DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus.

BACKGROUND: The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes. RESULTS: We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/µL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments. CONCLUSIONS: DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

DengueSeq: A pan-serotype whole genome amplicon sequencing protocol for dengue virus.

Background: The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes. Results: We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 101-102 RNA copies/µL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments. Conclusions: DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

Hydrodynamic shear dissipation and transmission in lipid bilayers.

Vital biological processes, such as trafficking, sensing, and motility, are facilitated by cellular lipid membranes, which interact mechanically with surrounding fluids. Such lipid membranes are only a few nanometers thick and composed of a liquid crystalline structure known as the lipid bilayer. Here, we introduce an active, noncontact, two-point microrheology technique combining multiple optical tweezers probes with planar freestanding lipid bilayers accessible on both sides. We use the method to quantify both fluid slip close to the bilayer surface and transmission of fluid flow across the structure, and we use numerical simulations to determine the monolayer viscosity and the intermonolayer friction. We find that these physical properties are highly dependent on the molecular structure of the lipids in the bilayer. We compare ordered-phase with liquid disordered-phase lipid bilayers, and we find the ordered-phase bilayers to be 10 to 100 times more viscous but with 100 times less intermonolayer friction. When a local shear is applied by the optical tweezers, the ultralow intermonolayer friction results in full slip of the two leaflets relative to each other and as a consequence, no shear transmission across the membrane. Our study sheds light on the physical principles governing the transfer of shear forces by and through lipid membranes, which underpin cell behavior and homeostasis.

Erythrocytosis in a Large Cohort of Trans Men Using Testosterone: A Long-Term Follow-Up Study on Prevalence, Determinants, and Exposure Years.

CONTEXT: Erythrocytosis is a known side effect of testosterone therapy that can increase the risk of thromboembolic events. OBJECTIVES: To study the prevalence and determinants in the development of erythrocytosis in trans men using testosterone. METHODS: A 20-year follow-up study in adult trans men who started testosterone therapy and had monitoring of hematocrit at our center (n = 1073). RESULTS: Erythrocytosis occurred in 11% (hematocrit > 0.50 L/L), 3.7% (hematocrit > 0.52 L/L), and 0.5% (hematocrit > 0.54 L/L) of trans men. Tobacco use (odds ratio [OR] 2.2; 95% CI, 1.6-3.3), long-acting undecanoate injections (OR 2.9; 95% CI, 1.7-5.0), age at initiation of hormone therapy (OR 5.9; 95% CI, 2.8-12.3), body mass index (BMI) (OR 3.7; 95% CI, 2.2-6.2), and pulmonary conditions associated with erythrocytosis and polycythemia vera (OR 2.5; 95% CI, 1.4-4.4) were associated with hematocrit > 0.50 L/L. In the first year of testosterone therapy hematocrit increased most: 0.39 L/L at baseline to 0.45 L/L after 1 year. Although there was only a slight continuation of this increase in the following 20 years, the probability of developing erythrocytosis still increased (10% after 1 year, 38% after 10 years). CONCLUSION: Erythrocytosis occurs in trans men using testosterone. The largest increase in hematocrit was seen in the first year, but also after the first years a substantial number of people present with hematocrit > 0.50 L/L. A reasonable first step in the care for trans men with erythrocytosis while on testosterone is to advise them to quit smoking, to switch to a transdermal administration route, and if BMI is high, to lose weight.

Explorative Prospective Evaluation of Short-Term Subjective Effects of Hormonal Treatment in Trans People-Results from the European Network for the Investigation of Gender Incongruence.

INTRODUCTION: Although many studies on the short- and long-term effects of hormonal treatment (HT) in trans people focus on objective changes such as body composition or bone density, few studies have evaluated self-reported effects of HT. AIM: To evaluate self-reported symptoms during the first year of HT in trans people. METHODS: This study is part of the European Network for the Investigation of Gender Incongruence, a multicenter prospective cohort study. For this study, 205 trans women and 193 trans men from the gender clinics of Amsterdam, Ghent, and Florence, who were >18 years of age and started hormonal treatment were included. Questionnaires, self-developed based on the Menopause Rating scale and clinical experiences, were completed, and changes in symptom scores were analyzed using linear mixed models. MAIN OUTCOME MEASURES: Self-reported psycho vegetative symptoms, as well as physical, cognitive, emotional, sexual and genital complaints, and pain were evaluated at baseline and after 3, 6, and 12 months of HT using a 4-point Likert scale (no, mild, moderate, or severe complaints). RESULTS: In trans men, with a median age of 23, transient increases were reported in night sweats, weight gain, and clitoral pain. Persistent increases were reported for hot flashes, balding, voice instability, acne, and increase in sexual desire, whereas emotional instability, fear, and menses decreased. For trans women, with a median age of 29, hot flashes, night sweats, fatigue, weight gain, changes in olfactory sense, brittle nails, emotional instability, mood swings, and breast tenderness increased persistently during 12 months of HT, whereas a decrease was observed for balding and sexual desire. Sleeping difficulties decreased temporarily. No changes were observed in palpitations, dizziness, abdominal complaints, anxiety, panic attacks, cognition, and pain, except for clitoral and breast pain. CLINICAL IMPLICATIONS: Knowledge on the occurrence of these self-reported, subjective effects and their course over time may help physicians informing trans people starting with and during HT. STRENGTHS & LIMITATIONS: This study was performed in a large cohort of trans people. The follow-up period was limited to 12 months. CONCLUSION: Changes in self-reported symptoms were mentioned in all investigated areas, except cognition. Most symptoms were as expected and even desired, whereas others may be considered unpleasant by some trans people. van Dijk D, Dekker MJHJ, Conemans EB, et al. Explorative Prospective Evaluation of Short-Term Subjective Effects of Hormonal Treatment in Trans People-Results from the European Network for the Investigation of Gender Incongruence. J Sex Med 2019;16:1297-1309.

[A woman with a red demarcation of the tongue]. / Een vrouw met een rode streep over de tong.

A 68-year-old woman had a red demarcation of the tongue, without any discomfort. Because of the use of corticosteroid inhalations, it was thought that this was caused by median rhomboid glossitis, based on Candida albicans. Oral antimycotic therapy made the demarcation disappear, which confirmed the diagnosis.