DNA extraction and polymerase chain reaction (PCR) are the basic techniques employed in the molecular laboratory. This short overview covers various physical and chemical methods used for DNA ...extraction so as to obtain a good-quality DNA in sufficient quantity. DNA can be amplified with the help of PCR. The basic principle and different variants of PCR are discussed.
BACKGROUND Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is superior to conventional transbronchial needle aspiration (cTBNA) in the staging of lung cancer. However, ...its efficiency in diagnosis of sarcoidosis when combined with endobronchial biopsy (EBB) and transbronchial lung biopsy (TBLB) has not been studied. This randomized controlled trial compares diagnostic yield of EBUS-TBNA vs cTBNA in combination with EBB and TBLB. METHODS Patients with clinical diagnosis of sarcoidosis were randomized 1:1 to EBUS-TBNA or cTBNA. All patients underwent TBLB and EBB. The primary outcome was detection of granulomas. The secondary end points were the individual and cumulative yields of various procedures, serious adverse events, and procedure time. RESULTS Of the 130 patients, sarcoidosis was diagnosed in 117 (62 cTBNA, 55 EBUS-TBNA). The two groups were similar at baseline. Granulomas were demonstrated in 104 (53 cTBNA, 51 EBUS-TBNA) patients and were similar in two groups (85.5% vs 92.7%, P = .34). Individually, EBUS-TBNA had the highest yield (41 of 55, 74.5%), which was better than cTBNA (30 of 62, 48.4%, P = .004) or EBB (40 of 111, 36.3%, P < .0001) but not TBLB (78 of 112, 69.6%, P = .54). Adding EBB/TBLB to cTBNA led to an increase in granuloma detection, whereas the addition of TBLB (but not EBB) significantly enhanced the yield of EBUS-TBNA. The procedure time was significantly longer with EBUS-TBNA. No major adverse events occurred. CONCLUSIONS Individually, EBUS-TBNA has the highest diagnostic yield in sarcoidosis, but it should be combined with TBLB for the optimal yield. The diagnostic yield of cTBNA (plus EBB and TBLB) is similar to EBUS-TBNA plus TBLB. TRIAL REGISTRY ClinicalTrials.gov ; No.: NCT01908868; URL: www.clinicaltrials.gov
Objective The differential diagnosis of sarcoidosis and tuberculosis is difficult, especially in countries with a high tuberculosis burden. We hypothesized that sonographic features on endobronchial ...ultrasonography (EBUS) would help in differentiating tuberculosis from sarcoidosis. In this study, the endosonographic features of tuberculosis and sarcoidosis are compared. Methods This was a retrospective analysis of prospectively collected data of patients with intrathoracic lymphadenopathy who underwent EBUS-guided transbronchial needle aspiration (TBNA), and were finally diagnosed with sarcoidosis or tuberculosis. Sonographic features such as size, shape (round or oval), margin (distinct or indistinct), echogenicity (heterogeneous or homogeneous), presence or absence of a central hilar structure, and coagulation necrosis sign were recorded and compared in the 2 groups. Results During the study period, 249 EBUS-guided TBNA procedures were performed and a diagnosis of sarcoidosis (n = 118) or tuberculosis (n = 47) was made in 165 patients. A total of 358 lymph node stations were examined. Heterogeneous echotexture (53.4% vs 12.6%, P < .001) and coagulation necrosis (26.1% vs 3.3%; P < .001) were significantly higher in tuberculous lymph nodes. A combination of a positive tuberculin skin test (TST) and either heterogeneous echotexture or coagulation necrosis sign had specificity of 98% and positive predictive value of 91% for a diagnosis of tuberculosis. Conclusions Sonographic features of heterogeneous echotexture or coagulation necrosis in the lymph nodes on EBUS are fairly specific for tuberculosis. Along with a positive TST, these features strongly favor a diagnosis of tuberculosis over sarcoidosis.
Background
To distinguish follicular adenoma (FA) and follicular carcinoma (FC) of thyroid in fine needle aspiration cytology (FNAC) is a challenging problem.
Aims and objectives
In this article, we ...attempted to build an artificial neural network (ANN) model from the cytological and morphometric features of the FNAC smears of thyroid to distinguish FA from FC.
Material and methods
The cytological features and morphometric analysis were done on the FNAC smears of histology proven cases of FA (26) and FC (31). The cytological features were analysed semi‐quantitatively by two independent observers (RS and PD). These data were used to make an ANN model to differentiate FA versus FC on FNAC material. The performance of this ANN model was assessed by analysing the confusion matrix and receiving operator curve.
Result
There were 39 cases in training set, 9 cases each in validation and test sets. In the test group, ANN model successfully distinguished all cases (9/9) of FA and FC. The area under receiver operating curve was 1.
Conclusion
The present ANN model is efficient to diagnose follicular adenoma and carcinoma cases on cytology smears without any error. In future, this ANN model will be able to diagnose follicular adenoma and carcinoma cases on thyroid aspirate. This study has immense potential in future. This is an open ended ANN model and more parameters and more cases can be included to make the model much stronger.
