PCR and Culture Analysis of Streptococcus pneumoniae Nasopharyngeal Carriage in Healthy Children

Invasive Streptococcus pneumoniae disease is preceded by asymptomatic nasopharyngeal carriage. Measuring carriage in healthy populations provides data on what serotypes are present in communities, which is of interest in the era of polyvalent pneumococcal conjugate vaccines. Nasopharyngeal swabs from a survey of 682 and 800 healthy children in 2016 and 2018, respectively, were analyzed by culture and Quellung reaction to determine rates of carriage and serotypes. All swabs from 2016 and 300 randomly selected swabs from 2018 were then analyzed using real-time semi-quantitative PCR (qPCR) to detect S. pneumoniae gene targets lytA, piaA, and SP2020 and determine serotype. There were 71 (10.4%) and 68 (8.5%) culture positive samples in 2016 and 2018, respectively. All of these were also positive by qPCR except one that was equivocal. In total, 46.0% of 2016 swabs were positive by qPCR. In 2018, results from the selected sample extrapolated to the complete sample showed 49.0% positive by qPCR. PCV13 serotypes were detected in 29.3% and 21.7% of S. pneumoniae qPCR positive samples from 2016 and 2018, respectively; compared with only 8.4% and 6.0% PCV13 serotypes detected by Quellung reaction in culture positive samples. Compared with culture, qPCR detected S. pneumoniae more frequently. Further, qPCR serotyping detected PCV13 serotypes in a larger proportion of samples than culture and Quellung reaction did, showing that, despite established universal childhood PCV13 immunization, vaccine serotypes can still be detected in a large proportion of young children.

Prevalence of target anaerobes associated with chronic periodontitis

Introduction. Periodontal diseases are a group of chronic infections that destroy tissues surrounding and supporting the teeth. Data on the anaerobes associated with periodontal infections in Kuwait is lacking. Aim. To investigate the target anaerobes associated with chronic periodontitis (CP) in patients admitted to Dental Clinics in Kuwait University Health Sciences Center, Kuwait. Methodology. Patients with CP (severe and moderate) were recruited into this study during a period of 15 months. Samples were collected directly from inside the gingival pockets and subjected to semi-quantitative PCR assays. Results. A total of 30 patients, stratified into moderate and severe CP and 31 healthy individuals, used as controls, were studied. Nine (30 %) of the 30 patients were in the 50–59-year age group. The detection rate of Aggregatibacter actinomycetemcomitans between the patients (9 : 30 %) versus the controls (5 : 16.1 %) was non-significant (P >0.05). Fusobacterium spp., were detected in all patients versus 29 (93.1 %) controls, (P >0.05). However, four target anaerobes were significantly associated with CP patients; Porphyromonas gingivalis was detected in ten (33.3 %) patients versus two (6.4 %) controls (P <0.0001); Tannerella forsythia 25 (83.3 %) versus 16 (51.6 %) controls (P <0.0001); Parvimonas micra 27 (90 %) versus 16 (51.6 %) controls (P <0.0001) and Treponema denticola, 18 (60 %) versus nine (29 %) controls (P <0.0001), respectively. Prevotella spp. were detected in 27 (90 %) patients and 30 (96.7 %) controls (P>0.5). There was no significant difference in the burden of Prevotella spp. between patients and controls determined by semi-quantitative PCR assays. Conclusion. Some (4/7) of the target anaerobes were significantly associated with CP in our study. P. gingivalis was the most strongly associated anaerobe with CP, although not the keystone bacteria, while Prevotella spp. was similar to the healthy controls.

Morphometric Evaluation of Spermatogenic Cells and Seminiferous Tubules and Exploration of Luteinizing Hormone Beta Polypeptide in Testis of Datong Yak

Histological examination of testes is essential for understanding infertility, sex development, and growth. Therefore, to understand the histomorphology of testes at different developmental stages, we performed hematoxylin and eosin staining of Yak testis. Our results revealed that the diameters of spermatogenic cells and their nuclei were significantly larger (p < 0.05) in the testis at six years compared to at six and 18 months. No significant difference was noted between 30 months and six years. The study was designed to compare the expression profile of LHB in Datong yak. The expression pattern of LHB was explored using quantitative PCR, semi-quantitative PCR, molecular bioinformatic, and Western blot analysis. Our observations indicated that expression of LHB was significantly higher (p < 0.05) in the testis of Datong yak. Western blotting indicated that the molecular mass of LHB protein was 16 kDa in yak. The protein encoded by yak LHB included conserved cysteine-knot domain regions. The high expression of LHB in testis indicated that LHB may be vital for the development of male gonads and the fertility of Datong yak.

