Mcl-1 is a key regulator of the ovarian reserve

A majority of ovarian follicles are lost to natural death, but the disruption of factors involved in maintenance of the oocyte pool results in a further untimely follicular depletion known as premature ovarian failure. The anti-apoptotic B-cell lymphoma 2 (Bcl-2) family member myeloid cell leukemia-1 (MCL-1) has a pro-survival role in various cell types; however, its contribution to oocyte survival is unconfirmed. We present a phenotypic characterization of oocytes deficient in Mcl-1, and establish its role in maintenance of the primordial follicle (PMF) pool, growing oocyte survival and oocyte quality. Mcl-1 depletion resulted in the premature exhaustion of the ovarian reserve, characterized by early PMF loss because of activation of apoptosis. The increasingly diminished surviving cohort of growing oocytes displayed elevated markers of autophagy and mitochondrial dysfunction. Mcl-1-deficient ovulated oocytes demonstrated an increased susceptibility to cellular fragmentation with activation of the apoptotic cascade. Concomitant deletion of the pro-apoptotic Bcl-2 member Bcl-2-associated X protein (Bax) rescued the PMF phenotype and ovulated oocyte death, but did not prevent the mitochondrial dysfunction associated with Mcl-1 deficiency and could not rescue long-term breeding performance. We thus recognize MCL-1 as the essential survival factor required for conservation of the postnatal PMF pool, growing follicle survival and effective oocyte mitochondrial function.Estimates of the human primordial follicle (PMF) reservoir, the size of which dictates the extent of the ovarian reserve, indicates the presence of at least half a million oocytes per ovary at birth.^{1, 2} The essential decision that PMFs face is either long-term arrest with a possibility of recruitment toward the growing pool, or death. Even upon recruitment to the growing pool, intricately orchestrated crosstalk of survival signals between ovarian somatic cells and oocytes facilitate the ovulation of a single oocyte in human in each cycle. Hence, the default fate for millions of ovarian germ cells is death, as only a small fraction survive till ovulation.³ Insufficient endowment during fetal development or excessive oocyte loss during postnatal life further limits the ovarian reserve and can result in an untimely exhaustion of the follicle pool leading to premature ovarian failure (POF); a syndrome that affects around 1% of all women, with a higher prevalence (up to 30%) in families with heritable traits of this condition.^{4, 5} Mechanisms responsible for maintenance of the follicular reserve are poorly understood, however, biological assessments and mathematical modeling reveal that progressive loss of follicles with age is non-linear and accelerates, especially after 38 years.^{6, 7} With a declining ovarian reserve, poor oocyte quality is an additional factor that contributes to the reduced fertility associated with increased maternal age. Oocytes and resulting embryos of older mothers have increased rates of aneuploidies likely due to defects in chromosomal cohesion and meiotic spindle stability, decreased DNA repair capacity, altered gene expression, impaired mitochondrial function and elevated cellular redox, all contributing to increased rates of cell death.^{8, 9, 10}The marked decline of oocyte number in mammalian ovaries has been attributed to oocyte loss via stage-specific modes of death. As yet, perinatal PMF loss in mice most frequently engages apoptotic cell death,^{11, 12} whereas within the postnatal ovary, oocytes in growing follicles undergo atresia, a less ''molecularly'' defined death, carrying hallmarks of both apoptosis and autophagy.^{13, 14, 15} It is thus surprising that no member of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) family has been identified with a definitive role in governing oocyte survival and the maintenance of the ovarian reserve. Bcl-2l2/Bcl-w and Bcl-2-l10/Diva deficiency had no apparent impact on the ovarian reserve, and although ablation of Bcl-2 led to a loss of one-third of the adult PMF pool, the growing follicle pool was not significantly impacted and these animals did not undergo POF.^{16, 17, 18, 19} Conditional Bcl-x (Bcl-2l1) inactivation led to increased primordial germ cell apoptosis in the embryo,²⁰ but postnatal inactivation of Bcl-x in oocytes did not compromise the ovarian reserve in young females.²¹ Bcl2a1a/Bfl-1/A1 was low to undetectable in fully grown germinal vesicle (GV) or ovulated murine oocytes,²² however, the impact of Bfl-1 deficiency on the ovarian reserve has not yet been analyzed to the best of our knowledge. Consequently, either various anti-apoptotic Bcl-2 members have overlapping roles in governing postnatal oocyte survival and maintenance of the adult ovarian reserve in mice, or the anti-apoptotic Bcl-2 member that regulates this decision has yet to be identified.     

