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Breast cancer (BC) remains the foremost cause of cancer-related mortality, with an estimated 2.3 million new cases anticipated globally. The timely diagnosis of BC is pivotal for effective treatment. Currently, BC diagnosis predominantly relies on Immunohistochemistry (IHC), a method known for its sluggishness, expense, and dependence on proficient pathologists for confident cancer typing. In this study, we introduce a novel approach to enhance the accuracy, speed, and cost-effectiveness of BC diagnosis. We employ multiplex Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) with touch-down methods, which consistently yield significantly lower Cycle Threshold (CT) values. The study evaluates gene expression profiles of HER2, PGR, ESR, and Ki67 genes across 61 samples representing four BC subtypes, using RPL13A as the endogenous control gene. The results demonstrate that our method offers remarkable precision, nearly equivalent to IHC, in detecting gene expressions vital for BC diagnosis and subtyping. Moreover, we explore the gene expression of Hif1A, ANG, and VEGFR genes involved in angiogenesis, shedding light on the metastatic potential of the tested BC tumours. Notably, numerous samples exhibit elevated levels of Hif1A and VEGFR, indicating their potential as valuable biomarkers for assessing metastatic status. Collectively, our RT-qPCR methodology emerges as a powerful diagnostic tool for swiftly identifying BC subtypes and can be complemented with other essential tumorigenic biomarker assessments, such as angiogenesis, to further refine cancer characterisation and inform personalised therapeutic strategies for BC patients. This innovation holds the promise of revolutionising BC diagnosis and treatment, offering expedited and reliable insights for improved patient care.

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Background: Bladder cancer is the most frequent malignancy that affects the urinary tract. Studies have shown different types of FGFR3 and HRAS genes mutations in human bladder cancer, with a comprehensive range of mutation number in various populations. This study aimed to determine the specific point mutations of these 2 genes among Iranian patients with bladder cancer. Methods: In this study, 100 specimens of patients with transitional cell carcinoma were analyzed. All samples were examined for FGFR3 and HRAS mutations using PCR and direct DNA sequencing methods. Results: A total of 9 pathogenic mutations and 9 polymorphisms were found in 2 exons (7 and 15) of the FGFR3 genes in patients with bladder cancer (S249Y, I633I, L645L, D646E, Y647*, D628V, P250T, Q263H, Y305H). However, no mutation was found in exon 10 of FGFR3 and exon 1 of HRAS genes. Conclusion: In this study, 5 mutations were found in FGFR3 gene that have not been detected previously. There was no mutation in exon 10 of FGFR3 and exon1 of HRAS. The results of this study confirmed the association of ethnic-genetic factors in the occurrence of bladder cancer, so that these variables may not be present in all ethnic groups.

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OBJECTIVE: To link congenital hearing loss with known loci to establish a platform for future research. METHODS: The cross-sectional study was conducted from February 2016 to March 2017 in Bannu, Khyber Pakhtunkhwa, Pakistan, and comprised families with Pashtun ethnicity having at least 2 individuals suffering from congenital hearing loss. Deoxyribonucleic acid from whole blood samples was extracted by salting-out method. Amplification was done through touchdown polymerase chain reaction to see any possible linkage to already reported deafness loci. Linkage analysis was carried out using microsatellite markers for each locus. Genotyping of the samples was done and haplotypes were accordingly generated to either include or exclude the linked / unlinked regions. RESULTS: Of the 4 families, family PKDF 1620 showed linkage with DFNB12/CDH23 (D10S1432, D10S606, and D10S1694) and family PKDF 1625 had linkage with DFNB3/MYO15A (D17S2196, D17S2207 and D17S2206). Families PKDF1623 and PKDF1624 showed no linkage with any of the prevalent reported loci in Pakistan . CONCLUSIONS: Linkage to DFNB12 and MYO 15 showed heterogeneity of congenital deafness.

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A rapid test method was developed for detecting mycoplasma contamination in veterinary biological products. The method reduces testing time by 2 weeks and shows comparable sensitivity to the current agar-based detection model. The primary goals for the development of the test were to reduce the testing time, incorporate a method that was easily adaptable across the veterinary biologics industry and reduce the subjective interpretation of results. We found that biological enrichment is necessary to maintain sensitivity of the detection method when compared to the standard culture-based test and that periodic sampling of enrichment cultures is essential to detect a wide variety of mycoplasma species that may be present as contaminants. The PCR detection method is comparable to the agar-based model and can reduce the overall testing time by up to 14 days.

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Culture-independent approaches to analyze metagenome are practical choices for rapid exploring useful genes. The mg-MSDH gene, acquired from the hot spring metagenomic, was retrieved full lengths of functional gene using semi-nest touch-down PCR. Two pairs of degenerate primers were used to separate seven conserve partial sequences by semi-nest touch-down PCR. One of them showed similarity with aldehyde dehydrogenase was used as a target fragment for isolating full-length sequence. The full-length mg-MSDH sequence contained a 1,473 bp coding sequence encoding a 490-amino-acid polypeptide and assigned an accession number JQ715422 in Genbank. The upstream sequences TAGGAG of the start codon (GTG), suggested that was a ribosome binding site. The coding sequence of mg-MSDH was ligated to pET-303 vector and the reconstructive plasmid was successfully overexpressed in E. coli. The purified recombinant mg-MSDH enzyme showed propionaldehyde oxidative activity of 3.0 U mg(-1) at 37 °C.

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The present study introduces four event detection algorithms for defining touch-down and foot-off during stair descent and stair ascent using segmental kinematics. For stair descent, vertical velocity minima of the whole body center-of-mass was used to define touch-down, and foot-off was defined as the instant of trail limb peak knee flexion. For stair ascent, vertical velocity local minima of the lead-limb toe was used to define touch-down, and foot-off was defined as the local maxima in vertical displacement between the toe and pelvis. The performance of these algorithms was determined as the agreement in timings of kinematically derived events to those defined kinetically (ground reaction forces). Data were recorded while 17 young and 15 older adults completed stair descent and ascent trials over a four-step instrumented staircase. Trials were repeated for three stair riser height conditions (85 mm, 170 mm, and 255 mm). Kinematically derived touch-down and foot-off events showed good agreement (small 95% limits of agreement) with kinetically derived events for both young and older adults, across all riser heights, and for both ascent and descent. In addition, agreement metrics were better than those returned using existing kinematically derived event detection algorithms developed for overground gait. These results indicate that touch-down and foot-off during stair ascent and descent of non-instrumented staircases can be determined with acceptable precision using segmental kinematic data.

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There are several methods for diagnosis of leprosy, including AFB stain, the measurement of PGL-1 (phenolic glycolipid - 1) antigen titer, and DNA-PCR. In this study, we have used the DNA-PCR amplifying the RLEP repetitive sequence. Our result showed that the RLEP primer offered the more sensitive detection and identification of M. leprae DNA in clinical specimens, compared with the other primer, for example, 18-kDa antigen gene. To screen the resistant M. leprae strain of MDT (Multi-Drug Therapy), we have used the TD (Touch-Down) PCR. We arranged and amplified sequences of the genes, folP, rpoB, gyr, 23S rRNA, in M. leprae involved in MDT-resistance, and could obtain the PCR product each gene, simultaneously. This method, based on annealing temperature, was useful to the detection for diagnosis and the screen of MDT-resistant strain of M. leprae, rapidly. Thus, we suggest that the RLEP primer and TD-PCR method are effective in assessing the diagnosis of leprosy and the identification of drug-resistant M. leprae.

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