Molecular and Functional Characterization of Human Sex-Determining Region on the Y Chromosome Variants Using Protamine 1 Promoter.

The male sex-determining gene, sex-determining region on the Y chromosome (SRY), is expressed in adult testicular germ cells; however, its role in regulating spermatogenesis remains unclear. The role of SRY in the postmeiotic gene expression was investigated by determining the effect of SRY on the promoter of the haploid-specific Protamine 1 (PRM1) gene, which harbors five distinct SRY-binding motifs. In a luciferase reporter assay system, SRY upregulates PRM1 promoter activity in vitro in a dose-dependent manner. Through a gel-shift assay involving a 31-bp DNA fragment encompassing the SRY element within the PRM1 promoter, the third SRY-binding site on the sense strand (-373/-367) was identified as crucial for PRM1 promoter activation. This assay was extended to analyze 9 SRY variants found in the testicular DNA of 44 azoospermia patients. The findings suggest that SRY regulates PRM1 promoter activity by directly binding to its specific motif within the PRM1 promoter.

ChAdOx1 nCoV-19 (AZD1222) vaccine candidate significantly reduces SARS-CoV-2 shedding in ferrets.

Vaccines against SARS-CoV-2 are likely to be critical in the management of the ongoing pandemic. A number of candidates are in Phase III human clinical trials, including ChAdOx1 nCoV-19 (AZD1222), a replication-deficient chimpanzee adenovirus-vectored vaccine candidate. In preclinical trials, the efficacy of ChAdOx1 nCoV-19 against SARS-CoV-2 challenge was evaluated in a ferret model of infection. Groups of ferrets received either prime-only or prime-boost administration of ChAdOx1 nCoV-19 via the intramuscular or intranasal route. All ChAdOx1 nCoV-19 administration combinations resulted in significant reductions in viral loads in nasal-wash and oral swab samples. No vaccine-associated adverse events were observed associated with the ChAdOx1 nCoV-19 candidate, with the data from this study suggesting it could be an effective and safe vaccine against COVID-19. Our study also indicates the potential for intranasal administration as a way to further improve the efficacy of this leading vaccine candidate.

Experimental and in silico evidence suggests vaccines are unlikely to be affected by D614G mutation in SARS-CoV-2 spike protein.

The 'D614G' mutation (Aspartate-to-Glycine change at position 614) of the SARS-CoV-2 spike protein has been speculated to adversely affect the efficacy of most vaccines and countermeasures that target this glycoprotein, necessitating frequent vaccine matching. Virus neutralisation assays were performed using sera from ferrets which received two doses of the INO-4800 COVID-19 vaccine, and Australian virus isolates (VIC01, SA01 and VIC31) which either possess or lack this mutation but are otherwise comparable. Through this approach, supported by biomolecular modelling of this mutation and the commonly-associated P314L mutation in the RNA-dependent RNA polymerase, we have shown that there is no experimental evidence to support this speculation. We additionally demonstrate that the putative elastase cleavage site introduced by the D614G mutation is unlikely to be accessible to proteases.

DNA Flow cytometric analysis of the human testicular tissues to investigate the status of spermatogenesis in azoospermic patients.

A single, rapid and reproducible diagnostic test to predict the type of azoospermia and outcome of sperm retrieval is not yet available. So the feasibility of employing DNA flow cytometry for rapid investigation of the status of spermatogenesis in the patients with azoospermia was investigated. Testicular biopsies of 44 patients with azoospermia undergoing sperm-retrieval surgery and 4 controls were analyzed by flow cytometry to ascertain their testicular germ-cell patterns. The observed germ-cell pattern was further confirmed by RT-PCR analysis of the cell-specific markers and histology for some patients. The patients with Obstructive Azoospermia (OA) exhibited normal spermatogenesis similar to the control fertile patients showing the presence of diploid, double-diploid and haploid cells. The non-obstructive azoospermia (NOA) patients exhibited disrupted spermatogenesis with arrest at the pre-meiotic (only diploid cells present) or meiotic (diploid and double-diploid cells present) stages. The germ-cell pattern, as ascertained by flow cytometry, provided a clear picture of the intra-testicular spermatogenesis and the presence of spermatozoa in the patients' testes, which was prognostic of their sperm-retrieval. DNA flow cytometry test to ascertain the testicular germ-cell pattern is simple in execution, analysis and interpretation, requires small amount of tissue and provides quantitative data about the status of spermatogenesis in patients. This test would allow comparable analysis of the status of spermatogenesis in patients across clinics and may form the basis for deciding future treatment and intervention strategies.

