Polycystic ovary syndrome: evidence for a primary disorder of ovarian steroidogenesis

Polycystic ovary syndrome (PCOS) is a very common endocrinopathy of uncertain aetiology in which the most consistent biochemical abnormality is hypersecretion of androgens. In this review, evidence is presented to support the view that a primary abnormality of ovarian androgen biosynthesis provides the basis for the syndrome. PCOS is a familiar disorder and we demonstrate, in molecular genetic studies, that CYP11a, the gene coding for P450 side chain cleavage, is a key susceptibility locus for development of hyperandrogenism.     

First Report of Phytoplasma (16SrI) Associated with Yellow Decline Disease of Royal Palms [Roystonea regia (Kunth) O. F. Cook] in Malaysia

Royal Palms (Roystonea regia) with symptoms such as severe chlorosis, stunting, collapse of older fronds and general decline were observed in the state of Selangor, Malaysia. Using polymerase chain reaction (PCR) amplification with phytoplasma universal primer pair P1/P7 followed by R16F2N/R16R2 and fU5/rU3 as nested PCR primer pairs, all symptomatic plants tested positively for phytoplasma. Results of phylogenetic and virtual RFLP analysis of the 16S rRNA gene sequences revealed that the phytoplasma associated with Royal Palm yellow decline (RYD) was an isolate of ‘Candidatus Phytoplasma asteris’ belonging to a new 16SrI‐subgroup. These results show that Roystonea regia is a new host for the aster yellows phytoplasma (16SrI). This is the first report on the presence of 16SrI phytoplasma on Royal Palm trees in Malaysia.     

Genotypic diversity of Iranian Cryptococcus neoformans using multilocus sequence typing (MLST) and susceptibility to antifungals

Molecular Biology Reports - Cryptococcus species is an opportunistic yeast pathogen and classified into different molecular types according to typing techniques including multilocus sequence typing...     

Prolonged viral shedding and antibody persistence in patients with COVID-19

SARS-CoV-2 as a new global threat has affected global population for one year. Despite the great effort to eradicate this infection, there are still some challenges including different viral presentation, temporal immunity in infected individuals and variable data of viral shedding. We studied 255 COVID-19 suspected individuals to assess the viral shedding duration and also the antibody development against SARS-CoV-2 among the cases. Real Time RT-PCR assay was applied to determine the virus presence and SARS-CoV-2 antibodies were evaluated using SARS-CoV-2 IgM and IgG kits. 113 patients were confirmed for COVID-19 infection. The patients were followed until negative PCR achieved. The median viral shedding among studied population was obtained 34.16 (±17.65) days which was not significantly associated with age, sex and underlying diseases. Shiver and body pain were found in prolonged form of the infection and also patients who had gastrointestinal problems experienced longer viral shedding. Moreover, IgG was present in 84% of patients after 150 days. According to this data, the median viral shedding prolongation was 34.16 days which indicates that 14 days isolation might not be enough for population. In addition, IgG profiling indicated that it is persistent in a majority of patients for nearly 6 months which has brought some hopes in vaccine efficacy and application.     

A dynamical systems model for combinatorial cancer therapy enhances oncolytic adenovirus efficacy by MEK-inhibition

Oncolytic adenoviruses, such as ONYX-015, have been tested in clinical trials for currently untreatable tumors, but have yet to demonstrate adequate therapeutic efficacy. The extent to which viruses infect targeted cells determines the efficacy of this approach but many tumors down-regulate the Coxsackievirus and Adenovirus Receptor (CAR), rendering them less susceptible to infection. Disrupting MAPK pathway signaling by pharmacological inhibition of MEK up-regulates CAR expression, offering possible enhanced adenovirus infection. MEK inhibition, however, interferes with adenovirus replication due to resulting G1-phase cell cycle arrest. Therefore, enhanced efficacy will depend on treatment protocols that productively balance these competing effects. Predictive understanding of how to attain and enhance therapeutic efficacy of combinatorial treatment is difficult since the effects of MEK inhibitors, in conjunction with adenovirus/cell interactions, are complex nonlinear dynamic processes. We investigated combinatorial treatment strategies using a mathematical model that predicts the impact of MEK inhibition on tumor cell proliferation, ONYX-015 infection, and oncolysis. Specifically, we fit a nonlinear differential equation system to dedicated experimental data and analyzed the resulting simulations for favorable treatment strategies. Simulations predicted enhanced combinatorial therapy when both treatments were applied simultaneously; we successfully validated these predictions in an ensuing explicit test study. Further analysis revealed that a CAR-independent mechanism may be responsible for amplified virus production and cell death. We conclude that integrated computational and experimental analysis of combinatorial therapy provides a useful means to identify treatment/infection protocols that yield clinically significant oncolysis. Enhanced oncolytic therapy has the potential to dramatically improve non-surgical cancer treatment, especially in locally advanced or metastatic cases where treatment options remain limited.     

An efficient in vitro refolding of recombinant bacterial laccase in Escherichia coli

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are important multicopper enzymes that are used in many biotechnological processes. A recombinant form of laccase from Bacillus sp. HR03 was overexpressed in Escherichia coli BL-21(DE3). Inclusion body (IB) formation happens quite often during recombinant protein production. Hence, developing a protocol for efficient refolding of proteins from inclusion bodies to provide large amounts of active protein could be advantageous for structural and functional studies. Here, we have tried to find an efficient method of refolding for this bacterial enzyme. Solubilization of inclusion bodies was carried out in phosphate buffer pH 7, containing 8 M urea and 4 mM β-mercaptoethanol and refolding was performed using the dilution method. The effect of different additives was investigated on the refolding procedure of denaturated laccase. Mix buffer (phosphate buffer and citrate buffer, 100 mM) containing 4 mM ZnSO₄ and 100 mM sorbitol was selected as an optimized refolding buffer. Also Kinetic parameters of soluble and refolded laccase were analyzed.     

