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Craniofacial development including palatogenesis is a complex process which requires an orchestrated and spatiotemporal expression of various genes and factors for proper embryogenesis and organogenesis. One such group of genes essential for craniofacial development is the homeobox genes, transcriptional factors that are commonly associated with congenital abnormalities. Amongst these genes, DLX4, HOXB3, and MSX2 have been recently shown to be involved in the etiology of non-syndromic cleft lip and palate. Hence, we investigated the gene and protein expression of these genes in normal and cleft affected mucosal tissue obtained from 22 children, along with analyzing their role in promoting local-site inflammation using NF-κB. Additionally, we investigated the role of PTX3, which plays a critical role in tissue remodeling and wound repair. We found a residual gene and protein expression of DLX4 in cleft mucosa, although no differences in gene expression levels of HOXB3 and MSX2 were noted. However, a significant increase in protein expression for these genes was noted in the cleft mucosa (p &lt, 0.05), indicating increased cellular proliferation. This was coupled with a significant increase in NF-κB protein expression in cleft mucosa (p &lt, 0.05), highlighting the role of these genes in promotion of pro-inflammatory environment. Finally, no differences in gene expression of PTX3 were noted.

Normalization of gene expression using internal controls or reference genes (RGs) has been the method of choice for standardizing the technical variations in reverse transcription quantitative polymerase chain reactions (RT-qPCR). Conventionally, ACTB and GAPDH have been used as reference genes despite evidence from literature discouraging their use. Hence, in the present study we identified and investigated novel reference genes in SK-BR-3, an HER2-enriched breast cancer cell line. Transcriptomic data of 82 HER2-E breast cancer samples from TCGA database were analyzed to identify twelve novel genes with stable expression. Additionally, thirteen RGs from the literature were analyzed. The expression variations of the candidate genes were studied over five successive passages (p) in two parallel cultures S1 and S2 and in acute and chronic hypoxia using various algorithms. Finally, the most stable RGs were selected and validated for normalization of the expression of three genes of interest (GOIs) in normoxia and hypoxia. Our results indicate that HSP90AB1, DAD1, PFN1 and PUM1 can be used in any combination of three (triplets) for optimizing intra- and inter-assay gene expression differences in the SK-BR-3 cell line. Additionally, we discourage the use of conventional RGs (ACTB, GAPDH, RPL13A, RNA18S and RNA28S) as internal controls for RT-qPCR in SK-BR-3 cell line.

Both chronic alcoholism and human herpesvirus-6 (HHV-6) infection have been identified as promoters of neuroinflammation and known to cause movement-related disorders. Substantia Nigra (SN), the dopaminergic neuron-rich region of the basal ganglia, is involved in regulating motor function and the reward system. Hence, we hypothesize the presence of possible synergism between alcoholism and HHV-6 infection in the SN region and report a comprehensive quantification and characterization of microglial functions and morphology in postmortem brain tissue from 44 healthy, age-matched alcoholics and chronic alcoholics. A decrease in the perivascular CD68+ microglia in alcoholics was noted in both the gray and white matter. Additionally, the CD68+/Iba1− microglial subpopulation was found to be the dominant type in the controls. Conversely, in alcoholics, dystrophic changes in microglia were seen with a significant increase in Iba1 expression and perivascular to diffuse migration. An increase in CD11b expression was noted in alcoholics, with the Iba1+/CD11b− subtype promoting inflammation. All the controls were found to be negative for HHV-6 whilst the alcoholics demonstrated HHV-6 positivity in both gray and white matter. Amongst HHV-6 positive alcoholics, all the above-mentioned changes were found to be heightened when compared with HHV-6 negative alcoholics, thereby highlighting the compounding relationship between alcoholism and HHV-6 infection that promotes microglia-mediated neuroinflammation.

Background Lower third molar extraction is usually associated with multiple stigmae that deters the patients in most cases to postpone or not seek appropriate surgical care. It is also one of the most feared procedure amongst dentists and dental students. However, evidence has been reported suggesting that the choice of suturing technique can have a profound effect on post-operation complications and thereby can improve the overall outlook and comfort of the patient. In the past, multiple techniques like Mattress, Continuous etc have been compared with tissue glues, staples however, a comparative clinical study amongst the simplest and most commonly used suturing techniques have not been reported. Material and Methods Sixty patients with impacted mandibular third molars fulfilled the inclusion criteria and were randomly divided into three groups of 20 each. All patients underwent third molar extraction and sutures were placed using different techniques – Simple Interrupted (Group A), Continuous (Group B) and Figure of eight (Group C). Patients were evaluated pre-operatively as well post-operatively (at different time points) for five parameters - Pain, Swelling, Trismus, Periodontal health of second molar and Wound infection. Kruskal-Wallis (non-parametric ANOVA) with post-hoc and effect size was used for statistical analysis with P < 0.05 as statistically significant. Results Statistically significant differences were obtained in terms of pain and trismus between Groups A and C post-operatively (P < 0.05). There were also significant differences in pain between Groups B and C post-surgery (P < 0.05). No significant differences were found between groups for swelling, periodontal health of second molar and wound infection. Conclusions Figure of eight suturing presents with better patient outlook and is associated with lower pain, swelling and trismus. Continuous and simple interrupted suturing can be preferred as second-in-line techniques. Final choice of technique shall be made based on wound anatomy, patient history and surgeon’s expertise.