Relationship between rainfall and Aedes larval population at two insular sites in Pulau Ketam, Selangor, Malaysia.

Two insular settlements (Kampung Pulau Ketam and Kampung Sungai Lima) were selected to study the population dynamics of Aedes aegypti and Aedes albopictus mosquitoes, vectors of dengue and chikungunya infections. Ovitrap surveillance was conducted between October 2007 and October 2008. There was an inverse negative association between ovitrap index and rainfall at the time of collection, probably because rainfall increased the number of available oviposition sites. Rainfall and ovitrap index were positively associates the 25th day after rainfall occurred. A minor, second peak was observed from the 38th to the 42nd day. The first peak was consistent with the minimum 18-day period between the hatching of eggs to the first oviposition. The second minor peak could be due to the second gonotrophic cycle of the female mosquitoes. Rainfall is an important environmental factor associated with Aedes breeding at the study sites.

A case of penile fracture at the crura of the penis without urethral involvement: Rare entity.

Penile fracture is a rare injury, most commonly sustained during sexual intercourse. We report the case of a 29-year-old man who presented with bilateral rupture of the crura of the cavernosa without urethral injury. This is the first case in the literature to present with this unique finding. Urgent surgical exploration was performed and the injuries repaired primarily. At follow-up, the patient reported satisfactory erectile function. This case highlights the importance of early diagnosis with unusual presentation and early surgical repair for better outcome.

Penile lengthening procedure with V-Y advancement flap and an interposing silicone sheath: A novel methodology.

Surgery to augment penile length has become increasingly common. Lack of standardization of this controversial procedure has led to a wide variety of poorly documented surgical techniques, with unconvincing results. The most commonly used technique involves release of the suspensory ligament, with an advancement of an infrapubic skin flap onto the penis via a V-Y plasty. This technique has a major drawback of the possibility of reattachment of the penis to the pubis. We describe a new technique of interposing a silicone sheath along with V-Y advancement flap that overrides this drawback and minimizes the loss of the gained length.

Semen analysis and sperm function tests: How much to test?

Semen analysis as an integral part of infertility investigations is taken as a surrogate measure for male fecundity in clinical andrology, male fertility, and pregnancy risk assessments. Clearly, laboratory seminology is still very much in its infancy. In as much as the creation of a conventional semen profile will always represent the foundations of male fertility evaluation, the 5th edition of the World Health Organization (WHO) manual is a definitive statement on how such assessments should be carried out and how the quality should be controlled. A major advance in this new edition of the WHO manual, resolving the most salient critique of previous editions, is the development of the first well-defined reference ranges for semen analysis based on the analysis of over 1900 recent fathers. The methodology used in the assessment of the usual variables in semen analysis is described, as are many of the less common, but very valuable, sperm function tests. Sperm function testing is used to determine if the sperm have the biologic capacity to perform the tasks necessary to reach and fertilize ova and ultimately result in live births. A variety of tests are available to evaluate different aspects of these functions. To accurately use these functional assays, the clinician must understand what the tests measure, what the indications are for the assays, and how to interpret the results to direct further testing or patient management.

Ovitrap surveillance of the dengue vectors, Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus Skuse in selected areas in Bentong, Pahang, Malaysia.

Ovitrap surveillance was conducted in methodically selected areas in Bentong, Pahang, Malaysia from June 2008 till December 2009 in order to identify insular sites with stable Aedes aegypti population. Eleven sites were surveyed in Bentong district, Pahang, and one of these locations (N3º33' E101º54') was found to have an ovitrap index of Ae. aegypti and Aedes albopictus ranging from 8%-47% and 37%-78% respectively, indicating that this site could be a high-risk area for dengue outbreak. Ae. aegypti larvae were found in both indoor and outdoor ovitraps (p>0.05) while significant difference between the populations of Ae. albopictus larvae from indoors and outdoors was observed (p<0.01). Data collected in this study could provide important entomological information for designing an effective integrated vector control programme to combat Aedes mosquitoes in this area.

Dengue vector surveillance in insular settlements of Pulau Ketam, Selangor, Malaysia.

