant-cell tumour is a locally aggressive tumour of long bones of epiphyseal region commonly occurring in adults aged 20-40 years. Most common location is distal femur, proximal tibia, and distal radius. Different treatment options being used are curettage with bone graft or bone cement, resection with arthrodesis, reconstruction, radiation, and chemotherapy. We are reporting a case of giant-cell tumour of right proximal radius in a 50-year-old female with posterior interosseous nerve palsy. It is very rare, and only four cases have been reported in the literature. It was treated by wide margin resection with fibular grafting, titanium elastic nail system along with cancellous bone graft reconstruction.

Disseminated hydatidosis with rare presentation as "cystovesical fistula".

Disseminated hydatidosis is a rare disease and may involve any organ of the human body. In this case, an elderly female got infected by Echinococcus and presented with disseminated disease. A fistulous communication developed between one of the hydatid cysts present in the retrovesical region and the bladder, because of which the patient presented with hydatiduria.

Rare Thoracolumbar Facet Synovial Cyst Presenting as Paraparesis.

Spinal cord compressing syndrome due to synovial cyst (SC) of the thoracolumbar spine is a rare clinical condition. In this report we aim to heighten awareness of the thoracolumbar facet synovial cyst as a possible cause of thoracic myelopathy. The SC was removed thoroughly by laminectomy. The patient had an excellent recovery. The etiological and therapeutic aspects are discussed.