Cloning, molecular characterization and expression of a DNA-ligase from a new bacteriophage: Phax1

DNA ligases join 3′ hydroxyl and 5′ phosphate ends in double stranded DNA and are necessary for maintaining the integrity of genome. The gene encoding a new Escherichia phage (Phax1) DNA ligase was cloned and sequenced. The gene contains an open reading frame with 1,428 base pairs, encoding 475 amino acid residues. Alignment of the entire amino acid sequence showed that Phax1 DNA ligase has a high degree of sequence homology with ligases from Escherichia (vB_EcoM_CBA120), Salmonella (PhiSH19 and SFP10), Shigella (phiSboM-AG3), and Deftia (phiW-14) phages. The Phax1 DNA ligase gene was expressed under the control of the T7lac promoter on the pET-16b (+) in Escherichia coli Rossetta gami. The enzyme was then homogeneously purified by a metal affinity column. Enzymatic activity of the recombinant DNA ligase was assayed by an in-house PCR-based method.     

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (K_m) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.     

Hospital factors associated with maternal and neonatal outcomes of deliveries to patients with a previous cesarean delivery: an ecological study

Background:Recommendations for deliveries of pregnant patients with a previous cesarean delivery and the type of hospitals deemed safe for these deliveries have evolved in recent years, although no studies have examined hospital factors and associated safety. We sought to evaluate maternal and neonatal outcomes among patients with a previous cesarean delivery by hospital tier and volume.Methods:We carried out an ecological study of singleton live births delivered at term gestation to patients with a previous cesarean delivery in all Canadian hospitals (excluding Quebec), 2013–2019. We obtained data from the Discharge Abstract Database of the Canadian Institute for Health Information. The primary outcomes were severe maternal morbidity or mortality (SMMM), and serious neonatal morbidity or mortality (SNMM). We used regression modelling to examine hospital tier (tier 4 hospitals being those that provide the highest level of care) and volume; we also identified hospitals with high rates of SMMM and SNMM using within-tier comparisons and comparisons with the overall rate.Results:We included 235 442 deliveries to patients with a previous cesarean delivery; SMMM and SNMM rates were 14.6 per 1000 deliveries and 4.6 per 1000 live births, respectively. Among patients with a parity of 1, SMMM rates were lower in tier 1 hospitals (adjusted incidence rate ratio [IRR] 0.68, 95% confidence interval [CI] 0.52–0.89) and higher in tier 4 hospitals (adjusted IRR 1.41, 95% CI 1.05–1.91) than in tier 2 hospitals; SNMM rates did not differ by hospital tier. Rates of SNMM increased with increasing hospital volume (adjusted IRR 1.02, 95% CI 1.00–1.04) and increasing rates of vaginal birth after cesarean delivery (adjusted IRR 1.02, 95% CI 1.01–1.04). Most hospitals had relatively low SMMM and SNMM rates, although a few hospitals in each tier and volume category had significantly higher rates than others.Interpretation:Adverse maternal and neonatal outcomes among patients with a previous cesarean delivery showed no clear pattern of decreasing SMMM and SNMM with increasing tiers of service and hospital volume. All hospitals, irrespective of tier or size, should continually review their rates of adverse maternal and neonatal outcomes.

The approach to delivery for patients with a previous cesarean delivery has undergone substantial changes over the last few decades in Canada, the United States and elsewhere. Rates of vaginal birth after cesarean delivery (VBAC) in Canada increased rapidly, from less than 5% of deliveries in the late 1970s to peak at more than 35% in the mid-1990s.^1–4 However, this trend reversed sharply after studies in the mid-1990s showed that attempted VBAC (as opposed to elective repeat cesarean delivery) was associated with higher rates of severe maternal morbidity and of fetal and infant morbidity and mortality.^5–9 The decline has partly reversed after the release of a guideline from the Society of Obstetricians and Gynaecologists of Canada (SOGC) in 2005, and the National Institutes of Health Consensus Development Conference in 2010, which affirmed that patients with 1 previous transverse lower-segment cesarean section and no contraindications could be offered a trial of labour with appropriate discussion of risks and benefits.^10,11Recommendations regarding hospitals deemed safe for delivering patients with a previous cesarean delivery have also evolved. The 1998 and 1999 guidelines of the American College of Obstetricians and Gynecologists (ACOG) required hospitals attempting trials of labour to have “ready availability of emergency care” or “immediate availability of emergency care.”^12,13 After the publication of these guidelines, about 30% of hospitals in the US stopped offering trial-of-labour services to patients with a previous cesarean delivery because they could not provide immediate surgical and anesthesia services, which compelled many patients who had opted for a trial of labour to travel to hospitals far from their homes and families.^14–16 More recently, guidelines have attempted to balance clinical safety with the challenges associated with such social disruption. The current ACOG guideline states that a trial of labour can be attempted in a level 1 maternity care facility (i.e., a hospital providing basic obstetric services), which has “the ability to begin emergency cesarean delivery within a time interval that best incorporates maternal and fetal risks and benefits.”¹⁷ Similarly, the current SOGC guideline states that hospitals providing trial-of-labour services should have “the resources to perform an emergency cesarean section.”¹⁸ This change in recommendations has led to an increase in the number of hospitals that offer trials of labour, though concerns about inadequate access to such delivery options persist.^19,20Although clinical guidelines regarding hospitals deemed safe for delivering patients with a previous cesarean delivery have changed in recent years, to our knowledge, no studies have evaluated hospital factors and associated safety issues. We sought to evaluate maternal and infant outcomes of deliveries to patients with a previous cesarean delivery by tier of obstetric service and hospital delivery volume.