A year-long ovitrap surveillance was conducted between November 2007 and October 2008 in two insular settlements (Kampung Pulau Ketam and Kampung Sungai Lima) within the Malaysian island of Pulau Ketam. Eighty standard ovitraps were placed indoors and outdoors of randomly selected houses/locations. Results demonstrated an endemic baseline Aedes population throughout the year without weekly large fluctuations. Kampung Pulau Ketam has high Aedes aegypti and Aedes albopictus population, but only Ae. aegypti was found in Kampung Sungai Lima. Aedes aegypti showed no preference for ovitraps placed indoor versus outdoor. However, as expected, significantly more outdoor ovitraps were positive for Ae. albopictus (p<0.05). Trends in Ae. albopictus and Ae. aegypti populations mirrored each other suggesting that common factors influenced these two populations.

Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples.

This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/µl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.

Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples

This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.

Susceptibility status of transgenic Aedes aegypti (L.) against insecticides

Two strains of Aedes aegypti, a RIDL® strain (MyRIDL513A) and a laboratory strain (MyWT), were used in the insecticide-susceptibility study. Two-to-five-day-old female mosquitoes from both strains were tested for their susceptibility against seven insecticides from the class of organophosphate, carbamate, pyrethroids and organochlorine. The adult bioassay was performed according to WHO standard procedures. The 50% lethal time (LT50) value was determined for each strain against the seven insecticides. Both the MyRIDL513A and MyWT strains were resistant to DDT, exhibiting mortality of 48% and 33% respectively, but were susceptible to malathion (5%), permethrin (0.75%), cyfluthrin (0.15%) and lambdacyhalothrin (0.05%). This study shows there is no evidence of altered susceptibility to insecticides in the RIDL strain compared to a WT strain of Ae. aegypti.

Clinical safety profile of sildenafil in Singaporean men with erectile dysfunction: pre-marketing experience (ASSESS-I evaluation).

Safety and tolerability of sildenafil citrate was assessed in a population subset of 60 Singaporean men with erectile dysfunction taken from the Asian Sildenafil Efficacy and Safety Study (ASSESS-I), a double-blind, placebo-controlled, flexible-dose study. The men, from two centres, with > or = 6 months' history of erectile dysfunction, were randomized to two treatment arms for 12 weeks. One group (30 patients) received sildenafil (initial dose 50 mg taken 1 h before sexual activity for the first 2 weeks, increased to 100 mg or decreased to 25 mg, according to efficacy and/or tolerability). The remaining 30 patients received a matching placebo. Incidence and type of adverse effects were evaluated at 2, 4, 8 and 12 weeks. Nine patients (30.0%) on sildenafil (33.1% in the full ASSESS-I study) and one patient (3.3%) on placebo (22.8% in the full ASSESS-I study) experienced treatment-related adverse events, the most frequent being headache in the sildenafil group (reported by five patients [16.7%]; 11.0% in the full ASSESS-I study). Flushing, visual disturbance, dizziness, insomnia, myalgia and back pain each occurred in one patient in the sildenafil group (3.3%); in the placebo group, one patient (3.3%) had headache. Importantly, the incidence of cardiovascular and respiratory system adverse events were relatively less than in the full ASSESS-I population (cardiovascular 3.3% in the present study versus 10.2% in the full ASSESS-I population; respiratory 3.3% versus 5.5%). All adverse events were transient and mild, and did not lead to treatment withdrawal. There was no effect on sitting blood pressure, heart rate or standard laboratory parameters; more importantly, there was no incidence of myocardial infarction, stroke or priapism. These results should reassure Singaporean patients and their physicians of the safety of sildenafil for erectile dysfunction.

A preliminary study on the possible role for luteinizing hormone in androgen independent growth of prostate.

The recent observation that LH receptors are found in the prostate and that LH can execute a mitogenic role in addition to its well documented role in stimulation of steroidogenesis prompted the evaluation of the role of LH in regulation of prostate growth. The results obtained in the present study reveal the presence of LH receptor transcript as well as receptor protein in the rat prostate. The LH receptor appears to be localized in the non-secretory epithelial cells of the prostate as assessed by I(125) LH binding and immunofluorescence. Addition of HCG to purified prostate cells resulted in an increase in bromodeoxyuridine incorporation only in the non-secretory epithelial cells. Using a castrated adult rat model system, it has also been observed that LH receptor is regulated by LH itself, and that LH also regulates protein synthesis in the prostate. In addition, it has been possible to demonstrate the presence of LH receptor in the prostate from cases of human benign prostate hyperplasia. These results suggest a possible role for LH in the regulation of prostate growth.