Introduction
Fallopian tube prolapse (FTP) is a rare complication of hysterectomy. The cytological features of the prolapsed fallopian tube in vault smears have been occasionally described in the ...literature.
Materials and methods
This was a retrospective study conducted to identify and describe the characteristic cytologic features of histopathologically confirmed cases of FTP in SurePath™ liquid‐based preparations. Additionally, the literature documenting cytologic features of the prolapsed fallopian tube in vault smears was also reviewed.
Results
A total of four corresponding vault cytology samples of FTP cases, reported on histopathology, were available. On cytologic examination, these cases demonstrated strips and papillaroid clusters of columnar‐shaped cells with mild nuclear enlargement, round to elongated nuclei, fine chromatin, inconspicuous nucleoli, and a moderate amount of wispy cytoplasm. Admixed inflammatory cells were also noted. Some of these cells demonstrated the presence of cilia and terminal bar toward the apical surface, indicative of tubal epithelium. The presence of three‐dimensional papillaroid clusters lined by columnar cells as well as strips of similar cells with cilia and terminal bars at the apical surface and fine nuclear chromatin were the most consistent cytologic features in these cases.
Conclusion
We conclude that a high index of clinical suspicion in post‐hysterectomy cases with knowledge of the characteristic cytologic features can help in suggesting a diagnosis of tubal prolapse in vault samples.
India has the highest burden of Tuberculosis (TB) and multidrug-resistant TB (MDR-TB) worldwide. Innovative technology is the need of the hour to identify these cases that remain either undiagnosed ...or inadequately diagnosed due to the unavailability of appropriate tools at primary healthcare settings. We developed and evaluated 3 kits, namely 'TB Detect' (containing BioFM-Filter device), 'TB Concentration and Transport' (containing Trans-Filter device) and 'TB DNA Extraction' kits. These kits enable bio-safe equipment-free concentration of sputum on filters and improved fluorescence microscopy at primary healthcare centres, ambient temperature transport of dried inactivated sputum filters to central laboratories and molecular detection of drug resistance by PCR and DNA sequencing (Mol-DST). In a 2-site evaluation (n = 1190 sputum specimens) on presumptive TB patients, BioFM-Filter smear exhibited a significant increase in positivity of 7% and 4% over ZN smear and LED-FM smear (p<0.05), respectively and an increment in smear grade status (1+ or 2+ to 3+) of 16% over ZN smear and 20% over LED-FM smear. The sensitivity of Mol-DST in presumptive MDR-TB and XDR-TB cases (n = 148) was 90% for Rifampicin (95% confidence interval CI, 78-96%), 84% for Isoniazid (95% CI, 72-92%), 83% for Fluoroquinolones (95% CI, 66-93%) and 75% for Aminoglycosides (95% CI, 35-97%), using phenotypic DST as the reference standard. Test specificity was 88-93% and concordance was ~89-92% (κ value 0.8-0.9). The patient-friendly kits described here address several of the existing challenges and are designed to provide 'Universal Access' to rapid TB diagnosis, including drug-resistant disease. Their utility was demonstrated by application to sputum at 2 sites in India. Our findings pave the way for larger studies in different point-of-care settings, including high-density urban areas and remote geographical locations.
Malignant transformation in leiomyoma is rare, with a few documented cases of sarcomatous transformation in leiomyomas. However, carcinosarcomatous transformation in leiomyomas is extremely ...infrequent. A 45-year-old female presented with a mass sensation per abdomen for the last five months. An abdominal ultrasound revealed multiple uterine fibroids. Contrast-enhanced magnetic resonance imaging revealed a large multiloculated abdominopelvic mass arising from the fundus of the uterus, along with multiple smaller subserosal fibroids. A total abdominal hysterectomy with bilateral salpingectomy was performed. Grossly, the uterus was bosselated with multiple subserosal fibroids. The larger degenerated fibroid showed a smooth outer surface; however, the cut surface was predominantly cystic and filled with necrotic material. Microscopically and immunohistochemically, the larger mass showed a high-grade biphasic tumor comprising carcinomatous and sarcomatous components with the peripheral solid areas showing compressed smooth muscle bundles representing the residual leiomyomatous areas. A final diagnosis of carcinosarcomatous transformation in leiomyoma was rendered. The index report highlights the significance of systematic gross and microscopic examination of all the uterine masses in patients with multiple leiomyomata.
What is the functional significance of Tex13b in male germ cell development and differentiation?
Tex13b regulates male germ cell differentiation by metabolic reprogramming during spermatogenesis.
...Studies in mice and humans suggest that TEX13B is a transcription factor and is exclusively expressed in germ cells.
We sequenced the coding regions of TEX13B in 628 infertile men and 427 ethnically matched fertile control men. Further, to identify the molecular function of Tex13b, we created a Tex13b knockout and conditional overexpression system in GC-1spg (hereafter, GC-1) cells.