Distension of the esophagogastric junction augments triggering of transient lower esophageal sphincter relaxation

Patients with gastroesophageal reflux disease show an increase in esophagogastric junction (EGJ) distensibility and in frequency of transient lower esophageal sphincter relaxations (TLESR) induced by gastric distension. The objective was to study the effect of localized EGJ distension on triggering of TLESR in healthy volunteers. An esophageal manometric catheter incorporating an 8-cm internal balloon adjacent to a sleeve sensor was developed to enable continuous recording of EGJ pressure during distension of the EGJ. Inflation of the balloon doubled the cross-section of the trans-sphincteric portion of the catheter from 5 mm OD (round) to 5 × 11 mm (oval). Ten healthy subjects were included. After catheter placement and a 30-min adaptation period, the EGJ was randomly distended or not, followed by a 45-min baseline recording. Subjects consumed a refluxogenic meal, and recordings were made for 3 h postprandially. A repeat study was performed on another day with EGJ distension status reversed. Additionally, in one subject MRI was performed to establish the exact position of the balloon in the inflated state. The number of TLESR increased during periods of EGJ distension with the effect being greater after a meal [baseline: 2.0(0.0-4.0) vs. 4.0(1.0-11.0), P=0.04; postprandial: 15.5(10.0-33.0) vs. 22.0(17.0-58.0), P=0.007 for undistended and distended, respectively]. EGJ distension augments meal-induced triggering of TLESR in healthy volunteers. Our data suggest the existence of a population of vagal afferents located at sites in/around the EGJ that may influence triggering of TLESR.     

Upper esophageal sphincter impedance as a marker of sphincter opening diameter

The measurement of the physical extent of opening of the upper esophageal sphincter (UES) during bolus swallowing has to date relied on videofluoroscopy. Theoretically luminal impedance measured during bolus flow should be influenced by luminal diameter. In this study, we measured the UES nadir impedance (lowest value of impedance) during bolus swallowing and assessed it as a potential correlate of UES diameter that can be determined nonradiologically. In 40 patients with dysphagia, bolus swallowing of liquids, semisolids, and solids was recorded with manometry, impedance, and videofluoroscopy. During swallows, the UES opening diameter (in the lateral fluoroscopic view) was measured and compared with automated impedance manometry (AIM)-derived swallow function variables and UES nadir impedance as well as high-resolution manometry-derived UES relaxation pressure variables. Of all measured variables, UES nadir impedance was the most strongly correlated with UES opening diameter. Narrower diameter correlated with higher impedance (r = -0.478, P < 0.001). Patients with <10 mm, 10-14 mm (normal), and ≥ 15 mm UES diameter had average UES nadir impedances of 498 ± 39 Ohms, 369 ± 31 Ohms, and 293 ± 17 Ohms, respectively (ANOVA P = 0.005). A higher swallow risk index, indicative of poor pharyngeal swallow function, was associated with narrower UES diameter and higher UES nadir impedance during swallowing. In contrast, UES relaxation pressure variables were not significantly altered in relation to UES diameter. We concluded that the UES nadir impedance correlates with opening diameter of the UES during bolus flow. This variable, when combined with other pharyngeal AIM analysis variables, may allow characterization of the pathophysiology of swallowing dysfunction.     

Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design

HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end complex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406-415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs.     

Out-of-Pocket Costs and Other Determinants of Access to Healthcare for Children with Febrile Illnesses: A Case-Control Study in Rural Tanzania

Objectives

To study private costs and other determinants of access to healthcare for childhood fevers in rural Tanzania.