Molecular Cloning and Characterization of SYCP3 and TSEG2 Genes in the Testicles of Sexually Mature and Immature Yak

Testis-specific genes play an essential part in the centromere union during meiosis in male germ cells, spermatogenesis, and in fertility. Previously, there was no research report available on the expression pattern of SYCP3 and TSEG2 genes in different ages of yaks. Therefore, the current research compared the expression profiling of SYCP3 and TSEG2 genes in testes of yaks. The expression pattern of SYCP3 and TSEG2 mRNA was investigated using qPCR, semi-quantitative PCR, western blot, immunohistochemistry, and molecular bioinformatics. Our findings displayed that SYCP3 and TSEG2 genes were prominently expressed in the testicles of yaks as compared to other organs. On the other hand, the protein encoded by yak SYCP3 contains Cor1/Xlr/Xmr conserved regions, while the protein encoded by yak TSEG2 contains synaptonemal complex central element protein 3. Additionally, multiple alignments sequences indicated that proteins encoded by Datong yak SYCP3 and TSEG2 were highly conserved among mammals. Moreover, western blot analysis specified that the molecular mass of SYCP3 protein was 34-kDa and TSEG2 protein 90-kDa in the yak. Furthermore, the results of immunohistochemistry also revealed the prominent expression of these proteins in the testis of mature yaks, which indicated that SYCP3 and TSEG2 might be essential for spermatogenesis, induction of central element assembly, and homologous recombination.

Expression Analysis of IZUMO1 Gene during Testicular Development of Datong Yak (Bos Grunniens)

The IZUMO1 gene has promising benefits for the national development of novel non-hormonal contraceptives and in the treatments of fertility. Understanding the function of IZUMO1, its mRNA, and protein expression is critical to gain insight into spermatogenesis and promote sperm-egg fusion during reproduction of Datong yak. Therefore, we estimated the IZUMO1 gene expression in different ages of Datong yak by using semi quantitative PCR, qPCR, and western blotting. The results of the qPCR, semi-quantitative PCR and western blotting revealed that the expression level of IZUMO1 mRNA was significantly (p < 0.05) higher in the testis of 30 months and 6 years old followed by 18 and 6 months old Datong yak, respectively. We also predicted secondary and tertiary protein structure of IZUMO1 by using bioinformatics software that the revealed presence of a signal peptide, Izumo domain, immunoglobulin (Ig) like domain, and transmembrane region. Moreover, immunostaining analysis also elucidated that IZUMO1 was more prominent in the testis of 30 months and 6 years old yak, which represented that the IZUMO1 gene expression might be higher during the peak breeding ages (6 to 7 years) of the yak, and play a potential role in spermatogenesis, fertility, and testicular development.

New Insight on Water Status in Germinating Brassica napus Seeds in Relation to Priming-Improved Germination

Seed priming is a pre-sowing method successfully used to improve seed germination. Since water plays a crucial role in germination, the aim of this study was to investigate the relationship between better germination performances of osmoprimed Brassica napus seeds and seed water status during germination. To achieve this goal, a combination of different kinds of approaches was used, including nuclear magnetic resonance (NMR) spectroscopy, TEM, and SEM as well as semi-quantitative PCR (semi-qPCR). The results of this study showed that osmopriming enhanced the kinetics of water uptake and the total amount of absorbed water during both the early imbibition stage and in the later phases of seed germination. The spin–spin relaxation time (T2) measurement suggests that osmopriming causes faster water penetration into the seed and more efficient tissue hydration. Moreover, factors potentially affecting water relations in germinating primed seeds were also identified. It was shown that osmopriming (i) changes the microstructural features of the seed coat, e.g., leads to the formation of microcracks, (ii) alters the internal structure of the seed by the induction of additional void spaces in the seed, (iii) increases cotyledons cells vacuolization, and (iv) modifies the expression pattern of aquaporin genes.

Modulation of TLR2 and TLR4 in macrophages following Trichinella spiralis infection

Summary Parasitic helminthes can suppress and/or regulate the host immune response to allow long-term survival and chronic infection where toll-like receptors (TLRs) expressed on macrophages play essential roles in response to parasitic infection. Semi-quantitative PCR and flow cytometry studies about the modulation of TLRs and cytokine profiles in macrophages following T. spiralis infection were performed. TLRs, MyD88 and NF-κB were up-regulated by T. spiralis infection and essential to the parasite life cycles. Cytokines profiles (IL-6, IL-10, IL-12, TNF-α) were modulated during T. spiralis infection. Results suggest that T. spiralis infection may regulate the expression of TLR4 on macrophages and TLR4/MyD88/NF-κB signaling pathways. This study provides further insights into the mechanisms of TLR-mediated post-inflammatory response during T. spiralis infection.