Our recent exome sequencing study identified novel candidate genes for male infertility. TEX13B was found to be one of the potential candidates, hence we explored the role of TEX13B in male infertility within a large infertile case-control cohort. We performed functional analyses of Tex13b in a GC-1 cell line using CRISPR-Cas9. We differentially labelled the cell proteins by stable isotope labelling of amino acids in cell culture (SILAC) and performed mass spectrometry-based whole-cell proteomics to identify the differential protein regulation in knockout cells compared to wild-type cells. We found that Tex13b knockout leads to downregulation of the OXPHOS complexes and upregulation of glycolysis genes, which was further validated by western blotting. These results were further confirmed by respirometry analysis in Tex13b knockout cells. Further, we also performed a conditional overexpression of TEX13B in GC-1 cells and studied the expression of OXPHOS complex proteins by western blotting.
We identified a rare variant, rs775429506 (p.Gly237Glu), exclusively in two non-obstructive-azoospermia (NOA) men, that may genetically predispose these men for infertility. Further, we demonstrated that Tex13b functions in the transcription regulation of OXPHOS complexes.
N/A.
We examined the function of Tex13b in GC-1 in vitro by knocking out and conditional overexpression, for understanding the function of Tex13b in germ cells. Unfortunately, this could not be replicated in either an animal model or in patient-derived tissue due to the non-availability of an animal model or patient's testis biopsies.
This study identified that Tex13b plays an important role in male germ cell development and differentiation. The findings of this study would be useful for screening infertile males with spermatogenic failure and counselling them before the implementation of assisted reproduction technique(s).
Funding was provided by the Council of Scientific and Industrial Research (CSIR) under the network project (BSC0101 and MLP0113) and SERB, the Department of Science and Technology, Government of India (J C Bose Fellowship: JCB/2019/000027). The authors do not have any competing interest.
Purpose
To identify the contribution of mutations in the Desert Hedgehog (
DHH
) gene to the disorders of sexual differentiation (DSD) and male infertility.
Methods
The study included a total 430 ...subjects, including 47 gonadal dysgenesis cases, 6 patients with undescended testis and infertility characterized by azoospermia, 125 infertile male patients characterized by oligoasthenozoospermia, 24 patients with oligoasthenoteratozoospermia, and 200 ethnically matched normozoospermic fertile men who had fathered a child in the last two years. Sequencing of the complete coding region of the
DHH
gene was undertaken to find its contribution to the DSD and male infertility.
Results
We observed four novel mutations in the
DHH
gene in the cases with different reproductive anomalies. A synonymous substitution, c. 543C>T (p.His181His) was observed in 6.6% oligoasthenozoospermic infertile males and 1.5% normozoospermic fertile control samples (RR = 4.4077, 95%CI 1.19–16.29). Another synonymous substitution, c.990G>A (p.Ala330Ala) was observed in an infertile patient with unilateral undescended testis (case #12). Insertion of G at c.1156insG (p.Arg385fs) was observed in a case with bilateral undescended testis and azoospermia (case #23). In gonadal dysgenesis category, two mutations, insertion of G at c.1156insG (p.Arg385fs) and c.997A>G (p.Thr333Ala) substitution were observed in one case (case #34). These mutations were completely absent in control samples.
Conclusion
Mutations in the
DHH
gene impact reproduction with mild mutations affecting fertility, and severe or multiple mutations resulting in gonadal dysgenesis.
Human spermatogenesis requires an orchestrated expression of numerous genes in various germ cell subtypes. Therefore, the genetic landscape of male infertility is highly complex. Known genetic ...factors alone account for at least 15% of male infertility. However, ~40% of infertile men remain undiagnosed and are classified as idiopathic infertile men. We performed exome sequencing in 47 idiopathic infertile men (discovery cohort), followed by replication study (40 variants in 33 genes) in 844 infertile men and 709 controls using Sequenom MassARRAY® based genotyping. We report 17 variants in twelve genes that comprise both previously reported (DNAH8, DNAH17, FISP2 and SPEF2) and novel candidate genes (BRDT, CETN1, CATSPERD, GMCL1, SPATA6, TSSK4, TSKS and ZNF318) for male infertility. The latter have a strong biological nexus to human spermatogenesis and their respective mouse knockouts are concordant with human phenotypes. One candidate gene CETN1, identified in this study, was sequenced in another independent cohort of 840 infertile and 689 fertile men. Further, CETN1 variants were functionally characterized using biophysical and cell biology approaches. We demonstrate that CETN1 variant- p.Met72Thr leads to multipolar cells, fragmented nuclei during mitosis leading to cell death and show significantly perturbed ciliary disassembly dynamics. Whereas CETN1-5' UTR variant; rs367716858 leads to loss of a methylation site and increased reporter gene expression in vitro. We report a total of eight novel candidate genes identified by exome sequencing, which may have diagnostic relevance and can contribute to improved diagnostic workup and clinical management of male infertility.
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