Methods

A case-control study was conducted in Tanzania to establish factors that determine access to a health facility in acute febrile illnesses in children less than 5 years of age. Carers of eligible children were interviewed in the community; cases were represented by patients who went to a facility and controls by those who did not. A Household Wealth Index was estimated using principal components analysis. A multivariable logistic regression analysis was performed to understand the factors which influenced attendance of healthcare facility including severity of the illness and household wealth/socio-demographic indicators. To complement the data on costs from community interviews, a hospital-based study obtained details of private expenditures for hospitalised children under the age of 5.

Results

Severe febrile illness is strongly associated with health facility attendance (OR: 35.76, 95%CI: 3.68-347.43, p = 0.002 compared with less severe febrile illness). Overall, the private costs of an illness for patients who went to a hospital were six times larger than private costs of controls ($5.68 vs. $0.90, p<0.0001). Household wealth was not significantly correlated with total costs incurred. The separate hospital based cost study indicated that private costs were three times greater for admissions at the mission versus public hospital: $13.68 mission vs. $4.47 public hospital (difference $ 9.21 (95% CI: 7.89 -10.52), p<0.0001). In both locations, approximately 50% of the cost was determined by the duration of admission, with each day in hospital increasing private costs by about 12% (95% CI: 5% - 21%).

Conclusion

The more severely ill a child, the higher the probability of attending hospital. We did not find association between household wealth and attending a health facility; nor was there an association between household wealth and private cost.     

Preparation and Characterization of a Novel Co-processed Excipient of Chitin and Crystalline Mannitol

A co-processed excipient was prepared from commercially available crystalline mannitol and α-chitin using direct compression as well as spray, wet, and dry granulation. The effect of the ratio of the two components, percentage of lubricant and particle size, on the properties of the prepared co-processed excipient has been investigated. α-Chitin forms non-hygroscopic, highly compactable, disintegrable compacts when co-processed with crystalline mannitol. The compaction properties of the co-processed mannitol–chitin mixture were found to be dependent upon the quantity of mannitol added to chitin, in addition to the granulation procedure used. Optimal physicochemical properties of the excipient, from a manufacturing perspective, were obtained using a co-processed mannitol–chitin (2:8, w/w) mixture prepared by wet granulation (Cop-MC). Disintegration time, crushing strength, and friability of tablets, produced from Cop-MC using magnesium stearate as a lubricant, were found to be independent of the particle size of the prepared granules. The inherent binding and disintegration properties of the compressed Cop-MC are useful for the formulation of poorly compressible, high-strength, and low-strength active pharmaceutical ingredients. The ability to co-process α-chitin with crystalline mannitol allows chitin to be used as a valuable industrial pharmaceutical excipient.     

Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics

Understanding the basis for differential responses to drug therapies remains a challenge despite advances in genetics and genomics. Induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to investigate the pharmacology of disease processes in therapeutically and genetically relevant primary cell types in vitro and to interweave clinical and basic molecular data. We report here the derivation of iPSCs from a long QT syndrome patient with complex genetics. The proband was found to have a de novo SCN5A LQT-3 mutation (F1473C) and a polymorphism (K897T) in KCNH2, the gene for LQT-2. Analysis of the biophysics and molecular pharmacology of ion channels expressed in cardiomyocytes (CMs) differentiated from these iPSCs (iPSC-CMs) demonstrates a primary LQT-3 (Na⁺ channel) defect responsible for the arrhythmias not influenced by the KCNH2 polymorphism. The F1473C mutation occurs in the channel inactivation gate and enhances late Na⁺ channel current (I_NaL) that is carried by channels that fail to inactivate completely and conduct increased inward current during prolonged depolarization, resulting in delayed repolarization, a prolonged QT interval, and increased risk of fatal arrhythmia. We find a very pronounced rate dependence of I_NaL such that increasing the pacing rate markedly reduces I_NaL and, in addition, increases its inhibition by the Na⁺ channel blocker mexiletine. These rate-dependent properties and drug interactions, unique to the proband’s iPSC-CMs, correlate with improved management of arrhythmias in the patient and provide support for this approach in developing patient-specific clinical regimens.     

Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution

Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, ɸ12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from ɸ6, ɸ8 and ɸ13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in ɸ8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in ɸ12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the ɸ12 enzyme.