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.     

Expression pattern of olfactory receptor genes in human cumulus cells as an indicator for competent oocyte selection

Odorant or olfactory receptors are mainly localized in the olfactory epithelium for the perception of different odors. Interestingly, many ectopic olfactory receptors with low expression levels have recently been found in nonolfactory tissues to involve in local functions. Therefore, we investigated the probable role of the olfactory signaling pathway in the surrounding microenvironment of oocyte. This study included 22 women in intracytoplasmic sperm injection cycle. The expression of olfactory target molecules in cumulus cells surrounding the growing and mature oocytes was evaluated by Western blotting and real-time polymerase chain reaction. Additionally, integrated bioinformatics analyses were carried out and 6 ectopic olfactory receptors were selected for further evaluation. The initiation of olfactory transduction cascade in cumulus cells of competent oocytes was confirmed by analyzing the expression of adenylyl cyclase type 3 and olfactory market protein. Moreover, the expression pattern of the selected olfactory receptors was evaluated and OR10H2 was selected due to a high level of expression in mature fertile oocytes. We suggested that OR10H2 could be considered as a reliable biomarker for oocyte selection in assisted reproduction technique programs. However, further studies are required to elucidate the role of olfactory transduction cascade in embryo quality and implantation.     

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.     

Effects of pioglitazone and metformin on beta-cell function in nondiabetic subjects at high risk for type 2 diabetes

Thiazolidinediones (TZDs) and metformin decreased the incidence of diabetes in subjects at risk for developing diabetes and improved peripheral or hepatic insulin sensitivity, respectively. Whether they also directly improved beta-cell function is not clear. In vitro studies showed improved beta-cell function in response to TZDs and metformin; however, the effects of TZDs or metformin on beta-cell function in humans are still uncertain. We hypothesized that both TZDs and metformin directly affect beta-cell function. We evaluated beta-cell function and insulin sensitivity (S(I)) in subjects with impaired glucose tolerance or a history of gestational diabetes using oral and intravenous glucose tolerance tests in addition to the glucose-potentiated arginine stimulation test. In contrast to metformin, pioglitazone improved S(I), glucose tolerance, and insulin-independent glucose disposal [glucose effectiveness (S(G))]. Neither pioglitazone nor metformin significantly improved beta-cell compensation for insulin resistance [disposition index (DI)], but the change in DI significantly correlated with baseline S(I). Insulin secretion in response to arginine at maximally potentiating glucose levels (AIR(max)) tended to increase after metformin and to decrease after pioglitazone; however, when adjusted for S(I), the changes were not significant. Our results demonstrate that, in nondiabetic subjects at risk for diabetes, pioglitazone, but not metformin, significantly improved glucose tolerance by improving S(I) and S(G). We did not find any evidence that either pioglitazone or metformin improved beta-cell function. Improved beta-cell compensation was observed primarily in the subgroup of subjects that had the lowest S(I) at baseline.     

De Novo Nonsense Mutations in KAT6A,a Lysine Acetyl-Transferase Gene,Cause a Syndrome Including Microcephaly and Global Developmental Delay

Chromatin remodeling through histone acetyltransferase (HAT) and histone deactylase (HDAC) enzymes affects fundamental cellular processes including the cell-cycle, cell differentiation, metabolism, and apoptosis. Nonsense mutations in genes that are involved in histone acetylation and deacetylation result in multiple congenital anomalies with most individuals displaying significant developmental delay, microcephaly and dysmorphism. Here, we report a syndrome caused by de novo heterozygous nonsense mutations in KAT6A (a.k.a., MOZ, MYST3) identified by clinical exome sequencing (CES) in four independent families. The same de novo nonsense mutation (c.3385C>T [p.Arg1129^∗]) was observed in three individuals, and the fourth individual had a nearby de novo nonsense mutation (c.3070C>T [p.Arg1024^∗]). Neither of these variants was present in 1,815 in-house exomes or in public databases. Common features among all four probands include primary microcephaly, global developmental delay including profound speech delay, and craniofacial dysmorphism, as well as more varied features such as feeding difficulties, cardiac defects, and ocular anomalies. We further demonstrate that KAT6A mutations result in dysregulation of H3K9 and H3K18 acetylation and altered P53 signaling. Through histone and non-histone acetylation, KAT6A affects multiple cellular processes and illustrates the complex role of acetylation in regulating development and disease.