Background MCF-7 breast cancer cell line is undoubtedly amongst the most extensively studied patient-derived research models, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, have previously identified suitable reference genes for qPCR normalization in MCF-7 cell line. However, over the course of identification of suitable reference genes, a comparative analysis comprising these genes together in a single study has not been reported. Furthermore, the expression dynamics of these reference genes within sub-clones cultured over multiple passages (p) has attracted limited attention from research groups. Therefore, we investigated the expression dynamics of 12 previously suggested reference genes within two sub-clones (culture A1 and A2) cultured identically over multiple passages. Additionally, the effect of nutrient stress on reference gene expression was examined to postulate an evidence-based recommendation of the least variable reference genes that could be employed in future gene expression studies. Results The analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as the least variable reference genes while for culture A2, GAPDH-RNA28S were identified. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization of AURKA and KRT19 with triplet pair GAPDH-CCSER2-PCBP1 yielded successful results. The triplet also proved its capability to handle variations arising from nutrient stress. Conclusions The variance in expression behavior amongst sub-clones highlights the potential need for exercising caution while selecting reference genes for MCF-7. GAPDH-CCSER2-PCBP1 triplet offers a reliable alternative to otherwise traditionally used internal controls for optimizing intra- and inter-assay gene expression differences. Furthermore, we suggest avoiding the use of ACTB, GAPDH and PGK1 as single internal controls.

Background MCF-7 cell line remains the most extensively studied patient derived model in breast cancer research, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, in the past have identified suitable reference genes in various breast cancer cell lines including MCF-7. However, over the course of identification of stable internal controls, a comparative analysis comprising these genes together in a single study have not been previously undertaken. Further, very little is known about how these identified reference genes are expressed in MCF-7 sub-clones and when the cell line is cultured over multiple passages (p), given the heterogenic expression behavior often associated with MCF-7 cell line. We investigated the expression dynamics of 12 previously reported and suggested endogenous reference genes using RT-qPCR, available algorithms (NormFinder, geNorm, BestKeeper etc.) and TCGA transcriptomic analysis within identically cultured two sub-clones (culture A1 and A2) of MCF-7 cell line cultured over multiple passages. Further candidate reference genes were used to normalize 4 genes of interest (2 simulated and 2 backed by qPCR data) to make an evidence-based recommendation of the least variable reference genes that could be used in MCF-7 cell line. Results The analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as least variable reference gene pair while for culture A2, GAPDH-RNA28S was recommended. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization with 3 genes and analyzing the combined dataset from culture A1 and A2 (supported by transcriptomic analysis), GAPDH-PCBP1-CCSER2 triplet was found to be least variable and hence potentially more well placed to handle any expression heterogeneity that may arise within sub-clones over multiple passages. Conclusions The variance in expression behavior amongst sub-clones shows the need for exercising caution while selecting and using reference genes for MCF-7. Further, using same reference genes amongst sub-clones can lead to misleading results arising from inaccurate normalization. GAPDH-PCBP1-CCSER2 triplet offers a reliable alternative to otherwise traditionally used internal controls in optimizing intra- and inter-assay gene expression differences.

Nosocomial fungal infections are an emerging global public health threat that requires urgent attention and proper management. With the limited availability of treatment options, it has become necessary to understand the emerging epidemiological trends, mechanisms, and risk factors. However, very limited surveillance reports are available in the Latvian and broader European context. We therefore conducted a retrospective analysis of laboratory data (2017–2020) from Pauls Stradinš Clinical University Hospital (PSCUH), Riga, Latvia, which is one of the largest public multispecialty hospitals in Latvia. A total of 2278 fungal isolates were analyzed during the study period, with Candida spp. comprising 95% of the isolates, followed by Aspergillus spp. and Geotrichum spp. Amongst the Candida spp., C. albicans and C. glabrata made up about 75% of the isolates. The Department of Lung Diseases and Thoracic Surgery had the highest caseload followed by Intensive Care Department. Majority of the fungal isolates were collected from the bronchoalveolar lavage (37%), followed by urine (19%) and sputum (18%) samples. A total of 34 cases of candidemia were noted during the study period with C. albicans being the most common candidemia pathogen. Proper surveillance of emerging epidemiological trends serve as the most reliable and powerful cornerstone towards tackling this emerging threat.

Cleft lip and palate are amongst the most common congenital malformations worldwide presenting with variable manifestations. Previous research has been primarily focused on the genetical aspects of its complex and multifactorial etiology. In the present study, we investigated the role of cytokines as mediators of epithelial–mesenchymal crosstalk and local site inflammation in cleft affected infants. Lip material was obtained from 12 children aged before primary dentition who suffered from orofacial clefting. The quantification of 12 cytokines (Interleukin-2,4,5,6,10,12,13,17A, Tumor Necrosis Factor-α, Interferon-γ, Transforming Growth Factor beta-1 and Granulocyte-Colony Stimulating Factor) was done using ELISA. Nonparametric Spearman Rho was used to ascertain the correlation between the expression levels of different cytokines. A significantly strong positive correlation was found between IL-2 and IFN-γ coupled with an IL4/IFN-γ ratio favoring IFN-γ. These findings indicate a shift towards the preferential activation of the Th1 differentiation pathway. Further, a pathological reduction in TGFβ-1 levels was noted, which may contribute to mucosal damage. IL-6 was more highly correlated to IFN-γ and IL-12 indicating its potential proinflammatory role in cleft affected tissues. This preferential activation of Th1 cell differentiation and consistent expression of IL-2,6,13 and TNF-α in cleft patients may indicate certain underlying mechanisms for inflammation mediation at the site of clefting.